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8043045 | Lyme disease. | In 1992, reported Lyme disease cases increased, but the majority remained clustered in the northeast and midwest, and in California. The clinical syndromes associated with infection are better understood, and this information can calm public anxiety. The concept of fibromyalgia occurring after infection with Borrelia burgdorferi allows more appropriate treatment in late Lyme disease. The polymerase chain reaction is being applied to gain insights into the pathogenic mechanisms of Lyme disease. Progress has been made toward developing a vaccine through the use of animal models. Together, these advances provide clinicians with more confidence in the diagnosis and treatment of Lyme disease. |
8043043 | Suppression of dyskinesias in advanced Parkinson's disease. II. Increasing daily clozapine doses suppress dyskinesias and improve parkinsonism symptoms. | We gave increasing daily doses of clozapine to six patients with advanced Parkinson's disease (PD) and levodopa-induced dyskinesias. Clozapine reduced the daily dyskinesia time five-fold, increased "on" time eight-fold, and doubled the serum [DOPA] producing half-maximal dyskinesia. Parkinsonism scores after overnight DOPA withdrawal improved with increasing daily clozapine intake, and there was no clozapine dose-related shift in levodopa dose response for relief of parkinsonism. Patients experienced sedation, sialorrhea, and orthostatic hypotension. Clozapine appears to be an effective agent for suppression of levodopa-induced dyskinesias in PD. |
8043040 | Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo. | The nef genes of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIVs) encode a protein (Nef) whose role in virus replication and cytopathicity remains uncertain. As an attempt to elucidate the function of nef, we characterized the nucleotide and corresponding protein sequences of naturally occurring nef genes obtained from several HIV-1-infected individuals. A consensus Nef sequence was derived and used to identify several features that were highly conserved among the Nef sequences. These features included a nearly invariant myristylation signal, regions of sequence polymorphism and variable duplication, a region with an acidic charge, a (Pxx)4 repeat sequence, and a potential protein kinase C phosphorylation site. Clustering of premature stop codons at position 124 was noted in 6 of the 54 Nef sequences. Further analysis revealed four stretches of residues that were highly conserved not only among the patient-derived HIV-1 Nef sequences, but also among the Nef sequences of HIV-2 and the SIVs, suggesting that Nef proteins expressed by these retroviruses are functionally equivalent. The "Nef-defining" sequences were used to evaluate the sequence alignments of known proteins reported to share sequence similarity with Nef sequences and to conduct additional computer-based searches for similar protein sequences. A gene encoding the consensus Nef sequence was also generated. This gene encodes a full-length Nef protein that should be a valuable tool in further studies of Nef function. |
8043041 | Location of CD4 dimerization site explains critical role of CDR3-like region in HIV-1 infection and T-cell activation and implies a model for complex of coreceptor-MHC. | CD4 cross-linking by antibodies or its natural ligands triggers a tyrosine kinase activity that is one of the necessary steps in the mechanism of human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and full Th-cell activation. In this study we mapped a part of the dimerization site of human CD4 to amino acids 87-98 using a bivalent CD4 immunoadhesin and a series of overlapping 12-mer peptides of the D1 domain. The dimerization site we found is part of the complementary determining region (CDR) 3-like region of CD4. Using the three-dimensional structure of other immunoglobulin dimers as a basis, a molecular modeling study was performed to dimerize the D1 domains of CD4. Both the peptide binding studies and molecular modeling studies independently led to the conclusion that the CDR3-like region is part of the CD4 dimerization site. The suggested dimerization of CD4 through its CDR3-like region explains the important role that has been ascribed to this region in Th-cell activation and HIV-1-mediated fusion. Based on this model of the CD4 dimer and published results of different mutational analysis studies, a model was proposed for the complex of the CD4 dimer with two MHC-II molecules. The CD4 dimer allows tight binding to a large surface of MHC-II and the complex of CD4 and MHC-II reconciles mutational analysis studies that were previously incompatible. Moreover, the complex suggests how CD4 can dimerize through ligand binding. |
8043038 | Surgical correction of upper eyelid retraction. | Over the last 7 years, I have used a "central aponeurosis disinsertion" technique to correct dysthyroid upper eyelid retraction in nine patients. Consistent and predictable results were achieved and maintained over follow-up periods ranging from 18 to 72 months. The technique is simple and effective, and does not involve extensive dissection or the use of a spacer. |
8043037 | Laparoscopic endocorporeal mobilization followed by extracorporeal sutureless anastomosis for the treatment of carcinoma of the left colon. | Surgery has become progressively more reliant on technology. The technique of colonic anastomosis utilizing the biofragmentable anastomotic ring (BAR) is one such example. The benefits of therapeutic laparoscopy have been applied to the arena of colorectal surgery. A case is presented that combines these two modalities in a patient with colon cancer, laparoscopic mobilization of the large bowel, exteriorized resection, and BAR anastomosis. |
8043036 | In vitro antimicrobial activity of CP-99,219, a novel azabicyclo-naphthyridone. | CP-99,219 is a trifluoronaphthyridone with significant antibacterial activity that includes the family Enterobacteriaceae (MICs for 90% of the strains tested [MIC90s], < or = 0.015 to 0.5 micrograms/ml), Moraxella catarrhalis, Haemophilus influenzae, and gonococci (MICs, < or = 0.015 micrograms/ml). Legionella spp. were also CP-99,219 susceptible, with MICs of 0.008 to 0.12 micrograms/ml. CP-99,219 demonstrated activity greater than that of ciprofloxacin, ofloxacin, or enoxacin against Pseudomonas aeruginosa (MIC90, 1 microgram/ml), Xanthomonas maltophilia (MIC90, 2 micrograms/ml), Staphylococcus haemolyticus (MIC90, 0.5 micrograms/ml), Enterococcus faecalis (MIC90, 1 microgram/ml), and pneumococci (MIC90, 0.12 micrograms/ml). Numerous ciprofloxacin-resistant isolates were susceptible to CP-99,219, a new compound showing potential value for further in vivo trials. |
8043035 | Chemiluminescence associated with doxorubicin-induced lipid peroxidation in rat heart mitochondria. | Chemiluminescence was observed in a rat heart mitochondrial suspension containing NADH, FeC1(3) and doxorubicin (Adriamycin) (DXR). There was good correlation between the total intensity of chemiluminescence and the total amount of thiobarbituric acid reactive substances (TBARS) produced during DXR redox cycling. Thus, the chemiluminescence was shown to be associated with lipid peroxidation. The chemiluminescence was quenched by superoxide dismutase (SOD), suggesting that superoxide anion radicals contributed to its production. Upon addition of 1,4-diazabicyclo[2,2,2]-octane (DABCO), a singlet oxygen emission enhancer, to the mitochondrial suspension emitting the chemiluminescence, the chemiluminescence intensity increased transiently, indicating the involvement of singlet oxygen. Furthermore, spectral analysis of the chemiluminescence showed it to be due to singlet oxygen and excited carbonyls. |
8043034 | Monoclonal antibodies reactive with a monoamine transporter preparation purified from bovine adrenal chromaffin granule membranes. | There have been no reports of monoclonal antibodies reactive with vesicular monoamine transporters from any source. Western blotting and ELISA data obtained using polyclonal serum from a mouse immunized with a highly purified bovine chromaffin granule monoamine transporter preparation yielded data consistent with the presence of antibodies to the transporter. Hybridomas produced by polyethylene glycol fusion of spleen cells from the mouse with X63/Ag8.653 myeloma cells were screened in an ELISA using partially purified transporter as coating agent. Of the 1142 wells containing colonies, 14 were positive in the initial screen. Hybridomas from wells testing positive were transferred to 24-well plates, grown up, and rescreened. Those still testing positive were subcloned, and the resulting positive wells containing single colonies were grown up and stocked. Of the 6 positive clones that tolerated freeze/thaw (0.5% of the wells tested), 1 was IgG1 kappa, 2 were IgG2a kappa, and 3 were IgG2b kappa isotypes. Ascites fluid was generated in pristane-primed BALB/c mice using hybridomas that had been cloned 2-3 times, and antibodies purified on immobilized Protein A. Immunoreactivity with a mixture of these antibodies, or with only one of them, coincided with dihydrotetrabenazine (TBZOH) binding activity in fractions eluted from all columns employed in the transporter purification. Antibody from at least one of the clones was capable of removing [3H]TBZOH binding activity from a partially purified preparation of transporter. Monoclonal antibodies exhibiting these properties have not been reported previously. |
8043033 | Characterization of specific binding sites for [3H]2-MeS-ADP on megakaryocytoblastic cell lines in culture. | Binding of [3H]2-methyl thio-adenosine 5' diphosphate ([3H]2-MeS-ADP), a stable analogue of adenosine 5' diphosphate (ADP) to DAMI and Meg-01, two megakaryocytoblastic cell lines, was time-dependent, reversible and saturable. Scatchard analysis of the saturation binding data indicated that [3H]2-MeS-ADP bound to one class of specific binding sites with high affinity (dissociation constants = 45.3 +/- 13.4 and 48.2 +/- 17.7 nM, and maximum binding capacities = 341.2 +/- 31.1 and 903 +/- 98 fmole/10(6) cells for DAMI and Meg-01, respectively) (N = 3). Unlabelled 2-MeS-ADP competitively and selectively inhibited the specific binding of [3H]2-MeS-ADP on DAMI and Meg-01 with inhibitory constant values of 118 +/- 11 and 38 +/- 11 nM, respectively (N = 3). ADP was 3 to 10 times less potent than 2-Mes-ADP in displacing [3H]2-MeS-ADP from its binding sites on DAMI and Meg-01, whereas other ADP analogues, such as AMP, GDP, UDP, adenosine or FSBA, did not interfere with the binding of [3H]2-MeS-ADP, suggesting that DAMI and Meg-01 contain ADP-specific receptors. |
8043032 | Strong inhibition of mammalian lipoxygenases by the antiinflammatory seleno-organic compound ebselen in the absence of glutathione. | Both human recombinant 5-lipoxygenase (EC 1.13.11.34) and 15-lipoxygenase (EC 1.13.11.33, mammalian enzyme) purified from rabbit reticulocytes were inhibited in the absence of glutathione (GSH) by submicromolar concentrations of the seleno-organic compound ebselen. These concentrations were comparable to those of the enzymes. Soybean lipoxygenase-1 (EC 1.13.11.33, plant enzyme) was not inhibited, whereas prostaglandin endoperoxide synthase-1 (EC 1.14.99.1) was inhibited only at much higher concentrations of ebselen (IC50 = 37.7 +/- 4.3 microM). The action of ebselen on reticulocyte 15-lipoxygenase (IC50 = 0.17 +/- 0.01 microM) was studied in detail. Inhibition occurred instantaneously and appeared to be reversible and was largely abolished by a 20-fold molar excess of GSH over ebselen. In the presence of 1 mM GSH 50% inhibition was observed only at ebselen concentrations as high as 234 +/- 27 microM. 13S-hydroperoxy-9Z, 11E-octadecadienoic acid, the lipoxygenase product formed from linoleic acid, augmented the inhibitory effect at low concentrations and caused a partial reversal at high concentrations. A variety of derivatives or structural analogues of ebselen were also tested and proved to be either inactive or weaker inhibitors of 15-lipoxygenase. We have concluded that the potent inhibition of 15-lipoxygenase by ebselen is due neither to GSH peroxidase-like activity nor to lowering of the hydroperoxide tone. The pharmacological implications of these unique characteristics of the action of ebselen on lipoxygenases are then discussed. |
8043031 | Expression of xenobiotic-metabolizing cytochrome P450 forms in human adult and fetal liver. | Expression of human cytochrome P450 (CYP) genes in human adult and fetal liver were studied using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. In adult liver mRNA of CYPs 1A1, 1A2, 2A6/2A7, 2B6/2B7, 2C8-19, 2D6, 2E1, 3A3/3A4 and 3A7 were detected while CYPs 2F1 and 4B1 were absent. In fetal liver mRNA of CYPs 2C8, 2D6, 3A3/3A4 and 3A7 were found but all other forms studied were undetectable. The results provide a comprehensive qualitative picture of the expression of CYP genes in families CYP1 through CYP4 in human adult and fetal liver. |
8043030 | Effects of a 2,3-oxidosqualene-lanosterol cyclase inhibitor 2,3:22,23-dioxidosqualene and 24,25-epoxycholesterol on the regulation of cholesterol biosynthesis in human hepatoma cell line HepG2. | N-[(1,5,9)-trimethyldecyl]-4 alpha,10-dimethyl-8-aza-trans-decal-3 beta-ol (8-azadecalin 1), a high-energy intermediate analogue for the 2,3-oxidosqualene-lanosterol cyclase, was found to be a powerful (IC50 approximately 0.1 microM) inhibitor of cholesterol biosynthesis in human hepatoma HepG2 cells. In analogy with other mammalian cells grown in the presence of cyclase inhibitors, the decrease in C27-sterol formation was accompanied by an accumulation of 2,3-oxidosqualene, 2,3:22, 23-dioxidosqualene, and by the formation of a compound characterized as 24,25-epoxycholesterol, a repressor of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase activity. In order to assess the cyclase as a potential pharmacological target for the design of hypocholesterolemic drugs, it is important to test whether inhibitors of this enzyme are able to act synergistically on the biosynthesis of cholesterol, i.e. by decreasing the amount of lanosterol formed and by repressing the regulatory HMG-CoA reductase via the formation of regulatory oxysterols. The accumulation of 24,25-epoxycholesterol in relationship to the decrease of C27-sterol biosynthesis and of HMG-CoA reductase activity showed only a partial correlation: e.g. at [1] = 100 x IC50 only a 50% reduction in enzyme activity could be attained. In contrast, when HepG2 cells were treated with 2,3:22,23-dioxidosqualene or 24,25-epoxycholesterol, excellent correlations were found between the inhibition of C27-sterol biosynthesis and the repression of HMG-CoA reductase activity, which was almost complete at the highest concentrations of these epoxides (10(-5) M). Altogether, our results suggest that treatment of HepG2 cells with a cyclase inhibitor such as 8-azadecalin (1) does not lead to an intracellular accumulation of repressor molecules high enough to fully trigger a regulatory pathway resulting in a complete down-regulation of HMG-CoA reductase. At intermediary concentrations of cyclase inhibitors (IC50), however, a synergistic mode of action of these inhibitors seems plausible. |
8043029 | Inhibition of purine nucleobase transport in human erythrocytes and cell lines by papaverine. Investigation of structure-activity relationship. | Papaverine was found to be an effective inhibitor of hypoxanthine transport not only in human erythrocytes, but also in the human cell lines HL60 (myeloic) and U937 (monocytic). IC50 values for inhibition of hypoxanthine influx ranged from 6 to 20 microM. In erythrocytes papaverine was found to be a non-competitive inhibitor of hypoxanthine equilibrium-exchange transport with a Ki value of approximately 13 microM, which is in close agreement with the respective IC50 values estimated for zero-trans influx of hypoxanthine. In addition papaverine also had a slight inhibitory effect on unmediated nucleobase transport, most likely due to a perturbation of the membrane lipid environment. Several papaverine analogs were tested for their inhibitory effect on nucleobase transport. Only ethaverine was as effective as papaverine. Drotaverine and berberine were moderately inhibitory while laudanosine had no inhibitory effect at all. Isoquinoline acted as a very weak inhibitor. |
8043028 | Ca2+ ionophore A23187-stimulated secretion of azurophil granules in human polymorphonuclear leukocytes is largely mediated by endogenously formed leukotriene B4. | The mode of action of the new leukotriene synthesis inhibitor BAY X1005 ((R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) and structurally-related quinoline derivatives is reflected by the binding to a high-affinity binding site presumably identical to FLAP (five lipoxygenase activating protein). In addition to FLAP, we have identified a second BAY X1005 (low-affinity) binding site localized in the granule fraction of human PMNL (polymorphonuclear leukocytes). Based on the hypothesis that the corresponding target protein might be involved in the regulation of granule release, the influence of the leukotriene synthesis inhibitors BAY X1005 and MK-886 and the direct 5-LOX (5-lipoxygenase, EC 1.13.11.34) inhibitor A-64077 on the A23187- and fMLP (N-formyl-methionyl-leucyl-phenylalanine)-stimulated release of beta-glucuronidase (as a marker for azurophil granules) and vitamin B12-binding protein (as a marker for specific granules) was investigated. In contrast to MK-886, neither BAY X1005 nor A-64077 significantly affected fMLP-stimulated granule release. This was also true for the A23187-stimulated release of specific granules; however, under the same conditions the A23187-stimulated release of azurophil granules was almost totally inhibited by all three compounds. No obvious relationship between the corresponding IC50 values and the ability of these compounds to compete for BAY X1005 binding at the low-affinity binding site existed. Instead, by extending these studies to additional inhibitors, a correlation between the IC50 values for inhibition of A23187-stimulated (i) beta-glucuronidase release and (ii) LTB4 (leukotriene B4) synthesis was found (r = 0.969, N = 7). This relationship was independent of the mode of action of the compounds, namely direct 5-LOX inhibition or indirect 5-LOX inhibition mediated via binding to FLAP. These results suggest that 5-LOX metabolites may be involved in A23187-stimulated azurophil granule release. Of the two main biologically active 5-LOX metabolites synthesized under these conditions (LTB4 and 5-hydroxyeicosatetraenoic acid), only LTB4 stimulated beta-glucuronidase release to nearly the same extent as A23187. In addition, this metabolite significantly enhanced A23187-stimulated beta-glucuronidase release, but only at A23187 concentrations (> or = 0.25 mumol/L) which by themselves were not sufficient to trigger LTB4 formation. Moreover, the inhibition of A23187-stimulated beta-glucuronidase release by BAY X1005 or A-64077 was totally reversed by the addition of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS) |
