Search PubMed⌕ Search

Biomedical subjects

W Zhong

Publications and source records attributed to W Zhong.

At least 73 records · Page 4Linked to original sources
PubMed1999

CUL-2 is required for the G1-to-S-phase transition and mitotic chromosome condensation in Caenorhabditis elegans.

The human cullin protein CUL-2 functions in a ubiquitin-ligase complex with the von Hippel-Lindau (VHL) tumour suppressor protein. Here we show that, in Caenorhabditis elegans, cul-2 is expressed in proliferating cells and is required at two distinct points in the cell cycle, the G1-to-S-phase transition and mitosis. cul-2 mutant germ cells undergo a G1-phase arrest that correlates with accumulation of CKI-1, a member of the CIP/KIP family of cyclin-dependent-kinase inhibitors. In cul-2 mutant embryos, mitotic chromosomes are unable to condense, leading to unequal DNA segregation, chromosome bridging and the formation of multiple nuclei.

Amino Acid Sequence↗
PubMed1999

A complex translational program generates multiple novel proteins from the latently expressed kaposin (K12) locus of Kaposi's sarcoma-associated herpesvirus.

The most abundantly expressed latent transcripts encoded by the Kaposi's sarcoma (KS)-associated herpesvirus derive from the genomic region surrounding open reading frame (ORF) K12 (kaposin A). Here we show that these transcripts, initially described as limited to ORF K12 itself, more frequently encompass upstream sequences spanning two sets of 23-nucleotide GC-rich direct repeats (DRs) (DR1 and DR2). Although the DRs lack AUG codons and were previously presumed to be noncoding, a monoclonal antibody raised to infected cells detected multiple polypeptides encoded by this region. These proteins are expressed during latency and upon induction of lytic viral replication in both primary effusion lymphoma (PEL) cell lines and KS tumors. Biochemical and genetic analyses reveal that these proteins are derived from variant translational initiation at CUG codons. The predominant translation product in the PEL cell line BCBL-1 derives from the 5'-most CUG codon in the transcript, resulting in a protein (termed kaposin B) which is encoded largely by the repeats themselves and which does not include K12 sequences. Other non-AUG codons in alternate reading frames are also used at lower efficiency, including one that initiates translation of a DR-K12 fusion protein (kaposin C) that is predicted to sort to a different subcellular locale than kaposin B. Thus, the products of the K12 region, which is the most abundantly transcribed region in latency, are surprisingly complex and may encompass multiple biological functions.

3T3 Cells↗
PubMed1999

[Transmission electron microscopic observations on dentin structures stained with silver].

The purpose of this study was to investigate the ultrastructure of silver deposit regions on human dentin. Three kinds of silver stained specimens were used in this study, the decalcified celloidin section stained with silver, the frozen section of decalcified tooth stained with silver, and the decalcified section of undecalcified tooth treated by the silver iontophorese method. After light microscopic observation of the specimens, these were embedded again in epoxy resin and ultra thin sections were made. Then these ultra thin sections were observed under transmission electron microscope, and the results were as follows: 1. By observing the decalcified celloidin section stained with silver, a deposition of silver particles was found on the surface and inside of the hypocalcified collagen fibrils. 2. By observing the section of decalcified tooth stained with silver, a deposition of silver particles was found on the hypercalcified collagen fibrils. According to the findings, it was concluded that the interglobular net and the dark-line of mineralized striation in dentin are hypercalcified regions. 3. By using the silver iontophorese method, large silver particles were found deposited among the hypocalcified collagen fibrils. 4. It was confirmed that the degree of mineralization of collagen fibrils in hard tissue can be estimated indirectly observing the morphological change of collagen fibrils in a decalcified section.

Collagen↗
PubMed1998

From ab initio quantum mechanics to molecular neurobiology: a cation-pi binding site in the nicotinic receptor.

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation-pi interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation-pi binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues-tryptophan-149 of the alpha subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of alpha tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position alpha149 produces a constitutively active receptor.

Binding Sites↗
PubMed1998

Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo.

