Biomedical subjects

S Zhang

Publications and source records attributed to S Zhang.

At least 361 records · Page 20Linked to original sources

PubMed2000

Protection against malignant progression of spontaneously developing liver tumors in transgenic mice expressing O(6)-methylguanine-DNA methyltransferase.

To study the effect of O(6)-methylguanine-DNA methyltransferase (MGMT) on carcinogenesis, we have previously generated MGMT transgenic mice overexpressing the bacterial MGMT gene, ada, and demonstrated that high MGMT levels in the liver suppress induction of liver tumors after treatment with an alkylating hepatocarcinogen. To examine the effects of life-long elevation of MGMT activity on mouse spontaneous liver tumor development, ada-transgenic and control non-transgenic mice were compared. We also examined mutations at codon 61 of the H-ras oncogene, reported as a hot spot in mouse liver tumors, using a direct DNA sequencing method. The results revealed no significant difference in tumor incidence or mutation spectrum, but interestingly, ada-transgenic mice were found to have fewer malignant tumors and survived longer, indicating a possible protective role of MGMT against malignant conversion.

Animals↗

PubMed2000

Three-dimensional mapping of spontaneous ventricular arrhythmias in a canine thrombotic coronary occlusion model.

INTRODUCTION: Ventricular tachycardia (VT) and ventricular fibrillation (VF) induced by thrombotic coronary occlusion were mapped in three dimensions in ten dogs. METHODS AND RESULTS: Thrombotic occlusion was induced using a wire to deliver current to the proximal left circumflex artery (LCX). In nine dogs, nonsustained VT (NSVT) arose from numerous focal sites. Sustained VT was initiated in six dogs (VT group) by a focus near or in the ischemic region. VT was maintained by a focus in the ischemic border in three dogs and by macroreentry that involved both the ischemic and nonischemic regions in the other three dogs. In five dogs, VT degenerated into VF due to intramural reentry in different locations. Mean total activation time (AT), the time for activation to traverse the ventricles, for a sinus beat when LCX current was first applied was 40 +/- 4 msec. In the four dogs in which VT occurred 3 to 7 minutes after total occlusion, sinus AT increased to 98 to 146 msec just before VT. Sinus AT in the four dogs without VT was always <98 msec. Mean AT of the first ten cycles of VT was significantly longer in those VTs that degenerated into VF (169 +/- 29 msec) than in those that did not (81 +/- 12 msec). CONCLUSION: Thrombotic LCX occlusion induced NSVT in 90%, VT in 60%, and VF in 50% of dogs. Focal mechanisms caused most NSVTs and VT initiation. VT was maintained by a focus near or in the ischemic region or by macroreentry involving both the ischemic and nonischemic regions. AT identified animals in which VT occurred soon after LCX occlusion and in which VT progressed to VF.

Animals↗

PubMed2000

Mycoplasmal infections alter gene expression in cultured human prostatic and cervical epithelial cells.

To better understand how infections by mycoplasmas affect gene expression in human cells, we quantitatively measured the transcripts of 38 cytokine genes in HPV E6- and E7-immortalized cervical and prostatic epithelial cells before and after infection by four human urogenital mycoplasmas, M. fermentans, M. genitalium, M. hominis and M. penetrans. Using the multi-probe RNase protection assay (RPA), 22 and 23 cytokine gene transcripts were detected in the non-infected control prostatic and cervical epithelial cells, respectively. Although there were no discernible changes in cell morphology and growth kinetics following 72 h of mycoplasmal infection, 55-74% of the cytokine genes expressed in the two human epithelial cell lines were altered. Most changes reflected an increased expression of these cytokine genes, while expression of some cytokine genes significantly decreased. The effects varied with host cell type and species of infecting mycoplasmas. These alterations in gene expression were more profound in the cervical epithelial cells than in the prostatic cells. M. fermentans produced the most significant effects, followed by M. penetrans, M. genitalium and M. hominis. Some alterations in the gene expression were transient, but most persisted over the course of chronic (9 months) mycoplasmal infection. Prolonged gene expression changes induced by chronic mycoplasmal infection may gradually alter important biological properties in the infected mammalian cells and produce a unique form of disease process.

