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Biomedical subjects

Y Dai

Publications and source records attributed to Y Dai.

At least 181 records · Page 10Linked to original sources
PubMed1995

Cytotoxicity of acetaminophen in human cytochrome P4502E1-transfected HepG2 cells.

Acetaminophen (APAP) when administered in excess can cause severe hepatic necrosis in vivo. To study the mechanism of APAP toxicity and the role of cytochrome P450, a previously established human hepatoma HepG2 subline, MVh2E1-9, that constitutively expresses human CYP2E1 was used as a model. At high concentrations (above 5 mM) and when intracellular reduced glutathione (GSH) was depleted, APAP caused severe cytotoxicity in MVh2E1-9, but not in MV-5 cells which lack CYP2E1. The APAP cytotoxicity was dependent on the concentration of APAP and time of exposure, and could be blocked by 4-methylpyrazole, ethanol, diallyl sulfide, N-acetylcysteine and N-t-butyl-alpha-phenylnitrone, but not by propylgallate, an inhibitor of lipid peroxidation. Significantly more 14C-labeled APAP protein adduct was detected in MVh2E1-9 cells than MV-5 cells, especially after depletion of GSH. The formation of the APAP adducts could be inhibited by the same agents which prevent APAP cytotoxicity. At a lower concentration (1-2 mM), APAP inhibited proliferation in both MVh2E1-9 and the control MV-5 cells to similar extents. This antiproliferative action of APAP did not require depletion of GSH as did the cytotoxic action of APAP. These data suggest that APAP has a dual toxic effect on MVh2E1-9 cells: a P450-independent antiproliferative effect and the CYP2E1-dependent cytotoxic effect. These results demonstrate the ability of human CYP2E1 to activate APAP to reactive metabolites which form covalent protein adducts and cause toxicity to a hepatoma cell line.

Acetaminophen↗
PubMed1995

Inactivation and degradation of human cytochrome P4502E1 by CCl4 in a transfected HepG2 cell line.

Treatment with CCl4 in vivo labilizes cytochrome P4502E1, inactivating the enzyme and enhancing its degradation. To investigate the mechanism of CCl4-induced degradation of human CYP2E1, a recently-established MVh2E1-9 cell line, which constitutively expresses the human CYP2E1 in HepG2 cells was used. CCl4 inhibited oxidation of p-nitrophenol in isolated microsomes from MVh2E1-9 cells suggesting that CCl4 could be metabolized in vitro by the system; however, CCl4 did not promote lipid peroxidation under these conditions. Treatment of the MVh2E1-9 cells in situ with 2 mM CCl4 for 24 hr caused a 30 to 50% loss of both enzyme activity and 2E1 protein. Treatment with cycloheximide at the same time to inhibit constitutive protein synthesis showed a more prominent loss of 2E1 activity and protein. CCl4-induced degradation of CYP2E1 could be prevented by ligands and substrates of 2E1. N-acetylcysteine, N-t-butyl-alpha-phenylnitrone or propylgallate did not significantly prevent CCl4-induced inactivation or degradation of 2E1. After treatment with 14C-labeled CCl4, there was increased radioactive adduct formation in MVh2E1-9 cells compared to control cells lacking CYP2E1. This increase was completely prevented by 4-methylpyrazole and ethanol indicating its dependence on CYP2E1. These results suggest that the human CYP2E1 expressed in the MVh2E1-9 cell line metabolizes CCl4, generating reactive species at the active site that directly inactivate the enzyme and also labilize P450 for degradation by proteases present in the HepG2 cells. Lipid peroxidation is not required for the CCl4-induced inactivation and degradation of CYP2E1 in these cells.

Acetaminophen↗
PubMed1995

Evidence for thrombin-induced human platelet secretion regulated by the cytoskeleton.

Human blood platelets store an abundance of growth factors, adhensive proteins, coagulation factors, platelet-specific proteins, calcium, serotonin, and adenine nucleotides in their secretory organelles. After exposure to stimuli (thrombin, collagen, ADP, etc.), the platelets undergo a rapid series of morphological change from characteristic disks to spheres with several filopodia, adhension to the inner surface of blood vessels, and aggregation among the platelets. Most of these changes are usually accompanied by secretion or release of dense bodies (release I) and alpha-granules (release II). The secreted products play essential parts in a variety of important physiological and/or pathological events, such as hematosis, thrombosis, blood coagulation, inflammation, and atherosclerosis. Little is known, however, for regulatory mechanism of platelet secretion. Platelets are a distinct kind of cells with many special organelles, but without a nucleus. It also contains a large amount of cytoskeleton. The numerous investigators have suggested that the platelet responses to stimuli are intimately linked to events involving cytoskeleton within the platelets. During platelet activation, as revealed by electron microscopy, the secretory organelles become centralized and enveloped by circular microtubules and a filamentous network. It is implied a possible relationship between cytoskeleton and platelet secretion. However, the precise role of cytoskeleton in platelet secretion remains uncertain. By using exposed GMP-140 (a platelet granule-membrane protein with MW of 140 kDa) as a specific signal of secretion, the present study was designed to investigate the effects of microtubular and microfilamental inhibitors on thrombin-induced platelet secretion.

