Comparative genomic analysis of dha regulon and related genes for anaerobic glycerol metabolism in bacteria

The dihydroxyacetone (dha) regulon of bacteria encodes genes for the anaerobic metabolism of glycerol. In this work, genomic data are used to analyze and compare the dha regulon and related genes in different organisms in silico with respect to gene organization, sequence similarity, and possible functions. Database searches showed that among the organisms, the genomes of which have been sequenced so far, only two, i.e., Klebsiella pneumoniae MGH 78578 and Clostridium perfringens contain a complete dha regulon bearing all known enzymes. The components and their organization in the dha regulon of these two organisms differ considerably from each other and also from the previously partially sequenced dha regulons in Citrobacter freundii, Clostridium pasteurianum, and Clostridium butyricum. Unlike all of the other organisms, genes for the oxidative and reductive pathways of anaerobic glycerol metabolism in C. perfringens are located in two separate organization units on the chromosome. Comparisons of deduced protein sequences of genes with similar functions showed that the dha regulon components in K. pneumoniae and C. freundii have high similarities (80-95%) but lower similarities to those of the Clostridium species (30-80%). Interestingly, the protein sequence similarities among the dha genes of the Clostridium species are in many cases even lower than those between the Clostridium species and K. pneumoniae or C. freundii, suggesting two different types of dha regulon in the Clostridium species studied. The in silico reconstruction and comparison of dha regulons revealed several new genes in the microorganisms studied. In particular, a novel dha kinase that is phosphoenolpyruvate-dependent is identified and experimentally confirmed for K. pneumoniae in addition to the known ATP-dependent dha kinase. This finding gives new insights into the regulation of glycerol metabolism in K. pneumoniae and explains some hitherto not well understood experimental observations.     

SH—SY5Y细胞的钙缓冲研究

目的：研究SH－SY5Y神经杂交瘤细胞的钙缓冲能力。方法：通过膜片钳手段,测量未分化的SH－SY5Y细胞钙离子通道电流;并应用显微荧光测量游离钙离子浓度和高钾去极化的方法,研究胞内Ca^2 浓度上升后浓度恢复的动力学过程。结果：未分化的SH－SY5Y细胞存在钙离子通道电流,在刺激时间间隔较短时（＜150s）,胞内钙浓度的恢复过程会由于缓冲机制的饱和而变慢;而时间间隔＞150s时,缓冲物质则可以基本恢复使得胞内钙的恢复过程基本保持不变。结论：钙缓冲蛋白在细胞内钙浓度的调节中起重要作用。     

Dynamic behavior and selective adsorption of a methanol/water mixture inside a cyclic peptide nanotube

Present molecular dynamics simulations indicate that the methanol component in a methanol/water mixture is more likely to be trapped in a cyclic peptide nanotube (CPNT), while water molecules tend to be present at the channel mouths as transient guests. Channel water resides mainly between methanol and the CPNT wall, resulting in a distinct decrease in the H-bond number per channel methanol. Six designed CPNTs with different channel diameters and outer surface characteristics all possess distinct selectivity to methanol over water. Of these, the amphipathic 8?×?(AQ)₄-CPNT exhibits the best performance. Results in this study provide basic information for the application of a CPNT to enrich methanol from a methanol/water mixture.

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Graphical Abstract Typical overview of water and methanol molecular distribution in cyclic peptide nanotubes

     

北黄海沉积物可培养产蛋白酶细菌分离鉴定

【目的】揭示北黄海沉积物中可培养产胞外蛋白酶细菌及蛋白酶多样性,增加人们对北黄海生态系统中产蛋白酶菌多样性的认识,为海洋产蛋白酶微生物的挖掘提供菌群资源。【方法】分别将5个北黄海沉积物样品梯度稀释涂布至酪蛋白明胶筛选平板,选择性分离产蛋白酶细菌;并通过分析基于16S rRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;分别测定胞外蛋白酶活性并对酶活较高的39株菌进行基于苯甲基磺酰氟(PMSF,丝氨酸蛋白酶抑制剂)、邻菲罗啉(o-phenanthroline,O-P,金属蛋白酶抑制剂)、E-64(半胱氨酸蛋白酶抑制剂)和pepstatin A(天冬氨酸蛋白酶抑制剂)4种抑制剂的酶活抑制实验以及所有菌株对3种底物(酪蛋白、明胶、弹性蛋白)的水解能力;分析这些细菌所产胞外蛋白酶的特性及多样性。【结果】从5个北黄海沉积物样品中分离获得66株产蛋白酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria、Actinobacteria和Firmicutes 4个门的7个属,其中Pseudoalteromonas(69.9%)、Sulfitobacter(12.1%)和Salegentibacter(10.6%)是优势菌群;沉积物中可培养的产蛋白酶细菌的丰度为104 CFU/g;蛋白酶酶活抑制实验表明所有测定菌株产生的胞外蛋白酶属于丝氨酸蛋白酶和/或金属蛋白酶,仅有少数菌株所产蛋白酶具有半胱氨酸蛋白酶或天冬氨酸蛋白酶活性。【结论】北黄海沉积物中可培养产蛋白酶细菌类群较为丰富,Pseudoalteromonas、Sulfitobacter和Salegentibacter菌株是优势菌群,测定菌株所产胞外蛋白酶主要是丝氨酸蛋白酶和/或金属蛋白酶。     