8043027 | Bradykinin-evoked release of [3H]noradrenaline from the human neuroblastoma SH-SY5Y. | Bradykinin (BK) evoked [3H]noradrenaline ([3H]NA) release from the human neuroblastoma SH-SY5Y and this was enhanced by pre-treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 8 min. This effect of BK was inhibited by 500 microM [D-Phe7]BK and 100 microM [Thi5,8,D-Phe7]BK but not by 500 microM [Des-Arg9,Leu8]BK. The BK (B1)-agonist [Des-Arg9]BK did not evoke [3H]NA release. This suggested that SH-SY5Y expressed BK (B2)-receptors coupled to the release of [3H]NA. BK acting at B2-receptors, also elevated intracellular calcium and depolarized SH-SY5Y cells. Although pre-treatment of SH-SY5Y cells with TPA enhanced BK-evoked [3H]NA release, the elevation of intracellular calcium [Ca2+]; was decreased by about 50%. BK-evoked release of [3H]NA in cells not pre-treated with phorbol ester was only 23% dependent on extracellular calcium. In comparison, following phorbol ester treatment approximately 40% of [3H]NA release was dependent on extracellular calcium. Nifedipine (5 microM), CoCl2 (1 mM) and NiCl2 (1 mM) inhibited NA release in SH-SY5Y cells pre-treated with TPA by 16.0, 47 and 44%, respectively. The results of this study showed that BK, acting at B2-receptors, activated [3H]NA release in SH-SY5Y. Part of this effect appeared to be due to activation of L-type calcium channels but the majority of BK-evoked [3H]NA release in SH-SY5Y cells appeared to depend on [Ca2+]i. |
8043026 | Inhibition of tryptophan hydroxylase by dopamine and the precursor amino acids. | Effects of dopamine and its precursor amino acids on the activity of tryptophan hydroxylase were examined. They inhibited the enzyme activity prepared from mastocytoma cells in terms of the biopterin cofactor and the substrate L-tryptophan. In relation to the biopterin, tryptophan hydroxylase was found to have two different kinetics, and dopamine inhibited the activity in a non-competitive way to both the components. Dopamine had the highest affinity to the enzyme, followed by L-DOPA and L-tyrosine, while D-tyrosine did not inhibit the activity. In terms of L-tryptophan, L-tyrosine, L-DOPA and dopamine inhibited the enzyme non-competitively and their affinity to the enzyme was in this order. These results indicate that the indoleamine metabolism may be regulated by catecholamines and their related amino acids in the brain. |
8043025 | Enhanced gastric acid secretion induced by gastrin can be suppressed by glucose injected into the portal vein in rats. | Gastric acid secretion induced by tetragastrin was examined after glucose injection into the portal vein in rats. The enhancement of acid secretion caused by gastrin was inhibited by glucose injection into the portal vein, and the acid response was dose dependent. The acid response due to portal glucose injection was not reproduced when the hepatic vagal branch was sectioned. These findings suggest that the portal glucose signal modulates gastric acid secretion controlled by gastrin. |
8043024 | Inhibitory effect of the nonpeptide angiotensin II receptor antagonist losartan and its active metabolite, E-3174, on cAMP phosphodiesterase: additional action of the antagonists. | The inhibitory effects of the nonpeptide angiotensin II (AII) receptor antagonist losartan and its active metabolite, E-3174, on bovine brain calcium/calmodulin-dependent 3':5'-cyclic nucleotide phosphodiesterase (cAMP PDE) were investigated. Losartan and E-3174 inhibited cAMP PDE activity competitively with an apparent Ki of 18.7 +/- 2 microM (N = 3) and 70.4 +/- 8 microM (N = 3) with respect to cAMP, respectively. With 1.2 mM cAMP as a substrate, cAMP PDE activities were inhibited by losartan and E-3174 in a concentration-dependent manner. The concentrations of losartan and E-3174 required to obtain 50% inhibition of the enzyme activity (IC50) were estimated to be 38.9 +/- 7 microM (N = 3) and 139.3 +/- 39 microM (N = 3), respectively. These results show that losartan is about four times more potent than E-3174 in inhibiting the enzyme. The Hill coefficient of -1.0 +/- -0.04 (N = 3) for losartan and -1.1 +/- -0.14 (N = 3) for E-3174 was obtained, indicating that one inhibitor binding site is available on cAMP PDE. This study demonstrated that losartan and E-3174 exert additional inhibitory action on cAMP PDE besides AII receptor antagonism. |
8043023 | Comparison of levels of aldehyde oxidase with cytochrome P450 activities in human liver in vitro. | Microsomal suspensions and 9000 g supernatant (S-9) fractions were prepared from the liver tissue of six human multiorgan donors. The S-9 fractions were characterized for cytosolic aldehyde oxidase (AO) activity, using three different substrates [N1-methylnicotinamide (NMN), benzaldehyde and 6-methylpurine]. In addition, human liver NMN oxidase activity was compared with that detected in rat, dog and monkey liver S-9 fractions. As expected, the rank order of NMN oxidase activity was monkey > rat > dog, and in five out of six subjects the activity was lowest in humans (< 2.0 nmol/min/mg). The variation in AO activity among the various human livers was greater for NMN oxidase (> 40 fold) than for 6-methylpurine and benzaldehyde oxidase (< or = 3.6 fold) activity. The corresponding microsomal preparations were characterized with respect to the levels of total cytochrome P450 (CYP) and six CYP-dependent mixed-function monooxygenase (MFO) activities. The variation was greatest for dextromethorphan O-demethylase (CYP2D6) and lowest for N,N-dimethylnitrosamine N-demethylase (CYP2E1) activity (147- vs. 1.4-fold). The inter-sample variation for the total CYP, CYP3A (erythromycin N-demethylase), CYP1A2 (7-ethoxyresorufin O-deethylase), CYP2A6 (coumarin 7-hydroxylase) and CYP2C9/10 (tolbutamide 4-methyl hydroxylase) was 2.2- to 5.3-fold. Furthermore, the levels of AO activity did not correlate with total (spectrally detectable) CYP or any of the CYP form-selective MFO activities. |
8043022 | Methoctramine binding sites sensitive to alkylation on muscarinic receptors from tracheal smooth muscle. | The binding of L-[benzilic-4,4'-3H]quinuclidinyl benzilate was studied in the plasma membrane fraction of bovine tracheal smooth muscle treated with the alkylating agent N-ethylmaleimide (NEM). It was found that NEM (2.5 mM) reduced significantly the Bmax from 1116 to 853 fmol/mg protein and increased the KD values of the muscarinic acetylcholine receptor (mAchR) activity from 36 to 61 pM. The mAchR subtypes in these plasma membranes were studied using competition experiments with selective antagonists. Pirenzepine displayed low competitive activity, having a pKi of 6.91 +/- 0.03, which was similar to that of AF-DX 116 (11[[2-[(diethylamino)methyl]- 1-piperidinyl]-acetyl]-5,11-dihydro-6H-pyrido[2,3- b][1,4]benzodiazepine-6-one); (pKi = 6.90 +/- 0.04), whereas hexahydrodifenidol (HDD) and its p-fluoro-derivative (p-FHHSiD) showed higher affinities than pirenzepine, having pKi values of 7.45 +/- 0.05 and 7.17 +/- 0.06, respectively. The antagonist 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) showed a pKi of 8.25 +/- 0.03, which did not differ significantly from the affinity shown by methoctramine (pKi = 8.00 +/- 0.04). These data indicate that the mAchR associated with the plasma membrane fraction isolated from bovine airway smooth muscle can be classified as an M2 subtype muscarinic receptor. NEM treatment altered the affinities of the mAchR towards specific antagonists, such as methoctramine (Ki increased 3 times), and the results indicated that the alkylated mAchR behaves as a chemically modified M2 subtype. This suggests the presence of thiol groups controlling the antagonist binding activity of this muscarinic receptor subtype. |
8043021 | Specificity of N-ethyl lysine of a monoclonal antibody to acetaldehyde-modified proteins prepared under reducing conditions. | A monoclonal antibody has been developed that recognizes only protein-acetaldehyde (AA) adducts prepared under reducing conditions: 5 mM AA with 30 mM sodium cyanoborohydride overnight at 37 degrees. This monoclonal antibody is a mouse IgG2b that has been designated RT1.1. The primary adduct formed when proteins are exposed to acetaldehyde under reducing conditions is N-ethyl lysine (NEL). To examine the epitope specificity of RT1.1, inhibition ELISAs were developed using NEL and other possible inhibitors, such as arginine, ethylamine, lysine and proteins modified with AA under non-reducing conditions. RT1.1 (at half-maximum optical density, 50 ng/mL) was inhibited only by NEL and was independent of the carrier or the pH of the buffer used in the ELISA. Further evidence indicating that NEL is the epitope recognized by RT1.1 was obtained using mouse and human epidermal growth factor (EGF). Both proteins contain one alpha amino group but only the human-EGF contains lysine residues with epsilon amino groups. In experiments where these two proteins were modified with AA under reducing conditions, RT1.1 reacted only with human-EGF. These studies demonstrate that RT1.1 is specific for NEL that is formed by the ethylation of proteins with acetaldehyde under reducing conditions. Additionally, these studies demonstrate that the procedures and methods used herein may be useful for characterizing other antibodies prepared to AA-modified proteins under a variety of defined in vitro chemical conditions. |
8043020 | Characterization of dextromethorphan N-demethylation by human liver microsomes. Contribution of the cytochrome P450 3A (CYP3A) subfamily. | In an effort to identify the human cytochromes P450 involved in the N-demethylation of dextromethorphan, the kinetics of 3-methoxymorphinan formation were studied in microsomal enzyme systems. Under initial rate conditions, 3-methoxymorphinan formation demonstrated single enzyme Michaelis-Menten kinetics using microsomes obtained from three human livers (Km: 0.52-0.71 mM; Vmax: 375-812 pmol/mg protein/min). B-lymphoblastoid cells expressing CYP3A4 incubated with 0.4 mM dextromethorphan catalyzed the formation of 3-methoxymorphinan at a rate of 22 pmol product/mg protein/min. Midazolam, a prototypic substrate for CYP3A4 and CYP3A5, competitively inhibited dextromethorphan N-demethylation by two human liver microsomal samples with Ki values of 46 +/- 10 and 63 +/- 8 microM. At a dextromethorphan concentration of 0.4 mM, gestodene (100 microM) inhibited 3-methoxymorphinan formation by approximately 50%. Immunoinhibition of dextromethorphan N-demethylation using rabbit anti-CYP3A4 antibodies resulted in a 60% decrease in 3-methoxymorphinan formation at a dextromethorphan concentration of 0.4 mM. Additional inhibition studies using furafylline, coumarin, sulfaphenazole, mephenytoin, quinidine, and diethyldithiocarbamic acid, which are selective inhibitors of CYP1A2, CYP2A6, CYP2C8/9, CYP2Cmp, CYP2D6, and CYP2E1, respectively, demonstrated no substantial inhibition of dextromethorphan N-demethylation. Correlation analysis was performed using the rate of 3-methoxymorphinan formation at a concentration of 1 mM dextromethorphan and immunoquantified levels of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 and their associated characteristic catalytic activities. A significant correlation was observed between dextromethorphan N-demethylase activity and midazolam 1'- and 4-hydroxylase activity (r2 = 0.77 and 0.69 respectively, N = 19, P < 0.01); the exclusion of those samples containing both CYP3A4 and CYP3A5 increased the correlation significantly (r2 = 0.87 and 0.91 respectively, N = 12, P < 0.01). In the absence of CYP3A5, a significant correlation was observed between 3-methoxymorphinan formation and the sample's erythromycin N-demethylase activity (r2 = 0.94, N = 12, P < 0.01), testosterone 6 beta-hydroxylase activity (r2 = 0.96, N = 7, P < 0.01) and relative immunoquantified levels of CYP3A4 (r2 = 0.96, N = 12, P < 0.01). Inclusion of those samples expressing CYP3A5 in addition to CYP3A4 reduced the magnitude of the observed correlation. No significant correlation between 3-methoxymorphinan formation and the sample's relative immunoquantified levels of or form-selective activity associated with CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19 (or CYP2Cmp), CYP2D6, and CYP2E1 was observed. In conclusion, dextromethorphan N-demethylation appears to be catalyzed primarily by CYP3A4 and to a lesser extent by CYP3A5 in vitro in humans.(ABSTRACT TRUNCATED AT 400 WORDS) |
8043019 | Induction and genetic regulation of mouse hepatic cytochrome P450 by cannabidiol. | Cannabidiol (CBD) has been shown to be a selective inactivator of cytochromes P450 (P450s) 2C and 3A in the mouse and, like many P450 inactivators, it can also induce P450s after repeated administration. The inductive effects of CBD on mouse hepatic P450s 2B, 3A, and 2C were determined using cDNA probes, polyclonal antibodies, and specific functional markers. P450 2B10 mRNA was increased markedly after repeated CBD administration and correlated well with increased P450 2B immunoquantified content and functional activity. On the other hand, although the 2-fold increase in P450 3A mRNA detected after repeated CBD administration was consistent with the increased immunoquantified P450 3A protein content, the lack of an observable increase in P450 3A-specific functional activity suggested subsequent inactivation of the induced P450 3A. Repeated CBD treatment increased P450 2C mRNA content 2-fold, but did not increase either the P450 2C immunoquantified content or its functional activity. The effect of CBD treatment on the ability of tetrahydrocannabinol (THC) to induce P450 2B was also determined. A THC dose that did not induce P450 2B significantly was administered alone or in combination with a CBD dose that markedly inactivated P450s 2C- and 3A but submaximally increased P450 2B functional activity. The combination of THC and CBD did not increase P450 2B-catalyzed activity significantly over that observed after CBD treatment alone. Thus, prior CBD-mediated P450 inactivation does not appear to increase the ability of THC to induce P450 2B. To further characterize the relationship between P450 inactivation and induction, several structurally diverse CBD analogs with varying P450 inactivating potentials were tested for their ability to induce P450 2B. At least one CBD analog that is an effective P450 inactivator failed to induce P450 2B, while at least one CBD analog that is incapable of inactivating P450 was found to be a very good P450 2B inducer. It therefore appears that inherent structural features of the CBD molecule rather than its ability to inactivate P450 determine P450 2B inducibility. The complex effects of CBD treatment on P450 inactivation and induction have the potential to influence the pharmacological action of many clinically important drugs known to be metabolized by these various P450s. The mechanism of CBD-mediated P450 induction remains to be elucidated but does not appear to be related to CBD-mediated P450 inactivation. |
8043018 | Contribution of hepatic cytochrome P450 systems to the generation of reactive oxygen species. | The rate of generation of reactive oxygen species (ROS) in hepatic microsomes was assayed using a fluorescent probe. This rate was stimulated in a manner proportional to the concentration of NADPH present. NADH could not be substituted for NADPH, and an inhibitor of mixed-function oxidases (SKF 525A) blocked stimulation by NADPH. This suggested the involvement of cytochrome P450 oxidase systems in ROS formation. Low molecular weight iron salts may not have been involved in the stimulated ROS formation since deferoxamine failed to eliminate the oxidative response to NADPH. Catalase only partially inhibited, and glutathione peroxidase did not significantly inhibit this response, implying that hydrogen peroxide does not play a key role. However, since NADPH-enhanced generation of reactive oxygen species was totally prevented by superoxide dismutase, superoxide was an obligatory intermediate. The presence of toluene, ethanol or phenobarbital did not enhance the production of NADPH-effected reactive oxygen species; free radical production was maximal in the absence of substrates subject to oxidation by cytochrome P450 enzymes. Hepatic cytochrome P450 oxidases are likely to contribute significantly to overall ROS formation, even under basal conditions where mixed-function oxidases are not induced. |
