Functional characterization of cysteine residues in GlpT,the glycerol 3-phosphate transporter of Escherichia coli

In Escherichia coli, the GlpT transporter, a member of the major facilitator superfamily, moves external glycerol 3-phosphate (G3P) into the cytoplasm in exchange for cytoplasmic phosphate. Study of intact cells showed that both GlpT and HisGlpT, a variant with an N-terminal six-histidine tag, are inhibited (50% inhibitory concentration approximately 35 microM) by the hydrophilic thiol-specific agent p-mercurichlorobenzosulfonate (PCMBS) in a substrate-protectable fashion; by contrast, two other thiol-directed probes, N-maleimidylpropionylbiocytin (MPB) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), have no effect. Use of variants in which the HisGlpT native cysteines are replaced individually by serine or glycine implicates Cys-176, on transmembrane helix 5 (TM5), as the major target for PCMBS. The inhibitor sensitivity of purified and reconstituted HisGlpT or its cysteine substitution derivatives was found to be consistent with the findings with intact cells, except that a partial response to PCMBS was found for the C176G mutant, suggesting the presence of a mixed population of both right-side-out (RSO) (resistant) and inside-out (ISO) (sensitive) orientations after reconstitution. To clarify this issue, we studied a derivative (P290C) in which the RSO molecules can be blocked independently due to an MPB-responsive cysteine in an extracellular loop. In this derivative, comparisons of variants with (P290C) and without (P290C/C176G) Cys-176 indicated that this residue shows substrate-protectable inhibition by PCMBS in the ISO orientation in proteoliposomes. Since PCMBS gains access to Cys-176 from both periplasmic and cytoplasmic surfaces of the protein (in intact cells and in a reconstituted ISO orientation, respectively) and since access is unavailable when the substrate is present, we propose that Cys-176 is located on the transport pathway and that TM5 has a role in lining this pathway.     

Exposure to the anti-TNF-alpha drug thalidomide induces apoptotic cell death in human T leukemic cells.

TNF-alpha is a pro-inflammatory cytokine that plays a key role in disorders due to HIV-1 infection and replication such as Kaposi sarcoma, wasting, aphthous ulcerations and progression to AIDS. The controversial drug thalidomide is anti-inflammatory, anti-angiogenic and a selective inhibitor of TNF-alpha that is being studied as a treatment for HIV-1-related disorders, immune disorders and cancer. The cellular and molecular mechanism of thalidomide is unclear despite renewed clinical interest in the drug. Previous data from this laboratory indicate that thalidomide decreases cell growth and cell-cell interactions of human T leukemic cells. The specific aim of the present study is to determine whether thalidomide administration induces cell death via apoptosis. Low dose thalidomide treatment of human T leukemic cells exhibited rapid increases in caspase-3 activity, annexin V-FITC binding and DNA disintegration that is characteristic of apoptosis. These data indicate that low doses of thalidomide signal human T leukemic cells to die by apoptosis, which is a possible method of altering inflammatory cells and inflammatory activities.     

Quantitative determination of a chemically modified hammerhead ribozyme in blood plasma using 96-well solid-phase extraction coupled with high-performance liquid chromatography or capillary gel electrophoresis

Versatile bioanalytical assays to detect chemically stabilized hammerhead ribozyme and putative ribozyme metabolites from plasma are described. The extraction protocols presented are based on serial solid-phase extractions performed on a 96-well plate format and are compatible with either IEX-HPLC or CGE back-end analysis. A validation of both assays confirmed that both the HPLC and the CGE methods possess the required linearity, accuracy, and precision to accurately measure concentrations of hammerhead ribozyme extracted from plasma. These methods should be of general use to detect and quantitate ribozymes from other biological fluids such as serum and urine.     

BONE MARROW RESTORATION AFTER LOCALIZED DEPLETION

Studies have been made of marrow restoration after localized depletion of the rabbit femur by dextran perfusion. Restoration was shown to involve an initial period of reorganization which blends with a more prolonged period of hemic cell repopulation. Cellularity returned to normal levels by 35 days, the recovery of myeloid cells being somewhat more rapid than that of the erythroid elements. In either case, the evolution of immature hemic cells was soon followed by the appearance of more mature forms even at the earliest stages of marrow repopulation. ³H-TdR uptake per cell increased rapidly to a level approximately twice normal after the first week. The augmented incorporation of thymidine, revealed by scintillation spectrometry and confirmed upon autoradiography, was shown to be due to an increase in DNA synthesis rate as well as in the fraction of participating cells. It is suggested that the enhanced cell production is brought about by a decrease in the proliferative cell cycle and an increase in the growth fraction. The origin of the repopulating cells remains a moot point. Cell migration from the epiphyseal marrow is apparently not involved. Irrespective of the source of stem-type cells, the stimulus for regeneration appears to be locally determined.