Nitric oxide and superoxide impair human placental amino acid uptake and increase Na+ permeability: implications for fetal growth

Based on evidence that thiol and tyrosine reagents inhibit some amino acid transporters, we tested the hypothesis that NO- and O2- -derived free radicals would impair nutrient uptake by the human placenta. Syncytiotrophoblast microvillous plasma membrane vesicles (MVM) and placental villous fragments were exposed to the drug SIN-1 in the presence or absence of superoxide dismutase (SOD) and hemoglobin (Hb). The uptake of [3H]arginine, [3H]taurine, and [3H]leucine; [14C]MeAIB; and 22Na was studied in MVM, whereas the uptake of [3H]taurine was examined in villous fragments. Nitrotyrosine formation was assessed by Western blotting and quantified by ELISA. In MVM, SIN-1 caused an inhibition of [3H]arginine, [3H]taurine, and [14C]MeAIB uptake but had no significant effect on equilibrium [3H]leucine uptake. These effects were prevented by SOD or Hb, implying that both NO and O2- radicals were essential. In contrast, 22Na+ uptake was significantly increased, and this effect was prevented by SOD. In villous fragments, SIN-1 impaired Na+-dependent [3H]taurine uptake, with no effect on Na+-independent uptake. Increased nitrotyrosine formation was observed in MVM after SIN-1 treatment. Endogenous NO- and O2- -derived free radicals may alter human placental nutrient transfer in vivo, with implications for fetal growth.     

Galpha protein dependent and independent effects of human RGS1 expression in yeast

Regulators of G-protein Signalling (RGS) regulate the functional lifetime of G-Protein Coupled Receptor (GPCR)-activated heterotrimeric G-protein by serving as GTPase Accelerating Proteins (GAPs) for the G(alpha) subunit. A number of mammalian RGSs can functionally replace the yeast RGS containing SST2 gene and inhibit GPCR signalling. Using yeast strains harbouring a G(betagamma)-responsive FUS1-LacZ reporter gene, we demonstrate that heterologously expressed mammalian RGS1 also serves to decrease basal signalling in the absence of agonist. Although this effect was dependent on the expression of a GPA1-encoded functional G(alpha) protein, the GPCR itself was nevertheless not required. Using the GAL1 inducible promoter to express RGS1, we further demonstrate that in addition to serving as a GAP for Gpa1p in yeast, RGS1 is a dosage-dependent inhibitor of growth. This effect is specific to RGS1 since growth is not altered in cells expressing either mammalian RGS2 or RGS5. We further demonstrate that neither of the two yeast G(alpha) proteins is responsible for mediating this growth inhibitory effect of RGS1. Taken together, our results indicate that RGS1 can function in both G-protein-dependent and -independent manners in yeast.     

A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults

Background

Many malaria vaccines are currently in development, although very few have been evaluated for efficacy in the field. Plasmodium falciparum multiple epitope (ME)– thrombospondin-related adhesion protein (TRAP) candidate vaccines are designed to potently induce effector T cells and so are a departure from earlier malaria vaccines evaluated in the field in terms of their mechanism of action. ME-TRAP vaccines encode a polyepitope string and the TRAP sporozoite antigen. Two vaccine vectors encoding ME-TRAP, plasmid DNA and modified vaccinia virus Ankara (MVA), when used sequentially in a prime-boost immunisation regime, induce high frequencies of effector T cells and partial protection, manifest as delay in time to parasitaemia, in a clinical challenge model.

Methods and Findings

A total of 372 Gambian men aged 15–45 y were randomised to receive either DNA ME-TRAP followed by MVA ME-TRAP or rabies vaccine (control). Of these men, 296 received three doses of vaccine timed to coincide with the beginning of the transmission season (141 in the DNA/MVA group and 155 in the rabies group) and were followed up. Volunteers were given sulphadoxine/pyrimethamine 2 wk before the final vaccination. Blood smears were collected weekly for 11 wk and whenever a volunteer developed symptoms compatible with malaria during the transmission season. The primary endpoint was time to first infection with asexual P. falciparum. Analysis was per protocol.DNA ME-TRAP and MVA ME-TRAP were safe and well-tolerated. Effector T cell responses to a non-vaccine strain of TRAP were 50-fold higher postvaccination in the malaria vaccine group than in the rabies vaccine group. Vaccine efficacy, adjusted for confounding factors, was 10.3% (95% confidence interval, −22% to +34%; p = 0.49). Incidence of malaria infection decreased with increasing age and was associated with ethnicity.

