Driving operators relevant: A feature of the Chern-Simons interaction.
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Journal Article↗
Biomedical subjects
W Chen
Publications and source records attributed to W Chen.
A new member of the ras superfamily, the rac1 homologue from Caenorhabditis elegans. Cloning and sequence analysis of cDNA, pattern of developmental expression, and biochemical characterization of the protein.
A new member of the ras superfamily, designated CErac1 has been identified. The CErac1 cDNA clone was isolated from a Caenorhabditis elegans mixed stage library and encodes a protein of 191 amino acids with 82 and 79% identity to human rac1 and rac2 proteins, respectively. The CErac1 cDNA maps to a position on C. elegans chromosome IV in close proximity to cha-1, a choline acetyltransferase gene. The CErac1 cDNA hybridizes to two mRNAs (1.7 and 0.9 kilobases). Their expression is developmentally regulated, that of the more abundant 1.7 kilobases being highest at the embryonic stage and decreasing dramatically during development with 10% of the embryonic level in adult nematodes. The glutathione-S-transferase/CErac1 fusion protein expressed in Escherichia coli binds GTP and exhibits intrinsic GTPase activity. The GTPase activity of the CErac1 protein is stimulated by human n-chimaerin, a GTPase-activating protein for p21 rac1. These data suggest a role of CErac1 in C. elegans early development. The conserved biochemical properties indicate that further characterization of CErac1 by genetic analysis will be helpful in elucidating not only its role in the signal transduction, but also the biological function of its mammalian homologues.
Amino Acid Sequence↗
Activation of protein kinase C in vitro and in intact cells or synaptosomes determined by acetic acid extraction of MARCKS.
MARCKS is a widespread cellular phosphoprotein that migrates at 80-87 kDa on polyacrylamide gels. It is phosphorylated apparently exclusively by protein kinase C (PKC) and its phosphorylation in intact cells can be used as an index of intracellular PKC activation. Most methods for the determination of its phosphorylation state in vitro or in intact cells rely on two-dimensional gel electrophoresis to detect the protein with a pI of 4.6; however, this does not readily allow for multiple samples to be simultaneously and rapidly processed. Here we utilize the acid solubility of MARCKS to develop a novel extraction procedure. MARCKS was found to be soluble in 40% acetic acid and can be extracted quantitatively and rapidly from phosphorylation mixtures in vitro or from labeled intact cells. Acetic acid has the advantage that it is volatile and readily removed and precipitates protein in the presence of SDS, and the extracted protein is more readily resuspended in sodium dodecyl sulfate (SDS) sample buffer. Two extraction methods were developed, one for extraction of MARCKS from [gamma-32P]ATP-labeled subcellular fractions or intact synaptosomes labeled with 32Pi and one for extraction from 32Pi-labeled cultured nucleated cells, the latter requiring an additional protein recovery and DNA removal step. With this procedure MARCKS phosphorylation in intact synaptosomes was shown to be reversibly stimulated upon depolarization and MARCKS phosphorylation was found to be an early event in the activation of cultured smooth muscle cells by angiotensin II.
Acetates↗
In vitro and in vivo study of the ectromelia virus homolog of the vaccinia virus K1L host range gene.
Ectromelia virus encodes a protein which is homologous to the product of the vaccinia virus host range gene, K1L, except for eight conservative and two non-conservative substitutions and an additional threonine residue at the carboxyl terminus. Unlike the vaccinia virus gene, the ectromelia virus homolog failed to support optimal virus replication in RK-13 cells and appeared to be expressed 20-fold less efficiently. This lower level of expression was not due to the genetic background of the virus, K1L RNA transcription, sequence of the K1L RNA leader, or stability of K1L RNA or protein. Infections of RK-13 cells with ectromelia or vaccinia virus mutants lacking an intact K1L gene resulted in transient expression of early genes followed by a rapid and irreversible cessation of both virus and host protein synthesis. Infections of the disease-susceptible ANCR or -resistant C57BL/6 mice with the K1L-lacking ectromelia virus yielded a pathogenesis pattern indistinguishable from wild-type, suggesting that the ectromelia virus homolog of vaccinia virus K1L is not important for ectromelia virus in vivo replication and spread.
Animals↗
Studies into the mechanism for MHV transcription.
Previous studies have demonstrated that the MHV genome is divided into seven transcriptional units which are transcribed from highly conserved intergenic start sites (UCU/CAAAC) into mRNA containing a common leader RNA at the 5' end and a coterminal 3' end. In this manuscript, we provide evidence that an additional transcriptional unit is encoded at the 3' end of the MHV genome and is transcribed from a perfect intergenic region into a leader-containing approximately 800 nt mRNA. This mRNA could potentially encode a small 17-18 kDa protein which is identical to the C-terminal third of the nucleocapsid gene.
