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Biomedical subjects

M Chen

Publications and source records attributed to M Chen.

At least 253 records · Page 14Linked to original sources
PubMed2000

Crystallization and preliminary crystallographic study of cucurmosin, a ribosome-inactivating protein from the sarcocarp of Cucurbita moschata.

Cucurmosin, a ribosome-inactivating protein purified from pumpkin, the sarcocarp of Cucurbita moschata, has been crystallized using polyethylene glycol as a precipitant. The crystals belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 41.91, b = 59. 48, c = 98.78 A. There is one molecule in the asymmetric unit. The diffraction data to 3.0 A resolution were collected on a MAR Research image-plate detector.

Crystallization↗
PubMed2000

Characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside Triphosphatase/Helicase domain of the hepatitis C virus nonstructural 3 protein.

We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors. Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371-1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.

Acid Anhydride Hydrolases↗
PubMed2000

Anti-HSP70 autoantibody formation by B-1 cells in Toxoplasma gondii-infected mice.

Formation of anti-Toxoplasma gondii HSP70 (TgHSP70) antibody cross-reactive to mouse HSP70 (mHSP70) was observed in the sera of BALB/c (a resistant strain) and C57BL/6 (B6; a susceptible strain) mice after peroral infection with T. gondii cysts of the Fukaya strain. The levels of anti-mHSP70 immunoglobulin G (IgG) autoantibody production in B6 mice were higher than those in BALB/c mice. The isotype and subclass of IgG of anti-TgHSP70 monoclonal antibodies cross-reactive to mHSP70 were mu and gamma3. Anti-mHSP70 autoantibody in T. gondii-infected BALB/c and B6 mice was shown to be produced by the CD5(+) subset of B cells (B-1a cells) but not by conventional B cells (B-2 cells). The epitopes recognized by anti-mHSP70 autoantibody were located primarily in the C-terminal fragment of mHSP70.

Animals↗
PubMed2000

Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions.

Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.

Aerobiosis↗
PubMed2000

Nondeletional T-cell receptor transgenic mice: model for the CD4(+) T-cell repertoire in chronic hepatitis B virus infection.

Chronicity after infection with the hepatitis B virus (HBV) can occur for a variety of reasons. However, once established, chronicity may be maintained by high levels of viral proteins circulating in the serum. To examine the characteristics of T cells capable of coexisting with the secreted hepatitis B e antigen (HBeAg), T-cell receptor (TCR) transgenic (Tg) mice were produced. To ensure that HBeAg-specific T cells would not be deleted in the presence of serum HBeAg, the TCR alpha- and beta-chain genes used to produce the TCR-Tg mice were derived from T-cell hybridomas produced from immunizing HBeAg-Tg mice. A TCR-Tg lineage (11/4-12) was produced that possessed a high frequency ( approximately 67%) of CD4(+) T cells that expressed a Tg TCR specific for the HBeAg. As predicted, when 11/4-12 TCR-Tg mice were bred with HBeAg-Tg mice no deletion of the HBeAg-specific CD4(+) T cells occurred in the thymus or the spleen. Functional analysis of the TCR-Tg T cells revealed that the HBeAg-specific CD4(+) T cells escaped deletion in the thymus and periphery by virtue of low avidity. Regardless of their low avidity, HBeAg-specific TCR-Tg T cells could be activated by exogenous HBeAg, as measured by cytokine production in vitro and T-helper-cell function for anti-HBe antibody production in vitro and in vivo. Furthermore, activated TCR-Tg HBeAg-specific T cells polarized to the Th1 subset were able to elicit liver injury when transferred into HBeAg or HBcAg-Tg recipients. Therefore, HBeAg-specific CD4(+) T cells that can survive deletion or anergy in the presence of circulating HBeAg nonetheless are capable of being activated and of mediating liver injury in vivo. The 11/4-12 TCR-Tg lineage may serve as a monoclonal model for the HBe/HBcAg-specific CD4(+) T-cell repertoire present in chronically infected HBV patients.

Adoptive Transfer↗
PubMed2000

Infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the E2 glycoprotein.

The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55(Gag) elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.

Adenovirus E2 Proteins↗
PubMed2000

Nckbeta adapter regulates actin polymerization in NIH 3T3 fibroblasts in response to platelet-derived growth factor bb.

