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8039712
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Transcriptional regulation of class-I major histocompatibility complex genes transformed in murine cells is mediated by positive and negative regulatory elements.
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The expression of class-I major histocompatibility complex (MHC) antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low or absent; a phenotype that correlates with the high tumorigenicity of these cell lines. In primary mouse embryonal fibroblasts (MEF) from class-I transgenic mice (PD1 transgenic mice), Ad12-mediated transformation results in down-regulation of both endogenous genes and the transgene. Functional analysis of class-I regulatory elements revealed that the suppression of a class-I promoter is mediated by two negative regulatory elements, one of which functions specifically in Ad12-transformed cells. In addition, Ad12-transformed cells produce only minute amounts of the nuclear factors that bind to the major class-I enhancer, RI (region I or H2TF1). A silencer element derived from the 5' region of the miniature swine class-I gene (PD1) is capable of competing for the binding of nuclear factors to a second enhancer, RII (region II or CREII), that is located upstream from RI in the class-I regulatory element (CRE). Based on these results, we propose that down-regulation of class-I genes in Ad12-transformed cells is mediated mainly by negative regulators.
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8039711
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Structure and chromosomal location of the gene encoding mouse corticosteroid-binding globulin: strain differences in coding sequence and steroid-binding activity.
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Corticosteroid-binding globulin (CBG) is a member of the serine proteinase inhibitor superfamily and is responsible for the plasma transport of glucocorticoids. The mouse Cbg gene structure has been deduced from two non-overlapping DNA fragments of a lambda EMBL-3 genomic library, as well as PCR amplification of the approx. 2 kb of genomic DNA that lies between them. Mouse Cbg comprises five exons that span a region of approx. 10.5 kb, and has been localized in tight linkage with the Aat (alpha 1-antitrypsin) and Spi (serine proteinase inhibitor) gene complex on chromosome 12, in a region syntenic with this genetic locus on human chromosome 14. Intron-specific oligodeoxyribonucleotide primers were also used to PCR-amplify Cbg coding regions from several mouse strains. No differences were found in the Cbg coding sequences of BALB/c and C57BL/6J-cpk/cpk mice, while two mutations were found within RIIIS/J Cbg that result in Lys201-->Glu and Ala357-->Thr substitutions in the mature mouse CBG polypeptide. To assess what impact these substitutions might have on the steroid-binding activity of RIIIS/J CBG, these mutations were introduced separately or together into a BALB/c mouse Cbg cDNA. Expression of these mutants in the MDCK cell line indicated that the Lys201-->Glu substitution accounts for the abnormal steroid-binding affinity of CBG in RIIIS/J mice.
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8039710
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Cloning and sequence analysis of a cDNA coding for the murine DNA ligase I enzyme.
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A complementary DNA (2961 bp) containing the complete coding sequence for murine DNA ligase I was isolated from a mouse fibroblast cDNA library using a cDNA encoding the human protein as a probe. An open reading frame of 2748 bp, encoding a protein of 916 amino acids (aa), was identified. Northern blot analysis of total RNA extracted from mouse fibroblasts showed a single band with a mobility corresponding to a size of 3.2 kb whose level increases upon serum stimulation of quiescent mouse NIH-3T3 cells. Alignment of the murine and human deduced aa sequences showed an overall 83% identity, that rises to 91% if only the sequence on the C-terminal portion of the protein containing the active site is considered.
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8039709
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A sensitive promoter assay based on the transcriptional activator Tat of the HIV-1 virus.
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We developed a sensitive vector system for the analysis of weak promoter activities. This promoter assay is based on the transcriptional activator protein, Tat, of human immunodeficiency virus type 1 (HIV-1). High-level expression of HIV requires activation in trans by Tat of the promoter in the long terminal repeat (LTR). Here we describe the construction of a promoterless pTat vector. Foreign promoter elements can be inserted upstream from the tat gene, and expression of Tat protein is measured in trans on a co-transfected LTR-CAT reporter plasmid. We show that this binary system is more sensitive than standard pCAT reporter assays.
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8039707
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Utilization of multiple polyadenylation signals in the human RHOA protooncogene.
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Little is known regarding the regulation of expression of the RHOA protooncogene, a member of the family of genes encoding Ras-related GTP-binding proteins. We have previously reported that the 3' untranslated region (UTR) of RHOA was contained within a genomic sequence which flanked the 5' end of the human glutathione peroxidase 1-encoding gene [J.A. Moscow et al., J. Biol. Chem. 267 (1992) 5949-5958]. Our previous studies revealed the presence of multiple (1.8 and 1.5 kb) RHOA mRNA species in breast cancer cell lines and of three putative polyadenylation signals in the RHOA 3' UTR. In this report, we have isolated several RHOA cDNAs from a multidrug-resistant MCF-7 human breast cancer cell line. Sequence analyses of these RHOA cDNA clones indicate that multiple polyadenylation signals are used to terminate RHOA transcripts. RNase-protection analysis demonstrated that all three polyadenylation signals are utilized in breast cancer cell lines and RNA stability studies demonstrated that RHOA RNA species with different 3' ends have equivalent stability. Since little is known about the RNA expression of RHOA in human tumors, and since both activated and non-activated RHOA genes possess transformation potential, we analyzed RHOA mRNA in lung and colon tumors by Northern blot and RNase-protection analyses. In all eight lung tumors examined, RHOA RNA levels were decreased relative to the level in normal surrounding tissue, whereas RHOA expression was decreased in only two of six colon tumors. We also found that lovastatin-induced cell cycle arrest resulted in increased RHOA RNA expression in breast cancer cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)
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8039708
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Insertion of the human cytomegalovirus enhancer into a myeloproliferative sarcoma virus long terminal repeat creates a high-expression retroviral vector.
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A new retroviral vector is characterized in which the enhancer in the long terminal repeat of myeloproliferative sarcoma virus (MPSV) is combined with the strong and universally active immediate-early human cytomegalovirus (hCMV) enhancer. We demonstrate that insertion of the hCMV enhancer increases the amount of vector-specific mRNA in various rodent cell lines and in human diploid fibroblasts, by at least 3-5-fold. The vector may be particularly useful if high expression of two genes is desired.
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8039706
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Cloning of a gene encoding a human leukocyte protein characterised by extensive heptad repeats.
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Complementary DNA (cDNA) clones, encoding a fusion protein that was recognised by an antiserum raised against a purified polypeptide fragment of a 180-kDa human leukocyte protein, were isolated from a lambda gt11 expressed library. The clones encoded a unique amino acid (aa) sequence interspersed with heptad repeats that typify coiled-coil proteins, and hybridised to a 5-kb transcript universally expressed in a panel of eight human tissues. Comparatively high levels of RNA expression were seen in testis, ovary and mitogen-activated peripheral blood leukocytes (PBLs). The deduced 1300-aa sequence reveals a protein with a typical signal peptide, a hydrophilic domain containing an N-terminal globular head with a nuclear localization signal sequence, a C-terminal region of coiled-coil structure, a candidate transmembrane domain, and a short 10-aa C-terminal domain. Rabbit polyclonal antisera raised against a truncated lambda gt11 fusion protein recognized a 150-170-kDa protein (non-reduced) in mitogen-activated PBLs. The protein designated here as CG-1 may exist as a homodimer destined for translocation to the nucleus, with a role in leukocyte differentiation and/or effector function.
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8039705
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Characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A using a baculovirus vector.
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Fabry disease is an X-linked inborn error of glycolipid metabolism caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (GalA; EC 3.2.1.22). In order to obtain large quantities of this human enzyme for physical characterization and for the development of new approaches for enzyme therapy, we constructed derivatives of the Autographa californica nuclear polyhedrosis virus that produce the human enzyme. The recombinant GalA (re-GalA) is produced at high levels, and is active with both the artificial substrate, 4-methylumbelliferyl-alpha-D-galactopyranoside, and the natural in vivo substrate, trihexosylceramide. The purified re-GalA is glycosylated and is taken up by normal and Fabry fibroblasts in cell culture. Mass spectral analysis of total monosaccharides released by hydrazinolysis indicates that it contains fucose, galactose, mannose and N-acetylglucosamine. Amino-acid sequence analysis of six proteolytic peptides corresponded to sequences predicted by the cDNA. The molecular masses of the purified enzyme, estimated by electrospray mass spectroscopy and laser desorption time-of-flight analysis are 46.85 and 46.62 kDa, respectively, approx. 10% greater than the polypeptide portion predicted by the cDNA. The recombinant enzyme retains significant catalytic activity after modification with poly(ethylene glycol), a treatment which decreases the immunogenicity and increases the circulation life of many proteins used therapeutically.
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8039704
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The tripartite DNA element responsible for diet-induced rat fatty acid synthase (FAS) regulation.
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We investigated which region of the 5'-flanking sequence of the rat fatty acid synthase (FAS)-encoding gene could be responsible for its nutritionally regulated expression. Diet-induced differences in chromatin structure were determined by DNase I treatment of intact nuclei from hepatic tissue. A low-fat diet results in a different pattern of DNase I-hypersensitive sites (HS) in the chromatin of the FAS promoter (pFAS) from that seen when the nuclear extract was prepared from the livers of normally fed rats. The protein-binding properties of the region defined by DNase I hypersensitivity were tested by gel retardation. A putative cis-acting element with a tripartite structure, 5'-GCCT, 6-bp spacer and a 3'-palindrome, could be localized between bp -518 to -495 in pFAS. Competition experiments with oligodeoxyribonucleotides (oligos) representing subfragments of this cis-element showed that the requirement for structure is stricter than that for sequence. This element could be one of the termini of the insulin-induced signal cascade.
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8039703
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The mouse hepatocyte growth factor-encoding gene: structural organization and evolutionary conservation.
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A mouse genomic phage library was screened by using a cDNA probe coding for mouse hepatocyte growth factor (HGF). Five overlapping genomic clones which contained the entire mouse HGF gene were isolated and characterized by restriction mapping, Southern hybridization and DNA sequencing. HGF spans about 65 kb and consists of 18 exons separated by 17 introns, similar to its human counterpart. The nucleotide (nt) sequences of the introns at the exon-intron junctions are GT-AG, analogous to those found in other eukaryotic genes. The exon-intron gene organization of HGF is highly homologous to that of several other genes encoding kringle-containing proteins, especially HGF-like protein and plasminogen. This result suggests that HGF probably evolved through gene duplication and/or exon shuffling events from an ancestral gene. Southern hybridization of genomic DNA from different species revealed that a high degree of homology exists among a variety of vertebrates, including chicken, when a mouse HGF cDNA was used as a probe. This evolutionary conservation of HGF strongly suggests that the protein may play an important role in normal cell physiology. Our current results on mouse HGF structure provide basic and detailed information to carry out further manipulation, such as gene targeting.
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8039702
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Alpha-crystallins are involved in specific interactions with the murine gamma D/E/F-crystallin-encoding gene.
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The promoter of the murine gamma E-crystallin (gamma E-Cry) encoding gene (gamma E-cry) was analyzed for specific interactions with lenticular proteins in a gel-retardation assay. A 21-bp fragment immediately downstream of the transcription initiation site (DOTIS) is demonstrated to be responsible for specific interactions with lens extracts. The DOTIS-binding protein(s) accept only the sense DNA strand as target; anti-sense or double-stranded DNA do not interact with these proteins. The DOTIS sequence element is highly conserved among the murine gamma D-, gamma E- and gamma F-cry and is present at comparable positions in the orthologous rat genes. Only a weak or even no protein-binding activity is observed if a few particular bases are changed, as in the rat gamma A-, gamma C- and gamma E-cry elements. DOTIS-binding proteins were found in commercially available bovine alpha-Cry preparations. The essential participation of alpha-Cry in the DNA-binding protein complex was confirmed using alpha-Cry-specific monoclonal antibody. The results reported here point to a novel function of alpha-Cry besides the structural properties in the lens.
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8039701
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Identification of negative-acting and protein-binding elements in the mouse alpha A-crystallin -1556/-1165 region.
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The mouse alpha A-crystallin-encoding gene (alpha A-cry) is expressed in a highly lens-preferred manner. To date, it has been shown that this lens-preferred expression is controlled by four proximal positive-acting transcriptional regulatory elements: DE1 (-111/-97), alpha A-CRYBP1 (-66/-57), PE1/TATA (-35/-19) and PE2 (+24/+43). The present study extends our knowledge of mouse alpha A-cry transcriptional regulatory elements to the far upstream region of that gene by demonstrating that the -1556 to -1165 region contains negative-acting sequence elements which function in transfected lens cells derived from mouse, rabbit and chicken. This is the first negative-acting regulatory region identified in mouse alpha A-cry. The -1556 to -1165 region contains sequences similar to repressor/silencer elements identified in other genes, including those highly expressed in the lens, such as the delta 1-crystallin (delta 1-cry) and vimentin (vim) genes. The -1480 to -1401 region specifically interacts with nuclear proteins isolated from the alpha TN4-1 mouse lens cell line. Contained within this protein-binding region and positioned at -1453 to -1444 is a sequence (RS1) similar to the chicken delta 1-cry intron 3 repressor, and which competes for the formation of -1480 to -1401 DNA-protein complexes. Our findings suggest that lens nuclear proteins bind to the mouse alpha A-cry RS1 region. We demonstrate that the chicken delta 1-cry intron repressor binds similar nuclear proteins in chicken embryonic lens cells and mouse alpha TN4-1 lens cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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8039700
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A gene-sized DNA molecule encoding the catalytic subunit of DNA polymerase alpha in the macronucleus of Oxytricha nova.
