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S Wee

Publications and source records attributed to S Wee.

34 records · Page 2Linked to original sources
PubMed1994

Characterization of a CD6 ligand(s) expressed on human- and murine-derived cell lines and murine lymphoid tissues.

CD6, a type I cell surface glycoprotein expressed predominantly by thymocytes and mature T lymphocytes, becomes phosphorylated on tyrosine residues following T cell activation and has been implicated as an accessory molecule in T cell activation. The purpose of this study was to identify cell lines and tissues which express CD6 ligand(s), determine the requirements for CD6 binding, and biochemically characterize the putative CD6 ligand(s). Binding studies with a CD6 immunoglobulin fusion protein, CD6-Rg, allowed the identification of a number of human cell lines which express a CD6 ligand(s). The binding to these cell lines was trypsin sensitive, in part required divalent cations, was blocked by an anti-CD6 mAb, and could be downregulated by tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma). Among the cell lines tested, the human breast carcinoma-derived cell line HBL-100 expressed the highest levels of CD6 ligand(s) and was used for immunoprecipitation studies. Following metabolic labeling, CD6-Rg immunoprecipitated glycoproteins of approximately 100, approximately 90, and approximately 45 kDa from HBL-100 cells. Using CD6-Rg we were able to show that murine thymus, lymph nodes, and skin express high levels of CD6 ligand(s) and that CD6-Rg bound to a murine thymic epithelial cell line and to cultured human epidermal keratinocytes.

Animals↗
PubMed1994

Characterization of cell surface material removed by water and NaCl washing of Paracoccus denitrificans grown under conditions of divalent cation deficiency and sufficiency.

Paracoccus denitrificans grown on complex medium deficient in Mg2+ and Ca2+ are rendered lysozyme susceptible by washing with NaCl, whereas cells grown in a succinate-salts medium (Mg2+ and Ca2+ sufficient) or complex medium supplemented with Mg(2+)+Ca2+ are not. The material released by water washing of cells grown on complex medium and complex medium supplemented with Mg2+ and Ca2+ was characterized by a high protein content. There was a high lipid: protein ratio and an appreciable amount of 3-deoxyoctulosonic acid in the material released by NaCl washing of cells grown under all conditions, indicating release of outer membrane material. The lipid ornithine: lipid phosphorous ratios of NaCl wash from cells grown on complex medium and complex medium supplemented with Mg2+ and Ca2+ were 0.54 and 0.34, respectively. Although NaCl washing removed outer membrane material from cells grown under all conditions, only divalent cation deficient cells were rendered lysozyme susceptible. This might be explained by the increased outer membrane ornithine-containing lipid to phospholipid ratio in these cells yielding a more permeable outer membrane.

Cations, Divalent↗
PubMed1993

Testicular temperature-labile cholesteryl ester hydrolase. Relationship to isoenzymes from other tissues, correlation with spermatogenesis, and inhibition by physiological concentrations of divalent cations.

Temperature-labile cholesterol ester hydrolase (TLCEH) was purified 2,000-fold from rat testis cytosol using sequential ammonium sulfate precipitation, cation exchange chromatography, and isoelectric focusing chromatography. the purified enzyme, which exhibited a single silver-stained band (66 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was inhibited 89% by the elevation of the temperature from 32 to 37 degrees C and 65% by treatment with alkaline phosphatase. Its amino acid composition and amino-terminal sequence differed markedly from those of isoenzymes from other tissues, although 6 of 20 residues were conserved. Polyclonal antibodies raised to TLCEH exhibited no cross-immunoreactivity with cytosolic proteins from other rat tissues and inhibited 70% of testis cytosolic CEH. Western blot analysis demonstrated a high correlation between immunoreactive protein and catalytic activity in the testis during maturation of the rat, with a marked increase at the onset of spermatogenesis. TLCEH was inhibited by physiological levels of Cu2+ (I50 = 0.60 microM) and Zn2+ (I50 = 0.75 microM) and by Cd2+ (I50 = 0.15 microM) but not by 0.5-5 mM Mn2+.

Aging↗
PubMed1993

Tyrosine phosphorylation of CD6 by stimulation of CD3: augmentation by the CD4 and CD2 coreceptors.

