Search PubMed⌕ Search

Biomedical subjects

J M Fisher

Publications and source records attributed to J M Fisher.

At least 37 records · Page 2Linked to original sources
PubMed1997

Pit-1/growth hormone factor 1 splice variant expression in the rhesus monkey pituitary gland and the rhesus and human placenta.

We have examined the expression of Pit-1 messenger RNA (mRNA) splice variants in the nonhuman primate pituitary and in rhesus and human placenta. Full-length complementary DNAs (cDNAs) representing Pit-1 and the Pit-1 beta splice variants were cloned from a rhesus monkey pituitary cDNA library and were readily detectable by RT-PCR with rhesus pituitary gland RNA. The Pit-1T variant previously reported in mouse pituitary tumor cell lines was not detectable in normal rhesus pituitary tissue, although two novel splice variants were detected. A cDNA approximating the rat Pit-1 delta 4 variant was cloned but coded for a truncated and presumably nonfunctional protein. Only by using a nested RT-PCR approach were Pit-1 and Pit-1 beta variants consistently detectable in both human and rhesus placental tissue. The Pit-1 beta variant mRNA was not detectable in JEG-3 choriocarcinoma cells unless the cells were stimulated with 8-Br-cAMP. Immunoblot studies with nuclear extracts from primary rhesus syncytiotrophoblast cultures or JEG-3 choriocarcinoma cells indicated that although mRNA levels were very low, Pit-1 protein was detectable in differentiated cytotrophoblasts, and levels increased after treatment with 8-Br-cAMP. Two major species of Pit-1 protein were detected that corresponded to the two major bands in rat pituitary GH3 cell nuclear extracts. Low levels of slightly larger bands also were seen, which may represent Pit-1 beta protein or phosphorylated species. We conclude that Pit-1 splice variants expressed in the primate pituitary gland differ from those in the rodent gland and that the Pit-1 and Pit-1 beta mRNAs expressed in the placenta give rise to a pattern of protein expression similar to that seen in pituitary cells, which is inducible by treatment with 8-Br-cAMP.

Amino Acid Sequence↗
PubMed1997

Psychosocial job factors associated with back and neck pain in public transit operators.

OBJECTIVES: This cross-sectional study examined associations between psychosocial job factors and the prevalence of nondisabling back and neck pain in professional drivers after physical work load was taken into account. METHODS: A total of 1449 transit vehicle operators completed a medical examination and a questionnaire yielding information on demographic and anthropometric variables, health status, and physical and psychosocial job factors. Company records were used to supplement information on employment history. Physical work load was measured in life-time years and current weekly hours of professional driving. The relation of psychosocial factors with back or neck pain was analyzed by logistic regression models adjusted for past and current physical work load, vehicle type, age, gender, body height, and weight. RESULTS: The main result of this study was that both physical work load and psychosocial factors were simultaneously and independently associated with back or neck pain. Psychosocial factors associated with back or neck pain included extended uninterrupted driving driving periods, frequency of job problems, high psychosocial demands, high job dissatisfaction, and low supervisory support. An analysis of specific job problems is provided which may be useful in setting priorities for research and intervention efforts in this high risk occupation. CONCLUSION: The results provide support for the role of psychosocial job characteristics in the etiology of back or neck pain in occupational settings.

Adult↗
PubMed1997

Cytochrome P450 2E1 is the principal catalyst of human oxidative halothane metabolism in vitro.