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We have isolated a gene-sized molecule encoding the catalytic subunit of DNA polymerase alpha from a macronuclear genomic library of Oxytricha nova, by using a 0.7-kb fragment of the corresponding human gene as a hybridization probe. Two different versions of the gene are present in the macronucleus, one with an EcoRI site (RI+) and one without an EcoRI site (RI-). The cloned RI- version has been characterized. It is 4938 bp in length, excluding telomeres. It consists of a 329-bp 5' leader, a 4479-bp coding region and a 130-bp 3' trailer. The deduced amino-acid sequence shares conserved regions with the yeast and human polypeptides. We also demonstrate by Southern analysis that gene-sized molecules of similar size, homologous to the isolated O. nova gene are present in the mac genome of closely and distantly related hypotrichs.
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8039698
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Assessing residents' performance in C-L psychiatry. Work in progress.
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The scores from the Psychiatry Resident In Training Examination (PRITE) provide useful information concerning residents' acquired knowledge about consultation-liaison (C-L) psychiatry. However, a comparable method for assessment of C-L residents' clinical skills has not been developed. A task force was commissioned by the Consultation-Liaison Psychiatry Section of the Association for Academic Psychiatry to develop such an assessment system. This paper presents the work of that task force and includes a methodology for assessing clinical performance, a prototype evaluation form, and instructions for its use.
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8039697
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Psychiatric comorbidity among patients with hypochondriasis.
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The purpose of this study was to determine the nature and extent of comorbidity among patients with DSM-III-R hypochondriasis and to examine the relationships between this disorder and coexisting psychiatric illness. For this purpose, patients seen in a general medicine clinic were screened using measures of hypochondriacal attitudes and somatic symptoms. Those scoring above an established cutoff were given a structured diagnostic interview. In this manner, 50 patients who met DSM-III-R criteria for hypochondriasis and 50 age- and sex-matched controls were identified. The presence of other psychiatric disorders (current and past) was determined by means of the same diagnostic interview. More hypochondriacal subjects (62.0%) had lifetime comorbidity than did controls (30.0%). Major depression, the most frequent comorbid disturbance, was usually current and most often had an onset after that of hypochondriasis. Panic disorder with agoraphobia, the most frequent anxiety disorder, was also current but often began before or at the same time as hypochondriasis. Few subjects met criteria for somatization disorder but a third qualified for a subsyndromal form of this disorder. The data show that, in medical outpatients with hypochondriasis, mood and anxiety disorders frequently coexist. This comorbidity is subject to varying interpretations including overlap of symptom criteria, treatment-seeking bias, and the possibility that hypochondriasis predisposes to or causes the comorbid disorder, as seems likely in the case of depression. In some instances hypochondriasis may be an associated feature of another illness.
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8039696
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The Substance Abuse Consultation Team. Addressing the problem of hospitalized substance abusers.
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The abuse of alcohol and other drugs represents a significant health care problem that has largely gone unrecognized and undiagnosed by physicians. A Substance Abuse Consultation (SAC) Team was initiated at a tertiary-care teaching hospital to provide expert diagnostic, interventional, and management services for hospitalized substance abusers. This preliminary study represents the first 100 consults received and reviewed. All 100 patients agreed to an interview and evaluation by the SAC Team. It was decided that 91 patients needed alcohol and other drug rehabilitation; 69% of these acknowledged having a substance abuse problem and accepted referral for treatment. The results of this report suggest the usefulness of a SAC Team for identification, evaluation, and intervention with hospitalized substance abusers. This program is easily replicable by other general hospitals and may provide a cost-effective, multidisciplinary approach to the abuse of alcohol and other drugs.
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8039694
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Lilliputian hallucinations and medical illness.
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Lilliputian hallucinations have been described in patients with delirium, schizophrenia, seizure disorders, visual disturbances, and brain tumors. The authors report two cases of patients with lilliputian hallucinations, one with AIDS-dementia complex and the other with dementia following head trauma. This is the first time that lilliputian hallucinations have been described in association with such medical conditions.
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8039693
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Hepatic encephalopathy presenting as delirium and mania. The possible role of bilirubin.
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The case of a woman who suffered from hepatic encephalopathy is described. The psychiatric symptoms presented first as a delirium, changing into a manic syndrome. As the neurotoxic direct fraction of bilirubin was greater than 100 times normal, increasing and decreasing in a parallel fashion with the psychiatric symptoms, direct bilirubin is assumed to play a role in the pathogenesis of this case. This is discussed in the context of bilirubin encephalopathy, very seldom diagnosed in adults.
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8039692
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Exacerbation of posttraumatic stress disorder symptoms with medical illness.
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Chronic posttraumatic stress disorder (PTSD) may increase the risk for associated psychiatric and medical illnesses. In turn, the onset of medical illness may result in an exacerbation of PTSD symptoms leading to excessive or maladaptive psychological and physiological reactions. Five combat veterans with PTSD and medical disease are presented to illustrate this potential for worsening of PTSD with concurrent medical illness. Health care workers in general hospital settings should be aware of unique psychological vulnerabilities in PTSD patients. Prospective studies are needed to assess the impact of medical comorbidity on the course of PTSD.
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8039691
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Psychiatric liaison on a bone marrow transplantation unit.
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Patients undergoing bone marrow transplantation experience some unique coping problems which differentiate them from other cancer patients. Problems structuring time are particularly difficult for patients while waiting in isolation for the success or failure of their transplant. The authors discuss the establishment of a psychiatric liaison to a bone marrow transplantation unit. As a result of the new liaison with this unit, psychiatric consultations to all cancer patients tripled within 1 year.
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8039690
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Alcohol abuse among general hospital inpatients according to the Munich Alcoholism Test (MALT).
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The aim of this study was to assess the prevalence of alcohol abuse in a general hospital inpatient population. The study population consisted of 1138 consecutive admissions to three surgical and two medical wards of the University Hospital Rotterdam during a 6-month period. According to the Munich Alcoholism Test (MALT), 7.7% of the patients were identified as having an alcohol problem, but only 0.6% had alcohol abuse recorded as a discharge diagnosis by the Central Medical Registration Unit. In comparison to nonalcoholic patients, alcoholics more often were male and less than 65 years old. They more frequently reported that they lived alone, had prematurely left school, and were unemployed. With regard to medical characteristics, alcoholic patients more often suffered from neurological diseases and intoxication. Compared with nonalcoholics they more frequently had a psychiatric history, and drug abuse and smoking were more prevalent in this group. The alcoholic and nonalcoholic patients did not differ with regard to use of psychotropic medication, although the former more often used vitamins. Based on the above-mentioned discriminating features, the percentages of correctly classified alcoholic patients and nonalcoholic patients were 74% and 73%, respectively, with an overall percentage of 73%.
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8039689
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Competency evaluations in a VA hospital. A 10-year perspective.
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The authors reviewed all 228 competency evaluations performed at the Baltimore VA Medical Center during a 10-year period. Between 1980 and 1984 and 1985 and 1989, the rate of inpatients who had competency evaluations increased from 0.20% to 0.42%, and the average number of competency evaluations per year doubled from 12 to 24.6. The percentage of all psychiatric consultations which were for competency evaluation nearly doubled from 5% to 9.4%. The rate of competency evaluations was slightly but not significantly higher for patients over age 65 than the rate for younger patients (0.44% vs. 0.33%). However, older patients were significantly more likely to be judged incompetent.
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8039688
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Workplace mental health consultation. A primer of organizational and occupational psychiatry.
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Though there are only a few hundred psychiatrists who identify themselves as specialists in organizational and occupational issues, the need for psychiatric skills in the workplace has increased dramatically in recent years. Crucial issues include distressed employees, the effects of organizational structure and change, job stress, psychiatric disability, substance abuse, and violence in the workplace. It is important for general psychiatrists to become more conversant in work and workplace-related mental health issues. Use of an occupational history with all patients, and knowledge of organizational structures and function, are just two ways to become more aware of these matters. With business and government increasingly attentive to mental health benefits and systems, appropriate psychiatric focus on organizational and occupational concerns becomes ever more important.
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8039685
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Case of a 29-year-old nurse with factitious disorder. The utility of psychiatric intervention on a general medical floor.
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A 29-year-old nurse was treated for septic arthritis of the knee. She was suspected of producing this infection and others. The consultation-liaison team intervened early, and the patient was treated in individual outpatient psychotherapy for 1 year. Her course was complicated by criminal behavior and a suicide attempt, prompting relocation to her home town. The patient demonstrated a strong need to be nurtured, as noted in other cases of factitious disorders. An overview of factitious disorders is presented, and guidelines for managing these difficult patients in the hospital are provided.
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8039684
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HIV-dementia and suicide.
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Suicide is a dangerous concomitant of HIV-dementia. Impulsivity, impaired judgment, lability, and disinhibition are associated with dementia. The authors describe two suicidal individuals with HIV-dementia.
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8039683
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Chlorpromazine prophylaxis of steroid-induced psychosis.
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We report a case of successful prophylactic treatment for psychiatric side effects of megadose glucocorticoid therapy by the administration of chlorpromazine. We review currently used prophylactic treatment strategies and discuss the rationale for adding neuroleptic treatment as an alternative to these strategies.
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8039682
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Quality of antidepressant medications prescribed at discharge to depressed elderly patients in general medical hospitals before and after prospective payment system.
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This study describes the quality of antidepressant medication use at hospital discharge for depressed elderly inpatients and compares quality of care before and after implementation of Medicare's Prospective Payment System (PPS). The study reviewed data from medical records of 2746 depressed, elderly, hospitalized patients in acute-care general medical hospitals in five U.S. states (pre-PPS period 1981-82; post-PPS period 1985-86). The majority were discharged on antidepressant medication both pre-PPS and post-PPS. After PPS' implementation, sedating medications were used less often in all treatment settings. In general medical wards, a higher percentage post-PPS (24%) than pre-PPS (14%) were discharged 48 hours or less after first starting an antidepressant medication. In both time periods, one-third of patients receiving antidepressant medications were prescribed daily dosages at discharge below recommended, minimum, therapeutic levels, whether treated in general medical wards or psychiatric units. Otherwise, patients previously treated in psychiatric units received higher quality of medication management than those treated in general medical wards. Over time, patients discharged on antidepressant medication were less likely to use sedating medication, suggesting improved quality of care. In general medical wards, however, patients were discharged more rapidly after starting medication, possibly suggesting lower quality of care. A substantial percentage of patients received subtherapeutic dosages of medication or sedating medications, suggesting that improved management of discharge antidepressant medication in the elderly is needed in general medical hospitals.
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8039681
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Family planning needs of male chronic mental patients in the general hospital psychiatry clinic.
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We investigated the parenting history, contraceptive behavior, and contraceptive needs of 35 male chronic mental patients seen at an urban outpatient psychiatric clinic. Most patients had fathered children, but 60% of the children less than 16 years of age were not being reared by their biological father. Forty-one percent of the patients who had sexual intercourse during the preceding year and had not wanted children reported that they or their sexual partner had not used contraception at the time of last intercourse. Patients were reportedly at significant risk of fathering unwanted children and sexually transmitted diseases. Strategies for preventing unwanted pregnancies within this population are discussed.
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8039680
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Medical psychiatry units and the roles of the inpatient psychiatric service in the general hospital.
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The medical-psychiatric unit concept has been the most important influence in the development of general hospital inpatient psychiatry in the last decade. This concept and style of unit organization is reviewed in the context of the history of general hospital inpatient psychiatry. It is suggested that elements of the medical-psychiatry unit model will become more important in the organization of general hospital psychiatric units. Modifications of the medical psychiatry unit concept are suggested to take into account the heterogeneity of many general hospital inpatient services. Implications of these factors are discussed in the context of unit design, models of staff organization, and the formation of networks of care.
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8039679
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The impact of psychiatric comorbidity on Medicare reimbursement for inpatient medical care.
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Funding for psychiatric consultation-liaison (C-L) services has been a difficult problem. It has been suggested that the identification of psychiatric co-morbidities in Medicare patients on medical services could generate incremental hospital revenue by moving patients from a lower to a higher paying Diagnostic Related Group (DRG). This increased revenue could be used as a means of supporting the psychiatric C-L service. This study documents the financial impact of screening for and documenting psychiatric co-morbidities on a general acute medical service. We clinically assessed 100 consecutive Medicare admissions and found 25 psychiatric co-morbidities in 20 patients. In only one case did the psychiatric diagnosis result in moving the case to a higher DRG. However, the need for psychiatric consultation remains evident as there was significant lack of recognition and documentation of the psychiatric diagnoses by the medical team. The authors discuss both the financial and clinical implications of screening medical inpatients for psychiatric co-morbidities and propose directions for further studies in this area.
