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Biomedical subjects

W Zhong

Publications and source records attributed to W Zhong.

At least 91 records · Page 5Linked to original sources
PubMed1997

Suppression of the malignant phenotype of human glioma cells by overexpression of manganese superoxide dismutase.

Manganese superoxide dismutase (MnSOD) has been previously shown to suppress the malignant phenotype of human melanoma and breast cancer cells. To test the possible role of MnSOD in glioma malignancy, MnSOD was overexpressed in wild type human glioma U118 cells and subcloned U118-9 cells by transfection of human MnSOD cDNA. The MnSOD-transfected cell lines demonstrated expression of exogenous (plasmid) MnSOD mRNA, increase in MnSOD immunoreactive protein, and a three- to eightfold increase in MnSOD enzymatic activity. The MnSOD overexpressing cell lines became less malignant as demonstrated by requiring a higher serum concentration to grow in vitro and much slower tumor growth in nude mice than the parental and neo control cell lines. These findings further support the hypothesis that MnSOD may be a tumor suppressor gene in a wide variety of human tumors.

Animals↗
PubMed1997

A novel herpes vector for the high-efficiency transduction of normal and malignant human hematopoietic cells.

Herpes simplex viruses (HSVs) would offer numerous advantages as vectors for gene transfer, but as yet they have not proved capable of transducing hematopoietic cells. Using a genetically inactivated form of HSV that is restricted to a single cycle of replication (disabled single-cycle virus, [DISC-HSV]), we have transduced normal human hematopoietic progenitor cells and primary leukemia blasts with efficiencies ranging from 80% to 100%, in the absence of growth factors or stromal support. Toxicity was low, with 70% to 100% of cells surviving the transduction process. Peak expression of transferred genes occurred at 24 to 48 hours after transduction with the DISC-HSV vector, declining to near background levels by 14 days. Despite this limitation, sufficient protein is produced by the inserted gene to permit consideration of the vector for applications in which transient expression is adequate. One example is the transfer of immunostimulatory genes, to generate leukemia immunogens. Thus, murine A20 leukemia cells transduced with a DISC-HSV vector encoding granulocyte-macrophage colony-stimulating factor were able to stimulate a potent antitumor response in mice, even against pre-existing leukemia. The exceptional transducing ability of the DISC-HSV vector should therefore facilitate genetic manipulation of normal and malignant human hematopoietic cells for biological and clinical investigation.

Animals↗
PubMed1997

Binding site on human C-reactive protein (CRP) recognized by the leukocyte CRP-receptor.

C-reactive protein (CRP), the prototypical inflammatory acute phase reactant in humans, interacts with monocytes and neutrophils via a specific receptor. To map the site on CRP recognized by the CRP receptor (CRP-R), synthetic peptides corresponding to the surface region on each of the five identical subunits were tested as competitors vs. [125I]-CRP for cell binding. A peptide of residues 27-38 (TKPLKAFTVCLH) efficiently inhibited CRP binding when compared to other nonoverlapping peptides. This peptide was termed the cell-binding peptide (CB-Pep). The F(ab')2 of an IgG Ab to the CB-Pep specifically inhibited CRP binding upon reacting with the ligand. Competitive binding studies with synthetic peptides truncated from either the NH2- or COOH-terminus of the CB-Pep revealed that the minimum length recognized by the CRP-R consisted of residues 31-36: KAFTVC. Conservative substitutions of residues within the CB-Pep indicated that the four residues AFTV were critical for CRP-R binding. The CB-Pep also inhibited induced superoxide generation by HL-60 granulocytes. The minimum length required for the inhibition was also KAFTVC; however, only Phe-33 and Leu-37 were critical residues in this assay. Anti-CB-Pep IgG Ab reacted more extensively with heat-modified CRP, suggesting that an altered conformation of CRP is preferentially recognized by the CRP-R. The results suggest that this contiguous sequence on a beta-strand on one face of each of five subunits of the CRP pentamer serves as a unique recognition motif for inflammatory leukocytes.

Amino Acid Sequence↗
PubMed1997

T helper cell priming of mice to Borrelia burgdorferi OspA leads to induction of protective antibodies following experimental but not tick-borne infection.

