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8040370
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Retrospective studies on the measurements, karyotyping and pathology of reproductive organs of bovine freemartins.
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A retrospective study of 217 cases of bovine freemartinism was made. Of these, 14 cases were diagnosed clinically, 141 were karyotyped and 62 were subjected to macroscopical examination of the reproductive tract. Of the 62 cases, 43 were assessed by measuring the reproductive organs (size and/or weight) and 41 were examined microscopically. Complete information on gross, histopathological and karyotyping analysis was available for only 10 animals. Freemartins showed reduced measurements of all reproductive organs. The mean lengths of vulva and vagina were 6.4 cm and 8.1 cm, respectively. Vesicular glands were observed in 30 freemartins. Histologically, these glands had more fibrous and less glandular tissue than the normal male vesicular glands. Uterine changes were variable, ranging from aplasia (two animals) to normal. Likewise, endometrial glands ranged from hypoplastic (decreased number and size) to normal. Glandular and myometrial cells showed prominent nuclei and less than normal cytoplasm, indicating a non-functional status. Gonadal size was reduced and an epididymis was present in seven animals. Rete ovarii were prominent and frequently occupied two-thirds of the gonad. Undifferentiated sex cords and abundant interstitial cells were salient features of the freemartin gonad. Oocytes were not observed. Ovarian follicles were seen in six cases. Karyotypic analyses demonstrated XY chimaerism in all freemartins.
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8040369
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Diet-induced lipofuscin and ceroid formation in growing pigs.
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Seven piglets, aged 2 weeks at the beginning of the experiment, were fed a diet lacking antioxidants (vitamin E and selenium) and enriched with thermally oxidated cod liver oil (the peroxide value of which was 310 meqv/kg). From one month before to 4 weeks after farrowing (weaning) the sow also received the experimental diet. One pig died at 9 days of age, due to degeneration of the parenchymal tissues. The oxidative stress produced was aggravated in four of the seven remaining pigs by giving them an injection of iron-dextran (300 mg) one day before the animals were killed for necropsy at the age of 2 months. The three other piglets were given an injection of a control saline solution. The nutritional myodegeneration produced ('white muscle disease') was characterized by a pale, yellowish colour and translucence of skeletal muscle, with degeneration, including swelling of the muscle fibres and slackening of the fibrillar pattern. The arrangement of nuclei in chains indicated repair. Ultrastructurally, skeletal muscle showed vacuolization of the sarcoplasmic reticulum, irregularities in the mitochondria and focal dissolution of myofibrils. The sarcoplasmic reticulum displayed condensation and lamellar formations. Hyaline degeneration of the heart muscle occurred both in iron-treated and control pigs, and in six animals a yellowish pigment resembling lipofuscin and ceroid was observed. Ultrastructurally, the ceroid deposits appeared as myelin-like figures. The livers were pale and brittle, four with centrilobular degeneration. By light microscopy, lipidosis was distinguished in four and ceroid accumulations in three pigs. Thiobarbituric-acid-reactive substances, creatine kinase activity and iron increased significantly in the serum as a result of the injection of iron-dextran.
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8040368
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Changes in arginase, aminotransferases and rhodanese in sera of domestic animals with experimentally induced liver necrosis.
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Changes in the serum concentrations of aspartic aminotransferase (AST), alanine aminotransferase (ALT), rhodanese and arginase were measured in dogs, sheep and cattle with hepatic necrosis induced by the oral administration of carbon tetrachloride. A new method for arginase assay was based on the determination of remaining arginine (after its conversion to urea and ornithine) by its reaction with p-nitrophenyl glyoxal (PNPG). In all species studied the serum arginase increased 6-12 h after liver damage, reached a peak value in 48 h and returned to normal thereafter. Rhodanese activity did not change in dogs but rose significantly in sheep and, to a lesser extent, in cattle. AST increased strikingly in sheep as compared with dogs and cattle and remained high for > 5 days. In dogs ALT rose sharply and remained elevated for > 10 days. No change in ALT was seen in sheep or cattle. The determination of arginase by a simple procedure such as the PNPG method, in conjunction with AST or ALT assay, may be of value in assessing the stage of liver necrosis.
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8040367
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Nitric oxide producing neurons in the monkey and human digestive system.
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Nitric oxide has been proposed as an inhibitory transmitter molecule that plays a role in muscle relaxation and vasodilation in the gastrointestinal tract. The present study analyzes the distribution of nitric-oxide-producing neurons in the monkey and human digestive system by means of nicotinamide-adenine-dinucleotide-phosphate-diaphorase histochemistry. This histochemical method is reliable and convenient for the visualization of neuronal nitric-oxide synthase, the enzyme responsible for nitric-oxide generation. In the gastrointestinal tract, nitric-oxide-synthase-related diaphorase activity was present in nerve fibers running throughout the muscular layer (circular > longitudinal) and in numerous ganglion cells and processes in the myenteric plexus of monkeys and humans. Labelled ganglion cells and fibers also were observed in the submucous plexus, although they were much less numerous than those seen in the myenteric plexus. In the submucosa, a few positive fibers were seen around blood vessels. In the mucosa, stained fibers were sparse at the base of the villi and crypts, whereas they were quite abundant in the muscularis mucosae, especially in the small intestine and colon. In the gallbladder (human), labelling was found in ganglion cells and processes of the innermost and outermost ganglionated plexuses. Stained fibers also were distributed to the muscular layer and, less abundantly, to the mucosa and vasculature. Labelled fibers were more abundant in the sphincter of Oddi (human) than in the gallbladder. In the monkey and human pancreas, nicotinamide-adenine-dinucleotide-diaphorase staining was seen mainly in ganglion cells and fibers of intrapancreatic ganglia, and in processes running among acini, around ducts and in the stroma. A moderate density of stained fibers also was distributed to the vasculature, whereas the islets showed few positive processes. Finally, double label experiments performed in the pancreas showed that the vast majority of neurons producing nitric oxide are immunoreactive for vasoactive intestinal peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
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8040366
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Undernutrition of rats during early life does not affect the total number of cortical neurons.
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Undernutrition during early life is known to cause deficits and distortions in brain structure. However, it remains uncertain whether this includes a diminution of the total numbers of neurons. Recent advances in stereological techniques have made it possible to obtain unbiased estimates of total numbers of cells in well-defined biological structures. Rats were undernourished from day 16 of gestation to 30 postnatal days of age by standardized procedures. These rats and well-fed control rats were anaesthetized and killed by intracardiac perfusion with fixatives at 70 days of age. The left cerebral hemisphere from each animal was embedded in Paraplast and serially sectioned. The sections were analyzed via the Cavalieri principle to obtain the total cortical volume and by the "disector" method to estimate the numerical density of neurons in the cortex. These values were later used to compute estimates of the total number of cortical neurons for each animal. Well-fed control rats had 26.9 million cortical neurons, while the previously undernourished animals had 24.8 million. The difference between these two groups was not statistically significant. It therefore appears that undernutrition of rats during early postnatal life does not affect the total numbers of neurons in the cerebral cortex.
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8040365
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Intrinsic cortical connections in macaque visual area V2: evidence for interaction between different functional streams.
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Area V2 of macaque visual cortex represents an important but poorly understood stage in visual processing. To provide a better understanding of the region, we studied the organization of its intrinsic cortical connections by making focal (200-300 microns) iontophoretic microinjections of the tracer biocytin. Alternate tissue sections were tested for biocytin, cytochrome oxidase (CO), or Cat-301 immunoreactivity to localize biocytin label relative to the three stripelike compartments that characterize this area. Biocytin-labeled pyramidal neurons of layers 2/3, and, to a lesser extent, layer 5, provided laterally spreading axon projections that terminated in discrete patches (250-300 microns diameter), primarily in layers 1-3. Any injected locus in V2 projected to 10-15 similarly sized patches, up to 4 mm from the injection site, and distributed in an elongated field orthogonal to the stripe compartments. We noted prominent patchy connections within, as well as between, individual compartments, perhaps reflecting functional substructures within stripes. Each stripe compartment projected to all three compartments but with different relative frequencies; CO-rich compartments projected mainly to other CO-rich compartments (75%), whereas CO-poor compartments projected equally to CO-rich and CO-poor compartments. We therefore emphasize the existence of substantial interconnections among all three V2 compartments. As further evidence for crosstalk between visual channels, we also noted an input to the V2 "thick" CO stripes from V1 cells in layer 4A as a distinct population in addition to the neurons of layer 4B. Thus, the CO stripe architecture may not be a marker for strictly segregated parallel visual pathways through V2.
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8040364
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Dendritic architecture of hypoglossal motoneurons projecting to extrinsic tongue musculature in the rat.
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The tracer, cholera toxin-horseradish peroxidase, was used to determine the dendritic architecture and organization of hypoglossal motoneurons in the rat. In 22 animals, the tracer was injected unilaterally into either the geniohyoid, genioglossus, hyoglossus, or styloglossus muscle. Within the hypoglossal nucleus, motoneurons innervating the extrinsic tongue muscles were functionally organized. Geniohyoid and genioglossus motoneurons were located within the ventrolateral and ventromedial subnuclei, respectively, while hyoglossus and styloglossus motoneurons were located within the dorsal subnucleus. Motoneurons located in all subnuclear divisions were found to have extensive dendrites that extended laterally into the adjacent reticular formation and medially to the ependyma. Less extensive extranuclear dendritic projections were found in the dorsal vagal complex and median raphe. Prominent rostrocaudal and mediolateral dendritic bundling was evident within the ventral subnuclei and dorsal subnucleus, respectively. Dendritic projections were also found extending inter- and intrasubnuclearly with a distinct pattern for each muscle. These data suggest that the varied and extensive dendritic arborizations of hypoglossal motoneurons provide the potential for a wide range of afferent contacts for, and interactions among, motoneurons that could contribute to the modulation of their activity. Specifically, the prominent dendritic bundling may provide an anatomic substrate whereby motoneurons innervating a specific muscle receive and integrate similar afferent input and are thus modulated as a functional unit. In contrast, the extensive intermingling of both inter- and intrasubnuclear dendrites within the hypoglossal nucleus may provide a mechanism for the coordination of different muscles, acting synergistically or antagonistically to produce a tongue movement.
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8040363
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Distribution of choline acetyltransferase immunoreactivity in the pigeon brain.
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We have investigated the distribution of cholinergic perikarya and fibers in the brain of the pigeon (Columba livia). With this aim, pigeon brain sections were processed immunohistochemically by using an antiserum specific for chicken choline acetyltransferase. Our results show cholinergic neurons in the pigeon basal telencephalon, the hypothalamus, the habenula, the pretectum, the midbrain tectum, the dorsal isthmus,the isthmic tegmentum, and the cranial nerve motor nuclei. Cholinergic fibers were prominent in the dorsal telencephalon, the striatum, the thalamus, the tectum, and the interpeduncular nucleus. Comparison of our results with previous studies in birds suggests some major cholinergic pathways in the avian brain and clarifies the possible origin of the cholinergic innervation of some parts of the avian brain. In addition, comparison of our results in birds with those in other vertebrate species shows that the organization of the cholinergic systems in many regions of the avian brain (such as the basal forebrain, the epithalamus, the isthmus, and the hindbrain) is much like that in reptiles and mammals. In contrast, however, birds appear largely to lack intrinsic cholinergic neurons in the dorsal ("neocortex-like") parts of the telencephalon.
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8040362
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Immunocytochemical localization of immunoglobulins in the rat brain: relationship to the blood-brain barrier.
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The central nervous system has been traditionally regarded as an immunologically privileged area. This feature has been in part attributed to the blood-brain barrier, which provides a restrictive interface to circulating immunoglobulins (IgG). Recent kinetic studies suggest that the barrier to immune proteins is not absolute, but rather may be regulated by a specific transfer mechanism. In this study, we confirm the presence of IgG in the central nervous system by immunocytochemistry and demonstrate a close anatomical relationship between the distribution of this protein and the blood-brain barrier. IgG was immunolocalized in the normal rat brain by using monoclonal and polyclonal antibodies to IgG and its subclasses. On the basis of an initial evaluation, the most appropriate antibodies and dilutions were selected for subsequent analyses. In the first study, IgG and albumin were immunolocalized in adjacent sections. In the second study, horseradish peroxidase (HRP) was given intravenously prior to sacrifice, in order to examine artifacts related to perfusion fixation. The distribution of HRP and IgG was then examined in adjacent sections. In the third study, IgG was immunolocalized in sections of brain after mild traumatic head injury. A monoclonal antibody to IgG2a and a polyclonal antibody to IgG were selected on the basis of specificity and consistent, mutual localization. Distinct, patchy, perivascular staining, infrequently associated with labeled neurons, was noted throughout the brain. Electron microscopy confirmed the perivascular localization; IgG was localized along the basal lamina of microvasculature and within the adjacent parenchyma. Albumin and HRP did not exhibit a similar pattern of perivascular immunostaining. After head injury, prominent immunostaining for IgG was observed in the injured hemisphere. In summary, these data indicate that the normal rat brain contains IgG, which dramatically increases after head injury. The distinct perivascular distribution in the normal brain suggests local microvascular permeability. This permeability is selective for IgG, since albumin does not share a similar perivascular localization. The neuronal staining which is closely associated with perivascular label may reflect one intracellular route for extravasated IgG.
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8040353
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Radioimmunoassay for the testosterone 5 alpha-reductase inhibitor turosteride in biological fluids.
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An antiserum against turosteride (code name FCE 26073), a potent testosterone 5 alpha-reductase inhibitor, has been raised in rabbits by immunization with an immunogen produced by conjugation of a derivative of FCE 26073 (FCE 27424) to bovine serum albumin. The antiserum was able to distinguish FCE 26073 from its derivatives modified at the 17 beta position and from all the endogenous steroids tested. A radioimmunoassay for the determination of FCE 26073 in human plasma and urine was developed using this antiserum and tritium labeled turosteride. FCE 26073 was extracted from 50 microliters of plasma or 25 microliters of urine using ethyl-ether with a recovery greater than 90%. Using this procedure it was possible to achieve a final limit of quantitation of 142 pg/ml in plasma and 284 pg/ml in urine. The assay was validated in terms of reproducibility, accuracy and precision in the range 3.9-250 pg/50 microliters of plasma and 25 microliters of urine. The plasma concentration of FCE 26073 in a healthy male volunteer who received 0.2 mg of the drug was measured using the radioimmunoassay.
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8040352
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Concentrations of thymulin in unextracted serum from pigs, sheep and cattle as measured by ELISA.
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These studies were conducted to develop an ELISA for measurement of thymulin concentrations in unextracted blood serum (or plasma) from domestic animal species (pigs, sheep and cows). This assay was quite variable (intraassay C.V. of 13.3 and 6.4% at 12.6 and 50.5 pg/mL and interassay C.V. of 24.2%). Serial dilutions of serum from these species produced inhibition curves parallel to the reference standard, suggesting that there were no substances in serum causing non-specific interference in the assay. In addition, none of the other thymic peptides tested resulted in problematic displacement of thymulin binding to the antiserum. Using this assay, it was found that somatotropin (ST) treatment had no effect on serum thymulin concentrations in either pigs or cows. Chromatographic separation of thymulin activity in sheep serum showed three peaks with approximate MW estimates of 95, 80 and 1 kDa. Serum thymulin concentrations in a sheep injected with thymulin was cleared from blood with a half-life (t1/2) of 10.3 +/- 0.6 min. Serum thymulin concentrations increased between birth and 6 mo old in pigs. These data indicate that a rapid and reliable ELISA has been developed to measure thymulin in blood of these domestic animals. This assay should be of value in the study of thymulin function and factors regulating its secretion.
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8040351
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Development and validation of a sensitive and specific radioimmunoassay for the determination of cicaprost in biological samples.
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Cicaprost is a potent, chemically and metabolically stable PGI2-mimetic. Pharmacodynamic effects were observed after oral administration of approximately 10 micrograms in man when plasma levels were in the low pg-range. The present report describes the development of a selective antiserum and a tracer with high specific activity and their use for the RIA determination of Cicaprost in biological samples. Cicaprost-[3H]-methylester with a specific activity of 819 GBq/mmol was used as a tracer. RIA-analyses were carried out with 0.05-0.5 ml plasma adjusted to pH 2 with 1 N HCl and extracted with 2.5 ml diethylether. Separation of antiserum bound and unbound Cicaprost was achieved by the charcoal method. Extraction recovery of Cicaprost was approximately 90% at pH approximately 2. The detection limit of the assay was 10-20 pg/ml plasma. Coefficients of variations were 6, 3 and 9% (within-day, n = 5) and 25, 12 and 10% (day-to-day, n = 11) at 50, 100 and 200 pg/ml. HPLC-chromatograms of plasma extracts did not reveal any peak apart from Cicaprost, demonstrating the specificity of the method. The present RIA for Cicaprost exhibits high specificity and sensitivity and will be used for further bioanalyses in pharmacokinetic study.
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8040349
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A competitive enzyme immunoassay Subclass for the determination of total IgG-subclass levels in human serum. Comparison with single radial immunodiffusion.