Conclusions

DNA/MVA heterologous prime-boost vaccination is safe and highly immunogenic for effector T cell induction in a malaria-endemic area. But despite having produced a substantial reduction in liver-stage parasites in challenge studies of non-immune volunteers, this first generation T cell–inducing vaccine was ineffective at reducing the natural infection rate in semi-immune African adults.     

Programmed cell death remodels lace plant leaf shape during development

Programmed cell death (PCD) functions in the developmental remodeling of leaf shape in higher plants, a process analogous to digit formation in the vertebrate limb. In this study, we provide a cytological characterization of the time course of events as PCD remodels young expanding leaves of the lace plant. Tonoplast rupture is the first PCD event in this system, indicated by alterations in cytoplasmic streaming, loss of anthocyanin color, and ultrastructural appearance. Nuclei become terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive soon afterward but do not become morphologically altered until late stages of PCD. Genomic DNA is fragmented, but not into internucleosomal units. Other cytoplasmic changes, such as shrinkage and degradation of organelles, occur later. This form of PCD resembles tracheary element differentiation in cytological execution but requires unique developmental regulation so that discrete panels of tissue located equidistantly between veins undergo PCD while surrounding cells do not.     

Phosphatidylinositol 4-OH kinase is a downstream target of neuronal calcium sensor-1 in enhancing exocytosis in neuroendocrine cells

Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-hand-containing Ca(2+) sensors. NCS-1/frequenin has been shown to enhance synaptic transmission in PC12 cells and Drosophila and Xenopus, respectively. However, the precise molecular mechanism for the enhancement of exocytosis is largely unknown. In PC12 cells, NCS-1 potentiated exocytosis evoked by ATP, an agonist to phospholipase C-linked receptors, but had no effect on depolarization-evoked release. NCS-1 also enhanced exocytosis triggered by ionomycin, a Ca(2+) ionophore that bypasses K(+) and Ca(2+) channels. Overexpression of NCS-1 caused a shift in the dose-response curve of inhibition of ATP-evoked secretion using phenylarsine oxide, an inhibitor of phosphatidylinositol 4-OH kinase (PI4K). Plasma membrane phosphatidylinositol 4,5-bisphosphate pools were increased upon NCS-1 transfection as visualized using a phospholipase C-delta pleckstrin homology domain-green fluorescent protein construct. NCS-1-transfected cell extracts displayed increased phosphatidylinositol-4-phosphate biosynthesis, indicating an increase in PI4K activity. Mutations in NCS-1 equivalent to those that abolish the interaction of recoverin, another EF-hand-containing Ca(2+) sensor, with its downstream target rhodopsin kinase, lost their ability to enhance exocytosis. Taken together, the present data indicate that NCS-1 modulates the activity of PI4K, leading to increased levels of phosphoinositides and concomitant enhancement of exocytosis.     

Functional and molecular identification of ERG channels in murine portal vein myocytes

Ion channels encoded byether-à-go-go-related genes (ERG) have been implicatedin repolarization of the cardiac action potential and also ascomponents of the resting membrane conductance in various cells. Theaim of the present study was to determine whether ERG channels wereexpressed in smooth muscle cells isolated from portal vein. RT-PCRdemonstrated the expression of murine ERG (mERG), and real-timequantitative PCR showed that the mERG1b isoform predominated over themERG1a, mERG2, and mERG3 in portal vein. Single myocytes from portalvein displayed membrane staining with an ERG1-specific antibody. Wholecell voltage-clamp experiments were performed to determine whetherportal vein myocytes expressed functional ERG channels. Large inwardcurrents with distinctive kinetics were elicited that were inhibitedrapidly by E-4031 (mean amplitude of the E-4031-sensitive current at120 mV was 205 ± 24 pA; n = 14). Deactivationof the E-4031-sensitive current was voltage dependent (mean timeconstants at 80 and 120 mV were 103 ± 9 and 33 ± 2 ms,respectively; n = 13). Because of the rapid kinetics ofmERG currents at more negative potentials, there was a substantialnoninactivating "window" current that reached a maximum of66 ± 10 pA at 70 mV. Complete portal veins exhibitedspontaneous contractile activity in isometric tension experiments, andthis activity was modified significantly by E-4031. These data showthat ERG channels are expressed in murine portal vein myocytes that maycontribute to the resting membrane conductance.