8043017 | Neutrophil "priming" induced by orthovanadate: evidence of a role for tyrosine phosphorylation. | The mechanism of neutrophil "priming" is unknown. In this study the level of tyrosine phosphorylation within intact neutrophils, using orthovanadate, have been manipulated. It has been demonstrated that this procedure both increased tyrosine phosphorylation of a number of protein substrates, including a prominent band at 74 kDa, and also primed the neutrophil oxidase response with a time and orthovanadate concentration-dependency, which were consistent with a role for tyrosine phosphorylation. No effect of orthovanadate on cytosolic-free Ca2+ concentration or actin polymerization was detected. Inhibition of tyrosine phosphorylation by genistein prevented "priming" by orthovanadate. This data thus provided evidence of a role for tyrosine phosphorylation in neutrophil "priming". |
8043016 | Differences in in vivo and in vitro sequence-specific sites of cisplatin-DNA adduct formation and detection of a dose-response relationship. | The cytotoxic and mutagenic properties of the anticancer drug cis-diammine-dichloroplatinum(II) (cisplatin) are mediated by bifunctional adducts between purines. Experiments performed in this study employed a new repetitive thermal-cycling technique to detect cisplatin adduct formation following exposure of cells in culture (in vivo) or following treatment of purified DNA (in vitro exposure). The initial goal of this study was to determine if cisplatin-DNA adduct formation could be measured accurately using phosphor-imaging over a broad concentration range. If this proved possible, it would then be feasible to determine if adduct formation differed within chromatin compared with purified DNA. There were no significant differences in the cisplatin-DNA adduct pattern induced in closed circular or linear double-stranded plasmids in vitro, suggesting that this type of tertiary structural change does not affect the formation of adduct sites. Sequence-specific DNA adduct formation within a human repetitive DNA target sequence, alphoid DNA, following cisplatin treatment of prostate cancer cells in culture (in vivo) and treatment of purified DNA in vitro revealed consistent increases in adduct formation over a broad concentration range, validating the experimental technique. Comparing preferences for cisplatin adduct site formation under these different conditions of exposure demonstrated statistically significant differences. Similar differences were detected for cisplatin repair-deficient Xeroderma pigmentosum cells treated in cell culture, indicating that in vivo/in vitro preferences for adduct site formation are not the result of DNA repair in vivo. |
8043015 | The distribution of non-specific carboxylesterases and glutathione S-transferases in different rat liver cells. Effects of vitamin A deficiency. | Non-specific carboxylesterases (carboxylesterases) and glutathione S-transferases (GSTs) are two groups of drug metabolizing enzymes responsible for hydrolysis and glutathione conjugation of xenobiotics. This study was conducted to determine the following: (1) the distribution of carboxylesterase and GST activities in different rat liver cells, (2) the effects of vitamin A deficiency (A-) on the absolute activities and on the distribution of carboxylesterases and GSTs in rat liver. Rat livers were fractionated into parenchymal and non-parenchymal cells by means of collagenase perfusion and differential centrifugation. Non-parenchymal cells were further fractionated by means of Percoll density gradient centrifugation into a layer of Kupffer cells and another layer containing stellate and endothelial cells. Carboxylesterase and GST activities were determined in these fractions. show that: (1) both carboxylesterases and GSTs were mainly localized in the parenchymal fraction, (2) there was no significant difference between male and female rats with regard total activity or distribution of carboxylesterases and GSTs in rat liver cells, (3) A- caused a highly significant reduction in carboxylesterase and GST activities in total liver homogenates and parenchymal cells. This reduction was not ameliorated by administration of retinoic acid 18 hr before sacrifice of animals. These results open up a new era of investigations about the potential role of vitamin A in the regulation of detoxification enzymes. |
8043014 | Modulation of pulmonary leukotriene formation and perfusion pressure by bestatin, an inhibitor of leukotriene A4 hydrolase. | We investigated the effects of bestatin, a prototype leukotriene A4 (LTA4) hydrolase inhibitor, on leukotriene (LT) formation and pulmonary artery perfusion pressure (Ppa) in isolated, perfused rat lungs. In lung parenchymal strips stimulated with a 10 microM concentration of the Ca2+ ionophore A23187, bestatin inhibited LTB4 formation with an IC50 = 10.4 +/- 30 microM (mean +/- SD, N = 4). It did not alter cysteinyl LT formation, confirming that it inhibited LTA4 hydrolase selectively, without inhibiting phospholipase, 5-lipoxygenase, or LTC4 synthase. In isolated, perfused lungs stimulated with 10 microM A23187, 300 microM bestatin inhibited LTB4 release by 72.2 +/- 10.6% (mean +/- SEM, N = 6, P < 0.01) but had no significant effect on LTE4 formation (P > 0.5). In these perfused lungs, bestatin did not alter the change in Ppa following stimulation with A23187. This effect is consistent with the insubstantial re-direction of LTA4 toward formation of vasospastic cysteinyl LTs. Separate experiments used lungs from rats treated with lipopolysaccharide endotoxin in vivo, prior to isolation, perfusion, and stimulation with 5 microM formyl-methionyl-leucyl-phenylalanine, in vitro. In these inflamed lungs, 750 microM bestatin inhibited LTB4 formation (P < 0.05) and increased LTE4 formation (P < 0.05), compatible with selective inhibited LTB4 hydrolase. The re-direction of LTA4 metabolism toward formation of cysteinyl LTs by inflamed, perfused lungs did not cause an increase in P(pa). |
8043013 | Reduction of DNA synthesis, pigment synthesis, pigmentation gene mRNA and resistance to UVB in human melanoma cells treated with analogues of a histamine (H2) agonist. | Two groups of S-[2-(N,N-dialkylamino)ethyl]isothiourea derivates which depigmented melanoma cells either with inhibition of tyrosinase (group 1, R = methyl, isopropyl) or without inhibition of tyrosinase (group 2, R = benzyl, phenyl) were studied. Treatment of human melanoma cells with non-lethal doses of group 1 drugs led to a reduction in the levels of mRNA for the pigmentation genes tyrosinase, tyrosinase-related protein-1 and Pmel 17. The group 1 drug S-[2-N,N-diisopropylamino)ethyl[isothiourea] (DINOR) (R = isopropyl) produced only moderate inhibition of DNA, RNA and protein synthesis in three cell lines during the first 24 hr of treatment, and there was no correlation between the extent of inhibition and long-term toxicity. A group 2 drug (R = benzyl) rapidly inhibited DNA synthesis in an amelanotic melanoma cell line (MM96E) sensitive to killing by the drug; association of the latter with inhibition of RNA or protein synthesis was less clear. MM96E cells were also sensitive to killing by reactive oxygen species. In pigmented melanoma cells (MM418), incorporation of [125I]thiouracil, a false precursor of melanin, increased during the first 24 hr of treatment with DINOR whereas a group 2 drug (R = phenyl) inhibited incorporation of [125I]thiouracil. Cells depigmented by treatment with drugs from either group suffered the same amount of DNA damage as pigmented cells after UVB irradiation, as judged by inhibition of DNA synthesis, but did not recover as well as pigmented cells, whether or not drug was present during recovery. The results suggested that (1) group 1 agents down-regulated message for several pigmentation genes, possibly at the transcriptional level; (2) the toxicity of group 2 drugs was related to reactive oxygen species; and (3) melanin protected cells from UVB by enhancing cellular recovery. |
8043012 | Differential expression of microsomal epoxide hydrolase gene by azole heterocycles in rats. | The effects of heterocycles including imidazole (IM), 1,2,4-triazole (TR) and thiazole (TH) on the expression of microsomal epoxide hydrolase (mEH) gene were examined in rats (200 mg/kg body weight/day, i.p.). Hepatic microsomes prepared from rats treated with IM for 3 days failed to exhibit an increase in mEH protein level whereas TR treatment resulted in an approximately 2- to 3-fold elevation in hepatic mEH levels relative to control, as assessed by both SDS-PAGE and immunoblot analyses. In contrast, thiazole-induced hepatic microsomes resulted in a substantial increase in mEH levels (i.e. approximately 5-fold). Slot and northern blot analyses, probed with an mEH cDNA, showed that the hepatic mEH mRNA levels in the animals treated with IM for 3 days were marginally increased by approximately 2-fold, as compared with untreated animals, whereas TR caused an approximately 8-fold increase in hepatic mEH mRNA levels after three consecutive daily treatments. TH treatment resulted in an approximately 22-fold increase in the mEH mRNA levels, demonstrating that TH is the most efficacious among these three azole heterocycles. Because TH was the most effective in increasing hepatic mEH protein and mRNA levels, the agent was chosen for further evaluation. Time course of mEH gene expression at early times after a single treatment with TH was determined and compared with that caused by pyrazine (PZ), a strong mEH inducer. Hepatic mEH mRNA levels were increased approximately 1-, 3-, 20- and 16-fold at 3, 6, 12 and 24 hr, respectively, following TH treatment, relative to control, whereas mEH mRNA levels were elevated approximately 1-, 1-, 22- and 18-fold, respectively, at the same time points after PZ treatment, as monitored by slot RNA hybridization analyses. Northern blot analyses using either total RNA or poly(A)+ RNA fractions exhibited comparable time courses in increasing mEH mRNA levels after TH or PZ treatment with maximal mRNA increases being noted at 12 hr post treatment. Although neither IM or TR failed to affect renal mEH gene expression to a notable extent, TH treatment caused 6- to 8-fold increases in kidney mEH mRNA levels, with a 2-fold increase in mEH protein detected. These results demonstrated that the azole heterocyclic compounds IM, TR and TH differentially induce mEH with TH as the most efficacious azole; and that the changes in mEH levels are primarily associated with increases in mRNA levels. |
8043011 | Equal inhibition of the replication of human immunodeficiency virus in human T-cell culture by ddA bis(SATE)phosphotriester and 3'-azido-2',3'-dideoxythymidine. | It is shown that ddA bis(SATE)phosphotriester is one of the most potent anti-HIV agents in cell culture. Compared with the parent nucleoside, ddA, an increase of 3 orders of magnitude was observed in the EC50, which makes this compound as active as AZT. This can be tentatively explained if one considers that direct ddAMP intracellular delivery shunts the well established ddA/ddI metabolism pathway. |
8043008 | Studies on the effect of an heterologous fatty acid-binding protein on acyl-CoA oxidase induction in Saccharomyces cerevisiae. | The participation of fatty acid-binding protein (FABP) in the induction of peroxisomal beta-oxidation of fatty acids was investigated in vivo in an heterologous system. Bovine heart FABP was expressed in Saccharomyces cerevisiae under the control of two different promoters: a constitutive one and an oleic acid-inducible one. Constructs were introduced into yeast cells on multicopy and integrating plasmids. The heterologous FABP was present in yeast cells in two isoforms having pI values of about 5 and was able to bind oleic acid. The heterologous FABP had no significant effect on acyl-CoA oxidase activity at various concentrations of the inducing agent. |
8043010 | Vaninolol: a new selective beta 1-adrenoceptor antagonist derived from vanillin. | The beta-adrenoceptor blocking properties of vaninolol ((+/-)4-[4'-(2-hydroxy-3-tert-butyl-aminopropoxy)-3'-methoxyphenyl]- 3-buten-2-one), derived from vanillin, were first investigated under in vivo and in vitro conditions. Vaninolol (0.1, 0.5, 1.0 mg/kg, i.v.), as well as propranolol, produced a dose-dependent bradycardia response and a sustained pressor action in urethane-anesthetized normotensive rats. Vaninolol inhibited the tachycardia effects induced by (-)isoproterenol, but had no blocking effect on the arterial pressor responses induced by phenylephrine. These findings suggested that vaninolol possessed beta-adrenergic blocking activity, but was without alpha-adrenergic blocking activity. In isolated guinea-pig tissues, vaninolol antagonized (-)isoproterenol-induced positive inotropic and chronotropic effects of the atria and tracheal relaxation responses in a concentration-dependent manner. The parallel shift to the right of the concentration-response curve of (-)isoproterenol suggested that vaninolol was a beta-adrenoceptor competitive antagonist. The effect of vaninolol was more potent on the atria than on tracheal tissues, indicating it had some beta 1-adrenoceptor selectivity. On the other hand, the order of the hydrophilicity was atenolol >> vaninolol > propranolol. In addition, vaninolol had a mild direct cardiac depression at high concentrations and was without intrinsic sympathomimetic activity (ISA). Furthermore, binding characteristics of vaninolol and other beta-adrenoceptor antagonists were evaluated in [3H]dihydroalprenolol binding to guinea-pig ventricular membranes. The order of potency of beta-adrenoceptor antagonists in competing for the binding sites was (-)propranolol >> vaninolol > or = atenolol. In conclusion, vaninolol was found to be a selective beta 1-adrenoceptor antagonist with relatively low lipophilicity in comparison with propranolol, devoid of ISA, and had a mild myocardial depressant effect. |
8043007 | Characterization of a metalloprotease from ovine chromaffin granules which cleaves a proenkephalin fragment (BAM12P) at a single arginine residue. | A metalloprotease has been identified in ovine chromaffin granules which cleaves the proenkephalin fragment BAM12P to produce adrenorphin-Gly. This cleavage occurs at a single arginine residue and is an intermediate step in the production of the opiate adrenorphin in vivo. The identity of the product was confirmed by reverse-phase and ion-exchange chromatography. The adrenorphin-Gly-generating enzyme (AGE) was determined by chromatofocusing to have a pI value of 5.2 and bound strongly to a metal-chelate affinity column. After purification by gel-filtration and ion-exchange chromatography AGE was free of contaminating activities, as cleavage of radiolabelled BAM12P generated a single product as judged by reverse-phase and ion-exchange chromatography. The enzyme has a molecular mass of approx. 45 kDa and a pH optimum of 8.6 in Mops, Taps and Hepes buffers, but was inhibited by phosphate buffers. It was inhibited by micromolar concentrations of copper and zinc ions, but not by millimolar concentrations of calcium or manganese ions. The addition of BAM22P, dynorphin 1-13 or dynorphin 1-8 to the incubation mixture inhibited the cleavage of radiolabelled BAM12P. The cleavage was also inhibited by the presence of catecholamines at concentrations similar to those found within the chromaffin granule. This may explain the known effect of reserpine on chromaffin cells of reducing catecholamine levels and simultaneously increasing adrenorphin levels. It may also indicate a function for AGE and adrenorphin as reporters of intragranular conditions. |
8043006 | Two calcium-binding sites mediate the interconversion of liver inositol 1,4,5-trisphosphate receptors between three conformational states. | Cytosolic Ca2+ biphasically regulates Ins(1,4,5)P3-stimulated Ca2+ mobilization in liver [Marshall and Taylor (1993) J. Biol. Chem. 268, 13214-13220]. We have investigated the mechanisms underlying this biphasic control of Ca2+ mobilization in permeabilized hepatocytes by comparing the effects of Sr2+, Ba2+ and Ca2+ on the liver Ins(1,4,5)P3 receptor. Both Ca2+ and Sr2+ increased the binding of [3H]Ins(1,4,5)P3 to liver membranes by converting receptors from a low-affinity (KD approximately 35 nM) to a high-affinity (KD approximately 5 nM) state. Ba2+ (< or = 20 microM) did not affect [3H]Ins(1,4,5)P3 binding. At concentrations similar to those that caused an enhancement of [3H]Ins(1,4,5)P3 binding, Sr2+ (EC50 = 570 nM) and Ca2+ (EC50 = 200 nM) increased the sensitivity of the intracellular Ca2+ stores to Ins(1,4,5)P3. Further modest elevations in [Ca2+] (EC50 = 1.5 microM) inhibited Ins(1,4,5)P3-stimulated Ca2+ mobilization, whereas Sr2+ caused inhibition only when its concentration was very substantially increased (EC50 approximately 900 microM). Sr2+ is therefore only 3-fold less potent than Ca2+ in causing sensitization of Ins(1,4,5)P3-stimulated Ca2+ release, but 600-fold less potent in causing inhibition. Ba2+ neither sensitized ([Ba2+] < or = 20 microM) nor inhibited ([Ba2+] < or = 1 mM) Ins(1,4,5)P3-stimulated Ca2+ release, and did not inhibit either the sensitization of Ca2+ release evoked by Sr2+ or the inhibition of Ca2+ release evoked by Ca2+. Our results suggest that two distinct Ca(2+)-binding sites, which differ in their selectivities for bivalent cations, mediate the interconversion of Ins(1,4,5)P3 receptors between at least three different conformational states. These two Ca(2+)-binding sites, which may reside either on the Ins(1,4,5)P3 receptor itself or on distinct regulatory proteins, can be distinguished by their different selectivities for bivalent cations. |
