Biomedical subjects

D Zhou

Publications and source records attributed to D Zhou.

At least 73 records · Page 4Linked to original sources

PubMed2001

[Induction and kinetic characterization of nitric oxide synthase in hepatocytes].

OBJECTIVE: To study the synergistic responses of nitric oxide synthase (NOS) induction in rat hepatocytes to LPS and various cytokines in vitro and the kinetic characteristics of iNOS. METHODS: The hepatocytes were isolated by in-situ pre-perfusion and collagenase circulatory perfusion of rat livers. The effects of LPS associated with IFN-r, TNF-alpha and IL-1beta or IL-6 on NOS activity, cGMP, and NO(2)(-)+NO(3)(-) were observed in hepatocytes, respectively. Also the kinetic characteristics of this enzyme and dose response of corticosteroids on the induction of iNOS were analyzed. RESULTS: The maximum induction of NOS activity was observed in hepatocytes treated by LPS in combination with IFN-r, TNF-alpha and IL-1beta or IL-6. The kinetic analysis of this iNOS demonstrated specific constants of Km=10.8 micromol/L, Vmax=263.2 pmol/min/mg protein(for L-Arg), and Ki of 0.56, 0.94 micromol/L for competitive inhibitor, L-NMMA and NNA, respectively. The time course of induction showed that iNOS activity peaked at 9 h; however, significant increase in release of NO(2)(-)+NO(3)(-) and cGMP sustained for at least 18 h. Dexamethasone and hydrocortisone dramatically inhibited the NOS induction in hepatocytes in vitro with IC50 of 3.5+/-10(-8)mol/L and 2.6+/-10(-6)mol/L, respectively. CONCLUSIONS: The expression of inductive NOS in hepatocytes requires specific synergetic action of cytokines, and the inducible characteristics may play an important pathogenesis in endotoxemia and septic shock.

Animals↗

PubMed2001

[Manufacture and study of porous poly(l-lactic acid) (PLLA)/beta-tricalcium phosphate (beta-TCP) composite].

A promising alternative to supply bone substitutes is to develop living tissue substitutes based on biodegradable materials, which is called bone tissue engineering. One of the research high-lights of bone tissue engineering is to design and manufacture scaffolds for cell attaching, migrating, and proliferating. A process which consists of a solvent casting stage, a compression molding stage and a leaching stage has been used to fabricate macroporous composites of poly(l-lactic acid) (PLLA) and beta-tricalcium phosphate (beta-TCP). The effects of the weight fraction of porogen--NaCl, of the weight ratio of PLLA to beta-TCP and of the diameters of beta-TCP on the porosities, the average pore diameters and the compressive yield strength and compressive modulus have been studied. The results showed that the porosities and the average pore diameters increased and the compressive yield strength and modulus decreased when the weight fraction went from 50% to 90%. The compressive yield strength and compressive modulus could be improved by changing the weight ratio of PLLA to beta-TCP and the diameters of beta-TCP in low-porosity composites (lower than 70%). But high-porosity composites (90%) were not reinforced by changing the weight ratio.

Biocompatible Materials↗

PubMed2001

[Effects of vascular endothelial growth factor on adhesion molecular expression of hepatocellular carcinoma HepG].

OBJECTIVE: To study the effect of vascular endothelial growth factor (VEGF) on homotypic adhesion and E-cadherin of hepatocellular carcinoma cells. METHODS: We measured the function of homotypic adhesion with (3)H-TdR infiltration and the expression of E-cadherin with laser scanning confocal microscope. RESULTS: After 60, 90, 120min, the values (dpm/min) of (3)H-TdR infiltration in HepG(2) cells induced by 1ng/ml, 5ng/ml VEGF were 1758.67+/-289.46, 1380.03+/-328.55, 2657.43 +/- 310.31 and 3124.30+/-262.14, 2245.60+/-273.24, 2091.52+/-213.84. The values by 10ng/ml VEGF were 1232.32+/-201.04, 2337.50+/-333.04, 2236.99 +/- 237.07, which were significantly lower than those in control group (P<0.05 or 0.01). The expression of E-cadherin in hepatocellular carcinoma cells induced by 5ng/ml VEGF was 352+/-56 and much lower than that in control group (757+/-103, P<0.01). CONCLUSIONS: VEGF can decrease homotopic adhesion of hepatocellular carcinoma HepG(2) cell, which is connected with the less expression of E-cadherin in hepatocellular carcinoma HepG(2) cell.

Cadherins↗

PubMed2001

[Genotypes of transfusion-transmitted virus].

