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.     

芒果APl同源基因的克隆及其生物信息学分析

我们采用RT-PCR方法克隆了2个APl同源基因全长cDNA,分别命名为MAPl-1(GenBank accession No.FJ529206)和MAPl-2(GenBank accession No.FJ529207).MAPl-1编码247个氨基酸,开放阅读框长度为741 bp,蛋白质分子量为28.54kD,等电点为8.31;MAPl-2编码248个氨基酸,开放阅读框长度为744 bp,蛋白质分子量为28.78 kD,等电点为8.70.同源性分析表明,它们的核苷酸序列与其它木本植物APl同源基因的一致性为72%～81%.实验分析表明,MAPl-1和MAPl-2第1至第61个氨基酸含有一个MADS盒结构域,第88至第178个为K盒结构域;两个基因均定位于细胞核,且功能位点分布存在着不同,推测这两个基因在花器官发育过程中的功能存在差异.蛋白二级结构预测显示,MAPl-1蛋白有12个a-螺旋,4个β折叠区,14个β-转角;而MAPl-2蛋白有11个a-螺旋,5个β折叠区,15个β-转角:其大多数氨基酸具有亲水性.本研究有助于进一步了解芒果的开花分子机理及成花的生物学发育阶段.     

小溪自然保护区非盐环境土壤中嗜盐和耐盐菌多样性

【目的】研究湖南小溪国家级自然保护区普通非盐环境(ordinary non-saline environment)土壤样品中可培养嗜盐及耐盐细菌(含放线菌)多样性。【方法】采用纯培养法和基于16S rRNA基因序列的系统发育分析对样品中嗜盐及耐盐细菌多样性进行研究。【结果】用补充5%-20%(w/v)NaCl的MA、ISP2、ISP5、NA和HAA培养基从土壤样品中分离到114株细菌,其中8株为中度嗜盐菌,19株为轻度嗜盐菌,87株为耐盐菌。根据形态观察和部分生理生化实验结果去冗余,选取61个代表性菌株进行基于16S rRNA基因序列的系统发育多样性分析。结果表明,这些菌株属于细菌域(Bacteria)的3个大的系统发育类群(门;phylum)(Actinobacteria,Firmicutes,Proteobacteria)的16个科、18个属,代表了41个物种。多数菌株属于Firmicutes门(38株,62.3%)和Actinobacteria门(18株,29.5%)。大多数菌株与其系统发育关系最密切的已知物种的典型菌株之间存在一定的遗传差异(16S rRNA基因序列相似性为96.9%-99.8%),其中有7个菌株(JSM070026,JSM081004,JSM081006,JSM081008,JSM083058,JSM083085,JSM084035)代表7个潜在新种(potential novel species)。【结论】研究结果表明,湖南小溪国家级自然保护区普通非盐环境土壤中存在较为丰富的可培养嗜盐及耐盐细菌多样性,并且潜藏着较多新的微生物类群(物种)。     

西双版纳热带雨林中两种生态型蕨类植物的光合特性比较研究

通过比较分布于西双版纳热带雨林林下生境中的附生鸟巢蕨（Neottopteris nidus）和地生网脉铁角蕨（Asplenium finlaysonianum）的光合特征和光合诱导特性,来研究不同生态型蕨类植物的光斑利用策略。研究结果表明,2种蕨类植物的最大净光合速率、暗呼吸速率、表观量子效率、光饱和点和光补偿点没有显著差异,但网脉铁角蕨的最大气孔导度远远高于鸟巢蕨,表明后者具有更强的光合水分利用效率。在暗处理3／J＇,时接着光照（光强为20I~mol-m-2,s。‘）30分钟后,网脉铁角蕨的初始气孔导度显著高于鸟巢蕨。连续照射饱和强光后,网脉铁角蕨达到最大净光合速率50％（T50％）和90％的时间（T90%）比鸟巢蕨短：网脉铁角蕨和鸟巢蕨的T50%分别为0．57和5．31分钟,T90％分别为5．85和26．33分钟。诱导过程中,气孔导度对强光的响应明显滞后于净光合速率。鸟巢蕨达到最大气孔导度的时间明显比网脉铁角蕨慢,但在光合诱导消失过程中2种蕨类植物的光合诱导维持能力却没有显著差异。上述结果表明,与大多数地生林下植物（如网脉铁角蕨）相比,附生鸟巢蕨的水分保护比碳获得更重要,但却限制了附生蕨对光斑的利用。     

