DmsD is required for the biogenesis of DMSO reductase in Escherichia coli but not for the interaction of the DmsA signal peptide with the Tat apparatus

The DmsD protein is essential for the biogenesis of DMSO reductase in Escherichia coli, and binds the signal peptide of the DmsA subunit, a Tat substrate. This suggests a role as a guidance factor to target pre-DmsA to the translocase. Here, we have analysed the export of fusion proteins in which the DmsA and TorA signal peptides are fused to green fluorescent protein. Both chimeras are efficiently exported to the periplasm in wild-type E. coli cells and we show that their export efficiencies are essentially identical in a mutant lacking DmsD. An authentic Tat substrate, TMAO reductase, is also efficiently exported in the dmsD mutant. The data indicate that DmsD carries out a critical role in DMSO reductase biogenesis/assembly but is not required for the functioning of the DmsA signal peptide.     

Antagonists of the human CCR5 receptor as anti-HIV-1 agents. Part 2: structure-activity relationships for substituted 2-Aryl-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-(piperidin-1-yl)butanes

(2S)-2-(3,4-Dichlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[spiro(2,3-dihydrobenzthiophene-3,4'-piperidin-1'-yl)]butane S-oxide (3) has been identified as a potent CCR5 antagonist lead structure having an IC50 = 35 nM. Herein, we describe the structure-activity relationship studies directed toward the requirement for and optimization of the C-2 phenyl fragment. The phenyl was found to be important for CCR5 antagonism and substitution was limited to small moieties at the 3-position (13 and 16: X= H, 3-F, 3-Cl, 3-Me).     

Hyperglycemia increases endothelial superoxide that impairs smooth muscle cell Na+-K+-ATPase activity

Nitric oxide (NO) plays an important role in the control of numerous vascular functions including basal Na+-K+-ATPase activity in arterial tissue. Hyperglycemia inhibits Na+-K+-ATPase activity in rabbit aorta, in part, through diminished bioactivity of NO. The precise mechanism(s) for such observations, however, are not yet clear. The purpose of this study was to examine the role of superoxide in modulating NO-mediated control of Na+-K+-ATPase in response to hyperglycemia. Rabbit aorta incubated with hyperglycemic glucose concentrations (44 mM) demonstrated a 50% reduction in Na+-K+-ATPase activity that was abrogated by superoxide dismutase. Hyperglycemia also produced a 50% increase in steady-state vascular superoxide measured by lucigenin-enhanced chemiluminescence that was closely associated with reduced Na+-K+-ATPase activity. Specifically, the hyperglycemia-induced increase in vascular superoxide was endothelium dependent, inhibited by L-arginine, and stimulated by N(omega)-nitro-L-arginine. Aldose reductase inhibition with zopolrestat also inhibited the hyperglycemia-induced increase in vascular superoxide. In each manipulation of vascular superoxide, a reciprocal change in Na+-K+-ATPase activity was observed. Finally, a commercially available preparation of Na+-K+-ATPase was inhibited by pyrogallol, a superoxide generator. These data suggest that hyperglycemia induces an increase in endothelial superoxide that inhibits the stimulatory effect of NO on vascular Na+-K+-ATPase activity.     

Nominal kinship cues facilitate altruism.

We investigated whether names in common promote altruistic behaviour, predicting that this would be especially so for relatively uncommon names, for surnames (which are better kinship cues than first names), and among women (who, although less willing than men to help strangers, according to prior research, are also the primary "kin keepers"). We solicited help from 2960 email addressees, with the request ostensibly coming from a same-sex person sharing both, either, or neither of the addressee''s first and last names. As anticipated, addressees were most likely to respond helpfully when senders shared both their names (12.3%) and least likely when they shared neither (2.0%), and this was especially true for relatively uncommon names. A shared surname was more effective than a shared first name only if it was relatively uncommon. Women were substantially more likely to reply than men. These results indicate that names elicit altruism because they function as salient cues of kinship.     

Prostaglandin H2 (PGH2) accelerates formation of amyloid beta1-42 oligomers

Epidemiologic evidence implicates cyclooxygenase activity in the pathogenesis of Alzheimer's disease, in which amyloid plaques have been found to contain increased levels of dimers and higher multimers of the amyloid beta peptide. The product of the oxygenation of arachidonic acid by the cyclooxygenases, prostaglandin H2 (PGH2), rearranges non-enzymatically to several prostaglandins, including the highly reactive gamma-keto aldehydes, levuglandins E2 and D2. We demonstrate that PGH2 markedly accelerates the formation of dimers and higher oligomers of amyloid beta1-42. This is associated with the formation of levuglandin adducts of the peptide. These findings provide the molecular basis for a hypothesis linking cyclooxygenase activity to the formation of oligomers of amyloid beta.     

Inhibition of mesangial cell nitric oxide in MRL/lpr mice by prostaglandin J2 and proliferator activation receptor-gamma agonists

MRL/Mp-lpr/lpr (MRL/lpr) mice develop immune complex glomerulonephritis similar to human lupus. Glomerular mesangial cells are key modulators of the inflammatory response in lupus nephritis. When activated, these cells secrete inflammatory mediators including NO and products of cyclooxygenase perpetuating the local inflammatory response. PGJ2, a product of cyclooxygenase, is a potent in vitro inhibitor of macrophage inflammatory functions and is postulated to function as an in vivo inhibitor of macrophage-mediated inflammatory responses. We hypothesized that in lupus, a defect in PGJ2 production allows the inflammatory response to continue unchecked. To test this hypothesis, mesangial cells were isolated from MRL/lpr and BALB/c mice and stimulated with IL-1beta or LPS plus IFN-gamma. In contrast to the 2- to 3-fold increase in PGJ2 production by stimulated BALB/c mesangial cells, supernatant PGJ2 did not increase in MRL/lpr mesangial cell cultures. NO production in stimulated MRL/lpr and BALB/c mesangial cells, was blocked by PGJ2 and pioglitazone. These studies suggest that abnormalities in PGJ2 production are present in MRL/lpr mice and may be linked to the heightened activation state of mesangial cells in these mice.     

Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo

We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.