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Biomedical subjects

W Chen

Publications and source records attributed to W Chen.

At least 19 recordsLinked to original sources
PubMed1992

A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate.

A fluorometric microplate assay was established for the detection of respiratory burst activity in phagocytic cells by assessing oxidation of 2',7'-dichlorofluorescin-diacetate (DCFH-DA). This method is based on flow cytometric studies by Bass et al. (J. Immunol. 130 (1983) p. 1910) describing intracellular detection of DCFH oxidation due to the presence of hydrogen peroxides. In the present study we have adapted the assay for use in microtiter plates to determine the amount of extracellular reactive oxidative products. DCFH-DA, granulocytes and stimuli (phorbol myristate acetate, n-formyl-methionyl-leucylphenylalanine, concanavalin A) were added to microtiter plates and after incubation at 37 degrees C, the development of fluorescence intensity was read in a fluorescence concentration analyzer (FCA, Baxter). Calibration of fluorescence units recorded by the FCA was achieved by comparison with defined amounts of fluorescent DCF. The change in measured fluorescence was linear with cell density over the range of 2 x 10(5)-1 x 10(6) cells/well. Cumulative DCF generation in individual wells could be recorded non-destructively at frequent intervals for time course measurements. Results from FCA measurements correlated perfectly with the FACS analysis of the same samples (r = 0.99). In conclusion, this assay can be useful for screening monoclonal antibodies recognizing cell surface structures possibly involved in signal transduction as well as for testing phagocytes for their capacity to release reactive oxidative intermediates.

Flow Cytometry↗
PubMed1992

Decreased expression of E-cadherin and increased invasive capacity in EBV-LMP-transfected human epithelial and murine adenocarcinoma cells.

The EBV-encoded membrane protein LMP is one of 9 viral proteins regularly expressed in virally immortalized B lymphocytes. It is expressed in EBV-carrying lymphoblastoid cell lines of normal origin and in the majority of the poorly differentiated nasopharyngeal carcinomas, but not in Burkitt lymphomas. LMP has been reported to transform rodent fibroblasts, to inhibit epithelial differentiation and to alter morphology and cytokeratin expression in an in vitro immortalized human keratinocyte cell-line, RHEK-I. We now report that an LMP-transfected mouse mammary carcinoma line, SHG, exhibits a similar morphological change to that previously described in the LMP-transfected RHEK-I. In the LMP-transfected RHEK-I and SHG cells, we observed a decreased expression of the calcium-dependent adhesion molecule E-cadherin. The LMP-transfected RHEK-I cells were capable of invading type-I collagen gels while the control cells were not. The LMP-transfected SHG cells showed a significantly higher invasive capacity than the original cell line.

Adenocarcinoma↗
PubMed1992

Interleukin 1: an important mediator of host resistance against Pneumocystis carinii.

The importance of endogenous interleukin 1 (IL-1) in resistance to Pneumocystis carinii infection was examined in a SCID mouse model. Naturally acquired pulmonary infection of P. carinii in SCID mice was completely cleared by reconstitution of the infected mice with immunocompetent spleen cells. IL-1 activity in the lung homogenate supernatant of these mice increased significantly after reconstitution and returned to baseline level after the clearance of P. carinii. Treatment of reconstituted SCID mice with 35F5, a monoclonal antibody against murine type I IL-1R almost completely inhibited the clearance of P. carinii. In contrast, treatment with control rat immunoglobulin G had no detectable effect. Further study revealed that for the complete clearance of P. carinii, IL-1 must be present at the early stage of immune responses induced by reconstitution, since clearance could be blocked by a single injection of 35F5 into SCID mice at 2 d, but not at either 8 or 13 d postreconstitution. Furthermore, pulmonary recruitment of neutrophils, macrophages, and lymphocytes was significantly inhibited in mice that received 35F5 treatment. These findings strongly suggest that, in reconstituted SCID mice, endogenous IL-1 is important in host resistance to P. carinii infection and that IL-1 may function early in the host response possibly by the recruitment of inflammatory cells into the lungs.

