Hydrogen sulfide acts as an inflammatory mediator in cecal ligation and puncture-induced sepsis in mice by upregulating the production of cytokines and chemokines via NF-kappaB

Increasing data suggest that oxidative stress, due to an increased production of reactive oxygen species and/or a decrease in antioxidants, is involved in the pathophysiology of pulmonary hypertension. Several antioxidant systems regulate the presence of oxidant species in vivo, and of primary interest are the superoxide dismutases (SOD) and catalase. However, little is known about the expression of antioxidant enzymes during the development of pulmonary hypertension. This study uses our lamb model of increased postnatal pulmonary blood flow, secondary to in utero aortopulmonary graft placement (shunt lambs), to investigate the expression patterns as well as activities of antioxidant enzymes during the early development of pulmonary hypertension. Protein levels of catalase, SOD1, SOD2, and SOD3 were evaluated by Western blot, and the activities of catalase and SOD were also quantified. In control lambs, protein expression and activities of catalase and SOD2 increased postnatally (P < 0.05). However, SOD1 and SOD3 protein levels did not change. In shunt lambs, catalase, SOD1, and SOD2 protein levels all increased over the first 8 wk of life (P < 0.05). However, SOD3 did not change. This was associated with an increase in the activities of catalase and SOD2 (P < 0.05). Compared with control lambs, catalase and SOD2 protein levels were decreased in 2-wk-old shunt lambs and this was associated with increased levels of hydrogen peroxide (H(2)O(2)) and superoxide (P < 0.05). Developmentally superoxide but not H(2)O(2) levels significantly increased in both shunt and control lambs with levels being significantly higher in shunt compared with control lambs at 2 and 4 but not 8 wk. These data suggest that the antioxidant enzyme systems are dynamically regulated postnatally, and this regulation is altered during the development of pulmonary hypertension secondary to increased pulmonary blood flow. An increased understanding of these alterations may have important therapeutic implications for the treatment of pulmonary hypertension secondary to increased pulmonary blood flow.     

The cyclin-dependent kinase Cdc28p regulates multiple aspects of Kar9p function in yeast

During mitosis in the yeast Saccharomyces cerevisiae, Kar9p directs one spindle pole body (SPB) toward the incipient daughter cell by linking the associated set of cytoplasmic microtubules (cMTs) to the polarized actin network on the bud cortex. The asymmetric localization of Kar9p to one SPB and attached cMTs is dependent on its interactions with microtubule-associated proteins and is regulated by the yeast Cdk1 Cdc28p. Two phosphorylation sites in Kar9p were previously identified. Here, we propose that the two sites are likely to govern Kar9p function through separate mechanisms, each involving a distinct cyclin. In the first mechanism, phosphorylation at serine 496 recruits Kar9p to one SPB. A phosphomimetic mutation at serine 496 bypasses the requirement of BIK1 and CLB5 in generating Kar9p asymmetry. In the second mechanism, Clb4p may target serine 197 of Kar9p for phosphorylation. This modification is required for Kar9p to direct cMTs to the bud. Two-hybrid analysis suggests that this phosphorylation may attenuate the interaction between Kar9p and the XMAP215-homologue Stu2p. We propose that phosphorylation at serine 197 regulates the release of Kar9p from Stu2p at the SPB, either to clear it from the mother-SPB or to allow it to travel to the plus end.     

Cortisol and stress responses during a game and practice in female collegiate soccer players

The purpose of this study was to compare the cortisol responses from a regular season game and a typical practice session in female National Collegiate Athletic Association Division I collegiate soccer players. Eighteen players were assigned to 2 groups, 10 starters and 8 nonstarters, depending on their playing time. Salivary cortisol concentration, as well as competitive sport anxiety (somatic and cognitive anxiety, self-confidence), was monitored before and after 1 regular season game and 1 typical practice session. Although salivary cortisol levels increased postgame for both starters (+250%) and nonstarters (+140%), they increased to a greater extent for the starters. Practice salivary cortisol did not significantly change (p > 0.05). Cognitive and somatic anxiety was greater pre- and postgame when compared with the pre- and postpractice scores, respectively. These data clearly demonstrate the psychological and physiological differences between soccer competition and practice in collegiate women. It appears that both physiological and psychological variables combine to contribute to the large stress hormone response to an actual competitive game.     