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8039677
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Irreversible formation of pseudohyphae by haploid Saccharomyces cerevisiae.
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Culturing haploid strains of Saccharomyces cerevisiae in liquid minimal medium with 2% ethanol and 2% leucine resulted in the formation of long anucleate pseudohyphae. This occurred only with the combination of ethanol as carbon source and leucine as nitrogen source and was independent of mating type. The transition to a pseudohyphal form observed under these conditions appears to be irreversible. These findings further extend our view of the developmental alternatives in this important model eukaryote.
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8039676
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A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12.
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Five different c-type cytochromes have been detected during anaerobic growth of various Escherichia coli strains in different media. None of these cytochromes was detectable in aerobically-grown cultures. Only a single, 43 kDa cytochrome was synthesized in response to the presence of trimethylamine-N-oxide: synthesis of this cytochrome was unaffected by the presence of nitrate or nitrite, was repressed by oxygen, but was dependent upon a functional tor operon located at minute 22 (coordinate 1070 kb) on the E. coli chromosome. The other four cytochromes, masses 16, 18, 24 and 50 kDa, were induced by nitrite coordinately with formate-dependent nitrite reductase activity, but repressed by oxygen and nitrate. As only the 18 kDa and 50 kDa cytochromes are encoded by the nrf operon located at minute 92 (coordinate 4366 kb), there must be other loci, possibly essential for formate-dependent nitrite reduction, encoding the 16 kDa and 24 kDa cytochromes. No other c-type cytochrome was detected under any growth condition tested.
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8039675
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Isolation of glucosinolate degrading microorganisms and their potential for reducing the glucosinolate content of rapemeal.
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A range of microorganisms were assessed for their ability to degrade glucosinolates, using sinigrin (2-propenyl glucosinolate) as a model compound. Eight different species capable of growing on sinigrin as a sole carbon source were isolated. These were predominantly Gram-positive bacteria which also degraded the natural glucosinolates within rapemeal. Growth of the majority of these organisms in a sinigrin/glucose liquid medium wass biphasic; glucose was utilised during the initial rapid phase of growth. The ability to degrade sinigrin was found to be unstable and was rapidly and irreversibly lost when organisms were cultured on sinigrin-free media.
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8039674
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Energy-dependent receptor activities of Escherichia coli K-12: mutated TonB proteins alter FhuA receptor activities to phages T5, T1, phi 80 and to colicin M.
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The activity of the FhuA receptor in the outer membrane of Escherichia coli is dependent on the TonB, ExbB and ExbD proteins which are anchored to the cytoplasmic membrane. Only infection by phage T5 occurs independently of TonB, ExbB and ExbD. In this paper we describe mutated FhuA proteins which displayed either an increased or decreased FhuA activity to phage T5 when combined with mutated TonB proteins. These results suggest conformational changes in FhuA by TonB which are recognized by phage T5. Similar results were obtained with colicin M and the phages T1 and phi 80. It is proposed that the FhuA mutant proteins assume conformations which are either improved or impaired by the TonB derivatives. For the direct interaction of FhuA with TonB regions which are located outside the TonB box of FhuA and the region around residue 160 of TonB are important.
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8039673
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Selective release of the periplasmic enzyme beta-lactamase from Escherichia coli with tetradecyl betainate.
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The periplasmic enzyme beta-lactamase was selectively released from Escherichia coli K12 by the amphiphilic quaternary ammonium compound tetradecyl betainate at certain concentration intervals. At low concentrations little enzyme was released, and at high concentrations enzyme inactivation occurred. Greater effects of tetradecyl betainate were seen both with respect to release and inactivation at higher pH. At intermediate concentrations of tetradecyl betainate high yields of beta-lactamase were obtained with no detectable contribution of the cytoplasmic marker beta-galactosidase. The highest yields of beta-lactamase activity were obtained when high concentrations of salt were added 1 min after permeation of the bacteria with tetradecyl betainate.
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8039672
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Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake.
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The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens, or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.
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8039671
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Biochemical and genetic heterogeneity of staphylococcal protein A.
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We investigated the biochemical and genetic heterogeneity of protein A from Staphylococcus aureus. SpA genes (spas) of various strains were heterogeneous when detected as DraI and EcoRV fragments of chromosomal DNA. Polymerase chain reaction using primers to detect DNA encoding the IgG-binding domains in spa revealed that they numbered between 2 and 5. Protein A from several S. aureus strains showed two types of reactivities to immunoglobulins in normal canine serum according to the number of active domains.
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8039670
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An improved method for detecting cytostatic toxin (emetic toxin) of Bacillus cereus and its application to food samples.
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We developed an improved HEp-2 cell assay method for the detection of Bacillus cereus toxin, which affects the proliferation of HEp-2 cells. The cytostatic toxin was stable upon exposure to heat, pH 2, pH 11 and trypsin, which suggests it is an emetic. Using the HEp-2 cell assay, we examined the distribution and contamination of B. cereus strains that produced an emetic toxin in various foods. Although there were 228 enterotoxin producers among 310 B. cereus strains obtained from foods, 16 of them produced the cytostatic type (emetic toxin). All of the strains that produced the cytostatic toxin were of the H.1 serotype.
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8039669
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Electrotransformation of Streptococcus agalactiae with plasmid DNA.
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A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli-Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-microliters aliquot containing about 5 x 10(9) colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 micrograms. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2 x 10(4) cfu micrograms-1. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae.
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8039668
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Nucleotide sequence and distribution of the pepPN gene from Lactobacillus helveticus CNRZ32.
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The Lactobacillus helveticus CNRZ32 gene encoding a di-/tri- pepidase with prolinase activity (pepPN) was sequenced. An open reading frame of 912 base pairs was identified corresponding to a peptide with a molecular mass of 35.04 kDa. Southern hybridization indicated that the gene sequence is well conserved in strains of lactobacilli and pediococci.
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8039667
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The nusG gene of Streptomyces griseus: cloning of the gene and analysis of the A-factor binding properties of the gene product.
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The nusG gene of Streptomyces griseus was cloned and the nucleotide sequence determined. It encodes a protein with an identity of 76% to the reported receptor (VbrA) for VB-C, an autoregulatory factor in Streptomyces virginae. NusG protein was expressed in Escherichia coli. However, no binding activity for A-factor, an butyrolactone autoregulator in S. griseus very similar to VB-C, could be detected. The nusG gene of S. griseus does not seem to encode the A-factor-binding protein.
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8039666
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The membrane destabilising action of the antibacterial agent chlorhexidine.
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The antibacterial agent chlorhexidine has long been used as an agent for medical antisepsis. This compound is a membrane active agent which probably has its major antibacterial action by interference with the function of cellular membranes. The results demonstrated an inhibition of oxygen utilisation by bacteria which was related to falls in cellular ATP levels. There was an effect on the outer membranes of Gram-negative bacteria which allowed the release of periplasmic enzymes. The inner membrane was not ruptured but its functionality was breached and there was an inhibition of active uptake of small molecules which did not appear to be related to cellular ATP levels.
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8039665
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Molecular characterization of ura1, a mutant allele for orotidine-5'- monophosphate decarboxylase in Schizophyllum commune.
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The basis of the auxotrophic ura1 phenotype in Schizophyllum commune has been investigated. Two point mutations causing changes in conserved amino acid positions 62 (from lysine to glutamate) and 79 (from leucine to phenylalanine) most likely are the cause for the observed phenotype, whereas the overall gene structure was unchanged. Since reversion rates in this locus are extremely low, a single point mutation could not be expected to be the cause for the mutation. Besides the two point mutations expected to be induced by UV mutagenesis, the two alleles investigated from independently isolated strains differ by approximately 7% in nucleic acid sequence and about 3% in amino acid sequence, indicating a distant relationship between the strains used.
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8039664
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Agar-entrapped bacteria as an in vitro model of biofilms and their susceptibility to antibiotics.
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A simple in vitro system was developed as a model structure of biofilms and to evaluate their susceptibility to antibiotics. Viable Escherichia coli cells were entrapped in agar gel layers and incubated for 2 days in a minimal salt medium supplemented with glucose. After subsequent culture for 3 weeks under metal ion depletion, the biomass distribution inside the gel layer was highly heterogeneous. The cell concentration reached 10(11) cfu/g gel in the outer regions of the agar structure whereas the inner gel areas were less colonized (10(9) cfu/g gel). Immobilized cells displayed enhanced resistance to latamoxef as compared with free microorganisms. Moreover, a 3-week-old immobilized-cell membrane was less susceptible to the antibiotic than a younger (2 days old) one. The exposure for 11 h to 64 micrograms/cm3 latamoxef killed about 90% of the bacteria entrapped in the older agar layer, whereas the number of killed cells was 100-fold higher in the younger structure. Effective diffusivity measurements showed that the diffusion of latamoxef in the biofilm-like agar structures was moderately restricted as compared to that in water, and independent of the immobilized-cell content.
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8039662
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Saccharomyces cerevisiae mutants sensitive to the antimalarial and antiarrhythmic drug, quinidine.
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Mutations at three loci in Saccharomyces cerevisiae have been shown to confer increased sensitivity to the antimalarial and antiarrhythmic alkaloid, quinidine. Two of these groups are composed of strains carrying recessive mutations, the other group contains two dominant alleles. The largest complementation group has been designated QDS1, for increased quinidine-sensitivity. Exposure of qds1 cells to lethal concentrations of quinidine results in a novel small-budded terminal morphology in about 70% of the cells in the culture. Strains which carry qds1 alleles share other pleiotropic phenotypes. qds1 mutants are incapable of mating as alpha but not a cells, due to a defect in alpha-factor production. Homozygous diploid qds1 strains cannot sporulate. Genetic evidence indicates that QDS1 is allelic to KEX2, a precursor processing protease. Loss of QDS1/KEX2 function results in quinidine sensitivity.
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8039663
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Adherence to human small intestines of capsulated Vibrio cholerae O139.
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Capsulated cells of V. cholerae O139 adhered to formalin-fixed or native mucosa of the small intestines from an adult and a child. The primary adherence target was mucus. Capsulated O139 cells adhered better to the antigen sampling cells (M cells) of ileal Peyer's patch than to the absorptive cells. O139 cells on the mucosa appeared as small aggregates. Similar organisms were found on the mucosa of duodenal biopsy samples from patients infected with V. cholerae O139. The findings indicated that capsulated cells of V. cholerae O139 tend to autoagglutinate and contribute to the effective adherence to the intestinal mucosa.
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8039661
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Anaerobic degradation of halogenated benzoic acids by photoheterotrophic bacteria.
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From light-exposed enrichment cultures containing benzoate and a mixture of chlorobenzoates, a pure culture was obtained able to grow with 3-chlorobenzoate (3-CBA) or 3-bromobenzoate (3-BrBA) as the sole growth substrate anaerobically in the light. The thus isolated organism is a photoheterotroph, designated isolate DCP3. It is preliminarily identified as a Rhodopseudomonas palustris strain. It differs from Rhodopseudomonas palustris WS17, the only other known photoheterotroph capable of using 3-CBA for growth, in its independence of benzoate for growth with 3-CBA and in its wider substrate range: if grown on 3-CBA, it can also use 2-CBA, 4-CBA or 3,5-CBA.
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8039659
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Rapid typing of group A streptococci by the use of DNA amplification and non-radioactive allele-specific oligonucleotide probes.
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Because of the allelic variations within the M protein gene (emm gene) of group A streptococci, reliable typing of this important human pathogen can be accomplished by the use of emm gene-specific oligonucleotide probes. Two technical modifications (a reverse dot blot and a reverse line blot hybridization assay) of a novel approach for the type-specific identification of emm genes have been developed. Both procedures involved amplification of an emm gene by polymerase chain reaction. The non-radioactively labeled amplicon was subsequently hybridized to a membrane carrying an array of immobilized emm gene-specific oligonucleotide probes, thus allowing the simultaneous analysis of the gene polymorphism in a single hybridization reaction. The feasibility of these rapid and easy to perform methods was shown for the unequivocal identification of reference strains and clinical isolates belonging to 16 different M serotypes.
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8039660
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Subdivision of Salmonella enteritidis PT 4 by pulsed-field gel electrophoresis: potential for epidemiological surveillance.
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Using pulsed-field gel electrophoresis (PFGE), nine profile types were identified in XbaI digests of a panel of strains of Salmonella enteritidis phage type 4 (PT 4) isolated in England and Wales between 1967 and 1992. When applied to 39 isolates from humans, chickens, poultry products and feed, strains belonging to pulsed-field profile (PFP) 1 predominated. DNA-DNA hybridization studies demonstrated that a band of approximately 57 kb was associated with the presence of the 38 MDa S. enteritidis 'serovar-specific' plasmid. It is concluded that PFGE provides a method for discriminating strains of S. enteritidis PT 4 suitable for epidemiological investigations.
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8039658
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The phylogeny of Neospora caninum and Toxoplasma gondii based on ribosomal RNA sequences.
