植物GPATs基因研究进展

甘油-3-磷酸酰基转移酶(Glycerol-3-phosphate acyltransferase, GPAT)是三酰甘油(Triacylglycerol, TAG)生物合成的限速酶, 催化TAG生物合成的起始步骤。GPATs主要负责将脂肪酰基从酰基-酰基载体蛋白(acyl-ACP)或酰基辅酶A(acyl-CoA)上转移到甘油-3-磷酸的(Glycerol-3-phosphate, G3P) sn-1位置上。有些成员还具有sn-2酰基转移活性。目前已经在多种植物中克隆得到了GPAT基因。这些GPAT基因编码的酶主要分为三类, 它们在细胞中分别定位于质体、线粒体和内质网上。这些酶参与三酰甘油、几丁质和软木脂等多种脂质的生物合成, 在植物的生长发育中发挥着非常重要的作用。文章介绍了植物GPAT基因的染色体定位和基因结构以及GPAT酶的亚细胞定位、sn-2酰基转移特异性、GPAT酶的底物选择性及其生理功能的最新研究进展。     

Gamma-glutamyl compounds: substrate specificity of gamma-glutamyl transpeptidase enzymes

Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites, and neuroactive compounds. Two cell surface enzymes that metabolize gamma-glutamyl compounds have been identified: gamma-glutamyl transpeptidase (GGT1) and gamma-glutamyl leukotrienase (GGT5). There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetic analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative, and conducted at physiological pH. We evaluated a series of gamma-glutamyl compounds as substrates for human GGT1 and human GGT5. The K_m value for reduced glutathione was 11 μM for both GGT1 and GGT5. However, the K_m values for oxidized glutathione were 9 μM for GGT1 and 43 μM for GGT5. Our data show that the K_m values for leukotriene C₄ are equivalent for GGT1 and GGT5 at 10.8 and 10.2 μM, respectively. This assay was also used to evaluate serine–borate, a well-known inhibitor of GGT1, which was 8-fold more potent in inhibiting GGT1 than in inhibiting GGT5. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism, and other pathways that involve gamma-glutamyl compounds.     

Stability Study of Crude Dextransucrase from <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> NRRL B-742

In the present work, the stability of crude dextransucrase from Leuconostoc citreum B-742 was evaluated in synthetic and in cashew apple juice culture broth. Optimum stability conditions for dextransucrase from L. citreum B-742 were different from the reported for its parental industrial strain enzyme (L. mesenteroides B-512F). Crude dextransucrase, from L. citreum B-742, produced using cashew apple juice as substrate, presented higher stability than the crude enzyme produced using synthetic culture medium, showing the same behavior previously reported for dextransucrase from L. mesenteroides B-512F. The crude enzyme presented good stability in cashew apple juice for 48 h at 25°C and pH 6.5.     

Density dynamics of the larvae ofMytilus trossulus mussels in plankton and their settling on collectors in Vostok Bay, Sea of japan

This paper presents the results of the analysis of density dynamics of the larvae of the Pacific mussel in plankton, and experimental data on their settling on collectors in Vostok Bay, Sea of Japan, obtained in May–September 1989. It was found that, in the summer-autumn season, complex demographic processes occur on the suspended anthropogenic substrata. These processes are caused by the primary settling of pelagic larvae and by the secondary settling of juvenile mussels.     

A general and direct method for the solid phase synthesis of intramolecularly quenched fluorogenic protease substrates using a bifunctional coumarin derivative

A general method for the solid phase preparation offluorogenic peptide substrates or intramolecularly quenchedones (IQFS) is presented, using the highly fluorescentbifunctional coumarin derivative 7-amino-4-coumarinyl-acetic acid. The key feature of this method is theconjugation of H–Aca–OH through its carboxyl group on theresin, followed by the development of the peptide chainthrough its amino group, using standard Fmoc-derived solidphase peptide synthesis methodology. The 2,4-dinitrophenylgroup was used as quencher and introduced directly to theresin-bound peptides. The IQFSDnp–Lys–Pro–Ile–Cys–Phe–Ile–Lys–Leu–Aca–OH (2) andfour Dnp–X-Lys–Pro–Ile–Cys–Phe–Ile–Lys–Leu–Aca–OH (3–6), where X = Val, Lys, Ser and Glu at P₆ position,potential substrates for cathepsin D, were synthesized forproving the utility of the method. The compoundsH–Ile–Lys–Leu–Aca–OH (7),H–Lys–Pro–Ile–Cys–Phe–Ile–Lys–Leu–Aca–OH (8),H–Leu–Aca–OH (9), Dnp–Leu–Aca–OH (10) and Dnp-Leu-OH (11) were also synthesized for comparisonpurposes. The fluorescence properties of compounds 9and 10 were measured.     