The factors controlling cationic liposome-DNA complex (CLDC)-based gene transfer in cells and in animals are poorly understood. We found that cell surface heparin/heparan sulfate-bearing proteoglycans mediate CLDC-based gene transfer and expression both in cultured cells and following intravenous gene delivery into animals. CLDC did not transfect Raji cells, which lack proteoglycans, but did efficiently transfect Raji cells stably transfected with the proteoglycan, syndecan-1. Fucoidan, heparin, or dextran sulfate, all of which are highly anionic polysaccharides, each blocked CLDC-mediated transfection both in cultured cells and following intravenous injection into mice, but had no effect on transfection by either recombinant adenovirus infection or electroporation. Intravenous pretreatment of mice with heparinases, which specifically cleave heparan sulfate molecules from cell surface proteoglycans, blocked intravenous, CLDC-mediated transfection in mice, confirming that proteoglycans mediate CLDC gene delivery in vivo. Modulation of proteoglycan expression may prove useful in controlling the efficiency of, as well as targeting the sites of, CLDC-based gene transfer in animals.

Adenoviridae↗
PubMed1998

Effect of human C-reactive protein on chemokine and chemotactic factor-induced neutrophil chemotaxis and signaling.

C-reactive protein (CRP) is a unique serum pentraxin and the prototype acute phase reactant. CRP is a ligand for specific receptors on phagocytic leukocytes, and mediates activation reactions of monocytes/macrophages, but inhibits the respiratory burst of neutrophils (PMN). Since CRP selectively accumulates at inflammatory sites in which IL-8 is also produced, we tested the effects of CRP on the responsiveness of PMN to IL-8 and the bacterial chemotactic peptide, FMLP-phenylalanine (FMLPP). Purified human CRP inhibited the chemotactic response of PMN to IL-8 and FMLPP. A mouse IgM mAb that was generated against the leukocyte CRP receptor (CRP-R) also inhibited the chemotactic response. Incubation of purified CRP with activated PMN generated CRP-derived peptides that also inhibited chemotaxis. A synthetic CRP peptide (residues 27-38) that binds to the CRP-R had weak chemotactic activity, whereas two other CRP synthetic peptides (residues 174-185 and 191-205) inhibited chemotaxis of PMNs to both IL-8 and FMLPP. CRP did not alter receptor-specific binding of IL-8, but exerted its effect at the level of signaling. CRP augmented both IL-8- and FMLPP-induced mitogen-activated protein kinase (extracellular signal-regulated kinase-2) activity. CRP at acute phase levels increased both agonist-induced and noninduced phosphatidylinositol-3 kinase activity. The results suggest a role for CRP as a regulator of leukocyte infiltration at inflammatory sites.

Amino Acid Sequence↗
PubMed1998

Differentiation of Lactobacillus Species by Molecular Typing.

A total of 64 type, reference, clinical, health food, and stock isolates of microaerophilic Lactobacillus species were examined by restriction fragment length polymorphisms. Of particular interest were members of six of the eight species most commonly recovered from the vaginas of healthy premenopausal women, namely, Lactobacillus jensenii, L. casei, L. rhamnosus, L. acidophilus, L. plantarum, and L. fermentum. Six main groupings were identified on the basis of ribotyping. This technique was able to classify fresh isolates to the species level. In the case of the ribotype A grouping for L. rhamnosus, differences between strains were evident by chromosome typing (chromotyping). Many isolates did not possess plasmids. Six L. rhamnosus strains isolated from four different health food products appeared to be identical to L. rhamnosus ATCC 21052. The molecular typing system is useful for identifying and differentiating Lactobacillus isolates. Studies of strains of potential importance to the urogenital flora should include molecular characterization as a means of comparing genetic traits with those of strains whose characteristics associated with colonization and antagonism against pathogens have been defined.

Journal Article↗
PubMed1998

Overexpression of manganese superoxide dismutase in DU145 human prostate carcinoma cells has multiple effects on cell phenotype.

BACKGROUND: Recent studies suggest that the gene for manganese superoxide dismutase (MnSOD) is a candidate tumor-suppressor gene. The present study was designed to study the effect of overexpression of MnSOD on cultured human prostate carcinoma cells. METHODS: DU145 human prostate carcinoma cells were transfected with the cDNA for manganese superoxide dismutase (MnSOD), and two clones overexpressing MnSOD activity were subsequently characterized by comparison with parental and plasmid control-transfected cells. RESULTS: One clone overexpressing MnSOD had no change in other antioxidant enzymes (AEs) (nonadapted), while a second clone showed an increase in catalase activity (adapted). Sensitivity of parental, plasmid control-transfected, and MnSOD cDNA-transfected cells to agents that generate oxidative stress correlated with AE profiles. Both clones overexpressing MnSOD activity showed increased reactive oxygen species levels under basal cell culture conditions. Both clones overexpressing MnSOD activity showed inhibition of cell growth in vitro and in vivo compared with parental and plasmid control-transfected cells. Flow cytometry studies using mitochondrial-specific probes showed equal mitochondrial mass in all cell lines, but altered mitochondrial membrane potential in MnSOD-overexpressing clones compared with parental or plasmid control-transfected cells. CONCLUSIONS: Our results suggest novel mechanisms by which MnSOD overexpression may modulate the malignant phenotype, with potential applications in developing new therapies for prostate cancer.

