PubMed Health⌕ Search

Biomedical subjects

D Zhang

Publications and source records attributed to D Zhang.

At least 469 records · Page 26Linked to original sources
PubMed1996

A method for the large scale isolation of high transformation efficiency fungal genomic DNA.

A procedure for isolation of genomic DNA from the zygomycete Cunninghamella elegans and other filamentous fungi and yeasts is reported. This procedure involves disruption of cells by grinding using dry ice, removal of polysaccharides using cetyltrimethylammonium bromide and by phenol extractions, and precipitation of DNA with isopropanol at room temperature. The isolation method produced large scale (approximate 1 mg DNA/5 g wet cells) and highly purified high molecular mass DNA. Sau3AI partially digested DNA showed high transformation efficiency (> 10(6)/100 ng DNA) when ligated to ZAP-express lambda vector.

Basidiomycota↗
PubMed1996

Binding of CD44 to hyaluronic acid can be induced by multiple signals and requires the CD44 cytoplasmic domain.

Human Jurkat T cells transfected with a human CD44H gene do not bind fluorescein-conjugated hyaluronic acid (F-HA). Activation of Jurkat CD44 transfectant is required for binding to F-HA. Binding can be induced by anti-CD3 monoclonal antibody (Mab), PMA, calcium ionophore, forskolin, or a monoclonal anti-CD44 Mab (F10-44-2). Cytochalasin D, an inhibitor of actin polymerization, inhibited both PMA-induced and anti-CD44 Mab-induced binding. In contrast, only PMA-induced binding was blocked by inhibitors of microtubule functions: colchicine or Taxol. Therefore, binding induced by PMA and anti-CD44 Mab involves different cytoskeletal proteins. The conclusion that interactions between CD44 and cytoskeletal proteins are important for binding was further supported by our observations that the cytoplasmic domain of CD44 is required for both PMA- and anti-CD44 Mab-induced binding. Jurkat CD44 mutant transfectant lacking the last 23 amino acids of the cytoplasmic domain can be induced to bind FHA. In contrast, Jurkat mutant CD44 transfectant lacking the last 57 amino acids of the cytoplasmic domain was unable to bind. Collectively, these data provide supportive evidence that binding activity of CD44 is under the regulation of multiple signal pathways and requires the presence of the cytoplasmic domain.

Amino Acid Sequence↗
PubMed1996

hprt mutation frequency among workers exposed to 1,3-butadiene in China.

Hypoxanthine-guanine phosphoribosyl transferase (hprt) mutation frequency (M(f)) was studied in workers at a polybutadiene rubber production facility in Yanshan, China. Exposed workers included for study were active either as process analysts, who sampled butadiene production process lines and analyzed product by gas chromatography, or as process operators, who did routine process control, minor maintenance and, as needed, major repair operations. For process analysts at the polymer and dimethyl formamide (DMF) facilities, the median air levels of BD were 1.0 and 3.5 ppm, respectively. Among process operators, air levels of 1.1 ppm were found during routine activities, while the median air level during pump repair and related operations was 45 ppm (6-h time-weighted average). Overall, M(f) was similar in unexposed (mean M(f) = 20.2 x 10(-6)) and butadiene-exposed (mean M(f) = 21.6 x 10(-6)) workers (P = 0.68). M(f) decreased with cloning efficiency, increased with age, and was moderately greater in women than in men. After adjustment by multiple regression analysis for mean age, sex, and cloning efficiency, the adjusted mean M(f)(Xadj) was 13.6 x 10(-6) in unexposed and 18.0 x 10(-6) in butadiene-exposed. This 32% difference was, however, not statistically significant (P = 0.13). Butadiene exposure was associated with a modest, if any, increase in hprt M(f) in this population of Chinese workers.

Adult↗
PubMed1996

Formation of mammalian metabolites of cyclobenzaprine by the fungus, Cunninghamella elegans.

