Quantitative determination of helicid in rat biosamples by liquid chromatography electrospray ionization mass spectrometry

A simple liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS) method with highly improved sensitivities for the determination of helicid in rat bile, urine, feces and most tissues was developed. The tissues and feces were firstly homogenized mechanically using deionized water as the media. Bile, urine, tissues and feces homogenates were extracted by liquid–liquid extraction with n-butyl alcohol for sample preparation. The subsequent analysis procedures were performed on a Shimadzu LCMS2010A system (electrospray ionization single quadrupole mass analyzer). A Luna C₁₈ column (150 mm × 2.00 mm, 5 μm) was used as the analytical column, while a mixture of acetonitrile and ammonium chloride water solution was used as the mobile phase. The proportions of mobile phase were changed timely according to gradient programs. Chlorinated adducts of molecular ions [M+Cl]^? at m/z 319.00 and 363.05 were used to quantify helicid and bergeninum (internal standard), respectively. The method was validated to be accurate, precise and rugged with good linearity. The proposed method was successfully applied to the preclinical tissue distribution and excretion studies of helicid in rats.     

Analysis of KaiA-KaiC protein interactions in the cyano-bacterial circadian clock using hybrid structural methods

The cyanobacterial circadian clock can be reconstituted in vitro by mixing recombinant KaiA, KaiB and KaiC proteins with ATP, producing KaiC phosphorylation and dephosphorylation cycles that have a regular rhythm with a ca. 24-h period and are temperature-compensated. KaiA and KaiB are modulators of KaiC phosphorylation, whereby KaiB antagonizes KaiA's action. Here, we present a complete crystallographic model of the Synechococcus elongatus KaiC hexamer that includes previously unresolved portions of the C-terminal regions, and a negative-stain electron microscopy study of S. elongatus and Thermosynechococcus elongatus BP-1 KaiA-KaiC complexes. Site-directed mutagenesis in combination with EM reveals that KaiA binds exclusively to the CII half of the KaiC hexamer. The EM-based model of the KaiA-KaiC complex reveals protein-protein interactions at two sites: the known interaction of the flexible C-terminal KaiC peptide with KaiA, and a second postulated interaction between the apical region of KaiA and the ATP binding cleft on KaiC. This model brings KaiA mutation sites that alter clock period or abolish rhythmicity into contact with KaiC and suggests how KaiA might regulate KaiC phosphorylation.     

Comparative proteome analysis of Aspergillus oryzae 3.042 and A. oryzae 100-8 strains: Towards the production of different soy sauce flavors

Aspergillus oryzae plays a central role in soybean fermentation, particularly in its contribution to the flavor of soy sauce. We present a comparative assessment of the intracellular differences between wild-type strain 3.042 and mutant strain A100-8, at the proteome level. 522 different protein spots were identified by MALDI-TOF MS, with 134 spots being confirmed by MALDI-TOF MS/MS. Of these, 451 were differentially expressed proteins (DEPs). There was at least a two-fold increase for 288 spots, and at least a two-fold decrease for 163 spots, in strain A100-8 when compared to 3.042. Further analysis showed that 63 of the more abundant proteins were involved in glycolysis and the citrate cycle; 43 more abundant proteins and 10 less abundant proteins were related to amino acid biosynthesis and metabolism; two of the more abundant proteins were involved in vitamin biosynthesis; and five of the more abundant proteins and four of the less abundant proteins were related to secondary metabolites. Moreover, quantitative real time PCR showed that the mRNA expression levels of six typical genes we selected were consistent with changes in protein expression. We postulate that there may be a relationship between DEPs and the flavor formation mechanism in A. oryzae.     

含甲肝病毒基因的重组痘苗病毒在动物体内产生针对甲肝病毒的中和抗体

用DNA重组技术得到的含甲肝病毒基因的重组痘苗病毒,可在家兔体内产生ELISA竞争抑制与中和抗体。基础免疫后,动物体内竞争抑制抗体滴度为10,加强免疫后达到80。由重组病毒产生的抗体中和指数比甲肝病毒产生者略低。     

共表达甲型流感病毒M1和HA抗原的重组杆状病毒的构建

为构建同时表达流感病毒M1和HA抗原的重组杆状病毒,采用PCR扩增流感病毒A/PR/8/34株的M1基因和去除信号肽的HA基因,将两基因克隆到杆状病毒转座载体pFastBac Dual的两个启动子下游的多克隆位点,筛选出阳性重组转座载体pFastBac Dual-M1-HA。将其转化含有杆状病毒穿梭载体(Bacmid)的DH10Bac感受态细胞,通过抗生素、蓝白斑筛选和PCR鉴定获得重组杆状病毒穿梭载体rBacmid-M1-HA,在脂质体介导下转染Sf9昆虫细胞,获得重组杆状病毒rBac-M1-HA。提取重组病毒基因组,通过PCR鉴定外源基因插入成功。间接免疫荧光和Western-blot检测表明,该重组杆状病毒在Sf9昆虫细胞中成功地表达了M1和HA。应用杆状病毒/昆虫细胞系统成功共表达流感病毒M1和HA抗原,为研究流感病毒VLP的形成机制和开发新型流感疫苗奠定了基础。     