Animals↗
PubMed1992

Resolution of Pneumocystis carinii pneumonia in CD4+ lymphocyte-depleted mice given aerosols of heat-treated Escherichia coli.

Mice were thymectomized and depleted of CD4+ lymphocytes by treatment with monoclonal antibody to induce Pneumocystis carinii (PC) pneumonia (PCP). These mice were then exposed to aerosols of heat-treated Escherichia coli three times a week. Aerosol treatment for 10 d caused a slight reduction in numbers of PC nuclei in the lungs of mice, and treatment for 22 d resulted in nearly complete resolution of PCP. Large numbers of macrophages, polymorphonuclear leukocytes, and lymphocytes accumulated in lungs of aerosol-treated mice. Depletion of either CD8+ lymphocytes or asialo GM1+ cells that remained in the mice after CD4+ cell depletion had no effect on the ability of the mice to resolve PCP after E. coli aerosol treatments. However, depletion of Thy-1+ lymphocytes in these mice abrogated their ability to resolve PCP and reduced the numbers of macrophages that accumulated in the lungs. In addition, it was found that resolution of PCP induced by heat-treated E. coli aerosol treatments was also abrogated when mice were treated with polyclonal antibodies against tumor necrosis factor alpha (TNF-alpha). Thus, resolution of PCP in CD4+ lymphocyte-depleted mice by heat-treated E. coli aerosols was not dependent on either CD8+ or asialo GM1+ cells but was dependent on Thy-1+CD4-CD8- lymphocytes and on the participation of TNF. These results indicate that heat-treated E. coli aerosols can act as an immune response modifier by inducing resolution of PCP in mice by a mechanism not dependent on the presence of CD4+ lymphocytes.

Aerosols↗
PubMed1992

Overexpression of the alpha-thyroid hormone receptor in avian cell lines. Effects on expression of the malic enzyme gene are selective and cell-specific.

The role of the alpha-thyroid hormone receptor (TR alpha) in regulation of transcription of the gene for chicken malic enzyme was analyzed in fibroblast cell lines normally unresponsive to triiodothyronine (T3). The gene for this transcription factor was introduced stably and overexpressed using a replication-competent retroviral vector. In chick embryo fibroblasts (CEF), overexpression of TR alpha decreased malic enzyme activity by 90% in the absence of T3. Addition of T3 almost completely restored malic enzyme activity to the level of similarly treated control CEF infected with virus lacking TR alpha. These TR alpha-induced changes in malic enzyme activity were mediated by alterations in transcription of the malic enzyme gene. Similar results were obtained when transcriptional activity of TR alpha was analyzed using a transient co-transfection system. Thus, the unliganded TR alpha is a transcriptional repressor of the malic enzyme gene; binding of T3 to the receptor abolishes this repression. In contrast, stable overexpression of TR alpha in QT6 cells had no effect on malic enzyme expression in the absence or presence of T3. Nuclear T3 binding was equally high in CEF and QT6 cells overexpressing TR alpha. These findings suggest that cell-specific factors control the ability of TR alpha to regulate the malic enzyme gene. Overexpression of TR alpha in CEF had no effect on the expression of fatty acid synthase and acetyl-CoA carboxylase, lipogenic enzymes that are stimulated by T3 in hepatocytes in culture. Thus, gene-specific factors also may control the transcriptional activity of TR alpha.

Acetyl-CoA Carboxylase↗
PubMed1992

T-cell immunity to the joining region of p210BCR-ABL protein.

