The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

Background  

The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues.     

Expression of Na+-D-glucose cotransporter SGLT2 in rodents is kidney-specific and exhibits sex and species differences

With a novel antibody against the rat Na(+)-D-glucose cotransporter SGLT2 (rSGLT2-Ab), which does not cross-react with rSGLT1 or rSGLT3, the ～75-kDa rSGLT2 protein was localized to the brush-border membrane (BBM) of the renal proximal tubule S1 and S2 segments (S1 > S2) with female-dominant expression in adult rats, whereas rSglt2 mRNA expression was similar in both sexes. Castration of adult males increased the abundance of rSGLT2 protein; this increase was further enhanced by estradiol and prevented by testosterone treatment. In the renal BBM vesicles, the rSGLT1-independent uptake of [(14)C]-α-methyl-D-glucopyranoside was similar in females and males, suggesting functional contribution of another Na(+)-D-glucose cotransporter to glucose reabsorption. Since immunoreactivity of rSGLT2-Ab could not be detected with certainty in rat extrarenal organs, the SGLT2 protein was immunocharacterized with the same antibody in wild-type (WT) mice, with SGLT2-deficient (Sglt2 knockout) mice as negative control. In WT mice, renal localization of mSGLT2 protein was similar to that in rats, whereas in extrarenal organs neither mSGLT2 protein nor mSglt2 mRNA expression was detected. At variance to the findings in rats, the abundance of mSGLT2 protein in the mouse kidneys was male dominant, whereas the expression of mSglt2 mRNA was female dominant. Our results indicate that in rodents the expression of SGLT2 is kidney-specific and point to distinct sex and species differences in SGLT2 protein expression that cannot be explained by differences in mRNA.     

A Substrate Binding Hinge Domain Is Critical for Transport-related Structural Changes of Organic Cation Transporter 1

Organic cation transporters are membrane potential-dependent facilitative diffusion systems. Functional studies, extensive mutagenesis, and homology modeling indicate the following mechanism. A transporter conformation with a large outward-open cleft binds extracellular substrate, passes a state in which the substrate is occluded, turns to a conformation with an inward-open cleft, releases substrate, and subsequently turns back to the outward-open state. In the rat organic cation transporter (rOct1), voltage- and ligand-dependent movements of fluorescence-labeled cysteines were measured by voltage clamp fluorometry. For fluorescence detection, cysteine residues were introduced in extracellular parts of cleft-forming transmembrane α-helices (TMHs) 5, 8, and 11. Following expression of the mutants in Xenopus laevis oocytes, cysteines were labeled with tetramethylrhodamine-6-maleimide, and voltage-dependent conformational changes were monitored by voltage clamp fluorometry. One cysteine was introduced in the central domain of TMH 11 replacing glycine 478. This domain contains two amino acids that are involved in substrate binding and two glycine residues (Gly-477 and Gly-478) allowing for helix bending. Cys-478 could be modified with the transported substrate analog [2-(trimethylammonium)-ethyl]methanethiosulfonate but was inaccessible to tetramethylrhodamine-6-maleimide. Voltage-dependent movements at the indicator positions of TMHs 5, 8, and 11 were altered by substrate applications indicating large conformational changes during transport. The G478C exchange decreased transporter turnover and blocked voltage-dependent movements of TMHs 5 and 11. [2-(Trimethylammonium)-ethyl]methanethiosulfonate modification of Cys-478 blocked substrate binding, transport activity, and movement of TMH 8. The data suggest that Gly-478 is located within a mechanistically important hinge domain of TMH 11 in which substrate binding induces transport-related structural changes.     

四种独立通风笼具（IVC）的检测

目的比较4种独立通风笼具（individually ventilated cages,IVC）的环境参数。方法参考GB14925-2001的检测方法对分别来自4个厂家生产的4种不同的IVC进行环境检测。结果4种IVC在空气洁净度、落下菌、噪声和照度等4个指标检测数据较为一致;在梯度压差、换气次数、气流速度等3个指标存在较大的差别;而温湿度等指标取决于IVC所处的外部环境。结论目前缺乏国家标准对IVC环境指标进行限定,各厂家所生产的IVC存在差异,需要在此方面加强研究,以促进IVC的标准化,更好的服务于实验动物行业。     

日光温室栽培对杏花及果实生长发育的影响

对日光温室内与露地栽培的金太阳杏的花期物候、花型及果实生长发育进行了系统观察与分析,结果表明,日光温室栽培比露地栽培的始花期提前33d,花期延长4d,不完全花比例上升33.25%;果枝上花的有效性顺序由露地时的中果枝>长果枝>短果枝>花束状果枝变为温室栽培的短果枝>花束状果枝>中果枝>长果枝;日光温室栽培使杏果实发育的第 、 阶段延长,第 阶段缩短,整个生育期延长15d。统计分析认为:日光温室栽培的第 阶段生长速率显著低于露地,第 阶段的累积生长量显著高于露地。较低的夜间温度是造成温室内杏果实第 阶段较长、生长较慢以及果个变大的原因。     

The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 +/- 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP +. ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 +/- 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.