     

Host location,survival and fecundity of the Oriental rat flea Xenopsylla cheopis (Siphonaptera: Pulicidae) in relation to black rat Rattus rattus (Rodentia: Muridae) host age and sex

Host choice and fecundity are two factors that may contribute to the variation in flea counts observed when assessing the potential risk of flea-borne transmission of pathogens from rodents to humans. Using the black rat, Rattus rattus Linnaeus, as host the effects of age and sex on host choice and fecundity of the Oriental rat flea, Xenopsylla cheopis Rothschild, were examined experimentally at 25 degrees C and 80% rh. During the first two days of emergence from cocoons, female fleas dominated the sex ratio by 4:1 but from the third day onwards this switched to a male-dominated sex ratio of 4:1. The sex of the flea did not influence their host-seeking behaviour. Newly emerged fleas of both sexes were not influenced by the rat's presence and at seven days old both sexes demonstrated similar levels of attraction toward the rat host. The sex of the rat did not affect flea host-seeking behaviour. There was a 50-70% decline in the initial number of adult fleas during the first week after their release onto a rat host, and this decline was greatest on juvenile rats. Flea fecundity was also significantly lower on juvenile rat hosts but no differences due to the sex of the rat were observed. This experimental study supports the hypothesis that differences in flea count due to host sex, reported in field surveys, result from sexual differences in host behaviour and not from discriminatory host-seeking behaviour by X. cheopis. Differences in flea count due to host age may be affected by differences in X. cheopis fecundity, which may itself be mediated by host behaviour such as grooming.     

Mechanics of tether formation in liposomes

It is well-known that a "tether" may be drawn out from a pressurized liposome by means of a suitably applied radial-outward force applied locally to the lipid bilayer. The tether is a narrow, uniform cylindrical tube, which joins the main vesicle in a short "transition region." A first-order energy analysis establishes the broad relationship between the force F needed to draw the tether, the radius R0 of the tether, the bending-stiffness constant B for the lipid bilayer and the membrane tension T in the pressurized liposome. The aim of the present paper is to study in detail the "transition region" between the tether and the main vesicle, by means of a careful application of the engineering theory of axisymmetric shell structures. It turns out that the well-known textbook "thin-shell" theory is inadequate for this purpose, because the tether is evidently an example of a thick-walled shell; and a novel ingredient of the present study is the introduction of elastic constitutive relations that are appropriate to the thick-shell situation. The governing equations are set up in dimensionless form, and are solved by means of a "shooting" technique, starting with a single disposable parameter at a point on the meridian in the tether, which can be adjusted until the boundary conditions at the far "equator" of the main vessel are satisfied. It turns out that the "transition region" between the tether and the main vessel is well characterized by only a few parameters, while the tether and main vessel themselves are described by very simple equations. Introduction of the thick-shell constitutive relation makes little difference to the conformation of and stress-resultants in, the main vessel; but it makes a great deal of difference in the tether itself Indeed, a kind of phase-change appears to take place in the "transition region" between these two zones of the liposome.     

Inhibition of somatostatin receptor 5-signaling by mammalian regulators of G-protein signaling (RGS) in yeast

Regulators of G-protein signaling (RGSs) are negative regulators of G-protein coupled receptor (GPCR)-mediated signaling that function to limit the lifetime of receptor-activated G(alpha)-proteins. Here we show that four mammalian RGSs differentially inhibit the activation of a FUS1--LacZ reporter gene by the STE2 encoded GPCR in yeast. In order to examine the role of the GPCR in modulating RGS function, we functionally expressed the human somatostatin receptor 5 (SST(5)) in yeast. In the absence of RGSs, FUS1--LacZ activation in response to somatostatin increased in a dose-dependent manner in cells expressing SST(5). In contrast to the results obtained with Ste2p, all RGSs completely inhibited SST(5)-mediated signaling even at concentrations of agonist as high as 10(minus sign5) M. The ability of RGSs to inhibit SST(5) signaling was further assessed in cells expressing modified Gpa1 proteins. Even though SST(5)-mediated FUS1--LacZ activation was 5-fold more efficient with a Gpa1p/G(i3alpha) chimera, response to somatostatin was completely abolished by all four RGSs. Furthermore, we demonstrate that RGS1, RGS2 and RGS5 have reduced ability to inhibit SST(5)-mediated activation of the RGS-resistant Gpa1p(Gly302Ser) mutant suggesting that the ability to interact with the G(alpha)-protein is required for the inhibition of signaling. Taken together, our results indicate that RGSs serve as better GAPs for Gpa1p when activated by SST(5) than when this G-protein is activated by Ste2p.