The fungus, Cunninghamella elegans, was used as a microbial model of mammalian drug metabolism to biotransform a tricyclic antidepressant, cyclobenzaprine. Seventy-five percent of this drug at a concentration of 1 mM was metabolized within 72 h by C. elegans grown on Sabouraud dextrose broth. Milligram amounts of fungal metabolites were isolated by reversed-phase high performance liquid chromatography (HPLC) and their structures were characterized by 1H NMR spectroscopy, mass spectrometry, and UV spectroscopy analyses. The major fungal metabolites of cyclobenzaprine were 2-hydroxycyclobenzaprine (59%), N-desmethylcyclobenzaprine (21%), cyclobenzaprine trans-10,11-dihydrodiol (5%), N-desmethyl-2-hydroxy-cyclobenzaprine (3%), 3-hydroxycyclobenzaprine (3%), and cyclobenzaprine N-oxide (1%). These fungal metabolites were used as standards to investigate the metabolism of cyclobenzaprine by rat liver microsomes. Rat liver microsomes also biotransformed cyclobenzaprine to produce similar metabolites as the fungus. The isotope labeling of 2-hydroxycyclobenzaprine by 18O2 and the trans-configuration of the dihydrodiol suggested that these reactions were catalyzed by cytochrome P-450 monooxygenases in C. elegans. These results also demonstrated that the fungal biotransformation system could be used to predict and synthesize the mammalian drug metabolites.

Amitriptyline↗
PubMed1996

Elevated parathyroid hormone-related peptide associated with lactation and bone density loss.

OBJECTIVE: To investigate the hypothesis that parathyroid hormone-related peptide (PRHrP) may be involved with bone loss and recovery as a means of providing adequate calcium and phosphate to infants. DESIGN: An 18-month prospective cohort study. SETTING: General community setting with recruitment occurring at birthing education classes. PARTICIPANTS: Volunteer sample of 115 postpartum healthy women aged 20 to 40 years, and 0 or 1 parity prior to parturition with no intent to breast-feed or intent to breast-feed at least 6 months. MAIN OUTCOME MEASURES: Parathyroid hormone-related peptide, prolactin, estradiol, 1,25-dihydroxyvitamin D, 24-hydroxyvitamin D, femoral bone mineral density, and bone turnover markers were measured in 115 postpartum women at 2 weeks, 2 months, 4 months, 6 months, 12 months, and 18 months postpartum. Lumbar bone mineral density was measured at 2 weeks, 6 months, 12 months, and 18 months postpartum. RESULTS: Elevated PTHrP values were significantly associated (P<.001) with breast-feeding status, elevated prolactin levels, and lower serum estradiol levels, conditions occurring during lactation. Furthermore, elevated PTHrP levels were negatively and significantly associated (P<.01) over time with bone mineral density change at both the spine and the femoral neck, even after accounting for prolactin levels, breast-feeding status, return of menstruation, estradiol levels, PTH levels, 1,25-dihydroxyvitamin D levels, dietary calcium intake, physical activity, and body size. CONCLUSION: These data clearly support the hypothesis that PTHrP is an alternative mechanism associated with bone loss and recovery during and subsequent to lactation.

Absorptiometry, Photon↗
PubMed1996

'Anaphase' and cytokinesis in the absence of chromosomes.

Anaphase and cytokinesis are key processes in the segregation of replicated chromosomes to the daughter cells: in anaphase, chromosomes move apart; in cytokinesis, a cleavage furrow forms midway between the separated chromosomes. Some evidence suggests that chromosomes may be involved both in controlling the timing of anaphase onset and in dictating the position of the cleavage furrow. Other evidence indicates that the controlling mechanisms are intrinsic to the spindle and the cell. Here we test these possibilities in grasshopper spermatocytes by observing spindles and cells after removal of chromosomes. We found that both anaphase and cytokinesis occur independently of chromosomes: stage-specific changes occur at an appropriate time and in the correct way, despite the absence of chromosomes. This finding is particularly noteworthy because chromosomes have an important impact on spindle microtubule assembly and the timing of anaphase onset in these cells.

Anaphase↗
PubMed1996

Manduca sexta hemolymph ferritin: cDNA sequence and mRNA expression.