The SH3-SH3-SH3-SH2 adapter Nck represents a two-gene family that includes Nckalpha (Nck) and Nckbeta (Grb4/Nck2), and it links receptor tyrosine kinases to intracellular signaling networks. The function of these mammalian Nck genes has not been established. We report here a specific role for Nckbeta in platelet-derived growth factor (PDGF)-induced actin polymerization in NIH 3T3 cells. Overexpression of Nckbeta but not Nckalpha blocks PDGF-stimulated membrane ruffling and formation of lamellipoda. Mutation in either the SH2 or the middle SH3 domain of Nckbeta abolishes its interfering effect. Nckbeta binds at Tyr-1009 in human PDGF receptor beta (PDGFR-beta) which is different from Nckalpha's binding site, Tyr-751, and does not compete with phosphatidylinositol-3 kinase for binding to PDGFR. Microinjection of an anti-Nckbeta but not an anti-Nckalpha antibody inhibits PDGF-stimulated actin polymerization. Constitutively membrane-bound Nckbeta but not Nckalpha blocks Rac1-L62-induced membrane ruffling and formation of lamellipodia, suggesting that Nckbeta acts in parallel to or downstream of Rac1. This is the first report of Nckbeta's role in receptor tyrosine kinase signaling to the actin cytoskeleton.

3T3 Cells↗
PubMed2000

Complex effects of naturally occurring mutations in the JAK3 pseudokinase domain: evidence for interactions between the kinase and pseudokinase domains.

The structure of Janus kinases (JAKs) is unique among protein tyrosine kinases in having tandem, nonidentical kinase and pseudokinase domains. Despite its conservation in evolution, however, the function of the pseudokinase domain remains poorly understood. Lack of JAK3 expression results in severe combined immunodeficiency (SCID). In this study, we analyze two SCID patients with mutations in the JAK3 pseudokinase domain, which allows for protein expression but disrupts the regulation of the kinase activity. Specifically, these mutant forms of JAK3 had undetectable kinase activity in vitro but were hyperphosphorylated both in patients' Epstein-Barr virus-transformed B cells and when overexpressed in COS7 cells. Moreover, reconstitution of cells with these mutants demonstrated that, although they were constitutively phosphorylated basally, they were unable to transmit cytokine-dependent signals. Further analysis showed that the isolated catalytic domain of JAK3 was functional whereas either the addition of the pseudokinase domain or its deletion from the full-length molecule reduced catalytic activity. Through coimmunoprecipitation of the isolated pseudokinase domain with the isolated catalytic domain, we provide the first evidence that these two domains interact. Furthermore, whereas the wild-type pseudokinase domain modestly inhibited kinase domain-mediated STAT5 phosphorylation, the patient-derived mutants markedly inhibited this phosphorylation. We thus conclude that the JAK3 pseudokinase domain is essential for JAK3 function by regulating its catalytic activity and autophosphorylation. We propose a model in which this occurs via intramolecular interaction with the kinase domain and that increased inhibition of kinase activity by the pseudokinase domain likely contributes to the disease pathogenesis in these two patients.

Amino Acid Sequence↗
PubMed2000

Complex adnexal masses: detection and characterization with MR imaging--multivariate analysis.

PURPOSE: To evaluate the accuracy of magnetic resonance (MR) imaging in the detection and characterization of complex adnexal masses, with particular reference to the findings predictive of malignancy, role of gadolinium-enhanced contrast material, and observer variability. MATERIALS AND METHODS: Preoperative MR imaging of the pelvis was performed in 128 consecutive patients with clinically or ultrasonographically detected complex adnexal masses. Histopathologic examination demonstrated 187 masses, 96 of which were malignant. MR imaging studies were prospectively and independently reviewed by two radiologists, one of whom reevaluated the studies after a 6-month interval. The predictive value of MR imaging findings was determined with multivariate logistic regression analysis. The value of gadolinium enhancement was assessed by using receiver operating characteristic analysis. Inter- and intraobserver variabilities were assessed by using weighted K statistics. RESULTS: Gadolinium-enhanced MR imaging depicted 176 (94%) of 187 adnexal masses, with an overall accuracy for the diagnosis of malignancy of 93%. The MR imaging findings that were most predictive of malignancy were necrosis in a solid lesion (odds ratio, 107) and vegetations in a cystic lesion (odds ratio, 40). Use of gadolinium-based contrast material contributed significantly to lesion characterization. Interobserver (K, 0.79-0.85) and intraobserver (K, 0.84-0.86) agreement were excellent. CONCLUSION: Gadolinium-enhanced MR imaging is highly accurate in the detection and characterization of complex adnexal masses, with excellent inter- and intraobserver agreement.

Adolescent↗
PubMed2000

Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle.

