Biomedical subjects

J Welsh

Publications and source records attributed to J Welsh.

At least 109 records · Page 6Linked to original sources

PubMed1992

1,25(OH)2D3 increases membrane associated protein kinase C in MDBK cells.

To determine whether 1,25-dihydroxycholecalciferol [1,25(OH)2D3] affects protein kinase C (PKC) activity in kidney, as has been demonstrated in HL-60 cells we measured 1,25(OH)2D3 binding, PKC activity and PKC immunoreactivity in Madin Darby bovine kidney (MDBK) cells, a normal renal epithelial cell line derived from bovine kidney. Our data demonstrate that MDBK cells exhibit specific high affinity binding for 1,25(OH)2D3, indicating the presence of the vitamin D receptor (VDR). Treatment of MDBK cells with 1,25(OH)2D3 for 24 h increased membrane PKC activity and immunoreactivity. The effect of 1,25(OH)2D3 was dose-dependent, with a peak effect observed at 10(-7)M 1,25(OH)2D3. The 1,25(OH)2D3 induced increase in membrane PKC was paralleled by a comparable decrease in cytosolic PKC activity and amount. Although time course studies were consistent with a VDR mediated effect of 1,25(OH)2D3 on PKC protein synthesis, total PKC activity was not increased by 1,25(OH)2D3, suggesting an effect on PKC translocation or localization. These results suggest that 1,25(OH)2D3 modulates PKC mediated events in kidney, a classic target for this steroid hormone.

Animals↗

PubMed1992

Genomic fingerprinting by arbitrarily primed polymerase chain reaction resolves Borrelia burgdorferi into three distinct phyletic groups.

The causative agent of Lyme disease, Borrelia burgdorferi, was first identified by Burgdorfer et al. in 1982 (W. Burgdorfer, A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis, Science 216:1317-1319, 1982) and was isolated by Barbour et al. in 1983 (A. G. Barbour, W. Burgdorfer, S. E. Hayes, O. Peter, and A. Aeschlimann, Curr. Microbiol. 8:123-126, 1983). Since then, a large number of isolates have been collected, and there have been questions regarding the relationships among the various strains. Using genomic fingerprinting by an arbitrarily primed polymerase chain reaction, we resolved into three groups a collection of Eurasian and North American isolates of spirochetes that are generally categorized as B. burgdorferi. Group I strains have been identified in both North America and Eurasia, while strains belonging to Borrelia groups II and III have been found only in Eurasia. These same three groups have also been delineated by Baranton et al. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:370-375, 1992) by independent methods. Two isolates are distinct from all of the other strains in our collection but are clearly members of the genus Borrelia.

Bacterial Typing Techniques↗

PubMed1992

PCR-amplified length polymorphisms in tRNA intergenic spacers for categorizing staphylococci.

The intergenic spacers between some adjacent tRNA genes were shown to be polymorphic in length when closely related Staphylococcus species were compared. A simple procedure was developed to detect and sequence these tRNA intergenic length polymorphisms (tRNA-ILPs). A comparison of homologous tRNA gene sequences flanking these ILPs in three Staphylococcus species was used to derive primers for high-stringency amplification of the ILPs by the polymerase chain reaction (PCR). The detection of tRNA-ILPs by PCR allowed the classification of virtually all strains from the five species of Staphylococcus that were examined. The procedure used to identify, sequence and derive primers for PCR detection of tRNA-ILPs in Staphylococcus should be applicable to many other genera of eubacteria. These primers could be used on uncultured material such as clinical samples.

Base Sequence↗

PubMed1992

Screening for drinking disorders in the elderly using the CAGE questionnaire.

OBJECTIVE: To assess the performance of the CAGE questionnaire in identifying elderly medicine outpatients with drinking problems. DESIGN: Cross-sectional design, with the alcohol module of the Diagnostic Interview Schedule as the criterion standard. SETTING: The outpatient medical practice of an urban university teaching hospital. PATIENTS: Consecutive patients 18 years or older who signed a consent form approved by the university's institutional review board. For this study, 323 patients greater than or equal to 60 years old. MAIN OUTCOME MEASURES: Sensitivity, specificity, receiver operating characteristics (ROC) curve and positive predictive value for CAGE scores of 0-4 for patients 60 years or older. RESULTS: Thirty-three percent of the sample group met study criteria for a history of drinking problems, including 63% of the male patients and 22% of the female patients. The sensitivity and specificity for a cut-off score of one for all patients was 86% and 78%, respectively, and 70% and 91% for a cut-off of two. The calculation of the area under the ROC curve was .86, and the positive predictive value of CAGE scores of 0-4 were 33%, 66%, 79%, 82%, and 94%, respectively. The predictive value for any score was higher in males than females, reflecting the higher prevalence of problems in the male population. CONCLUSIONS: The CAGE can effectively discriminate elderly patients with a history of drinking problems from those without such a history. The chosen cut-off score should consider the prevalence of drinking problems in the population being tested.