8043005 | Alternate-strand DNA triple-helix formation using short acridine-linked oligonucleotides. | We have used DNAse I footprinting to examine the formation of intermolecular DNA triple helices at sequences containing adjacent blocks of purines and pyrimidines. The target sites G6T6.A6C6 and T6G6.C6A6 were cloned into longer DNA fragments and used as substrates for DNAse I footprinting, which examined the binding of the acridine (Acr)-linked oligonucleotides Acr-T5G5 and Acr-G5T5 respectively. These third strands were designed to incorporate both G.GC triplets, with antiparallel Gn strands held together by reverse Hoogsteen base pairs, and T.AT triplets, with the two T-containing strands arranged antiparallel to each other. We find that Acr-T5G5 binds to the target sequence G6T6.-A6C6, in the presence of magnesium at pH 7.0, generating clear DNAse I footprints. In this structure the central guanine is not recognized by the third strand and is accessible to modification by dimethyl sulphate. Under these conditions no footprint was observed with Acr-G5T5 and T6G6.C6A6, though this triplex was evident in the presence of manganese chloride. Manganese also facilitated the binding of Acr-T5G5 to a second site in the fragment containing the sequence T6G6.C6A6. This represents interaction with the sequence G4ATCT6, located at the boundary between the synthetic insert and the remainder of the fragment, and suggests that this bivalent metal ion may stabilize triplexes that contain one or two mismatches. Manganese did not affect the interaction of either oligonucleotide with G6T6.A6C6. |
8043004 | The intracellular Ca(2+)-pump inhibitors thapsigargin and cyclopiazonic acid induce stress proteins in mammalian chondrocytes. | Primary cultures of mammalian articular chondrocytes respond to treatment with the intracellular Ca(2+)-pump inhibitors thapsigargin (TG) and cyclopiazonic acid by specific changes in protein synthesis consistent with a stress response. Two-dimensional gel electrophoresis of newly synthesized proteins confirmed that the response was consistent with the induction of glucose-regulated proteins. The effects of low-dose TG (10 nM), measured by changes in [35S]methionine labelling of newly synthesized proteins, can first be observed by 10 h and are maximal by 24 h. The pattern of changes induced by TG is shared with cyclopiazonic acid, but effects of both perturbants differ significantly from changes induced by heat shock. Upon removal of TG, normal protein synthesis is restored by 48 h. Immunoblots showed increased concentrations of the stress proteins HSP90, HSP72/73 and HSP60 in chondrocytes treated with TG, but induction of newly synthesized heat-shock proteins by TG was not apparent on [35S]methionine-labelled gels. The alterations in protein synthesis induced by Ca(2+)-pump inhibitors were unaffected by BAPTA-AM loading, which clamped cytosolic Ca2+ at resting levels. We conclude that inhibition of intracellular Ca(2+)-pump activity can elicit a stress response, which has important implications for the interpretation of chronic use of Ca(2+)-pump inhibitors. In particular, the activation of the cellular shock response should be considered in interpreting the regulation of protein synthesis and cell survival by Ca(2+)-pump inhibitors such as TG. |
8043003 | Expression of rat liver S-adenosylmethionine synthetase in Escherichia coli results in two active oligomeric forms. | A cDNA containing the complete coding sequence for rat liver S-adenosylmethionine synthetase was cloned into the prokaryotic expression vector pT7-7 and expressed in Escherichia coli BL21(DE3). A major additional band corresponding to a protein of 48 kDa was detected on SDS/PAGE after induction with isopropyl beta-D-thiogalactopyranoside. This protein was distributed in both the soluble and insoluble fractions and accounted for approx. 30% of the total bacterial protein. The soluble enzyme was fully active, as revealed by assays in vitro of S-adenosylmethionine synthetase activity. In addition, transformed bacteria exhibited highly increased levels of intracellular S-adenosylmethionine. Two active forms of the recombinant enzyme, with apparent molecular masses of 210 kDa and 110 kDa, were detected when cytosolic extracts of the transformed cells were fractionated by gel-filtration chromatography. It is concluded that the expressed S-adenosylmethionine synthetase polypeptide assemble as tetramers and dimers. |
8043001 | Identification of cytokine-induced neutrophil chemoattractants (CINC), rat GRO/CINC-2 alpha and CINC-2 beta, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization. | Four basic neutrophil chemotactic factors (chemokines) have been purified from conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in the rat. On the basis of their N-terminal amino acid sequences, one of the chemokines was identical with rat GRO/cytokine-induced neutrophil chemoattractant (CINC) which we reported previously, and another was identical with rat macrophage inflammatory protein-2 (MIP-2). Two other chemokines were novel chemoattractants related to MIP-2. The novel chemokines are referred to as rat GRO/CINC-2 alpha and CINC-2 beta, and consequently CINC and rat MIP-2 are renamed rat GRO/CINC-1 and CINC-3 respectively. The complete amino acid sequences of purified CINC-2 alpha and CINC-3 were determined by analysis of the fragments isolated from proteinase V8-treated CINCs. The cDNA for CINC-2 beta was cloned by reverse transcription/PCR amplification using specific primers starting with total RNA extracted from lipopolysaccharide-stimulated rat macrophages. A comparison of the amino acid sequence encoded by the cDNA with the N-terminal amino acid sequence of purified CINC-2 beta revealed that mature CINC-2 beta is a 68-residue chemoattractant produced by cleavage of a 32-residue signal peptide. The difference in amino acid sequences between CINC-2 alpha and CINC-2 beta consisted of only three C-terminal residues. Rat GRO/CINC-2 alpha is a major chemokine, and the four purified chemokines have similar chemotactic activity, suggesting that they contribute to neutrophil infiltration into inflammatory sites in rats. |
8043002 | Synthesis and characterization of a biotinylated organophosphorus ester for detection and affinity purification of a brain serine esterase: neuropathy target esterase. | We have synthesized a novel stable precursor, saligenin phosphorotrichloridate, which, on reaction with N-monobiotinyldiamines, generates a series of biotinylated covalent inhibitors of serine esterases. A homologue designated S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane] was selected to allow detection and rapid isolation of neuropathy target esterase (NTE). This enzyme is the primary target site for those organophosphorus esters (OPs) which cause delayed neuropathy. NTE comprises about 0.03% of the total protein in brain microsomal fractions and has resisted purification attempts over many years. S9B is a potent progressive inhibitor of NTE esteratic activity (second-order rate constant 1.4 x 10(7) M-1.min-1). Incubation of S9B with brain microsomes led to specific covalent labelling of NTE as determined by detection of a biotinylated 155 kDa polypeptide on Western blots. Specificity of S9B labelling was further demonstrated by inhibition with the neuropathic OP mipafox. Biotinyl-NTE in SDS-solubilized S9B-labelled microsomes was adsorbed on to avidin-Sepharose and subsequently eluted, yielding a fraction enriched approx. 1000-fold in NTE by a single step with recoveries of 30%. Essentially pure NTE was obtained after separation from two endogenous biotinylated polypeptides (120 and 70 kDa) in avidin-Sepharose eluates by preparative SDS/PAGE. Other biotinylated saligenin phosphoramidates derived from the same precursor may be useful for detection and isolation of other serine esterases and proteinases. |
8043000 | Activation of multiple pH-regulatory pathways in granulocytes by a phosphotyrosine phosphatase antagonist. | Activated phagocytes undergo a massive burst of metabolic acid generation, yet must be able to maintain their cytosolic pH (pHi) within physiological limits. Peroxides of vanadate (V(4+)-OOH), potent inhibitors of phosphotyrosine phosphatases, have recently been shown to produce activation of the respiratory burst in HL60 granulocytes. We therefore investigated the effects of V(4+)-OOH on pHi homoeostasis in HL60 granulocytes, using a pH-sensitive fluorescent dye. V(4+)-OOH stimulation induced a biphasic pH change: a transient cytosolic acidification followed by a significant alkalinization. The initial acidification was prevented by inhibition of the NADPH oxidase and was absent in undifferentiated cells lacking oxidase activity. Analysis of the alkalinization phase demonstrated the involvement of the Na+/H+ antiporter, and also provided evidence for activation of two alternative H(+)-extrusion pathways: a bafilomycin-sensitive component, likely reflecting vacuolar-type H(+)-ATPase activity, and a Zn(2+)-sensitive H(+)-conductive pathway. Our results indicate that V(4+)-OOH stimulation not only activated the NADPH oxidase but concomitantly stimulated H(+)-extrusion pathways, enabling the cells to compensate for the massive production of intracellular H+ associated with the respiratory burst. |
8042999 | Role of type 1 and type 2A phosphatases in signal transduction of platelet-activating-factor-stimulated rabbit platelets. | Calyculin A, the potent inhibitor of type 1 (PP1) and type 2A (PP2A) phosphatases, has been employed in order to investigate the role of endogenously activated PP1/PP2A in the signal-transduction pathway of platelet-activating-factor (PAF)-stimulated platelets. Calyculin A alone caused an increase in protein phosphorylation in unstimulated platelets, with the detection of a number of newly phosphorylated proteins, whereas in PAF-stimulated platelets phosphorylation of the major substrates of protein kinase C and myosin light-chain kinase were no longer transient, but phosphorylation was sustained. PP1/PP2A appear to play a role in Ca2+ homoeostasis, as inhibition of PP1/PP2A caused an inhibition of Ca2+ mobilization and Ca2+ influx through the plasma membrane in PAF-stimulated platelets. The effect of calyculin A on Ca2+ mobilization correlated with the observed inhibition of the production of the signal molecule Ins(1,4,5)P3. The release reaction (which is a Ca(2+)-dependent event) was also inhibited by calyculin A. The results are discussed in relation to the possible role of protein kinase C in mediating the events leading to the effects observed with calyculin A. |
8042997 | Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate. | Phosphodiesterase activity is a novel property of the still-enigmatic alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation constant (K0.5) of 1.9 mM and 3.9 mM respectively. The hydrolysis of p-nitrophenyl-5'-thymidine phosphate followed hyberbolic kinetics with a K0.5 of 500 microM. For p-nitrophenyl phenylphosphonate, site-site interactions [Hill coefficient (h) = 1.3] were observed in the range between 0.2 and 100 microM, and K0.5 was 32.8 mM. The hydrolysis of cyclic AMP by the enzyme followed more complex kinetics, showing site-site interactions (h = 1.7) and K0.5 = 300 microM for high-affinity sites. The low-affinity sites, representing 85% of total activity, also showed site-site interactions (h = 3.8) and a K0.5 of about 22 mM. ATP and cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phosphatase activity of the enzyme and Ki values (25 mM and 0.6 mM for cyclic AMP and ATP respectively) very close to those of the K0.5 (22 mM and 0.7 mM for cyclic AMP and ATP respectively), determined by direct assay, indicated that a single catalytic site was responsible for the hydrolysis of both substrates. Non-denaturing PAGE of detergent-solubilized enzyme showed coincident bands on the gel for phosphomonohydrolase and phosphodiesterase activities. Additional evidence for a single catalytic site was the similar pKa values (8.5 and 9.7) found for the two ionizing groups participating in the hydrolysis of bis-(p-nitrophenyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH optima, the requirement for bivalent metal ions and the inhibition by methylxanthines, amrinone and amiloride demonstrated that rat osseous-plate alkaline phosphatase was a type I phosphodiesterase. Considering that there is still confusion as to which is the physiological substrate for the enzyme, the present results describing a novel property for this enzyme could be of relevance in understanding the mineralization process. |
8042996 | Activation of human complement serine-proteinase C1r is down-regulated by a Ca(2+)-dependent intramolecular control that is released in the C1 complex through a signal transmitted by C1q. | The activation of human C1, a Ca(2+)-dependent complex proteinase comprising a non-enzymic protein, C1q, and two serine proteinases, C1r and C1s, is based primarily on the intrinsic property of C1r to autoactivate. The aim of the present study was to investigate the mechanisms involved in the regulation of C1r autoactivation, with particular attention to the role of Ca2+ ions. Spontaneous activation of proenzyme C1r was observed upon incubation in the presence of EDTA, whereas Ca2+ ions reduced markedly the activation process. Several lines of evidence indicated that Ca2+ inhibited the intramolecular activation reaction but had little or no effect on the intermolecular activation reaction. C1q caused partial release of this inhibitory effect of Ca2+. Complete stabilization of C1r in its proenzyme form was obtained upon incorporation within the Ca(2+)-dependent C1s-C1r-C1r-C1s tetramer, and a comparable effect was observed when C1s was replaced by its Ca(2+)-binding alpha-fragment. Both tetramers, C1s-C1r-C1r-C1s and C1s alpha-C1r-C1r-C1s alpha, readily associated with C1q to form 16.0 S and 14.7 S complexes respectively in which C1r fully recovered its activation potential. Both complexes showed indistinguishable activation kinetics, indicating that the gamma B catalytic region of C1s plays no role in the mechanism that triggers C1r activation in C1. The collagen-like fragments of C1q retained the ability to bind to C1s-C1r-C1r-C1s, but, in contrast with intact C1q, failed to induce C1r activation in the resulting complex at temperatures above 25 degrees C. On the basis of these observations it is proposed that activation of the serine-proteinase domain of C1r is controlled by a Ca(2+)-dependent intramolecular mechanism involving the Ca(2+)-binding alpha-region, and that this control is released in C1 by a signal originating in C1q and transmitted through the C1q/C1r interface. |
8042994 | Effects of tri-iodothyronine administration on the disposal of oral [1-14C]triolein, lipoprotein lipase activity and lipogenesis in the rat during lactation and on removal of the litter. | The effect of tri-iodothyronine (T3) administration on the utilization of dietary [14C]lipid by the mammary gland and adipose tissue of lactating and litter-removed rats was studied. (1) After an oral load of [1-14C]triolein, the lactating rats treated with T3 (50 micrograms/100 g body wt.) over 24 h showed an increase in 14CO2 production and a decrease in the total [14C]lipid transferred through the mammary gland that was paralleled by a decrease in tissue lipoprotein lipase (LPL) activity. (2) T3 administration decreased plasma prolactin in the lactating rats. Prolactin replacement in T3-treated rats restored LPL activity in the mammary gland, but did not increase the amount of dietary [14C]lipid transferred to the milk. (3) Chronic T3 administration (4 days) to lactating rats did not affect pup growth or the lipogenic rate in the mammary gland. (4) The administration of T3 to litter-removed rats inhibited the increase of LPL activity in white adipose tissue and decreased the accumulation of dietary [14C]lipid. This decrease was accompanied by increased 14CO2 production and [14C]lipid accumulation in skeletal muscle and heart. (5) It is concluded that hyperthyroidism depresses LPL activity in mammary gland and white adipose tissue, but not in muscle. The increased accumulation of [14C]lipid in muscle and increased production of 14CO2 in lactating and in litter-removed rats treated with T3 is in part due to the decreased total LPL in mammary gland and adipose tissue respectively, which are therefore less able to compete with muscle for the available plasma triacylglycerols. |
8042995 | Zinc and barium inhibit the phospholipase A2 from Naja naja atra by different mechanisms. | The mode of inhibition of the phospholipase A2 (PLA2) enzyme from the Chinese cobra (Naja naja atra) by Zn2+ is qualitatively different from inhibition by Ba2+. Inhibition by Ba2+ shows the kinetic characteristics of a conventional competitive inhibitor acting to displace Ca2+ from a single essential site, but Zn2+ has the paradoxical property of being more inhibitory at high than at low Ca2+ concentration. Kinetic analysis of the Ca(2+)-dependence of enzymic activity shows a bimodal response, indicating the presence of two Ca(2+)-binding sites with affinities of 2.7 microM and 125 microM respectively, and we propose that these can be identified with the two Ca(2+)-binding sites revealed by crystallographic analysis [White, Scott, Otwinowski, Gleb and Sigler (1990) Science 250, 1560-1563]. The results are consistent with the model that the enzyme is activated by two Ca2+ ions, one that is essential and can be displaced by Ba2+, and one that modulates the activity by a further 5-10-fold and which can be displaced by Zn2+. An alternative model is also presented in which the modulating Zn(2+)-binding site is a phenomenon of the lipid/water interface. |
8042993 | Catalytic mechanism of active-site serine beta-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad. | The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S)G] triad has been studied for a class A and a class C beta-lactamase by site-directed mutagenesis. Surprisingly, the disappearance of this functional group had little impact on the penicillinase activity of both enzymes. The cephalosporinase activity was much more affected for the class A S235A (Ser235-->Ala) and the class C T316V (Thr315-->Val) mutants, but the class C T316A mutant was less impaired. Studies were extended to beta-lactams, where the carboxy group on C-3 of penicillins or C-4 of cephalosporins had been modified. The effects of the mutations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those obtained with a similar mutant (T299V) of the Streptomyces R61 DD-peptidase. With this enzyme the mutation also affected the interactions with penicillins and severely decreased the peptidase activity. The strict conservation of the hydroxy group on the second residue of the KT(S)G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially those of class C. |