Antibodies to the outer surface lipoprotein A (OspA) of Borrelia burgdorferi confer protection to SCID mice against subsequent tick-borne or experimental infection. However, OspA-specific antibodies are hardly detectable in naturally infected humans, dogs, hamsters and mice. This is most probably due to limited expression of OspA on spirochetes transmitted from the vector to the host. Here we have tested whether T cell priming of mice would lead to the induction of protective OspA-specific antibodies upon infection. It is shown that AKR/N mice, previously immunized with either a single T helper cell peptide of OspA, or a mixture of 27 peptides spanning the entire molecule, develop OspA-specific IgM or IgG antibodies, including those to a prominent protective B cell epitope of OspA. LA-2, within 7 days of infection with low doses (10(3)) of culture-derived spirochetes. In marked contrast, the same groups of pre-sensitized mice failed to generate any detectable OspA-specific antibodies after tick-borne infection for more than 40 days after infection. All mice, irrespective of their state of T cell immunity to OspA or the mode of infection, produced similar levels of OspC-specific IgM and IgG antibodies as early as day 14 after infection. None of the mice previously immunized with OspA peptides were protected against experimental infection, in spite of the appearance of protective antibodies. It is clear from these data that, in contrast to culture-derived spirochetes, the naturally transmitted pathogen fails to express OspA within the mammalian host at levels sufficient for induction of B cell responses, even in the presence of pre-activated T helper cells. Together with the fact that OspA-specific antibodies are mainly operative by eliminating spirochetes from the vector during infestation, the data suggest that OspA-vaccination for T helper cell immunity alone is not sufficient to prevent Lyme disease.

Amino Acid Sequence↗
PubMed1997

Detection and preliminary characterization of circulating immune complexes in patients with Lyme disease.

To investigate whether circulating immune complexes can be used as a disease marker for assessment of the activity of Lyme disease and for monitoring patients response to treatment, we tested 104 sera from patients with different stages of Lyme disease using the C1q enzyme-linked immunosorbent assay (ELISA) and a modified Raji cell test. Among 62 sera of patients with clinically active disease 27 sera (43.5%) reacted positively in the C1q-ELISA and 21 sera (33.9%) positively in the Raji cell test. In contrast, serum circulating immune complexes were found in less than 10% of 42 sera after antibiotic treatment. Similar results were obtained by both tests in 35 cerebrospinal fluid samples from patients with neuroborreliosis. Most importantly, dot blot analysis revealed the presence of both Borrelia burgdorferi-specific antigen(s) and host-derived components in the isolated immune complexes from serum samples of patients with active Lyme disease. These results indicate that detection of circulating immune complexes may be an useful parameter for judging the activity of Lyme disease. Moreover, preliminary characterization of spirochete-specific immune complexes implies new pathophysiological aspects of Lyme disease.

Antibodies, Bacterial↗
PubMed1997

Sapporo-like human caliciviruses are genetically and antigenically diverse.

The Sapporo-like human caliciviruses (HuCVs) comprise one of three genogroups of HuCVs associated with acute gastroenteritis. Phylogenetic analysis has shown that Sapporo-like HuCVs are related more closely to animal caliciviruses than to other known HuCVs. We produced 3.2 kb cDNA fragments from the 3' end to three Sapporo-like HuCVs that were associated with acute gastroenteritis in children (Houston/86, Houston/90, and London/92). Sequence analysis of the 3.2 kb cDNAs showed that two of the three viruses had a genomic organization similar to that of other Sapporo-like strains and the third strain (London/92) lacked an open reading frame overlapping the 5' end of the capsid gene. Alignment of the capsid sequences of these three strains showed 44-78% amino acid identity among the three strains. Phylogenetic analysis of the aligned sequences indicated the three strains are related but each belongs to a distinct genetic cluster. The genetic differences are associated with antigenic differences in that an enzyme immune assay (EIA) specific for the prototype Sapporo/82 strain detected the Houston/86 strain, but not the Houston/90 and London/92 strains. In vitro transcription and translation of viral cDNA containing the predicted capsid gene of Houston/90 resulted in a protein of 63 K, which is immunoprecipitated by sera from children infected with the strain. Genetically and antigenically distinct strains in the Sapporo-like HuCVs have not been described previously and the occurrence of such diverse strains in the same community likely increases the importance of these strains as a cause of illness in children.

Amino Acid Sequence↗
PubMed1997

Kaposi's sarcoma-associated herpesvirus gene expression in endothelial (spindle) tumor cells.