Animals↗
PubMed1998

Development of an internally quenched fluorescent substrate for Escherichia coli leader peptidase.

Escherichia coli leader peptidase, an integral membrane protein, is responsible for the cleavage of the signal sequence of many exported proteins. Recent studies suggest that it is a novel serine protease that utilizes a serine-lysine catalytic dyad. In an effort to further understand the mechanism of this enzyme, an internally quenched fluorescent peptide substrate incorporating the leader peptidase cleavage site of maltose binding protein signal peptide, Y(NO2)-F-S-A-S-A-L-A-K-I-K(Abz) (anthraniloyl), was designed and synthesized. In the intact peptide, the fluorescence of the anthraniloyl group is quenched by the 3-nitrotyrosine. This quenched fluorescence is liberated upon cleavage of the peptide by the leader peptidase, resulting in increased fluorescence that could then be monitored fluorometrically. The designed substrate can be cleaved effectively by E. coli leader peptidase as detected by both HPLC and fluorescent spectroscopy. Mass spectra of cleavage products demonstrated that the cleavage occurs at the predicted site (A-K). The cleavage of the peptide substrate has a linear dependence on the enzyme concentration (0.1 to 1.9 microM) and the kcat/K(m) was calculated to be 71.1 M-1 s-1. These data are comparable with the unmodified peptide substrate. This report represents the first direct continuous assay based on fluorescence resonance energy transfer for E. coli leader peptidase.

Amino Acid Sequence↗
PubMed1998

Inhibition of cell growth in NIH/3T3 fibroblasts by overexpression of manganese superoxide dismutase: mechanistic studies.

NIH/3T3 mouse fibroblasts were transfected with the cDNA for manganese superoxide dismutase (MnSOD), and two clones overexpressing MnSOD activity were subsequently characterized by comparison with parental and control plasmid-transfected cells. One clone with a 1.8-fold increase in MnSOD activity had a 1.5-fold increase in glutathione peroxidase (GPX) activity (increased GPX-adapted clone), while a second clone with a 3-fold increase in MnSOD activity had a 2-fold decrease in copper, zinc superoxide dismutase (CuZnSOD) activity (decreased CuZnSOD-adapted clone). Increased reactive oxygen species (ROS) levels compared with parental or control plasmid-transfected cells were observed in nonsynchronous cells in the increased GPX-adapted clone, but not in the decreased CuZnSOD-adapted clone. The two MnSOD-overexpressing clones showed different sensitivities to agents that generate oxidative stress. Flow cytometry analysis of the cell cycle showed altered cell cycle progression in both MnSOD-overexpressing clones. During logarithmic growth, both MnSOD-overexpressing clones showed increased mitochondrial membrane potential compared with parental and control plasmid-transfected cells. Both MnSOD-overexpressing clones showed a decrease in mitochondrial mass at the postconfluent phase of growth, suggesting that mitochondrial mass may be regulated by MnSOD and/or ROS levels. Our results indicate that adaptation of fibroblasts to overexpression of MnSOD can involve more than one mechanism, with the resultant cell phenotype dependent on the adaptation mechanism utilized by the cell.

3T3 Cells↗
PubMed1998

Binding properties and stoichiometries of a palladium(II) complex to metallothioneins in vivo and in vitro.