两种热带木质藤本幼苗形态、生长和光合能力对光强和养分的响应

比较了两种不同攀援习性,卷须缠绕种薄叶羊蹄甲（Bauhinia tenuiflora）和茎缠绕种刺果藤（Byttneria aspera）,木质藤本植物的形态、生长及光合特性对不同光强（4％、35％和全光照）和土壤养分（高和低）的响应。两种藤本植物大部分表型特征主要受光照的影响,而受土壤养分的影响较小。弱光促进地上部分生长,弱光下两种植物均具有较大的比叶面积（specific leaf area,SLA）、茎生物量比（stem mass ratio,SMR）和平均叶面积比（mean leaf area ratio,LARm）。高光强下,两种植物的总生物量和投入到地下部分的比重增加,具有更大的根生物量比（root mass ratio,RMR）、更多的分枝数、更高的光合能力（maximum photosynthetic rate,Pmax）和净同化速率（net assimilation rate,NAR）,综合表现为相对生长速率（relative growth rate,RGR）增加。两种藤本植物的Pmax与叶片含氮量的相关性均未达显著水平,但刺果藤的Pmax与SU志间呈显著的正相关,而薄叶羊蹄甲的Pmax与SLA之间相关性不显著。在相同光照强度和土壤养分条件下,卷须缠绕种薄叶羊蹄甲的RGR显著高于茎缠绕种刺果藤。薄叶羊蹄甲的RGR与NAR呈显著正相关,其RGR与SLA、平均叶面积比（EARm）及Pmax之间相关性不显著。刺果藤的RGR与NAR呈显著的正相关,而与SLA存在显著的负相关。上述结果表明,与土壤养分相比,光照强度可能是决定木质藤本分布更为重要的生态因子。卷须缠绕种薄叶羊蹄甲由于具有特化的攀援器官,在形态上和生理上具有更大的可塑性,这使得卷须缠绕种木质藤本在与其它植物的竞争中更具优势。     

Identification of a new mutation in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

A mutation involving an A-to-G nucleotide replacement at position 985 of the medium-chain acyl-CoA dehydrogenase (MCAD) cDNA was found in homozygous form in 18 unrelated MCAD-deficient families and in heterozygous form in 4 families. By PCR amplification and sequencing of cDNA from a compound heterozygote, we have detected a new mutation in an MCAD-deficient patient in whom one MCAD allele produces mRNA that is missing 4 bp in the MCAD cDNA, while the other allele carries the A-to-G-985 mutation. The presence of this 4-bp deletion was confirmed in the patient's genomic DNA by dot-blot hybridization with allele-specific oligonucleotide probes and by restriction analysis of PCR products. A rapid screening test for this 4-bp deletion was developed, based on mismatched primer PCR amplification. The deletion created a new restrictive-enzyme site which yielded two DNA fragments. The 4-bp deletion was not found in the three remaining MCAD chromosomes not harboring the A-to-G-985 mutation, nor it was present in 20 chromosomes from 10 unrelated normal Caucasians. The PCR-based method for screening these two mutations can detect over 93% of all MCAD mutations.     