Base Sequence↗
Changes of the prevalence of endemic fluorosis after changing water sources in two villages in Guangdong, China.
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Adolescent↗
Mutagenic peroxides in diethyl ether.
Concentration of water-saturated commercial samples of diethyl ether by a stream of nitrogen yielded an aqueous residue mutagenic in S. typhimurium strains TA100 and TA102, but not TA1535. Reverse phase high-performance liquid chromatography resolved the mixture into 4 UV absorbing peaks, each of which was positive for peroxide and mutagenic in strain TA100 and TA102. One of these was identified as H2O2 based on HPLC retention time and sensitivity to decomposition by catalase, and another as 1-ethoxyethylhydroperoxide, based on its 1H-NMR spectrum. The mutagenic peroxides appeared to be stable in water between pH 3 and 6, but unstable above pH 7; they were less stable in methanol, acetonitrile, and least stable in chloroform, in which they partially decomposed after 24 h at 20 degrees C. Generalization of the results suggests that adverse effects observed in vitro and in vivo with chemicals containing ether linkages that actually have been caused by contaminant peroxides and reactive oxygen compounds.
Chromatography, High Pressure Liquid↗
High expression of the hormone binding active extracellular domain (1-294) of rat lutropin receptor in Escherichia coli.
Using the polymerase chain reaction (PCR) technique, the cDNA fragment corresponding to the receptor coding region for residues 1-294 was prepared from rat lutropin receptor (LHR) cDNA and subsequently subcloned into Escherichia coli expression vector pT7-7. This truncated receptor was efficiently expressed in E. coli as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The recombinant protein present in the inclusion bodies was solubilized in 6 M guanidine-HCl and purified in two successive steps of fast performance liquid chromatography (FPLC) using Superose-12 and Mono Q columns. Refolding of the purified recombinant protein was achieved in 1.5 M guanidine-HCl in the presence of an equimolar proportion of cysteine and cystine. The refolded soluble truncated LHR(1-294) had apparent molecular weights of 33 kDa and 140 kDa under reducing and nonreducing conditions, respectively. The multimeric nature of the extracellular domain of the receptor is believed to be due to its self-association by intermolecular disulfide bond formation since the 1-294 amino acid segment has nine cysteine residues and thus has one or possibly more free sulfhydryl groups. The purified truncated receptor had high binding affinity for human choriogonadotropin (hCG) as indicated by ligand blotting on SDS-PAGE and radioligand receptor assays. The amount of hCG required for 50% inhibition of binding of 125I-hCG to the soluble truncated receptor was 2.7 x 10(-10) M (or 12.25 ng). Similarly, the amount of soluble truncated receptor required for 50% inhibition of 125I-hCG binding to the rat ovarian receptor in the radioligand receptor assay was 58 ng.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
A tutorial introduction to stochastic simulation algorithms for belief networks.
Belief networks combine probabilistic knowledge with explicit information about conditional independence assumptions. A belief network consists of a directed acyclic graph in which the nodes represent variables and the edges express relationships of conditional dependence. When information about one variable's state is given to the network in the form of evidence, an update algorithm computes the posterior marginal probability distributions for the remaining variables in the network. Many algorithms for performing this inference task have been proposed. Exact algorithms report precise results for some classes of networks, but take exponential time (in the number of nodes) both in the worst case and for many interesting networks. Stochastic simulation algorithms estimate the posterior marginal probability distribution for many graph topologies that would require exponential time when using an exact algorithm. Nonetheless, for some belief networks, stochastic simulation algorithms are also known to have exponential worst case performance. This article describes at a tutorial level several stochastic simulation algorithms for belief networks, and illustrates them on some simple examples. In addition, the theoretical and empirical performance of the algorithms is briefly surveyed.
Algorithms↗
Studies on pharmacokinetics of sulfonated aluminum phthalocyanine in a transplantable mouse tumor by in vivo fluorescence.
The pharmacokinetic properties of sulfonated aluminum phthalocyanine (A1PCS) in mouse bearing transplantable S180 tumors were determined by an in vivo method. In vivo fluorescence measurements were made on the hind legs of mice, one leg bearing a tumor and the other, without a tumor, being used as a control. These in vivo data were compared with the results obtained from in vitro extraction fluorescence experiments. The results obtained by the two methods showed remarkable agreement, both procedures demonstrating that the concentration of A1PCS in the tumor was substantially higher than that in muscle. In both cases, the maximum tumor to muscle A1PCS concentration ratio occurred at 24-36 h after drug administration. The agreement between the in vivo and in vitro measurements shows that the in vivo fluorescence technique can be used successfully in pharmacokinetic studies of metallo-phthalocyanines in a superficial tumor model. The in vivo technique has the advantages of being rapid and convenient.