Cell Line, Transformed↗

PubMed2000

Cytotoxic activity of coagulase-negative staphylococci in bovine mastitis.

Secreted toxins play important roles in the pathogenesis of bacterial infections. In this study, we examined the presence of secreted cytotoxic factors of coagulase-negative staphylococci (CoNS) from bovine clinical and subclinical mastitis. A 34- to 36-kDa protein with cell-rounding cytotoxic activity was found in many CoNS strains, especially in Staphylococcus chromogenes strains. The protein caused cell detachment and cell rounding in several cell lines, including HEp-2, Int 407, CHO-K1, and Y-1 cells. Native protein recovered from nondenatured polyacrylamide gel electrophoresis showed both cytotoxic activity and casein hydrolysis activity. The purified protein had a pH optimal at 7.2 to 7.5 and a pI of 5.1 and was heat labile. The proteolytic activity could be inhibited by zinc and metal specific inhibitors such as 1, 10-phenanthroline and EDTA, indicating that it is a metalloprotease. Protein mass analysis and peptide sequencing indicated that the protein is a novel metalloprotease. Different bacterial strains expressed variable levels of 34- to 36-kDa protease, which may provide an indication of strain virulence.

Animals↗

PubMed2000

Characterization of the promoter elements for the staphylococcal enterotoxin D gene.

Deletion analysis of the promoter for the Staphylococcus aureus enterotoxin D determinant indicated that a 52-bp sequence, from -34 to +18, was sufficient for sed promoter function and agr regulation. A consensus -10 Pribnow box sequence, a less conserved -35 sequence, and a TG dinucleotide motif were present. Transcribed sequences (+1 to +18) are essential for promoter activity.

Bacterial Toxins↗

PubMed2000

Insulin increases NO-stimulated guanylate cyclase activity in cultured VSMC while raising redox potential.

Insulin acutely stimulates cyclic guanosine monophosphate (cGMP) production in primary confluent cultured vascular smooth muscle cells (VSMC) from canine femoral artery, but the mechanism is not known. These cells contain the inducible isoform of nitric oxide (NO) synthase (iNOS), and insulin-stimulated cGMP production in confluent cultured cells is blocked by the NOS inhibitor, N(G)-monomethyl-L-arginine (L-NMMA). In the present study, it is shown that iNOS is also present in freshly dispersed VSMC from this artery, indicating that iNOS expression in cultured VSMC is not an artifact of the culture process. Insulin did not stimulate NOS activity in primary confluent cultured cells because it did not affect citrulline or combined NO(-)(3)/NO(-)(2) production. To see whether insulin required the permissive presence of NO to stimulate cGMP production, iNOS and basal cGMP production were inhibited with L-NMMA, and the cells were incubated with or without 1 nM insulin and/or the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at a concentration (0.1 microM) that restored cGMP production to the basal value. In the presence of L-NMMA, insulin no longer affected cGMP production but when insulin was added to L-NMMA plus SNAP, cGMP production was increased by 69% (P < 0.05 vs. L-NMMA plus SNAP). Insulin, which increases glucose uptake by these cells, increased the cell lactate content and the lactate-to-pyruvate ratio (LPR) by 81 and 97%, respectively (both P < 0.05), indicating that the hormone increased aerobic glycolysis and the redox potential. The effects of insulin on LPR and cGMP production were blocked by removing glucose or by adding 2-deoxyglucose to the incubation media and were duplicated by the reducing substrate, beta-hydroxybutyrate. We conclude that insulin does not acutely affect iNOS activity in these VSMC but it does augment cGMP production induced by the NO already present in the cell while increasing aerobic glycolysis and the cell redox potential.

Aerobiosis↗

PubMed2000

Methodological and physiological variability within the ventilatory response to hypoxia in humans.