Muscle contraction causes an increase in activity of 5'-AMP-activated protein kinase (AMPK). This study was designed to determine whether chronic chemical activation of AMPK will increase mitochondrial enzymes, GLUT-4, and hexokinase in different types of skeletal muscle of resting rats. In acute studies, rats were subcutaneously injected with either 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mg/g body wt) in 0.9% NaCl or with 0.9% NaCl alone and were then anesthetized for collection and freezing of tissues. AMPK activity increased in the superficial, white region of the quadriceps and in soleus muscles but not in the deep, red region of the quadriceps muscle. Acetyl-CoA carboxylase (ACC) activity, a target for AMPK, decreased in all three muscle types in response to AICAR injection but was lowest in the white quadriceps. In rats given daily, 1 mg/g body wt, subcutaneous injections of AICAR for 4 wk, activities of citrate synthase, succinate dehydrogenase, and malate dehydrogenase were increased in white quadriceps and soleus but not in red quadriceps. Cytochrome c and delta-aminolevulinic acid synthase levels were increased in white, but not red, quadriceps. Carnitine palmitoyl-transferase and hydroxy-acyl-CoA dehydrogenase were not significantly increased. Hexokinase was markedly increased in all three muscles, and GLUT-4 was increased in red and white quadriceps. These results suggest that chronic AMPK activation may mediate the effects of muscle contraction on some, but not all, biochemical adaptations of muscle to endurance exercise training.

Adaptation, Physiological↗
PubMed2000

Relative potency of cellular and humoral immune responses induced by DNA vaccination.

DNA vaccines can prime broad-based immune responses in small animal models. In the present study, we sought to evaluate the relative ability of DNA vaccines to induce humoral and cellular immune responses. Using a DNA vaccine encoding HIV gag in mice, we observed that CD8+ T cell responses were primed more readily than were antibody responses, particularly at low doses of DNA. These CD8+ T cell responses were detected in spleen cells, as well as at local sites such as the lung and draining lymph nodes. The potency of the HIV gag DNA vaccine used was sufficient to prime strong CTL responses in macaques, but only low to undetectable antibody responses. Therefore, DNA vaccines appear able to prime strong, broad CTL but only modest antibody responses. These results may have implications on the development of vaccines against infectious diseases where both CTL and antibody responses are desired, such as HIV.

AIDS Vaccines↗
PubMed2000

Increased expression of lectin-like oxidized low density lipoprotein receptor-1 in initial atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits.

A novel lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) was recently identified in bovine aortic endothelial cells. It is strongly suggested to have a potential role in the initiation and development of atherosclerosis. In this study, we have isolated cDNA clones encoding the rabbit homologue of LOX-1 by screening a rabbit placenta cDNA library. In amino acid sequence and domain structure organization, the rabbit LOX-1 is highly conserved with the human counterpart. Transfection of rabbit LOX-1 cDNA to HEK-293 cells confers on them the activity to bind and internalize oxidized low density lipoprotein. Rabbit LOX-1 was identified as a 45-kDa protein by Western blot analysis with a specific monoclonal antibody. Notably, analyses by reverse transcription-polymerase chain reaction and Western blot revealed that LOX-1 was accumulated in 8-week-old Watanabe heritable hyperlipidemic rabbit aortas compared with normal rabbit aortas. Immunostaining confirmed that the augmented expression of LOX-1 was primarily localized within the intima at the earliest stages of atherogenesis. The most prominent staining was in the endothelial cells of lesions. Furthermore, the distinctive staining of LOX-1 was identified in the endothelium of non-lesion areas of Watanabe heritable hyperlipidemic rabbit aortas. Taken together, these findings support the possibility that LOX-1 might be involved in the initiation of atherosclerosis.

Amino Acid Sequence↗
PubMed2000

Toxoplasma gondii Hsp70 as a danger signal in toxoplasma gondii-infected mice.

Toxoplasma gondii Hsp70, T gondii Hsp30/bag1, and surface antigen 1 messenger RNAs were shown to be useful in analyzing stage conversion of T gondii between bradyzoites and tachyzoites. The high-level expression of T gondii Hsp70 was correlated with mortality in interferon-gamma knockout mice infected with T gondii. Tgondii Hsp70 inhibited the induction of nitric oxide release by peritoneal macrophages of T gondii-infected mice. These findings identify T gondii Hsp70 as a danger signal during lethal, acute T gondii infection.

Animals↗
PubMed2000

Nocturnal integrated gonadotropin concentrations in evaluating pubertal transition in girls.