代谢型谷氨酸受体在突触可塑性中的作用研究进展

突触可塑性是近 30年来神经科学领域的研究热点之一 ,它主要包括长时程增强 (long termpotentiation ,LTP)和长时程抑制 (long termdepression ,LTD)。以往的研究已经证实 ,离子型谷氨酸受体 (iGluRs)中的NMDA受体和AMPA受体 ,在LTP和LTD的诱导和维持中通过阳离子内流 ,引起细胞内的级联反应而起作用。新近的研究发现 ,代谢型谷氨酸受体 (mGluRs)与G蛋白偶联 ,通过细胞内的多种信使系统介导慢突触传递。本文主要就mGluRs在不同脑区LTP和LTD中的作用进行综述     

腔内大量盐水灌洗辅助取出乳房内聚丙烯酰胺水凝胶临床应用

目的：探讨聚丙烯酰胺水凝胶注射隆胸后取出新方法.方法：对48例注射聚丙烯酰胺水凝胶隆胸患者术前行双乳MRI或彩超检查结合触诊准确定位.全麻小切口开放直视下吸出水凝胶,再用大量生理盐水反复灌洗所有腔隙,直至手感探测不到硬结,盐水中无水凝胶为止.结果：48例患者术后无继发感染和出血等并发症.术前诸症状体征消失,无明显乳房变形,乳房形态术后恢复满意.术后双乳MRI复查未见明显异物残留.术后1 ～12月（6.2±0.3月）45例随访复查MRI亦未见异物残留,乳房修复完好.结论：注射隆乳后腔内大量盐水灌洗辅助取出水凝胶具有创伤小、出血少、操作简单、费用低廉的优点,是一种较好的、值得推广的凝胶取出术式.     

Alpha-melanocyte stimulating hormone protects retinal pigment epithelium cells from oxidative stress through activation of melanocortin 1 receptor–Akt–mTOR signaling

Patients with age related macular degeneration (AMD) will develop vision loss in the center of the visual field. Reactive oxygen species (ROS)-mediated retinal pigment epithelium (RPE) cell apoptosis is an important contributor of AMD. In this study, we explored the pro-survival effect of α-melanocyte stimulating hormone (α-MSH) on oxidative stressed RPE cells. We found that α-MSH receptor melanocortin 1 receptor (MC1R) was functionally expressed in primary and transformed RPE cells. RPE cells were response to α-MSH stimulation. α-MSH activated Akt/mammalian target of rapamycin (mTOR) and Erk1/2 signalings in RPE cells, which were inhibited by MC1R siRNA knockdown. α-MSH protected RPE cells from hydrogen peroxide (H₂O₂)-induced apoptosis, an effect that was almost abolished when MC1R was depleted by siRNA. α-MSH-mediated S6K1 activation and pro-survival effect against H₂O₂ was inhibited by Akt inhibitors (perifosine, MK-2206 and LY294002). Further, mTOR inhibition by rapamycin, or by mTOR siRNA knockdown, diminished α-MSH’s pro-survival effect in RPE cells. Thus, Akt and its downstream mTOR signaling mediates α-MSH-induced survival in RPE cells. In summary, we have identified a new α-MSH–MC1R physiologic pathway that reduces H₂O₂-induced RPE cell damage, and might minimize the risk of developing AMD.     

Leishmania major LmACR2 is a pentavalent antimony reductase that confers sensitivity to the drug pentostam

Arsenicals and antimonials are first line drugs for the treatment of trypanosomal and leishmanial diseases. To create the active form of the drug, Sb(V) must be reduced to Sb(III). Because arsenic and antimony are related metalloids, and arsenical resistant Leishmania strains are frequently cross-resistant to antimonials, we considered the possibility that Sb(V) is reduced by a leishmanial As(V) reductase. The sequence for the arsenate reductase of Saccharomyces cerevisiae, ScAcr2p, was used to clone the gene for a homologue, LmACR2, from Leishmania major. LmACR2 was able to complement the arsenate-sensitive phenotype of an arsC deletion strain of Escherichia coli or an ScACR2 deletion strain of Saccharomyces cerevisiae. Transfection of Leishmania infantum with LmACR2 augmented Pentostam sensitivity in intracellular amastigotes. LmACR2 was purified and shown to reduce both As(V) and Sb(V). This is the first report of an enzyme that confers Pentostam sensitivity in intracellular amastigotes of Leishmania. We propose that LmACR2 is responsible for reduction of the pentavalent antimony in Pentostam to the active trivalent form of the drug in Leishmania.