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A quantitative immunoassay has been developed for the determination of the total IgG subclass levels in human serum. The method is based on an enzyme immunoassay in which IgG subclass proteins IgGx in an unknown serum sample compete with a known quantity of peroxidase (PO)-labelled IgGx in fluid phase for subclass-specific monoclonal antibodies coated to the solid phase of microtiter wells. Using a series of human blood samples an excellent correlation was observed with the IgG-subclass levels determined by single radial immunodiffusion.
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8040350
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A new method for bovine pepsinogen purification. Preparation of a specific antibody.
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Bovine pepsinogen was purified from abomasum by ammonium sulphate precipitation and ionic exchange chromatography on DEAE-cellulose and Mono Q columns. Purified pepsinogen was shown to be homogeneous by analytical electrophoresis, having an estimated molecular mass of 46,000 Daltons. The isoelectric point, determined by analytical chromatofocusing was 4.6. Using this pepsinogen preparation to immunize rabbits, a specific antiserum of high titer was obtained.
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8040348
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New, sensitive and specific ELISA for the detection of neuropeptides in culture supernatants.
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Accurate and sensitive sandwich ELISA has been developed for the detection and identification of each of the three neuropeptides, namely, Vasoactive intestinal peptide, Somatostatin and Substance P. The neuropeptides conjugated with BSA and emulsified with Freund's adjuvant were used for immunisation of rabbits. Titres of polyclonal antibodies were checked by indirect immunofluorescence. The animals were bled when titres were high, sera separated, complement inactivated and IgG class of antibodies were purified using a protein G column. Purified IgG antibodies were used for coating the wells and for conjugation with HRPO and used for the detection of the synthetic neuropeptides in a standard solution or in the culture supernatant. The ELISA thus developed for the assay of each of the three neuropeptides had a sensitivity (0.01 ng - 12.8 ng/ml) equal to or better than that reported for these peptides by radioimmunoassay. The assay was highly specific and did not react with a panel of other neuropeptides tested. High level of sensitivity without compromising the specificity was achieved by using activated polyvinyl plates and using purified IgG from high titre rabbit anti-peptide sera. The non specific reaction was minimised by using 10,000 MW cut off amicon filtered supernatants.
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8040347
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Method for determining the affinity of monoclonal antibody using non-competitive ELISA: a computer program.
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A simple and reliable method based upon law of mass action for calculating affinity of a monoclonal antibody using non-competitive ELISA, is described. In this method, the binding of an antibody (Ab) with an antigen (Ag) is measured by ELISA using serial dilutions of both antigen (coated on the plate) as well as antibody. When the OD measured after the antigen antibody interaction was plotted against the concentration of Ab, added to the wells, a hyperbolic curve was obtained. The OD, at any point of the curve, was considered as a direct reflection of the amount of antibody bound to the antigen. The OD-100 denotes the occupancy of maximum no. of epitopes available on the antigen molecules, accessible to the antigen. The concentration of antibody (Ab, Ab') at corresponding levels of antigen concentration (Ag, Ag'), presents the value obtained at OD-50. The [Ag] and [Ag'] are not the true antigen concentrations but are the measurement of antigen density on the plate. The affinity constant K(aff) was calculated by using the formula K(aff) = (n - 1)/2(n[Ab'] - [Ab]), derived from law of mass action, where n = [Ag]/[Ag']. A computer program to calculate the affinity of antibody to the antigen using method described in this manuscript has been developed and discussed.
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8040345
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Apolipoprotein AI transgene corrects apolipoprotein E deficiency-induced atherosclerosis in mice.
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Apolipoprotein E (apo E)-deficient mice are severely hypercholesterolemic and develop advanced atheromas independent of diet. The C57BL/6 strain differs from most inbred strains by having lower HDL concentrations and a high risk of developing early atherosclerotic lesions when fed an atherogenic diet. The relative HDL deficiency and atherosclerosis susceptibility of the C57BL/6 strain are corrected with the expression of a human apolipoprotein AI (apo AI) transgene in this genetic background. To examine if increases in apo AI and HDL are also effective in minimizing apo E deficiency--induced atherosclerosis, we introduced the human apo AI transgene into the hypercholesterolemic apo E knockout background. Similar elevations of total plasma cholesterol occurred in both the apo E knockout and apo E knockout mice also expressing the human apo AI transgene. The latter animals, however, also showed a two- to threefold increase in HDL and a sixfold decrease in susceptibility to atherosclerosis. This study demonstrates that elevating the concentration of apo AI reduces atherosclerosis in apo E deficient-mice and suggests that elevation of apo AI and HDL may prove to be a useful approach for treating unrelated causes of heightened atherosclerosis susceptibility.
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8040344
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Genetic evidence for a common pathway mediating oxidative stress, inflammatory gene induction, and aortic fatty streak formation in mice.
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In a previous survey of inbred mouse strains on an atherogenic diet, we observed that the susceptibility to aortic atherosclerotic lesion formation was associated with the accumulation of lipid peroxidation products, induction of inflammatory genes, and the activation of NF-kB-like transcription factors (Liao, F., A. Andalibi, F. C. deBeer, A. M. Fogelman, and A.J. Lusis. 1993. J. Clin. Invest. 91:2572-2579). We hypothesized that the inflammation-related processes were stimulated by oxidized lipids, since injection of minimally oxidized LDL (MM-LDL) activated the same set of genes. We now report that the induction of inflammatory genes and activation of NF-kB-like transcription factors cosegregate with aortic atherosclerotic lesion formation in BXH recombinant inbred strains derived from parental C57BL/6J (susceptible) and C3H/HeJ (resistant) mice. In addition, the accumulation of hepatic conjugated dienes exhibited a significant correlation with inflammatory gene activation. These results provide strong evidence for the role of inflammatory mediators inducible by oxidative stress in atherogenesis. They also suggest that a major gene contributing to aortic lesion development in this mouse model, designated Ath-1, may control either the accumulation of lipid peroxides in tissues or the cellular responses to such lipid peroxides.
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8040343
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Differential in situ cytokine gene expression in acute versus chronic atopic dermatitis.
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The mechanisms involved in the initiation and maintenance of skin inflammation in atopic dermatitis (AD) are poorly understood. Recent data suggest that the pattern of cytokines expressed locally plays a critical role in modulating the nature of tissue inflammation. In this study, we used in situ hybridization to investigate the expression of interleukin 4 (IL-4), IL-5, and interferon-gamma (IFN-gamma) messenger RNA (mRNA) in skin biopsies from acute and chronic skin lesions of patients with AD. As compared with normal control skin or uninvolved skin of patients with AD, acute and chronic skin lesions had significantly greater numbers of cells that were positive for mRNA, IL-4 (P < 0.01), and IL-5 (P < 0.01), but not for IFN-gamma mRNA expressing cells. However, as compared with acute AD skin lesions, chronic AD skin lesions had significantly fewer IL-4 mRNA-expressing cells (P < 0.01), but significantly greater IL-5 mRNA (P < 0.01). T cells constituted the majority of IL-5-expressing cells in acute and chronic AD lesions. Chronic lesions also expressed significantly greater numbers of activated EG2+ eosinophils than acute lesions (P < 0.01). These data indicate that although acute and chronic AD lesions are associated with increased activation of IL-4 and IL-5 genes, initiation of acute skin inflammation in AD is associated with a predominance of IL-4 expression whereas maintenance of chronic inflammation is predominantly associated with increased IL-5 expression and eosinophil infiltration.
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8040342
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Apolipoprotein E associates with beta amyloid peptide of Alzheimer's disease to form novel monofibrils. Isoform apoE4 associates more efficiently than apoE3.
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Late-onset and sporadic Alzheimer's disease are associated with the apolipoprotein E (apoE) type 4 allele expressing the protein isoform apoE4. Apolipoprotein E binds avidly to beta amyloid (A beta) peptide, a major component of senile plaque of Alzheimer's disease, in an isoform-specific manner. The apoE4 isoform binds to A beta peptide more rapidly than apoE3. We observed that soluble SDS-stable complexes of apoE3 or apoE4, formed by coincubation with A beta peptide, precipitated after several days of incubation at 37 degrees C with apoE4 complexes precipitating more rapidly than apoE3 complexes. A beta(1-28) and A beta(1-40) peptides were incubated in the presence or absence of apoE3, apoE4, or bovine serum albumin for 4 d at 37 degrees C (pH 7.3). Negative stain electron microscopy revealed that the A beta peptide alone self-assembled into twisted ribbons containing two or three strands but occasionally into multistranded sheets. The apoE/A beta coincubates yielded monofibrils 7 nm in diameter. ApoE4/A beta coincubates yielded a denser matrix of monofibrils than apoE3/A beta coincubates. Unlike purely monofibrillar apoE4/A beta coincubates, apoE3/A beta coincubates also contained double- and triple-stranded structures. Both apoE isoforms were shown by immunogold labeling to be uniformly distributed along the A beta peptide monofibrils. Monofibrils appeared earlier in apoE4/A beta than in apoE3/A beta in time-course experiments. Thus apoE3 and apoE4 each interact with beta amyloid peptide to form novel monofibrillar structures, apoE4 more avidly, a finding consistent with the biochemical and genetic association between apoE4 and Alzheimer's disease.
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8040341
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The linked roles of nitric oxide, aldose reductase and, (Na+,K+)-ATPase in the slowing of nerve conduction in the streptozotocin diabetic rat.
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Metabolic and vascular factors have been invoked in the pathogenesis of diabetic neuropathy but their interrelationships are poorly understood. Both aldose reductase inhibitors and vasodilators improve nerve conduction velocity, blood flow, and (Na+,K+)-ATPase activity in the streptozotocin diabetic rat, implying a metabolic-vascular interaction. NADPH is an obligate cofactor for both aldose reductase and nitric oxide synthase such that activation of aldose reductase by hyperglycemia could limit nitric oxide synthesis by cofactor competition, producing vasoconstriction, ischemia, and slowing of nerve conduction. In accordance with this construct, N-nitro-L-arginine methyl ester, a competitive inhibitor of nitric oxide synthase reversed the increased nerve conduction velocity afforded by aldose reductase inhibitor treatment in the acutely diabetic rat without affecting the attendant correction of nerve sorbitol and myo-inositol. With prolonged administration, N-nitro-L-arginine methyl ester fully reproduced the nerve conduction slowing and (Na+,K+)-ATPase impairment characteristic of diabetes. Thus the aldose reductase-inhibitor-sensitive component of conduction slowing and the reduced (Na+,K+)-ATPase activity in the diabetic rat may reflect in part impaired nitric oxide activity, thus comprising a dual metabolic-ischemic pathogenesis.
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8040340
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The molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes angiokeratoma corporis diffusum with glycopeptiduria.
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Angiokeratoma corporis diffusum with glycopeptiduria is a recently recognized inborn error of glycoprotein catabolism resulting from the deficient activity of human alpha-N-acetylgalactosaminidase (E.C. 3.2.1.49; alpha-GalNAc). The first patient with this autosomal recessive disorder, a 46-yr-old consanguineous Japanese woman, presented with diffuse angiokeratoma, mild intellectual impairment, and peripheral neuroaxonal degeneration. Deficient alpha-GalNAc activity also has been reported in consanguineous brothers with an infantile-onset form of neuroaxonal dystrophy resulting from a missense mutation (designated E325K) in the alpha-GalNAc gene. To identify the mutation causing the phenotypically distinct adult-onset disorder, Southern and Northern hybridization analyses of DNA and RNA from the affected homozygote were performed which revealed a grossly normal alpha-GalNAc gene structure and normal transcript size and abundancy. Reverse transcription, amplification, and sequencing of the alpha-GalNAc transcript identified a single C to T transition at nucleotide (nt) 985 that predicted an arginine to tryptophan substitution in residue 329 (designated R329W) of the alpha-GalNAc polypeptide. This base substitution was confirmed by hybridization of PCR-amplified genomic DNA from family members with allele-specific oligonucleotides. Transient expression of an alpha-GalNAc construct containing the R329W mutation resulted in the expression of an immunoreactive polypeptide which had no detectable alpha-GalNAc activity. Comparison of the biosynthesis and stabilities of the transiently expressed and radiolabeled normal, E325K (infantile-onset) and R329W (adult-onset) alpha-GalNAc polypeptides in COS-1 cells indicated that both the mutant precursors were processed to the mature form; however, the E325K mutant polypeptide was more rapidly degraded than the R329W subunit, thereby providing a basis for the distinctly different infantile- and adult-onset phenotypes.
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8040339
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Bicarbonate transport along the loop of Henle. II. Effects of acid-base, dietary, and neurohumoral determinants.
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The loop of Henle contributes to renal acidification by reabsorbing about 15% of filtered bicarbonate. To study the effects on loop of Henle bicarbonate transport (JHCO3) of acid-base disturbances and of several factors known to modulate sodium transport, these in vivo microperfusion studies were carried out in rats during: (a) acute and chronic metabolic acidosis, (b) acute and chronic (hypokalemic) metabolic alkalosis, (c) a control sodium diet, (d) a high-sodium diet, (e) angiotensin II (AII) intravenous infusion, (f) simultaneously intravenous infusion of both AII and the AT1 receptor antagonist DuP 753, (g) acute ipsilateral mechanicochemical renal denervation. Acute and chronic metabolic acidosis increased JHCO3; acute metabolic alkalosis significantly reduced JHCO3, whereas chronic hypokalemic alkalosis did not alter JHCO3. Bicarbonate transport increased in animals on a high-sodium intake and following AII administration, and the latter was inhibited by the AII (AT1) receptor antagonist DuP 753; acute renal denervation lowered bicarbonate transport. These data indicate that bicarbonate reabsorption along the loop of Henle in vivo is closely linked to systemic acid-base status and to several factors known to modulate sodium transport.
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8040338
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Autocrine and paracrine effects of atrial natriuretic peptide gene transfer on vascular smooth muscle and endothelial cellular growth.
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In addition to the atria, recent evidence suggests that atrial natriuretic peptide (ANP) is also synthesized in other tissues. Of particular interest is the location of ANP mRNA in the vessel wall. We and others have shown that exogenously added ANP inhibited the growth of endothelial cells and vascular smooth muscle cells (VSMC) in culture. However, it is not known if the locally synthesized ANP would act similarly. Because cultured endothelial cells and VSMC have lost the ability to express the endogenous ANP gene, we have transfected cells in culture with an expression vector expressing rat ANP and have examined the effects on growth. Cultured endothelial cells transfected with an ANP expression vector synthesized and secreted high levels of ANP. These cells also showed significantly lower rates of DNA synthesis under basal and fibroblast growth factor (FGF)-stimulated conditions. Addition of conditioned medium from endothelial cells transfected with ANP vector to nontransfected endothelial cells resulted in the significant increase in cyclic GMP. Similarly, conditioned media collected from endothelial cells transfected with ANP vector also decreased DNA synthesis in VSMC. Coculture of ANP-transfected endothelial cells with quiescent VSMC showed that released ANP from endothelial cells inhibited DNA synthesis in VSMC. Finally, we examined the autocrine effect of direct transfection of ANP vector into VSMC. Transfection of the ANP vector decreased DNA synthesis in VSMC under basal and angiotensin II-stimulated conditions. Similarly, transfection of the ANP vector resulted in a decrease in the PDGF and serum (5%)-stimulated DNA synthesis of VSMC. These results demonstrate that endogenously produced ANP can exert autocrine and paracrine inhibitory effects on endothelial cell and VSMC growth. In vivo gene transfer of ANP may provide us with the opportunity of gene therapy for vascular diseases in which the abnormalities are vasoconstriction and pathological growth.
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8040337
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FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.
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Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants.
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8040336
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LAP (NF-IL6) transactivates the collagen alpha 1(I) gene from a 5' regulatory region.
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Although collagen is known to enhance hepatocyte differentiation and hepatocytes produce collagen in vivo, the transcriptional factors responsible for collagen type I gene expression in hepatic cells are not known. LAP (Liver Activator Protein) is a member of the C/EBP family, which in differentiated hepatocytes contributes to the high levels of liver-specific gene expression. In this study we show that LAP binds to the collagen alpha 1(I) promoter at both reverse CCAAT motifs and activates transcription. Furthermore, an upstream element, collagen element I (-370/-344), which shares homology with the LAP binding cis-element of the albumin promoter (9 of 13 bp) is described. This collagen element I stimulates transcription in both orientations and when placed in front of either a homologous or a heterologous chimeric report construct. These experiments suggest that LAP may be important in the expression of collagen in differentiated hepatocytes through both the promoter and a newly described upstream element.
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8040335
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Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8+ T cells.
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Antigen-specific, CD8+, cytolytic T lymphocytes (CTLs) could potentially provide resistance to several infectious and malignant diseases. However, the cellular requirements for the generation of specific CTLs in human lymphocyte cultures are not well defined, and repetitive stimulation with antigen is often required. We find that strong CD8+ CTL responses to influenza virus can be generated from freshly isolated blood T cells, as long as dendritic cells are used as antigen presenting cells (APCs). Small numbers of dendritic cells (APC:T cell ratio of 1:50-1:100) induce these CTL responses from most donors in 7 d of culture, but monocytes are weak or inactive. Whereas both dendritic cells and monocytes are infected with influenza virus, the former serve as effective APCs for the induction of CD8+ T cells while the latter act as targets for the CTLs that are induced. The strong CD8+ response to influenza virus-infected dendritic cells is accompanied by extensive proliferation of the CD8+ T cells, but the response can develop in the apparent absence of CD4+ helpers or exogenous lymphokines. CD4+ influenza virus-specific CTLs can also be induced by dendritic cells, but the cultures initially must be depleted of CD8+ cells. These findings should make it possible to use dendritic cells to generate human, antigen-specific, CD8+ CTLs to other targets. The results illustrate the principle that efficient T cell-mediated responses develop in two stages: an afferent limb in which dendritic cells are specialized APCs and an efferent limb in which the primed T cells carry out an immune response to many types of presenting cells.