OBJECTIVE: To investigate the genotypes of transfusion-transmitted virus (TTV) in South China. METHODS: One capture probe for all types of TTV from the conserved sequence nt2160 to nt2196 and six varied probes, heterogeneity more than 20% between each other for G1, G2, G3, G4, G5 and G6 type, respectively were synthesized. The primers coming from the conserved sequence of ORF1 were used to amplify TTV DNA from the serums of patients. Microplate sandwich hybridization-ELISA technique was used to detect the genotypes of TTV in 380 hepatitis patients with liver damage. RESULTS: Sixty-one of 380 patients, about 16.0%, were TTV DNA positive, in whom 44 were genotype I (G1), 5 were G2, 10 were mixed infection of G1 and G2, one was G3 and one was G4. Neither G5 nor G6 has been detected. The rate of TTV infection in chronic hepatitis patients was equal to that in acute hepatitis patients. CONCLUSIONS: TTV infection is common in South China. There are four types of TTV. The first genotype was G1, and the second was G2. G5 or G6 has not yet been detected. PCR microplate sandwich hybridization-ELISA has certain value in clinic diagnosis for its specific, sensitive and practicable characters.

DNA Virus Infections↗

PubMed2001

[Umbilical cord blood transplantation (UCBT) in thalassemia children].

OBJECTIVE: To evaluate the efficacy of allo-UCBT in thalassemia children. METHODS: Five patients with beta-thalassemia major (genotypes were homozygote of beta41-42, beta654 and double heterozygosities of beta41-42/beta654, beta41-42/-28, beta654/-28, respectively) were treated by allo-UCBT. The median age was 3 year and ten month old (31/2 - 7 2/12). Four donor-recipient pairs were HLA-identical and one 1 locus mismatch. The patients received a median UCB nucleated cells (NC) of 5.5 x 10(7)/kg body weight [range (4.7 - 7.7) x 10(7)/kg] and CD(34)(+)CD(38)(-) cells 2.8 x 10(5)/kg [range (0.6 - 4.5) x 10(5)/kg] and CFU-GM 1.09 x 10(5)/kg [range (0.24 - 230.00) x 10(5)/kg]. The conditioning regimen consisted ofbusalphan 16 - 20 mg/kg, cyclophosphamide 180 - 200 mg/kg, horse antithymocyte globulin (ATG) 90 mg/kg or rabbit anti-lymphocyte globulin (ALG) 25 mg/kg and melphalan of 90 mg/m(2) was added to four cases. Cyclosporine A (CsA) alone was administered in 3 cases, and a combination of CsA and methotrexate in the other 2 cases. RESULTS: Four cases were engrafted, but one rejected and recurred thalassemia state at day 60 past UCBT. One case had autologous reconstitution. The reconstitutions were found in all patients with neutrophils 0.5 x 10(9)/L at day 19 (14 - 22), platelets > 20 x 10(9)/L at day 37 (21 - 63). Two cases developed grade I and II acute GVHD respectively. None developed chronic GVHD. Three patients survived disease-freely (survival with ex-thalassemia state). The median follow-up was 18 months, the probability of survival was 100%. CONCLUSION: Allo-UCBT could reconstitute hematopoiesis with low frequency and mild grade of GVHD. It is an effective treatment for thalassemia.

Child↗

PubMed2001

[Transforming growth factor-beta can induce differentiation of hypertrophic chondrocytes into osteoblast-like cells in epiphyseal plate].

OBJECTIVE: To investigate the effect of transforming growth factor-beta (TGF-beta) on the differentiation of hypertrophic chondrocytes in epiphyseal plate in order to understand its role in the mechanism of bone and cartilage formation. METHODS: We observed the effect of TGF-beta in varied time and dosage on the differentiation of hypertrophic chondrocytes in embryonic chick femora which were cultured in serum-free medium, using histochemical staining to detect the expression of alkaline phosphatase (ALP) and immunohistochemical staining to detect the expression of type I collagen and fibronectin. RESULTS: TGF-beta enabled the chondrocytes to express type I collagen and fibronectin, and it enhanced the expression of ALP in hypertrophic zone. Time-effect and dosage-effect relationships were observed. CONCLUSION: TGF-beta can induce the differentiation of hypertrophic chondrocytes into osteoblast-like cells in epiphyseal plate of embryonic chick femora. The inducing effect might be closely related to time and dosage of TGF-beta.

