间断低氧对大鼠下丘脑超微结构及前增食欲素水平的影响

目的探讨睡眠中间断低氧对大鼠下丘脑前增食欲素及受体水平的影响以及下丘脑超微结构的变化。方法大鼠分成对照组、间断低氧组和持续低氧组,分别给予吸入空气,持续低氧和间断低氧气体,并在实验开始后1d、3d、1w和4w应用RT-PCR方法测定大鼠下丘脑前增食欲素及受体水平,分析其间的变化关系,电镜观察下丘脑的超微结构变化。结果与对照组和持续低氧组比较,间断低氧4w后大鼠下丘脑前增食欲素mRNA水平明显降低,受体水平升高,但在持续低氧和对照组之间无明显差异。在低氧后1d、3d、7d后大鼠下丘脑前增食欲素mRNA降低,受体水平升高,在4w后,持续低氧组则接近正常。急性持续低氧大鼠超微结构变化更严重,而慢性间断低氧变化更持久。结论慢性间断低氧可以引起下丘脑前增食欲素下降及受体水平升高,急性持续低氧也可引起上述变化,而慢性持续低氧未引起增食欲素改变;慢性间断低氧大鼠下丘脑超微结构表现为严重而持久的变化。     

Diversity surveys and evolutionary relationships of aoxB genes in aerobic arsenite-oxidizing bacteria

A new primer set was designed to specifically amplify ca. 1,100 bp of aoxB genes encoding the As(III) oxidase catalytic subunit from taxonomically diverse aerobic As(III)-oxidizing bacteria. Comparative analysis of AoxB protein sequences showed variable conservation levels and highlighted the conservation of essential amino acids and structural motifs. AoxB phylogeny of pure strains showed well-discriminated taxonomic groups and was similar to 16S rRNA phylogeny. Alphaproteobacteria-, Betaproteobacteria-, and Gammaproteobacteria-related sequences were retrieved from environmental surveys, demonstrating their prevalence in mesophilic As-contaminated soils. Our study underlines the usefulness of the aoxB gene as a functional marker of aerobic As(III) oxidizers.     

Surface-enhanced resonance Raman spectroscopy and spectroscopy study of redox-induced conformational equilibrium of cytochrome c adsorbed on DNA-modified metal electrode

The redox-induced conformational equilibrium of cytochrome c (cyt c) adsorbed on DNA-modified metal electrode and the interaction mechanism of DNA with cyt c have been studied by electrochemical, spectroscopic and spectroelectrochemical techniques. The results indicate that the external electric field induces potential-dependent coordination equilibrium of the adsorbed cyt c between its oxidized state (with native six-coordinate low-spin and non-native five-coordinate high-spin heme configuration) and its reduced state (with native six-coordinate low-spin heme configuration) on DNA-modified metal electrode. The strong interactions between DNA and cyt c induce the self-aggregation of cyt c adsorbed on DNA. The orientational distribution of cyt c adsorbed on DNA-modified metal electrode is potential-dependent, which results in the deviation from an ideal Nernstian behavior of the adsorbed cyt c at high electrode potentials. The electric-field-induced increase in the activation barrier of proton-transfer steps attributed to the rearrangement of the hydrogen bond network and the self-aggregation of cyt c upon adsorption on DNA-modified electrode strongly decrease the interfacial electron transfer rate. In addition, the strongly Coulombic interactions between DNA and cyt c only disturb the microenvironment of the heme, and do not affect the states of heme ligation and spin. The secondary structure of the adsorbed cyt c is retained, while the conformation of DNA is changed from the B form DNA to A form DNA.     

应用基因敲除快速构建大肠杆菌突变体改造脂肪酸代谢途径

【目的】基因敲除技术是研究基因功能的重要手段。我们试图建立一种快速、高效的大肠杆菌基因敲除方法。【方法】利用大肠杆菌(Escherichia coli)BW25113单基因缺失体Keio文库,将经典的Red同源重组技术与P1噬菌体转导技术相结合,对E.coli MG1655脂肪酸代谢基因进行快速敲除。【结果】获得了大肠杆菌β-氧化途径的缺失菌株△fadD、△fadE和△fadD-△fadE;脂肪酸合成途径缺失菌株△fabH、△fabF和△fabH-△fabF。敲除fadD和fadE对生长情况没有影响;敲除fabH后,生长速度明显减慢;敲除fabF对生长几乎没有影响。FadD、FadE及双敲缺失体的脂肪酸含量18.2 mg/L、20.0mg/L和19.2 mg/L,略高于野生型17.5 mg/L;FabH、FabF及双敲缺失体的含量分别为12.6 mg/L、15.2 mg/L和11.2 mg/L,明显低于野生型。【结论】在单基因突变体文库基础上,利用P1噬菌体转导、Red同源重组和抗性基因消除进行基因敲除,简化了构建大肠杆菌单基因和多重突变体的方法。     

Genetic and alkaloid analysis of Menispermum dauricum DC. by RAPD and HPLC

The aim of the present work was to determine whether dauricine could be used as a taxonomic marker for Menispermum dauricum DC., and to explore the correlation among RAPD, ecological markers and chemical markers. To this end, the chemical and genetic differences of 173 individual samples of M. dauricum from nine different sources were studied based on the relevant ecological factors including longitude, latitude, annual precipitation, mean temperature, annual accumulated temperature and mean sea level. The contents of dauricine in the sample rhizomes were assayed by HPLC with photodiode array detection. The leaves from the same sample were assayed using randomly amplified polymorphism DNA (RAPD). The genetic distances were then compared. Hierarchical cluster analysis and multiple linear stepwise regression analysis were used in the statistical analysis. The results indicated that the contents of dauricine were respectively correlated with the genetic distance (r = 1.000), longitude (r = 0.849), latitude (r = 0.861), annual precipitation (r = 0.903), mean temperature(r = 0.912), annual accumulated temperature (r = 0.919) and mean sea level (r = 0.925). It is concluded that the content of dauricine in M. dauricum is significantly correlated with genetic distance and ecological factors, and may be used as the taxonomic marker.     

Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115

Human serum albumin (HSA) and human parathyroid hormone (1-34) [PTH (1-34)] fusion protein [HSA/PTH (1-34)] is a promising long-acting form of PTH (1-34) for osteoporosis treatment. Secretory expression of intact HSA/PTH (1-34) in Pichia pastoris GS115 was accompanied by two degradation fragments, with molecular weights around 66 kDa, in addition to the well-known ~45 kDa HSA-truncated fragment, resulting in a low yield of intact protein. In this study, two internal cleavage sites were identified in the PTH (1-34) portion of the fusion protein by Western Blot analysis. To minimize proteolytic cleavages, several protease genes including PEP4 (encoding proteinase A), PRB1 (proteinase B) and seven YPSs genes (yapsin family members) were knocked out respectively by disruption of the individual genes and the selective combinations. Reduced degradation was observed by single disruption of either PEP4 gene or YPS1 gene, and the lowest level of degradation was observed in a pep4△yps1△ double disruptant. After 72 h of induction, more than 80 % of the HSA/PTH (1-34) secreted by the pep4△yps1△ double disruptant remained intact, in comparison to only 30 % with the wild-type strain.     

Gating of inward rectifier K+ channels by proton-mediated interactions of N- and C-terminal domains

Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO(2)/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO(2) and pH(i). These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K(+) channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator.     

Enhanced chemiluminescence enzyme‐linked immunoassay for the determination of DNA methyltransferase 1 in human serum

The occurrence of many diseases is closely related to the high expression of DNA methyltransferase 1 (DNMT1). However, most studies are focused on the detection of DNMT1 activity, a few are concerned with the detection of DNMT1 content. In this study, we developed a simple and highly sensitive chemiluminescence (CL) assay for the detection of DNMT1 content. In this method, anti‐DNMT1 monoclonal antibody was coated on a polystyrene microplate to capture DNMT1. Then anti‐DNMT1 polyclonal antibody and goat anti‐rabbit immunoglobulin G with horseradish peroxidase (IgG‐HRP) were respectively added to combine with captured DNMT1 to form a sandwich structure. Finally, the HRP could catalyze CL substrate and achieve CL signal response. Based on this novel sensitive strategy, the recovery percents were in the ranges from 71.5% to 91.0%. The precision of intra‐assays and inter‐assays were 5.45%–11.29% and 7.03%–11.25%, respectively. The method was successfully applied for the determination of DNMT1 in human serum. The detection results of serum samples showed that the proposed assay had a high correlation with enzyme‐linked immunosorbent assay (ELISA) kit. Compared with the ELISA kit (limit of detection = 0.1 ng/mL), the method has a lower limit of detection of 0.042 ng/mL. Therefore, our method has the potential for the detection of DNMT1 content in clinical diagnosis.     

黄瓜钝绥螨对茶黄螨雌成螨和腐食酪卵的功能反应

研究黄瓜钝绥螨Amblyseius cucumeris 对茶黄螨Polyphagotarsonemus latus (Banks)雌成螨和腐食酪螨Tyrophagus putrescentiae卵的功能反应。结果表明,黄瓜钝绥螨的第1若螨,第2若螨,雌成螨捕食茶黄螨雌成螨和腐食酪螨卵的功能反应均属于Holling II型,其中,雌成螨的捕食能力最强,对腐食酪螨卵和对茶黄螨雌成螨的攻击系数a大,处理时间th短,第2若螨也具有较强的捕食能力,对静态的腐食酪螨卵比对动态的茶黄螨捕食能力强,黄瓜钝绥螨对茶黄螨雌成螨具有很强的捕食能力。     

Activated PAK4 regulates cell adhesion and anchorage-independent growth

The serine/threonine kinase PAK4 is an effector molecule for the Rho GTPase Cdc42. PAK4 differs from other members of the PAK family in both sequence and function. Previously we have shown that an important function of this kinase is to mediate the induction of filopodia in response to activated Cdc42. Since previous characterization of PAK4 was carried out only with the wild-type kinase, we have generated a constitutively active mutant of the kinase to determine whether it has other functions. Expression of activated PAK4 in fibroblasts led to a transient induction of filopodia, which is consistent with its role as an effector for Cdc42. In addition, use of the activated mutant revealed a number of other important functions of this kinase that were not revealed by studying the wild-type kinase. For example, activated PAK4 led to the dissolution of stress fibers and loss of focal adhesions. Consequently, cells expressing activated PAK4 had a defect in cell spreading onto fibronectin-coated surfaces. Most importantly, fibroblasts expressing activated PAK4 had a morphology that was characteristic of oncogenic transformation. These cells were anchorage independent and formed colonies in soft agar, similar to what has been observed previously in cells expressing activated Cdc42. Consistent with this, dominant-negative PAK4 mutants inhibited focus formation by oncogenic Dbl, an exchange factor for Rho family GTPases. These results provide the first demonstration that a PAK family member can transform cells and indicate that PAK4 may play an essential role in oncogenic transformation by the GTPases. We propose that the morphological changes and changes in cell adhesion induced by PAK4 may play a direct role in oncogenic transformation by Rho family GTPases and their exchange factors.