When T cells are activated via the T cell receptor (TCR) complex a number of cellular substrates, including some cell surface proteins, become phosphorylated on tyrosine (Tyr) residues. Phosphorylation of cytoplasmic Tyr renders these cell surface receptors competent to interact with proteins that link cell surface receptors to protein in the intracellular signaling pathways. Here we show that Tyr residues in the cytoplasmic domain of CD6 become phosphorylated upon T cell activation via the TCR complex. Tyr phosphorylation was observed when the T cells were activated by crosslinking CD3 or by cocrosslinking CD3 with CD2 or CD4, but not when the cells were stimulated by crosslinking CD2, CD4, or CD28 alone. Unlike other Tyr kinase substrates, such as the phospholipase C gamma 1-associated pp35/36 protein, whose level of Tyr phosphorylation is highest when T cells are activated by cocrosslinking CD3 with CD2, the levels of CD6 Tyr phosphorylation are highest when T cells were activated by cocrosslinking CD3 with CD4.

Antigens, CD↗
PubMed1993

Monoclonal antibodies in organ transplantation.

Monoclonal antibody (mAb) technology has made possible the production of designer proteins, specifically reactive with almost any conceivable biological molecule. Using these reagents, the surface molecules on cells crucial for allograft rejection have been identified and described in detail. These structures can now be selectively targeted by mAb-based therapy in order to prevent rejection. For instance, the CD3 molecule, expressed on all mature T lymphocytes, triggers T cell activation, a key event in rejection. OKT3, an anti-CD3 mAb, disrupts T cell function and is now the agent of choice for the treatment of severe rejection episodes. MAbs targeting other T cell molecules are currently being investigated. Some of the most promising, the anti-CD4, anti-ICAM-1, and anti-interleukin 2 receptor mAbs, have already induced donor-specific tolerance in rodent models. These hosts accept permanently a genetically incompatible graft after only a limited period of mAb therapy. Interestingly, anti-ICAM-1 also diminishes the ischemic injury of preservation. The development of these new molecular agents, effectively directed to specific cellular targets, will likely play an increasingly important role in future clinical protocols, and perhaps finally provide a means to achieve long-term tolerance in human allograft recipients.

Journal Article↗
PubMed1988

Expression, isolation and properties of Fur (ferric uptake regulation) protein of Escherichia coli K 12.

The cloned fur (ferric uptake regulation) gene of Escherichia coli K12 was ligated to an expression vector which was inducible with nalidixic acid. The Fur protein was isolated in a single step by immobilized metal-ion-affinity chromatography over zinc iminodiacetate agarose. The amino acid composition of the isolated protein agreed with that predicted from the gene sequence and indicated post-transcriptional removal of the N-terminal methionine residue. All four cysteines were shown to be present as thiols. Proteolysis with trypsin and chymotrypsin yielded large fragments identifiable on polyacrylamide gel electrophoresis. Various divalent metal ions were found by a nitrocellulose filter binding assay to effect non-specific interaction of the Fur dimer with DNA with a dissociation constant of 7 x 10(-12) M. A much smaller value, 2.5 x 10(-17) M, was measured by gel mobility retardation assay for binding of Fur to a DNA fragment containing the operator sequences of the aerobactin promoter.

Amino Acid Sequence↗
PubMed1988

Increased outer membrane ornithine-containing lipid and lysozyme penetrability of Paracoccus denitrificans grown in a complex medium deficient in divalent cations.

Paracoccus denitrificans grown in a complex medium was highly susceptible to lysozyme, in contrast to cells grown in a complex medium supplemented with Mg2+ and Ca2+ or in a succinate-salts medium. The complex medium was deficient in divalent cations needed for optimum outer membrane stability. The major change in molecular compositions of outer membranes isolated from cells grown under the different conditions was a higher ratio of ornithine-containing lipid to phospholipid in complex-medium-grown cells (0.63) than in cells grown in complex medium with Mg2+ and Ca2+ (0.22) or in succinate-salts medium (0.14). We suggest that the dipolarionic ornithine-containing lipid is less dependent than acidic phospholipids on divalent cations for its incorporation into the outer membrane.

Calcium↗
PubMed1987

Hydrophobicity-hydrophilicity of staphylococci.