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Neospora caninum is a newly described cyst-forming coccidium which is the cause of severe neurological disease in dogs. The parasite is morphologically similar to Toxoplasma gondii, but the two species can be differentiated serologically. In order to define the phylogenetic position of N. caninum, we have determined 16S-like rRNA sequences from three members of the family of Sarcocystidae: N. caninum, T. gondii, and Sarcocystis fusiformis. The 16S-like rRNA genes from the three parasites were amplified by polymerase chain reaction and the sequences were determined by direct solid-phase sequencing. The sequences derived were computer aligned with several other 16S-like rRNA sequences from protozoan parasites to construct phylogenetic trees. The study confirmed that N. caninum should be classified as a member of the family Sarcocystidae. However, because of the close relationship to T. gondii it seems questionable that N. caninum should be placed in a new genus.
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8039656
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Typing of Staphylococcus aureus by amplification of the 16S-23S rRNA intergenic spacer sequences.
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The possibility of using polymorphisms in the spacer regions between 16S and 23S rRNA genes in order to type Staphylococcus aureus has been evaluated. To this purpose, DNA extracted from 74 independent isolates was amplified making use of a pair of primers complementary to conserved regions in the 16S and 23S genes. We have demonstrated that the method provides a good discrimination between unrelated isolates, giving better results when methicillin-sensitive strains are considered. Moreover, the amplification profiles were reproducible and all strains were typable. Given these results, and the technical simplicity of the process, we propose PCR-ribotyping to be taken into consideration as a method for typing S. aureus.
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8039657
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Structural changes induced by glycine on Streptomyces antibioticus.
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Germination and vegetative growth of Streptomyces antibioticus in liquid medium with different concentrations of glycine was examined. Both processes proved to be sensitive to the amino acid, being inhibited by 5 and 2.5% glycine, respectively. At concentrations of 5% or more, lysis of the vegetative mycelium occurred. Subinhibitory concentrations of glycine induced structural changes on germinating spores. These included an increase in the number of germ tubes produced by spore, in relation to the control. Moreover, soon after outgrowth the tubes bifurcate, giving rise to germinated spores with a characteristic aspect, and anomalous formation of cross-walls that appear both within the spores and in the newly formed germinative tubes, at or close to the region of outgrowth. The branching effect of glycine was also observed during vegetative growth of S. antibioticus.
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8039655
|
Genotypic and phenotypic relatedness of 80 strains of Branhamella catarrhalis of worldwide origin.
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80 clinical Branhamella catarrhalis strains of worldwide origin were examined for genotypic relatedness and phenotypic characteristics. Using a quantitative bacterial dot method for DNA-DNA hybridization the strains were found to form a homogeneous group with delta Tm-values ranging from 0.0-2.3 degrees C, In Minibact-N, an identification kit for oxidase positive, Gram-negative diplococci using eight phenotypic characteristics, all isolates were correctly identified and also demonstrated complete homogeneity except for beta-lactamase production. Type strains representing the genera Branhamella, Moraxella and Neisseria were also examined for comparison. B. catarrhalis strain NCTC 4103-known to be atypical-had a delta Tm-value of 5.7 degrees C and produced gamma-glutamylaminopeptidase, in contrast to all other B. catarrhalis strains. In GN MicroPlate, a kit which tests utilizable carbon sources, B. catarrhalis strains were found to be able to utilize up to 16 to 95 carbon sources.
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8039654
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Two membrane-bound c-type cytochromes of Thiobacillus ferrooxidans: purification and properties.
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Membrane-bound cytochrome c, cytochrome c-552 (m) was purified from Thiobacillus ferrooxidans. It showed an absorption peak at 410 nm in the oxidized form, and peaks at 552, 523 and 416 nm in the reduced form. Its molecular mass, Em,7 and isoelectric point were 22,300, +0.336 volt and 9.1, respectively. Another membrane-bound cytochrome c, cytochrome c-550 (m) was also purified. It showed an absorption peak at 408 nm in the oxidized form, and peaks at 550, 523 and 418 nm in the reduced form. Its molecular mass was estimated to be 51,000. Ferrocytochromes c-552 (m) and c-550 (m) were oxidized by cytochrome c oxidase of the bacterium. The reactivity with the oxidase of cytochrome c-550 (m) was higher than that of cytochrome c-552 (s) (soluble cytochrome) of the bacterium, while the reactivity of cytochrome c-552 (m) was greatly lower than that of cytochrome c-552 (s).
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8039653
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Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence.
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The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EIIMan) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EIIMan showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EIIGlc). Similar homology was shown between the C-terminal sequence of EIIMan and the E. coli glucose-specific enzyme III (EIIIGlc), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EIIMan contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIIIGlc, or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively.
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8039652
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Characteristics of a protease inhibitor produced by Prevotella intermedia.
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The characterization of a protease inhibitor produced by a strain of Prevotella intermedia is reported in the present study. The protease inhibitor was secreted into the culture medium during the exponential growth phase and was also present in significant amounts in the periplasmic space. The inhibitor was active against a wide variety of proteolytic enzymes, including the trypsin-like activity of Porphyromonas gingivalis. The inhibitory compound, a heat labile protein, was isolated by non-denaturing preparative polyacrylamide gel electrophoresis, using concentrated culture supernatant as starting material. The exact functions and the ecological roles of the protease inhibitor produced by P. intermedia remain to be determined.
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8039651
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Genetic analysis of the DnaA-dependent priming in the initiation of lagging strand DNA synthesis of plasmid pUB110 in Bacillus subtilis.
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Derivatives of Bacillus subtilis plasmid pUB110 lacking the major lagging strand replication origin (ssoU-) accumulate intracellular single-strand circular (SS(c)) DNA intermediates and are unable to propagate in dnaB and dnaD hosts. DnaA-dependent priming requires a DnaA box in a stable hairpin form; a higher copy number of a DnaA box is not sufficient as a signal for the conversion of the SS(c) into its dsDNA form. The introduction into the plasmid of a hairpin structure, whose stem carries a DnaA box, mediates conversion of SS(c) into dsDNA and makes plasmid replication independent of the B. subtilis dnaB function. This conversion signal has been termed ssoA.
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8039650
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The phylogenetic position of Hydrogenobacter acidophilus based on 16S rRNA sequence analysis.
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Hydrogenobacter acidophilus strain 3H-1 is a thermoacidophilic, obligately chemolithoautotrophic, hydrogen-oxidizer isolated from a Japanese solfataric field. Strain 3H-1 requires elemental sulfur for growth. We used PCR to amplify the 16S rRNA gene of strain 3H-1, and sequenced the amplification product directly. Phylogenetic analyses show strain 3H-1 is closely related to Aquifex pyrophilus and may be located in the deepest branch within the eubacterial phylogenetic tree. Sulfur-dependency of the ancestral eubacterium is also discussed.
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8039649
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Purification and characterization of chitinase from Candida albicans.
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A novel procedure was used to purify a cytosolic chitinase from Candida albicans to electrophoretic homogeneity. The results represent the first demonstration of the purification of a fungal intracellular chitinase using the criterion of a single band detected following silver-staining of a polyacrylamide gel run under denaturing conditions. Purified chitinase had pH and temperature optima of 5.0 and 50 degrees C, respectively. Inhibition of enzyme activity by allosamidin was pH-dependent occurring maximally at pH 8.0. Phospholipids had similar marked and highly specific effects on the activities of both the purified soluble enzyme and a solubilized microsomal chitinase from C. albicans. Evidence is provided for the existence of a complex chitinolytic system in this organism.
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8039648
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Microcin production by the yeast Cryptococcus humicola.
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Cryptococcus humicola strains secrete killer toxins inhibitory (at pH values ranging from 3 to 5.5) to many ascomycetous and basidiomycetous yeast-like fungi. RNA or DNA plasmids were not detected in the killers. The amino acid-containing toxins were of low M(r), soluble in methanol, resistant to proteolysis, thermostable, cellophane-diffusible and were specified as microcins. These findings show that the killer phenomenon in yeasts such as bacteriocinogeny may be due to excretion of two types of killer toxins: mycocins and microcins.
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8039633
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Genetic aspects and risk factors in alcoholism and alcoholic liver disease.
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There is a great deal of evidence for genetic predisposition to alcoholism; considerably less is known regarding predisposition to alcoholic liver disease. The specific genes involved in either disorder are not well understood, although the enzymes of alcohol metabolism appear to play some role. It will be interesting to determine whether genetic factors that alter the expression of these enzymes, in addition to altering the kinetics of the enzymes, could modify responses to drinking. Work in the next few years will include determination of which responses to alcohol are indeed genetically influenced in twin studies, testing additional candidate genes for alcohol-related traits in populations and families, as well as the application of genomic mapping methodologies to alcoholic pedigrees. The latter strategy will be integrated into the larger number of studies that will grow from the Human Genome Project. Animal studies with selectively bred lines of rodents that differ in voluntary alcohol consumption will lead the way to define the neuronal and behavioral substrates responsible for differences in alcohol-drinking behavior. The use of the quantitative trait locus (QTL) mapping approach in F2 intercross between two inbred strains of rodents with opposite alcohol-response characteristics and in recombinant inbred strains derived from F2 intercross already has and will continue to help identify chromosomal locations of genes relevant to voluntary alcohol consumption. Perhaps in the future selective breeding of rodents and QTL mapping strategies can also be used to determine the biology and genetics of alcohol-induced liver injury.
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8039632
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Pathophysiology of potassium absorption and secretion by the human intestine.
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When normal people ingest 90 mEq/day of K+ in their diet, they absorb about 90% of intake (81 mEq) and excrete an equivalent amount of K+ in the urine. Normal fecal K+ excretion averages about 9 mEq/day. The vast majority of intestinal K+ absorption occurs in the small intestine; the contribution of the normal colon to net K+ absorption and secretion is trivial. K+ is absorbed or secreted mainly by passive mechanisms; the rectum and perhaps the sigmoid colon have the capacity to actively secrete K+, but the quantitative and physiological significance of this active secretion is uncertain. Hyperaldosteronism increases fecal K+ excretion by about 3 mEq/day in people with otherwise normal intestinal tracts. Cation exchange resin by mouth can increase fecal K+ excretion to 40 mEq/day. The absorptive mechanisms of K+ are not disturbed by diarrhea per se, but fecal K+ losses are increased in diarrheal diseases by unabsorbed anions (which obligate K+), by electrochemical gradients secondary to active chloride secretion, and probably by secondary hyperaldosteronism. In diarrhea, total body K+ can be reduced by two mechanisms: loss of muscle mass because of malnutrition and reduced net absorption of K+; only the latter causes hypokalemia. Balance studies in patients with diarrhea are exceedingly rare, but available data emphasize an important role for dietary K+ intake, renal K+ excretion, and fecal K+ losses in determining whether or not a patient develops hypokalemia. The paradoxical negative K+ balance induced by ureterosigmoid anastomosis is described. The concept that fecal K+ excretion is markedly elevated in patients with uremia as an intestinal adaptation to prevent hyperkalemia is analyzed; we conclude that the data do not convincingly show the existence of a major intestinal adaptive response to chronic renal failure.
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8039631
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Interleukin 6-producing gastric carcinoma with fever, hypergammaglobulinemia, and plasmacytosis in bone marrow.
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A 42-year-old man with a remittent fever was found to have both para-aortic and hepatic tumors with generalized lymphadenopathy. The pathological findings from biopsy specimens from the para-aortic lymph node and hepatic tumor by laparotomy and from left supraclavicular lymphadenectomy showed undifferentiated carcinoma. However, the location of the primary lesion could not be determined. Chemotherapy temporarily reduced the size of the metastatic lymph nodes and the hepatic tumor and also suppressed the remittent fever. Fifteen months after onset, massive polyclonal hypergammapathy developed together with plasmacytosis (30% plasmacytes) in bone marrow, consisting of normal mature plasmacytes. Among cytokines, including interleukin (IL) 1 beta, IL-3, IL-4, IL-6, and tumor necrosis factor alpha, a high value of IL-6 was detected in the serum. Postmortem pathological examination showed the scirrhous type of gastric carcinoma with specific staining by polyclonal antibody against human IL-6. To our knowledge, this is the first report of a gastric carcinoma producing IL-6 associated with fever, hypergammaglobulinemia, and plasmacytosis in bone marrow.
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8039630
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Superficial thrombophlebitis, dysplasia, and cholangiocarcinoma in primary sclerosing cholangitis.
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Cholangiocarcinoma occurs in approximately 10% of patients with primary sclerosing cholangitis. Usually, liver failure, rapidly progressing jaundice, and an increase in alkaline phosphatase levels are suggestive diagnostic features. We report two cases of patients with primary sclerosing cholangitis who developed cholangiocarcinoma without jaundice and with no changes in their serum biochemistry. Both patients were taking ursodeoxycholic acid at the time of tumor diagnosis. Initial suspicion of malignancy was based on the development of superficial thrombophlebitis. Liver histology showed evidence of bile duct epithelial dysplasia in areas free from tumor in one patient, and in the other, bile duct epithelial dysplasia preceded the appearance of cholangiocarcinoma by at least 18 months. In one of the cases, the dysplastic epithelium stained positively for carcinoembryonic antigen. The histological finding of bile duct epithelial dysplasia in patients with primary sclerosing cholangitis may suggest either imminent or actual development of cholangiocarcinoma and may thus affect consideration of orthotopic liver transplantation. In addition, the development of superficial thrombophlebitis in patients with primary sclerosing cholangitis should arouse suspicion of the presence of cholangiocarcinoma even if there is no evidence of deterioration of the liver function or a dominant stricture on endoscopic retrograde cholangiography.
