Effects of Mutations in GrlA of Topoisomerase IV from Staphylococcus aureus on Quinolone and Coumarin Activity

The grlA genes of Staphylococcus aureus ISP794 (wild type), MT5224c4 (grlA [Phe-80]), MT5224c2 (grlA [Pro-116]), and MT111 (grlA [Glu-116]) were cloned in pSK950, a shuttle vector, and introduced into S. aureus strains derived from strain RN4220. The mutations at position 116 of GrlA (Ala→Pro or Glu) caused an increase in the level of fluoroquinolone resistance and a decrease in the level of coumarin susceptibility, whereas the mutation at position 80 (Ser→Phe) caused only an increase in the level of fluoroquinolone resistance. In multicopy alleles, both types of mutations were codominant for fluoroquinolone resistance, and mutations at position 116 were also codominant for coumarin resistance.     

Dose-response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates

Thrombopoietin (TPO) is the physiologic Mpl-ligand regulating platelet production. Pegylated human recombinant megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated polypeptide Mpl-ligand derivitized with poly-(ethylene glycol), induces megakaryocyte endoreduplication and proliferation in vitro and in vivo. In the present study, the dose-response effects of PEG-rHuMGDF on pharmacokinetics, megakaryocytopoiesis, platelet production, and platelet function were characterized for dosing 0.05, 0.10, 0.50, or 2.5 micrograms/kg/d in 22 baboons for 28 days. Daily subcutaneous injections of PEG-rHuMGDF produced linear log-dose responses in (1) steady-state trough plasma levels of PEG-HuMGDF (P < 10(-3)); (2) marrow megakaryocyte volume (P < 10(-3)), ploidy (P < 10(-4)), and number (P < .01); and (3) peripheral platelet concentrations (P < 10(- 4)) and platelet mass turnover (P < 10(-3)). Platelet morphology, life span, and recovery were normal, and peripheral leukocyte, neutrophil, and erythrocyte counts were not significantly affected by PEG-rHuMGDF (P > .1 in all cases). PEG-rHuMGDF at 0.5 micrograms/kg/d produced similar blood concentrations of Mpl-ligand and platelets as 10 times the dose of rHu-MGDF (5.0 micrograms/kg/d), reflecting the extended plasma half-life achieved through pegylation. Whereas PEG-rHuMGDF did not induce platelet aggregation in vitro, platelet aggregatory responsiveness induced by thrombin receptor agonist peptide (TRAP1-6) and collagen was transiently enhanced ex vivo during the initial few days of PEG-rHuMGDF administration. However, adenosine diphosphate (ADP)-induced platelet aggregation was not enhanced ex vivo by PEG- rHuMGDF therapy. 111In-platelet deposition on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts increased in direct proportion to the circulating platelet concentration (P < 10(-4) for both EA and VG); 125l-fibrin accumulation was not affected by PEG-rHuMGDF-induced increases in peripheral platelet counts. Changes in platelet production and function produced by PEG-rHuMGDF returned to baseline within 2 weeks after discontinuing treatment. Thus, in nonhuman primates, PEG- rHuMGDF increases platelet production in a linear log-dose-dependent manner by stimulating megakaryocyte endoreduplication and new megakaryocyte formation from marrow hematopoietic progenitors. These findings suggest that appropriate dosing of PEG-rHuMGDF therapy during periods of chemotherapy-induced marrow suppression may maintain hemostatic concentrations of peripheral platelets without increasing the risk of thrombosis.     