The volatile anesthetic halothane undergoes substantial biotransformation generating metabolites that mediate hepatotoxicity. Aerobically, halothane undergoes cytochrome P450-catalyzed oxidation to trifluoroacetic acid (TFA), bromide and a reactive intermediate that can acetylate liver proteins. These protein neo-antigens stimulate an immune reaction that mediates severe hepatic necrosis ("halothane hepatitis"). This investigation identified the human P450 isoform(s) that catalyze oxidative halothane metabolism. Halothane oxidation by human liver microsomes was assessed by TFA and bromide formation. Eadie-Hofstee plots of TFA and bromide formation were both nonlinear, suggesting the participation of multiple P450s. Microsomal TFA and bromide formation were inhibited 45 to 66% and 21 to 26%, respectively, by the P450 2A6 inhibitors 8-methoxypsoralen and coumarin, 84 to 90% by the P450 2E1 inhibitor 4-methylpyrazole and 55% by diethyldithiocarbamate, an inhibitor of both P450 2A6 and 2E1. Selective inhibitors of P450s 1A, 2B6, 2C9/10, 2D6 and 3A4 did not affect halothane oxidation. At saturating halothane concentrations (2.4 vol%) only cDNA-expressed P450 2A6 and 2B6 catalyzed significant rates of TFA and bromide formation, and P450 2E1 catalyzed comparatively minimal oxidation. Conversely, at subsaturating halothane concentrations (0.30 vol%), metabolism by P450 2E1 exceeded that by P450 2A6. Among a panel of human liver microsomes, there were significant linear correlations between halothane oxidation and P450 2A6 activity and protein content at saturating halothane concentrations (2.4 vol%), and a significant correlation between metabolite formation and P450 2E1 activity (but not P450 2A6 activity) at subsaturating concentrations (0.12 vol%). These experiments suggested P450 2A6 and 2E1 as the predominant catalysts at saturating and subsaturating halothane concentrations, respectively. Further kinetic analysis using cDNA-expressed P450 and liver microsomes clearly demonstrated that P450 2E1 is the high affinity/low capacity isoform (Km = 0.030-0.053 vol%) and P450 2A6 is the low affinity/high capacity isoform (Km = 0.77-1.2 vol%). Evidence was also obtained for substrate inhibition of P450 2E1. The in vitro clearance estimates (Vmax/Km) for microsomal P450 2E1 (4.3-5.7 ml/min/g) were substantially greater than those for microsomal P450 2A6 (0.12-0.21). These clearances, as well as rates of apparent halothane oxidation predicted from kinetic parameters in conjunction with plasma halothane concentrations measured during clinical anesthesia in humans, demonstrated that both P450 2E1 and P450 2A6 participate in human halothane metabolism, and that P450 2E1 is the predominant catalytic isoform.

Anesthetics, Inhalation↗
PubMed1997

Mechanisms of enhanced oral availability of CYP3A4 substrates by grapefruit constituents. Decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins.

Grapefruit juice increases the oral availability of a variety of CYP3A4 substrates. It has been shown that recurrent grapefruit juice ingestion results in a loss of CYP3A4 from the small bowel epithelium. We now show that the reduction in intestinal CYP3A4 concentration is rapid; a 47% decrease occurred in a healthy volunteer within 4 hr after consuming grapefruit juice. To identify the specific components of the juice responsible for this effect, we used a recently developed Caco-2 cell culture model of human intestinal epithelium that expresses catalytically active CYP3A4. We found that grapefruit oil and two furanocoumarin constituents (6', 7'-dihydroxybergamottin and a closely related dimer) caused a dose-dependent fall in CYP3A4 catalytic activity and immunoreactive CYP3A4 concentration. The effect was selective in that concentrations of CYP1A1 and CYP2D6 did not fall, consistent with previous results obtained in vivo. Assays of various juices confirmed that 6',7'-dihydroxybergamottin is the major furanocoumarin present and, although its concentration varies significantly among types and brands of grapefruit juice, it is consistently present in concentrations exceeding the IC50 (1 microM) for loss of midazolam 1'-hydroxylase activity determined in the Caco-2 cells. Studies with recombinant CYP3A4 revealed that 6', 7'-dihydroxybergamottin is a mechanism-based inactivator, which supports the idea that loss of CYP3A4 results from accelerated degradation of the enzyme. We conclude that the effect of grapefruit juice on oral availability of CYP3A4 substrates can be largely accounted for by the presence of 6',7'-dihydroxybergamottin although other furanocoumarins probably also contribute.

Biological Availability↗
PubMed1997

Characterization of interintestinal and intraintestinal variations in human CYP3A-dependent metabolism.