Rapid cold-hardening protects <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> from cold-induced apoptosis

The rapid cold-hardening (RCH) response increases the cold tolerance of insects by protecting against non-freezing, cold-shock injury. Apoptosis, or programmed cell death, plays important roles in development and the elimination of sub-lethally damaged cells. Our objectives were to determine whether apoptosis plays a role in cold-shock injury and, if so, whether the RCH response protects against cold-induced apoptosis in Drosophila melanogaster. The present study confirmed that RCH increased the cold tolerance of the adults at the organismal level. No flies in the cold-shocked group survived direct exposure to ‒7°C for 2 h, whereas significantly more flies in the RCH group survived exposure to ‒7°C for 2 h after a 2-h exposure to 5°C. We used a TUNEL assay to detect and quantify apoptotic cell death in five groups of flies including control, cold-shocked, RCH, heat-shocked (37.5°C, 30 min), and frozen (‒20°C, 24 h) and found that apoptosis was induced by cold shock, heat shock, and freezing. The RCH treatment significantly improved cell viability by 38% compared to the cold-shocked group. Cold shock-induced DNA fragmentation shown by electrophoresis provided further evidence for apoptosis. SDS-PAGE analysis revealed an RCH-specific protein band with molecular mass of ∼150 kDa. Western-blotting revealed three proteins that play key roles in the apoptotic pathway: caspase-9-like (apoptotic initiator), caspase-3-like (apoptotic executioner) and Bcl-2 (anti-apoptotic protein). Consequently, the results of this study support the hypothesis that the RCH response protects against cold-shock-induced apoptosis.     

Real-time PCR method for Salmonella spp. targeting the stn gene

AIM: To develop a real-time PCR assay for Salmonella spp. targeting the stn gene. METHODS AND RESULTS: The presence of stn in the Salmonella bongori genome was found by a BLAST with Salmonella enterica stn sequence. Manual alignment of stn sequences showed that Salm. bongori had 88% sequence identity with Salm. enterica. Two primers (stnL-433 and stnR-561) and a probe (stnP-452) were designed to target conserved regions in stn and meet the requirements of a 5'-nuclease assay. The primers and probe were evaluated against 353 isolates, including 255 Salm. enterica representing 158 serotypes, 14 Salm. bongori representing 12 serotypes and 84 non-Salmonella representing 56 species from 31 genera. All isolates were correctly identified, with the exception of three isolates of Citrobacter amalonaticus, which gave false positives. The limit of detection with cultured Salmonella was 3 CFU per reaction. CONCLUSIONS: The stn real-time PCR method had 100% inclusivity, 96.4% exclusivity and a level of detection of 3 CFU per reaction for cultured Salmonella spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed that stn is present in Salm. bongori and is a valid target for both species of Salmonella. The Salmonella s tn real-time PCR is a useful method for identifying Salmonella spp.     

西伯利亚蝗基因组DNA提取及RAPD分析条件的优化

以西伯利亚蝗Gomphocerus sibiricus(L.)为研究材料,利用改良的SDS法提取高质量的DNA,分别测试了dNTP浓度、镁离子浓度、TaqDNA聚合酶用量、模板DNA的量等因素对反应结果的影响。通过各因子的组合比较,建立了西伯利亚蝗RAPD优化体系:25μLPCR反应体系,10×buffer2·5μL;dNTP0·24mmol/L;MgCl22·0mmol/L;Taq DNA聚合酶1U;DNA模板45ng;引物30ng。扩增程序为:94℃预变性1min45s、94℃变性30s、35℃退火1min30s、72℃延伸2min,45个循环、72℃延伸10min。结果表明,利用优化的反应条件进行西伯利亚蝗基因组DNA分析,实验有着良好的重复性和稳定性。     

Crystal structure of the pregnane X receptor-estradiol complex provides insights into endobiotic recognition

The human nuclear pregnane X receptor (PXR) responds to a wide variety of xenobiotic and endobiotic compounds, including pregnanes, progesterones, corticosterones, lithocholic acids, and 17beta-estradiol. In response to these ligands, the receptor controls the expression of genes central to the metabolism and excretion of potentially harmful chemicals from both exogenous and endogenous sources. Although the structural basis of PXR's interaction with small and large xenobiotics has been examined, the detailed nature of its binding to endobiotics, including steroid-like ligands, remains unclear. We report the crystal structure of the human PXR ligand-binding domain (LBD) in complex with 17beta-estradiol, a representative steroid ligand, at 2.65 A resolution. Estradiol is found to occupy only one region of PXR's expansive ligand-binding pocket, leaving a notable 1000 A3 of space unoccupied, and to bridge between the key polar residues Ser-247 and Arg-410 in the PXR LBD. Positioning the steroid scaffold in this way allows it to make several direct contacts to alphaAF of the receptor's AF-2 region. The PXR-estradiol complex was compared with that of other nuclear receptors, including the estrogen receptor, in complexes with analogous ligands. It was found that PXR's placement of the steroid is remarkably distinct relative to other members of the nuclear receptor superfamily. Using the PXR-estradiol complex as a guide, the binding of other steroid- and cholesterol-like molecules was then considered. The results provide detailed insights into the manner in which human PXR responds to a wide range of endobiotic compounds.     

Electrical signals propagate unbiased in cortex

The greater spatial coherence of local field potentials (LFPs) compared with that of spiking activity has been attributed to frequency-dependent propagation of signals through the cortical medium. However, in this issue of Neuron, Logothetis and colleagues show that signal propagation within cortex is largely unbiased across different frequencies, thus suggesting a more functional and interpretable basis of LFP coherence.