Measurement of the acute hypoxic ventilatory response (AHVR) requires careful choice of the hypoxic stimulus. If the stimulus is too brief, the response may be incomplete; if the stimulus is too long, hypoxic ventilatory depression may ensue. The purpose of this study was to compare three different techniques for assessing AHVR, using different hypoxic stimuli, and also to examine the between-day variability in AHVR. Ten subjects were studied, each on six different occasions, which were >/=1 wk apart. On each occasion, AHVR was assessed using three different protocols: 1) protocol SW, which uses square waves of hypoxia; 2) protocol IS, which uses incremental steps of hypoxia; and 3) protocol RB, which simulates an isocapnic rebreathing test. Mean values for hypoxic sensitivity were 1.02 +/- 0.48, 1.15 +/- 0.55, and 0.93 +/- 0.60 (SD) l. min(-1). %(-1) for protocols SW, IS, and RB, respectively. These differed significantly (P < 0.01). The coefficients of variation for measurement of AHVR were 20, 23, and 36% for the three protocols, respectively. These were not significantly different. There was a significant physiological variation in AHVR (F (50,100) = 3.9, P < 0. 001), with a coefficient of variation of 26%. We conclude that there was relatively little systematic variation between the three protocols but that AHVR varies physiologically over time.

Adult↗

PubMed2000

Insulin inhibits migration of vascular smooth muscle cells with inducible nitric oxide synthase.

Vascular smooth muscle cell (VSMC) migration participates in atherosclerosis and arterial restenosis after balloon angioplasty. Because these processes are enhanced in insulin-resistant states, our goal was to determine whether insulin affects VSMC migration and, if so, how. The migration of primary cultured VSMCs from canine femoral artery was measured with the use of a wound migration assay and related to cGMP levels. Insulin (1 nmol/L) did not affect migration or cGMP production in control cells. When inducible nitric oxide synthase (iNOS) was induced by 24-hour preincubation with lipopolysaccharide and interleuken-1beta, basal migration decreased, cGMP production increased, and insulin inhibited migration by >90% and stimulated cGMP production by 3-fold. The nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine blocked the affect of insulin on the migration of VSMCs with iNOS. 8-Bromo-cGMP inhibited VSMC migration in control cells, and 1-H-1[1,2,4]oxadiazolo-[4, 3a]quinoxolin-1-one, a selective inhibitor of guanylate cyclase, blocked the inhibition by insulin of migration of cells with iNOS. We conclude that insulin does not normally affect cGMP production or the migration of these VSMCs. However, after the induction of iNOS, insulin stimulates cGMP production and inhibits migration via an NOS-and a cGMP-dependent mechanism.

Animals↗

PubMed2000

Leptin and leptin receptor expression in rat and mouse pituitary cells.

Leptin is a circulating hormone secreted mainly by adipose tissue. Recent studies have shown leptin production by other tissues, including the placenta, stomach, and mammary tissues. Various reports have suggested that the anterior pituitary may have a role in the regulatory effects of leptin. We recently localized leptin in the human anterior pituitary, but analysis of leptin in rodent pituitary has not been previously reported. In this study we examined rat and mouse pituitary tissues and various cell lines for leptin by RT-PCR, immunohistochemistry, and Western blotting. Leptin receptor messenger RNA was also examined in these tissues by RT-PCR. Leptin was present in a small percentage of rat (4.8 +/- 0.7%) and mouse (7 +/- 2%) pituitary cells. Colocalization studies with leptin and pituitary hormones showed leptin expression mainly in TSH cells (24 +/- 2% of TSH cells in the rat pituitary and 31 +/- 1% of TSH cells in the mouse pituitary). A folliculo-stellate (FS) cell line, TtT/GF, also expressed leptin. The long isoform of leptin receptor (OB-Rb) was present in normal pituitary and in various pituitary cell lines, including FS, GH3, and alphaT3-1 cells. Treatment of GH3 and FS cells with leptin (1 x 10(-8) M) inhibited cell proliferation assessed by [3H]thymidine incorporation in GH3, but not in FS, cells. These findings show for the first time that leptin is expressed in rat and mouse anterior pituitaries mainly by TSH cells and by a mouse FS cell line. The finding of leptin and of the long isoform of leptin receptor in normal rat and mouse pituitaries and in various cell lines implicates an autocrine/paracrine loop in the production and regulation of leptin and leptin receptor in the rodent pituitary.

Animals↗

PubMed2000

Left-right asymmetric expression of BbPtx, a Ptx-related gene, in a lancelet species and the developmental left-sidedness in deuterostomes.

