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Biomedical subjects

W Chen

Publications and source records attributed to W Chen.

At least 955 records · Page 53Linked to original sources
PubMed1995

Antibody to ras proteins in patients with colon cancer.

The current study examined sera from 160 colon cancer patients and 60 normal individuals to determine whether antibody to mutated p21 ras protein was present. Studies focused on the aspartic acid substitution at amino acid position 12 (denoted D12), one of the most common mutations in colon adenocarcinoma. IgA antibodies directed against mutated p21 ras-D12 protein were detected in 51 (32%) of 160 colon cancer patients, but only in 1 (2.5%) of 40 normal individuals. The greater incidence of antibody in cancer patients provides presumptive evidence that immunization to the ras proteins occurred as a result of the malignancy. Examination of sera for antibody reactivity to wild-type p21 ras protein (denoted p21 ras-G12) as well as p21 ras proteins bearing the D12, V12, S12, or L61 mutations showed that antibody detected was largely to normal segments of the p21 ras protein. Epitope mapping, using peptide neutralization assays with mutated or normal ras peptides as competitors, demonstrated that in 10 (67%) of 15 sera examined the antibody reactivity to p21 ras-G12 protein was neutralized by peptides near the carboxyl terminus of p21 ras protein, but not by peptides spanning the specific point mutation region. Antibody reactivities correlated with peripheral blood lymphocyte count, but did not correlate with patient age, sex, histology, stage, tumor locus, lymph node metastasis, or serum carcinoembryonic antigen.

Antibodies, Neoplasm↗
PubMed1994

The Caenorhabditis elegans small GTP-binding protein RhoA is enriched in the nerve ring and sensory neurons during larval development.

p21 Ras has been implicated in vulval differentiation in Caenorhabditis elegans. We now describe the characteristics during nematode development of the related p21 RhoA which has been ascribed a morphological role in mammals. The CeRhoA cDNA isolated in this study encodes a sequence of 192 amino acids residues with 87.6% identity to human RhoA. Genomic Southern analysis indicates the presence of a single Rho gene in C. elegans. Its 2-kilobase mRNA is expressed at the highest levels during embryogenesis and decreases gradually thereafter. However, the level of the 24-kDa protein detected by the anti-CeRhoA antibody is high at the larval stages but low in embryos. The glutathione S-transferase/CeRhoA fusion protein expressed in Escherichia coli displays conserved biochemical activities. Unlike its counterpart in mammalian cells which is predominantly cytosolic, most of CeRhoA is associated with the membrane and the cytoskeleton throughout development. Indirect immunofluorescence analysis indicates an ubiquitous expression of CeRhoA throughout development with a particular enrichment at larval stages in the pharyngeal nerve ring and at the tip of the head containing chemosensory and mechanosensory neurons. This suggests a stage-specific role for p21 RhoA in mediating the signaling pathway underlying the sensory circuitry in C. elegans post-embryonic development.

Amino Acid Sequence↗
PubMed1994

Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor.

The genetic loci agouti and extension control the relative amounts of eumelanin (brown-black) and phaeomelanin (yellow-red) pigments in mammals: extension encodes the receptor for melanocyte-stimulating hormone (MSH) and agouti encodes a novel 131-amino-acid protein containing a signal sequence. Agouti, which is produced in the hair follicle, acts on follicular melanocytes to inhibit alpha-MSH-induced eumelanin production, resulting in the subterminal band of phaeomelanin often visible in mammalian fur. Here we use partially purified agouti protein to demonstrate that agouti is a high-affinity antagonist of the MSH receptor and blocks alpha-MSH stimulation of adenylyl cyclase, the effector through which alpha-MSH induces eumelanin synthesis. Agouti was also found to be an antagonist of the melanocortin-4 receptor, a related MSH-binding receptor. Consequently, the obesity caused by ectopic expression of agouti in the lethal yellow (Ay) mouse may be due to the inhibition of melanocortin receptor(s) outside the hair follicle.

Adenylyl Cyclases↗
PubMed1994

Retroviral transduction of protein kinase C-gamma into tumor-specific T cells allows antigen-independent long-term growth in IL-2 with retention of functional specificity in vitro and ability to mediate tumor therapy in vivo.

