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Biomedical subjects

W Zhong

Publications and source records attributed to W Zhong.

At least 109 records · Page 6Linked to original sources
PubMed1996

Asymmetric localization of a mammalian numb homolog during mouse cortical neurogenesis.

During Drosophila neurogenesis, differential segregation of Numb is necessary for daughter cells of asymmetric divisions to adopt distinct fates, at least partly by biasing the Notch-mediated cell-cell interaction. We have isolated a highly conserved mammalian homolog of Drosophila numb, m-numb. During mouse cortical neurogenesis, m-Numb is asymmetrically localized to the apical membrane of dividing ventricular neural progenitors. Depending upon the orientation of the cleavage plane, m-Numb may be distributed into one or both of the daughter cells. When expressed in Drosophila embryos, m-Numb is localized asymmetrically in dividing neural precursors and rescues the numb mutant phenotype. Furthermore, m-Numb can physically interact with mouse Notch1. We propose that some shared molecular mechanisms, both cell-intrinsic and cell-extrinsic, generate asymmetric cell divisions during neurogenesis of vertebrates and invertebrates.

Amino Acid Sequence↗
PubMed1996

Lytic growth of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in culture.

Kaposi's sarcoma (KS) is the leading neoplasm of AIDS patients, and HIV infection is known to be a major risk factor for its development. However, KS can occur in the absence of HIV infection and the risk of KS development varies widely even among HIV-infected patients, with homosexual men with AIDS being 20 times more likely to develop KS than AIDS-afflicted children or hemophiliacs. These and other data strongly suggest that a sexually transmitted agent or co-factor may be involved in KS pathogenesis. Recently, DNA sequences corresponding to the genome of a novel member of the herpesvirus family have been identified within AIDS-KS biopsies, and several reports indicate that these sequences are also present in all forms of HIV-negative KS. These and other findings suggest this new agent, referred to as KS-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8), as a candidate for the putative etiologic cofactor. However, the role of this agent in KS remains hotly debated. Further progress in understanding its biology has been severely hampered by the lack of a cell culture system for virus growth. Here we report the development of a system for the lytic growth of this virus in a latently infected B cell line and present the first ultrastructural visualization of the virus. This system will facilitate the detailed study of the molecular biology of viral replication, the testing of antiviral drugs and the development of diagnostic tests for viral infection.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Combined chemokine and cytokine gene transfer enhances antitumor immunity.

The probability of producing a specific antitumor response should be increased by multiplying the number of T lymphocytes that encounter the malignant cells. We tested this prediction in a murine model, using a recently discovered T-cell chemokine, lymphotactin (Lptn). This chemokine increased tumor cell infiltration with CD4+ lymphocytes but generated little antitumor activity. Coexpression of the T-cell growth factor interleukin-2, however, greatly expanded the T lymphocytes attracted by Lptn, affording protection from the growth of established tumor in a CD4+ and CD8+ T cell-dependent manner. Lesser synergy was seen with GM-CSF. Hence coexpression of a T-cell chemokine and T-cell growth factor potentiates antitumor responses in vivo, suggesting a general strategy to improve cancer immunotherapy.

Animals↗
PubMed1996

The size and conformation of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) DNA in infected cells and virions.

The genome of a novel human herpesvirus has been detected in specimens of Kaposi's sarcoma (KS) and in several AIDS-related lymphoproliferative disorders. Here we examine the size and genomic conformation of the DNA of this virus (known as KS-associated herpesvirus or human herpesvirus 8) in latently and lytically infected cells and in virions. Pulsed-field gel electrophoresis of viral DNA shows that the viral genome is similar in size to those of other gammaherpesviruses (160 to 170 kb). As with Epstein-Barr virus, KS-associated herpesvirus DNA is stably maintained in latently infected B cells as episomal monomer circles and induction from latency is associated with the selective accumulation of linear genomic forms.

Cell Line↗
PubMed1996

Inhibition of cell growth and sensitization to oxidative damage by overexpression of manganese superoxide dismutase in rat glioma cells.

The effects of overexpression of human manganese superoxide dismutase (MnSOD) on cell proliferation and response to oxidative stress in rat glioma cells were studied. MnSOD-overexpressing cells had a 2- to 14-fold increase in MnSOD activity, but did not have consistent changes in the activities of CuZnSOD, catalase, or glutathione peroxidase. Cells with more than a 5-fold increase in MnSOD activity became more sensitive to radiation, 1,3-bis(2-chloroethyl)-1-nitrosourea, and buthionine sulfoximine and had a lower growth rate than parental and vector control cells. The sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea was partially reduced by pyruvate, a H2O2 scavenger. Our results suggest that overexpression of MnSOD can cause an imbalance of antioxidant enzymes, which we hypothesize results in an elevation of intracellular H2O2. Overexpression of MnSOD can either inhibit cell proliferation or increase cell death by oxidative agents, depending on the levels of peroxide-removing enzymes.

Animals↗
PubMed1996

Dose-response relations for unnatural amino acids at the agonist binding site of the nicotinic acetylcholine receptor: tests with novel side chains and with several agonists.