The long-standing question of how asymmetric development or asymmetric body structures in lancelets (amphioxus) are phylogenetically related to the body plan of other animals is still untouched. Three anterior structures, the preoral pit, club-shaped gland and mouth, are remarkable asymmetric features in developing lancelets that all open on the left side of the body. A Ptx-related gene, BbPtx is the first identified transcription factor gene with an asymmetrical expression pattern in lancelets similar to that in vertebrates, and thus it may provide a clue for the above question. Expression of the BbPtx gene is first detected at the dorsal margin of the blastopore in early mid-gastrulae and then becomes restricted to the left anterodorsal wall of the primitive gut and to the developing left somitocoelomic system. Expression continues on the left side in the developing preoral pit, club-shaped gland and mouth as well as in the mesoderm at the caudal end. Unlike D-Ptx1 in Drosophila, BbPtx is not coexpressed with a fork head gene in lancelets; instead the two genes are expressed in a complementary fashion on the left side of the embryo. The expression pattern of BbPtx is not compatible with the calcichordate hypothesis of Jefferies, in which the proposed ancestor of chordates rotated its tail 90 degrees counterclockwise in relation to the head/trunk. The expression of both BbPtx and vertebrate Pitx2 in tissues derived from the coelom implies that the left-right asymmetric development has a common origin between cephalochordates and vertebrates. Considering the development of the coelom in deuterostomes, however, left-right asymmetric development involving Pitx2-related genes is rather likely to be a primitive character shared among deuterostomes.

Amino Acid Sequence↗

PubMed2000

Morphological changes of Babesia gibsoni grown in canine red blood cell-substituted severe combined immune deficiency mice.

Canine red blood cell-substituted severe combined immune deficiency (Ca-RBC-SCID) mice were prepared for canine Babesia gibsoni infection. The Ca-RBC-SCID mice infected with B. gibsoni developed a high level of parasitemia, and showed clinical symptoms such as anemia and hemoglobinuria, which are similar to those observed in dogs infected with B. gibsoni. The B. gibsoni parasites grown in Ca-RBC-SCID mice showed marked morphological changes, including a significantly larger size of parasites than those in dogs and abundant RBCs containing 4, 8, 16, and 32 parasites. The multiple infection may have resulted from 1 parasite because the posterior end of each parasite in a multiply infected cell was connected. The parasites grown in SCID mice retained their infectivity and virulence to dogs and their morphology was dramatically restored to the original state when they were returned to dogs.

Animals↗

PubMed2000

Neovascularization grading methods in a rat model of retinopathy of prematurity.

PURPOSE: The method of counting cell nuclei above the internal limiting membrane in histologic sections is considered the standard when quantifying neovascularization (NV) in rodent oxygen-induced retinopathy (OIR). An alternative, more rapid method of counting clock hours in flatmounted adenosine diphosphatase (ADPase)-stained rat retinas is analogous to clinically scoring retinopathy of prematurity (ROP). In the present study, the validity of counting clock hours was evaluated by a direct comparison of these techniques. The intereye correlation of NV score and retinal vascular area were also studied. METHODS: Newborn Sprague-Dawley rats were exposed to cycles of O2 (80-10%) for 7 days, followed by 5 days of room air recovery. Preretinal NV was quantified by three masked observers counting clock hours in flatmounted ADPase-stained retinas of both eyes. Retinal vascular and total retinal areas were calculated using computer-assisted analysis. Representative retinas that had been scored positive (n = 10) and negative (n = 3) for NV and room air control retinas (n = 3) were embedded in paraffin. Each entire peripheral retinal quadrant was serially sectioned at 6 microm and stained with hematoxylin and eosin. Nuclei above the internal limiting membrane were then counted in a masked manner. The total number of nuclei counted per retina was defined as the nucleus count (704-938 sections per retina; 12,900 sections). Correlations were evaluated using Spearman rank coefficients. RESULTS: The nucleus count was 0 to 44 in room air control retinas, 0 to 40 in negative OIR retinas, and 250 to 5634 in positive OIR retinas. The nucleus count was highly correlated with the clock hour score (r(s) = 0.95, P = 0.0001). For the paired retinas, there was a significant correlation between right and left eyes in the severity of NV (clock hours; r(s) = 0.76, P = 0.0001) and the ratio of retinal vascular area to total retinal area (r(s) = 0.81, P = 0.0001). CONCLUSIONS: The more rapid method of counting clock hours in flatmounted ADPase-stained retinas is valid for quantifying NV in rat models of ROP. Incidence and severity of NV and vascularized areas were similar between left and right eyes, which permits the use of paired retinas for complementary research techniques.