The hallmark of chronic myelogenous leukemia is the translocation of the human c-abl protooncogene (ABL) from chromosome 9 to the specific breakpoint cluster region (bcr) of the BCR gene on chromosome 22. The t(9;22)(q34;q11) translocation results in the formation of a BCR-ABL fusion gene that encodes a 210-kDa chimeric protein with abnormal tyrosine kinase activity. The ABL and BCR genes are expressed by normal cells and thus the encoded proteins are presumably nonimmunogenic. However, the joining-region segment of the p210BCR-ABL chimeric protein is composed of unique sequences of ABL amino acids joined to BCR amino acids that are expressed only by malignant cells. The current study demonstrates that the joining region of BCR-ABL protein is immunogenic to murine T cells. Immunization of mice with synthetic peptides corresponding to the joining region elicited peptide-specific, CD4+, class II major histocompatibility complex-restricted T cells. The BCR-ABL peptide-specific T cells recognized only the combined sequence of BCR-ABL amino acids and not BCR or ABL amino acid sequences alone. Importantly, the BCR-ABL peptide-specific T cells could recognize and proliferate in response to p210BCR-ABL protein. The response of peptide-specific T cells to protein demonstrated that p210BCR-ABL can be processed by antigen-presenting cells so that the joining segment is bound to class II major histocompatibility complex molecules in a configuration similar to that of the immunizing peptide and in a concentration high enough to stimulate the antigen-specific T-cell receptor. Thus, BCR-ABL protein represents a potential tumor-specific antigen related to the transforming event and shared by many individuals with chronic myelogenous leukemia.

Amino Acid Sequence↗
PubMed1992

Increased serum concentration of type IV collagen peptide and type III collagen peptide in hyperthyroidism.

Serum concentration of type IV collagen peptide, the 7S domain of type IV collagen (type IV collagen 7S) and the amino terminal propeptide of type III procollagen (type III procollagen peptide) is thought to be a useful marker of progressive liver disease. In the present study, serum levels of these collagens in patients with thyroidal diseases with normal liver function were assayed. Increased levels in the hyperthyroid state and relatively decreased levels in the hypothyroid state were observed. The increased levels in hyperthyroidism was most prominent in type IV collagen peptide. The increased level became normal in the subsidence of hyperthyroidism by treatment with anti-thyroid drug. A positive correlation between serum type IV collagen peptide levels and serum thyroid hormone levels such as T4, T3, free T4 and free T3 was observed. These facts show that serum type IV collagen peptide may be influenced by not only liver disease but also serum thyroid hormone levels. Type IV collagen peptide may provide a useful biochemical marker of hyperthyroid state.

Collagen↗
PubMed1992

Suppression of human nasopharyngeal carcinoma cell growth in nude mice by the wild-type p53 gene.

Wild-type and mutant human p53 genes were transfected into the nasopharyngeal carcinoma (NPC) cell line CNE-3. Tumorigenicity in nude mice showed that the tumor resulting from the cells transfected with the wild-type p53 gene grew more slowly and was smaller than that from the cells transfected with mutant p53 gene and that from control CNE-3 cells. In contrast, the tumor from the cells transfected with the mutant p53 gene grew faster than that produced by cells transfected with the wild-type p53 gene and that produced by control CNE-3 cells. The results demonstrate that the wild-type p53 gene could inhibit the NPC cell growth in nude mice and the mutant p53 gene could enhance the NPC cell growth in nude mice. The p53 gene may also play an important role in the pathogenesis of NPC.

Animals↗
PubMed1992

Restricted replication of ectromelia virus in cell culture correlates with mutations in virus-encoded host range gene.

Ectromelia virus (strain Moscow) was shown to replicate poorly or not at all in cell lines derived from the rabbit or hamster. The failure of ectromelia virus to replicate in cell lines derived from the hamster suggested that the virus lacked a functional CHO host range (hr) gene required for multiplication in these cells. A DNA fragment which hybridized to the CHO hr gene was cloned from the ectromelia virus genome and shown by sequence analysis to be deleted of 506 bp within the ectromelia virus CHO hr homologue. Two additional ectromelia viruses (Hampstead and Mill Hill strains) were also shown to lack an intact CHO hr gene. Insertion of the CHO gene from cowpox virus into the ectromelia virus genome extended the host range of ectromelia virus in tissue culture. These results demonstrate that an intact CHO hr gene is not required for maintenance of ectromelia virus in nature and provide a partial explanation for ectromelia virus' narrow host range, as opposed to the broad host range of cowpox virus, which has a functional CHO hr gene.

