Down-regulation of calponin destabilizes actin cytoskeletal structure in A7r5 cells

The effects of changes in the expression levels of h1 calponin (CaP) on actin cytoskeletal organization were studied in control and phorbol-ester-treated A7r5 smooth muscle cells. Protein association and expression in control and stimulated A7r5 smooth muscle cells were evaluated by Western blotting, laser scanning confocal microscopy (LSCM), and fluorescence resonance energy transfer (FRET) microscopy in cells treated with either 2 x 10(-6 ) mol/L TGF-beta 1 or 2 x 10(-)5 mol/L PDGF-BB to alter h1 calponin expression. Single immunostained samples showed that CaP and alpha-actin, localized in fibers in unstimulated control A7r5 smooth muscle cells, were translocated to podosomes following treatment with phorbol-12,13-dibutyrate (PDBu). Confocal colocalization imaging and FRET analysis both indicated substantial association of CaP with alpha-actin in stress fibers of control cells and in podosomes of PDBu-treated cells. PKC alpha, which showed evidence of only slight association with CaP in control cells, exhibited markedly increased (293%) association in PDBu-contracted cells. Platelet-derived growth factor (PDGF)-BB down-regulated CaP to non-detectable levels, whereas transforming growth factor (TGF)-beta 1 up-regulated (424%) the expression of CaP without affecting the levels of alpha-actin or PKC alpha. PDGF-BB resulted in a significant loss in alpha-actin stress fibers (-47%) and reduced podosome formation (-69%). By comparison, TGF-beta 1 had no effect on stress fibers in control cells but also reduced (-70%) podosome formation. The results suggest that CaP could play a major role in the stabilization of actin stress fibers in resting cells and may contribute to podosome formation in PDBu-treated cells.     

Mutations in CUL4B, which encodes a ubiquitin E3 ligase subunit, cause an X-linked mental retardation syndrome associated with aggressive outbursts, seizures, relative macrocephaly, central obesity, hypogonadism, pes cavus, and tremor

We have identified three truncating, two splice-site, and three missense variants at conserved amino acids in the CUL4B gene on Xq24 in 8 of 250 families with X-linked mental retardation (XLMR). During affected subjects' adolescence, a syndrome emerged with delayed puberty, hypogonadism, relative macrocephaly, moderate short stature, central obesity, unprovoked aggressive outbursts, fine intention tremor, pes cavus, and abnormalities of the toes. This syndrome was first described by Cazebas et al., in a family that was included in our study and that carried a CUL4B missense variant. CUL4B is a ubiquitin E3 ligase subunit implicated in the regulation of several biological processes, and CUL4B is the first XLMR gene that encodes an E3 ubiquitin ligase. The relatively high frequency of CUL4B mutations in this series indicates that it is one of the most commonly mutated genes underlying XLMR and suggests that its introduction into clinical diagnostics should be a high priority.     

Association analysis of 9,560 prostate cancer cases from the International Consortium of Prostate Cancer Genetics confirms the role of reported prostate cancer associated SNPs for familial disease

Previous GWAS studies have reported significant associations between various common SNPs and prostate cancer risk using cases unselected for family history. How these variants influence risk in familial prostate cancer is not well studied. Here, we analyzed 25 previously reported SNPs across 14 loci from prior prostate cancer GWAS. The International Consortium for Prostate Cancer Genetics (ICPCG) previously validated some of these using a family-based association method (FBAT). However, this approach suffered reduced power due to the conditional statistics implemented in FBAT. Here, we use a case–control design with an empirical analysis strategy to analyze the ICPCG resource for association between these 25 SNPs and familial prostate cancer risk. Fourteen sites contributed 12,506 samples (9,560 prostate cancer cases, 3,368 with aggressive disease, and 2,946 controls from 2,283 pedigrees). We performed association analysis with Genie software which accounts for relationships. We analyzed all familial prostate cancer cases and the subset of aggressive cases. For the familial prostate cancer phenotype, 20 of the 25 SNPs were at least nominally associated with prostate cancer and 16 remained significant after multiple testing correction (p ≤ 1E ^?3) occurring on chromosomal bands 6q25, 7p15, 8q24, 10q11, 11q13, 17q12, 17q24, and Xp11. For aggressive disease, 16 of the SNPs had at least nominal evidence and 8 were statistically significant including 2p15. The results indicate that the majority of common, low-risk alleles identified in GWAS studies for all prostate cancer also contribute risk for familial prostate cancer, and that some may contribute risk to aggressive disease.     

Telomere Length Shows No Association with BRCA1 and BRCA2 Mutation Status

This study aimed to determine whether telomere length (TL) is a marker of cancer risk or genetic status amongst two cohorts of BRCA1 and BRCA2 mutation carriers and controls. The first group was a prospective set of 665 male BRCA1/2 mutation carriers and controls (mean age 53 years), all healthy at time of enrolment and blood donation, 21 of whom have developed prostate cancer whilst on study. The second group consisted of 283 female BRCA1/2 mutation carriers and controls (mean age 48 years), half of whom had been diagnosed with breast cancer prior to enrolment. TL was quantified by qPCR from DNA extracted from peripheral blood lymphocytes. Weighted and unweighted Cox regressions and linear regression analyses were used to assess whether TL was associated with BRCA1/2 mutation status or cancer risk. We found no evidence for association between developing cancer or being a BRCA1 or BRCA2 mutation carrier and telomere length. It is the first study investigating TL in a cohort of genetically predisposed males and although TL and BRCA status was previously studied in females our results don''t support the previous finding of association between hereditary breast cancer and shorter TL.     