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8040334
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Reconstitution of cultured intestinal epithelial monolayers with a mucosal-derived T lymphocyte cell line. Modulation of epithelial phenotype dependent on lymphocyte-basolateral membrane apposition.
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In vivo, epithelial cells which line the intestine are intimately associated with lymphocytes, termed intraepithelial lymphocytes. Previous studies have demonstrated that intraepithelial lymphocytes are present in the uninflamed mucosa, and become especially prominent in various human enteropathies including coeliac disease, tropical sprue, dermatitis herpetiformis, and giardiasis. Using the intestinal crypt cell line T84, and a previously well-defined human mucosa-derived lymphocyte (MDL) line with phenotypic features similar to (but not specific for) intraepithelial lymphocytes, we describe a co-culture model to study the functional sequellae of MDL-T84 cell interactions in vitro. A co-culture method was defined which permitted reconstitution of the paracellular spaces of physiologically confluent epithelial monolayers with MDL. Such co-cultures thus mimicked the correct geometry of intraepithelial lymphocytes-epithelial cell interactions. The presence of physiologically positioned MDL brought about specific and dramatic effects on intestinal epithelial monolayer function. In a dose-dependent fashion, the presence of MDL significantly attenuated barrier function (expressed as a decrease in monolayer resistance), decreased epithelial electrogenic Cl- secretion, and modulated epithelial-neutrophil interactions. Such effects were not reproduced in monolayers similarly reconstituted with inert polystyrene beads equivalent in size to MDL. These MDL-elicited effects on epithelial function specifically required direct MDL apposition to the epithelial basolateral membrane. Furthermore, this specific form of MDL-epithelial basolateral contact released soluble factors which were able to confer the MDL-reconstituted phenotype on virgin epithelial monolayers in the absence of MDL. We have previously shown that many aspects of the MDL converted epithelial phenotype described here can be induced by IFN-gamma. While IFN-gamma, a cytokine produced by many lymphocytes including intraepithelial lymphocytes, was detectable in conditioned supernatants from co-cultures, it existed at concentrations insufficient to fully explain the physiologic effects observed here.
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8040333
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Selective response of human airway epithelia to luminal but not serosal solution hypertonicity. Possible role for proximal airway epithelia as an osmolality transducer.
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The response of cultured human nasal epithelia to hypertonic bathing solutions was tested using ion-selective microelectrode and quantitative microscopy. Raised luminal, but not serosal, osmolality (+/- 150 mM mannitol) decreased Na+ absorption but did not induce Cl- secretion. Raised luminal osmolality increased cell Cl- activity, Na+ activity, and transepithelial resistance and decreased both apical and basolateral membrane potentials and the fractional resistance of the apical membrane; equivalent circuit analysis revealed increases in apical, basolateral, and shunt resistances. Prolonged exposure (10 min) to 430 mosM luminal solution elicited no regulation of any parameter. Optical measurements revealed a reduction in the thickness of preparations only in response to luminal hypertonic solutions. We conclude that (a) airway epithelial cells exhibit asymmetric water transport properties, with the apical membrane water permeability exceeding that of the basolateral membrane; (b) the cellular response to volume loss is a deactivation of the basolateral membrane K+ conductance and the apical membrane Cl- conductance; (c) luminal hypertonicity slows the rate of Na+ absorption but does not induce Cl- secretion; and (d) cell volume loss increases the resistance of the paracellular path. We speculate that these properties configure human nasal epithelium to behave as an osmotic sensor, transducing information about luminal solutions to the airway wall.
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8040332
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Pathophysiological concentrations of glucose promote oxidative modification of low density lipoprotein by a superoxide-dependent pathway.
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Oxidized lipoproteins may be important in the pathogenesis of atherosclerosis. Because diabetic subjects are particularly prone to vascular disease, and glucose autoxidation and protein glycation generate reactive oxygen species, we explored the role of glucose in lipoprotein oxidation. Glucose enhanced low density lipoprotein (LDL) oxidation at concentrations seen in the diabetic state. Conjugated dienes, thiobarbituric acid reactive substances, electrophoretic mobility, and degradation by macrophages were increased when LDL was modified in the presence of glucose. In contrast, free lysine groups and fibroblast degradation were reduced. Although loss of reactive lysine groups could be due to either oxidative modification or nonenzymatic glycation of apolipoprotein B-100, inhibition of lipid peroxidation by the metal chelator, diethylenetriamine pentaacetic acid, blocked the changes in free lysines. Thus, glycation of lysine residues is unlikely to account for the alterations in macrophage and fibroblast uptake of LDL modified in the presence of glucose. Glucose-mediated enhancement of LDL oxidation was partially blocked by superoxide dismutase and nearly completely inhibited by butylated hydroxytoluene. These findings indicate that glucose enhances LDL lipid peroxidation by an oxidative pathway involving superoxide and raise the possibility that the chronic hyperglycemia of diabetes accelerates lipoprotein oxidation, thereby promoting diabetic vascular disease.
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8040331
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Antimyenteric neuronal antibodies in scleroderma.
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The pathogenesis of gastrointestinal (GI) dysmotility in scleroderma is incompletely understood, although previous studies have proposed a neuropathic mechanism. We studied patients with scleroderma as compared with other connective tissue disease patients and normal controls for the presence of circulating antibodies to myenteric neurons. Serial dilutions of sera were overlaid on rat intestine, double-labeled with antineurofilament antibody as a myenteric plexus marker, and imaged using indirect immunofluorescence techniques. High titer sera (> or = 1:50) from 19 out of 41 scleroderma patients stained myenteric neurons, whereas none of 22 normals or 5 patients with idiopathic GI dysmotility were positive. Although 6 out of 20 SLE and 6 out of 10 mixed connective tissue disease patients' sera stained myenteric plexus neurons, when positive sera were absorbed with calf thymus extract to remove antinuclear antibody, 15 scleroderma sera, 0 SLE, and 2 mixed connective tissue disease patients retained positive staining of myenteric neurons. Western blotting using actin and neuronal intermediate filament preparations failed to show immunoreactivity with scleroderma sera containing antimyenteric neuronal antibodies. Paraneoplastic sera associated with GI dysmotility stained myenteric neurons in a different pattern than seen with scleroderma sera. A positive correlation between the presence of Raynaud's phenomenon and antimyenteric neuronal antibodies was observed in scleroderma patients. Our results indicate that IgG antibodies reacting with myenteric neurons are present in many patients with scleroderma. Although the neuronal antigen has not yet been identified, the presence of myenteric neuronal antibodies in patients with GI dysmotility and scleroderma suggests a neuropathic process.
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8040330
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Chronic exposure to tumor necrosis factor (TNF) in vitro impairs the activation of T cells through the T cell receptor/CD3 complex; reversal in vivo by anti-TNF antibodies in patients with rheumatoid arthritis.
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Experiments were designed to test the hypothesis that chronic exposure to tumor necrosis factor alpha (TNF) alters the function of activated T lymphocytes. Pretreatment of tetanus toxoid-specific T cell clones with TNF for up to 16 d impaired rechallenge proliferative responses to antigen in a dose- and time-dependent fashion. IL-2 and PHA responses were preserved. Prolonged treatment with TNF impaired production of IL-2, IL-10, IFN gamma, TNF, and lymphotoxin (LT) following stimulation with immobilized OKT3, and resulted in suboptimal expression of the IL-2R alpha chain (Tac) but not CD3, CD4, or HLA-DR antigens, when compared to untreated control cells. By contrast, pretreatment of T cells for prolonged periods in vitro with neutralizing anti-TNF monoclonal antibodies (mAb) enhanced proliferative responses, increased lymphokine production, and upregulated Tac expression following stimulation with OKT3. To determine whether TNF exerts immunosuppressive effects on T cells in vivo, we studied cell-mediated immunity in patients with active rheumatoid arthritis (RA), before and after treatment with a chimeric anti-TNF mAb. Treatment with anti-TNF restored the diminished proliferative responses of PBMC to mitogens and recall antigens towards normal in all patients tested. These data demonstrate that persistent expression of TNF in vitro and in vivo impairs cell-mediated immune responses.
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8040329
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Ca2+ homeostasis in Brody's disease. A study in skeletal muscle and cultured muscle cells and the effects of dantrolene an verapamil.
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Brody's disease, i.e., sarcoplasmic reticulum (SR) Ca(2+)-dependent Mg(2+)-ATPase (Ca(2+)-ATPase) deficiency, is a rare inherited disorder of skeletal muscle function. Pseudo-myotonia is the most important clinical feature. SR Ca(2+)-ATPase and Ca2+ homeostasis are examined in m. quadriceps and/or cultured muscle cells of controls and 10 patients suffering from Brody's disease. In both m. quadriceps and cultured muscle cells of patients, the SR Ca(2+)-ATPase activity is decreased by approximately 50%. However, the concentration of SR Ca(2+)-ATPase and SERCA1 are normal. SERCA1 accounts for 83 and 100% of total SR Ca(2+)-ATPase in m. quadriceps and cultured muscle cells, respectively. This implies a reduction of the molecular activity of SERCA1 in Brody's disease. The cytosolic Ca2+ concentration ([Ca2+]i) at rest and the increase of [Ca2+]i after addition of acetylcholine are the same in cultured muscle cells of controls and patients. The half-life of the maximal response, however, is raised three times in the pathological muscle cells. Addition of dantrolene or verapamil after the maximal response accelerates the restoration of the [Ca2+]i in these muscle cells. The differences in Ca2+ handling disappear by administration of dantrolene or verapamil concomitantly with acetylcholine. The reduced Ca2+ re-uptake from the cytosol presumably due to structural modification(s) of SERCA1 may explain the pseudo-myotonia in Brody's disease. Single cell measurements suggest a beneficial effect of dantrolene or verapamil in treating patients suffering from Brody's disease.
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8040328
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Alveolar type II epithelial cells produce interleukin-6 in vitro and in vivo. Regulation by alveolar macrophage secretory products.
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The aims of this study were (a) to determine if rat alveolar type II (ATII) cells and human pulmonary epithelial-derived cells (A549 cell line) could generate IL-6 in vitro, (b) to characterize the cytokine regulation of IL-6 gene and protein expression in these cells, and (c) to detect the in vivo expression of immunoreactive IL-6 by human ATII cells. Rat ATII cells in primary culture secreted bioactive IL-6 and immunostained with an anti-IL-6 antiserum. Spontaneous IL-6 secretion by rat ATII cells amounted to 5,690 +/- 770 pg/ml/10(6) cells (n = 12) and was fivefold higher than spontaneous rat alveolar macrophages IL-6 secretion (1,052 +/- 286 pg/ml/10(6) cells, n = 8, P = 0.001). Rat alveolar macrophage conditioned media (CM) increased IL-6 secretion by rat ATII cells through the effect of IL-1 and TNF. IL-6 gene expression and IL-6 secretion by A549 cells was induced by IL-1 beta, TNF alpha, and by human alveolar macrophages and THP1 cells CM. Induction was abolished when CM were preincubated with anti-IL-1 beta and anti-TNF alpha antibody. The combination of IFN gamma and LPS induced the expression of IL-6 mRNA by A549 cells whereas LPS alone had no effect. Immunohistochemical staining evidenced the expression of immunoreactive IL-6 by hyperplastic ATII cells in fibrotic human lung, a condition in which alveolar macrophages are known to be activated. ATII cells in normal human lung did not express immunoreactive IL-6. Our findings demonstrate that ATII cells may be an important source of IL-6 in the alveolar space thereby participating to the regulation of the intra-alveolar immune response.
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8040327
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Identification and upregulation of galactose/N-acetylgalactosamine macrophage lectin in rat cardiac allografts with arteriosclerosis.
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Using differential mRNA display to uncover potential mediators associated with chronic rejection, we identified a cDNA fragment induced in Lewis to F344 rat cardiac allografts with arteriosclerosis but not Lewis syngrafts. The full-length cDNA (1.4 kb) isolated from a rat cardiac allograft cDNA library was 99% identical to galactose/N-acetylgalactosamine (Gal/GalNAc) macrophage lectin, a cell-surface receptor. This cDNA hybridized in Northern analysis with total RNA from eight cardiac allografts but not with host hearts, syngrafts, or other organs. There was a significant allograft-specific increase in transcript levels measured by reverse transcriptase PCR at days 7, 14, 28, and 75 in comparison with paired F344 host hearts (subject to same circulation but histologically normal), day-0 hearts, and syngrafts (P < 0.008, n = 4 at each time). Transcript levels in cardiac allografts were higher than those in paired host spleens (a major source of inflammatory cells) (P < 0.0001), indicating the localized nature of Gal/GalNAc lectin induction. By in situ hybridization and immunostaining, Gal/GalNAc lectin expression localized to a subset of inflammatory cells in cardiac allografts. These findings link Gal/GalNAc macrophage lectin to the chronic rejection process, as a possible mediator of macrophage infiltration.
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8040326
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An extra cysteine in one of the non-calcium-binding epidermal growth factor-like motifs of the FBN1 polypeptide is connected to a novel variant of Marfan syndrome.
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We present here a family with a clinical phenotype resembling Marfan syndrome (MFS), and displaying joint contracture and episodes of knee joint effusions, but lacking the cardiovascular features of the syndrome. The phenotype of this family represents a unique mixture of connective tissue symptoms, some of which are found in classical MFS and some of which are typical of dominant ectopia lentis. Linkage analyses suggested a linkage (LOD score 2.4; theta = 0) between the phenotype of the family and a polymorphic marker in the vicinity of the fibrillin locus on chromosome 15 (FBN1). Furthermore, a novel transition mutation was identified in the FBN1 gene in all the affected members of the family. In contrast to the majority of fibrillin mutations reported so far, this mutation substitutes a cysteine for arginine, producing an extra cysteine in one of the non-calcium-binding EGF-like motifs of the fibrillin polypeptide, most probably disturbing the formation of one of the three disulfide bridges known to be essential for the normal conformation of this motif.
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8040325
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Messenger RNA levels of XPAC and ERCC1 in ovarian cancer tissue correlate with response to platinum-based chemotherapy.
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Nucleotide excision repair is a DNA repair pathway that is highly conserved in nature, with analogous repair systems described in Escherichia coli, yeast, and mammalian cells. The rate-limiting step, DNA damage recognition and excision, is effected by the protein products of the genes ERCC1 and XPAC. We therefore assessed mRNA levels of ERCC1 and XPAC in malignant ovarian cancer tissues from 28 patients that were harvested before the administration of platinum-based chemotherapy. Cancer tissues from patients whose tumors were clinically resistant to therapy (n = 13) showed greater levels of total ERCC1 mRNA (P = 0.059), full length transcript of ERCC1 mRNA (P = 0.026), and XPAC mRNA (P = 0.011), as compared with tumor tissues from those individuals clinically sensitive to therapy (n = 15). In 19 of these tissues, the percentage of alternative splicing of ERCC1 mRNA was assessed. ERCC1 splicing was highly variable, with no difference observed between responders and nonresponders. The alternatively spliced species constituted 2-58% of the total ERCC1 mRNA in responders (median = 18%) and 4-71% in nonresponders (median = 13%). These data suggest greater activity of the DNA excision repair genes ERCC1 and XPAC in ovarian cancer tissues of patients clinically resistant to platinum compounds. These data also indicate highly variable splicing of ERCC1 mRNA in ovarian cancer tissues in vivo, whether or not such tissues are sensitive to platinum-based therapy.
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8040323
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Altered activity of the system A amino acid transporter in microvillous membrane vesicles from placentas of macrosomic babies born to diabetic women.
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Fetal macrosomia (FM) is a well-recognized complication of diabetic pregnancy but it is not known whether placental transport mechanisms are altered. We therefore studied the activity of the system A amino acid transporter, the system L amino acid transporter, and the Na+/H+ exchanger in microvillous membrane vesicles from placentas of macrosomic babies born to diabetic women (FM group), from placentas of appropriately grown babies born to diabetic women (appropriate for gestational age group) and from placentas of appropriately grown babies of normal women (control group). Sodium-dependent uptake of [14C]-methylaminoisobutyric acid at 30 s (initial rate, a measure of system A activity) was 49% lower into FM vesicles than into control vesicles (P < 0.02); this effect was due to a decrease in Vmax of the transporter with no change in Km. There was no significant difference in system A activity between the appropriate for gestational age group and control or FM group. There was also no difference between system L transporter or Na+/H+ exchanger activity between the three groups. We conclude that the number of system A transporters per milligram of membrane protein in the placental microvillous membrane is selectively reduced in diabetic pregnancies associated with FM.
