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W M Ching

Publications and source records attributed to W M Ching.

34 records · Page 2Linked to original sources
PubMed1989

Heterogeneity of CD4-positive human T-cell clones which recognize the surface protein antigen of Rickettsia typhi.

Immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of T lymphocytes, including those which produce gamma interferon. Since the surface protein antigen (SPA) derived from typhus group rickettsiae has been shown to be an effective immunogen in animal models, human T-cell clones specific for the SPA of Rickettsia typhi were isolated and tested for their antigenic specificity, as well as for their ability to produce gamma interferon. Eighteen CD4-positive clones specific for the SPA of R. typhi exhibited considerable diversity in their response to the SPAs derived from two strains of Rickettsia prowazekii and from Rickettsia canada. The vast majority of clones also recognized the SPAs from R. prowazekii but not from R. canada. Two heteroclitic clones demonstrated significantly higher proliferative responses to the SPAs derived from one or both of the R. prowazekii strains than to the SPA of R. typhi, and one clone demonstrated a significantly higher response to the SPA of R. typhi than to the other SPAs. All 18 clones produced gamma interferon in response to SPA stimulation. We conclude that the SPAs from typhus group rickettsiae can elicit both a diverse T-cell response in humans and the efficient stimulation of gamma interferon-mediated immunity.

Antigens, Bacterial↗
PubMed1989

Selenium-containing tRNA(Glu) and tRNA(Lys) from Escherichia coli: purification, codon specificity and translational activity.

In response to low (approximately 1 microM) levels of selenium, Escherichia coli synthesizes tRNA(Glu) and tRNA(Lys) species that contain 5-methylaminomethyl-2-selenouridine (mnm5Se2U) instead of 5-methylaminomethyl-2-thiouridine (mnm5S2U). Purified glutamate- and lysine-accepting tRNAs containing either mnm5Se2U (tRNA(SeGlu), tRNA(SeLys] or mnm5S2U (tRNA(SGlu), tRNA(SLys] were prepared by RPC-5 reversed-phase chromatography, affinity chromatography using anti-AMP antibodies and DEAE-5PW ion-exchange HPLC. Since mnm5Se2U, like mnm5S2U, appears to occupy the wobble position of the anticodon, the recognition of glutamate codons (GAA and GAG) and lysine codons (AAA and AAG) was studied. While tRNA(SGlu) greatly preferred GAA over GAG, tRNA(SeGlu) showed less preference. Similarly, tRNA(SGlu) preferred AAA over AAG, while tRNA(SeLys) did not. In a wheat germ extract--rabbit globin mRNA translation system, incorporation of lysine and glutamate into protein was generally greater when added as aminoacylated tRNA(Se) than as aminoacylated tRNA(S). In globin mRNA the glutamate and lysine codons GAG and AAG are more numerous than GAA and AAA, thus a more efficient translation of globin message with tRNA(Se) might be expected because of facilitated recognition of codons ending in G.

Cell-Free System↗
PubMed1988

Recognition of typhus group rickettsia-infected targets by human lymphokine-activated killer cells.

Lymphokine-activated killer cells caused significant lysis of target cells infected with typhus group rickettsiae (TGR) but not cells infected with Rickettsia tsutsugamushi. Monoclonal and polyclonal antibodies against TGR or TGR-infected cells failed to bind specifically to infected targets, as determined by flow cytometry or by immunoprecipitation of radiolabeled cell surface proteins. Major unaltered antigenic determinants of TGR therefore cannot be detected on the surface of infected targets.

Cytotoxicity, Immunologic↗
PubMed1987

Purification of individual tRNAs using a monoclonal anti-AMP antibody affinity column.

A murine monoclonal anti-AMP antibody affinity matrix was used for isolation of individual species of amino acid transfer nucleic acids (tRNAs). The antibodies had been prepared using 5'-AMP covalently attached to bovine serum albumin as antigen and exhibited high affinity for 5'-AMP but greatly reduced affinity for 3'-AMP. Native uncharged tRNAs that terminate in a 5'-AMP group on the amino acid acceptor arm of the molecule bind tightly to the anti-AMP affinity matrix, whereas aminoacylated tRNAs are not retained. This allows separation of a particular tRNA species as its aminoacyl derivative from a complex mixture of uncharged tRNAs under very mild conditions.

Adenosine Monophosphate↗
PubMed1986

Characterization of selenium-containing tRNAGlu from Clostridium sticklandii.

A selenium-containing tRNA from Clostridium sticklandii has been shown to be an isoaccepting tRNAGlu (W.-M. Ching and T. C. Stadtman (1982) Proc. Natl. Acad. Sci. USA 79, 374-377). Not only is this tRNAGlu one of the most abundant selenium-containing tRNA species but it is also the major glutamate isoacceptor in this organism. The selenonucleoside, which is located at the first position of the anticodon, was identified as 5-methylaminomethyl-2-selenouridine (A. J. Wittwer, L. Tsai, W.-M. Ching, and T. C. Stadt (1984) Biochemistry 23, 4650-4655). Other modified nucleosides present in this tRNA include 4-thiouridine, pseudouridine, ribothymidine, modified guanosine, and two different modified adenosines. When this seleno-tRNAGlu is incubated in 1.0 M Tris X HCl, pH 8.5, partial deselenization occurs. Moreover, treatment with cyanogen bromide almost completely removes the selenium. The presence of selenium in this tRNAGlu is essential for its enzymatic acylation with glutamate. This seleno-tRNAGlu recognizes both GAA and GAG codons. However, at 10 mM magnesium, which is near the physiological range, the GAA codon is slightly favored. In a cell free translation system, the acylated seleno-tRNAGlu is a very active glutamate donor.