Aged↗

PubMed1992

Length polymorphisms in tRNA intergenic spacers detected by using the polymerase chain reaction can distinguish streptococcal strains and species.

Intergenic tRNA spacers from strains of streptococcal groups A, B, and G were amplified by using the polymerase chain reaction (PCR) at low stringency with consensus tRNA gene primers. Cloning and sequencing showed that many of the homologous intergenic spacers differed in length between species. The sequences of the tRNA genes that flank these polymorphic spacers were determined and used to synthesize fully complementary primers. With these primers at high stringency, PCR products which varied in lengths from 53 to 71 bp, depending on the species or strain, were obtained from streptococcal DNAs, even in the presence of a 1,000-fold mass excess of human DNA. PCR products, the lengths of which could also be used for classification, were obtained at high stringency from a few genera closely related to Streptococcus. No products were obtained from genomic DNAs from more distantly related genera. Production of species- or strain-specific tRNA intergenic length polymorphisms with primers that generate characteristic products from a variety of species within the same genus should be applicable to many organisms, including those that would otherwise be difficult to culture or identify.

Base Sequence↗

PubMed1991

Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers.

Polymorphisms in genomic fingerprints generated by arbitrarily primed PCR (AP-PCR) can distinguish between slightly divergent strains of any organism. Single oligodeoxyribonucleotide (oligo) primers have been used to generate such fingerprints, with the same primer being present at the 5' end of both strands for every PCR product. We used three arbitrary oligos, individually and in pairs, to generate six different genomic fingerprints of the same mouse genomic DNAs. Fewer than half of the products in genomic fingerprints generated using the oligos in pairs were the same as those produced by AP-PCR using one of the three oligos alone. Thus, a few oligos could be used in a very large number of single and pairwise combinations, each producing a distinct AP-PCR fingerprint with the potential to identify new polymorphisms. For example, 50 oligos can be used in a matrix of pairwise combinations to produce 2,500 fingerprints, in which at least half the data can be expected to be unique to each pair. We demonstrate this principle by using two oligos, alone and together, to generate three sets of fingerprints and map thirteen polymorphisms in the C57BL/6J x DBA/2J set of recombinant inbred mice.

Animals↗

PubMed1991

Genomic fingerprints produced by PCR with consensus tRNA gene primers.

The polymerase chain reaction using only a single 'consensus' tRNA gene primer, or a pair of primers facing outward from tRNA genes, amplifies a set of DNA fragments in bacterial, plant and animal genomic DNAs. Presumably, these PCR fingerprints are mainly derived from the regions between closely linked tRNA genes. The pattern of the PCR products is determined by which genomes and which primer(s) are used. Genomic fingerprints are largely conserved within a species and, in bacteria, most products in the fingerprint are conserved between closely related species. Thus, PCR with tRNA gene consensus primers helps to identify species and genera.

Base Sequence↗

PubMed1991

Polymorphisms generated by arbitrarily primed PCR in the mouse: application to strain identification and genetic mapping.

Polymorphisms in genomic fingerprints generated by arbitrarily primed PCR (AP-PCR) can distinguish between strains of almost any organism. We applied the technique to the mouse (Mus musculus). The characteristic differences in the AP-PCR genomic fingerprints between strains will be of value in strain identification and verification. Using one primer, we genetically mapped four polymorphisms in a set of C57BL/6J x DBA/2J recombinant inbreds. One of these polymorphisms is a length variant. The method will allow rapid genetic mapping of DNA polymorphisms without Southern blotting.

Animals↗

PubMed1991

Regulation of renal 25(OH)D3 1 alpha-hydroxylase: signal transduction pathways.

In vitro, activation of the cAMP signalling pathway stimulates, whereas activation of PKC inhibits, 1,25(OH)2D3 synthesis. Since PTH activates both pathways, the ultimate effect of PTH on 1 alpha-hydroxylation in vivo likely depends on other endocrine-autocrine factors that impinge onto these signal transduction cascades. For example, 1,25(OH)2D3, a known repressor of 1 alpha-hydroxylation, may increase renal PKC amount-activity, thereby enhancing the inhibitory arm and preventing PTH stimulation of the 1-OHASE. In contrast, studies with diabetic rats suggest that insulin may allow cAMP-mediated stimulation to override PKC-mediated inhibition of 1-OHASE activity. Analogous to models proposed for regulation of adrenal steroid hydroxylases, it is likely that regulation of renal vitamin D hydroxylation involves both acute (reversible phosphorylation) and chronic (modulation of gene expression) mechanisms. However, the molecular details of these regulatory mechanisms remain to be resolved.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1991

Vitamin D receptors and compensatory tissue growth in spontaneously diabetic BB rats.