8042992 | The mechanism of action of DD-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 DD-peptidase. | The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results. |
8042990 | Calsequestrin is a component of smooth muscles: the skeletal- and cardiac-muscle isoforms are both present, although in highly variable amounts and ratios. | Expression by smooth-muscle cells of calsequestrin (CS), the low-affinity/high-capacity Ca(2+)-binding protein of striated-muscle sarcoplasmic reticulum (SR), has been investigated in recent years with conflicting results. Here we report the purification and characterization from rat vas deferens of two CS isoforms, the first deemed skeletal muscle, the second cardiac type, on account of their N-terminal amino acids and other relevant biochemical and molecular properties. Compared with vas deferens, the smooth muscles from aorta and stomach, in that order, were found to express lower amounts of CS, whereas in the uterus and bladder the protein was not detectable. The ratio between the two CS isoforms was also variable, with the stomach and aorta predominantly expressing the skeletal-muscle type and the vas deferens expressing the two CSs in roughly similar amount. Because of the property of CSs to localize within the skeletal-muscle SR lumen not uniformly, but according to the distribution of their anchorage membrane proteins, the expression of the protein suggests the existence in smooth-muscle cells of discrete endoplasmic-reticulum areas specialized in the rapidly exchanging Ca2+ storage and release, and thus in the control of a variety of functions, including smooth-muscle contraction. |
8042991 | Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver. | GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database. |
8042989 | A possible route for the release of fatty acid from fatty acid-binding protein. | A simulation of the release of fatty acid from intestinal fatty acid-binding protein was attempted, starting with the crystallographic model and using molecular-dynamic processes at different temperatures. The release of the ligand was observed only at high temperature, which perhaps makes the process unreliable in detail. Nevertheless, the overall behaviour of the protein, also confirmed by the simulation performed at room temperature, strongly supports the idea that the fatty acid leaves the protein through an opening formed by alpha-helix II and turns beta C-beta D and beta E-beta F. Additionally, it suggests a role for the lack of hydrogen bonds between the main chains of beta-strands D and E: this feature, observed in all the protein structures of this family which have currently been determined, seems to provide the structure with great flexibility, allowing the barrel to open and close without disruption of the hydrogen-bond network. |
8042988 | Acyl-chain selectivity of the 85 kDa phospholipase A2 and of the release process in intact macrophages. | The selectivity of the intracellular 85 kDa phospholipase A2 (PLA2-85) towards fatty acids closely related to arachidonic acid has been investigated, using purified PLA2-85 from J774 cells and mixed phospholipids, dually acyl-chain-labelled in the sn-2 position. In parallel experiments, we assessed the acyl-chain selectivity of the release process in intact, dually labelled, peritoneal mouse macrophages responding to either calcium ionophore or zymosan beads in the presence of indomethacin and BSA. The results obtained in the two systems were very similar, which supports previous evidence that PLA2-85 is responsible for stimulus-induced release of eicosanoid precursor in mouse macrophages. In the in vitro system, PLA2-85 was found to exhibit a moderate selectivity towards C20 acyl chains differing in double-bond structure, while the sensitivity to acyl-chain length was more pronounced. Together with previous data, these results demonstrate a striking preference for C20 over either C18 or C22 unsaturated acyl chains. |
8042986 | Threonine-497 is a critical site for permissive activation of protein kinase C alpha. | Phosphorylation of the region containing Thr-494, Thr-495 and Thr-497, present in the catalytic domain of protein kinase C alpha (PKC alpha), is a preliminary event necessary for subsequent PKC activation [Cazaubon and Parker (1993) J. Biol. Chem. 268, 17559-17563]. To define the essential residues in this region, various combinations of alanine substitutions for threonine residues 494, 495 and 497 have been tested. These mutations yielded expressed polypeptides of 76 and 80 kDa in ratios that vary from 100% 80 kDa (wild-type kinase, active) to 100% 76 kDa (AAA mutant, inactive) with the hierarchy being wild-type PKC alpha (TTT), ATT, AAT, TTA, ATA, TAA, AAA (the nomenclature indicates the location of alanine residues substituted for Thr-494, Thr-495 and Thr-497 respectively). Only the mutants retaining Thr-497 displayed kinase activity in vitro. The results overall indicate that Thr-497 plays the dominant role in the regulation of PKC alpha activity but that in the wild-type protein, Thr-495 may also be important. Consistent with the need for phosphorylation in this region, an intrinsically active PKC alpha could be produced in bacteria by exchanging Thr-495 for a glutamic acid residue. |
8042987 | A mutant of Arabidopsis thaliana lacking the ability to transport glucose across the chloroplast envelope. | At the end of a 12-h day leaves of the mutant of Arabidopsis thaliana L., TC265, contained 4-5 times more starch than those of the wild type. During a subsequent 12-h night the decline in the starch content of the leaves of the mutant was at least 50% of that of the wild-type leaves. Starch labelled in the light in a 30-min pulse in 14CO2 was rapidly broken down in a subsequent 12-h chase in the dark in air in the leaves of both mutant and wild type. Chloroplasts from leaves of the wild type took up [32P]Pi and [U-14C]glucose at 12 and 1.6 mumol/h per mg of chlorophyll respectively; chloroplasts from the mutant showed a similar rate for [32P]Pi but no uptake of [U-14C]glucose. The glucose content of freshly isolated chloroplasts from the mutant was twice that of chloroplasts from the wild type; this difference was accentuated when the isolated chloroplasts were incubated in the dark. SDS/PAGE of preparations of chloroplast envelopes showed that those from the mutant were deficient in a protein band of approximate molecular mass 40 kDa. It is suggested that in mutant TC265 the primary lesion is in a hexose transporter in the chloroplast envelope, and that this transporter moves the products of starch breakdown that are destined for sucrose synthesis from the chloroplast to the cytosol. |
8042985 | Interaction of disintegrins with the alpha IIb beta 3 receptor on resting and activated human platelets. | Viper venom disintegrins contain the RGD/KGD motif. They inhibit platelet aggregation and cell adhesion, but show structural and functional heterogeneity. We investigated the interaction of four prototypic disintegrins with alpha IIb beta 3 expressed on the surface of resting and activated platelets. The binding affinity (Kd) of 125I-albolabrin, 125I-echistatin, 125I-bitistatin and 125I-eristostatin toward resting platelets was 294, 153, 48 and 18 nM respectively. The Kd value for albolabrin decreased 3-fold and 6-fold after ADP- or thrombin-induced activation. The Kd values for bitistatin and echistatin also decreased with ADP, but there was no further decrease with thrombin. In contrast, eristostatin bound with the same high affinity to resting and activated platelets. The pattern of fluorescein isothiocyanate (FITC)-eristostatin and FITC-albolabrin binding to resting and activated platelets was consistent with observations using radiolabelled material. Eristostatin showed faster and more irreversible binding to platelets, and greater potency compared with albolabrin in inducing conformational neo-epitopes in beta 3. The anti-alpha IIb beta 3 monoclonal antibody OP-G2 that is RGD-dependent inhibited disintegrin binding to activated platelets more strongly than binding to resting platelets and it inhibited the binding to platelets of albolabrin more strongly than eristostatin. The specificity of disintegrin interaction with alpha IIb beta 3 was confirmed by demonstrating cross-linking of these peptides to alpha IIb beta 3 on normal platelets, but not to thrombasthenic platelets deficient in alpha IIb beta 3. |
8042984 | Human macrophage-mediated oxidation of low-density lipoprotein is delayed and independent of superoxide production. | There is growing evidence that oxidatively modified low-density lipoprotein (LDL) accumulates in the atherosclerotic intima of arteries. Cells present in the intima (including the monocyte/macrophage) are capable of oxidizing LDL in vitro, but the mechanisms by which this occurs are unknown. Several reports have claimed a crucial role for superoxide as a cell-derived radical species capable of enhancing the rate of LDL oxidation. We have used a sensitive h.p.l.c. system with chemiluminescence detection to measure LDL cholesteryl ester hydroperoxides at early stages of LDL oxidation. During the initial stages of LDL oxidation, there is at least a 2 h delay before human monocyte-derived macrophages enhance this process. Stimulation of these cells to produce large fluxes of superoxide does not increase the rate of LDL oxidation or decrease the delay of its onset. Prior exposure of LDL to a high flux of superoxide does not increase its susceptibility to oxidation by human monocyte-derived macrophages. We also show that the thiobarbituric acid-reactive substances (TBARS) assay does not always correlate with more direct methods of assessing LDL oxidation and confirm recent reports that superoxide dismutase only partially inhibits cell-mediated LDL oxidation. We conclude that superoxide does not play a major role in human monocyte-derived macrophage-mediated LDL oxidation under the conditions that we describe. |
8042983 | Phosphatidylinositol 3,4,5-trisphosphate is formed from phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets. | Platelets accumulate PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to thrombin and thrombin-receptor-directed peptide in a GTP-dependent manner. These phosphoinositides are considered to be mediators of signaling events in a variety of cells. We have examined the metabolic route by which PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are synthesized by briefly (10 min) incubating platelets with high activities of [32P]Pi, followed by 20 or 60 s exposure to thrombin, and analysing the relative radioactivities of the individual phosphate groups in the resulting labelled PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The phosphate group possessing the highest specific activity under such non-equilibrium labelling conditions indicates the last one added in a metabolic sequence. The thrombin-stimulated rate of labelling of PtdIns(3,4)P2 was significantly slower than that of PtdIns(3,4,5)P3. Increased labelled PtdIns3P was not detected within 60 s. The measured relative radioactivities decreased in the order 3 > 5 > 4 >> 1 for PtdIns(3,4,5)P3 and 3 > 4 >> 1 for PtdIns(3,4)P2. On the basis of the results of both rate-of-labelling and specific radioactivity analyses we conclude that PtdIns(3,4,5)Pa is formed by 3-OH phosphorylation of PtdIns(4,5)P2, whereas PtdIns(3,4)P2, may be formed by 3-OH phosphorylation of PtdIns4P and/or dephosphorylation of PtdIns(3,4,5)P3. These findings point to the activation of phosphoinositide 3-kinase as a critical receptor-regulated step in thrombin-stimulated platelets. |
8042982 | Cloning and characterization of a cDNA representing a putative complement-regulatory plasma protein from barred sand bass (Parablax neblifer). | It has been demonstrated previously that plasma from a number of vertebrate species including the phylogenetically old barred sand bass possesses molecules that cleave the alpha'-chain of the activated third (C3b) and fourth (C4b) components of the human complement system. A specific protease and a cofactor protein were identified to be responsible for this cleavage. The cofactor activity in sand bass correlated with a 110 kDa polypeptide chain of a 360 kDa plasma protein. The evolutionary conservation was probed at the cDNA level and subsequently a cDNA clone of barred sand bass was isolated that represents a protein with structural similarity to mammalian complement-regulatory proteins. The cDNA (SB1) was identified by immunoscreening of a sand bass liver expression library using affinity-purified IgG antibodies raised against the isolated 110 kDa material. The cDNA is 3397 bp in size and the open reading frame represents a protein of 1053 amino acid residues with a hydrophobic signal peptide indicative of a secreted protein. The calculated mass of the mature protein (SBP1) is 115.2 kDa which is in good agreement with the molecular mass of 110 kDa determined for the sand bass serum protein. Similarly to mammalian complement-regulatory proteins, the protein deduced from the sand bass cDNA is organized into short consensus repeats (SCR). It consists of 17 SCRs, of which SCRs 2, 12 and 16 exhibit significant homology to SCRs 2, 15 and 19 of human factor H, and SCRs 11, 12 and 13 have homology to SCRs 1, 2 and 3 of human C4b-binding protein. For the first time a complete cDNA representing a putative complement-regulatory protein which is structurally related to mammalian complement proteins has been isolated from a bony fish. |
8042980 | The GLUT3 glucose transporter is the predominant isoform in primary cultured neurons: assessment by biosynthetic and photoaffinity labelling. | Cerebellar granule neurons in primary culture express increasing levels of two glucose transporter isoforms, GLUT1 and GLUT3, as they differentiate in vitro. We have determined the relative abundance of GLUT1 and GLUT3 in these neurons by three different labelling methods. (1) Photoaffinity cell surface labelling of neurons with an impermeant bis-mannose photolabel revealed 6-10-fold more GLUT3 than GLUT1 and dissociation constants (Kd) for the photolabel of 55-68 microM (GLUT3) and 146-169 microM (GLUT1). Binding to both transporters was inhibited by cytochalasin B. (2) Photoaffinity labelling of neuronal membranes with a permeant forskolin derivative showed 5.5-8-fold more GLUT3 than GLUT1, whereas in rat brain membranes containing both neuronal and glial membranes, GLUT3 and GLUT1 were detected in similar proportions. (3) Biosynthetic labelling of neurons with [35S]methionine and [35S]cysteine showed GLUT3 to be 6-10-fold more abundant than GLUT1. Thus GLUT3 is quantitatively the predominant glucose-transport isoform in cultured cerebellar granule neurons. |
8042981 | Iron release from ferritin and its sensitivity to superoxide ions differs among vertebrates. | The influence of the superoxide-generating system, xanthine oxidase, on the release of iron from various vertebrate ferritins was determined both in the presence and absence of superoxide dismutase. The initial rate of iron release in the presence of this system was higher for ferritins from human, trout and rat liver than for those from lamprey liver and horse spleen. The proportion of this iron release that was superoxide-dependent in the case of rat, human and trout ferritins was 92, 86 and 84% respectively, whereas no such superoxide-dependent iron release occurred from the ferritins of lamprey liver and horse spleen. On the other hand, the rate of superoxide-independent iron release was of comparable magnitude for all of the species examined. The rate of superoxide-dependent iron release was related neither to the iron: protein ratios nor to the subunit size of the ferritins. However, it is significant that the ferritins with a high rate of superoxide-dependent iron release came from tissues known to be susceptible to iron damage. It is thus proposed that the resistance of lamprey liver ferritin to the mobilization of iron by superoxide ions accounts in part for the tolerance of the lamprey liver to high iron loads. |
8042979 | Substrate specificity of L-delta-(alpha-aminoadipoyl)-L-cysteinyl-D-valine synthetase from Cephalosporium acremonium: demonstration of the structure of several unnatural tripeptide products. | Potential substrates for L-delta-(alpha-aminoadipoyl)-L-(cysteinyl)-D-valine (ACV) synthetase were initially identified using both the amino-acid-dependent ATP<-->pyrophosphate exchange reaction catalysed by the enzyme and the incorporation of 14C-radiolabelled cysteine and valine into potential peptide products. S-Carboxymethylcysteine was an effective substitute for alpha-aminoadipate and both allylglycine and vinylglycine could substitute for cysteine, indicating that the thiol group of cysteine is not essential for peptide formation. L-allo-Isoleucine but not L-isoleucine substituted effectively for valine. The structures of the presumed peptide products derived from these amino acids were confirmed by combined use of electrospray-ionization m.s. (e.s.m.s.) and 1H n.m.r. These results clearly indicate that, in common with other peptide synthetases, but in contrast with ribosomal peptide synthesis, ACV synthetase has a relatively broad substrate specificity. |