Animals↗

PubMed2000

Vaccination of high-risk breast cancer patients with mucin-1 (MUC1) keyhole limpet hemocyanin conjugate plus QS-21.

Our objective was to determine whether an immune response can be generated against MUC1 peptide and against tumor cell MUC1 after vaccination with MUC1-keyhole limpet hemocyanin (KLH) conjugate plus QS-21 in breast cancer patients. Nine patients with a history of breast cancer but without evidence of disease were treated with MUC1-KLH conjugate plus QS-21, containing 100 microg of MUC1 and 100 microg of QS-21. s.c. vaccinations were administered at weeks 1, 2, 3, 7, and 19. Peripheral blood was drawn at frequent intervals to assess antibody titers. Skin tests were placed at weeks 1, 3, 9, and 21 to determine delayed type hypersensitivity reactions. Common toxicities included a local skin reaction at the site of the vaccine, usually of 4-5 days' duration, and mild flu-like symptoms usually of 1-2 days' duration. High IgM and IgG antibody titers against synthetic MUC1 were detected. IgG antibody titers remain elevated from a minimum of 106-137 weeks after the first vaccination. Binding of IgM antibody to MCF-7 tumor cells was observed in seven patients, although there was minimal binding of IgG antibody. Two patients developed significant antibody titers post-high-dose chemotherapy and stem cell reinfusion. There was no evidence of T cell activation. This MUC1-KLH conjugate plus QS-21 was immunogenic and well tolerated in breast cancer patients. Additional trials are ongoing to determine the optimal MUC1 peptide for use in larger clinical trials. Further investigation of vaccine therapy in high-risk breast cancer is warranted.

Adjuvants, Immunologic↗

PubMed2000

Temperature-modulated high-performance liquid chromatography for detecting variation in human genome.

Recently, temperature modulated heteroduplex analysis (TMHA) based upon ion-pair reversed-phase high-performance liquid chromatography has been developed as a tool for polymorphisms and novel mutation detection in human genome. This new approach, with increased accuracy and throughput over traditional methods. This new approach is also known as denaturing high performance liquid chromatography(DHPLC). However, the terms of both TMHA and DHPLC should be considered carefully. Firstly, it is DNA molecular that is denatured in high performance liquid chromatography but not the method or the instrument. Secondly, the temperature in the liquid chromatograph is modulated according to Tm (melting temperature) of the DNA fragments. As it is known, Tm is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands, and under this temperature, half of the DNA molecules is still not denaturing. Thirdly, the targets for detection are both the homoduplexes and the mismatched heteroduplexes, which are double-stranded nucleic acid molecules and are annealing. The term of DHPLC may lead to confusion in annealing and denaturing. In addition, since TMHA can be carried out using gel electrophoresis and HPLC, there is a need to distinguish HPLC from gel electrophoresis. Therefore, we suggest a new term for this technology, which is TmHPLC (temperature-modulated high- performance liquid chromatography). This term not only provides a clear definition for this new method, but also includes both TMHA and HPLC. More importantly, TmHPLC implies that the temperature in HPLC is modulated according to Tm of the DNA fragments. We also review advances in detecting variation through TmHPLC. This new approach leads to significant reduction of sequencing efforts for searching novel mutations and polymorphisms, and because of its high sample throughput, to faster data generation. This implies that TmHPLC is a sensitive, accurate, and cost effective approach to screening sequence variation in human genome.

Chromatography, High Pressure Liquid↗

PubMed2000

A preliminary study of single nucleotide polymorphisms of lipoprotein lipase gene in coronary atherosclerotic heart disease.