Animals↗
PubMed1992

A simple and rapid method of preparing large fragments of dengue virus cDNA from replicative-form RNA using reverse transcriptase and PCR.

A method is described for cloning large fragments (1.5 kb to 2 kb) of dengue virus cDNA from replicative-form viral RNA. Aedes albopictus cells (C6/36 clone) infected with dengue virus contain double-stranded, replicative-form RNA molecules which were used as a template for an initial reverse transcription using a primer containing sequence homologous to regions of the genome at or near the 3' end of the gene being studied. The product was then used as a template for polymerase chain reaction (PCR) amplification using the same 3' primer and a second which hybridized to a region at the 5' end of the sequence to be cloned. Both primers were engineered to contain specific restriction enzyme cutting sites which enabled the PCR product to be cut and cloned directly into plasmids for sequencing and expression studies. We have used this method to construct clones of the envelope glycoprotein gene (E) and the non-structural genes 1 and 2a (NS1/2a) and 3 (NS3) of dengue type 2, Tonga 1974 strain, and E and NS1/2a from dengue type 3, H-87 strain, either as discrete genes or as constructs with long and short leader sequences, with or without anchor sequences. The method could be applied to the cloning of any gene from any flavivirus, directly from infected cell extracts, without the necessity for tedious virus purification steps.

Animals↗
PubMed1992

Polyclonal antibodies against the polypeptide and carbohydrate epitopes of recombinant human choriogonadotropin beta-subunit.

In our previous paper (Chen et al. (1991) J. Biol. Chem. 266, 4081-4087) we reported the preparation and characterization of recombinant human choriogonadotropin beta subunit (hCG beta) using the baculovirus-insect cell expression system. The rhCG beta was found to contain high mannose type N-linked carbohydrates and 3-4 serine-linked disaccharide chains. Despite the carbohydrate structural variation, the rhCG beta was similar to hCG beta in in vitro immunological and biological properties. In order to evaluate its in vivo immunological properties, rabbit antiserum against rhCG beta was produced. The antiserum was found to be almost identical to anti-hCG beta in binding to hCG beta as well as in its crossreactivity with human lutropin (hLH), hCG and human follitropin (hFSH) as indicated by radioimmunoassays using 125I-hCG beta as a tracer. Further characterization of the anti-rhCG beta antiserum revealed that there are three types of antibodies in terms of antigenic specificity present in the anti-rhCG beta antisera pool as shown by dot blot and radioimmunoassays. The carbohydrate-specific antibodies were separated by affinity chromatography using an ovalbumin-glycopeptide-Sepharose column. The antibodies held on the ovalbumin affinity adsorbent were specific for the high mannose type carbohydrates such as those present in rhCG beta, rhCG and thyroglobulin and failed to react with transferrin, alpha 1-acid glycoprotein and hCG alpha, all containing complex type carbohydrates. This was further supported by the fact that the recombinant unglycosylated hCG or periodate oxidized rhCG beta also did not show any reactivity with the carbohydrate specific antibodies. Two types of peptide epitopes seemed to be present in rhCG beta since when the flowthrough fraction from the ovalbumin-glycopeptide-affinity column was passed through the hCG beta-Sepharose column, the antibodies in the flowthrough from the latter column were specific to the unique antigenic determinants present only in the rhCG beta and not in hCG beta. The eluate from the hCG beta-Sepharose column contained the third type of antibodies, being the predominant ones, directed to the common epitopes between rhCG beta and hCG beta. The high mannose type specific antibodies are potentially useful in differentiating between the high mannose and complex type of N-linked carbohydrates present in a glycoprotein. Also, the antibody could provide an effective reagent in studying the intracellular processing of the N-linked oligosaccharides.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Effects of conditioning depolarization and repetitive stimulation on Q beta and Q gamma charge components in frog cut twitch fibers.