Relationship between zinc intake, physical activity, and blood levels of high-density lipoprotein cholesterol in a healthy elderly population

We investigated the relationship between level of exercise, ingestion of zinc supplements, and serum high-density lipoprotein (HDL) cholesterol levels in 270 healthy men and women over age 60. After controlling for sex, alcohol intake, and body mass, there was a significant positive correlation between level of exercise and serum HDL cholesterol in the 180 subjects not taking supplemental zinc (r = 0.26, P = .005) but not for those subjects taking supplemental zinc (r = -0.18, P = .14). Multiple regression analysis showed a significant interaction of zinc intake and activity level on HDL cholesterol (P = .04). In 22 subjects who were ingesting greater than 15 mg of supplemental, elemental zinc daily, cessation of zinc supplements for 8 weeks was associated with a significant increase in HDL cholesterol levels (2.0 mg/dL; P = .04). The change in HDL after stopping zinc was positively correlated with the level of exercise of the subjects (r = .41, P = .05). Thus supplemental zinc ingestion appears to block the exercise-induced increase in serum HDL cholesterol in a healthy population.     

Evaluation of bosentan,pinacidil and nitroprusside on human pulmonary arteries: comparison with rat pulmonary arteries*

Summary— Bosentan (endothelin ET_A/ET_B antagonist), pinacidil (potassium channel opener) and nitroprusside (nitric oxide donor) were examined on isolated ring preparations of human intralobar pulmonary artery (3rd-5th generation; internal radius > 1 mm), rat main pulmonary artery (1st generation; internal radius > 1 mm) and rat intralobar pulmonary artery (3rd generation; internal radius ≥ 0.1–0.3 mm). The potency of endothelin-1 was the same in all three artery types. In human intralobar artery and rat main pulmonary artery, bosentan (3 and 10 μM) shifted the endothelin-1 concentration response curve to a higher concentration range (endothelin-1 concentration ratios, in human intralobar and rat main pulmonary artery, respectively: 3 liM bosentan, 4.5 and 8.1; 10 /xM bosentan, 13.5 and 19.5), but caused no significant block of endothelin-1 in rat intralobar artery. The latter finding may be due to the reported presence of ET_B receptors in rat intralobar arteries and the higher potency of bosentan on ET_A than on ET_B receptors. In contrast, the potencies of nitroprusside and pinacidil (relaxation of submaximal contractions to the thromboxane-mimetic, U46619) agreed on human and rat intralobar arteries but were 6 to 16-fold lower than on rat main pulmonary artery. We conclude that data obtained on pulmonary arteries from rats can be useful in predicting the effects of vasoactive drugs in human pulmonary arteries but selection of the most appropriate rat artery for study will depend on the drug group under investigation. For potassium channel openers and nitric oxide donors, good agreement between human and rat data will be found when using pulmonary arteries from the same anatomical location even though they differ markedly in size. In contrast, for endothelin antagonists, agreement is more likely to be found in arteries of comparable size, despite their different anatomical locations.     

The effect of neonatal tolerance to bovine gamma globulin (BGG) on BGG-reactive CD4+ T lymphocytes.

Compelling evidence has been presented that tolerance to major histocompatibility complex (MHC) and other antigens expressed on cells of the thymus is due to clonal deletion. However, it has not been determined conclusively how tolerance to diverse antigens, which may initially interact with the immune system in the periphery, is induced and maintained. Considerable evidence exists to suggest that mechanisms other than deletion, such as clonal anergy or immunoregulation, may be involved. We have previously shown that the clonal deletion of CD4+ cells specific for bovine gamma globulin (BGG) does not occur during induction of tolerance to this soluble antigen in adult life. In this study we have extended our previous findings by examining unresponsiveness to BGG which had been established during early ontogeny, when natural tolerance of self-antigens is likely to develop. It is demonstrated here that BGG-reactive, CD4+8- T cells, which proliferate and secrete interleukin-2 on stimulation with BGG in vitro, can be obtained from mice rendered tolerant of BGG as neonates. Even though the mice from which they were derived were unable to respond to BGG, these BGG-reactive T cells, by the parameters tested here, could not readily be distinguished from the corresponding cells in BGG-immune and non-immune animals. It is therefore evident that this tolerant state is not simply the result of clonal deletion of BGG-reactive CD4+ T cells but is more likely to be due to a reversible mechanism which controls their responsiveness in vivo.