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8039629
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Neutrophil autoantibodies: a genetic marker in primary sclerosing cholangitis and ulcerative colitis.
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Perinuclear antineutrophil cytoplasmic antibodies (pANCA) were found at high frequency in patients with primary sclerosing cholangitis and ulcerative colitis. In this study, to accumulate further evidence for the importance of genetic factors in pathogenesis of inflammatory bowel disease, sera of patients with inflammatory bowel disease and primary sclerosing cholangitis and their unaffected family members were tested for pANCA. Three hundred twenty-seven sera from 11 families of patients with primary sclerosing cholangitis, 43 families of patients with ulcerative colitis, 11 families of patients with Crohn's disease, and 11 healthy families were tested for pANCA in immunofluorescence on cytospin slides with isolated neutrophils. pANCA were found in 82% of the patients with primary sclerosing cholangitis and in 25% of their relatives. In ulcerative colitis, 70% of the patients and 30% of their relatives had pANCA. pANCA were found only in low titers in 27% of patients with Crohn's disease and in 6% of their relatives. pANCA were not detected in members of healthy families. Only 16% of the patients with ulcerative colitis and their families and none of the patients with primary sclerosing cholangitis and their families were completely negative for pANCA. These data show that pANCA may be a genetic marker in families of patients with ulcerative colitis and primary sclerosing cholangitis.
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8039628
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Endogenous cholecystokinin stimulates pancreatic enzyme secretion via vagal afferent pathway in rats.
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Recently we showed that doses of cholecystokinin octapeptide (CCK-8) that produce physiological plasma CCK levels act via stimulation of afferent vagal pathway to mediate pancreatic enzyme secretion. In this study we investigated if endogenous CCK also acts via similar pathway. In anesthetized rats, plasma CCK levels were elevated by diversion of bile pancreatic juice and duodenal casein feeding. The effects of acute vagotomy as well as that of chemical ablation of the afferent vagal pathway on pancreatic enzyme secretion evoked by increased endogenous plasma CCK levels were investigated. Diversion of bile pancreatic juice elevated plasma CCK levels from a basal level of 0.6 +/- 0.1 pmol/L to 8.9 +/- 2.1 pmol/L and caused a more than twofold increase in pancreatic protein secretion. Similar increases in plasma CCK levels and pancreatic secretion were observed with duodenal administration of casein. Vagotomy or perivagal application of capsaicin, a sensory neurotoxin, abolished increases in pancreatic secretion but not plasma CCK levels in response to diversion of bile pancreatic secretion or duodenal administration of casein. In contrast, pancreatic protein responses to 2-deoxy-D-glucose, a central vagal stimulant, remained intact in rats with perivagal capsaicin treatment indicating capsaicin did not affect efferent vagal function. Endogenous CCK under physiological conditions act via stimulation of vagal afferent pathway to mediate pancreatic enzyme secretion.
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8039626
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nm23-H1 expression and disease recurrence after surgical resection of small hepatocellular carcinoma.
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The nm23-H1 gene is thought to act as a metastasis-suppressor gene. This study investigates the relationship between nm23-H1 messenger RNA (mRNA) expression and intrahepatic tumor recurrence after surgical resection of small hepatocellular carcinoma. Seventeen cirrhotic patients with solitary hepatocellular carcinoma < 5 cm underwent surgical resection. In 7 patients, the neoplasm recurred after a 12-month median follow-up, whereas the other 10 patients were free of disease after a 30-month median follow-up. Both groups were similar according to age, sex, etiology, status of the underlying liver, tumor size, and other pathological characteristics of the neoplasm. nm23-H1 mRNA levels were assessed in matched tumor and surrounding cirrhotic liver samples by Northern blot hybridization using a 900-base pair probe, which is a BamHI fragment of pnm23-H1 recombinant complementary DNA clone encoding the nm23-H1 human gene. Eight of the 10 patients without disease recurrence during follow-up showed nm23-H1 overexpression with an increase ranging between three- and 45-fold when compared with the nontumoral surrounding liver. Only 1 of the 7 patients with tumor recurrence showed higher nm23-H1 mRNA levels in the tumor than in the nonneoplastic sample (P = 0.013). nm23 mRNA overexpression in small solitary hepatocellular carcinoma is associated with a lower recurrence rate after surgical resection, suggesting that this gene may participate in the metastatic dissemination of this neoplasm.
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8039627
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Inhibition of beta-oxidation by 3-mercaptopropionic acid produces features of Reye's syndrome in perfused rat liver.
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The cause of Reye's syndrome has not been completely defined. The rate of ketogenesis in the liver is a key determinant of, and reciprocally related to, triglyceride secretion. In the present study, 3-mercaptopropionic acid (MPA), a known inhibitor of mitochondrial long-chain acyl coenzyme A (CoA) dehydrogenase, was used to investigate the relationship between ketone body production, triglyceride secretion, and triglyceride accumulation in perfused rat liver. Livers from fasted rats were perfused 225 minutes with or without MPA in the presence of [1-14C]oleic acid. Morphology was studied by light and electron microscopy. Inhibition of fatty acid oxidation by the liver with MPA resulted in a decrease in ketone body production. Treatment with MPA caused an accumulation of small-droplet triglycerides in liver, whereas the net secretion of triglyceride ceased after an initial period of increased secretion with continued decreased ketogenesis. At the end of the perfusion period, mitochondria in the MPA group appeared to be damaged. The rates of both ketogenesis and triglyceride secretion by the liver appear to be the major determinants of hepatic triglyceride content. In addition, the MPA-mediated biochemical and morphological findings are quite similar to those of Reye's syndrome.
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8039625
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Continuous versus intermittent therapy for chronic hepatitis C with recombinant interferon alfa-2a.
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Prolonged interferon administration to patients with chronic hepatitis C, although increasing the sustained response rate, is poorly accepted and may favor drug resistance. A pulse-treatment schedule would be preferred for compliance and costs. One hundred thirty-five patients with chronic hepatitis C received 6 MU units of interferon alfa-2a, three times weekly, continuously for 9 months (group 1: 66 patients) or for two 3-month cycles, separated by 6 months pause (group 2: 69 patients). At the end of therapy, 25 of 54 patients of group 1 (46.3%) and 28 of 60 of group 2 (46.7%) had normal serum aminotransferase levels. Six months after the end of treatment, sustained responders were still similar in the two groups (11 or 16.7% vs. 7 or 10.1%; NS). A loss of response before the end of therapy was seen in 10 patients of group 1 and 6 of group 2; interferon-neutralizing antibodies developed in 1 of 7 and 6 of 6 of such patients, respectively. The intermittent administration of interferon alfa-2a to patients with chronic hepatitis C shows a sustained response rate comparable with that achieved with continuous treatment at the same dosage. Hepatitis breakthroughs during pulse therapy appeared to be limited to interferon neutralizing antibodies, whereas a prolonged, continuous treatment is more likely to induce other forms of interferon resistance.
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8039624
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Hepatocellular Na+/H+ exchange is activated at transcriptional and posttranscriptional levels in rat biliary cirrhosis.
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Rat hepatocyte Na+/H+ exchange is activated in vitro by growth factors and in vivo following partial hepatectomy. This study explored by which mechanism(s) it is activated in a cirrhosis model characterized by chronic stimulation of hepatocyte proliferation. Rat hepatocytes were isolated 4 weeks after bile duct ligation or sham operation. Intracellular pH (pHi) was fluorimetrically determined, and plasma membranes and messenger RNA (mRNA) were prepared from isolated hepatocytes by standard methods. Resting pHi was higher in bile duct-ligated than in control rats (7.42 +/- 0.03 vs. 7.06 +/- 0.04; P < 0.001). Although plasma membrane lipid composition and intracellular buffering capacity were similar, initial Na+/H+ exchange-mediated rates of pHi recovery following acid loading were higher in bile duct-ligated than in control rats (0.098 +/- 0.011 vs. 0.055 +/- 0.005 pH units/min; P < 0.05). The antiporter's set point was shifted approximately 0.3 pH units towards more alkaline values and its steady-state mRNA levels were doubled after bile duct ligation. Hepatocellular Na+/H+ exchange is transcriptionally and posttranscriptionally activated in rat biliary cirrhosis further supporting a relationship between hepatocyte proliferation and Na+/H+ exchange activation.
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8039623
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Liver function improvement following increased portal blood flow in cirrhotic rats.
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Liver microcirculation in cirrhosis is characterized by development of intrahepatic shunts and capillarization of sinusoids secondary to cell necrosis and deposition of new collagen, resulting in both decreased drug elimination and increased vascular resistance with portal hypertension. The aim of this study was to examine the effects of increased portal blood flow on hepatic microcirculation and drug elimination in 13 perfused livers from cirrhotic rats. Intrahepatic resistance was assessed under basal conditions (21.2 +/- 0.3 mL/min) and 1 hour after doubling the flow (41.6 +/- 1.0 mL/min). A multiple indicator dilution technique was used at both flow rates to measure sinusoidal volume, albumin and sucrose extravascular volumes, and cellular water volume. Hepatic elimination of labeled taurocholate and propranolol was also measured, and the recovery of 15-microns microspheres was used to evaluate large intrahepatic shunts. After doubling the flow, intrahepatic resistance decreased by 31%. Sinusoidal and extravascular volume increased significantly without a change in microsphere recovery. However, there was a marked increase in taurocholate and propranolol elimination by cirrhotic livers. Moreover, during high flow, significant correlations were found between changes in albumin extravascular volume and taurocholate and propranolol elimination. Increased portal blood flow in cirrhotic rats induces a decrease in intrahepatic resistance without changes in intrahepatic shunting and improves drug elimination by the liver without deleterious effects on hepatocyte viability.
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8039622
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Screening for hemochromatosis: a cost-effectiveness study based on 12,258 patients.
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Current emphasis in hemochromatosis has focused on early detection and treatment to prevent permanent liver damage and hepatocellular carcinoma. Thus far, only normal population and high-risk groups have been screened but not patients seeking medical care. Serum iron levels were determined in consecutive fasting blood samples collected in the morning from 12,258 Mayo Clinic patients. One hundred twenty-seven patients had an initial serum iron concentration > or = 180 micrograms/dL. Eight patients (age, 38-71 years; 7 men and 1 woman) had transferrin saturation > or = 62% (range, 84-99) and serum ferritin value > or = 400 micrograms/L (range, 457-4004) with no other explanation for the abnormal iron test results. Three patients (2 male and 1 female) had markedly elevated hepatic iron concentration (range, 11,080-29,719 micrograms/g dry wt) and hepatic iron index (range, 2.9-8.4) indicative of homozygous hemochromatosis. One patient who refused liver biopsy had 7 g of iron removed by phlebotomy and is likely homozygous. Two patients had hepatic iron indices < 1.5 and are probably heterozygous. The genetic status of 1 patient is indeterminate, and 1 patient with normal hepatic iron concentration and hepatic iron index had chronic active hepatitis. None had cirrhosis, diabetes, or cardiomyopathy. No patients with hemochromatosis would have been detected without this study. The yield in this study, 0.33 cases of 1000 screened, is approximately one tenth of the predicted homozygote frequency by recent estimates. Even at this yield, screening appears cost-effective.
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8039621
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Psyllium augments the cholesterol-lowering action of cholestyramine in hamsters by enhancing sterol loss from the liver.
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Psyllium hydrophilic mucilloid is a nonabsorbable soluble fiber that lowers plasma cholesterol levels in several species, including humans. However, its mechanism of action has not been fully elucidated. Therefore, using a hamster model, experiments were performed to determine whether psyllium given alone or in combination with a submaximal dose of cholestyramine blocks intestinal cholesterol absorption. The efficiency of cholesterol absorption and concentrations of plasma and hepatic total cholesterol were measured in male hamsters fed a cholesterol-enriched chow diet (0.1%) that contained either avicel (cellulose) (7.5%), surfomer (3%), cholestyramine (1% or 3%), or psyllium (7.5%) as single agents or a fixed level of cholestyramine (1%) combined with variable levels of psyllium (2%, 4%, 6%, or 8%). Psyllium, cholestyramine, and surfomer, when given alone, markedly lowered plasma and hepatic cholesterol concentrations. Surfomer, and cholestyramine at the higher dose (3%), blocked cholesterol absorption by 54% and 75%, respectively, whereas psyllium had no effect. Combining psyllium with a submaximal dose of cholestyramine augmented the cholesterol-lowering action of the resin without effecting any marked change in the level of cholesterol absorption, except at the highest dose used. Psyllium, given either as a single agent or as an adjunct to treatment with cholestyramine, exerts a significant hypocholesterolemic effect by enhancing net negative sterol balance across the liver.
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8039620
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Prospective evaluation of early morphological changes in pelvic ileal pouches.