Adult↗
PubMed1995

[The interstitial response and postoperation recurrent mechanism in liver cancer].

Immunoactive cell group markers and glycoproteins of extracellular matrix and basement membrane (fibronectin, laminin, type IV collage) in 156 human hepatocellular carcinomas and their surrounding tissues were localized with immunoperoxidase technique. T lymphocytes were the major immunoactive cells in the regions of local invasion in liver cancer. The distributing patterns of the glycoproteins were similar to the results of previous reports, mainly localized in the surrounding cancer cells, interstitium and vessel walls or sinusoids in tumor, and tumor capsule. The patients, whose hepatocarcinomas showed stronger cell immunoactivity (T and help T cells were in the majority) and higher expression of glycoproteins of extracellular matrix and basement membrane had a longer tumor-free survival (63.2 to 12.8 months) and lower recurrences (P < 0.01). Of the 156 patients 92.9%, 82.6%, 65.4%, 44.2% and 30.8% and 1, 2, 3, 4 and 5 postoperative tumor-free years respectively with a total currence rate of 53.2% (83 patients). In the 83 recurrences, 65 were intrahepatic subclinical, which were promptly reresected 78.3%, recurrence related factors included tumor number and size, capsule infiltration, and portal veins involvement. Being different from those with single node, capsulated hepatocellular carcinomas and multiple/daughter ones had invisible tumor cells disseminated to the remnant liver. Those without capsule infiltration had a low recurrence rate, which agrees to the hypothesis that capsules can bar the dissemination of tumor cells. Additional results show that postoperative tumor recurrence is mainly pertinent to histopathological characteristics of the primary focus. According to the phenomenon that nearly 63.1% of recurrent liver carcinomas are located at the ipsilateral segment of the primary ones, we emphasize that the occurrence and recurrence of liver carcinoma are mainly unicentral.

Adolescent↗
PubMed1995

[Electrophoretic analysis of the protein patterns of the nuclear transplant rabbit embryos].

The qualitative patterns of protein synthesis in nuclear transplant rabbit embryos were examined by SDS-polyacrylamide gel electrophoresis followed by silver staining. The results indicated that the qualitative pattern of several protein synthesis in NT embryos was very different from the protein pattern of donor morulae or recipient oocytes, and also not as the same as the protein pattern obtained from fertilized ova at pronuclear formation stage. After fertilization or nuclear transfer, several maternal proteins were no longer synthesized or decreased in the embryo, and some new bands were observed and several protein synthesis were increased obviously. The most intriguing aspect of this study was the observation that all major changes in the protein pattern took place after fertilization or nuclear transfer and were rather similar. It is suggested that the gene activities of the donor nucleus from rabbit morulae are reprogrammed by the oocyte cytoplasm in current nuclear transfer technology, but the reprogramming is incomplete. This paper stresses on the paternal effect during fertilization on gene expression in nuclear transplant rabbit embryos.

Animals↗
PubMed1994

Mimosine inhibits viral DNA synthesis through ribonucleotide reductase.

The plant amino acid mimosine (beta-N(3-hydroxy-4-pyridone)-alpha-amino propionic acid) is toxic for animals and reversibly inhibits the replication of animal cells in vitro. We have found that mimosine inhibits the DNA synthesis of a variety of DNA viruses, including adenovirus, parvovirus, and papovavirus and the replication of vaccinia and herpes simplex virus 1 in cells in culture. However, mimosine did not inhibit the replication of SV40 DNA in a cell-free system. Because mimosine inhibition of viral DNA synthesis was reversible by iron in the medium, we hypothesized that mimosine was inhibiting ribonucleotide reductase through its capacity to chelate the iron required in the R2 subunit of this enzyme. In support of this hypothesis, we found that mimosine-treated cells had a reduction in the pools of dGTP and dATP and that mimosine inhibited ribonucleotide reductase in vitro in an iron-dependent manner.

DNA Primase↗
PubMed1994

Modulation of the chemotactic responsiveness of guinea pig neutrophils to hrIL-8 and fMLP.