8042978 | Oestradiol-induced changes in the composition of phospholipid classes of quail oviduct: specific replacement of arachidonic acid by docosahexaenoic acid in alkenylacyl-glycerophosphoethanolamine. | The phospholipid composition and the molecular species of the major subclasses of ethanolamine and choline glycerophospholipids were determined during the natural or oestradiol-induced development of the quail oviduct. The phospholipid concentration increased significantly during oviduct development, and the proportion of ethanolamine glycerophospholipids (EPL) remained constant while that of choline glycerophospholipids increased. The immature oviduct contained the majority of its endogenous arachidonic acid mass (71%) in EPL, mainly in alkenylacyl-glycerophosphoethanolamine (alkenylacyl-GPE) (49% of the total). Oestrogen treatment induced the depletion of 20:4,n-6 specifically from this pool, which indicates the biological importance of 20:4,n-6 molecular species in alkenylacyl-GPE as substrates for the oviduct phospholipases activated by oestradiol, and suggests that this EPL subclass is involved in the oestrogen-induced cell proliferation. Another striking result was the marked increase in 22:6,n-3 EPL molecular species following the oestradiol treatment and more particularly the strict substitution of 20:4,n-6 by 22:6,n-3 in alkenylacyl-GPE. We speculate that alkenylacyl-GPE molecular species containing 22:6,n-3 may participate in the arrest of oestrogen-induced proliferation. |
8042977 | Kinetic mechanism of adenosine 5'-phosphosulphate kinase from rat chondrosarcoma. | Biosynthesis of the activated sulphate donor adenosine 3'-phosphate 5'-phosphosulphate (PAPS) involves the sequential action of two enzyme activities. ATP-sulphurylase catalyses the formation of APS (adenosine 5'-phosphosulphate) from ATP and free sulphate, and APS is then phosphorylated by APS kinase to produce PAPS. Initial-velocity patterns for rat chondrosarcoma APS kinase indicate a single-displacement formal mechanism with KmAPS 76 nM and KmATP = 24 microM. Inhibition studies using analogues of substrates and products were carried out to determine the reaction mechanism. An analogue of PAPS, adenosine 3'-phosphate 5'-[beta-methylene]phosphosulphate, exhibited competitive inhibition with APS and non-competitive inhibition with ATP. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate was also competitive with APS and non-competitive with ATP. Adenosine 5'-[beta gamma-imido]triphosphate showed competitive inhibition with respect to ATP and produced mixed-type inhibition, with a pronounced intercept effect and a small slope effect, with respect to APS. These results are in accord with the formulation of the predominant pathway as a steady-state ordered mechanism with APS as the leading substrate and PAPS as the final product released. |
8042976 | Kinetic mechanism of ATP-sulphurylase from rat chondrosarcoma. | ATP-sulphurylase catalyses the production of adenosine 5'-phosphosulphate (APS) from ATP and free sulphate with the release of PPi. APS kinase phosphorylates the APS intermediate to produce adenosine 3'-phosphate 5'-phosphosulphate (PAPS). The kinetic mechanism of rat chondrosarcoma ATP-sulphurylase was investigated by steady-state methods in the physiologically forward direction as well as the reverse direction. The sulphurylase activity was coupled to APS kinase activity in order to overcome the thermodynamic constraints of the sulphurylase reaction in the forward direction. Double-reciprocal initial-velocity plots for the forward sulphurylase intersect to the left of the ordinate for this reaction. KmATP and Kmsulphate were found to be 200 and 97 microM respectively. Chlorate, a competitive inhibitor with respect to sulphate, showed uncompetitive inhibition with respect to ATP with an apparent Ki of 1.97 mM. Steady-state data from experiments in the physiologically reverse direction also yielded double-reciprocal initial-velocity patterns that intersect to the left of the ordinate axis, with a KmAPS of 39 microM and a Kmpyrophosphate of 18 microM. The results of steady-state experiments in which Mg2+ was varied indicated that the true substrate is the MgPPi complex. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate, was a linear inhibitor competitive with APS and non-competitive with respect to MgPPi. The simplest formal mechanism that agrees with all the data is an ordered steady-state single displacement with MgATP as the leading substrate in the forward direction and APS as the leading substrate in the reverse direction. |
8042975 | In vivo inhibition of beta-glucosidase and beta-mannosidase activity in rats by 2-deoxy-2-fluoro-beta-glycosyl fluorides and recovery of activity in vivo and in vitro. | 2-Deoxy-2-fluoro-beta-glucosyl and -beta-mannosyl fluorides administered to rats in a single dose (10 mg/kg) inhibited beta-glucosidase and beta-mannosidase activity respectively after 1 h in brain, spleen, liver and kidney tissues. This inhibition, presumably caused by accumulation of 2-deoxy-2-fluoroglycosyl-enzyme intermediates, indicates that intact 2-deoxy-2-fluoroglycosyl fluorides are distributed to these organs and, in the case of brain, that they cross the blood/brain barrier. beta-Glucosidase activity recovered completely or partially in brain, spleen, liver and kidney by 20-48 h. beta-Mannosidase activity partially recovered in all tissues by 48 h. beta-Galactosidase activity in brain and kidney was not significantly affected by administration of either the gluco or manno compounds at this dosage, indicating that these inhibitors are directed towards specific glycosidases. Observation of similar relatively rapid rates of beta-glycosidase re-activation in vivo and in tissue homogenates in vitro at 37 degrees C suggests that hydrolysis or transglycosylation of 2-deoxy-2-fluoroglycosyl-enzymes, not protein synthesis, are the primary mechanisms involved in the recovery of glycosidase activity inhibited by this class of compounds in vivo. |
8042974 | Analysis of the optical absorption and magnetic-circular-dichroism spectra of peanut peroxidase: electronic structure of a peroxidase with biochemical properties similar to those of horseradish peroxidase. | The electronic structures of the cationic isoenzyme of peanut peroxidase, horseradish peroxidase (isoenzyme C) and bovine liver catalase are compared through analysis of their optical absorption and magnetic c.d. (m.c.d.) spectral properties. The spectral data for the native resting states and compounds I and II of peanut peroxidase (PeP) are reported. The absorption and m.c.d. data for the native PeP exhibit bands characteristic of the high-spin ferric haem. The absorption spectrum of PeP compound I closely resembles that observed for the HRP compound I species. The m.c.d. data for PeP I clearly identifies that ring oxidation has occurred. One-electron reduction forms the PeP compound II species. The absorption and m.c.d. spectra recorded for PeP II exhibit the well-resolved spectral characteristics previously observed for both HRP compound II and catalase compound II. The spectral data of PeP with HRP and catalase are compared. The data clearly indicate that the m.c.d. spectral patterns of both plant peroxidases (PeP and HRP) are very similar and, therefore, the electronic structures of their resting states, and as well their primary and secondary compounds, must be similar. The m.c.d. data suggest that, while the compound I species of PeP and HRP belong to one electronic class, catalase compound I belongs to a different class. These data emphasize how the ground states of these two classes of oxidized haem, may be characterized as predominantly 2A2u (PeP I and HRP I) or 2A1u (catalase I). Peanut peroxidase is the second plant peroxidase for which the electronic structure of the compound I intermediate has been studied using the m.c.d. technique. The similarities with horseradish peroxidase allow us to suggest that plant peroxidases may operate by the same general mechanism, in spite of the low degree of sequence similarity between their polypeptide chains. |
8042973 | Platelet-derived growth factor A-chain gene transcription is mediated by positive and negative regulatory regions in the promoter. | Platelet-derived growth factor (PDGF) is a disulphide-linked heterodimer of two polypeptide chains, the A and B chains, which are encoded by genes on separate chromosomes. The A-chain gene is transcribed in a number of transformed and non-transformed cell lines and is inducible by a wide variety of growth factors, cytokines and other mitogenic agonists. To localize DNA elements that mediate basal transcription in the promoter regulatory region of the A-chain gene, we have employed 5'-endpoint deletion mutagenesis and transient expression analysis in the renal epithelial cell line BSC-1 (African green monkey). Studies conducted in this cell line, which expresses high concentrations of PDGF A-chain mRNA, reveal a positive regulatory element (PRE) in a GC-rich stretch of the A-chain promoter between -82 and -40, relative to the transcription start site. Two discrete regions of the promoter were identified as negative regulatory elements (NREs), located between -1029 and -880 (NRE1) and between -1800 and -1029 (NRE2). The -1800 to -812 region, which contains both NREs, functions as a potent NRE when relocated in either orientation adjacent to the herpes simplex virus thymidine kinase promoter, reducing transcription activity by 60% in the positive orientation and 85% in the negative orientation. Comparison of BSC-1 cells and Saos-2 cells (human osteogenic sarcoma), which do not express significant quantities of PDGF A-chain mRNA or protein, indicates that basal transcription of the gene is determined by enhancer activity mediated by the GC-rich region rather than through de-repression of the upstream NREs. Electrophoretic gel mobility shift assays reveal a complex pattern of nuclear protein binding to the GC-rich PRE (-73 to -46). Competition studies conducted with mutant oligonucleotides that alternately disrupt consensus binding sites for Sp-1 or Egr-1 demonstrate a requirement for the presence of an Sp1-like core sequence (GGCGGG) but not Egr-1/Krox-24 [GCG(G/T)-GGGCG] for the formation of specific DNA-protein complexes. Our observations suggest that basal transcription of the A-chain gene in renal epithelial cells is achieved through active enhancement, mediated by a GC-rich PRE and nuclear proteins that bind to Sp-1-like consensus DNA sequences. |
8042972 | Leishmanial glycosomes contain superoxide dismutase. | In this work we report that superoxide dismutase is entrapped in a microbody-like organelle, the glycosome, present in Leishmania spp. Studies on the sensitivity of the enzyme to various inhibitors indicated that glycosomal superoxide dismutase is predominantly of the Cu/Zu type. Localization of superoxide dismutase in glycosomes points to the major importance of this organelle in the survival of this pathogen, suggesting a new approach towards chemotherapy for leishmaniasis. |
8042971 | The relationship between treatment goals and recidivism among child molesters. | Preliminary findings from a longitudinal outcome study of sex offender treatment are presented. Ss included 76 child molesters who were treated in a comprehensive relapse prevention program, and 79 molesters who were randomly assigned to a control (no treatment) condition. Three sets of findings are reported: survival analyses of time to reoffense; in-treatment change data relevant to the program's treatment goals; and the relationships between treatment measures and risk of reoffense. Over an average followup period of 38 months, there was a trend for treatment Ss to be at lower risk for both sex and violent crimes than were controls. Treatment Ss showed significant progress towards treatment goals of increased acceptance of personal responsibility for their crimes and decreased levels of deviant sexual arousal. Measures of personal responsibility, however, were not related to risk of rearrest for new sex crimes. Treatment Ss with high levels of both deviant and nondeviant sexual arousal were more likely to commit new sex offenses, but not other violent offenses. The strongest predictor in the study was a measure of the Ss' skills in applying the relapse prevention model, with highly skilled Ss being less likely to commit new sex crimes. |
8042970 | Relationships between conflict, affect and deviant sexual behaviors in rapists and pedophiles. | The aim of the current study was to determine the relationship in sexual offenders between conflict, affective states and particular sexual behaviors (fantasies and masturbatory activities while having such fantasies). To this end we developed the "Fantasy Report", a self-assessment method for recording affective components and sexual behaviors. Thirteen rapists and 9 pedophiles filled out the Fantasy Report every 2 days for a period of 60 days. In rapists, negative mood and the presence of conflicts coincided with both overwhelming deviant sexual fantasies and increased masturbatory activities while having such fantasies. Furthermore, the emotions most frequently reported by rapists following conflicts were loneliness, humiliation, anger and feelings of inadequacy and rejection. Affective components, however, were not associated with nondeviant sexual behaviors. For the pedophiles, the data revealed a significant relationship only between negative moods and deviant sexual fantasies. These data are interpreted to mean that, in sexual offenders, negative affect is a crucial component in the chain that leads to deviant sexual behaviors. |
8042969 | Process evaluation of a group therapy component designed to enhance sex offenders' empathy for sexual abuse survivors. | A process evaluation was conducted to assess the extent to which a specialized treatment group might enhance the offenders' empathy for sexual abuse survivors. Therapeutic efficacy was assessed through pre- and post-treatment administration of a battery of paper-and-pencil inventories. Pedophiles demonstrated greater empathy at pre-treatment and post-treatment than rapists. Surprisingly, pedophiles and rapists did not differ in pre-treatment endorsement of cognitive distortions hypothetically related to rape. Post-treatment psychological inventories revealed significant changes on a variety of empathetic qualities. The results suggests that highly-specialized interventions can enhance sex offenders' empathy for sexual abuse victims and decrease endorsement of distortions justifying sexualized violence. Follow-up is underway to determine if these changes are associated with long-term reductions in recidivism. |
8042967 | The effects of the z-score transformation on measures of relative erectile response strength: a re-appraisal. | In studies of stimulus control of sexual arousal, some researchers transform raw data to z-scores. Earls, Quinsey and Castonguay [Archives of Sexual Behavior, 16, 493-500 (1987)] have argued that the proportion of variance due to stimulus presentations was greater in z-scores than in the raw data or in a percent score. We present analyses of sexual arousal data that show that z-score transformations may distort the information inherent in the raw data and may increase random error. In addition, we present Monte Carlo analyses indicating that the z-score transformation compromises the estimates of Type I error, and, depending on specific circumstances, sometimes increases and sometimes decreases the power of the statistical test. Transformation of raw data to percent of full erection does not distort the information in the raw data, does not compromise the data analyses and, therefore, is the preferred transformation. |
8042966 | Differentiating rapists and non-offenders using the rape index. | Some studies have suggested that phallometrically derived rape indices can differentiate groups of rapists and non-offenders. There are other studies however which cast doubt on this assertion, at least in so far as it applies to all but those few rapists who are sadistic. These studies have used rape indices which are derived from rapists' sexual responses to audiotaped descriptions of mutually consenting sex and brutal sexual assaults. It was hypothesized however, that stimuli which put more emphasis on the degrading and humiliating elements of rape would improve discriminability. Stimuli were compiled to test this hypothesis. The results indicate that neither rape indices derived from the physically brutal elements nor the degrading elements of rape were able to discriminate between rapists and non-offenders. Furthermore, these indices were not related to offence histories. The results are discussed in terms of their implications for the assessment of rapists and theoretical considerations of their behaviour. |
8042965 | The relationship between phallometrically measured deviant sexual arousal and clinical characteristics in juvenile sexual offenders. | The current study examined the relationship between clinical characteristics and a phallometrically derived deviance quotient index in two samples of 44 and 54 juvenile sex offenders. Results support an association between higher measured deviant arousal and having a male victim only, consistent with the literature on adult child molesters. However, results reflected greater fluidity in the offense patterns of the juvenile offenders, and generally less correspondence between measured arousal and offense histories than what has been cited for adults. The authors review whether there is empirical support for a conditioning model of deviant arousal onset in juveniles, and suggest caution in the interpretation of phallometric data with this population. |
8042964 | Intrusive memories of childhood abuse during depressive episodes. | A sample of adult women with major depression who reported childhood sexual or physical abuse completed a measure of the extent to which they were experiencing intrusive memories of the abuse and their efforts to avoid these memories. The majority of women in the sample reported high levels of disturbing intrusive memories, and high levels of avoidance. Those abused women with particularly high levels of intrusions and more avoidance were also more severely depressed than both non-abused women and abused women with low levels of intrusions and avoidance. Higher levels of intrusions and avoidance were also associated with repeated childhood abuse, sexual abuse involving intercourse and sexual abuse involving a primary caregiver. |