A cDNA clone encoding a subunit of the tobacco hornworm Manduca sexta (Ms) hemolymph (serum) ferritin (Fer) has been identified and sequenced. The deduced amino acid (aa) sequence shows approx. 50% similarity to vertebrate Fer subunit sequences, and the nucleotide sequence contains a stem-loop structure in the 5' untranslated region that could serve as an iron-responsive element (IRE). The stem-loop of this putative IRE exhibits high identity to vertebrate IRE that play an essential role in the control of Fer synthesis. The Ms Fer subunit lacks one of the three Tyr residues required for the rapid biomineralization of iron shown in vertebrate heavy-chain Fer. In addition, aa residues that comprise the putative ferroxidase centers generally are not conserved, suggesting that the Ms Fer subunit more closely resembles the vertebrate light-chain subunit. Northern blot analyses indicate that the fer mRNA is expressed in the midgut, fat body and hemocytes, with the greatest expression in the midgut.

Amino Acid Sequence↗
PubMed1996

Mapping the binding site pocket of the serotonin 5-Hydroxytryptamine2A receptor. Ser3.36(159) provides a second interaction site for the protonated amine of serotonin but not of lysergic acid diethylamide or bufotenin.

Like other amine neurotransmitters that activate G-protein-coupled receptors, 5-hydroxytryptamine (5-HT) binds to the 5-HT2A receptor through the interaction of its cationic primary amino group with the conserved Asp3.32(155) in transmembrane helix 3. Computational experiments with a 5-HT2A receptor model suggest that the same functional group of 5-hydroxytryptamine also forms a hydrogen bond with the side chain of Ser3.36(159), which is adjacent in space to Asp3.32(155). However, other 5-HT2A receptor ligands like lysergic acid diethylamide (LSD), in which the amine nitrogen is embedded in a heterocycle, or N,N-dimethyl 5-HT, in which the side chain is a tertiary amine, are found in the computational simulations to interact with the aspartate but not with the serine, due mainly to steric hindrance. The predicted difference in the interaction of various ligands in the same receptor binding pocket was tested with site-directed mutagenesis of Ser3.36(159) --> Ala and Ser3.36(159) --> Cys. The alanine substitution led to an 18-fold reduction in 5-HT affinity and the cysteine substitution to an intermediate 5-fold decrease. LSD affinity, in contrast, was unaffected by either mutation. N,N-Dimethyl 5-HT affinity was unaffected by the cysteine mutation and had a comparatively small 3-fold decrease in affinity for the alanine mutant. These findings identify a mode of ligand-receptor complexation that involves two receptor side chains interacting with the same functional group of specific serotonergic ligands. This interaction serves to orient the ligands in the binding pocket and may influence the degree of receptor activation.

Amino Acid Sequence↗
PubMed1996

Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics.

The filamentous fungus Cunninghamella elegans has the ability to metabolize xenobiotics, including polycyclic aromatic hydrocarbons and pharmaceutical drugs, by both phase I and II biotransformations. Cytosolic and microsomal fractions were assayed for activities of cytochrome P450 monooxygenase, aryl sulfotransferase, glutathione S-transferase, UDP-glucurono-syltransferase, UDP-glucosyltransferase, and N-acetyltransferase. The cytosolic preparations contained activities of an aryl sulfotransferase (15.0 nmol min-1 mg-1), UDP-glucosyltransferase (0.27 nmol min-1 mg-1) and glutathione S-transferase (20.8 nmol min-1 mg-1). In contrast, the microsomal preparations contained cytochrome P450 monooxygenase activities for aromatic hydroxylation (0.15 nmol min-1 mg-1) and N-demethylation (0.17 nmol min-1 mg-1) of cyclobenzaprine. UDP-glucuronosyltransferase activity was detected in both the cytosol (0.09 nmol min-1 mg-1) and the microsomes (0.13 nmol min-1 mg-1). N-Acetyltransferase was not detected. The results from these experiments provide enzymatic mechanism data to support earlier studies and further indicate that C. elegans has a broad physiological versatility in the metabolism of xenobiotics.

Biotransformation↗
PubMed1996

Immunologic basis of transplant-associated arteriosclerosis.