Neutrophils elicited in the peritoneal cavity of guinea pigs were purified on Percoll gradients and their chemotactic response to hrIL-8 and fMLP measured in vitro. hrIL-8 and fMLP were effective chemoattractants with optimal concentrations of 6 x 10(-9) and 1 x 10(-7) M, respectively. Scatchard analysis revealed approximately 205,000 IL-8 receptors/cell and 34,000 fMLP receptors/cell with KD values of 4.1 x 10(-9) and 3.3 x 10(-8) M, respectively. At suboptimal concentrations of chemoattractants the response was inhibited by dibutyryl cyclic AMP, histamine, and adenosine in the presence of a phosphodiesterase inhibitor. IL-8 and fMLP induced an increase in cellular cyclic AMP and the response to optimal concentrations of chemoattractants was inhibited by Calphostin C and Ro 31-8220, inhibitors of protein kinase C (PKC). Our results indicate that the chemoattractants activate the same PKC-dependent pathway that is down-regulated by cyclic AMP-dependent mechanisms.

Amino Acid Sequence↗
PubMed1994

Examination of rat salivary glands for the presence of the aquaporin CHIP.

The aquaporin CHIP, AQP-CHIP, is a 28-kDa integral membrane protein that functions as a water channel in kidney and red blood cells. We have examined rat salivary glands for the presence of this molecule. In Northern blot analyses an AQP-CHIP cDNA hybridized to rat kidney mRNA approximately 20-fold more than to mRNA from the rat parotid gland. Western blot analyses, using antisera against human AQP-CHIP, showed that AQP-CHIP was present in crude membranes from rat salivary glands to a much lesser extent than in rat kidney membranes. A 2.8 kb cDNA encoding AQP-CHIP was also isolated from an adult rat parotid gland cDNA library. Immunolocalization studies using thin and ultrathin cryosections and light and electron microscopy, however, revealed that no glandular epithelium was labeled. In contrast, significant immunolabeling was present in microvascular endothelial cells. Non-fenestrated endothelia of capillaries and venules exhibited labeling on both their apical and basolateral membranes. The present study demonstrates that AQP-CHIP is not involved directly in fluid secretion from salivary epithelial cells. Further, this study provides strong evidence that AQP-CHIP is an endothelial cell water channel found within salivary gland tissue which may contribute to water permeability in the capillary beds.

Animals↗
PubMed1994

Desensitisation of neutrophil responses by systemic interleukin 8 in cystic fibrosis.

BACKGROUND: Inflammation associated with neutrophil infiltration is a commonly observed feature of children with cystic fibrosis. Production of the major neutrophil chemotactic cytokine interleukin 8 (IL-8) is potentially of great importance in the pathology of cystic fibrosis. Concentrations of IL-8 in both sputum and bronchoalveolar lavage fluid have been found to be higher in children with cystic fibrosis than in controls. The IL-8 induced chemotactic response and numbers of IL-8 receptors on peripheral neutrophils obtained from children with cystic fibrosis have been compared with a control group of children. METHODS: Cells were isolated from 18 patients with cystic fibrosis (aged 4-20 years) and 13 controls (aged 5-12 years) by dextran centrifugation followed by separation on Lymphoprep. Chemotaxis was assayed using multiwell microchemotaxis chambers and 5 microns polycarbonate filters. Filters were fixed and stained with Haema-Gurr for counting. Results were expressed as numbers of neutrophils per high power field (HPF). RESULTS: At the optimum concentration (1 x 10(-8) mol/l) the number of cells migrating were similar for controls (150 (12)/HPF) and for the cystic fibrosis group (140 (14)/HPF)). At lower concentrations the numbers of neutrophils migrating were lower for the cystic fibrosis group. Scatchard analysis of 125I-labelled IL-8 binding revealed lower numbers of receptors on neutrophils from patients with cystic fibrosis (22,000 per cell) than from controls (75,000 per cell). CONCLUSIONS: Reduced responsiveness to IL-8 of neutrophils from patients with cystic fibrosis is associated with receptor desensitisation as a result of exposure to high systemic levels of IL-8.

Adolescent↗
PubMed1994

Intra-A chain disulfide bond (A6-11) of insulin is essential for displaying its activity.