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Little is known about the evolution of morphological changes in pelvic ileal-pouch mucosa. This study evaluates prospectively the sequence of early morphological, histochemical, and phenotypic features in pouch mucosal biopsy specimens. Twenty-two patients with pelvic ileal pouches constructed after total colectomy for chronic ulcerative colitis had biopsies performed at the time of ileostomy closure and after 6 weeks and 6 months of pouch function and were evaluated to assess the type and degree of inflammation, villus atrophy, Paneth's cell hyperplasia, mucin histochemical changes, the mucosal proliferative activity using the murine monoclonal antibody 1 (MIB-1), and the expression of the enzyme sucrase-isomaltase. Early changes (6 weeks) were characterized by neutrophilic and eosinophilic inflammation, mild villus atrophy, Paneth's cell hyperplasia, a partial transition to colonic mucin phenotype, and an increased MIB-1 proliferation index. These features remained relatively stable after 6 months, except for a greater degree of mononuclear infiltration, a progressive increase in the degree of eosinophilic inflammation and a new higher steady state level of crypt epithelial kinetics. Expression of sucrase-isomaltase remained stable. Pelvic ileal pouches develop inflammatory, phenotypic, and kinetic changes early in the course of function but have only a limited potential for colonic type metaplasia. The persistence of these changes is evidence in support of an adaptive response to a new luminal environment.
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8039619
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Effect of free glutamine and alanyl-glutamine dipeptide on mucosal proliferation of the human ileum and colon.
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Glutamine (Gln) is considered a trophic factor for small intestinal epithelia, which is important during severe illness. Its use in parenteral nutrition is precluded by its instability, a problem that may be overcome by use of the stable dipeptide L-alanyl-L-glutamine (Ala-Gln). The hypothesis was tested that Gln or Ala-Gln may stimulate cell proliferation not only in the ileum but also in the proximal and distal colon and, thus, may contribute to the gut barrier function. Biopsy samples from the normal human ileum, proximal colon, and rectosigmoid were incubated for 4 hours with Gln (2 mmol/L), Ala-Gln (2 mmol/L), and saline (control). Cells in S phase were labeled with bromodeoxyuridine. In longitudinal crypt sections labeled and quiescent cells were counted. Gln as well as Ala-Gln stimulated crypt cell proliferation in the ileum, proximal colon, and rectosigmoid colon. In ileal specimens, labeling was greater in the entire crypt, whereas in both colonic regions, the trophic effect was confined to the basal crypt compartments. Gln and Ala-Gln have trophic effects not only in the ileum, but also in the proximal and distal colon. This could be important during parenteral nutrition when mucosal atrophy may weaken the gut barrier.
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8039618
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Molecular genetic profiles of colitis-associated neoplasms.
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A major long-term risk for patients with chronic idiopathic colitis is the development of colorectal dysplasia and adenocarcinoma. The presence or absence of specific genetic changes in these lesions will provide important insights into the relationship of colitis-associated dysplasia and the development of carcinoma. A case-study approach was used to develop detailed molecular genetic profiles of advanced dysplasias and carcinomas from six patients with ulcerative colitis or Crohn's colitis. Numerous genetic alterations were identified in each of the dysplasias and carcinomas profiled. These genetic alterations involved many of the same targets found in sporadic colorectal tumors and included multiple sites of allelic deletion, microsatellite instabilities, and mutations of the K-ras, p53, and APC genes. The progression of dysplasia to carcinoma was often accompanied by an accumulation of these mutations. Genetic alterations are common in colitis-associated neoplasia, just as in sporadic colorectal neoplasia. This could have important implications for the evaluation and treatment of patients with colitis.
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8039617
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Antroduodenal motility and transpyloric fluid movement in patients with diabetes studied using duplex sonography.
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To elucidate the relationship between diabetic autonomic neuropathy and gastrointestinal motility, antroduodenal motility was studied in patients with diabetes using duplex sonography. Antroduodenal motility, transpyloric fluid movement, and velocity curves of fluid flow were studied using duplex sonography in 32 patients with diabetes and 10 healthy subjects after their ingestion of a meat soup. The frequency of antroduodenal coordination was significantly reduced in patients with diabetes with both early and definite autonomic neuropathy compared with healthy subjects (P < 0.05 and P < 0.01, respectively). The frequency and duration of end-cycle reflux episodes were also significantly reduced in patients with early and definite autonomic neuropathy compared with healthy subjects (P < 0.05 and P < 0.01, respectively). The frequency of end-cycle reflux episodes was closely correlated with the frequency of antroduodenal coordination in both healthy subjects (r = 0.859; P = 0.002) and patients with diabetes (r = 0.929; P = 0.0001). There was a significant correlation between fasting plasma glucose concentrations and the frequency of antroduodenal coordination in patients with diabetes (r = -0.361; P = 0.039). These findings suggest that reduced frequency and duration of end-cycle reflux episodes may be an early indicator of diabetic gastroparesis that may be related mainly to autonomic neuropathy but also in part to acute hyperglycemia.
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8039616
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Effect of aging, position, and temperature on the threshold volume triggering pharyngeal swallows.
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Swallows triggered by direct stimulation of pharyngeal structures may help to prevent aspiration by emptying the pharynx. The aims of this study were to compare the biomechanical events of the pharyngeal and primary swallow, determine the threshold volume of liquid required to trigger the pharyngeal swallows, and determine the effect of aging, position, and temperature on this threshold volume. Concurrent manometry, video fluoroscopy, and video endoscopy were used to study young and elderly healthy volunteers. During pharyngeal swallows, in contrast to primary swallows, the free portion of the tongue did not make contact with the hard palate. In addition, pharyngeal swallows did not result in oral bolus clearance. All other biomechanical events, including deglutitive glottal function, were similar in both types of swallows. The threshold volume for pharyngeal swallows in young volunteers was significantly smaller than in the elderly (P < 0.01). Temperature and position did not have significant effects on threshold volume. Swallowing is readily induced by water stimulation of the pharynx. Pharyngeal swallows do not induce lingual peristalsis or clearance of oral content. The threshold volume of the pharyngeal swallow is significantly higher in the elderly than in the young, but it is not affected by body position or bolus temperature.
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8039615
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Mechanism by which glucose stimulates the passive absorption of small solutes by the human jejunum in vivo.
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Active absorption of glucose stimulates passive absorption of small solutes. Part of this effect may be caused by glucose-induced water absorption. Increased water absorption can enhance passive solute absorption by solvent drag and by passive diffusion if the luminal solute concentration increases as water is removed from the lumen. The purpose of this research was to quantitate the contribution of these two forces when glucose enhances the absorption of L-xylose. The effect of solvent drag on L-xylose absorption was determined by measuring the effect of water absorption stimulated by hypotonicity on L-xylose absorption when the L-xylose concentration was constant. The effect of diffusion on L-xylose absorption was determined by measuring the effect of L-xylose concentration on L-xylose absorption when water absorption was nil. Glucose increased L-xylose absorption by 1.8 mmol.h-1 x 30 cm-1 (from 1.4 to 3.2 mmol.h-1 x 30 cm-1). The increase attributable to solvent drag was 1.03 mmol.h-1 x 30 cm-1; the increase attributable to passive diffusion was 0.75 mmol.h-1 x 30 cm-1. When glucose stimulates the passive absorption of L-xylose, 57% of the increase can be attributed to solvent drag and 42% to passive diffusion. Because the combined effect of these two forces can account for 99% of the observed effect, virtually all of the glucose effect on L-xylose absorption can be explained by glucose-induced water absorption.
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8039614
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Mutations in the p53 gene: an early marker of neoplastic progression in ulcerative colitis.
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In long-term extensive ulcerative colitis, aneuploidy occurs earlier and loss of heterozygosity for p53 (p53 LOH) later during histological progression towards carcinoma. This study determined the time of onset of p53 mutation in this progression. We developed a rapid, sensitive screening assay for p53 mutations at codon 248. The geographic distribution of this p53 mutation was mapped in two fresh colectomy specimens with mutations of codon 248 (1 cancer, 1 dysplasia) and correlated with patterns of clonal expansion, histological progression, and allelic loss. Numerous samples from throughout both colons were analyzed (216 for histology, 142 for DNA content, 104 for mutation, and 41 for p53 LOH). p53 mutation correlated highly with histological grade and was distributed more extensively than p53 LOH. Mutation, but not LOH, was also found in diploid, nondysplastic colonic mucosa adjacent to dysplastic areas. These findings suggest that p53 mutation appears to be an early genetic event that precedes p53 LOH. The very close correlation of p53 mutation with aneuploidy (P > 0.0001) emphasizes the role of normal p53 at the G1 checkpoint to help prevent entry of genetically damaged cells into the cell cycle.
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8039613
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Lipids of human gastric mucosa: effect of Helicobacter pylori infection and nonalcoholic cirrhosis.
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Gastric mucosa phospholipids play an important protective role against exogenous and endogenous toxic agents. Recently, we described a significant alteration of phospholipid profile in patients with chronic atrophic gastritis without Helicobacter pylori infection. The aim of the present study was to assess the phospholipid composition of gastric biopsy specimens in 41 subjects with chronic gastritis in relation to H. pylori infection (no. 26) and nonalcoholic cirrhosis (no. 18). Phospholipids were extracted from homogenate mucosal samples using Folch's method, purified, and separated by thin-layer chromatography, while bound fatty acids were analyzed by gas liquid chromatography. The amounts of five gastric phospholipid classes, their rank order, and percent distribution of the principal ones (phosphatidylcholine [PC] 58%, phosphatidylethanolamine [PE] 26%, and phosphatidylinositol 11% vs. values of 49, 19, and 14, respectively, in the earlier study) were confirmed in chronic gastritis without H. pylori infection. H. pylori infection induced a dramatic reduction (about 30%) in the absolute amount of total phospholipids (24.2 micrograms/mg protein versus 35.1 of the H. pylori-negative group; P < 0.01), PC and PE being the most affected (-36% and -26%, respectively), while bound fatty acids remained unchanged. There was no difference in cirrhotic vs. noncirrhotic subjects. (1) The development of gastritis is characterized by an alteration of the lipid mucosal pattern that can change with the different etiologies, the most dramatic variations being observed in the presence of H. pylori infection; and (2) cirrhosis does not affect further the alteration in the phospholipid profile of the antral mucosa caused by chronic gastritis.
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8039612
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Treatment of Crohn's disease by lymphocyte apheresis: a randomized controlled trial. Groupe d'Etudes Thérapeutiques des Affections Inflammatoires Digestives.
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Several uncontrolled trials suggest that lymphapheresis improves the clinical course of patients with Crohn's disease; this study was designed to assess the efficacy of lymphapheresis in preventing early relapses of Crohn's disease in patients in clinical remission after steroid treatment for an acute attack. Twenty-eight patients in clinical remission at the end of 3-7 weeks of steroid therapy were included in this randomized multicenter prospective trial. Before starting steroid tapering, patients were randomly assigned either to the lymphapheresis group (9 procedures within 4-5 weeks) or to the control group. The primary judgement criterion was the cumulated recurrence rate after steroid discontinuation. All the patients treated by lymphapheresis (12 of 12) were successfully withdrawn from prednisolone and only 10 of 15 in the control group (NS). At the end of the 18-month follow-up period, the cumulated relapse rate was 83% in the lymphapheresis group and 62% in the control group. Although there was a trend towards a diminished incidence of corticosteroid dependence, lymphapheresis did not prevent the occurrence of early relapses.
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8039611
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Propionate-initiated changes in intracellular pH in rabbit colonocytes.
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Because regulation of intracellular pH (pHi) is critical to basic cell functions, most cells have evolved mechanisms to closely regulate intracellular acid-base balance. Short-chain fatty acids (SCFAs), the predominant luminal anion in the colon, acidify the cell interior in several cell systems, but their effect on their "natural target," the colonocytes, has not been examined thoroughly. We monitored the pHi response to a model SCFA, propionate, in isolated cells and epithelial sheets from rabbit proximal colon loaded with the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and -6)carboxyfluorescein. SCFAs induced a characteristic pHi response curve in colonocytes: an immediate acidification and a recovery phase returning to baseline in 100-200 seconds. Acidification was altered by increasing concentrations of SCFAs, by increasing SCFA chain length, extracellular osmolarity, and intracellular pH, and finally, Na+ removal. The recovery phase was slowed by amiloride and 4-alpha-OH cinnamate, an inhibitor of proton-monocarboxylate cotransport. Physiological concentrations of SCFAs have profound effects on intracellular pH. Simple diffusion of the SCFA may not explain the complexities of propionate-induced protonated acidification; the pH recovery phase may involve multiple processes including Na(+)-H+ exchange and H(+)-SCFA cotransport. Luminal constituents such as SCFAs may have significant effects on the intracellular pH and function of colonocytes.
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8039610
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Persistent lack of somatostatin receptors in gastric mucosa of healing ulcers in rats.