Structure-function relations in the nicotinic acetylcholine receptor are probed using a recently developed method based on chemical synthesis of nonsense suppressor tRNAs with unnatural amino acid residues, site-directed incorporation at nonsense codons in Xenopus laevis oocytes, and electrophysiological measurements. A broad range of unnatural amino acids, as many as 14 at a given site, are incorporated at three sites, alpha 93, alpha 190, and alpha 198, all of which are tyrosine in the wild-type receptor and are thought to contribute to the agonist binding site. Confirming and expanding upon earlier studies using conventional mutagenesis, the three tyrosines are shown to be in substantially different structural microenvironments. In particular, a crucial role is established for the hydroxyl group of alpha Tyr93, whereas a variety of substituents are functional at the analogous position of alpha Tyr198. Interestingly, consideration of three different agonists (acetylcholine, nicotine, and tetramethylammonium) does not discriminate between these two best-characterized binding site residues. In addition, double-mutation studies establish the independent effects of mutations at the pore region (second transmembrane region) and at the agonist binding site, and this observation leads to a novel strategy for adjusting EC50 values. These results establish the broad generality and great potential of the unnatural amino acid methodology for illuminating subtle structural distinctions in neuroreceptors and related integral membrane proteins.

Amino Acids↗
PubMed1995

Cationic liposome-mediated intravenous gene delivery.

Systemic gene transfer provides new opportunities for the analysis of gene function and gene regulation in vivo, as well as for human gene therapy. We used the chloramphenicol acetyltransferase reporter gene to examine several parameters important for the development of efficient, cationic liposome-mediated, intravenous (IV) gene transfer in mice. We then demonstrated that this approach can produce high level expression of biologically important genes. Specifically, we assessed the relationship of expression vector design to the level of systemic gene expression produced, and compared transfection levels produced by intravenously injecting DNA alone versus DNA-liposome complexes. We found that both the position of the heterologous intron, and the promoter element used in the expression plasmid, significantly affected the level of systemic gene expression produced. Although intravenous injection of plasmid DNA alone transfected every tissue analyzed, liposome-mediated delivery was much more efficient. We also established that repeated i.v. injection of DNA-liposome complexes produced high level systemic transfection. The second injection of DNA-liposome complexes produced levels of gene expression at least as high as those following a single i.v. injection. Thus, unlike some viral vectors, a neutralizing host-immune response does not limit re-expression, following reinjection of DNA-liposome complexes. Finally, we showed that the expression vectors which produced the highest levels of chloramphenicol acetyltransferase reporter gene expression could also produce high level expression of two colony stimulating factor genes in mice. Specifically, i.v. injection of liposomes complexed to expression vectors into which we had inserted either the murine granulocyte-macrophage-colony stimulating factor cDNA or the human granulocyte-CSF cDNA, produced circulating levels of the corresponding colony stimulating factor gene product comparable to levels which have been shown previously to be both biologically and therapeutically significant.

Animals↗
PubMed1995

A chemiluminescent immunoassay for the identification of functional single-chain antibodies.

Random mutagenesis of a template single-chain Fv (scFv) antibody generates a mutant library with members of varying ligand affinities; however, only a small population of these mutants exhibit strong (Kd approximately 1 nM) or improved affinities. We have therefore designed a chemiluminescent sandwich assay which can identify strong ligand-binding mutants expressed by Escherichia coli. A model system involving scFv 43C9 and selected mutants with varying hapten affinities was used to demonstrate the application of this methodology. Both the plaque and colony lifts of these scFvs were screened for hapten-binding activities with the chemiluminescent assay, and only strong binders were detected.

Antibodies, Bacterial↗
PubMed1995

The nucleotide sequence of chromosome I from Saccharomyces cerevisiae.

Chromosome I from the yeast Saccharomyces cerevisiae contains a DNA molecule of approximately 231 kbp and is the smallest naturally occurring functional eukaryotic nuclear chromosome so far characterized. The nucleotide sequence of this chromosome has been determined as part of an international collaboration to sequence the entire yeast genome. The chromosome contains 89 open reading frames and 4 tRNA genes. The central 165 kbp of the chromosome resembles other large sequenced regions of the yeast genome in both its high density and distribution of genes. In contrast, the remaining sequences flanking this DNA that comprise the two ends of the chromosome and make up more than 25% of the DNA molecule have a much lower gene density, are largely not transcribed, contain no genes essential for vegetative growth, and contain several apparent pseudogenes and a 15-kbp redundant sequence. These terminally repetitive regions consist of a telomeric repeat called W', flanked by DNA closely related to the yeast FLO1 gene. The low gene density, presence of pseudogenes, and lack of expression are consistent with the idea that these terminal regions represent the yeast equivalent of heterochromatin. The occurrence of such a high proportion of DNA with so little information suggests that its presence gives this chromosome the critical length required for proper function.

Base Sequence↗
PubMed1995

Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells.