This paper will be the first to discuss the in vivo and in vitro properties of a Pd(II) complex, K2PdCl4, interacting with metallothioneins (MTs). In vivo experiments revealed that intraperitoneal injections of K2PdCl4 into rabbits led to the simultaneous synthesis of Pd-MT in the kidney and Zn7MT in the liver. The renal Pd-MT complex contains 3.6 +/- 0.3 Pd, 2.1 +/- 0.2 Zn, and 1.0 +/- 0.1 Cu per mole protein. It was found that pre-treatment with Zn(NO3)2 before K2PdCl4 injections significantly enhanced renal Pd-MT level. The same pre-treatment also increases hepatic Zn-MT levels. These results strongly suggest that Pd(II) ions can be bound in vivo by MT existing in the rabbit kidneys to form Pd-MT. Gel-filtration chromatographic studies after the incubation of either native Cd5Zn2MT2 or Zn7MT2 with K2PdCl4 in vitro demonstrate that Pd(II) ions promote the non-oxidative oligomerization of native MTs. Increasing the level of Pd(II) relative to MT led to a concomitant increase in the apparent yield of MT oligomers. At relatively low Pd-MT ratio, Pd(II) is found predominantly in the oligomers while the monomeric products are chiefly composed of the reactants, Cd5Zn2MT2 or Zn7MT2. Based on our experimental data, the mechanisms of the reactions between Pd(II) and MTs in vivo and in vitro are discussed.

Animals↗
PubMed1998

Identification and characterization of an RNA-dependent RNA polymerase activity within the nonstructural protein 5B region of bovine viral diarrhea virus.

Nonstructural protein 5B (NS5B) of bovine viral diarrhea virus (BVDV) contains sequence motifs that are predictive of an RNA-dependent RNA polymerase activity. We describe the expression and purification of the BVDV NS5B protein derived from an infectious cDNA clone of BVDV (NADL strain). BVDV NS5B protein was active in an in vitro RNA polymerase assay using homopolymeric RNA or BVDV minigenomic RNA templates. The major product was a covalently linked double-stranded molecule generated by a "copy-back" mechanism from the input template RNA. In addition, a nucleotide-nonspecific and template-independent terminal nucleotidyl transferase activity was observed with the BVDV NS5B preparation.

Animals↗
PubMed1998

[Culture of chondrocytes seeded onto chitin scaffoldings coating with different materials: experimental study].

OBJECTIVE: To find the possibility of chitin used as scaffolds in tissue engineering. METHOD: Chondrocytes were cultured when they were seeded onto chitin meshcoated with polylactic acid (PLA) lecithin (LEC) and poly-l-lysine (PLYS). With light microscope and scanning electron microscope, the scaffolds in hydrophilia and absorptivity to chondrocytes and the function of the cells were observed. RESULT: LEC can change the hydrophilia of scaffolds. PLYS can strengthen the absorptivity of the scaffolds. and promote the matrices production of chondrocytes. CONCLUSION: The chitin coated with LEC and PLYS can be used as scaffolds in tissue engineering.

Animals↗
PubMed1997

Therapeutic passive vaccination against chronic Lyme disease in mice.

Passive and active immunization against outer surface protein A (OspA) has been successful in protecting laboratory animals against subsequent infection with Borrelia burgdorferi. Antibodies (Abs) to OspA convey full protection, but only when they are present at the time of infection. Abs inactivate spirochetes within the tick and block their transmission to mammals, but do not affect established infection because of the loss of OspA in the vertebrate host. Our initial finding that the presence of high serum titers of anti-OspC Abs (5 to 10 microg/ml) correlates with spontaneous resolution of disease and infection in experimentally challenged immunocompetent mice suggested that therapeutic vaccination with OspC may be feasible. We now show that polyclonal and monospecific mouse immune sera to recombinant OspC, but not to OspA, of B. burgdorferi resolve chronic arthritis and carditis and clear disseminated spirochetes in experimentally infected C.B.-17 severe combined immunodeficient mice in a dose-dependent manner. This was verified by macroscopical and microscopical examination of affected tissues and recultivation of spirochetes from ear biopsies. Complete resolution of disease and infection was achieved, independent of whether OspC-specific immune sera (10 microg OspC-specific Abs) were repeatedly given (4x in 3- to 4-day intervals) before the onset (day 10 postinfection) or at the time of fully established arthritis and carditis (days 19 or 60 postinfection). The results indicate that in mice spirochetes constitutively express OspC and are readily susceptible to protective OspC-specific Abs throughout the infection. Thus, an OspC-based vaccine appears to be a candidate for therapy of Lyme disease.

Animals↗
PubMed1997

CD40 ligand induces an antileukemia immune response in vivo.