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Growth factors and their receptors play a role in healing ulcers of the rat. Somatostatin is a putative growth factor in the gastric mucosa with multiple functions mediated by specific receptors. Therefore, the distribution of somatostatin receptors was evaluated in the gastric mucosa of healing ulcers. Somatostatin receptors were measured in the gastric mucosa of healthy rats and of rats with cryoulcers and acetic acid-induced ulcers, using somatostatin receptor autoradiography with 125I-[Tyr3]-octreotide or 125I-[Leu8, D-Trp22, Tyr25]-somatostatin-28 as radioligands. Epidermal growth factor receptors were measured on adjacent sections with 125I-epidermal growth factor. High-affinity somatostatin receptors are present in the gastric fundic mucosa of healthy rats. In contrast, in healing ulcers, the somatostatin receptors are almost lacking in the ulcer edge or scar at 3, 7, 28, 49, and even 84 days after ulcer induction (83%-91% reduction). Similar results are obtained with cryoulcers and with acetic acid-induced ulcers. For comparison, the number of epidermal growth factor receptors are increased in the same healing ulcers. The persistent absence of somatostatin receptors for several months after ulcer induction may be of pathophysiological significance in ulcer repair and healing.
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8039609
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HCO3- secretion by rat distal colon: effects of inhibitors and extracellular Na+.
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The large intestine secretes HCO3- via a Cl-/HCO3- exchange mechanism located in the apical membrane of colonocytes. However, an additional transport system(s) must facilitate HCO3- (OH-) entry or H+ exit across the basolateral cell surface. The aim of this study was to determine that mechanism(s). A modified Ussing apparatus was used to measure net HCO3- secretion in segments of rat distal colon. When added to the serosal solution, 10 mmol/L 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS), 1 mmol/L SITS and 0.1 mmol/L diisothiocyanostilbene-2,2'-disulfonic acid, inhibited HCO3- secretion by 88%, 51%, and 30%, respectively. However, the Na+/H+ exchange inhibitors, amiloride (1 mmol/L), dimethylamiloride (0.1 mmol/L), ethylisopropylamiloride (0.1 mmol/L), failed to affect HCO3- secretion. Acetazolamide (1 mmol/L) blocked HCO3- secretion by approximately 60% when in the serosal solution but had little effect when in the mucosal solution. Ion substitution studies showed that HCO3- secretion required Na+ in the serosal solution (K0.5 approximately 12 mmol/L). HCO3- secretion was unaffected by depolarizing the basolateral membrane potential with K(+)-rich medium. These data are consistent with Na+ linked HCO3- transport across the colonocyte basolateral membrane, which appears to be electroneutral.
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8039608
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Dexamethasone-induced hyperglycemia in obese Avy/a (viable yellow) female mice entails preferential induction of a hepatic estrogen sulfotransferase.
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Sex steroid sulfotransferases (ST) sulfurylate and thus inactivate estrogens or androgens, producing an androgenized or estrogenized state in the liver. The expression of diabetes in a number of animal models is sexually dimorphic and has been associated with steroidal states. Although the viable yellow (Avy) mutation produces an insulin-resistant obesity syndrome in mice of both sexes, only males develop chronic hyperglycemia. Hyperglycemia was rapidly induced in Avy/a females by dexamethasone (dex). This treatment completely suppressed both endogenous plasma corticosterone and hepatic corticosterone-binding globulin (CBG) mRNA within 24 h. Hyperglycemia in dex-implanted Avy/a females was accompanied by aberrant shifts in hepatic androgen/estrogen balance. This was effected by induction of estrogen sulfotransferase (EST) mRNA together with a > 10-fold increase in enzymatic activity. Similar dex-induced increases in androgen ST or phenol ST were not observed. Prior implantation of estrogen prevented development of hyperglycemia. The time-dependent spontaneous reversal of dex-induced hyperglycemia correlated with re-expression of CBG mRNA transcripts and reduced levels of EST transcripts and enzyme activity. Although dex-induced hyperglycemia was limited to Avy/a females, dex elicited hyperinsulinemia in lean a/a control mice of both sexes and exacerbated constitutive hyperinsulinemia in Avy/a males and females. In summary, dex-induced hyperglycemia in Avy/a females was associated with increased catabolism of hepatic estrogens mediated by induction of EST.
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8039607
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Autoimmune diabetes induced by the beta-cell toxin STZ. Immunity to the 60-kDa heat shock protein and to insulin.
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Administered at a suitably low dose, the toxin streptozotocin (STZ) can trigger an autoimmune process leading to destruction of the beta-cells of the pancreatic islets. In this study, we examined specific immunological reactions in mice before and during the development of STZ-induced autoimmune diabetes. We now report that the development of spontaneous autoantibodies to insulin can serve as a marker of susceptibility to a low dose of STZ. Susceptible male mice of the C57BL/KsJ strain manifested such anti-insulin antibodies, and resistant female mice did not. Administration of a low dose of STZ (five daily doses each of 30 mg/kg) induced transient hyperglycemia approximately 20-30 days later, which temporarily remitted but was followed by intractable diabetes approximately 2.5 months later. The diabetogenic process triggered by the low dose of STZ was associated with an increase in the level of anti-insulin antibodies bearing the Dana and Micha (DM) idiotype, later followed by the appearance of anti-idiotypic antibodies that peaked before the onset of diabetes. Antibodies and T-cells reactive to hsp60 (heat shock protein) were triggered by the low-dose STZ administration and persisted throughout the period that preceded clinical diabetes. T-cells reactive to the p277 peptide of hsp60 were also observed. Finally, active immunization to hsp60 caused transient hyperglycemia by itself and also aggravated the hyperglycemia induced by low-dose STZ. Thus, autoantibodies to insulin can indicate susceptibility to a toxic trigger of diabetes, and a low dose of a toxin can activate the insulin and hsp60 autoimmunity that has been detected previously in the spontaneous autoimmune diabetes of NOD strain mice.
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8039606
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Intracellular mechanisms involved in D-glucose-mediated amplification of agonist-induced Ca2+ response and EDRF formation in vascular endothelial cells.
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Prolonged treatment of vascular endothelial cells with pathologically high D-glucose amplifies autacoid-induced Ca2+ mobilization and thus formation of nitric oxide. This study investigated the Ca2+ source for the change in endothelial CA2+ response on agonist stimulation. Pretreatment with high D-glucose (44 vs. 5 mM) enhanced release of intracellular Ca2+ by bradykinin as a result of a 2.0-fold increased formation of inositol 1,4,5-trisphosphate. High D-glucose also amplified Ca2+ influx (2.0-fold). In high D-glucose preincubated cells, stimulation with bradykinin significantly increased transplasmalemmal 45Ca2+ flux (3.2-fold) and caused a 2.0-fold increase in permeability to Mn2+, a surrogate for endothelial plasma membrane Ca2+ channels. A significant 2.0-fold increase occurred in the maximal slope, suggesting a higher rate of Mn2+ (Ca2+) influx. Ca2+ influx, stimulated by an inositol phosphate-independent depletion of intracellular Ca2+ stores with 2,5-di-(tert-butyl)-hydroquinone was also significantly increased 2.4-fold by high D-glucose, with no effect on intracellular Ca2+ release. D-glucose failed to modulate resting or stimulated cAMP levels. We suggest that prolonged exposure to pathologically high D-glucose increases formation of inositol polyphosphates, thus increasing Ca2+ release. Ca2+ entry is increased by amplification of unknown signal transduction mechanisms triggered by Ca2+ store depletion.
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8039605
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Genetic variants in promoters and coding regions of the muscle glycogen synthase and the insulin-responsive GLUT4 genes in NIDDM.
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To examine the hypothesis that variants in the regulatory or coding regions of the glycogen synthase (GS) and insulin-responsive glucose transporter (GLUT4) genes contribute to insulin-resistant glucose processing of muscle from non-insulin-dependent diabetes mellitus (NIDDM) patients, promoter regions and regions of importance for translation, as well as coding sequences of the two genes, were studied using single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. The genetic analyses were performed in subgroups of 52 Caucasian NIDDM patients and 25 age-matched healthy volunteers. By applying inverse polymerase chain reaction and direct DNA sequencing, 532 base pairs (bp) of the GS promoter were identified and the transcriptional start site determined by primer extension. SSCP scanning of the promoter region detected five single nucleotide substitutions, positioned at 42, -16, -43, -143, and -250. The three most common variants could be excluded for having major impact on allele-specific GS mRNA expression in muscle. Scanning of GS cDNA revealed one frequent silent polymorphism at codon 342. Moreover, SSCP analysis of approximately 900 bp of the promoter, the 5'-untranslated region, and the coding region of the GLUT4 gene showed four polymorphisms, all single nucleotide substitutions, positioned at -581, 1, 30, and 582. None of the three changes in the regulatory region of the gene had any major influence on expression of the GLUT4 gene in muscle. The variant at 582 in the GLUT4 cDNA was a silent polymorphism at codon 130. Southern blotting of both gene loci did not detect any major abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)
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8039604
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Identification of glutamic acid decarboxylase autoantibody heterogeneity and epitope regions in type I diabetes.
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Glutamic acid decarboxylase (GAD) is an autoantigen of the islet cell antibodies (ICAs) present in type I diabetes. GAD autoantibodies are also found in patients with stiffman syndrome and in certain ICA-positive individuals who rarely develop diabetes on long-term follow-up. This latter subset of ICA has been termed restricted or beta-cell-specific ICA because the antibodies react with only the beta-cells of the islet. By immunoprecipitation of recombinant GAD65 and GAD67 protein and protein fragments, 83% of sera from individuals with new-onset diabetes or prediabetes (n = 30) had GAD65 autoantibodies, but only 26% had GAD67 autoantibodies. In contrast, all restricted ICA sera (n = 6) had both GAD65 and GAD67 autoantibodies. In both types of sera, the binding of GAD67 autoantibodies could be blocked by preincubation of the serum with GAD65 and GAD67, but the binding of GAD65 autoantibodies could not be blocked by preincubation with GAD67. The titer of GAD65 autoantibodies was much higher in the restricted ICA sera (titer > 1:1,000) than in the sera from individuals with new-onset diabetes or prediabetes (titer < 1:100) and was reflected by the greater amount of GAD65 protein immunoprecipitated by restricted ICA sera (2.61 +/- 1.39 U) compared with sera from individuals with new-onset diabetes (0.51 +/- 0.34 U). The restricted ICA sera immunoprecipitated equimolar amounts of GAD65 protein fragments, suggesting a non-conformational or linear epitope; epitope mapping localized the major epitope region to amino acids 361-442 and a second minor epitope region to amino acids 1-195.(ABSTRACT TRUNCATED AT 250 WORDS)
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8039602
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Aldose reductase catalysis and crystallography. Insights from recent advances in enzyme structure and function.
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Enhanced metabolism of glucose via the polyol pathway may play an important role in the pathogenesis of diabetic retinopathy, neuropathy, and nephropathy. Aldose reductase catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step in the polyol pathway. Interruption of the polyol pathway by inhibition of aldose reductase holds considerable promise as a therapeutic measure to prevent or delay the onset and severity of these late complications of diabetes. Dramatic advances in our understanding of the molecular biology, enzymology, and three-dimensional structure of aldose reductase have occurred in recent years, providing new and challenging insights into the enzyme's catalytic mechanism. Recent developments in structure determination of aldose reductase and the implications for evaluation and development of aldose reductase inhibitors are summarized.
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8039603
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NIDDM and its metabolic control predict coronary heart disease in elderly subjects.
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The aim of this study was to evaluate whether noninsulin-dependent diabetes (NIDDM) and its metabolic control and duration predict coronary heart disease (CHD) events during a 3.5-year follow-up in a randomly selected Finnish population sample 65-74 years of age at baseline. Of 1,298 subjects participating in the baseline study, 1,069 were nondiabetic and 229 had NIDDM. During the follow-up, 3.4% of nondiabetic and 14.8% of NIDDM subjects died from CHD or had a nonfatal myocardial infarction (MI). The impact of NIDDM on CHD mortality and morbidity was more marked in women than in men. Odds ratios (ORs) and their 95% confidence intervals for CHD death and nonfatal MI in women with NIDDM compared with women with normal glucose tolerance were 11.7 (3.8-36.4) and 4.7 (3.6-6.1). In men, the corresponding ORs were 0.43 (0.1-1.9) and 1.4 (0.6-3.2). In multiple logistic regression analyses including all study subjects, NIDDM (P < 0.01), male sex (P < 0.05), and previous MI (P < 0.01) predicted CHD death (n = 45). NIDDM (P < 0.01), male sex (P < 0.05), previous MI (P < 0.05), current smoking (P < 0.001), systolic blood pressure (P < 0.001), and low high-density lipoprotein cholesterol (P < 0.01) predicted all CHD events (CHD death or nonfatal MI) (n = 107). In NIDDM subjects, only glycated hemoglobin A1c (GHbA1c) at baseline (P < 0.01) and duration of diabetes (P < 0.05) predicted CHD death (n = 15) and all CHD events (n = 33). There was a significant increase in the risk of CHD death and all CHD events in NIDDM subjects with GHbA1c levels higher than 7.0% compared with diabetic subjects with lower GHbA1c (ORs 4.3 [1.1-16.7] and 2.2 [1.0-5.1]). In conclusion, NIDDM and its metabolic control and the duration of diabetes are important predictors of CHD in elderly subjects, particularly in women.