We assessed the utility of measuring the physiological levels of gonadotropins as a diagnostic tool for pubertal onset in girls. Two methods of gonadotropin measurements were compared: the standard frequent sampling method, in which blood samples were drawn every 20 min, and the multiple integrated sampling method, in which samples were obtained continuously at 30 min intervals by a withdrawal pump. The two methods were examined simultaneously overnight in a group of eight girls at different stages of puberty. The following parameters of both LH and FSH secretion, calculated by PULSAR program, were highly correlated between these methods: area under the curve (AUC), mean levels, mean from smoothed baseline and mean peak height. The diagnostic value of multiple integrated sampling of gonadotropins (performed over 6 h) was then assessed in five prepubertal girls and six girls at early puberty (Tanner stages 2 and 3), in whom peak gonadotropins levels in response to GnRH stimulation test were in the prepubertal range. Several parameters of LH (but none of FSH) were significantly higher (p<0.05) in early pubertal compared to prepubertal girls: AUC (5.61 +/- 2.40 vs 2.39 +/- 1.41), mean levels (0.52 +/- 0.21 vs 0.23 +/- 0.14), smoothed mean level (0.43 +/- 0.18 vs 0.18 +/- 0.11) and peak area (0.27 +/- 0.08 vs 0.11 +/- 0.06). We conclude that the technically simple method of multiple integrated sampling is useful in detecting pubertal transition and is superior to the GnRH-stimulation test. This method can be used in selective cases when the stimulation test yields equivocal results.

Adolescent↗
PubMed2000

The effect of the Taiwan motorcycle helmet use law on head injuries.

OBJECTIVES: This study evaluated the effect of the motorcycle helmet law implemented in Taiwan on June 1, 1997. METHODS: Collecting data on 8795 cases of motorcycle-related head injuries from 56 major Taiwanese hospitals, we compared the situation 1 year before and after implementation of the helmet law. RESULTS: After implementation of the law, the number of motorcycle-related head injuries decreased by 33%, from 5260 to 3535. Decreases in length of hospital stay and in severity of injury and better outcome were also seen. The likelihood ratio chi 2 test showed that severity decreased after the law's implementation (P < .001). Full helmets were found to be safer than half-shell helmets. CONCLUSION: The helmet law effectively decreased the mortality and morbidity from motorcycle-related head injuries.

Accidents, Traffic↗
PubMed2000

A role of carboxy-terminal region of Toxoplasma gondii-heat shock protein 70 in enhancement of T. gondii infection in mice.

We investigated the role of recombinant Toxoplasma gondii heat shock protein (rT.g.HSP) 70-full length, rT.g.HSP70-NH2-terminal region, or rT.g.HSP70-carboxy-terminal region in prophylactic immunity in C57BL/6 mice perorally infected with Fukaya cysts of T. gondii. At 3, 4, 5, and 6 weeks after infection, the number of T. gondii in the brain tissue of each mouse was measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene. Immunization with rT.g.HSP70-full length or r.T.g.HSP70-carboxy-terminal region increased the number of T. gondii in the brain tissue after T. gondii infection, whereas immunization with rT.g.HSP70-NH2-terminal region did not. These results suggest that T.g.HSP70-carboxy-terminal region as well as T.g.HSP70-full length may induce deleterious effects on the protective immunity of mice infected with a cyst-forming T. gondii strain, Fukaya.

Animals↗
PubMed2000

A comparative analysis of sextant and an extended 11-core multisite directed biopsy strategy.

PURPOSE: The 3 tumor locations unsampled by conventional sextant biopsies that have been identified on composite 3-dimensional reconstruction of 180 radical prostatectomy specimens are the anterior transition zone, midline peripheral zone and inferior portions of the anterior horn in the peripheral zone. We evaluated an 11-core multisite directed biopsy scheme incorporating these alternate areas and conventional sextant biopsies in 362 patients from 2 institutions. MATERIALS AND METHODS: Patients without a prior diagnosis of cancer underwent ultrasound guided 11-core biopsies which included conventional sextant and 3 alternate sites. All specimens were separated for specific location identification. Biopsy was performed in 183 patients at MD Anderson Cancer Center (group 1) and in 179 at Toronto General Hospital (group 2). All group 2 and 54% of group 1 patients (98 of 183) had a prior biopsy negative for cancer. RESULTS: Median prostate specific antigen was higher in group 2 than in group 1 patients (11.5 versus 9.5 ng./ml., p = 0.016). Overall a 33% increase (36 of 110 patients) in cancer detection was observed when biopsy technique included the alternate areas (p = 0.0021). The anterior horn was the most frequently positive biopsy site followed by the transition zone and midline sites. The 11-core technique had significantly better cancer detection rates when digital rectal examination and transrectal ultrasound were normal, and in men with serum prostate specific antigen between 4.1 and 10 ng./ml. CONCLUSIONS: Biopsies of the alternate sites suggested by our simulation studies are feasible and reproducible. This new strategy significantly enhanced (p = 0.0075) prostate cancer detection compared to conventional sextant biopsies in men undergoing a repeat procedure.

Adult↗