Cytochrome P450 3A (CYP3A) metabolizes a diverse array of clinically important drugs. For some of these (e.g., cyclosporine, verapamil, midazolam), CYP3A in the intestinal mucosa contributes to their extensive and variable first-pass extraction. To further characterize this phenomenon, we measured CYP3A content and catalytic activity toward the probe substrate midazolam in mucosa isolated from duodenal, jejunal and ileal sections of 20 human donor intestines. For comparison, the same measurements were performed for 20 human donor livers, eight of which were obtained from the same donors as eight of the intestines. Excellent correlations existed between homogenate and microsomal CYP3A content for the three intestinal regions. Median microsomal CYP3A content was greatest in the duodenum and lowest in the ileum (31 vs. 17 pmol/mg of protein). With respect to midazolam 1'-hydroxylation kinetics, the median Km for each intestinal region was similar to the median hepatic Km, approximately 4 microM. In contrast, the median Vmax decreased from liver to duodenum to jejunum to ileum (850 vs. 644 vs. 426 vs. 68 pmol/min/mg). Intrinsic clearance (Vmax/Km) followed a similar trend for the intestinal regions; median duodenal intrinsic clearance was comparable to hepatic intrinsic clearance (157 and 200 microl/min/mg, respectively). Vmax correlated with CYP3A content for all tissues except the ileum. Duodenal and jejunal Vmax and CYP3A content varied by >30-fold among donors. Microsomes prepared from every other 1-foot section of six intestines were also analyzed for CYP3A as well as for two coenzymes. In general, CYP3A activity, CYP3A content and CYP reductase activity rose slightly from duodenum to middle jejunum and then declined to distal jejunum and ileum. Cytochrome b5 content and cytochrome b5 reductase activity varied little throughout the intestinal tract. Regional intrinsic midazolam 1'-hydroxylation clearance was greatest for the jejunum, followed by the duodenum and ileum (144, 50 and 19 ml/min, respectively). Collectively, these results demonstrate that the upper small intestine serves as the major site for intestinal CYP3A-mediated first-pass metabolism and provides a rationale for interindividual differences in oral bioavailability for some CYP3A substrates.

Aryl Hydrocarbon Hydroxylases↗
PubMed1996

Neutrophil rolling altered by inhibition of L-selectin shedding in vitro.

The L-selectin adhesion molecule is involved in guiding leukocytes to sites of inflammation. L-selectin is cleaved by an unusual proteolytic activity at a membrane-proximal site resulting in rapid shedding from the cell surface. Although it has been demonstrated that L-selectin mediates, in part, the early event of leukocyte rolling under hydrodynamic flow, the contribution of shedding to L-selectin function has remained unknown. Here we show that hydroxamic acid-based metalloprotease inhibitors block L-selectin downregulation from the cell surface of stimulated neutrophils, without affecting Mac-1 mobilization or general neutrophil activation, and inhibit cleavage of L-selectin in a cell-free system. Unexpectedly, the hydroxamic acid-based inhibitors reduced neutrophil rolling velocity under hydrodynamic flow, resulting in increased neutrophil accumulation. These results suggest that L-selectin is cleaved in seconds--much faster than previously suspected--during the process of rolling under hydrodynamic flow, and that shedding of L-selectin may contribute significantly to the velocity of leukocyte rolling. L-selectin shedding during rolling interactions may be physiologically important for limiting leukocyte aggregation and accumulation at sites of inflammation.

Cell Adhesion↗
PubMed1996

Identification of a nuclear-specific cyclophilin which interacts with the proteinase inhibitor eglin c.

We have identified a novel human cyclophilin (hCyP-60) which interacts with the proteinase inhibitor eglin c using the yeast two-hybrid system. A cDNA isolated from a Raji B lymphocyte library reveals a domain showing sequence similarity to known cyclophilins flanked by unique N- and C-terminal residues. In addition, hCyP-60 contains a tyrosine residue (Tyr 389) instead of a tryptophan residue found in most eukaryotic cyclophilins at a position important for cyclosporin binding. Northern and Western analysis reveal widespread expression with considerable tissue-specific variation. Specifically, the highest levels of mRNA are detected in the thymus, pancreas, testis, and K-562 cell line, while the most protein is detected in the kidney. Immunohistochemistry indicates a nuclear-specific localization both in transfected cells and tissue sections. hCyP-60's specific subcellular localization and conserved amino acid sequence suggest that it may play a specific role in the nucleus.

Amino Acid Isomerases↗
PubMed1996

Development of a PCR-based allele-specific assay from an RFLP probe linked to resistance to cereal cyst nematode in wheat.

The RFLP locus Xglk605 identified by the probe Tag605 maps to a proximal position on the long arm of wheat chromosome 2B about 7 cM away from a gene conditioning resistance to cereal cyst nematode in the wheat line AUS10894. The clone Tag605 was partially sequenced and the PCR primer set AWP1 was designed. The 292-bp product, which showed no polymorphism between varieties, was cloned and sequenced. A single base difference was found in the sequence of the AWP1 products amplified and cloned from the wheat lines AUS10894 and "Spear'. PCR primers were designed with 3' termini that corresponded to the two alleles. A dual-PCR system was developed in which the primer sets AWP2 and AWP3 produced allele-specific amplification. The concentration of the oligonucleotide primers and the sequence of the primer-template mismatches were critical to the success of discriminatory allele amplification.

Alleles↗
PubMed1995

Occupational and nonoccupational correlates of alcohol consumption in urban transit operators.