The aim of our study was to examine the potential usefulness of transducing the protein kinase C-gamma (PKC-gamma) cDNA gene into tumor-specific T cells as a technique for facilitating the generation of large numbers of functional Ag-specific T for tumor therapy. Murine CD8+, F-MuLV gag-specific CTL clones, and CD4+, F-MuLV env-specific Th clones, as well as bulk-cultured T cell lines with defined Ag specificity to FBL-3, a Friend murine leukemia virus (F-MuLV)-induced tumor, were transduced with a retroviral vector pZipNeoPKC-gamma and selected in G418. The results demonstrated that PKC-gamma-transduced clones remained activated in culture, as evidenced by continued expression of up-regulated levels of IL-2R, which were as high after 6 mo in culture without Ag restimulation as 24 h after Ag stimulation. In vitro functional studies demonstrated that PKC-gamma-transduced CD8+ T cell clones maintained specific cytolytic activity to FBL-3, and PKC-gamma-transduced CD4+ T cell clones maintained specific proliferative activity to FBL-3 or F-MuLV Ag presented by irradiated syngeneic APC. Short-term bulk-cultured T cells specific to FBL-3 were also transduced and could be grown long term in vitro with maintenance of functional specificity. In vivo study showed that PKC-gamma-transduced CD4+ T cells were able to proliferate in response to Ag plus IL-2 stimulation in vivo in a similar pattern as the parental T cells. Therapy with adoptively transferred PKC-gamma-transduced T cell clones and lines into syngeneic mice, with or without FBL-3 tumor, showed that the PKC-gamma-transduced T cells were not tumorigenic and were effective in curing mice with disseminated FBL-3.

Animals↗
PubMed1994

Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues.

The contribution of major histocompatibility complex (MHC) class I-peptide affinity to immunodominance of particular peptide antigens (Ags) in the class I-restricted cytotoxic T lymphocyte (CTL) response is not clearly established. Therefore, we have compared the H-2Kb-restricted binding and presentation of the immunodominant ovalbumin (OVA)257-264 (SIINFEKL) determinant to that of a subdominant OVA determinant OVA55-62 (KVVRFDKL). Immunodominance of OVA257-264 was not attributable to the specific T cell repertoire but correlated instead with more efficient Ag presentation. This enhanced Ag presentation could be accounted for by the higher affinity of Kb/OVA257-264 compared with Kb/OVA55-62 despite the presence of a conserved Kb-binding motif in both peptides. Kinetic binding studies using purified soluble H-2Kb molecules (Kbs) and biosensor techniques indicated that the Kon for association of OVA257-264-C6 and Kbs at 25 degrees C was integral of 10-fold faster (5.9 x 10(3) M-1 s-1 versus 6.5 x 10(2) M-1 s-1), and the Koff approximately twofold slower (9.1 x 10(-6) s-1 versus 1.6 x 10(-5) s-1), than the rate constants for interaction of OVA55-62-C6 and Kbs. The association of these peptides with Kb was significantly influenced by multiple residues at presumed nonanchor sites within the peptide sequence. The contribution of each peptide residue to Kb-binding was dependent upon the sequence context and the summed contributions were not additive. Thus the affinity of MHC class I-peptide binding is a critical factor controlling presentation of peptide Ag and immunodominance in the class I-restricted CTL response.

Amino Acid Sequence↗
PubMed1994

Origin of delayed outward ionic current in charge movement traces from frog skeletal muscle.

1. Non-linear membrane ionic current was studied in highly stretched cut frog twitch fibres in a double Vaseline-gap voltage clamp chamber, with the internal solution containing 0.1 mM EGTA and the external solution containing Cl- as the major anion. After the Na+ currents was abolished by TTX in the external solution and the K+ currents were suppressed by external TEA+ and Rb+ and internal Cs+, a delayed outward ionic current with a time course similar to that of the delayed rectifier current was observed during depolarization. 2. The delayed outward ionic current was resistant to 1 mM 3,4-diaminopyridine (3,4-DAP) in the external solution and was unaltered when a fraction of the internal Cs+ was replaced by K+ or Na+, suggesting that the current was not carried by cations flowing through the delayed rectifiers. 3. The delayed outward ionic current was greatly reduced by replacing the external Cl- with CH3SO3-,SO4(2-), glutamate or gluconate, indicating strongly that the current was carried by Cl- flowing through anion channels. The current was also suppressed by 1 mM external 9-anthracenecarboxylic acid (9-ACA). 4. The delayed outward ionic current was reduced by blockers of calcium-dependent Cl- channels, such as SITS and frusemide (furosemide), in a dose- and voltage-dependent manner and by increasing intracellular [EGTA] to 20 mM, suggesting that part of the Cl- current in the muscle fibres could be calcium dependent. 5. The total Cl- current could be dissected into calcium-dependent and calcium-independent components. Each component accounted for roughly half of the total Cl- current. The maximum slope conductance of the calcium-dependent Cl- channels was 60.9 +/- 6.0 microS microF-1 (mean +/- S.E.M., n = 4).

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1994

Cbp1p is required for message stability following 5'-processing of COB mRNA.

Cbp1p is a nuclear encoded protein required for the stabilization of mitochondrial COB mRNA, which codes for apocytochrome b in the yeast Saccharomyces cerevisiae. The COB gene is cotranscribed with the upstream tRNA(Glu) gene. Release of tRNA(Glu) from the initial transcript generates a precursor mRNA with a 5'-end at position -1098. The 5'-end of mature COB message is generated by cleavage of the pre-mRNA at nucleotide -955 or -954. Previous work indicated that Cbp1p acts through cis-elements near these cleavage sites. Here we have tested whether Cbp1p stabilizes COB mRNA solely by stimulating the processing of COB precursor RNA at nucleotide -955/-954. Yeast strain TG955 was constructed such that the -955 COB mRNA 5'-processing site coincides with the upstream tRNA 3'-endonuclease site at position -1098, allowing the 5'-end of COB mRNA to be formed by the tRNA 3'-processing enzyme. Respiratory growth and stability of COB mRNA in TG955 are Cbp1p-dependent. Therefore, we conclude that Cbp1p is important for the stabilization of COB mRNA after 5'-processing.