The recent discovery of DNA sequences of a new human herpesvirus in Kaposi's sarcoma (KS) has fueled speculation that this virus might cause KS. The mere presence, however, of a virus in a complex multicellular tumor like KS could just as well be construed as evidence of a passenger agent. We sought stronger evidence linking the KS-associated herpesvirus (KSHV) to tumor formation by using in situ hybridization to investigate the specificity, constancy, and timing of KSHV gene expression in KS tumor cells. Here we document expression of a 700-nucleotide viral RNA in every KS tumor examined, from the earliest histologically recognizable stage to advanced tumors in which the vast majority of identifiable spindle tumor cells contain this transcript. Two other KSHV RNAs were also detected in a smaller fraction of the tumor cells in all but the earliest lesion. These viral RNAs were expressed to relatively low levels in this subset; because one of these RNAs encodes a major viral capsid protein, these cells may be producing KSHV. We did not find these KSHV genes expressed in a variety of other tumors and proliferative processes, but we did detect viral gene expression in prostatic tissue, supporting a possible mechanism for sexual transmission of KSHV. The close relationship between KS and KSHV gene expression is consistent with the hypothesis that KSHV is directly involved in the etiology and pathogenesis of KS.

AIDS-Related Opportunistic Infections↗
PubMed1997

Characterization of ribonucleoprotein complexes containing an abundant polyadenylated nuclear RNA encoded by Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8).

Infection with Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) is strongly linked to all forms of Kaposi's sarcoma. We have previously identified two polyadenylated KSHV transcripts that are actively transcribed in Kaposi's sarcoma (KS) tumors and in KSHV-infected B-lymphoma cells. One of these RNAs (termed T1.1 or nut-1 RNA) is a 1.1-kb transcript present in a subpopulation of KS tumor cells. This RNA is localized to the nucleus of infected cells and has no open reading frames longer than 62 codons, suggesting that it may not function as an mRNA in vivo. Here we demonstrate that nut-1 RNA is a lytic-cycle gene product that is found in high-molecular-weight ribonucleoprotein complexes in infected cell nuclei. The transcript lacks the trimethylguanosine (TMG) cap found in many U-like small nuclear RNAs, but a subpopulation of nut-1 RNAs can associate with Sm protein-containing small nuclear ribonucleoproteins, as judged by immunoprecipitation analyses using monoclonal anti-Sm and anti-TMG antibodies. This interaction does not require other viral gene products, and deletion of the sole candidate Sm binding site on nut-1 RNA does not ablate this association. This finding suggests an indirect interaction with Sm-containing structures, and models for such associations are presented.

Base Sequence↗
PubMed1997

Differential expression of mammalian Numb, Numblike and Notch1 suggests distinct roles during mouse cortical neurogenesis.

During Drosophila neurogenesis, asymmetric cell divisions are achieved by differential segregation of Numb (d-Numb) into one of the daughter cells to cause a bias in the Notch mediated cell-cell interaction. We have isolated a second mammalian gene with significant sequence similarity to d-numb, mouse numblike. When expressed in dividing neural precursors in Drosophila, Numblike is symmetrically distributed in the cytoplasm, unlike endogenous d-Numb or expressed mouse Numb (m-Numb), both of which are asymmetrically localized to one half of the cell membrane. In d-numb loss-of-function mutant embryos, expression of Numblike allows both daughter cells of a neural precursor to adopt the fate of the cell that normally inherits d-Numb. In mice, numblike mRNA is preferentially expressed in adult and embryonic nervous system. In the developing neocortex, Numblike is expressed in postmitotic neurons in the cortical plate, but not in progenitors within the ventricular zone where m-Numb and Notch1 are expressed. We have also found that, in dividing cortical progenitors, Notch1 is distributed around the entire membrane, unlike m-Numb which is asymmetrically localized to the apical membrane. We propose that an interplay between cell-intrinsic mechanisms (executed by m-numb and numblike) and cell-extrinsic mechanisms (mediated by Notch1) may be involved in both progenitor cell proliferation and neuronal differentiation during mammalian cortical neurogenesis.

Amino Acid Sequence↗
PubMed1996

Restricted expression of Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genes in Kaposi sarcoma.