Although immunosuppressive therapy minimizes the risk of graft failure due to acute rejection, transplant-associated arteriosclerosis of the coronary arteries remains a significant obstacle to the long-term survival of heart transplant recipients. The participation of specific inflammatory cell types in the genesis of this lesion was examined in a mouse model in which carotid arteries were transplanted across multiple histocompatibility barriers into seven mutant strains with immunologic defects. An acquired immune response--with the participation of CD4+ (helper) T cells, humoral antibody, and macrophages--was essential to the development of the concentric neointimal proliferation and luminal narrowing characteristic of transplant arteriosclerosis. CD8+ (cytotoxic) T cells and natural killer cells were not involved in the process. Arteries allografted into mice deficient in both T-cell receptors and humoral antibody showed almost no neointimal proliferation, whereas those grafted into mice deficient only in helper T cells, humoral antibody, or macrophages developed small neointimas. These small neointimas and the large neointimas of arteries grafted into control animals contained a similar number of inflammatory cells; however, smooth muscle cell number and collagen deposition were diminished in the small neointimas. Also, the degree of inflammatory reaction in the adventitia did not correlate with the size of the neointima. Thus, the reduction in neointimal size in arteries allografted into mice deficient in helper T cells, humoral antibody, or macrophages may be accounted for by a decrease in smooth muscle cell migration or proliferation.

Animals↗
PubMed1996

STAT3 participates in transcriptional activation of the C-reactive protein gene by interleukin-6.

Interleukin-6 (IL-6) is the major cytokine inducing transcription of human C-reactive protein (CRP) during the acute phase response. STAT (signal transducers and activators of transcription) family members, recently shown to be important mediators of the effects of many cytokines including IL-6, generally induce their effects by binding to palindromic sequences with TT(N)5AA motifs. We report an IL-6 responsive element in the proximal region of the human CRP 5'-flanking region that bears a TT(N)4AA motif, which we have termed CRP acute phase response element (CRP-APRE). In Hep3B cells, IL-6 but not interferon-gamma was capable of activating CAT constructs driven by the CRP promoter containing CRP-APRE. Overexpressed STAT3 was able to transactivate CRP-chloramphenicol acetyltransferase constructs through the CRP-APRE and was able to enhance endogenous CRP mRNA accumulation in response to IL-6. STAT3 (or an antigenically related molecule) bound to the CRP-APRE in response to IL-6. Overexpression of STAT3 in the presence of IL-6 was capable of inducing expression of a construct consisting of the CRP-APRE and a minimal thymidine kinase promoter lacking a C/EBP site. Taken together, these findings indicate that STAT3 participates in the transcriptional activation of CRP in response to IL-6.

Acute-Phase Proteins↗
PubMed1996

Batimastat, a potent matrix mealloproteinase inhibitor, exhibits an unexpected mode of binding.

Matrix metalloproteinase enzymes have been implicated in degenerative processes like tumor cell invasion, metastasis, and arthritis. Specific metalloproteinase inhibitors have been used to block tumor cell proliferation. We have examined the interaction of batimastat (BB-94) with a metalloproteinase [atrolysin C (Ht-d), EC 3.4.24.42] active site at 2.0-angstroms resolution (R = 16.8%). The title structure exhibits an unexpected binding geometry, with the thiophene ring deeply inserted into the primary specificity site. This unprecedented binding geometry dramatizes the significance of the cavernous primary specificity site, pointing the way for the design of a new generation of potential antitumor drugs.

Amino Acid Sequence↗
PubMed1996

Crystal structure of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae at 2.3 A resolution.

The crystal structure of pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate-dependent enzyme isolated from Saccharomyces cerevisiae, has been determined and refined to a resolution of 2.3 A. Pyruvate decarboxylase is a homotetrameric enzyme which crystallizes with two subunits in an asymmetric unit. The structure has been refined by a combination of simulated annealing and restrained least squares to an R factor of 0.165 for 46,787 reflections. As in the corresponding enzyme from Saccharomyces uvarum, the homotetrameric holoenzyme assembly has approximate 222 symmetry. In addition to providing more accurate atomic parameters and certainty in the sequence assignments, the high resolution and extensive refinement resulted in the identification of several tightly bound water molecules in key structural positions. These water molecules have low temperature factors and make several hydrogen bonds with protein residues. There are six such water molecules in each cofactor binding site, and one of them is involved in coordination with the required magnesium ion. Another may be involved in the catalytic reaction mechanism. The refined model includes 1074 amino acid residues (two subunits), two thiamin diphosphate cofactors, two magnesium ions associated with cofactor binding and 440 water molecules. From the refined model we conclude that the resting state of the enzyme-cofactor complex is such that the cofactor is already deprotonated at the N4' position of the pyrimidine ring, and is poised to accept a proton from the C2 position of the thiazolium ring.