Cloned Defective Interfering Influenza Virus Protects Ferrets from Pandemic 2009 Influenza A Virus and Allows Protective Immunity to Be Established

Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In addition there are periodic outbreaks in domestic poultry, horses, pigs, dogs, and cats. Infections of domestic birds can be fatal for the birds and their human contacts. Control in man operates through vaccines and antivirals, but both have their limitations. In the search for an alternative treatment we have focussed on defective interfering (DI) influenza A virus. Such a DI virus is superficially indistinguishable from a normal virus but has a large deletion in one of the eight RNAs that make up the viral genome. Antiviral activity resides in the deleted RNA. We have cloned one such highly active DI RNA derived from segment 1 (244 DI virus) and shown earlier that intranasal administration protects mice from lethal disease caused by a number of different influenza A viruses. A more cogent model of human influenza is the ferret. Here we found that intranasal treatment with a single dose of 2 or 0.2 µg 244 RNA delivered as A/PR/8/34 virus particles protected ferrets from disease caused by pandemic virus A/California/04/09 (A/Cal; H1N1). Specifically, 244 DI virus significantly reduced fever, weight loss, respiratory symptoms, and infectious load. 244 DI RNA, the active principle, was amplified in nasal washes following infection with A/Cal, consistent with its amelioration of clinical disease. Animals that were treated with 244 DI RNA cleared infectious and DI viruses without delay. Despite the attenuation of infection and disease by DI virus, ferrets formed high levels of A/Cal-specific serum haemagglutination-inhibiting antibodies and were solidly immune to rechallenge with A/Cal. Together with earlier data from mouse studies, we conclude that 244 DI virus is a highly effective antiviral with activity potentially against all influenza A subtypes.     

A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression

Here we report on the construction of a tetracycline inducible expression vector that allows a tightly regulable gene expression in Corynebacterium glutamicum which is used in industry for production of small molecules such as amino acids. Using the green fluorescent protein (GFP) as a reporter protein we show that this vector, named pCLTON1, is characterized by tight repression under non-induced conditions as compared to a conventional IPTG inducible expression vector, and that it allows gradual GFP synthesis upon gradual increase of anhydrotetracycline addition.     

Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death

Phospholipase A(2) (PLA(2)) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [(3)H]-AA released into the extracellular medium following NMDA receptor stimulation (100 microM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 microM) or by removal of extracellular Ca(2+). Neither diacylglycerol lipase inhibition (RHC-80267; 10 microM) nor selective inhibition of Ca(2+)-independent PLA(2) [bromoenol lactone (BEL); 10 microM] alone had an effect on NMDA receptor-stimulated release of [(3)H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 microM) and AACOCF(3) (1 microM), inhibitors of both cytosolic PLA(2) (cPLA(2)) and Ca(2+)-independent PLA(2) isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 microM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA(2)alpha. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA(2)alpha, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF(3) nor cultures derived from cPLA(2)alpha null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.     

Moraxella catarrhalis M35 is a general porin that is important for growth under nutrient-limiting conditions and in the nasopharynges of mice

Moraxella catarrhalis is a gram-negative respiratory pathogen that is an important causative agent for otitis media and exacerbations of chronic obstructive pulmonary disease. We have previously predicted the outer membrane protein M35 to be a general porin, and in the current study, we have investigated the function of M35 and its importance for survival of M. catarrhalis in vivo. Lipid bilayer experiments reveal that refolded M35 functions as a channel that is typical of gram-negative bacterial porins. M35 forms wide and water-filled channels with a single-channel conductance of about 1.25 nS in 1 M KCl solution and has only a small selectivity for cations over anions. When the in vitro growth characteristics of two M35 deletion mutant strains of M. catarrhalis were compared to the wild-type parent isolates, the growth of the mutant strains was inhibited only under nutrient-poor conditions. This growth defect could be eliminated by additional glutamic acid, but not additional aspartic acid, glycine, sucrose, or glucose. The mutant strains compensated for the lack of M35 by enhancing their uptake of glutamic acid, and this enhanced rate of glutamic acid uptake was attributed to the compensatory upregulation of a protein of approximately 40 kDa. M35 was also found to be essential for nasal colonization of mice, demonstrating that its presence is essential for survival of M. catarrhalis in vivo. These results suggest that M35 is a general porin that is necessary for the uptake of important energy sources by M. catarrhalis and that it is likely that M35 is an essential functional protein for in vivo colonization.     

3.6 kb genomic sequence from Takifugu capable of promoting axon growth-associated gene expression in developing and regenerating zebrafish neurons

Unlike mammals, fish have the capacity for functional adult CNS regeneration, which is due, in part, to their ability to express axon growth-related genes in response to nerve injury. One such axon growth-associated gene is gap43, which is expressed during periods of developmental and regenerative axon growth, but is not expressed in CNS neurons that do not regenerate in adult mammals. We previously demonstrated that cis-regulatory elements of gap43 that are sufficient for developmental expression are not sufficient for regenerative expression in the zebrafish. Here we have identified a 3.6kb genomic sequence from Fugu rubripes that can promote reporter gene expression in the nervous system during both development and regeneration in zebrafish. This compact sequence is advantageous for functional dissection of regions important for axon growth-associated gene expression during development and/or regeneration. In addition, this sequence will also be useful for targeting gene expression to neurons during periods of growth and plasticity.