BACKGROUND: The influence of occupation and the worksite has emerged as an important area of study in research on alcohol consumption. Occupational and nonoccupational factors were studied in relation to alcohol consumption using data from a 1983-1985 cross-sectional study of transit operators. METHODS: A total of 1.853 operators underwent a medical examination for driver's license renewal (including information on age, ethnicity, gender, education). Of these operators, 1,448 completed a questionnaire about occupational (e.g., time of shift, job stressors) and nonoccupational (e.g., personality, life stressors) factors. From either the medical examination or the questionnaire, weekly alcohol consumption was available for 1,820 operators. Variables related to alcohol consumption in previous studies, or theoretically linked to consumption, were analyzed in relation to heavy ( > or = 15 drinks/week) and average weekly consumption. RESULTS: Heavy and average consumption were both related to several nonoccupational variables, including demographic (age, ethnicity, gender, marital status), personality (depression, anger expression), and life stress variables (i.e., life events). Heavy and average consumption were also related to several occupational variables, including job history (number of years driving, specific worksite) and job stressors. Neither measure was related to subjective job content (job demand, decision latitude). CONCLUSIONS: Variability in consumption by demographic factors among this population reflects that seen in society as a whole. However, occupational factors may influence consumption, since consumption was strongly related to (a) specific worksite and time of shift and (b) reported job stressors. Clarifying the exact influence of occupational and worksite factors on alcohol consumption will depend on the convergence of findings from different research designs (e.g., cross-sectional, longitudinal, ethnographic).

Adult↗
PubMed1995

Trisomy 18: studies of the parent and cell division of origin and the effect of aberrant recombination on nondisjunction.

We have studied the mechanism of origin of 63 cases of trisomy 18. In 2 the additional chromosome was paternal in origin, and in the remaining 61 it was maternal in origin. Both paternal cases were attributable to a postzygotic mitotic (PZM) error. Among the 54 maternal cases for which the cell division of error was established, only 16 were attributable to an error at the first meiotic division (mat MI), whereas no fewer than 35 were due to an error at the second meiotic division (mat MII), the remaining 3 being the result of a PZM error involving the maternal chromosome 18. A standard map of chromosome 18 was constructed and compared with the nondisjunctional map. Approximately one-third of the mat MI errors were associated with complete absence of recombination, whereas in the remaining two-thirds and in all the mat MII errors recombination in the nondisjoined chromosomes appeared to be normal. All the maternal errors were associated with an increased maternal age, although this reached significance only for the mat MII category of nondisjunction. Our observations on chromosome 18 are compared with those on other chromosomes for which there are comparable data.

Adult↗
PubMed1994

On hormonal control of moulting in Aphelenchus avenae (Nematoda: Aphelenchida).

Ligatures applied to juveniles of A. avenae at particular developmental times and positions limited the moult to the anterior or posterior portion of the nematode. To partition the moult, ligatures needed to be applied from about 4 h after receipt of the stimulus for moulting up till the onset of lethargus. Nematodes which were ligated before the stimulus failed to moult, while nematodes which were ligated after the onset of lethargus moulted on both sides of the ligature. If, at the appropriate time, ligatures were placed in the anterior third of the nematode, then the posterior portion of the nematode moulted; if the ligatures were placed in the posterior half, then the anterior portion of the nematode moulted. If the ligatures were placed between third and half the length of the nematode, then either the anterior or posterior portion moulted, or moulting was prevented. Extracts of lyophilised samples of mixed populations of A. avenae induced about 20% pupation in Musca domestica bioassays giving a weight of 80-320 ng ecdysteroid/g dry weight of nematodes. Externally applied 20-OH ecdysone at 2 x 10(-3) M stimulated moulting in the non-moulted portion of ligated nematodes but 2 x 10(-7) M did not.

Animals↗
PubMed1993

Isoforms of agrin are widely expressed in the developing rat and may function as protease inhibitors.

The agrin family of extracellular matrix proteins may be important in the formation of the neuromuscular junction. Using in situ hybridization with a probe recognizing all agrin isoforms, we demonstrate that it is widely expressed during mammalian embryogenesis. In the developing rat, particularly high levels of expression are found in the dorsal root and cranial ganglia, gut, whisker rudiments, penis, snout, teeth, retina, hippocampus, cerebral cortex and the lining of brain ventricles. Functional analysis of the recombinant rat protein shows that it is a potent inhibitor of the proteases trypsin, chymotrypsin and plasmin but not thrombin or the plasminogen activators. We conclude that agrin and its isoforms may play multiple roles in mammalian development including the regulation of proteolysis in the extracellular matrix.

