Thrombin causes subsecond changes in protein phosphorylation of platelets

We have developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 40-kd proteins has been analyzed during the first five seconds of platelet response to thrombin from 0.1 to 5.0 U/mL and compared with the progress of aggregation and serotonin secretion. The onset time for aggregation and phosphorylation of both proteins was less than one second, although with lowest (less than 0.5 U/mL) thrombin levels, a lag of up to 0.6 seconds occurred before 40K phosphorylation increased. The thrombin sensitivity of aggregation and 20K phosphorylation was approximately twice that of 40K phosphorylation, with Ka values of 0.51 and 0.53 v 1.10 U/mL, respectively. External calcium was necessary for maximal 20K phosphorylation, since EDTA inhibited this by 30%. The 40K phosphorylation was not affected by EDTA. Platelet activation by thrombin thus induced biochemical changes well before one second. The quenched-flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet function.     

Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation.

Cells cultured from most patients suffering from the sunlight-sensitive hereditary disorder xeroderma pigmentosum are defective in the ability to excise ultraviolet light (UV)-induced pyrimidine dimers from their DNA. There is, however, one class of these patients whose cells are completely normal in this excision repair process. We have found that these cells have an abnormality in the manner in which DNA is synthesized after UV-irradiation. The time taken to convert initially low-molecular-weight DNA synthesized in UV-irradiated cells into high-molecular-weight DNA similar in size to that in untreated cells is much greater in these variants than in normal cells. Furthermore, this slow conversion of low to high-molecular-weight newly synthesized DNA is drastically inhibited by caffeine, which has no effect in normal cells. Two cell lines from classes of xeroderma pigmentosum that are defective in excision-repair show intermediate effects, with regard to both the time taken to convert newly synthesized DNA to high molecular weight and the inhibition of this process by caffeine.     

Energy expenditure and body composition in growth hormone deficient adults on exogenous growth hormone

OBJECTIVES We assessed whether the obesity observed in growth hormone deficient adults is maintained by a reduction in energy expenditure. We studied the effects of exogenous growth hormone on energy expenditure and body composition. DESIGN We performed an open study with growth hormone administered at 0 5 units per kilogram ideal body weight per week for 3 months. PATIENTS Seven growth hormone deficient adults were studied. Thirty-eight healthy volunteers had their resting metabolic rate measured, with seven of them proceeding to have their total energy expenditure assessed. MEASUREMENTS Total energy expenditure was measured by the doubly labelled water method (D₂0¹⁸), resting metabolic rate by ventilated hood indirect calorimetry, and fat free mass from the dilution volume of oxygen-18. Body composition and components of energy expenditure were assessed before, at 2 weeks and at the end of the 3-month treatment period on exogenous growth hormone. RESULTS Growth hormone deficient adults did not have a low total energy expenditure compared to healthy controls (13 12 vs 12 75 MJ/24 h) with only one patient expending less than 10 MJ/24 h. None had a resting metabolic rate lower than the 95% confidence limits of normality. The amount of energy expended on physical activity and thermogenesis was significant (6 54 MJ/24 h) and was similar to healthy controls (6 47 MJ/24 h). Resting metabolic rate increased by 15 9% after 14 days on exogenous growth hormone and was elevated 12-1% after 3 months treatment but the ratio to fat-free mass remained unaltered. Total energy expenditure increased by 13 4% after 14 days therapy. Fat-free mass increased significantly after 3 months treatment by (mean) 4 5 kg with no change in fat mass and no loss in body weight. CONCLUSIONS Obesity maintenance in growth hormone deficient adults is not a consequence of reduced total energy expenditure or a reduced exercise energy output. There was also no evidence for an energy sparing mechanism. Energy expenditure was increased by exogenous growth hormone but was not associated with a loss in fat mass or body weight suggesting the need for dietetic advice for those already obese at the outset of therapy.     

Control by cations of opioid binding in guinea pig brain membranes.