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8039600
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The role of liver glucosensors in the integrated sympathetic response induced by deep hypoglycemia in dogs.
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The significance of the portohepatic glucosensors for counterregulation in deep hypoglycemia (i.e., glycemia < 2.8 mM) was studied in chronically cannulated male mongrel dogs in the conscious state. A total of 16 experiments were carried out on 6 dogs using the liver clamp technique under hyperinsulinemic conditions (insulin infusion, 39 pmol.min-1.kg-1, 0-150 min). The level of glycemia presented to the liver was made to differ from the systemic arterial glucose level via portal glucose infusion. Tracer-determined rates of glucose clearance and hepatic glucose output (HGO) were assessed using D-[3-3H]glucose (0.26 microCi.min-1). Three protocols were used. In protocol I, liver clamp, systemic hypoglycemia at 2.60 +/- 0.09 mM, and liver glycemia at 3.86 +/- 0.05 mM were achieved with portal glucose infusion (28.2 +/- 3.0 mumol.min-1.kg-1). For protocol II, glucose was infused peripherally (18.2 +/- 4.3 mumol.min-1.kg-1), while systemic and liver glycemia were sustained at deep hypoglycemia, 2.50 +/- 0.08 mM. In protocol III, via peripheral glucose infusion (62.9 +/- 5.8 mumol.min-1.kg-1), systemic and liver glycemia were maintained at a level matched to the liver glycemia during protocol I (3.98 +/- 0.05 mM, P > 0.10). When compared with protocols I and III, the catecholamine response above basal was significantly greater during protocol II with liver and systemic deep hypoglycemia (7.30 +/- 1.51 and 2.89 +/- 0.5 nM for epinephrine and norepinephrine, respectively, P < 0.005). These values reflect net increases in the catecholamine responses of 100% and 85% for epinephrine and norepinephrine when compared with protocol I.(ABSTRACT TRUNCATED AT 250 WORDS)
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8039599
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Clinical and histological correlations of decline in renal function in diabetic patients with proteinuria.
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In 47 patients with diabetic nephropathy (29 type I, 18 type II) renal function and blood pressure (BP) (treated with or without an angiotensin-converting enzyme [ACE] inhibitor, enalapril [10 mg], in 38 hypertensive patients) were followed over 4 years. A percutaneous renal biopsy was performed in all patients initially and repeated in a representative 19 patients with treated hypertension after 4 years. Mean glomerular volume (MGV), interstitial fibrosis (IF), capillary volume, and sclerosed glomeruli (GS) were measured histomorphometrically. Mean fall in creatinine clearance (CCr) was 11.8% after 4 years with no difference between treatment groups or type of diabetes. BP both initially and during treatment correlated with initial and final serum creatinine and CCr (P < 0.01). There were no histomorphometric differences between type I and type II patients or hypertension treatment groups. Initial IF correlated with initial and final serum creatinine and CCr (P < 0.05) in all patients and type I patients alone, MGV correlated inversely with CCr in type I patients (P < 0.05). After 4 years, IF (24.8 vs. 30.0%, P < 0.01) and GS (26 vs. 37%, P < 0.05) increased significantly, and increase in IF correlated with fall in CCr (P < 0.01). Proteinuria and HbA1 did not correlate with indexes of function or structure. In this longitudinal study of patients with diabetic nephropathy, there was a close relation between BP and renal function but no difference between treatment with enalapril and other hypertensive agents. The correlations between renal function and histology at entry and after 4 years suggest that IF is a co-determinant of renal function in diabetic nephropathy.
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8039597
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High glucose attenuates peptide agonist-evoked increases in cytosolic free [Ca2+] in rat aortic smooth muscle cells.
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Incubation of cultured rat aortic smooth muscle cells (ASMCs) in a medium containing high glucose concentrations (25 mM) did not affect the basal cytosolic free calcium ([Ca2+]i) but led to significant reductions in peak [Ca2+]i response evoked by arginine vasopressin, angiotensin II, and endothelin-1 (ET-1). This was observed in both the presence and absence of extracellular Ca2+. Maintenance of rat ASMCs in a medium containing mannose (an osmotic control for high glucose) did not affect either the basal or peptide agonist-evoked increase in [Ca2+]i. However, pretreatment with either the nonselective protein kinase C (PKC) inhibitor staurosporine or the selective PKC inhibitor 2,6-diamino-N-([1-(1-oxotridecyl)-2 piperidinyl] methyl) hexanamide reversed the attenuating effect of high glucose on peak [Ca2+]i response evoked by ET-1. Also, short-term incubation of ASMCs with the active phorbol ester, phorbol 12-myristate 13-acetate, led to a reduction in peak [Ca2+]i response to all three agonists, whereas the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, which does not activate PKC, had no such effect. Although high-glucose treatment of rat ASMCs led to significant reductions in the maximal number of binding sites to the extent of 39% of [125I]ET-1 specific binding, no significant differences in the affinity (Kd approximately 110 pM) characteristics were evident between control and high-glucose treatment groups. It is proposed that incubation of rat ASMCs with high glucose enhances the de novo synthesis of diacylglycerol and activates membrane-bound PKC and that this, in turn, impairs agonist-mediated intracellular Ca2+ mobilization.
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8039598
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High glucose concentration causes a decrease in mesangium degradation. A factor in the pathogenesis of diabetic nephropathy.
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Mesangium enlargement is a constant feature of diabetic nephropathy and is likely to be important in the pathogenesis of this diabetic complication. Whether decreased degradation of mesangium plays any role in causing the enlargement is uncertain. We developed a system of preparing radioactively labeled mesangium matrix from mesangial cell cultures to be used as substrates for studies of mesangium degradation. Degradation is commenced by growing mesangial cells on the labeled matrix and monitored by the release of radioactivity into the culture medium. High glucose concentration (30 mM), whether present 1) when the matrix is being made or 2) when the degradation is taking place, reduces the rate of mesangium degradation. The second but not the first of these two phenomena was abolished by aminoguanidine. Phorbol 12-myristate 13-acetate, added in a manner to antagonize the action of protein kinase C, inhibited mesangium degradation and was not able to nullify the effect of high glucose. Thus it appears unlikely that a high glucose concentration inhibits mesangium degradation by increasing mesangial cell protein kinase C activity. We conclude that decreased degradation of mesangium as a result of hyperglycemia may play a role in causing the mesangium enlargement that occurs in diabetic nephropathy.
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8039596
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Endothelial-dependent vascular effects of insulin and insulin-like growth factor I in the perfused rat mesenteric artery and aortic ring.
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Insulin and insulin-like growth factor I (IGF-I) exhibit vasoactivity. To examine the role of the endothelium in mediating the vascular responses to insulin and IGF-I, we exposed both isolated intact rat mesenteric arteries and rat aortic rings to these growth factors in the presence and absence of endothelium. Perfusion of rat mesenteric arteries with insulin, IGF-I, or IGF-II resulted in the potentiation of arginine vasopressin (AVP)-induced vasoconstriction. Of these growth factors, IGF-I was the most potent, with a significant effect at 0.6 nM and maximal effects at 6.0 nM, followed by IGF-II and insulin. Endothelial denudation or addition of cycloheximide prevented the growth-factor effects. Tissue cGMP levels in the mesenteric artery were minimally affected by growth factors. Insulin and IGF-I vascular effects were not inhibited by BQ123, an endothelin (ET) antagonist that blocked ET-1 enhancement of AVP response. Perfusion of mesenteric arteries with IGF-I for 1 h did not alter vessel ET-1 or ET-1 mRNA contents. Addition of indomethacin markedly inhibited the IGF-I effect on AVP contraction. Thus, the mesenteric vascular effect of insulin and IGF-I is not associated with ET-1 release but appears to link to an increased release of an endothelial-derived contracting factor or the decreased production of an endothelial-derived relaxing factor from the cyclooxygenase pathway. In contrast to their action in the mesenteric artery, insulin (exceeding 100 nM) and IGF-I (1-30 nM) attenuated AVP- and norepinephrine-induced contraction in rat aortic rings. Endothelial-denudation abolished this effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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8039595
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Insulin sensitivity in cystic fibrosis.
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Cystic fibrosis (CF) patients demonstrate a spectrum of pancreatic beta-cell abnormalities. Those with no exocrine insufficiency (NEXO) have normal insulin secretion. Exocrine-insufficient CF patients with overt diabetes (EXO-IT) have impaired insulin secretion and fasting hyperglycemia. Exocrine-insufficient patients without diabetes (EXO) have impaired insulin secretion but maintain normoglycemia. We postulated that EXO individuals compensate for insulin deficiency by increasing insulin sensitivity and investigated glucose utilization in CF. To examine hepatic and peripheral insulin sensitivity, euglycemic-hyperinsulinemic clamp studies were performed by using the hot GINF isotope dilution technique. Insulin was sequentially infused at 0.25, 1.0, and 10.0 mU.kg-1.min-1. Glucose-mediated glucose uptake (GMGU) was assessed on another day with hyperglycemic clamp studies, during which insulin and somatostatin were infused to hold insulin-mediated glucose uptake constant between the two clamp studies. Skeletal muscle GLUT4 levels were assessed in EXO and control patients with Western blotting. Three patterns of peripheral and hepatic insulin sensitivity were seen that were related to the degree of pancreatic beta-cell dysfunction. NEXO individuals had normal peripheral and hepatic insulin sensitivity. EXO individuals had enhanced peripheral insulin sensitivity that was not associated with a change in skeletal muscle glucose transporter abundance compared with control patients; paradoxically, EXO subjects demonstrated hepatic insulin resistance. EXO-IT had peripheral and hepatic insulin resistance. GMGU was diminished in both EXO and EXO-IT subjects. The unique combination of increased hepatic glucose production and increased peripheral glucose utilization seen in EXO may be a metabolic adaptation to increased peripheral energy needs.(ABSTRACT TRUNCATED AT 250 WORDS)
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8039594
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Reduced plasma peroxyl radical trapping capacity and increased susceptibility of LDL to oxidation in poorly controlled IDDM.
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Oxidation of low-density lipoproteins (LDLs) has been postulated to play an important role in atherogenesis. Because oxidant stress may be increased and antioxidant defenses reduced in diabetes, the susceptibility of LDL to oxidative modification and total peroxyl radical trapping potential (TRAP) of plasma were evaluated in subjects with poorly controlled insulin-dependent diabetes mellitus (IDDM). The lag phase of conjugated diene formation after initiation of LDL oxidation by the addition of copper was shorter in diabetic subjects than in normal control subjects (126 +/- 11 vs. 165 +/- 15 min [means +/- SE], P < 0.05). This could not be attributed to the presence of oxidation-susceptible, small, dense LDL particles in the diabetic subjects, whose lipoprotein particle distribution did not differ from the control subjects. However, the total TRAP of plasma, a measure of antioxidant defense, was reduced (626 +/- 34 vs. 877 +/- 41 microM, P < 0.0001) in diabetes. Of the plasma antioxidants measured, only uric acid and vitamin A were decreased in diabetes (P < 0.01), and both levels correlated with TRAP (r = 0.75, P < 0.001; r = 0.54, P < 0.001, respectively). The correlation between uric acid levels and TRAP persisted when the diabetes and control groups were analyzed separately. The reduced TRAP of plasma and the increased susceptibility of LDL to oxidative modification observed is consistent with a role for lipoprotein oxidation in the pathogenesis of atherosclerosis in IDDM.
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8039593
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High level of concordance between assays for glutamic acid decarboxylase antibodies. The First International Glutamic Acid Decarboxylase Antibody Workshop.
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Glutamic acid decarboxylase antibodies (GADAbs) are being increasingly used in clinical and research programs for the prediction and classification of insulin-dependent diabetes mellitus (IDDM). A number of different assay formats for the measurement of GADAbs have been reported, but the degree of concordance between assays is unknown. In this study, GADAbs were measured on 16 coded sera in 34 assays to examine concordance between GADAb assays and establish the feasibility of an international GADAb standard of measurement unit. The 16 lyophilized coded samples consisted of sera from healthy control subjects (n = 2), IDDM patients (n = 3), a patient with polyendocrine autoimmunity (n = 1), and duplicate dilutions of plasmapheresis serum from a patient with stiff-man syndrome (SMS). A high level of concordance was found in the ranking of GADAb levels (P = 0.99, Friedman's test) in the samples. Thirteen (38%) assays could reproducibly distinguish dilutions of SMS serum and detect GADAbs in all IDDM and polyendocrine autoimmunity sera tested. Although assessed on only four samples, disease specificity was 100% in 29 assays. The majority of assays that immunoprecipitated radiolabeled GAD gave high results for sensitivity and specificity. Enzyme-linked immunosorbent assays and assays using immunofluorescence were generally less sensitive. Several assays, in particular those measuring GAD enzymatic activity immunoprecipitated in fluid phase from rat brain homogenate, showed a prozone-like phenomenon in the SMS dilution curve. Interpolation of results from a standard curve into workshop units resulted in relatively low scatter in samples with lower levels of GADAbs. Hence, the use of an international reference serum to enable comparison of results between laboratories appears feasible.(ABSTRACT TRUNCATED AT 250 WORDS)
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