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. Steady-state inactivation of charge movement was studied by changing the holding potential from -90 mV to a level ranging from -70 to -30 mV. Q beta and Q gamma at each holding potential were separated by fitting the Q-V plot with a sum of two Boltzmann distribution functions. At -70 mV Q beta and Q gamma were inactivated to 54.0% (SEM 2.2) and 82.7% (SEM 3.0) of the amounts at -90 mV. At holding potentials greater than or equal to -60 mV, more Q gamma was inactivated than Q beta, and at -30 mV Q gamma was completely inactivated but Q beta was not. There was no holding potential at which Q beta was unaffected and Q gamma was completely inactivated. The differences between the residual fractions of Q beta and Q gamma are significant at all holding potentials (P less than 0.001-0.05). The plot of the residual fraction of Q beta or Q gamma versus holding potential can be fitted well by an inverted sigmoidal curve that is a mirror image of the activation curve of the respective charge component. The pair of curves for Q gamma correlates well with those for tension generation or Ca release obtained by other investigators. The time courses of the inactivation of Q beta and Q gamma were studied by obtaining several Q-V plots with conditioning depolarizations lasting 1-20 s and separating each Q-V plot into Q beta and Q gamma components by fitting with a sum of two Boltzmann distribution functions. The inactivation time constant of Q beta was found to be 5-10 times as large as that of Q gamma. During repetitive stimulation, prominent I gamma humps could be observed in TEST-minus-CONTROL current traces and normal Q gamma components could be separated from the Q-V plots, whether 20 or 50 mM EGTA was present in the internal solution, whether 2 or 10 stimulations were used, and whether the stimuli were separated by 400 ms or 6 s. Repetitive stimulation slowed the kinetics of the I gamma hump and could shift the Q-V curve slightly in the depolarizing direction in some cases, resulting in an apparent suppression of charge at the potentials that fall on the steep part of the Q-V curve.

Animals↗
PubMed1992

Separation of Q beta and Q gamma charge components in frog cut twitch fibers with tetracaine. Critical comparison with other methods.

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. 25 microM tetracaine had very little effect on the maximum amounts of Q beta and Q gamma but slowed the kinetics of the I gamma humps in the ON segments of TEST-minus-CONTROL current traces, giving rise to biphasic transients in the difference traces. This concentration of tetracaine also shifted V gamma 3.7 (SEM 0.7) mV in the depolarizing direction, resulting in a difference Q-V plot that was bell-shaped with a peak at approximately -50 mV. 0.5-1.0 mM tetracaine suppressed the total amount of charge. The suppressed component had a sigmoidal voltage distribution with V = -56.6 (SEM 1.1) mV, k = 2.5 (SEM 0.5) mV, and qmax/cm = 9.2 (SEM 1.5) nC/microF, suggesting that the tetracaine-sensitive charge had a steep voltage dependence, a characteristic of the Q gamma component. An intermediate concentration (0.1-0.5 mM) of tetracaine shifted V gamma and partially suppressed the tetracaine-sensitive charge, resulting in a difference Q-V plot that rose to a peak and then decayed to a plateau level. Following a TEST pulse to greater than -60 mV, the slow inward current component during a post-pulse to approximately -60 mV was also tetracaine sensitive. The voltage distribution of the charge separated by tetracaine (method 1) was compared with those separated by three other existing methods: (a) the charge associated with the hump component separated by a sum of two kinetic functions from the ON segment of a TEST-minus-CONTROL current trace (method 2), (b) the steeply voltage-dependent component separated from a Q-V plot of the total charge by fitting with a sum of two Boltzmann distribution functions (method 3), and (c) the sigmoidal component separated from the Q-V plot of the final OFF charge obtained with a two-pulse protocol (method 4). The steeply voltage-dependent components separated by all four methods are consistent with each other, and are therefore concluded to be equivalent to the same Q gamma component. The shortcomings of each separation method are critically discussed. Since each method has its own advantages and disadvantages, it is recommended that, as much as possible, Q gamma should be separated by more than one method to obtain more reliable results.