Aluminum↗
Induction of T cells specific for the mutated segment of oncogenic P21ras protein by immunization in vivo with the oncogenic protein.
Many malignancies harbor mutated ras proto-oncogenes encoding 21 kDa proteins with single amino acid substitutions. Previous studies have shown that the aberrant p21ras proteins are potential tumor-specific antigens in that CD4+ class II major histocompatibility complex-restricted T cells specific for the mutated segment of various oncogenic p21ras proteins can be elicited by immunization in vivo with synthetic peptides corresponding to the mutated segment. T-cell recognition of an antigenic peptide within a protein may be influenced substantially, either positively or negatively, by flanking amino acid sequences as well as by more distal immunogenic or tolerogenic epitopes within the same protein. This study examined whether T cells specific for the mutated segment of an oncogenic p21ras protein can be elicited by immunization in vivo with the protein. The results showed that p21ras protein bearing the transforming single amino acid substitution of leucine for glutamine at residue 61 could elicit T cells specifically reactive to the mutated region of the protein in C3H/HeN mice. Thus, an abnormal p21ras protein specifically associated with malignant transformation can be immunogenic in vivo. These results predict that in some circumstances, mutated p21ras proteins expressed by malignancies might elicit detectable mutation-specific T-cell responses.
Amino Acid Sequence↗
Efficiency of diagnostic criteria for attention deficit disorder: toward an empirical approach to designing and validating diagnostic algorithms.
Using structured psychiatric interviews, 73 attention deficit disorder (ADD) patients, 26 psychiatric control patients, 26 normal controls, and all available first degree relatives of these index children were examined. ADD subgroups with and without comorbid psychiatric disorders did not differ on rates of specific ADD symptoms. The construct of ADD is internally consistent as measured by Cronbach's alpha. The diagnostic efficiency of individual items is presented. A receiver operating characteristic-based procedure is used to create an ADD diagnostic algorithm that is more efficient in discriminating ADD children from controls than the DSM-III-based clinical diagnosis. Cross-validation with family study data shows this procedure to be superior to the procedure used for the DSM-III-R diagnosis. The results show that proponents of conditional probability and receiver operating characteristic analyses are correct in asserting that the examination of symptom combinations may result in better diagnostic algorithms.
Algorithms↗
Immunoreactivity and protective effects in mice of a recombinant dengue 2 Tonga virus NS1 protein produced in a baculovirus expression system.
The double-stranded replicative form of dengue 2 virus (DEN-2) RNA (Tonga strain) was used as a substrate to produce DNA clones of the NS1-NS2A genes via reverse transcriptase synthesis of full length cDNA followed by polymerase chain reaction amplification of the NS1-NS2A region. Products were cloned into pTZ18R for sequencing and into baculovirus for expression studies. The deduced amino acid sequence of the NS1-NS2A was almost identical to that of the S1 attenuated strain of DEN-2 Puerto Rico 159, differing in only four amino acids. The NS1 protein expressed in insect cells [Spodoptera frugiperda (Sf-9)] from the baculovirus recombinant was indistinguishable from authentic NS1 of DEN-infected Aedes albopictus cells in glycosylation, dimerization, cellular presentation and antigenicity. Mice injected with the expressed protein developed NS1-specific complement-fixing antibodies and were partially protected against neurological residua after intracranial challenge. The protective effect was mouse strain- and gender-specific.
Amino Acid Sequence↗
Reorientation dynamics of ferroelectric liquid-crystal molecules near the smectic-A-smectic-C* transition.
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Journal Article↗
Interleukin-6 production in a murine model of Pneumocystis carinii pneumonia: relation to resistance and inflammatory response.
