Enantioselective lipase-catalyzed kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine

The enzyme (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester. Reaction conditions are optimized to enhance the enantioselectivity. The effects of various racemic alkyl esters, substrate concentration, operating temperature, pH of the aqueous medium and organic solvents on activity and enantioselectivity of BSL2 for kinetic resolution are also studied. A high enantiomeric ratio (E = 60.7) is reached in diisopropyl ether/water (10%, v/v) and the enantioselectivity is about 22-fold higher than that in pure buffered aqueous solution. The results show that the reaction medium greatly influences BSL2 reaction and its enantioselectivity in the hydrolysis of racemic methyl ester.     

Amino acid residues in transmembrane domain 10 of organic anion transporting polypeptide 1B3 are critical for cholecystokinin octapeptide transport

Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are multispecific transporters that mediate uptake of amphipathic organic compounds into hepatocytes. The two OATPs contain 12 transmembrane domains (TMs) and share 80% amino acid sequence identity. Besides common substrates with OATP1B1, OATP1B3 specifically transports cholecystokinin octapeptide (CCK-8). To determine which structural domains and/or residues are important for the substrate selectivity of OATP1B3, we constructed a series of chimeric proteins between OATP1B3 and 1B1, expressed them in HEK293 cells, and determined rates of uptake of CCK-8 along with surface expression of the proteins. Replacing TM10 in OATP1B3 with TM10 of OATP1B1 resulted in a dramatically reduced degree of CCK-8 transport, indicating that TM10 is crucial for recognition and/or translocation of CCK-8. Using site-directed mutagenesis, we identified three key residues within TM10, namely, Y537, S545, and T550. When we replaced these residues with the corresponding amino acid residues found in OATP1B1, the level of CCK-8 transport was similarly low as for the replacement of the whole TM10. Kinetic experiments showed that the K m values for CCK-8 transport in the TM10 replacement and triple mutant were only 1.3 and 1.1 microM, respectively, as compared to 16.3 microM for wild-type OATP1B3. Similarly, the V max values dropped from 495.5 pmol (normalized mg) (-1) min (-1) for wild-type OATP1B3 to 13.3 and 19.0 pmol (normalized mg) (-1) min (-1) for the TM10 replacement and triple mutant, respectively. Molecular modeling indicated that two of the three identified residues might form hydrogen bonds with CCK-8. In conclusion, we have identified three amino acid residues (Y537, S545, and T550) in TM10 of OATP1B3 that are important for CCK-8 transport.     

套式PCR检测体系扩增效率的分析及对假阳性的控制

用扩增效率等量化的指标来控制PCR检测过程中出现的假阳性,调查了受假阳性污染的试剂在特定的PCR检测系统和环境中产生假阳性的PCR循环数界限,采用模板量与扩增倍数的线性回归关系分析系统的扩增效率并以此测定计算了两个不同循环数系统（C27＋15）和C27＋20）的检测界限分别为0．78kg和0．065fg,提出以此检测界限的1／2量作为假阳性耐受量,两个不同循环数系统的假阳性耐受量分别为580个和48个拷贝。     

单核细胞促进人外周血淋巴细胞离体增殖的自由基机理

在人外周血淋巴细胞培养体系中加入不同数量的单核细胞共同培养,以~3H-TdR掺入法和细胞色素C还原法分别测定淋巴细胞增殖能力和培养介质中O_2~-浓度;加入NADPH氧化酶抑制剂DPI实验性地下调O_2~-浓度,观察淋巴细胞增殖能力的变化。结果显示加入单核细胞可增加培养介质中O_2~-浓度,进而促进淋巴细胞增殖,提示O_2~-浓度的升高是淋巴细胞和单核细胞共培养体系中淋巴细胞增殖的重要因素。     

锯缘青蟹精巢发育的组织学观察

组织学观察表明,锯缘青蟹（Scylla serrata)精巢发育周期可以分为精原细胞期,精母细胞期,精子细胞期,精子期和休止期5个时期。在划分精巢发育期的基础上,结合生精小管直径的成熟节段比例这2个指标,可以较准确地反映锯缘青解精巢的发育状况。而成熟节段比例可以作为锯缘青蟹精巢发育的表征指标。     

Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps.

