Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

Insulin receptor substrate-4 enhances insulin-like growth factor-I-induced cell proliferation.

The insulin receptor substrates (IRSs)-1-4 play important roles in signal transduction emanating from the insulin and insulin-like growth factor (IGF)-I receptors. IRS-4 is the most recently characterized member, which has been found primarily in human cells and tissues. It interacts with SH2-containing proteins such as phosphatidylinositol 3'-kinase (PI3K), Grb2, Crk-II, and CrkL. In this study, we transfected IRS-4 in mouse NIH-3T3 cells that overexpress IGF-I receptors. Clones expressing IRS-4 showed enhanced cellular proliferation when cells were cultured in 1% fetal bovine serum without added IGF-I. Addition of IGF-I enhanced cellular proliferation in cells overexpressing the IGF-I receptor alone but had an even greater proliferative effect in cells overexpressing both the IGF-I receptors and IRS-4. When etoposide and methylmethane sulfonate (MMS), both DNA damaging agents, were added to the cells, they uniformly induced cell cycle arrest. Fluorescence-activated cell sorter analysis demonstrated that the arrest of the cell cycle occurred at the G(1) checkpoint, and furthermore no significant degree of apoptosis was demonstrated with the use of either agent. In cells, overexpressing IGF-I receptors alone, IGF-I addition enhanced cellular proliferation, even in the presence of etoposide and MMS. In cells overexpressing IGF-I receptors and IRS-4, the effect of IGF-I in overcoming the cell cycle arrest was even more pronounced. These results suggest that IRS-4 is implicated in the IGF-I receptor mitogenic signaling pathway.     

Involvement of the galactosyl-1-phosphate transferase encoded by the Salmonella enterica rfbP gene in O-antigen subunit processing.

rfbT of Salmonella enterica LT2 was previously thought, together with rfaL, to be involved in the ligation of polymerized O antigen to core-lipid A, and three mutants were known. We report the mapping of the mutations to rfbP, the galactosyl-1-phosphate transferase gene, which is now shown to encode a bifunctional protein. The mutations which have the former rfbT phenotype are referred to as rfbP(T). We also show that rfbP(T) mutants are not blocked in the ligation step as previously believed but in an earlier step, possibly in flipping the O-antigen subunit on undecaprenyl pyrophosphate from the cytoplasmic to periplasmic face of the cytoplasmic membrane.     

Immunosuppressive activity of T cell clones generated from human T cells stimulated with autologous TPHA cells

T cell clones were generated from human T cells stimulated with autologous phytohemagglutinin (PHA)-activated T (TPHA) cells. Characterization of three T cell clones originated from donor SF and one from donor JM showed that they proliferated when stimulated with autologous TPHA cells, non-T cells, and peripheral blood mononuclear cells, but did not proliferate when stimulated with allogeneic TPHA cells, non-T cells, and mononuclear cells, with autologous and allogeneic resting T cells, and with PHA. These results in conjunction with the blocking of the proliferation by anti-histocompatibility leukocyte antigen class II monoclonal antibodies indicate that these class II antigens are involved in the proliferation of T cell clones stimulated with autologous lymphoid cells. The four T cell clones are cytotoxic neither to autologous lymphoid cells nor to a panel of cultured human cell lines. The four T cell clones display immunosuppressive activity, since they inhibit the proliferation of autologous and allogeneic cells stimulated with antigens and mitogens and the secretion of immunoglobulin by B cells stimulated with pokeweed mitogen in presence of T cells. Furthermore, the four T cell clones display differential inhibitory activity on the proliferation of cultured human cell lines. The immunosuppressive activity is species-specific, since the T cell clones do not inhibit the proliferation of murine cells. The suppression is mediated by a factor(s) with an apparent m.w. of 13,000 to 16,000. The suppressor activity is labile at alkaline pH and is lost following incubation with pronase (100 U/ml) for 30 min at 37 degrees C.