The nonsense codon suppression method for unnatural amino acid incorporation has been applied to intact cells and combined with electrophysiological analysis to probe structure-function relations in the nicotinic acetylcholine receptor. Functional receptors were expressed in Xenopus oocytes when tyrosine and phenylalanine derivatives were incorporated at positions 93, 190, and 198 in the binding site of the alpha subunit. Subtle changes in the structure of an individual side chain produced readily detectable changes in the function of this large channel protein. At each position, distinct features of side chain structure dominated the dose-response relation, probably by governing the agonist-receptor binding.

Animals↗
PubMed1995

Expression of mRNAs of multiple growth factors and receptors by neuronal cell lines: detection with RT-PCR.

Neurons and glia are capable of both secreting and responding to a large variety of growth factors. However, information on multiple expression of growth factors and their receptors was usually obtained from uncorrelated observations, using cells from various animals of origin, developmental stages, growth phases, culture ages and culture conditions. Because of its specificity and extreme sensitivity, reverse transcription-polymerase chain reaction (RT-PCR) is uniquely suitable to study a large panel of growth factors and their receptors from a limited cell sample, free of these intervening variables. In this paper we evaluate the expression of mRNA of a total of 35 growth factor-related proteins by conducting RT-PCR on three neuronal cell lines: the PC12 rat pheochromocytoma line, the MAH rat sympathoadrenal progenitor line, and the N18 mouse neuroblastoma line. Three types of results are presented. The first confirms the existing knowledge such as the presence of Trk-A (NFG receptor) in PC12. The second consists of new information that expands and extends earlier observations, such as the presence of CNTF receptor complex in PC12, which explains our previous report that CNTF enhances the biological effects of NGF on these cells. The third consists of novel information that leads the way to further experimentation by the more conventional methods. These include the strong expression of Trk-B by MAH, predicting the biological responsiveness of MAH to BDNF and NT-4, and the expression of CNTF receptor in N18. Our results also suggest that CNTF is an autocrine factor for PC12 and MAH, since both lines express the growth factor as well as the receptor. Thus, RT-PCR is a valuable tool in growth factor research that can be used in complement to, and interactively with, other approaches such as bioassay, receptor binding, and immunochemical determination. It will be particularly useful for screening a large number of growth factors in minute areas of the brain in patients suffering from neurodegenerative diseases such as Parkinson's and Alzheimer's.

Animals↗
PubMed1995

Expression of mRNAs of multiple growth factors and receptors by astrocytes and glioma cells: detection with reverse transcription-polymerase chain reaction.

1. Although glial cells in culture are known to secrete growth factors and are also known to be responsive to some of them, detailed comparisons are difficult because the bulk of information was based on various animals of origin, developmental stages, growth properties, culture age, and culture conditions. 2. To present a unified picture of the growth factors and their receptors found in glial cells, we surveyed the expression of messenger RNAs of a panel of growth factors and receptors, using reverse transcription-polymerase chain reaction (RT-PCR), in three common glial cell types: rat astrocytes in primary culture, rat glioma line C6, and human glioma line A172. 3. We observed that normal and neoplastic glial cells in culture express multiple growth factors and also possess most of the receptors to the factors, suggesting multiple autocrine functions. In addition, glia produce growth factors known to be capable of acting on neurons, implicating paracrine function involving glia-neuron interaction. Glial cells also produce growth factors and receptors that are capable of communicating with hematopoietic cells, suggesting neuroimmunologic interaction. What is most interesting is that glial cells express receptors for growth factors previously thought to be acting on neurons only. 4. The current study demonstrates the feasibility of screening from a small sample a large number of growth factors and receptors. The method portends future clinical application to biopsy or necropsy samples from brain tumors or pathologic brains suffering from degenerative diseases such as Alzheimer's or Parkinson's disease.

Animals↗
PubMed1995

Induction of B cell apoptosis by TH0, but not TH2, CD4+ T cells.

Engagement of the T cell receptor molecules with MHC-antigen complexes presented by B cells ascertains antigen specificity in T cell-dependent help. Ligation of MHC molecules on the surface of B cells, however, has not only been implicated in antigen-specific T-B cell interaction, but has also been linked to the induction of B cell apoptosis. To examine the role of T helper cells in either induction of immunoglobulin synthesis or B cell apoptotic death, we have facilitated T cell receptor-MHC interaction through a bacterial superantigen. CD4+ T cell clones could be categorized into two clearly distinct subsets based upon their ability to promote B cell help in the presence of superantigen. One subset of T cell clones supported immunoglobulin synthesis, and thus functioned as effective helper cells. B cells interacting with the second subset of T cells did not differentiate into antibody-secreting cells, but underwent apoptosis. Both types of helper cells were able to provide contact help after anti-CD3 stimulation. Induction of apoptosis was a dominant phenomenon; the addition of the superantigen suppressed immunoglobulin production in B cells activated by anti-CD3-stimulated helper T cells, indicating that the T cells delivered an apoptotic signal to the B cell. T cell clones providing effective MHC restrictive B cell help could be distinguished from T cells facilitating B cell apoptosis based on their lymphokine secretion profile. Induction of B cell apoptosis was a feature of T cells with a TH0 lymphokine pattern. Promotion of MHC-restricted B cell help was associated with a TH2 lymphokine profile. TH1-derived cytokines alone could not substitute for apoptosis-inducing T cells.

Antigens, CD↗