Apoproteins↗
PubMed1994

Purification and characterization of glutathione peroxidase from human blood platelets. Age-related changes in the enzyme.

Platelet glutathione peroxidase (GPx) is known to play a pivotal role in controlling the level of lipid hydroperoxides, especially those resulting from the 12-lipoxygenase activity. GPx was purified from the cell cytosol by more than 700-fold using an exchange chromatography, FPLP, gel filtration and covalent fixation. Isoelectric focusing revealed a peak activity at pH 5.1. The molecular mass of the enzyme was found between 90 and 100 kDa by gel filtration, and was approximating at 23 kDa by SDS-PAGE. A polyclonal antibody raised against commercial bovine erythrocyte GPx recognized the human platelet enzyme. It is concluded that human platelet GPx is likely a homotetramer of 92 kDa as described for most other sources. We have also found that the decreased platelet GPx activity observed in platelets from elderly people is associated with a lower content of the immunoreactive enzyme.

Adult↗
PubMed1994

Donor T cells can be induced to grow and survive long term in vivo without previous host immunosuppression.

To examine whether donor T cells can grow and survive long term in hosts without previous immunosuppression, cultured T cells (B6/Thy 1.1) specific to FBL-3 tumor were adoptively transferred into normal B6/Thy 1.2 mice and induced to proliferate in vivo by specific stimulation with irradiated FBL-3. Donor T cells residing in the spleen and ascites of hosts were identified and quantified by use of Ab to the Thy-1 allele. The results demonstrated that on day 7 after transfer, donor T cells in greater numbers than input (1.2-fold) could be recovered. By day 35, donor T cells had decreased to approximately 10% of T cells input. Administration of IL-2 at doses of 2,500 or 25,000 U/day for 7 days preferentially increased the growth of Ag-activated donor T cells rather than host lymphocytes, and increased both the short term growth on day 7 (6.6-fold and 14.5-fold greater than input, respectively) and the long term survival on day 35 (0.75-fold and 3.15-fold of input, respectively) of donor T cells. The combination of CY pretreatment plus low dose IL-2 (2,500 U/day) increased donor T cell growth over and above that of either manipulation alone. However, with higher dose IL-2 to 25,000 U/day, donor T cell growth was equivalent in CY-pretreated and normal hosts (18-fold vs 16-fold increase, respectively; p > 0.05). With either dose of IL-2 there was no significant difference in the survival of donor T cells on day 35 in CY-pretreated vs normal hosts. Functional assay confirmed that specific cytolytic function of donor T cells could be maintained in hosts without previous immunosuppression. Accordingly, established disseminated FBL-3 leukemia could be cured without CY treatment, in a regimen using 10(7) cultured immune T cells plus IL-2 (25,000 U/day x 7 days). Thus, adoptively transferred donor T cells can be grown to large numbers and survive long term in vivo with maintenance of substantial function without the necessity of previous host immunosuppression.

Animals↗
PubMed1994

Co-ligation of CD31 and Fc gamma RII induces cytokine production in human monocytes.

The CD31 (PECAM-1) cell surface glycoprotein is considered to be involved in intercellular recognition and adhesion. Cytokines play a major role in cellular interactions, and therefore it was of interest to study whether engagement of CD31 affects synthesis and release of proadhesive cytokines. Here we demonstrate that immobilized CD31 mAb 1B5 induces the release of TNF-alpha, IL-1 beta, and IL-8 from human PBMCs. CD11b mAb VIM12 and HLA-D mAb VID1, both of which are of the same Ig subclass as mAb 1B5 (IgG1), as well as nonbinding isotype control mAb VIAP, were ineffective. That the effect was caused by the mAb, but not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and coculture with polymyxin B, which did not abolish TNF-alpha release. Cytokine production through intact mAb 1B5 was completely blocked by soluble F(ab) fragments of anti-IgG Fc gamma RII mAb IV.3, suggesting a significant contribution of that FcR. Cross-linking of neither CD31 nor Fc gamma RII molecules with the respective F(ab) fragments induced TNF-alpha release, but nonbinding control IgG1 Ab was able to restore the response of PBMC to 1B5 F(ab) fragments, when both Ab preparations were coated concomitantly. Therefore, only coligation of CD31 and Fc gamma RII appears to transduce activation signals leading to cytokine production. Our findings thus indicate a novel functional aspect of CD31 molecules that might play an important role in the propagation of an ongoing immune response as well as in the regulation of cell-cell interactions during inflammatory reactions.

Antibodies, Monoclonal↗