The mutant proinsulin gene was constructed with the codons for A6 and A11 Cys changed to Ser to delete intra-A chain disulfide bond. After expression and purification, the mutations in the protein were further confirmed by amino acid composition. Electrophoretic mobility of the mutant proinsulin is similar to that of human proinsulin, so are the products of tryptic digestions. The mutant proinsulin, which retains its full radioimmuno activity, shows only 5.4% of receptor binding activity of human proinsulin. This suggests that though the intra-A chain disulfide bond disappears, the other two inter-chain disulfide bonds are still correctly paired, and hence the three dimensional structure has not been altered significantly. This intra-chain disulfide bond is essential for insulin displaying its activity.

Amino Acid Sequence↗
PubMed1994

Comparison of polymerase chain reaction and immunohistochemistry for the detection of Mycoplasma pulmonis in paraffin-embedded tissue.

The polymerase chain reaction (PCR) is a reliable method of detecting specific DNA sequences. The purpose of this study was to develop a PCR method of detecting Mycoplasma pulmonis in paraffin-embedded tissue sections and to compare the sensitivity of this method with that of the avidin-biotin complex (ABC) immunohistochemistry technique. We infected mice intranasally with 10(7) M. pulmonis and sacrificed them 28 days later. Using a published oligonucleotide primer sequence for M. pulmonis, we examined 8-microns paraffin sections by PCR for a 710 base-pair segment of the genome. A sequential 8-microns paraffin section was stained for M. pulmonis, using standard ABC immunohistochemistry methods. By PCR and dot blot hybridization, 60 of 62 paraffin-embedded lungs were positive for M. pulmonis, whereas only 17 of 62 lungs were positive, using the ABC method. In this study, we demonstrated that PCR of paraffin-embedded tissues followed by dot blot hybridization of the PCR products was much more sensitive than the ABC method in identifying mouse lungs infected with M. pulmonis.

Animals↗
PubMed1994

[New endoscopic laser treatment for unilateral vocal cord paralysis after pulmonary resection].

In two patients who had developed aspiration due to left recurrent laryngeal nerve injury during radical resection for lung cancer, low power Nd:YAG laser irradiation of the paralyzed vocal cord was performed through a bronchofiberscope. With irradiation, the paralyzed vocal cords of both patients swelled and became fixed at the median portion of the glottis. Thereafter, aspiration disappeared. Thus, this new application of Nd:YAG laser irradiation is apparently quite useful in treating aspiration due to unilateral vocal cord paralysis immediately after pulmonary resection.

Bronchoscopy↗
PubMed1993

Stable expression of human cytochrome P4502E1 in HepG2 cells: characterization of catalytic activities and production of reactive oxygen intermediates.

Experiments were carried out to stably and constitutively express the coding sequence of the human cytochrome P4502E1 in HepG2, a human-hepatoma-derived cell line, by recombinant retroviral expression. Southern blot analysis showed a successful integration of a single copy of unaltered viral DNA into the genome of each transduced clone tested. Northern blot analysis showed that the transduced clones produced an RNA species which hybridized to the CYP2E1 cDNA probe. Western blot analysis using anti-human P4502E1 IgG indicated that the transduced clones produced a protein band with molecular weight of 54 000. Microsomes from transduced clones were catalytically active with p-nitrophenol, dimethylnitrosamine, aniline, and ethanol as substrates; little or no activity was found with control clones. Oxidation of p-nitrophenol was inhibited by anti-human P4502E1 IgG, diethyl dithiocarbamate, 4-methylpyrazole, and ethanol. ESR spectroscopy showed that microsomes from clone MV2E1-9 produced superoxide radical. Rates were an order of magnitude higher than that for control microsomes, most likely reflecting the loose coupling associated with P4502E1. The rate of H2O2 production by microsomes from MV2E1-9 was 2-fold greater than that of control clones. The elevated rate of H2O2 production in clone MV2E1-9 is about half the rate of superoxide radical production, suggesting that this H2O2 is largely derived from superoxide radical dismutation. Microsomal lipid peroxidation was determined using ferric-ATP as the iron catalyst. When the concentration of iron was "high" (0.025 mM), rates of production of thiobarbituric acid reactive components were identical for microsomes from MV2E1-9 and control clones. However, when the concentration of iron was lowered to 0.005 mM, control clones did not display lipid peroxidation, whereas microsomes from MV2E1-9 were reactive. This peroxidation was sensitive to antioxidants such as trolox, propyl gallate, and glutathione but not to catalase or superoxide dismutase. Rates of superoxide and H2O2 production and of lipid peroxidation were 7-20-fold higher on a per nanomole of P450 basis with clone MV2E1-9 compared to human liver microsomes, indicating that the human P4502E1 is especially reactive in production of reactive oxygen intermediates and in catalysis of lipid peroxidation.

