Crystal structure of the E. coli dipeptidyl carboxypeptidase Dcp: further indication of a ligand-dependent hinge movement mechanism

Dcp from Escherichia coli is a 680 residue cytoplasmic peptidase, which shows a strict dipeptidyl carboxypeptidase activity. Although Dcp had been assigned to the angiotensin I-converting enzymes (ACE) due to blockage by typical ACE inhibitors, it is currently grouped into the M3 family of mono zinc peptidases, which also contains the endopeptidases neurolysin and thimet oligopeptidase (TOP). We have cloned, expressed, purified, and crystallized Dcp in the presence of an octapeptide "inhibitor", and have determined its 2.0A crystal structure using MAD methods. The analysis revealed that Dcp consists of two half shell-like subdomains, which enclose an almost closed two-chamber cavity. In this cavity, two dipeptide products presumably generated by Dcp cleavage of the octapeptide bind to the thermolysin-like active site fixed to side-chains, which are provided by both subdomains. In particular, an Arg side-chain backed by a Glu residue, together with two Tyr phenolic groups provide a charged anchor for fixing the C-terminal carboxylate group of the P2' residue of a bound substrate, explaining the strict dipeptidyl carboxypeptidase specificity of Dcp. Tetrapeptidic substrates are fixed only via their main-chain functions from P2 to P2', suggesting a broad residue specificity for Dcp. Both subdomains exhibit very similar chain folds as the equivalent but abducted subdomains of neurolysin and TOP. Therefore, this "product-bound" Dcp structure seems to represent the inhibitor/substrate-bound "closed" form of the M3 peptidases, generated from the free "open" substrate-accessible form by a hinge-bending mechanism. A similar mechanism has recently been demonstrated experimentally for ACE2.     

Single-Cell Sequencing of iPSC-Dopamine Neurons Reconstructs Disease Progression and Identifies HDAC4 as a Regulator of Parkinson Cell Phenotypes

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L-serine degradation in Escherichia coli K-12: cloning and sequencing of the sdaA gene.

A new mutant of Escherichia coli K-12 unable to grow with L-serine, glycine, and L-leucine has been isolated by lambda plac Mu insertion and shown to be deficient in L-serine deaminase activity. The corresponding gene, sdaA, has been cloned from a prototrophic strain, and the clone has been characterized and sequenced. The evidence is consistent with the hypothesis that sdaA is the structural gene for L-serine deaminase. However, other possibilities are also considered. No significant homology with previously reported DNA or protein sequences was detected.     

3-Hydroxyflavone inhibits endogenous Aurora B and induces growth inhibition of cancer cell line

The Aurora kinases play a critical role in mitosis and have been suggested as promising targets for cancer therapy due to their frequent overexpression in a variety of tumors. Compared with established inhibitors of cell division such as the anti-tubulins, novel agents target mitotic enzymes and show similar efficacy but with fewer side effects. Several small-molecule inhibitors of Aurora kinases have been developed as anticancer agents, some of which have progressed to early clinical evaluation. Here we identified 3-hydroxyflavone as a novel Aurora B inhibitor through high throughput screening. 3-Hydroxyflavone showed potent inhibition to Aurora B with the IC₅₀ on a nanomolar basis in the enzyme-based kinase activity assay. In the cell-based western blotting analysis, 3-hydroxyflavone dramatically decreased the phosphorylation level of Histone H3 on the site of serine 10, demonstrating the potent endogenous Aurora B activity inhibition in cell level. The followed cell image analysis provided the consist result. To make it clear whether 3-hydroxyflavone inhibited Aurora B by direct binding or not, SPR analysis was carried out to measure the affinity of interaction between Aurora B protein and 3-hydroxyflavone and the result proved the binding with high affinity. Usually Aurora activity suppression induced cancer cell proliferation inhibition. Colony formation and cell viability with/without treatment of 3-hydroxyflavone were measured using CCK-8. The growth suppression under 3-hydroxyflavone present and the growth recovery after being released gave strong evidence that presence of 3-hydroxyflavone efficiently inhibited the fast growth of cancer cells.     

转昆虫抗冻蛋白基因增强甘薯抗冻能力

为明确昆虫抗冻蛋白基因转入甘薯(Ipomoea batatas)后是否能提升其抗冻能力,进而为培育甘薯抗冻育种材料奠定基础,将黄粉虫(Tenebrio molitor)抗冻蛋白基因TmAFP导入植物基因表达质粒,经农杆菌介导的遗传转化获得抗冻甘薯新材料。以甘薯品种Huachano为受体材料建立甘薯植株高效再生体系,并采用不同成分的体细胞胚成熟培养基培养胚性悬浮细胞。胚性愈伤组织对除草剂的敏感性测试结果表明,转基因阳性植株筛选的最适培养基为MS+0.2 mg·L–12,4-D+0.8 mg·L^–1 GAP+100 mg·L^–1 Carb。将表达质粒分别转化Huachano后共获得7个胚性愈伤团并最终获得42株再生抗性植株,其中转pSUIBEV3-AFP有23个株系,转pCAMBIA-AFP有19个株系,经PCR、Southern杂交和RT-PCR检测后证实TmAFP基因已整合至甘薯基因组中并获得表达。将转基因甘薯及对照植株在–1℃下处理15小时后转移至室温,结果表明,转基因甘薯植株的抗冻能力显著提升。     

Modification of the polar head group of platelet-activating factor: influence on the biological activity

Racemic analogues of platelet-activating factor and its lyso derivatives have been prepared in which one methyl of the trimethylammonium group has been replaced by ethyl, propyl, allyl, or carboxymethylene. The influence of chemical modification on the biological activity was assessed by measuring platelet aggregation and desensitization. The results point to a crucial role of a positively charged polar head group for the expression of biological activity of platelet-activating factor. There are also some indications of a more non-specific interaction of the polar head group of platelet-activating factor with its platelet binding sites.     

Complete genome sequence of an H10N8 avian influenza virus isolated from a live bird market in Southern China

An H10N8 avian influenza virus (AIV), designated A/Duck/Guangdong/E1/2012 (H10N8), was isolated from a duck in January 2012. This is first report that this subtype of AIV was isolated from a live bird market (LBM) in Guangdong Province in southern China. Furthermore, the complete genome of this strain was analyzed. The availability of genome sequences is helpful to further investigations of epidemiology and molecular characteristics of AIV in southern China.     

Searching for a needle in the haystack: comparing six methods to evaluate heteroplasmy in difficult sequence context

Mitochondrial DNA (mtDNA) mutations have been involved in disease, aging and cancer and furthermore exploited for evolutionary and forensic investigation. When investigating mtDNA mutations the peculiar aspects of mitochondrial genetics, such as heteroplasmy and threshold effect, require suitable approaches which must be sensitive enough to detect low-level heteroplasmy and, precise enough to quantify the exact mutational load. In order to establish the optimal approach for the evaluation of heteroplasmy, six methods were experimentally compared for their capacity to reveal and quantify mtDNA variants. Drawbacks and advantages of cloning, Fluorescent PCR (F-PCR), denaturing High Performance Liquid Chromatography (dHPLC), quantitative Real-Time PCR (qRTPCR), High Resolution Melting (HRM) and 454 pyrosequencing were determined. In particular, detection and quantification of a mutation in a difficult sequence context were investigated, through analysis of an insertion in a homopolymeric stretch (m.3571insC).