Animals↗
PubMed1992

Importance of endogenous tumor necrosis factor alpha and gamma interferon in host resistance against Pneumocystis carinii infection.

C.B-17 scid/scid (SCID) mice that have acquired natural pulmonary infection with Pneumocystis carinii clear these organisms by 19 days after reconstitution with spleen cells from immunocompetent mice and therefore serve as a model for studying the pathogenesis of and immunity to P. carinii pneumonia. The present study examined the importance of endogenous tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) in the clearance of P. carinii by treatment of reconstituted SCID mice with anti-TNF-alpha and anti-IFN-gamma immunoglobulin G (IgG). Treatment of reconstituted mice with monospecific rabbit anti-TNF-alpha IgG almost completely inhibited the clearance of P. carinii from the lungs. In contrast, treatment with either anti-IFN-gamma antibody (polyclonal or monoclonal) or control IgG had no detectable effect on the clearance of P. carinii. The importance of endogenous TNF-alpha in the clearance of P. carinii was further supported by the finding of TNF-alpha but not IFN-gamma in lung homogenate supernatants from reconstituted SCID mice. Further study revealed that for the complete clearance of P. carinii, TNF-alpha must be present at the early stage of reconstitution, since clearance could be blocked by a single injection of anti-TNF-alpha IgG into SCID mice at day 0 but not at day 6 and/or day 12 after reconstitution. These results strongly suggest that, in reconstituted SCID mice, endogenous TNF-alpha is important in host resistance against P. carinii infection, whereas IFN-gamma appears not to play a significant role.

Analysis of Variance↗
PubMed1992

Resistance of severe combined immunodeficient mice to infection with Cryptosporidium parvum: the importance of intestinal microflora.

Cryptosporidium parvum is a protozoan parasite which colonizes intestinal epithelium, causing transient diarrheal illness in immunocompetent hosts and severe chronic disease in immunocompromised hosts. We examined the resistance of severe combined immunodeficient mice, either bearing intestinal flora or germfree, to intestinal infection with C. parvum. Infection was not readily detected in flora-bearing adult severe combined immunodeficient mice until 5 to 7 weeks following oral challenge with C. parvum. In contrast, germfree adult severe combined immunodeficient mice were heavily infected 3 weeks following challenge. These data support the hypothesis that resistance of adult mice to C. parvum infection does not require a specific immune response but can be mediated by nonspecific mechanisms associated with the presence of intestinal flora.

Animals↗
PubMed1992

The vaccinia virus B1R gene product is a serine/threonine protein kinase.

The nucleotide sequence of the vaccinia virus open reading frame B1 predicts a polypeptide with significant sequence similarity to the catalytic domain of known protein kinases. To determine whether the B1R polypeptide is a protein kinase, we have expressed it in bacteria as a fusion with glutathione S-transferase. Affinity-purified preparations of the fusion protein were found to undergo autophosphorylation and also phosphorylated the exogenous substrates casein and histone H1. Mutation of lysine 41 to glutamine within the conserved kinase catalytic domain II abrogated protein kinase activity on all three protein substrates, supporting the notion that the protein kinase activity is inherent to the B1R polypeptide. Casein and histone H1 were phosphorylated on serine and threonine residues. The B1R fusion protein was phosphorylated on a threonine residue(s) by an apparently intramolecular mechanism. The autophosphorylation reaction resulted in phosphorylation of the glutathione S-transferase portion of the fusion and not the protein kinase domain. The protein kinase activity of B1R was specific for ATP as the phosphate donor; GTP was not utilized to a detectable extent. Immunoblotting experiments with anti-B1R antiserum showed that the protein kinase is located in the virion particle. Chromatography of virion extracts resulted in separation of the B1R protein kinase from the bulk of the total protein kinase activity, indicating that multiple protein kinases are present in the virion particle and that B1R is distinct from the previously described vaccinia virus-associated protein kinase.

Adenosine Triphosphate↗