Untreated diabetic (BB) rats exhibited compensatory intestinal growth which was associated with hyperplasia and was accompanied by an increase in unoccupied vitamin D receptors. Although vitamin D receptors were increased, low circulating 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] prevented amplification of the action of 1,25(OH)2D3, as evidenced by reductions in calbindin D-9K and alkaline phosphatase activity in the BB rat intestine compared to control. In the kidney, a lesser degree of compensatory growth was observed which was not associated with hyperplasia, and no significant effect of diabetes on vitamin D receptors or calbindin D-28K was observed. These studies suggest tissue-specific changes in 1,25(OH)2D3 metabolism during spontaneous diabetes which may be related to the hyperplasia which occurs during compensatory tissue growth.

Animals↗

PubMed1990

Fingerprinting genomes using PCR with arbitrary primers.

Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).

Animals↗

PubMed1990

A report of three multiclinic trials evaluating arbaprostil in arthritic patients with ASA/NSAID gastric mucosal damage. The Upjohn Company Arbaprostil ASA/NSAID Gastric Mucosal Damage Treatment Study Groups.

Three randomized, placebo-controlled multiclinic trials involving arbaprostil dosages of (a) 10 micrograms; (b) 25 micrograms; and (c) 10, 25, or 50 micrograms orally for 4 wk in patients older than 18 yr with rheumatoid arthritis or osteoarthritis who had endoscopically documented nonsteroidal antiinflammatory drug-associated gastric mucosal damage were conducted in the United States. All patients continued taking the nonsteroidal antiinflammatory drugs and were reendoscoped after 4 wk of therapy. Success at that time was defined as complete resolution of all gastric mucosal damage. Six hundred fifty-eight patients were enrolled in the three trials. Significantly more patients experienced healing in the arbaprostil treatment groups in all trials compared with those who received placebo. The healing rates in the various trials were 68% and 32% (10 micrograms vs. placebo; p = 0.007); 77% and 23% (25 micrograms vs. placebo; p less than 0.001); and 52%, 46%, 35%, and 16% (50, 25, and 10 micrograms vs. placebo; p less than 0.001, less than 0.001, and 0.002, respectively). Diarrhea, mostly of a mild nature, was the only arbaprostil-associated side effect and was found with the 25- and 50-microgram dosages (33% and 59%, respectively). No exacerbation of arthritis signs or symptoms was found. Arbaprostil at doses with varying effects on gastric acid secretion (25 and 50 micrograms) was documented in these trials to be an effective and safe agent for healing gastric mucosal damage associated with aspirin or other nonsteroidal antiinflammatory drugs in patients with either rheumatoid arthritis or osteoarthritis without adversely affecting joint symptomatology.

Administration, Oral↗

PubMed1990

Cloning of PCR-amplified total cDNA: construction of a mouse oocyte cDNA library.

We describe a general method for the synthesis and cloning of cDNA, applicable to cases in which the availability of biological material for mRNA extraction is extremely limited. A protocol allowing amplification of a heterogeneous mixture of cDNAs by the polymerase chain reaction has been devised and applied successfully to the construction of an apparently representative cDNA library, using as a model of a scarce RNA source 50 mouse ovulated eggs that can yield a maximum of 1.75 ng of poly(A)+ RNA. However, about 5% of the material obtained after amplification was adequate for cloning. Using the cloned sequences, we have derived a preliminary indirect measurement of the sequence complexity of the maternal poly(A)+ RNA in this mammalian oocyte.

Animals↗

PubMed1990

Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7:K1 strain VW187.

We recently cloned biosynthesis genes for the O7-lipopolysaccharide (O7-LPS) side chain from the Escherichia coli K-1 strain VW187 (M. A. Valvano, and J. H. Crosa, Infect. Immun. 57:937-943, 1989). To characterize the O7-LPS region, the recombinant cosmids pJHCV31 and pJHCV32 were mutagenized by transposon mutagenesis with Tn3HoHo1, which carries a promoterless lac operon and can therefore generate lacZ transcriptional fusions with target DNA sequences. Cells containing mutated plasmids were examined for their ability to react by coagglutination with O7 antiserum. The LPS pattern profiles of the insertion mutants were also investigated by electrophoresis of cell envelope fractions, followed by silver staining and immunoblotting analysis. These experiments identified three phenotypic classes of mutants and defined a region in the cloned DNA of about 14 kilobase pairs that is essential for O7-LPS expression. Analysis of beta-galactosidase production by cells carrying plasmids with transposon insertions indicated that transcription occurs in only one direction along the O7-LPS region. In vitro transcription-translation experiments revealed that the O7-LPS region encodes at least 16 polypeptides with molecular masses ranging from 20 to 48 kilodaltons. Also, the O7-LPS region in VW187 was mutagenized by homologous recombination with subsets of the cloned O7-LPS genes subcloned into a suicide plasmid vector. O7-LPS-deficient mutants of VW187 were complemented with pJHCV31 and pJHCV32, confirming that these cosmids contain genetic information that is essential for the expression of the O7 polysaccharide.