The hydrophobicity-hydrophilicity of various strains of Staphylococcus has been studied by a technique involving partitioning of the cells between aqueous and hydrocarbon phases. S. aureus was typically hydrophobic, and to a greater degree in stationary- than in exponential-phase cultures. Mutants that lacked teichoic acid, protein A or coagulase production were hydrophobic, indicating that none of these factors was responsible for hydrophobicity. The presence of a capsule rendered strains hydrophilic. Thus, determination of hydrophobicity may be a useful additional test for capsulation. However, a non-capsulate S. aureus strain was hydrophilic. Trypsin treatment converted strains from hydrophobic to hydrophilic. Isolated bacterial cell wall preparation, either crude or purified, and peptidoglycan were hydrophilic. These results indicate that the determinant of hydrophobicity is a protein or protein-associated molecule localised at the cell surface of the organism, i.e., a component of either the cell wall, cell membrane, or both. Twenty-five strains of twelve coagulase-negative species were examined and most (18) were hydrophobic, again indicating that protein A is not a major determinant of hydrophobicity in these staphylococci. Four of seven hydrophilic strains were capsulate; three strains of S. sciuri were hydrophilic but non-capsulate.

Bacterial Proteins↗
PubMed1987

Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor.

The promoter region of the pColV-K30-encoded operon specifying biosynthesis and transport of the siderophore aerobactin was subjected to deletion analysis to determine the smallest DNA sequence affording iron regulation of a iucA'-'lacZ gene fusion. A 78-base-pair (bp) region containing the main (P1) promoter retained the character of inducibility under iron starvation. A 250-bp fragment carrying this sequence was examined for protection against DNase I by the Fur protein, the product of a gene (fur) required for negative control of several iron-regulated functions. The DNase I footprints, in the presence of various divalent heavy-metal ions added as corepressors, revealed two contiguous binding sites with different lengths and affinities for Fur. Increased concentrations of the protein appeared to elicit formation of repressor oligomers which bind to the upstream and downstream regions of the P1 promoter in a metal-dependent fashion, but with a presently undefined stoichiometry. The primary site for Fur binding spans 31 bp and contains two overlapping symmetry dyads which share the sequence 5'-TCATT-3'. It also contains extensive homology with a 19-bp consensus sequence for iron-regulated genes as deduced from comparison with the fhuA and fepA putative promoter sequences.

Bacterial Proteins↗
PubMed1986

Safety and immunogenicity of bovine rotavirus vaccine RIT 4237 in 3-month-old infants.

To assess the safety and immunogenicity of bovine rotavirus vaccine, we administered attenuated strain RIT 4237 to 54 inner-city infants randomized to one of three groups in a double-blind fashion to receive a dose at 3 and 5 months of age of either placebo, vaccine virus at 10(7) TCID50/ml, or vaccine virus at 10(8) TCID50/ml. Vaccination began in early fall 1984, and continued through spring 1985. Forty-nine infants received one dose of vaccine or placebo; 43 received both doses of vaccine or placebo. At 2 and 3 months after vaccination, homologous geometric mean neutralizing antibody titers were significantly higher in children who received either dose of vaccine compared with placebo recipients. Cumulative seroconversion to bovine rotavirus after either dose of vaccine virus was 87% at 6 months of age. Seroconversion was significantly higher (P less than 0.01) in both vaccine groups compared with the placebo group. No ill effects were associated with vaccine administration. RIT 4237 vaccine appears to be safe and immunogenic when administered to young infants living in the United States.

Animals↗
PubMed1986

Iron-dependent production of a heat-modifiable, 23,000-Mr outer membrane protein in Paracoccus denitrificans.

Production of a 23,000-Mr major outer membrane protein of Paracoccus denitrificans ATCC 13543 was dependent upon the addition of iron to a succinate-salts medium. The 23,000-Mr protein was not produced in an iron-deficient medium, but production of five outer membrane proteins in the 85,000- to 72,000-Mr range and of catechol were induced. The 23,000-Mr protein was not produced in a complex medium even when ferric citrate was added to the medium. Production of the protein was influenced by the carbon source and was decreased by peptone.

Bacterial Outer Membrane Proteins↗
PubMed1981

Removal of Paracoccus denitrificans outer membrane material by sodium chloride.

The effects of water washing and NaCl treatment on the cell surface of P. denitrificans were studied. Both treatments caused a release of material from cells. Chemical studies showed that NaCl treatment released material containing components characteristic of outer membrane. This treatment also increased the susceptibility of the organism to lysozyme. Scanning electron microscopy was used to monitor the effects of water washing and NaCl treatment on the cell surface. Both treatments were shown to alter the appearance of the cell surface. The disruptive effects of these procedures were found to be dependent upon the age of the culture.

Cell Membrane↗