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8040324
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Overexpression of the high affinity choline transporter in cortical regions affected by Alzheimer's disease. Evidence from rapid autopsy studies.
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Cholinergic deficits in Alzheimer's disease are typically assessed by choline acetyltransferase, the enzyme that synthesizes acetylcholine. However, the determining step in acetylcholine formation is choline uptake via a high affinity transporter in nerve terminal membranes. Evaluating uptake is difficult because regulatory changes in transporter function decay rapidly postmortem. To overcome this problem, brain regions from patients with or without Alzheimer's disease were frozen within 4 h of death and examined for both choline acetyltransferase activity and for binding of [3H]-hemicholinium-3 to the choline transporter. Consistent with the loss of cholinergic projections, cerebral cortical areas exhibited marked decreases in enzyme activity whereas the putamen, a region not involved in Alzheimer's disease, was unaffected. However, [3H]hemicholinium-3 binding was significantly enhanced in the cortical regions. In the frontal cortex, the increase in [3H]hemicholinium-3 binding far exceeded the loss of choline acetyltransferase, indicating transporter overexpression beyond that necessary to offset loss of synaptic terminals. These results suggest that, in Alzheimer's disease, the loss of cholinergic function is not dictated simply by destruction of nerve terminals, but rather involves additional alterations in choline utilization; interventions aimed at increasing the activity of cholinergic neurons may thus accelerate neurodegeneration.
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8040322
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Differential control of band 3 lateral and rotational mobility in intact red cells.
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Measurements of integral membrane protein lateral mobility and rotational mobility have been separately used to investigate dynamic protein--protein and protein-lipid interactions that underlie plasma membrane structure and function. In model bilayer membranes, the mobilities of reconstituted proteins depend on the size of the diffusing molecule and the viscosity of the lipid bilayer. There are no direct tests, however, of the relationship between mechanisms that control protein lateral mobility and rotational mobility in intact biological membranes. We have measured the lateral and rotational mobility of band 3 in spectrin-deficient red blood cells from patients with hereditary spherocytosis and hereditary pyropoikilocytosis. Our data suggest that band 3 lateral mobility is regulated by the spectrin content of the red cell membrane. In contrast, band 3 rotational mobility is unaffected by changes in spectrin content. Band 3 lateral mobility and rotational mobility must therefore be controlled by different molecular mechanisms.
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8040321
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Individual and synergistic effects of rabbit granulocyte proteins on Escherichia coli.
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Affinity purification of crude acid extracts of rabbit polymorphonuclear leukocytes using Escherichia coli (J5) as adsorbent yields the bactericidal/permeability-increasing protein (BPI), two 15-kD species (p15s), and the two most potent (cationic) defensin species (neutrophil peptides [NP] -1 and -2). Tested in buffered isotonic medium, the relative antibacterial potency of these proteins against E. coli J5 is BPI (IC50 0.2 nM) > p15A (10 nM) > NP -1 (400 nM). Sublethal doses of p15A or NP-1 can synergize with BPI to decrease the dose required to inhibit the growth of E. coli by up to 50-fold. BPI and p15A display similar features of antibacterial action distinct from defensin NP-1, but NP-1 acts synergistically only with BPI and not with p15A. All aspects of the combined action of BPI and NP-1 resemble those observed with higher concentrations of BPI alone, implying that NP-1 enhances BPI potency. Neither NP-1 nor p15A alter the amount of BPI binding to E. coli but BPI enhances binding of p15A to E. coli, raising the possibility that synergy between these two proteins may occur at least partially at the level of binding. The potent synergistic actions of these proteins can also be demonstrated against serum-resistant clinical isolates of encapsulated E. coli tested in whole blood and plasma ex vivo, suggesting that such combined action may contribute to host defense in vivo.
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8040320
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Stimulation of lung epithelial liquid clearance by endogenous release of catecholamines in septic shock in anesthetized rats.
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Exogenous administration of beta-adrenergic agonists has previously been reported to increase lung liquid clearance by stimulation of active sodium transport across the alveolar epithelium. We hypothesized for this study that endogenous release of epinephrine in septic shock would stimulate liquid clearance from the airspaces in rats. Liquid clearance from the air spaces was measured by the concentration of protein over 4 h in a test solution of 5% albumin instilled into one lung. Bacteremic rats developed severe systemic hypotension and metabolic acidosis that was associated with a 100-fold rise in plasma epinephrine levels. There was a 100% increase in liquid clearance from the airspaces of the lung in the bacteremic compared with control rats. To determine the mechanisms responsible for this accelerated lung liquid clearance, amiloride (10(-3) M), a sodium transport inhibitor, was added to the air spaces. Amiloride prevented the increase in liquid clearance from the airspaces, indicating that this effect depended on increased uptake of sodium across the lung epithelium. The addition of propranolol (10(-4) or 10(-5) M) to the instillate also prevented the acceleration in alveolar liquid clearance in the bacteremic rats. We conclude that the release of endogenous catecholamines associated with septic shock markedly stimulates fluid clearance from the distal airspaces of the lung by a beta-adrenergic mediated stimulation of active sodium transport across the epithelial barrier. This data provides evidence for a previously unrecognized mechanism that can protect against or hasten the resolution of alveolar edema in pathological conditions, such as septic shock, that are associated with the endogenous release of catecholamines.
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8040319
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Synthetic fibronectin peptides suppress arthritis in rats by interrupting leukocyte adhesion and recruitment.
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In an experimental model of arthritis, increased leukocyte adhesion is associated with the evolution of acute and chronic synovial inflammation. Whereas peripheral blood mononuclear cells (PBMC) from control animals bind minimally to fibronectin matrices, PBMC from animals receiving arthropathic doses of bacterial cell walls demonstrate increased integrin mRNA expression and enhanced adhesion. To determine whether this augmented adhesion was causal in the development of synovial pathology, peptides synthesized from several fibronectin domains which inhibited leukocyte adhesion in vitro were administered to arthritic animals either as free peptides or coupled to a carrier molecule. Not only were peptides containing either the RGD or CS-1 cell-binding domains inhibitory to chronic synovial pathology (articular index = 10.5 +/- 0.3 for untreated animals compared to 1.25 +/- 0.25 for RGD and 2.5 +/- 0.7 for CS-1), but three peptides synthesized from the carboxy-terminal 33-kD heparin-binding domain of fibronectin were also found to significantly inhibit leukocyte recruitment and the evolution of arthritis. Based on these data, which are the first to explore the therapeutic potential of heparin-binding fibronectin peptides in chronic inflammation, it appears that antagonism of cellular adhesion and recruitment by fibronectin peptides may provide an important mechanism for modulating the multi-step adhesion process and attenuating aberrant inflammatory responses.
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8040318
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Differential effects of metalloporphyrins on messenger RNA levels of delta-aminolevulinate synthase and heme oxygenase. Studies in cultured chick embryo liver cells.
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The acute porphyrias in relapse are commonly treated with intravenous heme infusion to decrease the activity of delta-aminolevulinic acid synthase, normally the rate-controlling enzyme in heme biosynthesis. The biochemical effects of heme treatment are short-lived, probably due in part to heme-mediated induction of heme oxygenase, the rate-controlling enzyme for heme degradation. In this work, selected nonheme metalloporphyrins were screened for their ability to reduce delta-aminolevulinic acid synthase mRNA and induce heme oxygenase mRNA in chick embryo liver cell cultures. Of the metalloporphyrins tested, only zinc-mesoporphyrin reduced delta-aminolevulinic acid synthase mRNA without increasing heme oxygenase mRNA. The combination of zinc-mesoporphyrin and heme, at nanomolar concentrations, decreased delta-aminolevulinic acid synthase mRNA in a dose-dependent manner. The combination of zinc-mesoporphyrin (50 nM) and heme (200 nM) decreased the half-life of the mRNA for delta-aminolevulinic acid synthase from 5.2 to 2.5 h, while a similar decrease was produced by heme (10 microM) alone (2.2 h). The ability of zinc-mesoporphyrin to supplement the reduction of delta-aminolevulinic acid synthase mRNA by heme, in a process similar to that observed with heme alone, provides a rationale for further investigation of this compound for eventual use as a supplement to heme therapy of the acute porphyrias and perhaps other conditions in which heme may be of benefit.
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8040316
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Mutations of CpG dinucleotides located in the triiodothyronine (T3)-binding domain of the thyroid hormone receptor (TR) beta gene that appears to be devoid of natural mutations may not be detected because they are unlikely to produce the clinical phenotype of resistance to thyroid hormone.
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Thyroid hormone receptor (TR) beta gene mutations identified in patients with resistance to thyroid hormone (RTH) revealed two clusters ("hot" areas) of mutations (RTHmut) in the triiodothyronine (T3)-binding domain. Furthermore, 45% of RTHmuts and 90% of recurring mutations are located in CpG dinucleotides ("hot spots"). To investigate why the region between the two hot areas lacks RTHmuts, we produced 10 artificial mutant TR beta s (ARTmut) in this "cold" region according to the hot spot rule (C-->T or G-->A substitutions in CpGs). The properties of ARTmuts were compared with those of six RTHmuts. Among all RTHmuts, R320H manifesting a mild form of RTH showed the least impairment of T3-binding affinity (Ka). In contrast, Ka was normal in six ARTmuts (group A), reduced to a lesser extent than R320H in three (group B), and one that was truncated (R410X) did not bind T3. All RTHmuts had impaired ability to transactivate T3-responsive elements and exhibited a strong dominant negative effect on cotransfected wild-type TR beta. Group B and A ARTmuts had minimally impaired or normal transactivation and weak or no dominant negative effect, respectively. R410X showed neither transactivation nor dominant negative effect. Natural mutations expected to occur in the cold region of TR beta should fail to manifest as RTH (group A) or should escape detection (group B) since the serum thyroid hormone levels required to compensate for the reduced binding affinity should be inferior to those found in subjects with R320H. R410X would manifest RTH only in the homozygote state. The cold region of the putative T3-binding domain is relatively insensitive to amino acid changes and, thus, may not be involved in a direct interaction with T3.
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8040317
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A novel mobile element inserted in the alpha spectrin gene: spectrin dayton. A truncated alpha spectrin associated with hereditary elliptocytosis.
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Nonviral retrotransposons, retropseudogenes, and short interspersed nuclear elements (SINEs) are mobile DNA segments capable of transposition to new genomic locations, where they may alter gene expression. De novo integration into specific genes has been described in both germ and somatic cells. We report a family with hereditary elliptocytosis and pyropoikilocytosis associated with a truncated alpha-spectrin protein. We present the biochemical characteristics of this abnormal protein and show that the alpha-spectrin gene is disrupted by a mobile element resulting in exon skipping. This element causes duplication of the insertion site and is terminated by a long poly-A tail downstream of multiple consensus polyadenylation signals. Southern blot analysis of human genomic DNA, using this element as probe, reveals one to three copies per individual. This element has no homology to any previously reported sequence and therefore appears to be a member of a novel family of mobile elements.
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8040315
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Abnormal expression of laminin suggests disturbance of sarcolemma-extracellular matrix interaction in Japanese patients with autosomal recessive muscular dystrophy deficient in adhalin.
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Dystrophin is associated with several novel sarcolemmal proteins, including a laminin-binding extracellular glycoprotein of 156 kD (alpha-dystroglycan) and a transmembrane glycoprotein of 50 kD (adhalin). Deficiency of adhalin characterizes a severe autosomal recessive muscular dystrophy prevalent in Arabs. Here we report for the first time two mongoloid (Japanese) patients with autosomal recessive muscular dystrophy deficient in adhalin. Interestingly, adhalin was not completely absent and was faintly detectable in a patchy distribution along the sarcolemma in our patients. Although the M and B2 subunits of laminin were preserved, the B1 subunit was greatly reduced in the basal lamina surrounding muscle fibers. Our results raise a possibility that the deficiency of adhalin may be associated with the disturbance of sarcolemma-extracellular matrix interaction leading to sarcolemmal instability.
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8040314
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Interleukin 6 promotes murine lupus in NZB/NZW F1 mice.
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To investigate the role of IL-6 in systemic lupus erythematosus (SLE), we selectively inhibited IL-6 in lupus-prone NZB/NZW F1(B/W) mice by chronic administration of a rat mAb to mouse IL-6. Anti-IL-6 alone elicited an anti-rat response that blocked its biologic effects. To circumvent this problem, we rendered B/W mice tolerant to the rat mAb by administration of anti-CD4 concurrent with the first dose of anti-IL-6. Thereafter, the mice received weekly injections of anti-IL-6 alone. There were two control groups: one group received the tolerizing regimen of anti-CD4 along with a control rat IgG1 mAb (GL113) instead of anti-IL-6; the other control group received PBS. Mice that received anti-CD4 were tolerant to the rat mAb for 6 mo. Throughout this period, treatment with anti-IL-6 prevented production of anti-dsDNA, significantly reduced proteinuria, and prolonged life. Mice that received anti-IL-6 without anti-CD4 developed an immune response to the rat mAb and then developed anti-dsDNA antibodies, proteinuria, and mortality comparable with control mice. These findings establish that IL-6 promotes autoimmunity in B/W mice. They further indicate that, although mAb to IL-6 can suppress murine lupus, the development of host immunity to the mAb abrogates its beneficial effects. Finally, this is the first study to demonstrate that a brief course of anti-CD4 can induce tolerance to another therapeutic mAb, in this case an anti-cytokine mAb.
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8040313
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Continuous inhalation of nitric oxide protects against development of pulmonary hypertension in chronically hypoxic rats.
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Exposure to hypoxia and subsequent development of pulmonary hypertension is associated with an impairment of the nitric oxide (NO) mediated response to endothelium-dependent vasodilators. Inhaled NO may reach resistive pulmonary vessels through an abluminal route. The aim of this study was to investigate if continuous inhalation of NO would attenuate the development of pulmonary hypertension in rats exposed to chronic hypoxia. In conscious rats previously exposed to 10% O2 for 3 wk, short-term inhalation of NO caused a dose-dependent decrease in pulmonary artery pressure (PAP) from 44 +/- 1 to 32 +/- 1 mmHg at 40 ppm with no changes in systemic arterial pressure, cardiac output, or heart rate. In normoxic rats, acute NO inhalation did not cause changes in PAP. In rats simultaneously exposed to 10% O2 and 10 ppm NO during 2 wk, right ventricular hypertrophy was less severe (P < 0.01), and the degree of muscularization of pulmonary vessels at both alveolar duct and alveolar wall levels was lower (P < 0.01) than in rats exposed to hypoxia alone. Tolerance to the pulmonary vasodilator effect of NO did not develop after prolonged inhalation. Brief discontinuation of NO after 2 wk of hypoxia plus NO caused a rapid increase in PAP. These data demonstrate that prolonged inhalation of low concentrations of NO induces sustained pulmonary vasodilation and reduces pulmonary vascular remodeling in response to chronic hypoxia.
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8040312
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Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo.
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Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis.
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8040311
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Expression of the murine plasma cell nucleotide pyrophosphohydrolase PC-1 is shared by human liver, bone, and cartilage cells. Regulation of PC-1 expression in osteosarcoma cells by transforming growth factor-beta.
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A bone and cartilage enzyme with both 5'-nucleotide phosphodiesterase I and nucleotide pyrophosphohydrolase (NTPPPH) activity modulates physiologic mineralization and pathologic chondrocalcinosis by generating inorganic pyrophosphate. We hypothesized that, as for alkaline phosphatase, expression of an NTPPPH gene can be shared by cells from bone, cartilage, and liver and by certain leukocytes. Recently, we demonstrated the hepatocyte and murine plasma cell membrane glycoprotein PC-1 to have both 5'-nucleotide phosphodiesterase I and NTPPPH activity. We detected polypeptides cross-reactive with PC-1 in human U20S osteosarcoma cells, articular chondrocytes, homogenized human knee cartilages, human knee synovial fluids, hepatoma cells, and murine plasmacytoma cells. Constitutive low abundance PC-1 mRNA expression was detected in U20S cells and chondrocytes by a nested RNA-PCR assay and by Northern blotting. TGF beta is known to substantially increase NTPPPH activity in primary osteoblast cultures. We demonstrated that TGF beta 1 increased NTPPPH activity and the level of PC-1 mRNA and immunoprecipitable [35S]-methionine-labeled PC-1 polypeptides in U20S cells. The identification of PC-1 as an NTPPPH expressed in cells derived from bone and cartilage may prove useful in furthering the understanding of the role of NTPPPH i n physiologic and pathologic mineralization.
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8040310
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Association of neonatal myasthenia gravis with antibodies against the fetal acetylcholine receptor.
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The specificities of autoantibodies directed against the acetylcholine receptor (AChR) for embryonic and adult muscle AChR were studied in 22 mothers with myasthenia gravis (MG) and in their newborns using human fetus and normal adult muscle AChR preparations. 12 mothers had transmitted MG to their neonates with, in three cases, antenatal injury. A clear correlation was found between occurrence of neonatal MG (NMG) and the high overall level of anti-AChR antibodies (embryonic or adult muscle AChR). However, a strong correlation was also found between occurrence of NMG and the ratio of anti-embryonic AChR to anti-adult muscle (Te/Ta) AChR antibodies (P < 0.0002). Taken together, these data suggest that autoantibodies directed against the embryonic form of the AChR could play a predominant role in the pathogenesis of NMG. Paradoxically, the three cases with antenatal injury presumably the most severe form of NMG, were not associated with high Te/Ta. At the clinical level, these observations could prove helpful in the prediction of transmission of NMG.