OBJECTIVE: This study inquired into the relationship between single nucleotide polymorphisms(SNPs) of lipoprotein lipase gene (LPL) and coronary atherosclerotic heart disease(CHD). METHODS: Exon 6 and fragment of intron 6 in LPL gene from 102 cases of CHD and 110 normal subjects were analysed by polymerase chain reaction amplification, temperature-modulated high-performance liquid chromatography (TmHPLC) and DNA sequencing. RESULTS: Three new SNPs (4212t/c, 4509t/c, 4576a/c) were detected in the intron 6 of LPL gene. There was significant difference in frequencies of 4212t/c, and 4576a/c between health group and CHD group(P<0.05). CONCLUSION: This study has accumulated additional data on SNPs in LPL gene and provided new data for exploring the mechanism of CHD.

Adult↗

PubMed2000

Analysis of temperature-modulated high-performance liquid chromatography as a tool for detection of single nucleotide polymorphism.

OBJECTIVE: To establish and evaluate temperature-modulated high-performance liquid chromatography(TmHPLC) as a rapid and efficient technique of detecting single nucleotide polymorphism(SNP). METHODS: Several TmHPLC parameters, such as the eluting gradient ranges and column temperatures used for analysing the amplicons have been studied. The size of DNA fragments suitable for analysis of SNP was studied. Also the sensitivity of the TmHPLC was evaluated. RESULTS: The suitable chromatographic condition of TmHPLC was found out. The elution peaks of dNTP and primers could be separated from those of polymerase chain reaction products of the genomic DNA. The heterozygotes of SNP from the sequenced DNA fragment with 178bp, 269bp and 272bp could be distinguished from the homozygotes of SNP clearly. CONCLUSION: The authors have established and evaluated the TmHPLC method, which is an efficient, sensitive and rapid method for screening out SNP in the DNA fragments of larger size.

Chromatography, High Pressure Liquid↗

PubMed2000

Fluidity and fatty acid components of erythrocyte membranes in diabetic retinopathy.

BACKGROUND: Recently, numerous anomalies of erythrocyte rheology and function have been reported in diabetic patients. These changes are thought to be related to the alteration of erythrocyte membrane fluidity and may play an important role in diabetic retinopathy (DR). Nonetheless, the principal factors contributing to fluidity change remain undetermined. The influence of erythrocyte membrane fatty acid components on membrane fluidity and retinopathy was evaluated in patients with type 2 diabetes. METHODS: The fatty acid components and fluidity of erythrocyte membranes in 63 patients with type 2 diabetes with and without retinopathy were examined using high-performance liquid chromatography and fluorescence polarization. RESULTS: The content and composition of erythrocyte membrane arachidonate (C20:4n-6) was significantly lower in diabetics than in controls, and diabetics had significantly increased erythrocyte membrane microviscosity compared with controls. Furthermore, membrane microviscosity was higher in diabetics with DR than those without DR. In the diabetic patients, arachidonic acid contents of erythrocyte membranes were significantly, inversely correlated with erythrocyte membrane microviscosity, fasting blood glucose, glycosylated hemoglobin, triglycerides and cholesterol, and positively correlated with the insulin sensitivity index. CONCLUSIONS: The results suggest that erythrocyte membrane fatty acid components may contribute to alterations in membrane fluidity in patients with type 2 diabetes. Alterations in membrane fluidity may play an important role in the development of diabetic microangiopathy.

Aged↗

PubMed2000

Gene variation of 5'-NCR and core region in serum samples collected from patients with HCV infection.

OBJECTIVE: To study the variation of HCV 5'-NCR and core gene in serial sera collected from patients with HCV infection. METHODS: Serial sera were collected from 4 plasma-donors with HCV infection. 5'-NCR and core gene were amplified, sequenced and analyzed using software. RESULTS: All the sequences obtained from the serial sera of patients with HCV infection were 1b and 2a subtypes. The variation of 5'-NCR only related to the genotype, and not associated with patients and time of serum collection. However, the sequences of core gene were not identical in HCV strains isolated from different patients. The sequences of those two regions of the same genotype isolated from the same patient did not change with time. CONCLUSION: 5'-NCR is more conserved than the core region and the genotype is the major cause of gene variation. No change in sequences of those two regions is found at the different points of time.

5' Untranslated Regions↗