Animals↗

PubMed2000

Altered ligand binding and transcriptional regulation by mutations in the PML/RARalpha ligand-binding domain arising in retinoic acid-resistant patients with acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by a specific translocation, t(15;17), that fuses the promyelocytic leukemia (PML) gene with the RA receptor RARalpha. Pharmacologic doses of retinoic acid (RA) induce differentiation in human APL cells and complete clinical remissions. Unfortunately, APL cells develop resistance to RA in vitro and in vivo. Recently, mutations in PML/RARalpha have been described in APL cells from patients clinically resistant to RA therapy. The mutations cluster in 2 regions that are involved in forming the binding pocket for RA. These mutant PML/RARalpha proteins have been expressed in vitro, which shows that they cause a diversity of alterations in binding to ligand and to nuclear coregulators of transcription, leading to varying degrees of inhibition of retinoid-induced transcription. This contrasts with the nearly complete dominant negative activity of mutations in PML/RARalpha previously characterized in cell lines developing RA resistance in vitro. Current data from this study provide additional insight into the molecular mechanisms of resistance to RA and suggest that alterations in the ability of mutants to interact with coregulators can be determinant in the molecular mechanism of resistance to RA. In particular, ligand-induced binding to the coactivator ACTR correlated better with transcriptional activation of RA response elements than the ligand-induced release of the corepressor SMRT. The diversity of effects that are seen in patient-derived mutations may help explain the partial success to date of attempts to overcome this mechanism of resistance in patients by the clinical use of histone deacetylase inhibitors.

Amino Acid Substitution↗

PubMed2000

Androgen receptor-immunoreactivity in the forebrain of the Eastern Fence lizard (Sceloporus undulatus).

Androgen receptor (AR) distribution in the lizard forebrain and optic tectum was examined using PG21 immunohistochemistry. In the male Eastern Fence lizard, AR-immunoreactive (-ir) nuclei were observed in the medial preoptic area, ventromedial and arcuate hypothalamic nuclei, periventricular hypothalamus, premammillary nucleus, bed nucleus of the stria terminalis, and ventral posterior amygdala. Punctate immunostaining of neuronal processes (axons and/or dendrites) was concentrated in the cortex, hypothalamus, and optic tectum. AR-ir nuclei in the female brain were confined to the ventral posterior amygdala and ventromedial hypothalamic nucleus. The AR distribution in the lizard brain is similar to that reported for other vertebrate classes. Sex differences in AR-immunoreactivity may contribute to sex-specific behaviors in the Eastern Fence lizard.

Animals↗

PubMed2000

Biophysical characterization of SipA, an actin-binding protein from Salmonella enterica.

An essential step in the pathogenesis of Salmonella enterica infections is bacterial entry into non-phagocytic cells of the intestinal epithelium. Proteins injected by Salmonella into host cells stimulate cellular responses that lead to extensive actin cytoskeleton reorganization and subsequent bacterial uptake. One of these proteins, SipA, modulates actin dynamics by directly binding to F-actin. We have biophysically characterized a C-terminal fragment, SipA(446-684), which has previously been shown to retain activity. Our results show that SipA(446-684) exhibits an elongated shape with a predominantly helical conformation and predict the existence of a coiled-coil domain. We suggest that the protein is able to span two adjacent actin monomers in a filament and propose a model that is consistent with the observed effects of SipA(446-684) on actin dynamics and F-actin stability and morphology.

Actins↗

PubMed2000

Differences of four catechins in cell cycle arrest and induction of apoptosis in LoVo cells.

(-)-Epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epicatechin (EC) were found to have different cytotoxicities to human colon carcinoma LoVo cells. EGCG and EGC suppressed the growth of LoVo cells in dose-dependent manner; ECG and EC, however, had no obvious effects. At lower concentrations, EC did seem to promote slight proliferation. Similarly, EGCG and EGC, rather than ECG and EC, were found to induce apoptosis in LoVo cells. Moreover, EGCG, EGC, and ECG arrested G(1) phase in the cell cycle progression, whereas EC resulted in S phase arrest. We suggest that there are differences in the cytotoxicities of the four catechins to LoVo cells. They exert the effects by inducing apoptosis and regulating the cell cycle.

Apoptosis↗

PubMed2000

Cytoskeletal disruption accelerates caspase-3 activation and alters the intracellular membrane reorganization in DNA damage-induced apoptosis.