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8040309
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Substitution of arginine-839 by cysteine or histidine in the androgen receptor causes different receptor phenotypes in cultured cells and coordinate degrees of clinical androgen resistance.
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We aim to correlate point mutations in the androgen receptor gene with receptor phenotypes and with clinical phenotypes of androgen resistance. In two families, the external genitalia were predominantly female at birth, and sex-of-rearing has been female. Their androgen receptor mutation changed arginine-839 to histidine. In a third family, the external genitalia were predominantly male at birth, and sex-of-rearing has been male: their codon 839 has mutated to cysteine. In genital skin fibroblasts, both mutant receptors have a normal androgen-binding capacity, but they differ in selected indices of decreased affinity for 5 alpha-dihydrotestosterone or two synthetic androgens. In transiently cotransfected androgen-treated COS-1 cells, both mutant receptors transactivate a reporter gene subnormally. The His-839 mutant is less active than its partner, primarily because its androgen-binding activity is more unstable during prolonged exposure to androgen. Adoption of a nonbinding state explains a part of this instability. In four other steroid receptors, another dibasic amino acid, lysine, occupies the position of arginine-839 in the androgen receptor. Androgen receptors with histidine or cysteine at position 839 are distinctively dysfunctional and appear to cause different clinical degrees of androgen resistance.
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8040308
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Gene expression and secretion of atrial natriuretic peptide by murine macrophages.
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An increased expression of atrial natriuretic peptide (ANP) has been reported in activated macrophages of the acutely involuted rat thymus. We communicate here that ANP may reflect a common constituent of macrophages, as mRNA coding for ANP is present in peritoneal- as well as in bone marrow-derived macrophages (PM, BMM). Furthermore, both types of macrophages synthesize and release ANP which was found to mainly represent the biologically active fragment ANP99-126. ANP expression in macrophages is regulated by compounds affecting the activity of these immune cells. For example, incubation of PM or BMM in vitro with LPS and zymosan, respectively, increased ANP-mRNA up to sixfold as determined by competitive PCR quantification. Exposure of macrophages to dexamethasone (Dex, 10(-7) M) elicits moderate effects (1.4-fold), while PMA (10(-7) M) failed to affect its abundance. These findings are complemented by data regarding ANP synthesis and secretion. Incubation of macrophages with LPS, Dex or a combination of both results in an up to 3.5-fold increase of intracellular ANP99-126 (basal 10 fmol/mg protein), and an up to 6.6-fold increase of its secretion (basal 40 fmol/mg protein, 24 h). Since macrophages synthesize and release ANP, the peptide may be involved in the complex mechanisms of host defense, a major function of these immune cells.
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8040307
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Molecular analysis of the human immunoglobulin V lambda II gene family.
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The 8.12 idiotype characterizes a subpopulation of anti-DNA antibodies in patients with systemic lupus erythematosus (SLE). The idiotype is present on lambda light chains and has previously been shown to be exclusively encoded by V lambda II light chains. RFLP analysis of the V lambda II gene family has shown the family to consist of 10 to 15 members. Thus far, the sequences of seven V lambda II germline genes are reported in the literature with one of these a pseudogene. To identify the V lambda II genes that encode 8.12 positive antibodies and to further characterize the V lambda II family, germline V lambda II clones were derived from a patient with SLE. Two libraries were constructed: a genomic DNA library and a library of PCR-derived V lambda II gene products obtained using a conserved V lambda II leader region primer and a primer for the nonamer region 3' of the coding sequence. We now describe seven new germline genes, two of which are pseudogenes. Comparison of V lambda II germline genes to sequences of 8.12 positive light chains produced by EBV-transformed B cell lines show that all 8.12 positive light chains are encoded by a limited number of highly homologous members of the V lambda II family. 8.12 negative V lambda II encoded light chains also derive from a limited number of V lambda II genes, suggesting that only a subset of the apparently available V lambda II genes are commonly expressed.
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8040306
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Large deletion of the peroxisomal acyl-CoA oxidase gene in pseudoneonatal adrenoleukodystrophy.
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We have cloned the cDNA encoding human peroxisomal acyl-CoA oxidase, the first enzyme in the peroxisomal beta-oxidation of very long chain fatty acids. Its nucleotide sequence was found to be highly homologous (85%) to the rat cDNA counterpart. An 88% homology between rat and human was found in the COOH-terminal end of the cDNA which includes the Ser-Lys-Leu peroxisomal targeting signal common to many peroxisomal proteins. The gene spans approximately 30-40 kb and is poorly polymorphic. Southern blot analyses were performed in two previously reported siblings with an isolated peroxisomal acyl-CoA oxidase deficiency (pseudoneonatal adrenoleukodystrophy). A deletion of at least 17 kb, starting down-stream from exon 2 and extending beyond the 3' end of the gene, was observed in the two patients. These observations provide a molecular basis for the observed acyl-CoA oxidase deficiency in our family. In addition, our study will enable the characterization of the genetic defect in unrelated families with suspected acyl-CoA oxidase disorders.
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8040305
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The Yaa gene abrogates the major histocompatibility complex association of murine lupus in (NZB x BXSB)F1 hybrid mice.
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To investigate the specific contribution of select MHC class II genes on the development of murine lupus, H-2 congenic (NZB x BXSB)F1 hybrid mice bearing either H-2b/b, H-2d/b, or H-2d/d haplotypes were generated. We compared the clinical development (autoantibody production and glomerulonephritis) of systemic lupus erythematosus (SLE) in these three F1 hybrids in the presence or absence of the mutant gene, Yaa (Y chromosome-linked autoimmune acceleration), which normally accelerates the progression of murine SLE. (NZB x BXSB)F1 hybrid female mice bearing either the H-2b/b or H-2d/b haplotype developed a rapid course of severe SLE, while the appearance of disease was markedly delayed in H-2d/d hybrid females. However, in the presence of the Yaa gene, H-2d/d F1 males developed SLE as severe as H-2b/b and H-2d/b F1 males. These data indicate that (a) the conventional H-2b is a haplotype leading to susceptibility for murine SLE, while H-2d is a relatively resistant haplotype; (b) the H-2b haplotype exhibits a dominant effect on autoimmune responses, similar to the classical MHC-linked Ir gene effect; and (c) most strikingly, the Yaa gene totally abrogates the MHC effect on murine lupus in (NZB x BXSB)F1 hybrid mice.
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8040303
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Genetic analysis of 29 kindreds with generalized and pituitary resistance to thyroid hormone. Identification of thirteen novel mutations in the thyroid hormone receptor beta gene.
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Resistance to thyroid hormone (RTH), with elevated serum free thyroid hormones and nonsuppressed thyrotropin levels, is either relatively asymptomatic, suggesting a generalized disorder (GRTH) or associated with thyrotoxic features, indicating possible selective pituitary resistance (PRTH). 20 GRTH and 9 PRTH cases, sporadic or dominantly inherited, were analyzed. Affected individuals were heterozygous for single nucleotide substitutions in the thyroid hormone receptor beta gene, except for a single case of a seven nucleotide insertion. With one exception, the corresponding 13 novel and 7 known codon changes localized to and extended the boundaries of two mutation clusters in the hormone-binding domain of the receptor. 15 kindreds shared 6 different mutations, and haplotype analyses of the mutant allele showed that they occurred independently. The majority (14 out of 19) of the recurrent but a minority (1 out of 10) of unique mutations were transitions of CpG dinucleotides. Mutant receptor binding to ligand was moderately or severely impaired and did not correlate with the magnitude of thyroid dysfunction. There was no association between clinical features and the nature or location of a receptor mutation. These observations suggest that GRTH and PRTH are phenotypic variants of the same genetic disorder, whose clinical expression may be modulated by other non-mutation-related factors.
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8040304
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Identification of mutations in the putative ATP-binding domain of the adrenoleukodystrophy gene.
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The recently identified adrenoleukodystrophy (ALD) gene is predicted to encode a peroxisomal protein of 745 amino acids that includes one domain for ATP-binding, termed nucleotide-binding fold (NBF). To determine whether mutations occur in the putative NBF of ALD protein, we analyzed by denaturing gradient gel electrophoresis (DGGE) exon 6 and 8 that encode most part of this domain in 50 ALD patients. Four amino acid substitutions, three frameshift mutations leading to premature termination signal, and a splicing mutation were identified. These amino acid substitutions occurred at residues highly conserved in other ATP-binding cassette (ABC) proteins. In addition, a nonsense mutation was detected in exon 4.
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8040302
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Gene transfer into the rat renal glomerulus via a mesangial cell vector: site-specific delivery, in situ amplification, and sustained expression of an exogenous gene in vivo.
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To evaluate the pathophysiological function of specific molecules in the renal glomerulus, selective, sustained, and modifiable expression of such molecules will be required. Towards achieving this end, we devised a gene transfer system using the glomerular mesangial cell as a vector for gene delivery. A reporter gene which encodes bacterial beta-galactosidase was introduced into cultured rat mesangial cells, and the stable transfectants were transferred into the rat kidney via the renal artery, leading to selective entrapment within the glomeruli. In the normal kidney, the reporter cells populated into 57 +/- 13% of glomeruli site specifically, and the expression of beta-galactosidase was sustained for 4 wk and declined thereafter. Within the glomerulus, some of the reporter cells remained in the glomerular capillaries, while others repopulated the mesangial area and, in part, extended their cytoplasmic processes toward the surrounding capillaries. When the cells were transferred into glomeruli subjected to transient mesangiolysis induced by monoclonal antibody 1-22-3, in situ expression of beta-galactosidase was amplified 7-12-fold, and the enhanced level of expression continued for up to 8 wk. The mesangial cell vector system thus achieves site-specific delivery of an exogenous gene into the glomerulus and is amenable to in situ amplification and sustained expression by preconditioning of the target site.
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8040301
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EWS-erg and EWS-Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations.
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We have determined the frequency of EWS fusion transcripts in a series of primary Ewing's sarcomas and peripheral primitive neuroectodermal tumors and cells lines. Type 1 and 2 EWS-Fli1 fusions were demonstrated in 8 cell lines and 14 patient samples. Five patients with cytogenetically characterized rearrangements of chromosome 22 that did not involve chromosome 11 were included in these studies. A novel EWS-Fli1 in-frame isoform fusing EWS to exon 8 of Fli1 was isolated from a tumor with a variant t(12;22;22)(q14;p1;q12) translocation. Three in-frame isoforms of a novel hybrid transcript derived from the fusion of EWS with the ETS domain of the human erg gene were identified in patient samples and a cell line with cytogenetically unidentified or cryptic translocations involving chromosomes 21 and 22. Interphase analysis by fluorescent in situ suppression hybridization using two overlapping erg yeast artificial chromosome clones demonstrated disruption of the erg gene on chromosome 21 in a patient sample with monosomy 22. Our results provide new information about the involvement of EWS in small round cell tumors involving exchange of its putative RNA-binding domain with DNA-binding domains derived from different members of the ETS family of transcription factors. These studies emphasize the utility of reverse transcriptase PCR analysis and fluorescent in situ hybridization as additional diagnostic tools for differential diagnosis among small round cell tumors.
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8040300
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Studies of thrombin-induced proteoglycan release in the degradation of human and bovine cartilage.
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Because fibrin is commonly observed within arthritic joints, studies were undertaken to determine whether purified coagulation and fibrinolytic proteases degrade cartilage in vitro and to seek evidence for the activation of coagulation in arthritic joints through measurements of the levels of inhibitor-enzyme complexes and several other proteins associated with coagulation and fibrinolysis. The concentrations of 13 plasma proteins and complexes of thrombin and Factor Xa with antithrombin III were measured in synovial fluids recovered at the time of knee replacement surgery. All zymogens necessary to constitute the coagulation cascade were present. Thrombin and the combination of prothrombin plus prothrombinase induced proteoglycan release from both normal and arthritic cartilages. Factor Xa and plasmin induced release from diseased cartilage only, and urokinase, tissue plasminogen activator, and activated protein C were without effect at the levels used. At saturating levels of thrombin (> or = 2.0 microM) 80% of the proteoglycan content of normal cartilage was released within 24 h. Thrombin, which is cationic, reversibly binds cartilage with Kd = 7.0 +/- 1.0 microM and Bmax = 820 +/- 70 ng/mg of human cartilage. Levels of thrombin-antithrombin III complexes in synovial fluids and arthritis were 4-fold higher in osteo (OA) and 43-fold higher in rheumatoid (RA) than in controls (0.98 nM). Factor Xa-antithrombin III complex levels were threefold lower in OA and fivefold higher in RA than in controls (0.24 nM). These elevated levels of enzyme-inhibitor complexes imply a history of activation of coagulation within the joint, especially in RA. Since thrombin degrades cartilage in vitro and had been generated in vivo, as inferred by the existence of thrombin-antithrombin III complexes, intraarticular activation of coagulation may both contribute to the pathology of arthritis and comprise a target for therapy and diagnosis.
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8040296
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Identification and characterization of a receptor for tissue ferritin on activated rat lipocytes.
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Hepatic iron overload causes lipocyte activation with resultant fibrogenesis. This study examines whether rat lipocytes express ferritin receptors, which could be involved in paracellular iron movement and in cellular regulation. Lipocytes from normal rat liver were cultured on plastic and incubated with 125I-labeled rat liver ferritin (RLF) +/- a 100-fold excess of either unlabeled RLF or human heart ferritin, human liver ferritin, human recombinant H-ferritin, a mutant human recombinant L-ferritin, or a variety of nonspecific proteins. Specific binding sites for ferritin were demonstrated by displacement of 125I-RLF by RLF (64.5 +/- 4.3%) and by other ferritins (55-60%), but not by recombinant L-ferritin. Scatchard analysis demonstrated a single class of binding sites with a Kd of 5.1 +/- 2.9 x 10(-10) M, maximum binding capacity of 4.7 +/- 1.3 x 10(-12) M, and 5,000-10,000 receptor sites/cell. Ferritin receptor expression was observed only in activated lipocytes. Internalization of RLF was observed within 15 min using FITC-RLF and confocal microscopy. This study demonstrates that (a) activated lipocytes express a specific high affinity ferritin receptor; (b) the binding appears to be dependent on the H-ferritin subunit; and (c) lipocytes internalize ferritin. Expression of ferritin receptors in activated lipocytes suggests that the receptor may either be involved in the activation cascade or may be a marker of activation.
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8040295
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Pseudo-acylceramide with linoleic acid produces selective recovery of diminished cutaneous barrier function in essential fatty acid-deficient rats and has an inhibitory effect on epidermal hyperplasia.
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Pseudo-acylceramides with different acyl properties were investigated for their capacity to restore diminished barrier function in essential fatty acid-deficient rats. Daily topical applications of synthetic pseudo-acylceramides containing ester-linked linoleic acid caused a dose-dependent, significant reduction of transepidermal water loss (TEWL). Both other pseudo-acylceramides with ester-linked oleic acid or saturated alkyl chains and ordinary ceramides exhibited a poor effect on recovery of TEWL. Furthermore, pseudoceramide containing ether-linked linoleic acid, which is biologically inactive in terms of degradation by hydrolytic enzymes, also induced a significant and similar increase in the barrier function. This restoration of barrier function by pseudo-acylceramides with linoleic acid was accompanied by suppressed DNA synthesis in the EFAD rat epidermis. In UVB-irradiated guinea pig skin, topical applications of the pseudo-acylceramides with linoleic acid immediately after the exposure significantly reduced epidermal hyperplasia, secondary to markedly diminished barrier disruption, whereas linoleic acid itself did not. A comparison of both the anti-hyperplasia and the barrier recovery effects in the series of pseudo-ceramide derivatives examined revealed that the suppressive effect on the induced epidermal hyperplasia was paralleled by the recovery of the barrier defect in EFAD rats. These findings directly suggest that acylceramide with an ester-linked linoleic acid has an essential role in the epidermal permeability barrier.
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8040294
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Distinct populations of basal keratinocytes express stromelysin-1 and stromelysin-2 in chronic wounds.
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Wound repair involves cell migration and tissue remodeling, and these ordered and regulated processes are facilitated by matrix-degrading proteases. We reported that interstitial collagenase is invariantly expressed by basal keratinocytes at the migrating front of healing epidermis (Saarialho-Kere, U. K., E. S. Chang, H. G. Welgus, and W. C. Parks, 1992. J. Clin. Invest. 90:1952-1957). Because of the limited substrate specificity of collagenase, principally for interstitial fibrillar collagens, other enzymes must also be produced in the wound environment to effectively restructure tissues with a complex matrix composition. Stromelysins-1 and -2 are closely related, yet distinct metalloproteinases, and both can degrade many noncollagenous connective tissue macromolecules. Using in situ hybridization and immunohistochemistry, we found that both stromelysins are produced by distinct populations of keratinocytes in a variety of chronic ulcers. Stromelysin-1 mRNA and protein were detected in basal keratinocytes adjacent to but distal from the wound edge in what probably represents the sites of proliferating epidermis. In contrast, stromelysin-2 mRNA was seen only in basal keratinocytes at the migrating front, in the same epidermal cell population that expresses collagenase. Stromelysin-1-producing keratinocytes resided on the basement membrane, whereas stromelysin-2-producing keratinocytes were in contact with the dermal matrix. Furthermore, stromelysin-1 expression was prominent in dermal fibroblasts, whereas no signal for stromelysin-2 was seen in any dermal cell. These findings demonstrate that stromelysins-1 and -2 are produced by different populations of basal keratinocytes in response to wounding and suggest that these two matrix metalloproteinases serve distinct roles in tissue repair.