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model

We describe a procedure to construct an artificial corneal epithelium from cryopreserved limbal stem cells (LSCs) for corneal transplantation. The LSCs were separated from limbal tissue of male goats. The primary LSCs were identified by flow cytometry and were expanded. They were examined for stem cell-relevant properties and cryopreserved in liquid nitrogen. Cryopreserved LSCs were thawed and then transplanted onto human amniotic membrane, framed on a nitrocellulose sheet, to construct corneal epithelium sheets. The artificial corneal epithelium was transplanted into the right eye of pathological models of total limbal stem cell deficiency (LSCD). Then, the effects of reconstruction were evaluated by clinical observation and histological examination. Polymerase chain reaction analysis was used to detect the SRY gene. The data showed that transplantation of cryopreserved LSCs, like fresh LSCs, successfully reconstructed damaged goat corneal surface gradually, but the SRY gene expression from male goat cells could only be detected in the first 2 months after transplantation. The therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of cryopreserved LSCs. This study provides the first line of evidence that cryopreserved LSCs can be used for reconstruction of damaged corneas, presenting a remarkable potential source for transplantation in the treatment of corneal disorders.     

Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux

The NOD-like receptor family, pyrin domain–containing protein 3 (NLRP3) inflammasome plays an important role in the development of atherosclerosis. The activated NLRP3 inflammasome has been reported to promote macrophage foam cell formation, but not all studies have obtained the same result, and how NLRP3 inflammasome is involved in the formation of foam cells remains elusive. We used selective NLRP3 inflammasome inhibitors and NLRP3-deficient THP-1 cells to assess the effect of NLRP3 inflammasome inhibition on macrophage foam cell formation, oxidized low-density lipoprotein (ox-LDL) uptake, esterification, and cholesterol efflux, as well as the expression of associated proteins. Inhibition of the NLRP3 inflammasome attenuated foam cell formation, diminished ox-LDL uptake, and promoted cholesterol efflux from THP-1 macrophages. Moreover, it downregulated CD36, acyl coenzyme A: cholesterol acyltransferase-1 and neutral cholesterol ester hydrolase expression; upregulated ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression; but had no effect on the expression of scavenger receptor class A and ATP-binding cassette transporter G1. Collectively, our findings show that inhibition of the NLRP3 inflammasome decreases foam cell formation of THP-1 macrophages via suppression of ox-LDL uptake and enhancement of cholesterol efflux, which may be due to downregulation of CD36 expression and upregulation of ABCA1 and SR-BI expression, respectively.     

定向进化技术的最新进展

筛选是制约酶定向进化改造的瓶颈。为解决这一难题,近年来一系列基于组合活性中心饱和突变(Combinatorial active-site saturation test,CAST)及迭代饱和突变(Iterative saturation mutagenesis,ISM)的半理性设计新方法被开发出来,包括单密码子饱和突变(Single code saturation mutagenesis,SCSM)、双密码子饱和突变(Double code saturation mutagenesis,DCSM)和三密码子饱和突变(Triple code saturation mutagenesis,TCSM)。通过构建"小而精"的高质量突变体文库,对特定靶点进行组合突变,并成功应用于多种生物催化剂的立体/区域选择性及催化活力等多参数的改造。文中综述了近年来定向进化技术的最新进展及其在生物催化剂定向改造中的应用。     

蛋白G IgG Fc段结合域的克隆表达及功能研究

旨在研究蛋白G IgG Fc段结合域(PGFB)的克隆、表达及其抗体结合功能,用于抗体的纯化.根据PGFB的氨基酸序列,选择大肠杆菌偏爱的密码子,设计并合成了4个寡核苷酸片段.通过重叠延伸PCR方法合成了PGFB DNA片段,测序鉴定后克隆至原核表达系统pET-28a-c(+)上,转化大肠杆菌,获得表达菌株;IPTG诱导表达PGFB,经Ni+-NTA琼脂糖凝胶层析纯化后偶联到琼脂糖凝胶6B上,用其纯化多克隆抗体.结果显示,PGFB在大肠杆菌BL21(DE3)中获得高效表达,纯化后纯度达到90%以上,相对分子量为12.25 kD,与预期值相符.此外,偶联产物纯化多克隆抗体达到了良好的效果,每毫升基质可结合20 mg抗体.本研究克隆构建并高效表达了具有较好抗体亲和能力的PGFB,为多克隆抗体的快速纯化提供了方便.     

Nitrite uptake and metabolism and oxidant stress in human erythrocytes

Nitric oxide, when released into the bloodstream, is quicklyscavenged by Hb in erythrocytes or oxidized to nitrite. Nitrite canalso enter erythrocytes and oxidize Hb. The goals of this work were todetermine the mechanism of erythrocyte nitrite uptake and whether thisuptake causes oxidant stress in these cells. Erythrocytes took up 0.8 mM nitrite with a half-time of 11 min. Nitrite uptake was sensitive totemperature and to the pH and ionic composition of the medium but wasnot inhibited by the specific anion-exchange inhibitor DIDS. About 25%of nitrite uptake occurred on the sodium-dependent phosphatetransporter and the rest as diffusion of nitrous acid or other speciesacross the plasma membrane. Methemoglobin formation increased inproportion to the intracellular nitrite concentration. Nitritereacted with erythrocyte ascorbate, but ascorbate loading of cellsdecreased nitrite-induced methemoglobin formation only at high nitriteconcentrations. In conclusion, nitrite rapidly enters erythrocytes andreacts with oxyhemoglobin but does not exert a strong oxidant stress onthese cells.

     

Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection

In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heme to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knockout mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.1–2.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-B nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate-induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-A1 (50 µM), and by bilirubin (1 µM), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stress-related glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium. endothelium; carbon monoxide; bilirubin; injury; reactive oxygen species; heme oxygenase