Kaposi sarcoma (KS) is the leading neoplasm of HIV-infected patients and is also found in several HIV-negative populations. Recently, DNA sequences from a novel herpesvirus, termed KS-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8) have been identified within KS tissue from both HIV-positive and HIV-negative cases; infection with this agent has been proposed as a possible factor in the etiology or pathogenesis of the tumor. Here we have examined the pattern of KSHV/HHV-8 gene expression in KS and find it to be highly restricted. We identify and characterize two small transcripts that represent the bulk of the virus-specific RNA transcribed from over 120 kb of the KSHV genome in infected cells. One transcript is predicted to encode a small membrane protein; the other is an unusual polyadenylylated RNA that accumulates in the nucleus to high copy number. This pattern of viral gene expression suggests that most infected cells in KS are latently infected, with lytic viral replication likely restricted to a much smaller subpopulation of cells. These findings have implications for the therapeutic utility of currently available antiviral drugs targeted against the lytic replication cycle.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Manganese-containing superoxide dismutase overexpression causes phenotypic reversion in SV40-transformed human lung fibroblasts.

Manganese superoxide dismutase (MnSOD) has been found to be low in a wide range of tumor cells as well as in vitro-transformed cell lines and has been implicated as a new type of tumor suppressor gene. The relationship between MnSOD activity and the malignant phenotype was studied by transfection of MnSOD cDNA into the SV40-transformed human fibroblast cell line WI-38 VA13 subline 2RA. The integration and expression of the exogenous MnSOD cDNA was confirmed in three selected clones with a 2-3.5-fold increase in MnSOD activity. The effect of elevated expression of MnSOD on the cell phenotype was determined by observing growth characteristics. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had a slower growth rate, lower plating efficiency, increased anchorage dependence, and morphological differences. These changes were correlated strongly with the level of MnSOD activity. The results suggest that an increase of MnSOD activity can reverse part of the malignant phenotype in SV40-transformed human fibroblast cells. A possible mechanism is that overexpression of MnSOD might alter the intracellular redox state by modulation of the balance of reactive oxygen species.

Cell Division↗
PubMed1996

Increased responsiveness of rheumatoid factor-producing B cells in seronegative and seropositive rheumatoid arthritis.

OBJECTIVE: To compare the frequencies and responsiveness of rheumatoid factor (RF)-producing B cells in the peripheral blood of patients with seronegative and seropositive rheumatoid arthritis (RA). METHODS: Frequencies of IgM+, IgG+, and RF+ B cells were determined by limiting-dilution analysis of purified peripheral blood B cells from 6 patients with seropositive RA, 8 patients with seronegative RA, and 7 normal controls. B cell help was provided by cloned T helper cells, which were stimulated by either anti-CD3 or the bacterial superantigen staphylococcal enterotoxin D (SED). IgM and IgG antibodies and RF in culture supernatants were detected by enzyme-linked immunosorbent assay. RESULTS: In the presence of anti-CD3-stimulated T helper cells, 2-10% of B cells from normal individuals secreted IgM and IgG antibodies. The frequency of RF+ B cells was low and ranged from 1:182 to 1:885 (RF+: IgM+) B cells. In patients with seropositive RA, the numbers of Ig-producing B cells were reduced by a factor of 2, while the fraction of RF+ B cell precursors was expanded by more than 50-fold (7-20% of IgM+ B cells; P = 0.004). Patients with seronegative RA had higher frequencies of RF-producing B cells (1.5-6% of IgM+ B cells) than normal individuals (P = 0.002), but not to the same extent as seropositive patients (P = 0.002). Stimulation of B cells using SED preferentially induced RF+ B cells in normal controls and in patients with seronegative and seropositive RA. CONCLUSION: B cell precursors with the potential to secrete RF were detectable in high frequencies in normal individuals and in patients with seropositive and seronegative RA. In all donors, these B cells could be stimulated with the bacterial superantigen SED. In normal individuals, RF+ B cells remained nonresponsive to help provided by anti-CD3-activated T cells, but were responsive in RA patients. Seronegative and seropositive RA form a continuous spectrum of disease, with a higher number of RF-secreting B cells in the seropositive patients.

Adult↗
PubMed1996

Plasmid DNA and protein vaccination of mice to the outer surface protein A of Borrelia burgdorferi leads to induction of T helper cells with specificity for a major epitope and augmentation of protective IgG antibodies in vivo.