In actinomycin D (AD)-induced apoptosis, caspase-3 activation and DNA cleavage in human megakaryoblastic leukemia CMK-7 cells were greatly accelerated by tubulin and actin polymerization inhibitors [e.g., colcemid (CL) and cytochalasin D (CD), respectively], but the acceleration was not found with Taxol or phalloidin. A decrease in mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of procaspase-9 to its active form preceded the activation of caspase-3 and, moreover, all of these events began earlier and/or proceeded faster in cells treated with AD plus CL or CD than in cells treated with AD only. These results suggest that cytoskeletal disruption in the apoptotic cells promotes damage of the mitochondrial membrane, resulting in the enhanced release of cytochrome c necessary for the activation of caspase-9 that initiates the caspase cascade. On the other hand, apoptotic bodies were rapidly formed from cells treated with AD and CL, but were suppressed when treated with AD and CD. Intracellular membranes and the actin system were reorganized to surround the nuclear fragments in the AD- and CL-treated cells, but such a membrane system was not formed in the presence of CD, implying that the apoptotic bodies are formed via reorganization of intracellular membranes under regulation by actin polymerization. Thus, the cytoskeletal change in CMK-7 cells has a strong effect on the early biochemical process as well as on the later morphologic process in AD-induced apoptosis.

Actins↗

PubMed2000

Striking a balance: modulation of the actin cytoskeleton by Salmonella.

Salmonella spp. have evolved the ability to enter into cells that are normally nonphagocytic. The internalization process is the result of a remarkable interaction between the bacteria and the host cells. Immediately on contact, Salmonella delivers a number of bacterial effector proteins into the host cell cytosol through the function of a specialized organelle termed the type III secretion system. Initially, two of the delivered proteins, SopE and SopB, stimulate the small GTP-binding proteins Cdc42 and Rac. SopE is an exchange factor for these GTPases, and SopB is an inositol polyphosphate phosphatase. Stimulation of Cdc42 and Rac leads to marked actin cytoskeleton rearrangements, which are further enhanced by SipA, a Salmonella protein also delivered into the host cell by the type III secretion system. SipA lowers the critical concentration of G-actin, stabilizes F-actin at the site of bacterial entry, and increases the bundling activity of the host-cell protein T-plastin (fimbrin). The cellular responses stimulated by Salmonella are short-lived; therefore, immediately after bacterial entry, the cell regains its normal architecture. Remarkably, this process is mediated by SptP, another target of the type III secretion system. SptP exert its function by serving as a GTPase-activating protein for Cdc42 and Rac, turning these G proteins off after their stimulation by the bacterial effectors SopE and SopB. The balanced interaction of Salmonella with host cells constitutes a remarkable example of the sophisticated nature of a pathogen/host relationship shaped by evolution through a longstanding coexistence.

Actins↗

PubMed2000

The beta 1,3-galactosyltransferase beta 3GalT-V is a stage-specific embryonic antigen-3 (SSEA-3) synthase.

We have previously reported the molecular cloning of beta1, 3-galactosyltransferase-V (beta3GalT-V), which catalyzes the transfer of Gal to GlcNAc-based acceptors with a preference for the core3 O-linked glycan GlcNAc(beta1,3)GalNAc structure. Further characterization indicated that the recombinant beta3GalT-V enzyme expressed in Sf9 insect cells also utilized the glycolipid Lc3Cer as an efficient acceptor. Surprisingly, we also found that beta3GalT-V catalyzes the transfer of Gal to the terminal GalNAc unit of the globoside Gb4, thereby synthesizing the glycolipid Gb5, also known as the stage-specific embryonic antigen-3 (SSEA-3). The SSEA-3 synthase activity of beta3GalT-V was confirmed in vivo by stable expression of the human beta3GalT-V gene in F9 mouse teratocarcinoma cells, as detected with the monoclonal antibody MC-631 by flow cytometry analysis and immunostaining of extracted glycolipids. The biological relation between SSEA-3 formation and beta3GalT-V was further documented by showing that F9 cells treated with the differentiation-inducing agent retinoic acid induced the expression of both the SSEA-3 epitope and the endogenous mouse beta3GalT-V gene. This study represents the first example of a glycosyltransferase, which utilizes two kinds of sugar acceptor substrates without requiring any additional modifier molecule.

Animals↗

PubMed2000

Designing small-molecule switches for protein-protein interactions.

Mutations introduced into human growth hormone (hGH) (Thr175 --> Gly-hGH) and the extracellular domain of the hGH receptor (Trp104 --> Gly-hGHbp) created a cavity at the protein-protein interface that resulted in binding affinity being reduced by a factor of 10(6). A small library of indole analogs was screened for small molecules that bind the cavity created by the mutations and restore binding affinity. The ligand 5-chloro-2-trichloromethylimidazole was found to increase the affinity of the mutant hormone for its receptor more than 1000-fold. Cell proliferation and JAK2 phosphorylation assays showed that the mutant hGH activates growth hormone signaling in the presence of added ligand. This approach may allow other protein-protein and protein-nucleic acid interactions to be switched on or off by the addition or depletion of exogenous small molecules.

