Medical sequencing at the extremes of human body mass

Body weight is a quantitative trait with significant heritability in humans. To identify potential genetic contributors to this phenotype, we resequenced the coding exons and splice junctions of 58 genes in 379 obese and 378 lean individuals. Our 96-Mb survey included 21 genes associated with monogenic forms of obesity in humans or mice, as well as 37 genes that function in body weight-related pathways. We found that the monogenic obesity-associated gene group was enriched for rare nonsynonymous variants unique to the obese population compared with the lean population. In addition, computational analysis predicted a greater fraction of deleterious variants within the obese cohort. Together, these data suggest that multiple rare alleles contribute to obesity in the population and provide a medical sequencing-based approach to detect them.     

Regulation of Ins(3,4,5,6)P(4) signaling by a reversible kinase/phosphatase

Regulation of Cl(-) channel conductance by Ins(3,4,5,6)P(4) provides receptor-dependent control over salt and fluid secretion, cell volume homeostasis, and electrical excitability of neurones and smooth muscle. Ignorance of how Ins(3,4,5,6)P(4) is synthesized has long hindered our understanding of this signaling pathway. We now show Ins(3,4,5,6)P(4) synthesis by Ins(1,3,4,5,6)P(5) 1-phosphatase activity by an enzyme previously characterized as an Ins(3,4,5,6)P(4) 1-kinase. Rationalization of these phenomena with a ligand binding model unveils Ins(1,3,4)P(3) as not simply an alternative kinase substrate, but also an activator of Ins(1,3,4,5,6)P(5) 1-phosphatase. Stable overexpression of the enzyme in epithelial monolayers verifies its physiological role in elevating Ins(3,4,5,6)P(4) levels and inhibiting secretion. It is exceptional for a single enzyme to catalyze two opposing signaling reactions (1-kinase/1-phosphatase) under physiological conditions. Reciprocal coordination of these opposing reactions offers an alternative to general doctrine that intracellular signals are regulated by integrating multiple, distinct phosphatases and kinases.     

p38 kinase inhibitors for the treatment of arthritis and osteoporosis: thienyl, furyl, and pyrrolyl ureas

Inhibitors of the MAP kinase p38 are potentially useful for the treatment for osteoporosis, arthritis, and other inflammatory diseases. A series of thienyl, furyl, and pyrrolyl ureas has been identified as potent p38 inhibitors, displaying in vitro activity in the nanomolar range.     

Sensitivity of Shiga toxin-producing Escherichia coli (STEC) strains for colicins under different experimental conditions

Twenty Escherichia coli strains producing well-characterised colicins were tested for their inhibitory activity against five Shiga toxin-producing E. coli (STEC) strains using different media under aerobic and anaerobic conditions. The five STEC strains used were of serotype O26, O111, O128, O145 and O157:H7 which are frequently isolated serotypes associated with disease in humans. The main route of infection for humans is through the eating of badly cooked or handled beef. The major reservoir for STEC strains in cattle is the rumen. To mimic the situation in the rumen of cattle, overlay assays were also performed under anaerobic conditions in the presence of 30% rumen fluid. Colicins E1, E4, E8-J, K and S4 are most active against STEC strains under anaerobic conditions in the absence or presence of rumen fluid. These colicins will be used in future experiments with the aim to eradicate the presence of STEC in cattle.     

Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes

A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8(+) T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2x MVA.HIVA) (n=8) or two doses of placebo (2x placebo) (n=4). The second group received 2x pTHr.HIVA followed by one dose of MVA.HIVA (n=8) or 3x placebo (n=4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-gamma) ELISPOT (group mean, 210 spot-forming cells/10(6) cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2x MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-gamma ELISPOT assay, positive responses mainly mediated by CD4(+) T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2x MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4(+) T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8(+) T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1 beta. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.     

Moving towards a better understanding of chemotaxis

Eukaryotic cells are thought to move across supporting surfaces through a combination of coordinated processes: polarisation; extension of dynamic protrusions from a leading edge; adhesion-associated stabilisation of some protrusions; centripetal pulling against those leading adhesions; and de-adhesion at the rear. Gradients of extracellular ligands can be detected by cells and then used to guide them either towards the source (in the case of a chemoattractant) or away from the source (in the case of a chemorepellent)--such migration is termed chemotaxis. Recent work suggests that chemotaxis probably emerges from the ability of cells to spatially encode extracellular gradients of ligands, a process for which phosphoinositide 3'-kinase (PI3K) signals alone are insufficient, and to use that vectorial information to bias movement by enhancing the survival, and not the formation, of the protrusions that experience the greatest stimulation.     

Highly sensitive method for quantitative determination of bilirubin in biological fluids and tissues

Unconjugated bilirubin (UCB) exhibits potent antioxidant and cytoprotective properties, but causes apoptosis and cytotoxicity at pathologically elevated concentrations. Accurate measurement of UCB concentrations in cells, fluids and tissues is needed to evaluate its role in redox regulation, prevention of atherosclerotic and malignant diseases, and bilirubin encephalopathy. In the present study, we developed and validated a highly sensitive method for tissue UCB determinations. UCB was extracted from rat organs with chloroform/methanol/hexane at pH 6.2 and then partitioned into a minute volume of alkaline buffer that was subjected to HPLC using an octyl reverse phase (RP) column. Addition of mesobilirubin as an internal standard corrected for losses of UCB during extraction. Recoveries averaged 75+/-5%. The detection limit was 10pmol UCB/g wet tissue. Variance was +/-2.5%. When used to measure UCB concentrations in tissues of jaundiced Gunn rats, this procedure yielded UCB levels directly comparable to published methods, and accurately determined very low tissue bilirubin concentrations (相似文献   

Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37

Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.