An efficient Fmoc strategy for the rapid synthesis of peptide para-nitroanilides

A new strategy has been developed for the rapid synthesis ofpeptide para-nitroanilides (pNA). The method involves derivatization of commercially available tritylchloride resin(TCP-resin) with 1,4-phenylenediamine, subsequent coupling withdesired amino acids by the standard Fmoc protocol, and oxidationof the intermediate para-aminoanilides (pAA) with Oxone®. This procedure allows easy assembly of the desired para-aminoanilides (pAA) on standard resin and efficient oxidation and purification of the corresponding para-nitroanilides (pNA). The method allows easy access to any desired peptide para-nitroanilides, which are useful substrates for the characterization and study of proteolytic enzymes.     

A multi-lake comparison of epilithic diatom communities on natural and artificial substrates

In a multi-season, multi-lake study of epilithic diatom communities, glass slide artificial substrate samplers provided poor representation of communities on natural substrates. Percent similarities between the two communities averaged only 37 over the course of the study. Overall, natural substrates exhibited greater species richness than artificial samplers, particularly during the summer, although this difference was slight. Patterns of selection and inhibition by artificial substrates of individual diatom species varied by both season and lake. Members of the genus Cymbella, particularly C. microcephala, appeared to be the most consistently inhibited, while Achnanthes minutissima was often selected for. In spite of the great differences between the two substrate types, replicability of artificial substrates was very high, and could prove to be a more important quality in monitoring applications than accurate representation of the natural community.     

Determination of the pH dependence,substrate specificity,and turnovers of alternative substrates for human ornithine aminotransferase

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.     

混合碳源发酵生产头孢菌素C过程优化

本文对头孢菌素C（Cephalosporin C,CPC）发酵过程中碳源补料控制策略进行了优化研究,提出了一种基于DO—Stat的混合碳源流加策略,提高了发酵整体性能。在7L发酵罐上对使用该策略和传统补油策略的头孢菌素发酵性能进行比较,结果表明,采用补加混合碳源（葡萄糖＋豆油）策略时,CPC终浓度最高,达到36．99g／L,CPC得率也从使用传统单纯补油策略时的11．39％提高到22．19％,代谢副产物去乙酰氧头孢菌素C（DAOC）的积累量少,DAOC／CPC只有0．38％,达到生产要求。     

Structure and physiological importance of angiotensin converting enzyme domains

Somatic angiotensin converting enzyme (ACE) consists of two homologous catalytic domains (N- and C-domain), exhibiting different biochemical properties. The catalytically active ACE isoforms consisted of just one domain have been also detected in mammals. Substantial progress in ACE domain research was achieved during the last years, when their crystal structures were determined. The crystal structures of domains in complex with diverse potent ACE inhibitors provided new insights into structure-based differences of the domain active sites. Physiological functions of ACE are not limited by regulation of the cardiovascular system. Recent evidence suggests that the ACE domains may be also involved into control of different physiological functions. The C-terminal catalytic domain plays an important role in the regulation of blood pressure: it catalyzes angiotensin I cleavage in vivo. The N-domain contributes to the processing of other bioactive peptides for which it exhibits high affinity. The role of the N-domain is not ultimately associated with functioning of the rennin-angiotensin system and it contributes processing of other bioactive peptides for which it exhibits high affinity (goralatide, luliberin, enkephalin heptapeptide, beta-amyloid peptide). Domain-selective inhibitors selectively blocking either the N- or C-domain of ACE have been developed.