Amino Acid Sequence↗

PubMed2000

Oxidative stress and vanadate induce tyrosine phosphorylation of phosphoinositide-dependent kinase 1 (PDK1).

Phosphoinositide-dependent kinase (PDK1) regulates a number of pathways involved in responses to stress and in growth factor signaling; however, little is known concerning the mechanisms governing the activity of PDK1. In this report, we find that oxidative stress (H(2)O(2)) and vanadate induce tyrosine phosphorylation of PDK1. These effects of H(2)O(2) and vanadate were found in 293T cells and CH310T1/2 cells expressing exogenous PDK1 and in A20 lymphoma cells expressing endogenous PDK1. Exogenously expressed PDK1 was also tyrosine-phosphorylated in response to NGF treatment of 293T expressing TrkA. H(2)O(2) induced a more rapid tyrosine phosphorylation of PDK1 relative to vanadate, and only vanadate-induced tyrosine phosphorylation of PDK1 was sensitive to pretreatment of cells with wortmannin. In vitro, PDK1 could be tyrosine-phosphorylated by both the c-Src and Abl tyrosine kinases. Both H(2)O(2) and vanadate treatments increased the activity of PDK1 when the serum/glucocorticoid regulated kinase (SGK) was used as substrate. Vanadate treatment appeared to bypass the requirement for phosphatidylinositol 3,4,5-trisphosphate when Akt was used as substrate for PDK1. Tyrosine phosphorylation of PDK1 by the Abl tyrosine kinase also increased the activity of PDK1 toward SGK and Akt. These data suggest a novel mechanism through which PDK1 activity may be regulated.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2000

Acquisition of intact allogeneic human leukocyte antigen molecules by human dendritic cells.

In an attempt to transduce monocyte-derived dendritic cells (DCs) by a retroviral vector coding for a cell surface marker, we were confronted by the observation of high transfer of the surface molecule in the absence of vector proviral DNA in the treated cells. Indeed, DCs acquired the surface marker by a mechanism independent of the vector machinery, requiring cell-to-cell contact and involving transfer of lipids and a variety of intact membrane proteins. Most important, this property of DCs also includes acquisition of foreign human leukocyte antigen (HLA) molecules. Consequently, DCs become immunological hybrids as they display their own and foreign HLA molecules. The newly acquired HLA is fully functional because it allows recognition by allo-specific T lymphocytes and the binding and presentation of antigen peptides.

Cell Communication↗

PubMed2000

Secretion and purification of recombinant beta1-4 galactosyltransferase from insect cells using pFmel-protA, a novel transposition-based baculovirus transfer vector.

The palette of transfer vectors available for generation of recombinant baculoviruses based on transposition-mediated recombination has been enlarged by constructing the pFmel-protA vector. The pFmel-protA plasmid includes the honeybee melittin secretion signal and a Staphylococcus aureus protein A fusion protein tag, which allows the secretion and purification of recombinant proteins. Using this system, the human beta1-4 galactosyltransferase-I protein was expressed in Sf9 insect cells at a level ranging from 22 to 28 U (4.8 to 6.0 mg)/L. The protein A tag enabled a simple monitoring of recombinant protein expression by enzyme-linked immunosorbent assay and Western blotting. Single step purification was achieved by immunoglobulin G affinity chromatography achieving a recovery yield of 28% and a specific activity of 1.9 U per mg of recombinant protein.

Amino Acid Sequence↗

PubMed2000

Alkaline treatment of the cellulose fiber affecting membrane column behaviour for high-performance immunoaffinity chromatography.

The original cellulose fibers and those treated by alkaline solution were both used to prepare the acrylic membranes. The two kinds of membranes were packed into the columns for high-performance immunoaffinity chromatography by the immobilization of protein A on them. It was observed that the alkaline treatment of the cellulose fiber decreased the pressure resistance of the membrane to the mobile phases and greatly increased the accessible volume to the proteins, but affected the adsorption capacity of human IgG on the protein A membrane columns less. There is little difference between those two kinds of membranes on the adsorption capacities of HIgG, which means that the alkaline treatment of the cellulose fiber only significantly changes the void volume inter-membrane, and the porosity and surface area of membrane less. Alkaline treatment of the cellulose fiber reduced the membrane-column efficiency significantly. Some typical examples for the immunoaffinity analysis of IgG from human and dog plasma on the protein A membrane columns are illustrated.

Alkalies↗