8042963 | Correlates of post-traumatic stress at 30 months: the Herald of Free Enterprise disaster. | Although exposure to a traumatic event is thought to be the main aetiological factor in the development of post-traumatic stress disorder, there remain large unexplained individual differences in the severity and chronicity of symptoms. The aim of the present study was to assess the relative contribution of a number of social and psychological factors which are thought to determine symptoms. Crisis support and life-events subsequent to the disaster are the two best predictors of general psychological well-being, whereas a sense of helplessness during the disaster and bereavement are the two best predictors of intrusive symptomatology. |
8042962 | The experimental induction of depersonalization and derealization in panic disorder and nonanxious subjects. | The present study evaluated the efficacy of three tasks in inducing depersonalization (DP) and derealization (DR) in three different groups: (a) panic disorder patients who report these symptoms while panicking (PD + DD; n = 10); (b) panic disorder patients never experiencing these symptoms during panic attacks (PD; n = 10); and (c) nonanxious controls (NC; n = 10). Clinical features of the PD+DD and PD Ss were compared as well. Relative to PD Ss, PD + DD Ss evidenced higher levels of depression, trait anxiety, more fear of panic, and had a briefer duration of their disorder. A substantial proportion of NC Ss reported past DP and DR experiences. DP and DR induction procedures were the following: staring at a dot on the wall, staring in a mirror, and silent repetition of one's name. Results indicated two tasks (mirror and dot) successfully elicited these sensations above baseline levels with DP reported more frequently and intensely than DR for all Ss. The PD + DD Ss evidenced greater baseline-to-task increases in DP and DR relative to the other two groups and exhibited a differential fear response, particularly on the dot task, with 30% of these Ss intentionally distracting themselves or terminating the induction. |
8042961 | The effects of stress, mood, and coping on blood glucose in NIDDM: a prospective pilot evaluation. | Eight subjects (Ss) with non-insulin-dependent diabetes (NIDDM) monitored their stress, blood glucose (BG), food intake, activity (via pedometer), mood, and coping responses for 8 days. They alternated 2 daily, self-selected ADA food-exchange diets to control for the effects of stress on adherence to diet. BG was significantly higher on high-stress compared to low-stress days. This effect was at least partially mediated by the effect of stress on activity; Ss were significantly less active on high-stress days. Further analyses suggested idiosyncratic relationships between mood and BG, and some evidence was found to suggest a relation between stress, coping, and BG. |
8042959 | The outcome problem in psychotherapy: what have we learned? | The outcome problem in psychotherapy has usually been studied without much regard to the theories underlying the methods used. It is suggested that theories are vital to scientific advancement, and that without them we cannot even specify criteria to judge outcomes. Numerous studies since the 1950s have in essence failed to disconfirm the view that various forms of psychotherapy do not show greater effectiveness than spontaneous remission or placebo treatment. An effort is made to clarify the nature of spontaneous remission and placebo treatment, and to discuss the consequences of the many empirical findings and meta-analyses published over the past 50 years. A theory is suggested linking spontaneous remission, placebo treatment, psychotherapy and behaviour therapy, leading to a discussion of ethical considerations and cost-effectiveness issues. |
8042948 | Heterogeneity in the phenotypic expression of the mutation in the mitochondrial tRNA(Leu) (UUR) gene generally associated with the MELAS subset of mitochondrial encephalomyopathies. | Point mutations in the mitochondrial (mt) genome underlie a number of neurological disorders. Some are well defined including the myoclonus epilepsy ragged red fibre syndrome (MERRF) and the mitochondrial encephalopathy lactic acidosis stroke like episode syndrome (MELAS). However, other clinical phenotypes are less distinctive and mitochondrial studies are often included in the workup in complex neurological syndromes of uncertain aetiology. We investigated 27 consecutive patients with varied clinical phenotypes referred to our laboratory for mtDNA studies to determine the incidence of recognised point mutations in a patient group with a range of phenotypes including many where mt disease was possible but did not fall into a classical syndrome. The recognised point mutations were detected by amplification of the appropriate DNA fragment by PCR followed by restriction-endonuclease digestion of the normal and mutant species. The A-G base substitution mutation at nucleotide (nt)3243 in the tRNA(Leu) gene of mtDNA which is present in the majority of cases of MELAS syndrome, was detected in four cases, only one of whom had typical MELAS symptoms. Their clinical manifestations ranged from mild deafness to a mixture of chronic progressive external ophthalmoplegia symptoms (CPEO) and stroke like episodes. The nt3243 mutation was also identified in one of seven mtDNA deletion negative CPEO cases. The presentation of the mtDNA mutation at nt3243 appears therefore to be quite variable with some mild phenotypes as well as severe phenotypes observed. In general, the chance of finding a mitochondrial point mutation in a patient with an atypical clinical phenotype is small. |
8042947 | A comparison of hepatitis C virus (HCV)-RNA and--antibody as markers of infection and predictors of infectivity. | The diagnosis of hepatitis C virus (HCV) infection currently relies on the detection of antibody to HCV (anti-HCV). However, anti-HCV positivity may indicate past infection, current infection or possibly non-specific reactivity. For confirmation of current infection the virus needs to be assayed directly and this is possible by the polymerase chain reaction (PCR). The aims were to compare HCV RNA and anti-HCV as markers of infection in two groups of individuals: (i) a heterogeneous group with suspected HCV infection and (ii) a small group of blood and bone marrow donors, and their respective recipients. Serum samples were tested for alanine aminotransferase (ALT) as part of a liver function screen, for anti-HCV by ELISA II, and HCV RNA was detected by PCR. Single round and nested PCR was performed using primers designed from the sequence of the 5'-untranslated region of the HCV genome. Of the 36 subjects in the heterogeneous group, 19/22 anti-HCV-positive patients with chronic non-A non-B hepatitis (NANBH) were viraemic, and the majority (17/19) demonstrated elevated ALT. However, HCV RNA was undetected in seven anti-HCV-positive patients, four of whom suffered autoimmune hepatitis Type I and three were low risk blood donors. Of the remaining subjects (seven/36) who were anti-HCV-negative, three/seven were HCV-RNA-positive and included two with acute post-transfusion (PT) NANBH and a recent needlestick victim who contracted HCV. In the second group, four individuals (donors), including a mother with a history of drug use, were implicated in transmission to three recipients. ALT levels were normal in all donors but raised in two of the recipients. PCR determined which of two anti-HCV-negative blood donors was infectious, confirmed transmission between a bone marrow donor and recipient, and indicated that anti-HCV detected in a newborn child represented passive transfer of antibody. Anti-HCV detected by ELISA II is a useful marker of chronic HCV infection, particularly in association with raised ALT. However, HCV RNA is a superior marker of acute HCV infection, a more reliable predictor of infectivity and is more specific. |
8042946 | Acute treatment of paroxysmal tachycardia by adenosine. | Adenosine has proven efficacy in clinical trials and in the electrophysiological laboratory for the treatment of paroxysmal tachycardia. To evaluate the efficacy and safety of adenosine administered in a clinical setting by non-consultant staff. Incremental doses of adenosine were administered intravenously to five children and 32 adults during 39 episodes of paroxysmal tachycardia in a clinical setting. Structural heart disease was present in 43% of patients. Of 35 episodes of narrow complex tachycardia, adenosine terminated 26 of 28 episodes of supraventricular re-entrant tachycardia (SVRT), one episode of ectopic atrial tachycardia, and induced transient atrioventricular block to reveal atrial arrhythmias in four. Termination of SVRT occurred at a mean (SD) dose of 9.2 (4.0) mg in adults and 0.09 (0.04) mg/kg in children, but two patients had later spontaneous reinitiation of SVRT. Two patients with narrow complex tachycardia who failed to respond to adenosine were subsequently found to have ventricular tachycardia. Adenosine was therapeutic or diagnostic in three of four episodes of broad complex tachycardia. Overall, by intention to treat by the clinician, adenosine was therapeutic or diagnostic in 34 of 39 episodes (87%). Breathlessness (26%), chest tightness (18%) and flushing (18%) occurred transiently. There were no episodes of hypotension. Adenosine was given safely to 15 patients in whom verapamil was considered contraindicated. Adenosine is a safe treatment for both narrow and broad complex tachycardias; usually effective for the former and diagnostic for the latter. |
8042945 | Asthma severity and morbidity in a population sample of Sydney school children: Part I--Prevalence and effect of air pollutants in coastal regions. | In two regions of Sydney where sewage treatment facilities with high temperature sludge burning incinerators are installed, there was concern that the resultant emissions were causing a local increase in symptoms of asthma and other allergic diseases. To investigate whether living in a region with high temperature sludge burning incinerators was associated with an increased prevalence of childhood asthma or allergy. We studied 713 children aged eight-12 years in two regions close to incinerators and 626 children in a control region with no sludge burning incinerator. We measured respiratory illness in the previous year by questionnaire, airway hyper-responsiveness (AHR) by histamine inhalation test, and atopy by skin prick tests. 'Current asthma' was defined as AHR and recent wheeze. Recordings of oxides of nitrogen and sulphur, hydrogen sulphide, ozone and particulates during the study period showed that the level of pollutants did not vary in any major way between the study regions and the control region. The prevalence of current asthma, atopy, symptom frequency or any category of severity of asthma illness was not significantly different between the control and study regions. This suggests that factors other than intermittent or industrial air pollutants are responsible for the high prevalence of asthma symptoms, asthma medication use, asthma morbidity and AHR in the study of children. |
8042944 | The cost-effectiveness of treatment of supraventricular arrhythmias related to an accessory atrioventricular pathway: comparison of catheter ablation, surgical division and medical treatment. | Treatment alternatives for patients with incapacitating supraventricular arrhythmias related to an accessory atrioventricular pathway include transcatheter radiofrequency (RF) ablation, surgical division and long-term antiarrhythmic therapy (medical). The aim of this study was to compare in terms of cost and efficacy, transcatheter, surgical and medical treatment of patients with incapacitating supraventricular arrhythmias resulting from an accessory pathway. The study population consisted of 52 patients who underwent transcatheter RF ablation (20 consecutive patients), surgical treatment (20) and medical treatment (12). Two types of economic analysis were used. In all groups, a resource based costing method was used and in the medical and surgical treatment groups, a diagnostic related group (DRG) based costing method was used. Eighteen out of 20 (90%) patients who underwent catheter ablation remained asymptomatic during 8.4 +/- 1.6 months of follow-up. All surgically treated patients remained asymptomatic during 54 +/- 15 months of follow-up. Only one of the 12 patients in the medical treatment group remained completely free of symptoms during the mean 58 +/- 23 month follow-up period. The mean cost (1992 Australian dollars) per patient, calculated on the basis of actual resources used (with a DRG based costing given in brackets), was $2746 +/- $800 for catheter ablation, $12141 +/- $4465 ($12880 +/- $3998) for surgical treatment and $1713 +/- $748 ($1967 +/- $33) for medical treatment. The total cost of management over 20 years is estimated to be: $2911 for catheter ablation, $17467 for surgery and $4959 for medical treatment. In the long term transcatheter RF ablation is the most cost-effective treatment strategy for patients with incapacitating supraventricular arrhythmias related to an accessory pathway. |
8042943 | Mechanism of stroke complicating cardiopulmonary bypass surgery. | Stroke is a devastating complication of cardiopulmonary bypass (CPB) surgery which occurs in 1 to 5% of cases. Strategies to reduce its incidence require a knowledge of the underlying pathology and aetiology. To determine the incidence, pathology and aetiology of stroke complicating CPB. Prospective review of clinical, operative and cranial CT scan findings in all cases of stroke complicating CPB procedures in our institution over an 18 month period. Twenty-one (1.6%, 95% CI 0.9-2.3%) cases of stroke were identified from 1336 CPB procedures. Cranial CT scan, performed in all but one patient, was normal in three patients or consistent with ischaemic stroke in 17 patients. There were no cases of haemorrhagic infarction or intracerebral haemorrhage. It was difficult to differentiate embolic and borderzone infarcts in two cases. After considering the clinical, operative and CT scan features together, 12 (57%, 95% CI 36-78%) of the cases were felt to be embolic in origin and nine (43%, 95% CI 22-64%) due to hypoperfusion in a borderzone. This study demonstrates that stroke remains an important complication of CPB procedures with an incidence in our series of 1.6%. The pathologic type of stroke is predominantly ischaemic in nature due to either cerebral embolism or borderzone infarction. Strategies for stroke prevention in patients undergoing CPB should be targeted primarily at these two mechanisms. |
8042942 | Sclerosing cholangitis in children with inflammatory bowel disease. | Primary sclerosing cholangitis (PSC) with inflammatory bowel disease (IBD) has been rarely reported in children. To describe the clinical presentation, sequential liver function test abnormalities, radiological bile duct anomalies and liver histology in four children with PSC and IBD. Over a period of 18 years, four of 130 patients with IBD developed abnormal liver function tests. Three of the four patients had ulcerative colitis and the other Crohn's disease. All four patients had baseline and follow-up liver function tests, percutaneous transhepatic cholangiography and a needle biopsy of the liver. The four patients at presentation had minimal symptoms or signs of liver disease. All had elevation of serum transaminases, gamma glutamyl transferase and/or alkaline phosphatase. Three had the typical onion skin fibrosis of bile ducts. Percutaneous transhepatic cholangiography demonstrated irregularity and beading of the hepatic and common bile ducts in three patients. The other with normal cholangiography had fibrosing cholangitis on liver biopsy and was considered to have small duct disease. We conclude that yearly biochemical assessment of liver function should be performed on all children with IBD, and if abnormal should raise the suspicion of PSC. The latter diagnosis can be confirmed by liver biopsy and cholangiography. |
8042941 | Transjugular intrahepatic portal-systemic shunts (TIPS)--initial experience and clinical outcome. | The clinical role of the transjugular intrahepatic portal-systemic shunt (TIPS) has not been fully defined. To determine the technical results of TIPS and the clinical outcome of patients undergoing the procedure. Retrospective audit of the results of the first 31 procedures performed in Melbourne. Thirty procedures were performed for variceal haemorrhage, one procedure was for ascites. The aetiology of the liver disease was cirrhosis due to alcohol in 20, cryptogenic in five, chronic viral infection in four, and autoimmune chronic active hepatitis in one. Nodular regenerative hyperplasia was present in one patient. Seventy-seven per cent of procedures were considered successful based on the angiographic demonstration of shunt patency at the end of the procedure. The in-hospital mortality in all patients undergoing TIPS was 45% and was 42% in patients undergoing technically successful TIPS. Only age could be identified as predictive of death in hospital. In patients leaving hospital, we found a rebleeding rate of 57% with one patient dying of bleeding, one requiring balloon tamponade and two requiring variceal sclerotherapy. Hepatic trauma was documented in six cases, shunt thrombosis in four cases, stent displacement in two cases and severe hepatic encephalopathy in one case. TIPS has the potential to decompress the portal venous system, but the procedure is technically complex and should be performed in the knowledge that mortality and morbidity can be relatively high, particularly in patients whose condition is poor. |