Agrin↗
PubMed1993

Cloning of four growth hormone/chorionic somatomammotropin-related complementary deoxyribonucleic acids differentially expressed during pregnancy in the rhesus monkey placenta.

The close homology of the chorionic somatomammotropin (CS) genes with pituitary GH is unique to primates. Southern blots of rhesus genomic DNA probed with a human CS cDNA demonstrated that the rhesus gene family consists of at least five EcoRI fragments between 2.3-9.5 kilobases. Rhesus pituitary and placental cDNA libraries were screened for hCS-hybridizing clones. A pituitary GH cDNA was isolated that was 95% and 96% identical to human GH at the mRNA and protein levels, respectively. Thirteen placental clones representing four different cDNAs were identified and sequenced. The relationships of the rhesus placental clones among themselves were similar to those among human placental mRNAs. Two cDNAs (mCS1 and mCS2) differed by two bases and coded for identical proteins. A third cDNA (mCS3) was 94% homologous to mCS1/2 in mRNA sequence as well as in deduced amino acid sequence. A fourth cDNA (mGH-V) was 84-86% homologous to the other placental cDNAs at the mRNA and protein levels, similar to the homology between human placental GH-V and CS mRNAs. To determine the relative expression of placental mRNAs, we developed a quantitative reverse transcription-polymerase chain reaction assay using diagnostic NlaIV restriction enzyme sites in cDNAs amplified from the placental mRNAs. All four mRNAs were expressed in the placenta throughout most of pregnancy, with the mCS1 and mGH-V mRNAs generally expressed at 2- to 10-fold higher levels than mCS2 and mCS3. Additionally, mCS3 and mGHV were expressed at relatively higher levels during the second and third trimesters than during the first trimester.

Amino Acid Sequence↗
PubMed1993

The use of 33P-labeled oligonucleotides for in situ hybridization of vertebrate embryo frozen sections.

In situ hybridization was performed on frozen sections of whole E16 rat embryos or whole E7 chick embryos using oligonucleotide probes tagged with a 33P-poly A tail. We used two antisense oligonucleotide probes: a 57-mer rat agrin oligo and a 60-mer chicken B-cadherin oligo. After exposure to Hyperfilm beta max for three days, mRNA was detected in distinct organs and tissues of the embryo. After dipping the slides in liquid emulsion and exposing them for two weeks, the grains could be localized to a specific cellular layer, for example, the ganglion layer of the retina or the luminal epithelium of the gizzard. It is concluded that 33P autoradiography is excellent for defining gross areas or cellular layers of mRNA in a vertebrate embryo with a resolution of about 20 microns.

Agrin↗
PubMed1993

Molecular studies of trisomy 18.

We have determined the parental origin of 50 cases of trisomy 18. In 48 cases the additional chromosome was maternal in origin, and in 2 cases it was paternal in origin. Seven cases, including both those with an additional paternal chromosome, appeared to be the result of postzygotic error. In contrast to the situation in nondisjunction involving chromosomes 21 and X, there was no evidence for nullochiasmate nondisjunction.

Adolescent↗
PubMed1992

Traffic volume and collisions involving transit and nontransit vehicles.

This study reports an analysis of collisions occurring between public transit vehicles operated by the San Francisco Municipal Railway System (Muni), the public transit agency for the City of San Francisco, and nontransit vehicles. The analysis, focusing on weekday collisions during 1987, demonstrated a strong association between hourly transit collisions rates and hourly traffic volume. The collision rate varied from 0.01 per 1,000 Muni vehicle-hours of operation during the interval 5 A.M. to 6 A.M., a time of very low traffic volume, to 0.93 (approximately 1 collision per 1,000 Muni vehicle-hours of operation) during the interval 5 P.M. to 6 P.M., a time of very high traffic volume. Using a power function to predict either the total number of collisions, or the rate of collisions per 1,000 Muni vehicle-hours, almost 90% of total variation was accounted for by traffic volume. A very similar pattern was found for collisions judged either avoidable or unavoidable. A peak in the collision rate between 2 A.M. and 3 A.M. could not be accounted for by traffic volume alone. That peak occurred in the one-hour interval following the 2 A.M. closing of bars in San Francisco, and was composed entirely of a sharp increase in unavoidable collisions. Increasing traffic volume appeared to operate through two mechanisms: (i) an increase in the number of opportunities for a collision, defined as a quantity proportional to the product of the number of Muni and non-Muni vehicles; (ii) an increase in the probability of a collision occurring between any given pair of vehicles.

Accidents, Occupational↗