The rockefeller university

P>Secretins are a family of large bacterial outer membrane channels that serve as exit ports for folded proteins, filamentous phage and surface structures. Despite the large size of their substrates, secretins do not compromise the barrier function of the outer membrane, implying a gating mechanism. The region in the primary structure that forms the putative gate has not previously been determined for any secretin. To identify residues involved in gating the pIV secretin of filamentous bacteriophage f1, we used random mutagenesis of the gene followed by positive selection for mutants with compromised barrier function ('leaky' mutants). We identified mutations in 34 residues, 30 of which were clustered into two regions located in the centre of the conserved C-terminal secretin family domain: GATE1 (that spanned 39 residues) and GATE2 (that spanned 14 residues). An internal deletion constructed in the GATE2 region resulted in a severely leaky phenotype. Three of the four remaining mutations are located in the region that encodes the N-terminal, periplasmic portion of pIV and could be involved in triggering gate opening. Two missense mutations in the 24-residue region that separates GATE1 and GATE2 were also constructed. These mutant proteins were unstable, defective in multimerization and non-functional.

Voltage sensors regulate the conformations of voltage-dependent ion channels and enzymes. Their nearly switchlike response as a function of membrane voltage comes from the movement of positively charged amino acids, arginine or lysine, across the membrane field. We used mutations with natural and unnatural amino acids, electrophysiological recordings, and x-ray crystallography to identify a charge transfer center in voltage sensors that facilitates this movement. This center consists of a rigid cyclic "cap" and two negatively charged amino acids to interact with a positive charge. Specific mutations induce a preference for lysine relative to arginine. By placing lysine at specific locations, the voltage sensor can be stabilized in different conformations, which enables a dissection of voltage sensor movements and their relation to ion channel opening.

Objectives The OPRL1 gene encodes the nociceptin/orphanin FQ receptor, which plays a role in regulating tolerance and behavioral responses to morphine. there is limited information on whether variants of are associated with vulnerability to develop opiate addiction. In this study, we examined five variants of OPRL1 and their role in determining vulnerability to develop opiate addiction. Methods We recruited 447 individuals: 271 former severe heroin addicts and 176 healthy controls. Using a 5'-fluorogenic exonuclease assay, we genotyped individuals at five variants in OPRL1. It was then determined whether there was a significant association of allele, genotype, or haplotype frequency with vulnerability to develop opiate addiction. Results When the cohort was stratified by ethnicity, we found that, in Caucasians but not in African-Americans or Hispanics, the allele frequency of rs6090041 and rs6090043 were significantly associated point-wise with opiate addiction (P=0.03 and 0.04, respectively). Of the haplotypes formed by these two variants, one haplotype was found to be associated with protection from developing opiate addiction in both African-Americans (point-wise P=0.04) and Caucasians (point-wise P=0.04), and another haplotype with vulnerability to develop opiate addiction in Caucasians only (P=0.020). Conclusion This study provides evidence for an association of two variants of the OPRL1 gene, rs6090041 and rs6090043, with vulnerability to develop opiate addiction, suggesting a role for nociceptin/orphanin FQ receptor in the development of opiate addiction. Psychiatr Genet 20:65-72 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

The annulus is a higher order septin cytoskeletal structure located between the midpiece and principal piece regions of the sperm tail. The annulus has been hypothesized to generate the diffusion barrier that exists between these two membrane domains. We tested this premise directly on septin 4 knockout mice, whose sperm are viable but lack an annulus, by following the diffusing membrane protein basigin. Basigin is normally confined to the principal piece domain on testicular and caput sperm, but undergoes relocation into the midpiece during sperm epididymal transit. On Sept4(-/-) sperm, domain confinement was lost, and basigin localized over the entire plasma membrane. Both immunofluorescence and immunoblotting further revealed reduced levels of basigin expression on sperm from the knockout. Testicular immunohistochemistry showed similar basigin expression and tail targeting in wild-type (WT) and Sept4(-/-) tubules until step 15 of spermatid development, at which point basigin was redistributed throughout the plasma membrane of Sept4(-/-) spermatids. The basigin outside of the tail was subsequently lost around the time of sperm release into the lumen. The redistribution in the knockout coincides with the time in WT sperm when the annulus completes its migration from the neck down to the midpiece-principal piece junction. We posit that basigin may not diffuse freely until after the annulus arrives at the midpiece-principal piece junction to restrict lateral movement. These results are the strongest evidence to date of a mammalian septin structure establishing a membrane diffusion barrier.