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8040293
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Extracellular matrix modulates epidermal growth factor receptor activation in rat glomerular epithelial cells.
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To understand how glomerular epithelial cell (GEC) proliferation may be regulated in health and disease, we studied the effects of type I collagen extracellular matrices (ECM) on EGF receptor (EGF-R) activation in cultured rat GEC. EGF stimulated proliferation of GEC adherent to ECM, but not of GEC on a plastic substratum. Significant and prolonged EGF-R tyrosine autophosphorylation (which reflects receptor kinase activation) was induced by EGF in GEC adherent to collagen, but EGF did not stimulate EGF-R autophosphorylation in GEC on plastic (at 37 degrees C). However, EGF-R autophosphorylation increased significantly in plastic-adherent GEC that were stimulated with EGF at 4 degrees C or in the presence of vanadate, an inhibitor of phosphotyrosine phosphatases. Furthermore, dephosphorylation of EGF-R was enhanced in GEC on plastic as compared with collagen. At 4 degrees C, [125I]EGF binding was not different between substrata, and there was negligible accumulation of intracellular [125I]EGF (which reflects EGF-R internalization). At 37 degrees C, EGF-R internalization was reduced significantly in collagen-adherent GEC as compared with GEC on plastic. Thus, contact with ECM facilitates proliferation and EGF-R activation in GEC. The enhanced activity of EGF-R tyrosine kinase may be due to ECM-induced reduction in EGF-R internalization and dephosphorylation by phosphotyrosine phosphatase(s). Signals from ECM to growth factor receptors may regulate cell turnover in the glomerulus under normal conditions and during immune glomerular injury.
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8040292
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Absorption of pathogenic autoantibodies by the extracellular domain of pemphigus vulgaris antigen (Dsg3) produced by baculovirus.
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Pemphigus vulgaris (PV) is an autoimmune blistering disease, in which autoantibodies against PV antigen (PVA or Dsg3) play a pathogenic role in inducing blister formation. Bacterial fusion proteins of PVA failed to absorb pathogenic autoantibodies from PV patients' sera probably because they did not represent the proper conformation. Therefore, a chimeric protein, PVIg, consisting of the whole extracellular domain of PVA and the constant region of human IgG1, was produced in either in COS7 or in insect Sf9 eucaryotic cells. Both PVIg-COS7 and PVIg-Sf9 were recognized by all of the 35 PV sera tested, but not by any of 10 pemphigus foliaceus (PF), 16 Brazilian PF, 10 bullous pemphigoid, or five normal control sera. Incubation of PV patients' sera with PVIg-Sf9 removed heterogeneous autoantibodies and significantly reduced their immunofluorescence titers on normal human epidermis, although PVIg-Sf9 did not affect the titers of PF sera at all. Furthermore, PVIg-Sf9 absorbed pathogenic autoantibodies from patients' sera and prevented gross blister formation in a neonatal mouse model for pemphigus. These results indicate that this baculovirus product has the proper conformation of the authentic PVA and that its conformation is important in pathogenicity of pemphigus.
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8040291
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Expression of platelet-derived growth factor receptors in normal and diseased human kidney. An immunohistochemistry and in situ hybridization study.
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We studied the expression of PDGF-alpha and -beta receptors in 10 normal and 40 pathologic human kidneys (five minimal change disease, five membranous nephropathy, 25 IgA nephropathy, five lupus nephritis), by both immunohistochemistry and in situ hybridization techniques. In normal-appearing kidneys, both PDGF-alpha and -beta receptors were expressed at the glomerular and interstitial level, the latter receptor more intensely than the former. The distribution and degree of expression of both receptors in nonproliferative glomerulonephritides were comparable with those found in normal-appearing kidneys. PDGF-beta receptor gene and protein expression were upregulated in proliferative nephritides both at the glomerular and the interstitial level and strictly correlated with the grade of histologic lesions. Finally, PDGF beta receptor expression was observed at a low level in normal-appearing renal vessels, and strikingly increased in injured arteries. Diseased kidneys displayed only a slight increase of PDGF-alpha receptor expression, chiefly at the interstitial level. Noteworthy, a few cases of lupus nephritis showed a moderate increase of PDGF-alpha receptor also at the glomerular level. These data establish PDGF-beta receptor activation as a candidate for driving glomerular and interstitial proliferation and, probably, expansion of extracellular matrix in proliferative glomerulonephritis, while the role of PDGF-alpha receptor activation at the renal level remains to be elucidated.
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8040290
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Chlamydia trachomatis from individuals in a sexually transmitted disease core group exhibit frequent sequence variation in the major outer membrane protein (omp1) gene.
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60 cervical Chlamydia trachomatis infections identified by antigen detection from 51 prostitute women in Nairobi, Kenya were evaluated for sequence polymorphism in the major outer membrane protein (omp1) gene. DNA from clinical specimens was amplified by the polymerase chain reaction and cycle sequenced through variable domains (VD) 1, 2, and 4.37 (63%) samples had variant VD sequences, 19 (32%) samples had prototype VD sequences, and 4 (6%) samples had prototype VD sequences, and 4 (6%) samples contained omp1 sequences from two or more C. trachomatis strains. Among the 37 variant strains, 18 had two or fewer nucleotide substitutions in one or two VDs and represented point mutational drift variants. 19 strains had a larger number of nucleotide changes and displayed mosaic omp1 sequences that may have been generated by omp1 VD recombination. We conclude that the prevalence of C. trachomatis omp1 DNA polymorphism is substantial among prostitute women in Nairobi, Kenya and that this is the likely result of immune selection pressure.
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8040289
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Early and specific antibody response to OspA in Lyme Disease.
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Borrelia burgdorferi (Bb), the cause of Lyme disease, has appeared not to evoke a detectable specific antibody response in humans until long after infection. This delayed response has been a biologic puzzle and has hampered early diagnosis. Antibody to the abundant organism-specific outer surface proteins, such as the 31-kD OspA, has rarely been detected less than 6 mo after infection. Antibody to a less organism-specific 41-kD flagellin protein, sharing common determinants with other bacteria and thus limiting its diagnostic potential, may appear after 4 to 6 wks. To investigate our hypothesis that specific antibody to OspA may actually be formed early but remain at low levels or bound in immune complexes, we analyzed serum samples from patients with concurrent erythema migrans (EM). This is the earliest sign of Lyme disease and occurs in 60-70% of patients, generally 4-14 d after infection. We used less conventional but more sensitive methods: biotin-avidin Western blots and immune complex dissociation techniques. Antibody specificity was confirmed with recombinant OspA. Specific complexed antibody to whole Bb and recombinant OspA was detected in 10 of 11 of the EM patients compared to 0 of 20 endemic area controls. IgM was the predominant isotype to OspA in these EM patients. Free IgM to OspA was found in half the EM cases. IgM to OspA was also detected in 10 of 10 European patients with EM who also had reactive T cells to recombinant OspA. In conclusion a specific antibody response to OspA occurs early in Lyme disease. This is likely to have diagnostic implications.
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8040288
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Neutralization of endogenous IL-1 receptor antagonist exacerbates and prolongs inflammation in rabbit immune colitis.
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Administration of exogenous interleukin-1 receptor antagonist (IL-1ra) is effective in reducing the severity of disease in animal models of acute inflammation. However, the function of endogenous IL-1ra in this process, is not yet known. We investigated the pathophysiological role of IL-1ra in a rabbit model of formalin-immune complex colitis. This model has previously been shown to be IL-1 mediated and a reduction in disease severity is observed with exogenous IL-1ra treatment. Colonic IL-1ra was found to be elevated subsequent to IL-1, and exceeded IL-1 levels 10-fold. Peak levels of IL-1ra preceded both the resolution of colitis and a significant decrease in IL-1 production. Administration of specific neutralizing antibodies against rabbit IL-1ra increased mortality and prolonged intestinal inflammatory responses. A significant increase in IL-1 alpha colonic tissue levels was also measured as a result of exogenous anti-IL-1ra treatment. These studies are the first demonstration that endogenous IL-1ra may play an important role in regulating the host's inflammatory response by counteracting the deleterious and possibly lethal effects of IL-1 produced during acute inflammation.
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8040287
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Activation of an imprinted allele of the insulin-like growth factor II gene implicated in rhabdomyosarcoma.
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The insulin-like growth factor II (IGF2) gene is exclusively silent at the maternal allele in the mouse as well as in normal human tissues and is expressed at a high level in rhabdomyosarcoma (RMS). We report here that the normally imprinted allele of the IGF2 gene is activated in RMS tumors as well as in one RMS cell line. Since overexpression of IGF2 has been shown to be important in the pathogenesis of RMS, our data suggest that loss of imprinting (LOI) may lead to overexpression of IGF2 and play an important role in the onset of RMS. Furthermore, embryonal RMS usually has loss of heterozygosity (LOH) with paternal disomy of the IGF2 locus. One informative embryonal RMS tumor evaluated in this study was heterozygous at the IGF2 allele and had LOI, raising the possibility that LOI may be the functional equivalent of LOH in this tumor with both events leading to overexpression of IGF2.
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8040286
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Induction of cell cycle progression by hepatitis B virus HBx gene expression in quiescent mouse fibroblasts.
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The HBx gene of hepatitis B virus has been shown to induce hepatic tumors in transgenic mice and is implicated in hepatocarcinogenesis in human hepatitis B virus infection. To further characterize the role of HBx gene in carcinogenesis, we established mouse fibroblast cell lines in which the expression of HBx gene could be controlled by glucocorticoid hormone and examined the effect of HBx gene expression on cell growth in vitro. Along with the expression of HBx gene, most cells in the G0/G1 phase moved into the S phase in 24 h, and the cell cycle progressed further toward 48 h. Induction of DNA synthesis was also demonstrated by bromo-deoxyuridine labeling analysis. These results indicate that HBx gene has a function to trigger the synthesis of cellular DNA and suggest that HBx gene may play a role in hepatocarcinogenesis in human infection by driving deregulated cell cycle progression.
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8040285
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Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions.
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Oxidatively modified lipoproteins have been implicated in atherogenesis, but the mechanisms that promote oxidation in vivo have not been identified. Myeloperoxidase, a heme protein secreted by activated macrophages, generates reactive intermediates that oxidize lipoproteins in vitro. To explore the potential role of myeloperoxidase in the development of atherosclerosis, we determined whether the enzyme was present in surgically excised human vascular tissue. In detergent extracts of atherosclerotic arteries subjected to Western blotting, a rabbit polyclonal antibody monospecific for myeloperoxidase detected a 56-kD protein, the predicted molecular mass of the heavy subunit. Both the immunoreactive protein and authentic myeloperoxidase bound to a lectin-affinity column; after elution with methyl mannoside their apparent molecular masses were indistinguishable by nondenaturing size-exclusion chromatography. Peroxidase activity in detergent extracts of atherosclerotic lesions likewise bound to a lectin column and eluted with methyl mannoside. Moreover, eluted peroxidase generated the cytotoxic oxidant hypochlorous acid (HOCl), indicating that enzymatically active myeloperoxidase was present in lesions. Patterns of immunostaining of arterial tissue with antihuman myeloperoxidase antibodies were similar to those produced by an antimacrophage antibody, and were especially prominent in the shoulder region of transitional lesions. Intense foci of myeloperoxidase immunostaining also appeared adjacent to cholesterol clefts in lipid-rich regions of advanced atherosclerotic lesions. These findings identify myeloperoxidase as a component of human vascular lesions. Because this heme protein can generate reactive species that damage lipids and proteins, myeloperoxidase may contribute to atherogenesis by catalyzing oxidative reactions in the vascular wall.
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8040284
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Mapping of a quantitative trait locus for blood pressure on rat chromosome 2.
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A genetic map for rat chromosome 2 that includes five candidate genes for blood pressure regulation was constructed in a region containing a quantitative trait locus (QTL) for blood pressure. Two F2 populations of male rats raised on high salt (8% NaCI) diet from weaning were studied: F2(WKY x S), derived from a cross of Dahl salt-sensitive rats (S) and Wistar-Kyoto rats (WKY); and F2(MNS x S), derived from a cross of S rats and Milan normotensive strain (MNS). In both populations a blood pressure QTL was localized between Na+,K(+)-ATPase alpha 1 isoform and calmodulin-dependent protein kinase II-delta loci. The LOD score for existence of this blood pressure QTL based on the combined populations (n = 330) was 5.66 and accounted for 9.2% of the total variance and 26% of the genetic variance.
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8040283
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Deficient type I protein kinase A isozyme activity in systemic lupus erythematosus T lymphocytes.
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Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by a dysfunctional cellular immune response. We have previously identified a metabolic disorder of the adenylate cyclase/cAMP/protein kinase A (AC/cAMP/PKA) pathway characterized by impaired cAMP-inducible, PKA-catalyzed protein phosphorylation in intact T lymphocytes from subjects with severe SLE disease activity. Because this metabolic disorder may contribute to abnormal T cell immune effector functions, we tested the hypothesis that impaired PKA-dependent protein phosphorylation is the result of a PKA isozyme deficiency in SLE T lymphocytes. Compared with healthy and rheumatoid arthritis (RA) controls, subjects with severe SLE activity exhibited reduced PKA-catalyzed phosphorylation of proteins in the T lymphocyte plasma membrane where the type I isozyme of PKA (PKA-I) is predominantly localized. Both silver staining and biosynthetic labeling of membrane-associated proteins with [35S]methionine demonstrated that reduced protein phosphorylation was not due to either an altered distribution of or absence of proteins. Moreover, phosphorylation of SLE membrane-associated proteins with the PKA catalytic (C) subunit showed a similar distribution and extent of phosphorylation compared with membrane proteins from healthy T cells, suggesting that SLE T cell membrane proteins could be phosphorylated. Sequential column chromatography of the type I and type II isozymes of PKA (PKA-I, PKA-II) demonstrated a deficiency of PKA-I isozyme activity. Compared with a ratio of PKA-I to PKA-II activity of 4.2:1 in healthy T cells, the activity ratio in T cells from subjects with severe SLE disease activity was 0.99:1 (P = 0.01, SLE versus healthy controls for PKA-I). The deficient PKA-I activity was associated with a significant increase of free C-subunit activity (P = 0.04, SLE versus healthy controls for C-subunit). T cells from subjects with mild/moderate SLE disease activity also exhibited diminished PKA-I activity, yielding a ratio of PKA-I to PKA-II activity of 2.4:1. By contrast, T cells from RA controls possessed increased PKA-I, PKA-II, and free C-subunit activities compared with healthy controls, resulting in a ratio of PKA-I to PKA-II activity of 3.6:1. We conclude that the reduced PKA-catalyzed protein phosphorylation in the plasma membrane of SLE T cells is the result of deficient PKA-I isozyme activity. This is the first identification of a deficiency of PKA activity in SLE T lymphocytes; the deficiency, resulting in diminished protein phosphorylation, may alter cellular homeostasis, contributing to the cellular immune dysfunctions observed in SLE.
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8040282
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Compensatory lung growth occurs in adult dogs after right pneumonectomy.
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We investigated the structural changes in the left lung of five adult male foxhounds 5 mo (n = 2) or 16 mo (n = 3) after right pneumonectomy (approximately 54% of lung resected) and five sex- and age-matched foxhounds 15-16 mo after right thoracotomy without pneumonectomy. Lungs were fixed by intratracheal instillation of glutaraldehyde and analyzed by standard morphometric techniques. After right pneumonectomy, volume of the left lung increased by 72%. Volumes of all septal structures increased significantly and were more pronounced at 5 than at 16 mo after pneumonectomy. At 16 mo, the relative increases in volume with respect to the control left lung were as follows: epithelium 73%, interstitium 100%, endothelium 55%, and capillary blood volume 43%. Surface areas of alveoli and capillary increased significantly by 52% and 34%, respectively. At 5 mo after pneumonectomy, harmonic mean thickness of the tissue-plasma barrier was significantly greater but at 16 mo it was not different from controls. There was a significant increase in diffusing capacity for oxygen (33% above controls) at 16 mo after pneumonectomy. These data suggest that, in contrast to previous findings after left pneumonectomy, compensatory lung growth does occur in adult dogs after resection of > 50% of lung.
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8040280
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Mechanism of compensatory hyperinsulinemia in normoglycemic insulin-resistant spontaneously hypertensive rats. Augmented enzymatic activity of glucokinase in beta-cells.