Bacterial Proteins↗
PubMed1985

A selenium-containing nucleoside at the first position of the anticodon in seleno-tRNAGlu from Clostridium sticklandii.

In previous studies, the single selenonucleoside component of a selenium-containing tRNAGlu isolated from Clostridium sticklandii has been shown to be 5-methyl-aminomethyl-2-selenouridine. Here, we show that this selenonucleoside is most likely located at the "wobble" position of the anticodon of the clostridial seleno-tRNAGlu. Nuclease T1 digestion of this seleno-tRNAGlu generated one major selenium-containing oligonucleotide (25 bases long). The selenium-containing residue within this oligonucleotide was located by sequence analysis of the oligonucleotide before and after removal of selenium by treatment with cyanogen bromide. The sequence of this oligonucleotide, A-A-C-C-G-C-C-C-U-U+-U-C-A+C-G-G-C-G-G-U-A-A-C-A-G, is homologous to that of the Escherichia coli tRNAGlu2 from residues 27 to 50, including the anticodon region and the variable loop, except that the E. coli tRNA has 5-methylaminomethyl-2-thiouridine instead of the selenonucleoside.

Anticodon↗
PubMed1984

Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-[(methylamino)methyl]-2-selenouridine.

Escherichia coli, Clostridium sticklandii, and Methanococcus vannielii synthesize 75Se-labeled amino acid transfer ribonucleic acids [( 75Se]tRNAs) when grown with low levels (approximately equal to 1 microM) of 75SeO32-. When E. coli [75Se]tRNA was digested to nucleosides and analyzed by reversed-phase high-performance liquid chromatography, a single selenonucleoside accounted for 70-90% of the 75Se label in the bulk tRNA. This nucleoside was shown to be indistinguishable in a number of its properties from authentic 5-[(methylamino)methyl]-2-selenouridine. Preparation of the authentic selenonucleoside was accomplished and the synthetic compound characterized by its UV and 1H NMR spectral properties. The new selenonucleoside also accounted for 40-60% of the 75Se found in [75Se]tRNA from C. sticklandii or M. vannielii. Each of these anaerobic bacteria contains one additional selenonucleoside in their tRNA populations distinct from 5-[(methylamino)methyl]-2-selenouridine. Pure seleno-tRNAGlu isolated from C. sticklandii contains one 5-[(methylamino)methyl]-2-selenouridine and one 4-thiouridine per tRNA molecule.

Escherichia coli↗
PubMed1984

Occurrence of selenium-containing tRNAs in mouse leukemia cells.

Selenium incorporation into the polynucleotide structures of tRNAs has been documented in several microorganisms. In the present study, selenium-containing species were isolated from bulk tRNA preparations from 75Se-labeled mouse leukemia cells. The major 75Se-labeled species was similar in size and exhibited the same sensitivity to ribonuclease as did Escherichia coli tRNAs. The chromatographic properties of the intact major selenium-containing tRNA species indicated it to be very hydrophobic in character. The selenium component that is unstable at neutral-to-alkaline pH but is relatively stable at acid pH is not an esterified selenoamino acid. HPLC analysis of enzymic digests of the major selenium-containing species detected selenium-containing hydrophobic products (probably selenonucleosides ). These properties strongly suggest that the selenium in the mouse leukemia-cell tRNAs is present in the form of a selenium-modified nucleoside.

Animals↗
PubMed1982

Selenium-containing tRNAGlu from Clostridium sticklandii: correlation of aminoacylation with selenium content.

Selenium-containing amino acid tRNAs are normal components of several bacterial tRNA populations. In Clostridium sticklandii seleno-nucleotides occur in at least four different tRNA species which account for 5--8% of the total tRNA population. One of these has been isolated in a highly purified form and shown to be an isoaccepting tRNAGlu. Experimental evidence indicates that the presence of the seleno-nucleotide in this tRNAGlu is essential for its acylation with glutamate.

Clostridium↗
PubMedDate not supplied

DNA breakage by methyl methanesulfonate and its repair in brain and liver cells cultured from fetal rat and mouse.

DNA strand breakage and repair following methyl methanesulfonate (MMS) treatment of primary cell cultures from 14-day fetal Sprague-Dawley rat brain and liver and 12-day fetal C57BL/6 mouse brain and liver, were studied using alkaline sucrose density gradient analysis. Cells were incubated with MMS (7 mM or 14 mM) for 20 min and harvested for alkaline sucrose gradients 40 min or 24 h later. The extent of initial damage in fetal rat and fetal mouse cells was comparable. Fetal mouse brain and liver and rat liver showed nearly complete repair 24 h after treatment. However, fetal rat brain cells showed comparatively little repair after 24 h. The possible significance of a repair deficit in cultured rat fetal brain cells and the striking neurogenic organotropism of transplacentally administered direct-acting alkylating agents in the rat is discussed.

Animals↗
PubMedDate not supplied

Identification of a murine CD4+ T-lymphocyte response site in hepatitis C virus core protein.

The T cell response to a recombinant HCV truncated core protein (cp1-10) was measured in a proliferation assay. Based on a 10-fold greater response to this truncated core protein than to its shorter form (cp1-8), a predominant epitope was mapped to the carboxyl quarter of this sequence. This epitope was further mapped to a synthetic peptide corresponding to amino acids 121-140 of the core protein. The peptide was antigenic for T cells of all three H-2 types tested, H-2 r, b and d, and the proliferating T cells were CD4+. Besides inducing specific proliferation in vitro, peptide aa121-140 can prime helper T cells in vivo. When boosted with core protein, mice primed with peptide produced 64-fold higher antibody titer than without priming in 1 week. The identification of a broadly immunogenic T cell helper epitope on core protein may be important for vaccine design against HCV.

Amino Acid Sequence↗