The culture and demonstration of putative nanobacteria (NB) and calcifying nanoparticles (CNP) from human and animal tissues has relied primarily on the use of a culture supplement consisting of FBS that had been gamma-irradiated at a dose of 30 kGy (gamma-FBS). The use of gamma-FBS is based on the assumption that this sterilized fluid has been rid entirely of any residual NB/CNP, while it continues to promote the slow growth in culture of NB/CNP from human/animal tissues. We show here that gamma-irradiation (5-50 kGy) produces extensive dose-dependent serum protein breakdown as demonstrated through UV and visible light spectrophotometry, fluorometry, Fourier-transformed infrared spectroscopy, and gel electrophoresis. Yet, both gamma-FBS and gamma-irradiated human serum (gamma-HS) produce NB/CNP in cell culture conditions that are morphologically and chemically indistinguishable from their normal serum counterparts. Contrary to earlier claims, gamma-FBS does not enhance the formation of NB/CNP from several human body fluids (saliva, urine, ascites, and synovial fluid) tested. In the presence of additional precipitating ions, both c-irradiated serum (FBS and HS) and c-irradiated proteins (albumin and fetuin-A) retain the inherent dual NB inhibitory and seeding capabilities seen also with their untreated counterparts. By gel electrophoresis, the particles formed from both gamma-FBS and gamma-HS are seen to have assimilated into their scaffold the same smeared protein profiles found in the gamma-irradiated sera. However, their protein compositions as identified by proteomics are virtually identical to those seen with particles formed from untreated serum. Moreover, particles derived from human fluids and cultured in the presence of gamma-FBS contain proteins derived from both gamma-FBS and the human fluid under investigation-a confusing and unprecedented scenario indicating that these particles harbor proteins from both the host tissue and the FBS used as feeder. Thus, the NB/CNP described in the literature clearly bear hybrid protein compositions belonging to different species. We conclude that there is no basis to justify the use of gamma-FBS as a feeder for the growth and demonstration of NB/CNP or any NB-like particles in culture. Moreover, our results call into question the validity of the entire body of literature accumulated to date on NB and CNP.

Staphylococcus aureus is the causative agent of several serious infectious diseases. The emergence of antibiotic-resistant S. aureus strains has resulted in significant treatment difficulties, intensifying the need for new antimicrobial agents. Toward this end, we have developed a novel chimeric bacteriophage (phage) lysin that is active against staphylococci, including methicillin-resistant S. aureus (MRSA). The chimeric lysin (called ClyS) was obtained by fusing the N-terminal catalytic domain of the S. aureus Twort phage lysin with the C-terminal cell wall-targeting domain from another S. aureus phage lysin (phiNM3), which displayed Staphylococcus-specific binding. ClyS was expressed in Escherichia coli, and the purified protein lysed MRSA, vancomycin-intermediate strains of S. aureus (VISA), and methicillin-sensitive (MSSA) strains of S. aureus in vitro. In a mouse nasal decolonization model, a 2-log reduction in the viability of MRSA cells was seen 1 h following a single treatment with ClyS. One intraperitoneal dose of ClyS also protected against death by MRSA in a mouse septicemia model. ClyS showed a typical pattern of synergistic interactions with both vancomycin and oxacillin in vitro. More importantly, ClyS and oxacillin at doses that were not protective individually protected synergistically against MRSA septic death in a mouse model. These results strongly support the development of ClyS as an attractive addition to the current treatment options of multidrug-resistant S. aureus infections and would allow for the reinstatement of antibiotics shelved because of mounting resistance.

Low serotonin(1A) receptor (5-HT1AR) binding is a risk factor for anxiety and depression, and deletion of the 5-HT1AR results in anxiety-like behavior in mice. Here we show that anxiety-like behavior in mice also can be caused, independently of the offspring's own 5-HT1AR genotype, by a receptor deficit in the mother: a nongenetic transmission of a genetic defect. Some of the nongenetically transmitted anxiety manifestations were acquired prenatally and linked to a delay in dentate gyrus maturation in the ventral hippocampus of the offspring. Both the developmental delay and the anxiety-like phenotype were phenocopied by the genetic inactivation of p16(ink4a) encoding a cyclin-dependent kinase inhibitor implicated in neuronal precursor differentiation. No maternal 5-HT1AR genotype-dependent anxiety developed when the strain background was switched from Swiss Webster to C57BL/6, consistent with the increased resilience of this strain to early adverse environment. Instead, all anxiety manifestations were caused by the offspring's own receptor deficiency, indicating that the genetic and nongenetic effects converge to common anxiety manifestations. We propose that 5-HT1AR deficit represents a dual risk for anxiety and that vulnerability to anxiety associated with genetic 5-HT1AR deficiency can be transmitted by both genetic and nongenetic mechanisms in a population. Thus, the overall effect of risk alleles can be higher than estimated by traditional genetic assays and may contribute to the relatively high heritability of anxiety and psychiatric disorders in general.

Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.

Building on mode-coupling-theory calculations, we report a novel scenario for multiple glass transitions in a purely repulsive spherical potential: the square shoulder. The liquid-glass transition lines exhibit both melting by cooling and melting by compression as well as associated diffusion anomalies, similar to the ones observed in water. Differently from all previously investigated models, we find for small shoulder widths a glass-glass line that is disconnected from the liquid phase. Upon increasing the shoulder width such a glass-glass line merges with the liquid-glass transition lines, featuring two distinct end point singularities that give rise to logarithmic decays in the dynamics. We analytically explain these findings by considering the interplay of different repulsive length scales.

Histone proteins often come in different variants serving specialized functions in addition to their fundamental role in packaging DNA. The metazoan histone H3.3 has been most closely associated with active transcription. Its role in histone replacement at active genes and promoters is conserved to the single histone H3 in yeast. However, recent genetic studies in flies have challenged its importance as a mark of active chromatin, and revealed unexpected insights into essential functions of H3.3 in the germline. With strikingly little. amino acid sequence difference to the canonical H3, H3.3 therefore accomplishes a surprising variety of cellular and developmental processes.