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The cause of compensatory hyperinsulinemia in normoglycemic insulin-resistant states is unknown. Using spontaneously hypertensive rats (SHR), we tested the hypothesis that a lowered beta-cell set-point for glucose causes a hypersecretion of insulin at a normal glucose level. Islets isolated from normoglycemic hyperinsulinemic SHR were compared to age-matched (12 wk old) Wistar-Kyoto (WK) rats. The ED50 for glucose-induced insulin secretion was 6.6 +/- 1.0 mM glucose in SHR versus 9.6 +/- 0.5 mM glucose in WK (P < 0.02). Glucokinase enzymatic activity was increased 40% in SHR islets (P < 0.02) without any change in the glucokinase protein level by Western blot. The level of the beta-cell glucose transporter (GLUT-2) was increased 75% in SHR islets (P < 0.036). In summary, the beta-cell sensitivity for glucose was increased in these normoglycemic insulin resistant rats by an enhanced catalytic activity of glucokinase. We have identified a regulatory system for glucokinase in the beta-cell which entails variable catalytic activity of the enzyme, is modulated in response to variations in whole-body insulin sensitivity, and is not dependent on sustained changes in the plasma glucose level.
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8040279
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A comparison of the antiatherogenic effects of probucol and of a structural analogue of probucol in low density lipoprotein receptor-deficient rabbits.
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The efficacies of probucol and a close structural analogue as antioxidants in the prevention of atherogenesis in LDL receptor-deficient rabbits were compared. The antioxidant potency of the analogue in vitro was equal to that of probucol. Its biological availability was much greater: almost comparable concentrations in total plasma were achieved by feeding 1% probucol (wt/wt) and 0.05% analogue (wt/wt). Total plasma concentrations were comparable, but the concentration of probucol within the LDL fraction was about twice that of the analogue. Probucol slowed lesion progression by almost 50%, confirming earlier reports; the analogue, however, showed no detectable inhibitory effect on atherogenesis. Resistance of LDL to oxidation was measured at the end of the study by incubating it with Cu2+ and measuring the rate of diene conjugation. Probucol prolonged diene conjugation lag time from the control value of 130 min to values > 1,000 min. The analogue approximately tripled the lag time (mean, 410 min) and yet failed to slow the atherogenic process. The results suggest that LDL resistance to oxidation must reach some threshold level before there is significant protection against atherogenesis. However, probucol has additional biological effects, possibly not shared by the analogue, that could contribute to its antiatherogenic potential.
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8040278
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Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway.
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The trefoil peptides, a recently recognized family of protease-resistant peptides, expressed in a regional specific pattern throughout the normal gastrointestinal tract. Although these peptides have been hypothesized to act as growth factors, their functional properties are largely unknown. Addition of recombinant trefoil peptides human spasmolytic polypeptide (HSP), rat and human intestinal trefoil factor (RITF and HITF) to subconfluent nontransformed rat intestinal epithelial cell lines (IEC-6 and IEC-17), human colon cancer-derived cell lines (HT-29 and CaCO2) or nontransformed fibroblasts (NRK and BHK) had no significant effect on proliferation. However addition of the trefoil peptides to wounded monolayers of confluent IEC-6 cells in an in vitro model of epithelial restitution resulted in a 3-6-fold increase in the rate of epithelial migration into the wound. Stimulation of restitution by the trefoil peptide HSP was enhanced in a cooperative fashion by the addition of mucin glycoproteins purified from the colon or small intestine of either rat or man, achieving up to a 15-fold enhancement in restitution. No synergistic effect was observed by the addition of nonmucin glycoproteins. In contrast to cytokine stimulation of intestinal epithelial cell restitution which is mediated through enhanced TGF beta bioactivity, trefoil peptide, and trefoil peptide-mucin glycoprotein stimulation of restitution was not associated with alteration in concentrations of bioactive TGF-beta and was not affected by the presence of immunoneutralizing anti-TGF beta antiserum. Collectively, these findings suggest that the trefoil peptides which are secreted onto the lumenal surface of the gastrointestinal tract may act in conjunction with the mucin glycoprotein products of goblet cells to promote reestablishment of mucosal integrity after injury through mechanisms distinct from those which may act at the basolateral pole of the epithelium.
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8040277
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Pathogenesis of periodontitis: a major arginine-specific cysteine proteinase from Porphyromonas gingivalis induces vascular permeability enhancement through activation of the kallikrein/kinin pathway.
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To elucidate the mechanism of production of an inflammatory exudate, gingival crevicular fluid (GCF), from periodontal pockets in periodontitis, we examined the vascular permeability enhancement (VPE) activity induced by an arginine-specific cysteine proteinase, Arg-gingipain-1 (RGP-1), produced by a major periopathogenic bacterium, Porphyromonas gingivalis. Intradermal injections into guinea pigs of RGP-1 (> 10(-8) M), or human plasma incubated with RGP-1 (> 10(-9) M), induced VPE in a dose- and activity-dependent manner but with different time courses for the two routes of production. VPE activity induced by RGP-1 was augmented by kininase inhibitors, inhibited by a kallikrein inhibitor and unaffected by an antihistamine drug. The VPE activity in human plasma incubated with RGP-1 also correlated closely with generation of bradykinin (BK). RGP-1 induced 30-40% less VPE activity in Hageman factor-deficient plasma and no VPE in plasma deficient in either prekallikrein (PK) or high molecular weight kininogen (HMWK). After incubation with RGP-1, plasma deficient in PK or HMWK, reconstituted with each missing protein, caused VPE, as did a mixture of purified PK and HMWK, but RGP-1 induced no VPE from HMWK. The VPE of extracts of clinically isolated P. gingivalis were reduced to about 10% by anti-RGP-1-IgG, leupeptin, or tosyl-L-lysine chloromethyl ketone, which paralleled effects observed with RGP-1. These results indicate that RGP-1 is the major VPE factor of P. gingivalis, inducing this activity through PK activation and subsequent BK release, resulting in GCF production at sites of periodontitis caused by infection with this organism.
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8040276
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Selective reovirus infection of murine hepatocarcinoma cells during cell division. A model of viral liver infection.
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Reovirus type 1, strain Lang (1/L), can infect hepatocytes in vivo only after hepatocellular damage is induced by hepatotoxins, surgical trauma, resection, or profound immunosuppression. To examine the role of cell cycle and cellular differentiation on liver cell susceptibility to reovirus infection, a murine hepatocarcinoma cell line, Hepa 1/A1, was infected with reovirus and assayed for the presence of infectious virus or reovirus antigen in cells. Despite a > 95% binding of reovirus to hepatocarcinoma cells as indicated by cytometric analysis; only 10% of hepatoma cells contained infectious virus by infectious center assay. In comparison, 100% of L cells were infected. Analysis of intracellular reovirus antigen revealed its presence in dividing but not in quiescent hepatocytes. This correlation of cellular division and cell capacity to support viral replication suggests that induction of hepatocyte proliferation may be a mechanism for liver susceptibility to reovirus infection.
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8040275
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Cytokine-induced neutrophil chemoattractant mediates neutrophil influx in immune complex glomerulonephritis in rat.
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Chemokines are a family of cytokines whose participation in inflammation in vivo remains to be established. Using the rat model of anti-glomerular basement membrane (GBM) nephritis, we found that mRNA for the chemokine CINC (cytokine-induced neutrophil chemoattractant) was induced in the kidney, and the corresponding protein was elaborated by isolated inflamed glomeruli. Production of CINC by glomeruli was unaffected by complement- or leukocyte-depletion prior to disease induction. Cytokines which induce CINC expression in renal cells (TNF-alpha and IL-1 beta) were also expressed in the kidney during glomerular inflammation. TNF-alpha production, unlike CINC, was complement and leukocyte dependent. In vivo administration of anti-CINC, but not anti-human IL-8, IgG selectively attenuated the influx of PMNs into the glomerulus and commensurately diminished proteinuria. The participation of CINC was not tissue-specific: anti-CINC IgG also diminished the influx of PMNs in dermal immune complex inflammation. In sum, we propose that glomerular immune complex deposition/complement activation leads to cytokine production which results in CINC expression by endogenous glomerular cells. The CINC produced plays a contributory role in the influx of PMNs into the glomerulus in the context of the activation of other inflammatory mediators. These results suggest a potential role for CINC homologues, IL-8 and the GRO family of chemokines, in human immune complex-mediated disease.
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8040274
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Regulation of hormone-sensitive calcium influx by the adenylyl cyclase system in renal epithelial cells.
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To study signaling pathways regulated by alpha s and alpha i1 in renal epithelial cells, we expressed mutant, activated forms of alpha s and alpha i1 in a continuous proximal tubule cell line (MCT cells). alpha sQ227L increased cAMP production, and alpha ilQ204L reduced forskolin-sensitive cAMP production. alpha ilQ204L increased and alpha sQ227L decreased bradykinin-induced Ca influx across the cell membrane, but neither mutant affected bradykinin-stimulated intracellular Ca release or basal Ca influx. Bradykinin-stimulated Ca influx was reduced by dibutyryl cAMP, isoproterenol, and forskolin. Expression of a mutant regulatory type I subunit for cAMP-dependent protein kinase with reduced affinity for cAMP and treatment with KT-5720, a specific cAMP-dependent protein kinase inhibitor, enhanced Ca influx to a degree similar to that in cells expressing alpha ilQ204L. Bradykinin-stimulated c-fos mRNA expression is partially dependent on extracellular Ca. alpha sQ227L reduced and alpha ilQ204L enhanced bradykinin-stimulated c-fos expression. Consequently, in bradykinin-stimulated cells, the adenylyl cyclase system regulates Ca influx through cAMP-dependent protein kinase, but not intracellular Ca release. Furthermore, the Ca influx mechanism acts as an integrator of two signaling pathways such that Ca-dependent signals are damped by activators of adenylyl cyclase and enhanced by inhibitors of adenylyl cyclase.
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8040273
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A reappraisal of the role of insulin-like growth factor I in the regulation of human hematopoiesis.
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IGF-I has been reported to increase hematopoietic progenitor cell cloning efficiency. To investigate this phenomenon, we studied the IGF-I responsiveness of human marrow cells expressing IGF-I receptor (IGF-IR), a direct strategy not used previously. IGF-IR+ and control CD34+ marrow cells were isolated using immunoaffinity methods. Then, the cells were cloned in methylcellulose containing variable amounts of serum- and lineage-appropriate growth factors supplemented with recombinant human IGF-I. In contrast to CD34+ cells, IGF-IR+ cells never gave rise to CFU-Blast, CFU-Mix, CFU-GM, BFU-E, or CFU-E. To substantiate the suggestion that CD34+ and IGF-IR+ cells were distinct populations, we used reverse transcription PCR to detect IGF-I, EpO, and KIT receptor mRNAs in these cells. The mRNA phenotype of CD34+ cells was EpO (+), KIT (+), and IGF-IR (-), while IGF-IR+ cells were IGF-IR (+), EpO (-), and KIT (-). These results suggested that IGF-IR is either not expressed or expressed at low levels on normal hematopoietic progenitor cells. Functional significance of the latter possibility was tested by exposing CD34+ cells to IGF-IR antisense oligodeoxynucleotides. Colony formation was unaffected by oligodeoxynucleotide disruption of IGF-IR, suggesting that, even if expressed at low level, the receptor's functional significance was doubtful. Nevertheless, when cultured in the presence of IGF-I, IGF-IR+ cells elaborated an activity with mild BFU-E stimulatory effects. Accordingly, if IGF-I plays a role in hematopoietic colony formation, it is probably and results from stimulation of IGF-IR-positive ancillary cells to secrete growth factors. Studies carried out with human leukemia cells yielded similar results.
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8040272
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Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses.
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We tested the hypothesis that pulmonary surfactant-associated lectins--surfactant proteins A and D (SP-A, and -D)--contribute to initial protective mechanisms against influenza A viruses (IAVs). SP-D potently inhibited hemagglutination activity of several strains of IAV as well as causing viral aggregation. SP-D enhanced neutrophil binding of IAV and neutrophil respiratory burst responses to the virus. Neutrophil dysfunction resulting from IAV exposure was diminished when the virus was pre-incubated with SP-D. Each of these effects was mediated by the calcium-dependent carbohydrate-binding property of SP-D. Native SP-D preparations of both human and rat origin, as well as recombinant rat SP-D, had similar activity. SP-A also inhibited IAV hemagglutination activity. We have previously reported that related mammalian serum lectins (mannose-binding lectin [MBL] and conglutinin) have similar effects. SP-D was at least 10-fold more potent at causing hemagglutination inhibition than were SP-A or MBL. SP-D was shown to contribute to potent anti-IAV activity of human bronchoalveolar lavage fluid. These results suggest that SP-D--alone, and in conjunction with SP-A and phagocytic cells--constitutes an important component of the natural immune response to IAV infection within the respiratory tract.
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8040271
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Increased angiotensin-I converting enzyme gene expression in the failing human heart. Quantification by competitive RNA polymerase chain reaction.
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Local activation of the components of the renin angiotensin system in the heart is regarded as an important modulator of cardiac phenotype and function; however, little is known about their presence, regulation, and potential activation in the human heart. To investigate the gene expression of major angiotensin-II-forming enzymes in left ventricles of normal (n = 9) and failing human hearts (n = 20), we established a competitive RNA-polymerase chain reaction (PCR) for mRNA quantification of angiotensin-I converting enzyme (ACE) and human heart chymase. For each gene, competitor RNA targets with small internal deletions were used as internal standards to quantify the original number of transcripts and to control reverse transcription and PCR. In PCR, each target and the corresponding competitor were amplified by competing for the same primer oligonucleotides. The variability of ACE RNA-PCR was 11% indicating a high reproducibility of this method. In addition, ACE mRNA levels obtained by competitive RNA-PCR correlated favorably with traditional slot blot hybridization (r = 0.69, n = 10; P < 0.05). Compared with nonfailing hearts, the number of ACE transcripts referred to 100 ng of total RNA was increased threefold in patients with chronic heart failure (4.2 +/- 2.5 vs. 12.8 +/- 6 x 10(5); P < 0.0005). In contrast, no significant difference was found in chymase gene expression between normal and failing hearts. Thus, the expression of the cardiac ACE but not of human heart chymase is upregulated in failing human heart indicating an activation of the cardiac renin-angiotensin system in patients with advanced heart failure.
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8040269
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Glycogen synthase: a putative locus for diet-induced hyperglycemia.
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Inbred mouse strains fed a diabetogenic diet have different propensities to develop features analogous to type 2 diabetes mellitus. To define chromosomal locations that control these characteristics, recombinant inbred strains from diabetes-prone C57BL/6J (B/6J) and diabetes-resistant A/J strains were studied. Insulin levels and hyperglycemia correlated with two different regions of mouse chromosome 7 (two point LOD scores > 3.0). For insulin levels, 15 of 16 recombinant inbred strains were concordant with a region that contains the tubby mutation that results in hyperinsulinemia. For hyperglycemia, 19 of 23 strains were concordant with the D7Mit25 marker and 20 of 23 strains with the Gpi-1 locus on proximal mouse chromosome 7. Using more stringent criteria for hyperglycemia, 10 of 11 strains characterized as A/J or B/6J like were concordant with D7Mit25. This putative susceptibility locus is consistent with that of the glycogen synthase gene (Gys) recently suggested as a candidate locus by analyses of type 2 diabetes patients. Fractional glycogen synthase activity in isolated muscle was significantly lower in normal B/6J diabetic-prone mice compared with normal diabetic-resistant A/J mice, a finding similar to that reported in relatives of human patients with type 2 diabetes. These data, taken together, raise the possibility that defects in the Gys gene may in part be responsible for the propensity to develop type 2 diabetes.
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8040268
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Platelet factor 4 modulates the mitogenic activity of basic fibroblast growth factor.
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Basic fibroblast growth factor (bFGF) has been shown to stimulate cell proliferation after vascular injury. The mitogenic activity of bFGF requires interactions with both a high affinity receptor and a cell-surface heparan sulfate proteoglycan. We tested the ability of platelet factor 4 (PF 4) and other platelet heparin-binding proteins to modulate bFGF-stimulated [3H]thymidine incorporation into fibroblasts. The supernatant of thrombin-stimulated platelets contained an inhibitor of bFGF-induced mitogenesis; this activity coeluted with PF 4 upon gel filtration, heparin-agarose, and ion-exchange chromatography. Purified thrombospondin and beta-thromboglobulin did not inhibit the mitogenic activity of bFGF. PF 4 inhibited the activity of 5 pM bFGF with 50% inhibitory concentration of 75 nM. Purified PF 4 also inhibited the basal incorporation of [3H]thymidine into 3T3 fibroblasts and the increased [3H]thymidine incorporation occurring after wounding of a cell monolayer. PF 4 did not affect the mitogenic activity of serum. Inhibition of bFGF activity by PF 4 could be overcome by exogenous heparin or chondroitin-4-sulfate, suggesting that inhibition of mitogenesis is caused by binding of PF 4 to cell-surface glycosaminoglycans. These results indicate that an important role of PF 4 released at sites of vascular injury and platelet activation is to control cellular proliferation caused by the release of bFGF from ruptured cells.
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8040267
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Interleukin-10 inhibits apoptotic cell death in infectious mononucleosis T cells.