NOX4-Derived Reactive Oxygen Species Drive Apelin-13-Induced Vascular Smooth Muscle Cell Proliferation via the ERK Pathway

Apelin is the endogenous ligand of the G-protein-coupled receptor, apelin–angiotensin receptor-like 1 (APJ). Vascular smooth muscle cells express both apelin and APJ, which are important regulatory factors in the cardiovascular system. Apelin-13 significantly stimulated vascular smooth muscle cell proliferation. However, little is known about the precise cellular mechanisms responsible for vascular smooth muscle cell proliferation induced by apelin-13. Here, we present novel data that indicate the key role of NADPH oxidase 4-derived reactive oxygen species in proliferation of vascular smooth muscle cells treated with apelin-13. Apelin-13 stimulated reactive oxygen species production in a concentration- and time-dependent manner. Furthermore, DPI impaired apelin-13-induced reactive oxygen species generation and vascular smooth muscle cell proliferation. Apelin-13-treatment increased the expression of NADPH oxidase 4 in a dose-dependent manner. Down-regulation of NADPH oxidase 4 using siRNA prevented apelin-13-induced reactive oxygen species generation and vascular smooth muscle cell proliferation. An increase in reactive molecules can trigger the activation of ERK stress-sensitive signaling pathways. Additionally, siRNA-NOX4 and DPI reversed the phosphorylation of ERK induced by apelin-13. Apelin-13 induced vascular smooth muscle cell proliferation by NOX4-derived ROS via the ERK signaling pathway.     

Aptamer selection for the detection of Escherichia coli K88

In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.     

The influence of dietary lipid composition on liver mitochondria from mice following 1 month of calorie restriction

To investigate the role mitochondrial membrane lipids play in the actions of CR (calorie restriction), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and the CR groups were fed diets containing soya bean oil (also in the control diet), fish oil or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak and H₂O₂ production were measured in liver mitochondria following 1 month of CR. The results indicate that mitochondrial phospholipid fatty acids reflect the PUFA (polyunsaturated fatty acid) profile of the dietary lipid sources. CR significantly decreased the capacity of ROS (reactive oxygen species) production by Complex III but did not markedly alter proton leak and ETC (electron transport chain) enzyme activities. Within the CR regimens, the CR-fish group had decreased ROS production by both Complexes I and III, and increased proton leak when compared with the other CR groups. The CR-lard group showed the lowest proton leak compared with the other CR groups. The ETC enzyme activity measurements in the CR regimens showed that Complex I activity was decreased in both the CR-fish and CR-lard groups. Moreover, the CR-fish group also had lower Complex II activity compared with the other CR groups. These results indicate that dietary lipid composition does influence liver mitochondrial phospholipid composition, ROS production, proton leak and ETC enzyme activities in CR animals.     

Solid Phase Synthesis of Phosphopeptides Incorporating 2,2-Dimethyloxazolidine Pseudoproline Analogs: Evidence for <Emphasis Type="Italic">trans</Emphasis> Leu-Pro Peptide Bonds in Stat3 Inhibitors

To answer the question of whether the conformation of the Leu-Pro bond is cis or trans in Ac-pTyr-Leu-Pro-Gln-Thr-Val-NH₂ when complexed with the SH2 domain of Stat3, we substituted 2,2-dimethyloxazolidines derived from serine (Ser(Ψ^Me,Mepro)) and threonine (Thr(Ψ^Me,Mepro)) for proline. The 2,2-dimethyloxazolidine and 2,2-dimethylthiazolidine pseudoproline (ΨPro) analogs induce predominantly cis Xxx-ΨPro peptide bonds. As these ΨPro analogs are acid-labile, the phosphopeptides were synthesized using Fmoc-based SPPS using unprotected phosphotyrosine and 4-hydroxybenzoate as the linker that allowed release from the support by alkaline ammonolysis, conditions that kept the oxazolidine rings intact. Incorporation of Ser(Ψ^Me,Mepro) resulted in 69% cis Leu-ΨPro bond content in aqueous solution whereas that for Thr(Ψ^Me,Mepro) analog was 63%. Affinities for Stat3 were 3–5 fold lower than the lead compound and were inversely correlated with cis content. Thus we conclude that the Leu-Pro peptide bond is trans when the peptide is bound to Stat3.