In membrane suspensions from guinea pig brain or cerebellum, NaCl, LiCl, NH4Cl, and KCl inhibit the equilibrium binding at 25 degrees C of the selective mu-agonist [3H][2-D-alanine,4-methylphenylalanine,5-glycinol]enkephalin ([D-Ala2,MePhe4,Gly-ol5]EK), the selective delta-agonist [3H][2-D-penicillamine,5-D-penicillamine]enkephalin ([D-Pen2,D-Pen5]-EK), and the selective kappa-agonist [3H]dynorphin A-(1-9). Choline chloride inhibits mu- and kappa-binding but not delta-binding. The relative activities of these monovalent salts and the slopes of the dose-response curves are site-dependent. Binding at the kappa-binding site is also inhibited by CaCl2, MnCl2, and MgCl2. On the other hand, these divalent salts potentiate delta-binding, and MnCl2 and MgCl2 have both potentiating and inhibitory effects on mu-binding; CaCl2 inhibits but does not potentiate mu-binding. Thus, the mechanisms by which monovalent cations inhibit opioid binding differ from those of divalent cations, and the mechanisms of action of both monovalent and divalent cations may differ at each site. When the antagonist [3H]naloxone, rather than the agonist [3H][D-Ala2,MePhe4,Gly-ol5]EK, is used to label the mu-binding site, the main effect of NaCl is to potentiate binding; a 22-fold higher concentration of LiCl is required to inhibit binding. The effects of NH4Cl, KCl, MnCl2, MgCl2, CaCl2, and choline chloride are little changed when [3H]naloxone is the ligand.     

Granulocyte-macrophage colony-stimulating factor augmentation of T-cell receptor-dependent and T-cell receptor-independent thymocyte proliferation

The effects of granulocyte-macrophage colony-stimulating factor (GM- CSF) are not confined to cells of the myeloid lineage. GM-CSF has been shown to have effects on mature T cells and both mature and immature T- cell lines. We therefore examined the GM-CSF responsiveness of murine thymocytes to investigate whether GM-CSF also affected normal immature T lymphocytes. The studies presented here indicate that GM-CSF augments accessory cell (AC)-dependent T-cell receptor (TCR)-mediated proliferation of unseparated thymocyte populations. To identify the GM- CSF responsive cell type, thymic AC and T cells were examined for GM- CSF responsiveness. We found that GM-CSF augmentation of TCR-induced thymocyte proliferation appears to be mediated via augmentation of AC function, and not via direct effects on mature single-positive (SP) thymocytes. Enriched double-negative (DN) thymocytes were also tested for GM-CSF responsiveness. GM-CSF induced the proliferation of adult and fetal DN thymocytes in an AC-independent and TCR-independent single- cell assay. Thus, in contrast to the SP thymocytes, a DN thymocyte population was directly responsive to GM-CSF. GM-CSF therefore may play a direct role in the expansion of DN thymocytes and an indirect role in the expansion of SP thymocytes.     

Detection of anti-HTLV-I Tax antibodies in HTLV-I enzyme-linked immunosorbent assay-negative individuals

The HTLV-I tax gene protein (Tax) is not packaged within the mature viral particle from which the proteins for the commercially available enzyme-linked immunosorbent assay (ELISA) are derived. Screening of 162 individuals within a cohort of white intravenous (IV) drug abusers, previously identified as having an increased incidence of HTLV-I infection, demonstrated that seven of them had antibodies to the HTLV-I Tax protein but tested negative in HTLV-I ELISAs and Western blots prepared from purified virion proteins. Three out of 35 individuals in other behaviorally defined high-risk groups also displayed this limited pattern of reactivity to HTLV-I proteins. The presence of the anti-HTLV- I p40/Tax antibodies was determined by radioimmunoprecipitation assay (RIPA), which also revealed low levels of anti-env reactivity. The specificity of the anti-p40 reactivity was confirmed on specific Tax ELISAs and Western blots prepared from recombinantly produced Tax. In vitro gene amplification by the polymerase chain reaction (PCR) was used to establish the presence of sequences homologous to HTLV-I proviral DNA in four/four of these HTLV-I ELISA negative, Tax ELISA/Tax western blot/RIPA positive individuals. These data suggest that the true incidence of HTLV-I infection within high-risk cohorts is greater than previously reported.