Silver crucian carp (Carassius auratus gibelio) is a unique triploid bisexual species that can reproduce by gynogenesis. As all other gynogenetic animals, it keeps its chromosome integrity by inhibiting the first meiosis division (no extrusion of the first pole body). To understand the molecular events governing this reproduction mode, suppression subtractive hybridization was used to identify the genes differentially expressed in fully-grown oocytes of the gynogenetic and gonochoristic crucian carp (gyno-carp and gono-carp). From two specific subtractive cDNA libraries, the clones screened out by dot blots and virtual Northern blots were chosen to clone full-length cDNA by RACE. Four differentially expressed genes were obtained. Two are novel genes and are expressed specifically in the oocytes. The gyno-carp stores much more mRNA of cyclin A2, a new member of the fish A-type cyclin gene, in its fully-grown oocyte than in the gono-carp. The last gene is histone H2A. The histone H2As of these two closely related crucian carps are quite different in the C-terminus. Preliminary characterization of the four genes has been analyzed by nucleotide and deduced amino acid sequence and Northern analysis.     

ALBUMIN CAN REVERSE THE RELEASE OF POTASSIUM FROM HUMAN ERYTHROCYTES TREATED WITH THE NON-IONIC DETERGENT,BRIJ 58

Exchange of erythrocyte intracellular (i/c) K⁺for extracellular (e/c) Na⁺in human erythrocytes treated with sub-CMC concentrations of the non-ionic detergent Brij 58 can be stopped by reincubation in serum or albumin containing solutions. The progressive equilibration of the K⁺contents of detergent-treated human erythrocytes with the incubation medium was reversed by an albumin-mediated withdrawal of detergent molecules from the cell. Re-establishment of near normal [K⁺] in terms of K⁺/kg water proceeds in two ways: (i) a metabolism-dependent net accumulation of K⁺ions; and (ii) a metabolism-independent shrinkage of erythrocytes, this being the more significant factor.     

Metformin's antitumour and anti‐angiogenic activities are mediated by skewing macrophage polarization

Beneficial effects of metformin on cancer risk and mortality have been proved by epidemiological and clinical studies, thus attracting research interest in elucidating the underlying mechanisms. Recently, tumour‐associated macrophages (TAMs) appeared to be implicated in metformin‐induced antitumour activities. However, how metformin inhibits TAMs‐induced tumour progression remains ill‐defined. Here, we report that metformin‐induced antitumour and anti‐angiogenic activities were not or only partially contributed by its direct inhibition of functions of tumour and endothelial cells. By skewing TAM polarization from M2‐ to M1‐like phenotype, metformin inhibited both tumour growth and angiogenesis. Depletion of TAMs by clodronate liposomes eliminated M2‐TAMs‐induced angiogenic promotion, while also abrogating M1‐TAMs‐mediated anti‐angiogenesis, thus promoting angiogenesis in tumours from metformin treatment mice. Further in vitro experiments using TAMs‐conditioned medium and a coculture system were performed, which demonstrated an inhibitory effect of metformin on endothelial sprouting and tumour cell proliferation promoted by M2‐polarized RAW264.7 macrophages. Based on these results, metformin‐induced inhibition of tumour growth and angiogenesis is greatly contributed by skewing of TAMs polarization in microenvironment, thus offering therapeutic opportunities for metformin in cancer treatment.     

Production optimization for concentration and volume-limited fed-batch reactors in biochemical processes

Bioprocess and Biosystems Engineering - Since a very slight violation of constraint could cause process safety and product quality problems in biochemical processes, an adaptive approach of...