Transmembrane distribution of sterol in the human erythrocyte

The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.     

Contractile responses of canine isolated pulmonary lobar arteries and veins to norepinephrine, serotonin, and tyramine.

Isolated helical strips of canine intrapulmonary lobar arteries and veins (about 4 mm in diameter) undergo dose-related tension development when exposed to increasing concentrations (10(-8) - 10(-3) M) of norepinephrine (NE), serotonin or 5-hydroxytryptamine (5-HT) and tyramine (Tyr). Venous segments were generally more sensitive while the maximum tension development was greater in the arterial strips, probably owing to their greater thickness. Both strips were more sensitive to 5-HT than NE and only responded to Tyr at high concentrations. Norepinephrine and 5-HT were nearly equally efficacious, whereas Tyr was less so. Responses to the latter were slow to develop, exhibited tachyphylaxis, and were greatly inhibited by phentolamine (10(-8) M), an alpha-adrenergic blocker. Exposure to cocaine (10(-5) M) enhanced submaximal NE responses, inhibited Tyr contractions and had no consistent effect on 5-HT responses. Phentolamine (10(-8) M) was also found to inhibit NE responses without altering 5-HT probably acts on other receptors. Tyramine may, in part, act directly on alpha-adrenergic receptors but may also release NE from surviving adrenergic nerve terminals in the preparation. Cocaine inhibits this effect and potentiates responses to lower levels of NE, presumably by blocking NE uptake into nerve terminals although a post-junctional action cannot be excluded.     

Actions of prostaglandins E1 and F2alpha on isolated intrapulmonary vascular smooth muscle.

The effects of PGE1 and PGF2alpha were studied on isolated strips of intrapulmonary arteries and veins from dog, sheep, swine and man. PGF2alpha contracted human arterial strips in a dose-dependent fashion, relaxed slightly sheep arteries and had no effect on dog arteries. Canine, sheep and human venous strips were contracted by PGF2alpha. PGE1 relaxed slightly both veins and arteries from dog and sheep. Human arteries usually contracted slightly and human veins usually relaxed slightly to PGE1. In a limited number of experiments, swine arteries and veins failed to respond to PGF2alpha or PGE1. All the vascular strips contracted well when exposed to NE. These results suggest that the responses of intrapulmonary vessels to PGF2alpha and PGE1 are species-dependent. PGF2alpha generally exhibits a contractile action, especially on veins. PGE1 usually relaxes intrapulmonary vessels. With regard to vessels from man, PGF2alpha is a powerful stimulant while PGE1 produces only small, variable effects.     

Effects of indomethacin on venoconstrictor responses to bradykinin and norepinephrine

In order to determine whether the venoconstrictor response to BK was dependent on prostaglandin (PG) synthesis, effects of indomethacin (INDO) on responses to bradykinin (BK) and norepinephrine (NE) were studied in canine lateral saphenous vein. Cumulative dose-response curves (10⁻⁹-10⁻⁶M BK or NE) were done in the presence and absence of INDO (10⁻⁶M). In the presence of INDO, responses to BK were markedly enhanced while responses to NE were unchanged. After prolonged periods in the bath, responses to BK were enhanced in control strips while responses of strips which had been treated with INDO were depressed. These results suggest that BK does not normally cause venoconstriction by stimulating synthesis of a venoconstrictor PG and that the increase in response to BK after prolonged periods in the bath may be related to changes in PG synthetase.     

Mechanisms of stable serum resistance of Neisseria gonorrhoeae

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent sera from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by immunoglobulin G (IgG) or F(ab)₂ isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, contained most of the blocking activity in IgG. In addition, immune convalescent DGI serum, which did not exhibit bactericidal activity, was restored to killing by selective immunodepletion of protein III antibodies. Blocking IgG or F(ab)₂ prepared from IgG, partially inhibited binding of bactericidal antibody to N. gonorrhoeae. Also, binding of a monoclonal antibody recognizing N. gonorrhoeae outer membrane protein PIII was almost completely inhibited by blocking F(ab)₂.Presensitization of N. gonorrhoeae with increasing concentrations of blocking IgG or F(ab)₂ before incubation with bactericidal antibody and an antibody free source of complement, increased consumption and deposition of the third component of human complement (C3) and the ninth component of complement (C9) but inhibited killing in dose-related fashion.     

Reactivation of koi herpesvirus infections in common carp Cyprinus carpio

Two co-habitation studies with common carp were conducted to determine whether latent infections of koi herpesvirus (KHV) exist. Fish were exposed to KHV using 2 different temperature profiles, which induced low and high initial mortality. Subsequently, certain groups of fish were co-habited with naive fish while others were not. Koi herpesvirus was reactivated in fish from 3 of the 5 experimental tanks. Reactivation of the virus occurred regardless of the initial mortality associated with the virus or whether fish were co-habited with naive fish. The reactivation of the virus in our experiments occurred several months after the initial exposure to KHV and appeared to be temperature dependent.     

Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma

The intracellular protozoan Toxoplasma gondii lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this essential lipid from the host environment. In this study, we demonstrated that T. gondii diverts cholesterol from low-density lipoproteins for cholesteryl ester synthesis and storage in lipid bodies. We identified and characterized two isoforms of acyl-CoA:cholesterol acyltransferase (ACAT)-related enzymes, designated TgACAT1alpha and TgACAT1beta in T. gondii. Both proteins are coexpressed in the parasite, localized to the endoplasmic reticulum and participate in cholesteryl ester synthesis. In contrast to mammalian ACAT, TgACAT1alpha and TgACAT1beta preferentially incorporate palmitate into cholesteryl esters and present a broad sterol substrate affinity. Mammalian ACAT-deficient cells transfected with either TgACAT1alpha or TgACAT1beta are restored in their capability of cholesterol esterification. TgACAT1alpha produces steryl esters and forms lipid bodies after transformation in a Saccharomyces cerevisiae mutant strain lacking neutral lipids. In addition to their role as ACAT substrates, host fatty acids and low-density lipoproteins directly serve as Toxoplasma ACAT activators by stimulating cholesteryl ester synthesis and lipid droplet biogenesis. Free fatty acids significantly increase TgACAT1alpha mRNA levels. Selected cholesterol esterification inhibitors impair parasite growth by rapid disruption of plasma membrane. Altogether, these studies indicate that host lipids govern neutral lipid synthesis in Toxoplasma and that interference with mechanisms of host lipid storage is detrimental to parasite survival in mammalian cells.