Plasmid DNA-based vaccination is an efficient way to evoke various forms of protective immunity in laboratory animals. Our previous experiments have shown that mice immunized with either plasmid DNA encoding the outer surface lipoprotein A (pOspA) of Borrelia burgdorferi or the respective lipoprotein (Lip-OspA) produce protective antibodies against subsequent challenge with virulent spirochetes. In the present study, we compared the specificity and function of T cells generated in AKR/N mice previously immunized to either pOspA or Lip-OspA. T cell populations derived by either of the two protocols consistently responded by proliferation in vitro to one (residues 186-203; B4) out of a panel of 27 overlapping 20-mer peptides spanning the entire OspA molecule of strain ZS7. B4 was shown to express allele-specific ligand motifs for I-Ek. Most of the other peptides produced variable and much less pronounced or marginal proliferative T cell responses. T cells reactive to B4 as well as to some minor epitopes were CD4+CD8- T cells which produced IFN-gamma but no detectable IL-4 upon antigen stimulation in vitro. Priming of AKR/N mice with B4 but not with inactive peptides of OspA led to an enhanced production of IgG antibodies, mainly of the IgG1 isotype, including those to a prominent protective epitope (LA-2) upon subsequent challenge with Lip-OspA or intact spirochetes. The data demonstrate that both plasmid DNA and protein immunization with OspA results in T cell responses with specificity for a dominant OspA epitope and suggest that priming of mice with immunodominant peptides accelerates the appearance of protective antibodies in vivo. The identification of T helper cell epitopes relevant for the induction of protective antibodies will also facilitate the design of more potent vaccines against Lyme disease.

Amino Acid Sequence↗
PubMed1996

Protective immunization with plasmid DNA containing the outer surface lipoprotein A gene of Borrelia burgdorferi is independent of an eukaryotic promoter.

Plasmid DNA encoding the outer surface lipoprotein A (OspA) of Borrelia burgdorferi under the control of either strong eukaryotic/viral or its own bacterial promoter was injected intramuscularly (m. tibialis anterior) or intradermally into BALB/c and AKR/N mice. OspA-specific antibodies and OspA-reactive T helper 1 cells (Th1) were induced only with those plasmids containing the ospA structural gene including its own regulatory control region immediately upstream. In the absence of the ospA promoter, no or only marginal immune responses to OspA were obtained, even when strong eukaryotic promoter/enhancer elements were present. Together with the finding that the ospA promoter is active in a mouse B-lymphoma line, the data suggest that spirochetes are able to express at least part of their genes in the mammalian environment. Mice previously vaccinated with the relevant ospA plasmid DNA were protected against subsequent experimental challenge with a virulent strain of B. burgdorferi, as measured by the appearance of antibodies to a prominent protective epitope (LA-2) and the failure to re-isolate spirochetes from ear biopsies. In addition, C.B-17 severe-combined immunodeficient mice could be protected against infection by passive transfer of immune sera from ospA plasmid DNA-inoculated normal mice. Protective LA-2-related antibody titers obtained after repeated immunization persisted for 200 days and longer. This simple procedure of immunization using plasmid DNA consisting of a prokaryotic gene under the control of its own promoter holds great promise for the development of alternative subunit vaccines against bacterial infections, including Lyme disease. In addition, the availability of this novel prokaryotic promoter element now allows the study of the basis for the differential expression of bacterial genes in prokaryotic and eukaryotic environments.

Animals↗
PubMed1996

Reaction of metallothionein with ethylenediamine-N,N,N',N'-tetraacetic acid.

The reactions of various rabbit MTs (Cd7Th, Zn7Th, Hg7Th, Bi7Th, Pb7Th) with EDTA were studied at different pH and EDTA concentrations, and were determined to be pseudo-first-order reactions. The reactions were all divided into two steps: one faster and EDTA-dependent, and the other EDTA-independent. The mechanism is also described in detail. Based on obtained results, the varied induction behaviors of MT formation for those metals are discussed.

Animals↗
PubMed1996

Determinants of nicotinic receptor gating in natural and unnatural side chain structures at the M2 9' position.

A nonsense suppression method was employed to incorporate a total of four natural and six unnatural residues at the 9' position of the M2 region in the beta, gamma, and delta subunits of muscle nicotinic receptors. In 33 pairwise comparisons of functional properties as influenced by structural features including side chain length, branching, and substitution of oxygen for methylene carbons, it is concluded that increased polarity in the side chains at the 9' position consistently increases the sensitivity to acetylcholine. In addition, the stereochemistry of the side chain can have marked influences on the EC50, primarily because of changes in the single-channel open time. For the case of isoleucine versus allo-isoleucine in the delta subunit, these changes are themselves modified by mutations at the 9' position in other subunits. The data suggest an especially strong interaction between the beta and delta subunits in the pore region, leading in turn to a suggested arrangement of subunits within the pentamer.

Animals↗