3T3 Cells↗
PubMed1993

Dynamic observations on NK cell activity and IL-2 and IFN gamma production in vitro in patients on maintenance hemodialysis.

In order to study the immune function of patients on maintenance hemodialysis (MHD), we assayed NK cell cytotoxicity against K562 targets in 40 patients on MHD, and the production of IL-2 and IFN in peripheral blood mononuclear cells (PBMC) after PHA stimulation, in contrast to those in normal controls. The results showed that NK cell activity and IL-2 and IFN levels were markedly lower in the patients than in the controls. After a single dialysis, NK cell activity as well as IL-2 and IFN levels were elevated to different extent. But there was no significant change in patients after long-term dialysis. There was a positive correlation between the NK cell activity and IL-2 and IFN activity in the controls, but no such correlation was found in the patients on MHD. There was a positive correlation between the NK cell activity and IL-2 activity in patients after dialysis, suggesting that immune function were impaired in the patients on MHD, with a decline in the activity of NK cell and IL-2 and IFN, and a disorder of immune regulation cycle. These abnormal immune impairments in the patients could be partly corrected by hemodialysis. However, long-term hemodialysis is not much helpful in the improvement of patient's immune function.

Adult↗
PubMed1993

Correlative factors of left ventricular hypertrophy in end-stage renal disease.

The correlative factors of left ventricular hypertrophy (LVH) in 287 end-stage renal disease (ESRD), admitted from 1983 to 1992, were analyzed. 52% of patients had LVH including 54% of patients on hemodialysis, 75% of patients with peritoneal dialysis and 38% of transplanted patients. Single factor analysis indicated that age, blood pressure, serum creatinine and BUN, hemoglobin, serum alkaline phosphatase, aortic valve disease and present DSRD therapy were related to LVH. Multiple logistic regression analysis indicated that the most important factors which independently related to LVH in all patients studied, were management of present ESRD treatment, age, hypertension and high serum alkaline phosphatase. In a subset of patients with severe LVH, high serum alkaline phosphatase level, high systolic blood pressure and age were the predictive factors. In patients on dialysis, the most important variable were age and high serum alkaline phosphatase. Hypertension was the sole predictor of LVH in the transplant group.

Adult↗
PubMed1993

Relationship between muscarinic receptor occupancy and response in rat parotid acinar cells.

To determine whether spare muscarinic cholinergic receptors (mAChRs) exist in rat parotid acinar cells, we examined the effect of propylbenzilylcholine mustard (PBCM) on agonist (carbachol)-stimulated inositol trisphosphate (IP3) formation and on mAChR number, using l-[N-methyl-3H]scopolamine methyl chloride (NMS)-binding assays. Treatment with PBCM (1, 3, 10, 30, 50 nM) for 15 min caused a 5, 22, 60, 66, and 72% decrease, respectively, in maximal IP3 formation stimulated by carbachol as well as a large reduction in the potency of carbachol in eliciting this response. Using these data, equilibrium constants (Ka) for activation of the mAChRs by carbachol were calculated. These Ka values agreed well with Kd values of high-affinity mAChR binding sites determined from carbachol displacement of [3H]NMS binding in parotid acinar cells. Reduction in mAChR number after PBCM treatment was determined by Scatchard analysis of specific [3H]-NMS binding sites and compared with the expected reduction (q values) calculated from dose-response curves for carbachol-stimulated IP3 formation before and after PBCM treatment. PBCM (1, 3, 10, 30 nM) decreased mAChR maximal binding in cells 47.5, 68.9, 82.4, and 85.3%, respectively, which did agree with the approximately 38, 70, 90, and 92% decrease in receptor number expected from the calculated q values. Data demonstrate that PBCM irreversibly inactivates mAChRs in rat parotid cells, and the decrease in receptor number, measured directly from [3H]NMS binding or calculated from receptor theory, is greater than that observed for stimulated IP3 production. These results suggest that a modest (30-40%) population of spare receptors exists for mAChR-mediated IP3 production in rat parotid glands.

Animals↗
PubMed1993

Signaling mechanisms that regulate saliva formation.

The precipitating event in the formation of saliva is the binding of neurotransmitter molecules to cell surface receptor proteins. The principal neurotransmitters involved are acetylcholine and norepinephrine that bind, respectively, to muscarinic-cholinergic, and alpha- and beta-adrenergic receptors. The transduction of the extracellular signal requires an integral membrane protein capable of binding GTP, a G protein, that specifically interacts with the receptor. The components of G protein transduction systems are fairly well studied, but the pathways by which signals are routed are just being recognized. Delineation of such routing pathways is essential to understanding the regulation of saliva formation.

Animals↗