Chromosome Mapping↗

PubMed1990

Vitamin D receptors in intestine, kidney and thymus of streptozotocin diabetic rats.

Our data confirms previous reports of intestinal and kidney compensatory growth, low plasma 1,25(OH)2D3 and up regulation of intestinal VDRs during untreated diabetes. All of these parameters were normalized in diabetic rats treated with exogenous insulin. There were no alterations in VDR numbers in kidneys or thymus of untreated diabetic animals, indicating that up-regulation of VDRs during untreated diabetes did not occur in all vitamin D target tissues. Compensatory growth of the intestine during untreated diabetes was associated with both hypertrophy and hyperplasia, whereas diabetic kidney growth appeared to be associated with hypertrophy without hyperplasia. Diabetes did not affect somatic index of thymus. The data suggests that the up regulation of VDRs during untreated diabetes may be unique to the intestine and further that VDR up regulation may be related to hyperplasia. Calbindin D-9K was significantly lower in diabetic intestine, suggesting that low circulating 1,25(OH)2D3 prevented amplification of 1,25(OH)2D3's action despite up regulation of intestinal VDRs.

Alkaline Phosphatase↗

PubMed1990

1,25 Dihydroxycholecalciferol and the genesis of hypocalcaemia in magnesium-deficient chicks.

The role of 1,25 dihydroxycholecalciferol (1,25 DHCC) in the genesis of hypocalcaemia of magnesium (Mg) deficiency was studied in chicks fed control or Mg-deficient diets with varying calcium (Ca) content. In one study, Ca and Mg deficiencies were induced simultaneously whereas in a second study, exposure to low dietary Ca followed induction of Mg deficiency. In both studies, Mg-deficient chicks fed adequate dietary Ca were hypocalcaemic yet had normal circulating 1,25 DHCC, whereas Mg-deficient chicks fed low dietary Ca maintained normocalcaemia and had increased 1,25 DHCC. Bone Ca content, which was raised in Mg-deficient chicks fed adequate Ca, decreased in association with increased 1,25 DHCC and normalization of plasma Ca during exposure to low dietary Ca. The results suggest that the inappropriately normal 1,25 DHCC observed in conventional Mg deficiency may contribute to the pathological hypocalcaemia. In addition, these studies indicate that hypomagnesaemia per se does not limit 1,25 DHCC production during Mg deficiency.

Animals↗

PubMed1988

Synthesis and properties of novel psoralen derivatives.

We have synthesized a set of new trimethylpsoralen derivatives that are characterized by a chain extending from the 4'-position of the furan ring and linked to this ring by an aminomethylene group. The nature of the side chain can be varied widely. In these derivatives, the chains contain either amino or ethylene oxide units for enhanced water solubility and allow the introduction of a thiol or amine group to nucleic acids. These compounds represent the first set of thiolated psoralen derivatives, and their usefulness is demonstrated in several nucleic acid cross-linking experiments. The reagents can be used to create both intraduplex reversible cross-links between the two single-strand partners in a DNA double helix and interduplex reversible cross-links between two DNA double helices.

Cross-Linking Reagents↗

PubMed1988

The regulation of adenylate cyclase in liver membranes of lean and obese mice.

The modulation of adenylate cyclase by guanosine triphosphate (GTP) and hormones was examined in liver membranes of lean and ob/ob mice, to determine whether a defective regulation of cyclase similar to that found in adipocyte membranes was present. In conjunction with GTP, glucagon was a powerful stimulant of cyclase in both types of membranes. In contrast, GTP alone or in conjunction with isoproterenol and norepinephrine stimulated significantly less in the membranes of the lean than in those of the obese mouse. In addition, low concentrations of norepinephrine elicited an inhibitory response in membranes of the lean mouse, but not in those of the obese. This inhibitory effect of norepinephrine was abolished by the alpha 2-antagonist yohimbine and by treatment with pertussis toxin, but not by propranolol or treatment with cholera toxin. These data show that it is possible to demonstrate inhibitory effects of guanine nucleotides on cyclase in the membranes from lean but not those from obese mice and suggest a defect in inhibitory regulation in the tissue of the obese.

Adenylate Cyclase Toxin↗