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T lymphocytes from patients with acute EBV-induced infectious mononucleosis rapidly die by apoptosis in vitro. Because human and viral IL-10 are likely to be induced during acute EBV infection and display a variety of functions on human T cells, we examined IL-10 effects on infectious mononucleosis T cell death. After 12 h of incubation in medium alone, only 35.6 (+/- 8.2%) of the originally seeded infectious mononucleosis T cells were viable. Addition of human IL-10 (100 U/ml) to T cell cultures significantly improved recovery of viable cells (71.3 +/- 6.2%, P = 0.0156). Viral IL-10 had comparable effects to human IL-10 in this system. Protection from death by human and viral IL-10 (100 U/ml) was dose dependent and continued over a 6-d culture period. The human IL-10 effect was neutralized by the anti-human IL-10 mAb 19F1. Morphology and analysis of DNA after separation on agarose gels showed that IL-10 inhibits loss of cell volume, chromatin condensation, and DNA fragmentation, characteristics of death by apoptosis. As assessed by [3H]thymidine incorporation, the T cells were not induced to proliferate by IL-10 above the level exhibited when first removed from blood. T cells protected from death by IL-10 proliferated to IL-2 and spontaneously killed sensitive targets as effectively as medium-precultured T cells. Thus, IL-10 promotes the survival of infectious mononucleosis T cells otherwise destined to die by apoptosis and may be critical for the establishment of immunologic memory after resolution of the illness.
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8040266
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Tumor necrosis factor-alpha-induced inhibition of phosphatidylcholine synthesis by human type II pneumocytes is partially mediated by prostaglandins.
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TNF alpha seems to play an important role in the pathogenesis of adult respiratory distress syndrome. We studied the effect of TNF alpha on phospholipid synthesis by isolated type II pneumocytes and attempted to characterize the role of arachidonate metabolites and the influence of pentoxifylline on such an effect. Lung tissue obtained from both multiple organ donors (n = 14) and lung cancer patients (n = 11) was used for cell isolation. Surfactant synthesis was measured by the incorporation of D-[U-14C]glucose into phosphatidylcholine (PC). The basal PC synthesis was higher in the donor group than in the malignant group (3.44 +/- 0.19 vs 2.15 +/- 0.15 pmol/microgram protein x 120 min, P < 0.01), and, in the presence of 100 ng/ml of TNF alpha, the incorporation of labeled glucose into PC was reduced significantly in both donor (1.13 +/- 0.11 vs 3.44 +/- 0.19 pmol/microgram protein x 120 min, P < 0.01) and cancer (0.99 +/- 0.11 vs 2.15 +/- 0.15 pmol/microgram protein x 120 min, P < 0.01) groups. Indomethacin was able to completely block the cytokine-induced decrease in PC synthesis by pneumocytes from the malignant group and to attenuate the inhibitory effect of TNF alpha in those from donors, nordihydroguaiaretic acid having a similar effect. The TNF alpha effect can be blocked by pentoxifylline (100 micrograms/ml), a substance which can even succeed in reverting the basal secretory inhibition of cancer patients' pneumocytes to levels similar to those of the donor group. TNF alpha may contribute to the pathophysiology of adult respiratory distress syndrome by inhibiting the synthesis of surfactant. TNF alpha might be produced in lung tumors, resulting in chronic paracrine or systemic exposure of pneumocytes to low concentrations of the cytokine. The TNF alpha effect was not prevented completely by the blockage of the arachidonic acid metabolism, hence other mediators should also be implicated.
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8040265
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Renin and renin mRNA in proximal tubules of the rat kidney.
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The present study was undertaken to assess the presence of renin enzymatic activity and renin mRNA in proximal tubules of rat kidneys, and to determine the effect of converting enzyme inhibition (CEI) on proximal tubule renin gene expression. Proximal convoluted tubules (PCT), proximal straight tubules (PST), outer medullary collecting ducts (OMCD), and glomeruli (Gloms) were isolated by microdissection. Renin activity was measured in sonicated segments by radioimmunoassay. Renin mRNA levels were assessed using a quantitative PCR. Renin activity in PCT averaged 51 +/- 15 microGU/mm compared to 405 +/- 120 microGU/glomerulus. No measurable renin activity was found in PST and OMCD. Renin activity in both glomeruli and tubules had the same pH optimum, between 7.0 and 7.5. Renin mRNA was consistently detectable in cDNA prepared from PCT and PST, although its abundance per mm tubule was about 1/500th that found in one glomerulus. Renin mRNA was not detectable in OMCD. Tubular renin PCR product identity was confirmed by restriction digestion. CEI administration increased glomerular renin activity and renin mRNA, but not proximal tubular renin. The absence of a stimulatory effect of CEI on proximal tubule renin gene expression suggests the operation of different intracellular signals in control of renin synthesis in the proximal tubule than in the vascular compartment.
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8040263
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Alpha 1 adrenergic receptor-induced c-fos gene expression in rat aorta and cultured vascular smooth muscle cells.
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While growth of blood vessels is important in hypertension, relatively little is known about the contribution of catecholamines. Using isolated rat aorta and cultured smooth muscle cells, we examined adrenergic stimulation of gene expression. Phenylephrine, a selective alpha 1 adrenergic receptor agonist, caused a rapid and transient increase in c-fos mRNA accumulation which was inhibited by prazosin, an alpha 1 receptor antagonist. Similarly, phenylephrine stimulated c-jun and c-myc mRNA accumulation. Chloroethyl-clonidine, a compound which irreversibly blocks alpha 1B receptors, completely blocked the phenylephrine-induced increase in c-fos mRNA. RNase protection experiments demonstrated that rat aorta prominently expressed mRNA for alpha 1B and alpha 1A/D receptors. Phenylephrine-induced c-fos mRNA was partially inhibited by H-7, a protein kinase C inhibitor, and by nifedipine, a Ca2+ channel blocker; these two compounds together had additive effects. In situ hybridization showed that expression of c-fos mRNA induced by phenylephrine was localized to aorta's medial layer. These results suggest that alpha 1 receptor-induced increase in c-fos mRNA in aorta is mediated by a chloroethyl-clonidine-sensitive receptor subtype signaling via increasing intracellular Ca2+ concentrations and activating protein kinase C.
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8040262
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Characterization of dermal dendritic cells in psoriasis. Autostimulation of T lymphocytes and induction of Th1 type cytokines.
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Local activation of T lymphocytes is regarded as an important immunological component of psoriatic skin lesions. Within psoriatic plaques (PP) there are large numbers of dermal dendritic cells (DDCs) immediately beneath the hyperplastic epidermis surrounded by T cells. In this study we investigated the ability of DDCs isolated from PP skin to support immune responses to resting peripheral blood T cells. For comparison, other dendritic cells were obtained from blood of the same psoriatic patients, as well as DDCs from skin of normal healthy individuals (designated NN skin). All dendritic cells studied had high surface expression of HLA-DR, B7, and lymphocyte function associated antigen-1 molecules. T cell proliferative responses and cytokine production profiles to these various dendritic cells were measured in the absence and presence of PHA or bacterial-derived superantigens. In the absence of exogenous mitogens, PP skin-derived DDCs were much more effective stimulators of spontaneous T cell proliferation compared with either psoriatic blood-derived or NN skin-derived dendritic cells. Antibody blocking studies revealed involvement of HLA-DR, B7, and lymphocyte function associated antigen-1 on PP skin-derived DDCs. Cytokine profiles revealed that in the absence of exogenous stimuli PP skin-derived DDCs mediated a T cell response with high levels of IL-2 and IFN-gamma, but not IL-4 or IL-10. NN skin-derived DDCs produced a similar qualitative response, but quantitative amounts of all cytokines measured were lower. Upon addition of PHA or superantigens, both PP skin-derived and NN skin-derived DDCs mediated high levels of IL-2 and IFN-gamma production, with induction of IL-4 particularly evident for PHA reactions. Addition of conditioned medium from psoriatic dermal fragments did not enhance the autostimulatory capacity of blood-derived dendritic cells. These findings highlight the potent autostimulatory potential of PP skin-derived DDCs and suggest an important immunological contribution for these previously overlooked cell types contained within lesional skin sites.
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8040259
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The central role of chromatin in autoimmune responses to histones and DNA in systemic lupus erythematosus.
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To gain insight into the mechanisms of autoantibody induction, sera from 40 patients with systemic lupus erythematosus (SLE) were tested by ELISAs for antibody binding to denatured individual histones, native histone-histone complexes, histone-DNA subnucleosome complexes, three forms of chromatin, and DNA. Whole chromatin was the most reactive substrate, with 88% of the patients positive. By chi-square analysis, only the presence of anti-(H2A-H2B), anti-[(H2A-H2B)-DNA], and antichromatin were correlated with kidney disease measured by proteinuria > 0.5 g/d. SLE patients could be divided into two groups based on their antibody-binding pattern to the above substrates. Antibodies from about half of the patients reacted with chromatin and the (H2A-H2B)-DNA subnucleosome complex but displayed very low or no reactivity with native DNA or the (H3-H4)2-DNA subnucleosome complex. An additional third of the patients had antibody reactivity to chromatin, as well as to both subnucleosome structures and DNA. Strikingly, all sera that bound to any of the components of chromatin also bound to whole chromatin, and adsorption with chromatin removed 85-100% of reactivity to (H2A-H2B)-DNA, (H3-H4)2-DNA, and native DNA. Individual sera often bound to several different epitopes on chromatin, with some epitopes requiring quaternary protein-DNA interactions. These results are consistent with chromatin being a potent immunogenic stimulus in SLE. Taken together with previous studies, we suggest that antibody activity to the (H2A-H2B)-DNA component signals the initial breakdown of immune tolerance whereas responses to (H3-H4)2-DNA and native DNA reflect subsequent global loss of tolerance to chromatin.
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8040260
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Effect of chronic ethanol feeding on glutathione and functional integrity of mitochondria in periportal and perivenous rat hepatocytes.
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Chronic ethanol feeding selectively impairs the translocation of cytosol GSH into the mitochondrial matrix. Since ethanol-induced liver cell injury is preferentially localized in the centrilobular area, we examined the hepatic acinar distribution of mitochondrial GSH transport in ethanol-fed rats. Enriched periportal (PP) and perivenous (PV) hepatocytes from pair- and ethanol-fed rats were prepared as well as mitochondria from these cells. The mitochondrial pool size of GSH was decreased in both PP and PV cells from ethanol-fed rats either as expressed per 10(6) cells or per microliter of mitochondrial matrix volume. The rate of reaccumulation of mitochondrial GSH and the linear relationship of mitochondrial to cytosol GSH from ethanol-fed mitochondria were lower for both PP and PV cells, effects observed more prominently in the PV cells. Mitochondrial functional integrity was lower in both PP and PV ethanol-fed rats, which was associated with decreased cellular ATP levels and mitochondrial membrane potential, effects which were greater in the PV cells. Mitochondrial GSH depletion by ethanol feeding preceded the onset of functional changes in mitochondria, suggesting that mitochondrial GSH is critical in maintaining a functionally competent organelle and that the greater depletion of mitochondrial GSH by ethanol feeding in PV cells could contribute to the pathogenesis of alcoholic liver disease.
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8040258
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Mechanism of apical and basolateral Na(+)-independent Cl-/base exchange in the rabbit superficial proximal straight tubule.
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The present study was undertaken to determine the magnitude and mechanism of base transport via the apical and basolateral Na(+)-independent Cl-/base exchangers in rabbit isolated perfused superficial S2 proximal tubules. The results demonstrate that there is an apical Na(+)-independent Cl-/base exchanger on both membranes. HCO3- fails to stimulate apical Cl-/base exchange in contrast to the basolateral exchanger. Inhibition of endogenous HCO3- production does not alter the rate of apical Cl-/base exchange in Hepes-buffered solutions. Both exchangers are inhibited by H2DIDS and furosemide; however, the basolateral anion exchanger is more sensitive to these inhibitors. The results indicate that the apical and basolateral Cl-/base exchangers differ in their transport properties and are able to transport base equivalents in the absence of formate. The formate concentration in rabbit arterial serum is approximately 6 microM and in vitro tubule formate production is < 0.6 pmol/min per mm. Formate in the micromolar range stimulates Jv in a dose-dependent manner in the absence of a transepithelial Na+ and Cl- gradient and without a measurable effect on Cl(-)-induced equivalent base flux. Apical formic acid recycling cannot be an important component of any cell model, which accounts for formic acid stimulation of transcellular NaCl transport in the rabbit superficial S2 proximal tubule. We propose that transcellular NaCl transport in this nephron segment is mediated by an apical Na+/H+ exchanger in parallel with a Cl-/OH- exchanger and that the secreted H+ and OH- ions form H2O in the tubule lumen.
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8040257
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Regulation of acetylcholine receptor alpha subunit variants in human myasthenia gravis. Quantification of steady-state levels of messenger RNA in muscle biopsy using the polymerase chain reaction.
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Myasthenia gravis (MG) is an autoimmune disease mediated by auto-antibodies that attack the nicotinic acetylcholine receptor (AChR). To elucidate the molecular mechanisms underlying the decrease in AChR levels at the neuromuscular junction, we investigated the regulation of AChR expression by analyzing mRNA of the two AChR alpha subunit isoforms (P3A+ and P3A-) in muscle samples from myasthenic patients relative to controls. We applied a quantitative method based on reverse transcription of total RNA followed by polymerase chain reaction (PCR), using an internal standard we constructed by site-directed mutagenesis. An increased expression of mRNA coding for the alpha subunit of the AChR isoforms was observed in severely affected patients (P < 0.003 versus controls) but not in moderately affected patients, independently of the anti-AChR antibody titer. Study of mRNA precursor levels indicates a higher expression in severely affected patients compared to controls, suggesting an enhanced rate of transcription of the message coding for the alpha subunit isoforms in these patients. We have also reported that mRNA encoding both isoforms are expressed at an approximate 1:1 ratio in controls and in patients. We have thus identified a new biological parameter correlated with disease severity, and provide evidence of a compensatory mechanism to balance the loss of AChR in human myasthenia gravis, which is probably triggered only above a certain degree of AChR loss.
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8040256
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Inhibition of hypercholesterolemia-induced atherosclerosis in the nonhuman primate by probucol. I. Is the extent of atherosclerosis related to resistance of LDL to oxidation?
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Lipoprotein oxidation is believed to play an important role in atherogenesis. To investigate whether inhibition of oxidation of low density lipoprotein (LDL) would alter atherogenesis in the nonhuman primate, we administered probucol, a potent antioxidant, to Macaca nemestrina fed a high-fat, high-cholesterol diet. Probucol was administered to half of the 16 monkeys 14 wk after starting the hypercholesterolemic diet, and was given daily until they were sacrificed after 11 mos. To evaluate the antioxidant effect of probucol, the resistance of isolated plasma LDL to in vitro oxidation was evaluated. Probucol significantly increased the resistance of LDL to oxidative modification, as shown by an increase in the lag time required for conjugated diene formation. Lesions in the probucol-treated animals appeared less mature, and increased accumulation of lipid was observed in smooth muscle cells. Comparison of all control and probucol-treated monkeys demonstrated that intimal lesion areas in the thoracic aortas of the probucol-treated monkeys were reduced by 43% (P < 0.0001), but no significant difference in lesion area was found in the abdominal aortas or in the iliac arteries. However, the lag phase of conjugated diene formation was not prolonged in 2 of the 8 probucol-treated animals. A plot of intimal lesion size versus lag phase of all 16 animals showed a trend that lesion size was inversely related to oxidation resistance for all anatomic sites. The strong inverse relationship between intimal lesion size and resistance of LDL to oxidation supports a role for lipoprotein oxidation in the development and progression of lesions of atherosclerosis. The possibility that some of the effect is due to other biological properties of probucol cannot be ruled out.
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8040255
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Quantitative differences in biosynthesis and extracellular deposition of fibrillin in cultured fibroblasts distinguish five groups of Marfan syndrome patients and suggest distinct pathogenetic mechanisms.
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Pulse-chase studies of [35S]cysteine-labeled fibrillin were performed on fibroblast strains from 55 patients with Marfan syndrome (MFS), including 13 with identified mutations in the fibrillin-1 gene and 10 controls. Quantitation of the soluble intracellular and insoluble extracellular fibrillin allowed discrimination of five groups. Groups I (n = 8) and II (n = 19) synthesize reduced amounts of normal-sized fibrillin, while synthesis is normal in groups III (n = 6), IV (n = 18), and V (n = 4). When extracellular fibrillin deposition is measured, groups I and III deposit between 35 and 70% of control values, groups II and IV < 35%, and group V > 70%. A deletion mutant with a low transcript level from the mutant allele and seven additional patients have the group I protein phenotype. Disease in these patients is caused by a reduction in microfibrils associated with either a null allele, an unstable transcript, or an altered fibrillin product synthesized in low amounts. In 68% of the MFS individuals (groups II and IV), a dominant negative effect is invoked as the main pathogenetic mechanism. Products made by the mutant allele in these fibroblasts are proposed to interfere with microfibril formation. Insertion, deletion, and exon skipping mutations, resulting in smaller fibrillin products, exhibit the group II phenotype. A truncated form of fibrillin of 60 kD was identified with specific fibrillin antibodies in one of the group II cell culture media. Seven of the nine known missense mutations, giving rise to abnormal, but normal-sized fibrillin molecules, are in group IV.
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