Leukemia cells may express tumor specific antigens in association with Class I and II major histocompatability complex (MHC) molecules. However, lack of expression of conventional costimulator molecules means that these cells tend to induce specific T-cell anergy rather than activation. CD40 ligand (CD40L) is a costimulator molecule that directly activates T cells and may promote antigen presentation by CD40-expressing cells, which include professional antigen presenting cells and B-acute lymphoblastic leukemia (ALL) cells from many patients. We determined whether transgenic expression of CD40L could enhance an antileukemia immune response using a CD40+ murine lymphoblastic (A20) leukemia and a CD40- myeloblastic (WEHI-3) leukemia in a tumor treatment model. Injection of otherwise nonimmunogenic A20 cells in the presence of CD40L induced an immune response active against preexisting A20 tumor at a distant site. Moreover, concomitant local secretion of transgenic interleukin-2 (IL-2) further amplified the antileukemic response induced and increased protection against preexisting tumor. In ex vivo studies, CD40 activation of A20 cells enhances the antigen presenting potential of A20 cells by upregulating expression of B7.1 (CD80), Class I and II MHC molecules, and increases expression of fas antigens. The importance of CD40 activation to the resulting antitumor response is further emphasized by the failure of transgenic CD40L to protect against the CD40- WEHI myeloblastic leukemia. Depletion studies showed the protective effects against A20 cells to be mediated by a combination of CD4+ and CD8+ T lymphocytes and by natural killer (NK) cells. These results suggest a means by which CD40+ leukemia cells may be rendered immunogenic in vivo.

Animals↗
PubMed1997

Interaction of sodium chloroplatinate and iproplatin with metallothionein in vivo.

The abilities of platinum(IV) complexes to induce the biosynthesis of metallothionein (MT) were investigated in rabbits given injections s.c. of sodium chloroplatinate (Na2PtCl6) and iproplatin (cis-dichloro-bis-isopropylamine-trans-dihydroxylplatinum IV). It is revealed for the first time that both complexes can induce MT synthesis in the liver and the kidney, but the induction ability was weaker compared to Zn2+ compounds. The induced MT was purified and identified. The hepatic MT resulting from Na2PtCl6 injection only contained Zn, whereas the hepatic MT from iproplatin injection and the renal MT from injection of both complexes contained 4-5 Zn and 1-2 Pt per mole of protein, and the renal MT also contained 1-2 Cu per mole of protein. The oxidation state of platinum in the MT is +2 as determined by X-ray photoelectron spectroscopic measurements. Pretreatment with Zn(NO3)2 elevated the levels of MT, but the binding of Pt to MT was significantly less compared to that without Zn(NO3)2 pretreatment. The data obtained from the amino acid composition analysis were consistent with the theoretical values. Upon these bases, the role of MT in relation to its involvement in the metabolism of Pt(IV) complexes and the mechanism of drug resistance to the Pt(IV) complexes as antitumor agents are discussed.

Animals↗
PubMed1997

Reaction of a platinum(IV) complex with native Cd,Zn-metallothionein in vitro.

The first observation of a redox process following a substitution reaction between a platinum(IV) complex K2PtCl6 with rabbit liver native Cd,Zn-MT is presented. The reaction features and products are studied by UV-visible and circular dichroism spectroscopy, chromatography, and X-ray photoelectron spectroscopic measurements. It is a significant complicated reaction comprising redox and substitution reactions. The reaction generates monomeric and dimeric products, and higher oligomers precipitate with intra- or intra- and intermolecular CyS-SCy linkages. Pt(IV) is reduced to Pt(II), which then binds to the monomeric and dimeric products, and may also bind to higher oligomers. The beta-cluster is more reactive than the alpha-cluster, and reacts first with K2PtCl6. Cd5Pt2 and Cd4Pt4 were found when native Cd,Zn-MT reacted with 2 and 4 molar equivalents of Pt(IV) for 2 h in which four Cd ions were located in the alpha-cluster. The amounts of Cd and Pt ions decreased in both monomeric and dimeric products when the reaction was prolonged and intramolecular CyS-SCy linkages increased. Besides the oligomers which precipitated, only dimeric products were formed when the reaction molar ratio of Pt(IV) to MT was more than 10:1. Cd3Pt6 and Cd1Pt8 were obtained when the reaction occurred for 2 and 72 h, respectively. The structure of the clusters may exist when native Cd, Zn-MT reacts with substoichiometric quantities of K2PtCl6 (< 0.5 K2PtCl6 per MT thiolate) for a short time (2 h), but may be partly disrupted with stoichiometric or excess quantities of K2PtCl6 (> or = 0.5 K2PtCl6 per MT thiolate) for a long time. The disruption of the cluster structures results in an increase of the nonbridge thiolate and an increase of the binding sites to Pt ions. The mechanism of the antitumor activity and developing drug resistance of Pt(IV) complex drugs is discussed.

Animals↗