The production of interleukin-6 (IL-6) and its possible relationship to host resistance and inflammatory response to Pneumocystis carinii infection were examined in mice with severe combined immunodeficiency (SCID mice). IL-6 activity was detected in the serum and lungs of P. carinii-infected mice but not in mice free of P. carinii. Moreover, the IL-6 levels in P. carinii-infected mice increased markedly after spleen cell reconstitution but then decreased to an undetectable level after the clearance of P. carinii. However, neutralization of IL-6 activity in spleen cell-reconstituted SCID mice by treatment with anti-IL-6 immunoglobulin G (IgG) resulted in no significant effect on the clearance of P. carinii (P > 0.05). Both the serum and lungs of treated mice contained an excess amount of anti-IL-6 IgG and lacked detectable IL-6. These results suggest that failure to inhibit the P. carinii clearance by anti-IL-6 treatment was not due to insufficient administration of antibody or incomplete neutralization of IL-6 activity. However, compared with mice receiving rat control IgG, mice treated with anti-IL-6 IgG had significantly higher numbers of neutrophils and lymphocytes (particularly CD8+ cells) in the lung lavage fluids (P < 0.05 for both) at day 19 after reconstitution. In addition, the levels of both total IgG (P < 0.001) and P. carinii-specific antibodies (P < 0.05) in the serum of mice treated with anti-IL-6 were significantly higher than those in control mice. These results indicate that although P. carinii infection causes both local and systemic production of IL-6 in SCID mice, IL-6 does not appear to play a crucial role in the clearance of P. carinii. However, it appears that during resolution of P. carinii pneumonia, IL-6 plays a role in the regulation of pulmonary inflammation and antibody responses.
Acute-Phase Reaction↗
Latency is not an inevitable outcome of infection with Pneumocystis carinii.
Severe combined immunodeficiency (SCID) mice resolve naturally acquired Pneumocystis carinii pneumonia after reconstitution with immunocompetent spleen cells and can therefore be used as a model to study latent P. carinii infection. Neither P. carinii nor amplified P. carinii DNA was detected in the lungs of SCID mice killed 21 days after spleen cell reconstitution. Furthermore, SCID mice that recovered from P. carinii infection failed to reactivate the infection after they were either depleted of CD4+ cells for up to 84 days or depleted of CD4+ cells and treated with corticosteroid for 35 days. These results indicate that an immune response to P. carinii can completely clear the organism from the host. This supports the hypothesis that P. carinii pneumonia that develops in immunocompromised patients may be a new infection resulting from exposure to an exogenous source of P. carinii and not necessarily from reactivation of latent infection.
Animals↗
Gamma interferon functions in resistance to Cryptosporidium parvum infection in severe combined immunodeficient mice.
Severe combined immunodeficient (SCID) adult mice are relatively resistant to Cryptosporidium parvum infection, even though they are deficient in both T- and B-cell function. The requirement for gamma interferon (IFN-gamma) in this resistance was examined by treatment of these mice with monoclonal antibody to IFN-gamma. SCID mice injected intraperitoneally with monoclonal anti-IFN-gamma 4 h before and three times weekly after challenge with C. parvum had heavy intestinal infections 3 weeks postchallenge. SCID mice similarly injected with irrelevant antibody were not infected. Furthermore, SCID mice receiving a single injection of anti-IFN-gamma either 2 h before or 18 h after challenge were also susceptible to infection. Although IFN-gamma was not detected in SCID mouse intestinal samples, it was found in the supernatant of SCID mouse splenocyte cultures after stimulation with C. parvum antigens. On the other hand, SCID mice receiving multiple injections of antibodies against tumor necrosis factor remained resistant to infection. These data indicate that the resistance of SCID mice to C. parvum infection is IFN-gamma dependent, whereas tumor necrosis factor appears not to play a significant role.
Animals↗
Requirements for CD4+ cells and gamma interferon in resolution of established Cryptosporidium parvum infection in mice.
The importance of CD4+ cells and gamma interferon (IFN-gamma) in the resolution of established Cryptosporidium parvum infection was investigated with a murine model of cryptosporidiosis in severe combined immunodeficient (SCID) mice. C. parvum-infected SCID mice were reconstituted with spleen cells from immunocompetent donors. The recipients were able to resolve their C. parvum infection by 17 days postreconstitution. Treatment of reconstituted SCID mice with either anti-CD4 monoclonal antibodies to deplete them of CD4+ cells or with anti-IFN-gamma to neutralize IFN-gamma activity reduced or eliminated their ability to resolve C. parvum infection whereas treatment with either anti-CD8 monoclonal antibodies or anti-asialo-GM1 antibodies had no effect. We also found C. parvum-specific antibodies in serum samples from two of four reconstituted SCID mice killed on postreconstitution day 17 but not in unreconstituted SCID mice. Moreover, anti-CD4-treated mice had no detectable specific antibodies to C. parvum, whereas all mice treated with either anti-CD8 or anti-asialo-GM1 had substantial levels of specific antibodies in their serum. Although the role of the specific antibody is not known, these findings clearly indicate that resolution of an established C. parvum infection in immunologically reconstituted SCID mice is dependent on both CD4+ cells and IFN-gamma.
Animals↗