Amino Acid Sequence↗
PubMed1996

Genetic and environmental influences on insulin levels and the insulin resistance syndrome: an analysis of women twins.

Multiple factors may determine insulin resistance and the insulin resistance syndrome. The contributions of genes and environment to the distribution of fasting insulin levels and to the associations of fasting insulin with elements of the syndrome were evaluated in the second examination of the Kaiser Permanente Women Twins Study (Oakland, California, 1989-1990). Subjects included 556 white women (165 monozygous twin pairs, 113 dizygous pairs; 455 women with normal glucose tolerance, 75 with impaired glucose tolerance, and 26 with non-insulin-dependent diabetes by World Health Organization criteria). The intraclass correlation coefficients for log fasting insulin for monozygous and dizygous twin pairs were 0.64 and 0.40, respectively. After adjustment for age, behavioral factors, and body mass index, the estimated classic heritability was 0.53 (p = 0.003). Commingling analysis of fasting insulin indicated the presence of four distributions (p < 0.001), consistent with at least one, and perhaps two, genes influencing this trait. In an unmatched multiple regression model among women from monozygous twin pairs only, log fasting insulin was independently associated with body mass index (p < 0.0001), waist/hip ratio (p = 0.02), and glucose intolerance (p = 0.04), but not with triglycerides, high density lipoprotein cholesterol, or hypertension. After removal of genetic influences by analysis of monozygous intrapair differences, only body mass index (p < 0.0001) remained independently related to fasting insulin. The authors conclude that, in addition to significant genetic influences on fasting insulin, environmental or behavioral factors (particularly nongenetic variation in obesity) are important determinants of fasting insulin and the insulin resistance syndrome.

Adult↗
PubMed1996

Adherence to single daily dose of aspirin in a chemoprevention trial. An evaluation of self-report and microelectronic monitoring.

A consecutive sample of 64 healthy adults (33 female and 31 male) were recruited at the University of Michigan Medical Center, Ann Arbor. Data were available for analysis on 57 subjects. The participants were asked to take a single daily dose of aspirin ranging from 0 to 640 mg. Adherence to the daily aspirin ingestion was measured by self-report and the Medication Event Monitoring System (MEMS, Aprex Corp, Fremont, Calif); adherence rate for the study population was 35%. The adherence rates for all dosing errors between self-report and Medication Event Monitoring System were significantly different (P = .002). There was no significant gender difference in adherence rates. Adherence to regular aspirin ingestion was poor in healthy, paid subjects despite explicit, written and verbal instructions. Patient self-report alone is not a reliable measure of adherence.

Adult↗
PubMed1996

Expression of neurotrophin trk and p75 receptors in quail embryos undergoing gastrulation and neurulation.

We have previously demonstrated the presence of mRNA for the full-length neurotrophin receptors trkA, trkB and trkC in quail embryos from stages 1 through 6 using reverse transcription followed by the polymerase chain reaction (RT-PCR; Yao et al. [1994] Dev. Biol. 165: 727-730). Furthermore, we showed that mRNA for the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 was present from stage 1 onward, while nerve growth factor mRNA began to be expressed at stage 5. In the present study, wholemount in situ hybridization was used to localize full-length trk mRNA in embryos from stages 3 through 10. Structures expressing trkC mRNA included the primitive streak and Hensen's node, the neural plate or notochord, somites and the rostral neural tube. trkA and trkB mRNA were expressed at much lower levels than trkC mRNA; however, staining was detected on the primitive streak and Hensen's node. In addition to trk mRNA, we have also demonstrated the presence of full-length Trk protein in embryos from stages 3 through 11, suggesting that the trk mRNA detected at these early stages is translated into functional cell surface receptors. To support this hypothesis, we have shown that neurotrophins can induce phosphorylation of Trk on tyrosine residues, at least at stage 11. We also detected mRNA and protein for the nontyrosine kinase neurotrophin receptor, p75, at similar stages. The presence of neurotrophin receptors, as well as neurotrophin mRNA, in embryos undergoing gastrulation and neurulation leads to speculation that neurotrophins may be playing a role in these processes.

Animals↗