8042940 | Transjugular intrahepatic portosystemic stent-shunt (TIPSS) for variceal haemorrhage: initial results in 28 patients. | Endoscopic sclerotherapy is an effective form of treatment of bleeding varices in patients with cirrhosis. However, the mortality in patients who rebleed is high. Recently, transjugular intrahepatic portosystemic stent-shunt (TIPSS) has been developed as an alternative to surgical shunt formation in patients who have failed sclerotherapy. To review the early experience with TIPSS at a teaching hospital. Twenty-eight patients underwent TIPSS on 30 occasions between September 1991 and June 1993 for bleeding oesophageal or gastric varices. The majority had alcoholic liver disease. TIPSS was performed successfully in all patients. Immediate control of bleeding was achieved, but one patient rebled within 24 hours. Complications related to the procedure occurred in 30%, but no patient died from these. Thirty-day mortality was 11% (three of 28), two patients dying from progressive liver failure and one from sepsis. A further three patients died from six weeks to two months following TIPSS, due to liver failure in one, spontaneous bacterial peritonitis in the second and in the third after a fall. This represents an overall mortality of 21%. Three patients have rebled at mean follow-up of 11.3 months. One of these had repeat TIPSS while the other two had balloon dilatation of the stent with control of bleeding. Four patients developed mild chronic encephalopathy which was readily controlled with medical therapy. TIPSS is an effective means for control of bleeding from oesophageal and/or gastric varices not responding to other methods. Further follow-up is required with regard to rates of rebleeding, encephalopathy and survival. |
8042938 | Control and dispositional style among the hearing-impaired in communication situations. | Forty hearing-impaired subjects were exposed to three stressful communication situations in a laboratory setting. The communication patterns used and the extent to which the subjects succeeded in coping with the situations were studied. Measures, such as the life orientation test measuring dispositional style, a hearing questionnaire measuring the ability to cope with different hearing situations, degree of control and pure-tone audiometry were performed. Coping behaviour and expressed emotion during communication situations were also observed. The results showed that controllability together with dispositional style and aspects of expressed emotion played an important role in explaining the overall success rates of hearing-impaired individuals. |
8042937 | Speech recognition and just-follow-conversation tasks for normal-hearing and hearing-impaired listeners with different maskers. | Speech recognition (SRT) and just-follow-conversation (JFC) tasks were performed by normal-hearing and hearing-impaired listeners matched for age and sex using three masking backgrounds: speech spectrum random noise, continuous forward speech and reversed speech. In the SRT task, the signal-to-noise (S/N) ratio for 50% correctly repeated words of short, 5-word, low-redundancy sentences was determined. In the JFC task, the listener adjusted the speech level until he felt he could just understand what was being said. The listeners needed a higher S/N ratio for the JFC task than for the SRT task. The speech maskers gave less masking than the random noise. No difference between forward and reversed speech was observed. The hearing-impaired listeners needed a higher S/N ratio than the normal-hearing subjects. Correlations between results obtained with the SRT and the JFC techniques were significant with speech but not with random noise as masker. The highest correlations between pure-tone hearing thresholds at different frequencies and results in the SRT and JFC tests were seen at 0.5 and 1 kHz. |
8042936 | Click-evoked oto-acoustic emissions in very-low-birth-weight infants: a cross-sectional data analysis. | For the purposes of studying the phenomenon of evoked oto-acoustic emissions (EOAEs) in very-low-birth-weight (VLBW) infants, and the conditions affecting the utility of EOAE ear screening in this population, click EOAEs were repeatedly recorded in ears of 144 VLBW infants, at different postconceptional ages of the infants and at two different test sites, i.e. in the neonatal high-care unit (ward), or at the neonatal outpatient clinic. The postconceptional age of the infants examined in the ward was 30-49 weeks and 37-66 weeks for the infants examined at the outpatient clinic. Overall 840 recording attempts were done. In the ward 86% of these attempts (388) were successful against 60% (of 452 attempts) at the outpatient clinic. In the latter group of infants the success rate of recording was only 33% at the corrected age of 6 months, which is significantly less than the 66% until the corrected age of 3 months. For a cross-sectional analysis of age effects one ear of each successfully recorded infant was selected. Analysis of the 127 successful recordings revealed that the EOAE prevalence was 71% in the ward (54% for infants receiving extra oxygen per naso) and 91% at the outpatient clinic. Compared with healthy newborns, VLBW infants are much more difficult to test, especially at the outpatient clinic. However, the EOAE prevalence at this test site is the highest and approaches that in healthy newborns. At the outpatient clinic response levels of EOAEs recorded approach levels found in healthy newborns. The higher success rate of recording in the ward and the lower EOAE prevalence are two counteracting factors as to the utility of EOAE-based ear screening of VLBW infants. |
8042935 | Effect of stimulus duration on stapedius reflex threshold in electrical stimulation via cochlear implant. | The effect of stimulus duration on the threshold of the contralateral stapedius reflex was investigated in patients supplied with a Vienna cochlear implant (analog stimulation via CI) and compared to results of a normal-hearing reference group in case of acoustic stimulation. Changes in reflex threshold were determined at four frequencies (125, 500, 1,000 and 2,000 Hz in case of electrostimulation and at 500, 1,000, 2,000 and 4,000 Hz for acoustic stimulation) and for five durations (30, 50, 100, 300 and 500 ms). The comparison of the two stimulation modes was accomplished by using the same instrumentation and procedure. Reflex threshold was evaluated according to an objective criterion based on the individual noise distribution of recordings without reflex and by subjective judgement. For both stimulation modes a strong effect of stimulus duration on reflex threshold was observed (p < 0.001). The amount of temporal integration reflected by the threshold difference between 500 and 50 ms was approximately 2-4 dB for electrical stimulation via CI and 6 dB for acoustic stimulation in normal-hearing individuals. In case of electrostimulation, the reflex threshold for stimuli of 30 ms was most often above the limit of uncomfortable loudness sensation; the increase in reflex threshold for acoustic stimulation between 500 and 30 ms was approximately 14 dB. There was no evidence of frequency effect on reflex threshold nor an interaction between frequency and stimulus duration for either stimulation mode. |
8042934 | Cochlear delays and traveling waves: comments on 'Experimental look at cochlear mechanics'. | In a recent publication [Audiology 1992;31:301-312], A. Dancer argued that direct and indirect measures of basilar membrane motion are more consistent with theories of cochlear resonance than with the traveling-wave theory. The present communication reviews empirical evidence that contradicts Dancer's argument. Such evidence--recordings of mechanical responses of the basilar and Reissner's membranes to sound--strongly supports the existence of displacement waves that propagate on the basilar membrane from the base of the cochlea toward its apex. |
8042933 | Lupus erythematosus and Miller-Fisher syndrome. | To compare the clinical course of an unusual case of Miller-Fisher syndrome in systemic lupus erythematosus with therapeutic interventions, in particular with plasma exchanges. The clinical state and laboratory and electrophysiologic parameters were controlled for over a year and related to therapeutic attempts with immunoglobulins, steroids, and plasma exchanges. Medical intensive care unit of a university hospital. A 17-year-old black female student with known systemic lupus erythematosus who developed ataxia, are flexia, and ophthalmoplegia (Miller-Fisher syndrome) and later became tetraplegic and required full mechanical ventilatory support. High-dose immunoglobulin treatment combined with corticosteroid pulse therapy was not beneficial. However, plasma exchange (performed five times over a period of 4 months) was followed by a striking clinical improvement within hours after each plasma exchange. Plasma exchange appears to remove a yet unknown agent producing a distal motor nerve conduction block and is efficacious in severe neuropathy associated with Miller-Fisher syndrome in lupus erythematosus. |
8042932 | Remote memory and lexical retrieval in a case of frontal Pick's disease. | To examine the status of remote memory and lexical retrieval processes in a patient with frontal lobe-type dementia studied serially during an 18-month period. Longitudinal single-case study. University hospital. A 67-year-old man presented to the hospital with progressive frontal lobe dysfunction confirmed by neuropsychological testing and single photon emission-computed tomographic scan. Postmortem brain examination demonstrated focal atrophy of the orbitofrontal region of the frontal lobe and medial temporal structures with abundant Pick's bodies and Pick's cells. Anterograde memory was assessed on a range of free recall and recognition tests using verbal and nonverbal material. Remote memory was assessed by Famous Faces and Famous Events tests and the Crovitz test of remote personal memory. Lexical retrieval was examined using initial-letter and semantic category-based fluency tasks. Initially there was a dissociation between anterograde memory, which was severely impaired, and relatively spared remote memory. Within retrograde memory there was evidence of selective difficulty in retrieving contextually rich and time-specific personal memories and in dating personal and public memories. In contrast, retrieval of names of famous persons and recognition of famous events was relatively normal. As the disease progressed, performance on all remote memory tests worsened, but without a temporally graded pattern. Lexical retrieval was markedly impaired and affected to an equivalent degree performance on initial-letter and category fluency tasks. These findings are interpreted in the context of a general retrieval deficit resulting from the interruption of frontostriatal circuits. |
8042931 | Teaching neurology residents in the outpatient setting. | To determine how residency programs are responding to the shift of neurological practice into the outpatient setting. A nine-item questionnaire was sent to the directors of all US neurology residency programs. Each item had two parts, the first describing the current program, and the second describing an "ideal" program designed to optimize resident education. The same questionnaire was also sent to all house officers and faculty associated with a single residency program to assess variability in perceptions. United States neurology residency programs (mail survey). Directors of neurology residency programs and all house officers and faculty members at a single residency program. Eighty-one (70%) of the 116 questionnaires distributed were returned. There were four areas of general consensus among the program directors: (1) more time should be devoted to outpatient care during residency training; (2) more continuity at the resident level should be provided for patients seen in subspecialty clinics; (3) faculty should provide more supervision of residents when they see follow-up patients; and (4) conferences specifically directed at outpatient management issues should be developed. Neurology residency directors agree that current approaches to teaching in the outpatient setting fall short of an educationally ideal system. Four areas of perceived deficiency have been identified. Creative solutions will be necessary to correct these perceived deficiencies. |
8042930 | Astasia without abasia due to peripheral neuropathy. | To describe an unusual symptom characterized by an inability to stand still despite the ability to walk in eight patients with paraparesis due to peripheral neuropathy. Case series during the past 18 years. Referral center. Six patients with acute or subacute polyneuropathies recovering from flaccid paralysis of the lower limbs and two patients with chronic progressive polyneuropathy for more than 10 years were studied. Weakness around the ankle joints was profound, while muscle strength around the hip joints was well recovered or preserved. Standing and walking were recorded and reviewed on videotape or motion pictures. Spectral content of postural sway was analyzed in three recent cases. The symptom was transient in acute or subacute cases and was continual in chronic cases. The patients were compelled to take a series of steps forward and backward while standing until they initiated locomotion. They swayed rapidly around the hip joints before stepping. The anteroposterior component of postural sway in three patients had frequency peaks around 1 Hz. We have termed this symptom astasia without abasia, or stilts phenomenon, in which maintenance of the body mass depends on a moving base of support. Both an abnormal pattern of postural movements and defective somatosensory feedback for postural stabilization may be responsible for the symptom. |
8042929 | Stimulus timing effects on Wada memory testing. | To determine the effects of presenting Wada memory stimuli at different times after intracarotid amobarbital injection on Wada memory asymmetries. Wada memory asymmetries from three timing series were related to the laterality of eventual temporal lobectomy. Academic institution epilepsy surgery program. Forty-three patients with complex partial seizures who later underwent anterior temporal lobectomy (left temporal lobectomy, 24 patients; right temporal lobectomy, 19 patients). No patient included had abnormalities on magnetic resonance imaging scans to suggest a lesion other than gliosis. Memory performance for objects whose presentation began approximately 45 seconds after amobarbital administration differentiated laterality of seizure onset. Memory for items presented later and after partial return of language (on average 3 minutes 40 seconds postinjection) also differed as a function of ipsilateral vs contralateral injection, but at a lower level of statistical significance. Memory for items presented last during the procedure (on average 6 minutes postinjection) discriminated seizure groups at a still lower level of statistical significance. When used to predict lateralized temporal lobe impairment in individual patients, early object memory performance was significantly better than memory performance employing either middle (56%) or late (43%) stimulus presentation timings. The results of early object memory testing are superior to those obtained from stimulus presentation later in the procedure in documenting temporal lobe dysfunction associated with a lateralized seizure onset. |
8042928 | beta-Amyloid protein immunoreactivity in skin is not a reliable marker of Alzheimer's disease. An autopsy-controlled study. | As a possible diagnostic marker for Alzheimer's disease (AD), we investigated beta-amyloid protein (beta/A4) immunoreactivity in skin. Furthermore, we studied the presence of beta-amyloid precursor protein 695 immunoreactivity in skin. Lifetime skin biopsy specimens were stained for beta/A4 and beta-amyloid precursor protein 695. The follow-up period was 12 months. We determined the correlation between beta/A4 immunoreactivity in skin and brain in patients with a neuropathologic diagnosis. All patients with dementia were hospitalized; most of them had moderate to severe dementia. Aged nondemented controls were residents of a nursing home. The Down's syndrome (DS) group included both hospitalized and ambulatory patients. Young nondemented controls were medical students or staff members who volunteered for the study. The study included a total of 111 subjects. Thirty-five patients had probable AD, nine had possible AD, 15 had multi-infarct dementia, one had idiopathic Parkinson's disease, and one had Parkinson's disease and possible AD. There were also 19 elderly nondemented controls, 23 patients with DS, and eight young nondemented controls. Immunohistochemical detection of beta/A4 in skin and correlation to the diagnosis of AD. Immunopositivity for beta/A4 antibody was present in and around the endothelium of dermal blood vessels in a proportion of patients with AD and multi-infarct dementia as well as elderly controls. The patients with sporadic AD displayed beta/A4 immunoreactivity significantly more frequently than did patients with familial AD, patients with multi-infarct dementia, and controls. The beta/A4 immunopositivity in skin was rare in the patients with DS and not present in young controls. Instead, 48% of patients with DS but none of other groups had beta-amyloid precursor protein 695 immunoreactivity in skin. Only four (31%) of 13 patients with neuropathologically confirmed AD had shown endothelial beta/A4 immunopositivity in skin biopsy specimens while alive. Our results do not support beta/A4 as a diagnostic marker for AD. |
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