手性化合物酶法制备中辅酶再生体系的构建与应用进展

辅酶的再生与反复利用对氧化还原型生物催化剂在手性化合物中的生产具有重要作用。辅酶的酶催化法再生具有高效、专一的特点,根据其催化环境可分为单细胞内辅酶再生体系、双细胞耦合体系及胞外酶耦合体系。本文详细阐述了酶法再生中各种辅酶再生体系的构建方法,特别指出了辅酶再生体系使用中可能遇到的问题并给出了解决方法,如利用离子液体解决有机底物低水溶性的问题,利用共价固定方法解决小分子辅酶的留存问题,无细胞抽提液中内源性辅酶再生酶的利用等。除此之外,对每种辅酶再生方法与体系在工业催化中应用的优势与局限性作了评述,特别关注了辅酶再生体系反复多批次利用的问题。

卵黄萤荧光素再生酶基因的克隆与序列分析

目的克隆和分析荧光素再生酶基因(LRE)。方法通过GeneBank中已知的荧光素再生酶基因保守区段设计引物,利用5’RACE(rapid-amplification of cDNA ends)和3’RACE技术克隆了来自云南省西双版纳州的卵黄萤(Luciola ovalis)荧光素再生酶基因cDNA和全基因序列。通过GeneBank、National Center for Biotechnology Information和ProDom at the ExPASy Server软件和数据库进行序列分析。结果卵黄萤荧光素再生酶的cDNA序列和基因序列存在2个不同碱基位点,但是它们编码的荧光素再生酶是相同的。卵黄萤荧光素再生酶基因全长(从起始密码子到终止密码子)为1131bp,包含5个外显子4个内含子,其cDNA序列为1008bp,包含924bp的荧光素酶基因开放阅读框和84bp的3’UTR序列。卵黄萤荧光素酶基因的开放阅读框编码1个307个氨基酸的蛋白质。它与北美萤火虫(Photinus pyralis)荧光素再生酶在碱基序列和氨基酸序列上分别有61.8%和53.3%的相似性。结论成功地克隆了荧光素再生酶的cDNA和基因序列,为其在基因工程中的应用奠定了基础。

丹麦提炼出废纸再生酶

丹麦现代北欧公司提炼出一种废纸再生酶,它可将废旧纸张中的墨迹和油墨分离出来。 其生产工艺流程是:向磨碎的纸浆中加入强碱,而后按一吨200-300毫升的比例向纸浆中添加废纸再生酶。经过这样处理以后,黑色的油墨沉淀到纸浆池底。

磁性固定化PLA_1的制备及其在脱胶应用中的研究

本文目的是研究磷脂酶A1的固定化条件并将固定化酶用于脱胶过程。以磁性Fe3O4/SiOx-g-P(GMA)复合粒子为载体,考察不同的固定化条件对固定化酶相对酶活的影响,然后将制得的磁性固定化PLA1用于大豆油脱胶中,并对其酶活再生进行有益研究。结果表明,最优固定化条件为:酶液添加量20mL,pH6.0,反应时间5h,此时酶活力最高;最佳脱胶条件为:反应时间6h、反应温度55℃、加酶量0.12g/kg、油相起始pH6.0。在最优条件下进行脱胶试验,测定脱胶油中的磷含量为12.5mg/kg,脱胶效果较好。将再生的磁性固定化磷脂酶A1再用于大豆油脱胶中,延长了磁性固定化磷脂酶A1的利用时间,降低了大豆油酶法脱胶的生产成本。

有机磷等农药残留生物监测技术研究进展

综述了有机磷等农监测技术的最新进展,简述和比较了国内外各种最新生物监测技术的原理和应用,为农业和环境保护领域的监测提供参考,同时也为我国的在线生物监测技术的发展作出铺垫。

Expression of phosphatase regenerating liver 3 is an independent prognostic indicator for gastric cancer

AIM： To investigate the prognostic significance of phosphatase regenerating liver 3 （PRL-3） protein expression in gastric cancer.METHODS： PRL-3 expression in paraffin-embedded tumor specimens from 293 patients with gastric cancer was studied retrospectively by immunohistochemistry. Nonoclonal antibody specifically against PRL-3, 3B6, was obtained with hybridoma technique.RESULTS： Positive PRL-3 expression was detected in 43.3% （227 of 293） of gastric cancer cases. High expression of PRL-3 was positively correlated with tumor size, depth of invasion, vascular/lymphatic invasion, lymph node metastasis, high TNM stage and tumor recurrence. Patients with positive PRL-3 expression had a significantly lower 5-year survival rate than those with negative expression （28.3% vs 52.9%, P 〈 0.0001）. Patients who received curative surgery, and with positive PRL-3 expression had a significant shorter overall survival and disease-free disadvantage over patients with negative expression （hazard ratio of 16.7 and 16.6, respectively; P 〈 0.0001 for both）. Multivariate analysis revealed that PRL-3 expression was an independent prognostic indicator for overall and disease-free survival of gastric cancer patients, particularly for survival in TNM stage Ⅲ patients. CONCLUSION： PRL-3 expression is a new independent prognostic indicator to predict the potential of recurrence and survival in patients with gastric cancer at the time of tumor resection,

Expression of tumor necrosis factor-alpha converting enzyme in liver regeneration after partial hepatectomy

AIM:To study the expression of tumor necrosis factor- alpha converting enzyme (TACE) and evaluate its significance in liver regeneration after partial hepatectomy in vivo. METHODS: Male SD rats underwent 70% partial hepatec- tomy. The remaining liver and spleen tissue samples were collected at indicated time points after hepatectomy. TACE expression was investigated by Western blotting, immunohistochemistry, and serial section immunostaining. RESULTS: Expression of TACE in liver and spleen tissues after partial hepatectomy was a time-dependent alteration, reaching a maximal level between 24 and 48 h and remaining elevated for more than 168 h. TACE protein was localized to mononuclear cells (MNC), which infiltrated the liver from the spleen after hepatectomy. The kinetics of TACE expression was in accordance with the number of TACE-staining MNCs and synchronized with those of transforming growth factor-α (TGFα). In addition, TACE-staining MNC partially overlapped with CD3+ T lymphocytes. CONCLUSION: TACE may be involved in liver regenera- tion by pathway mediated with TGFα-EGFR in the cell- cycle progressive phase in vivo. TACE production and effect by paracrine may be a pathway of involvement in liver regeneration for the activated CD3+ T lymphocytes.

NAD(P)H再生及其依赖型酶在工程菌中研究进展

许多氧化还原型酶都属于辅酶依赖型酶类,在生物转化中添加外源性辅酶将增加生产成本,而辅酶的缺乏又将直接影响生物转化效率,因此构建能实现辅酶再生的基因工程菌是辅酶依赖型基因工程菌走向工业化的有效手段之一。该综述对目前已报道的辅酶依赖型酶常用的还原态辅酶和氧化态辅酶再生的脱氢酶体系进行整理,并简述了在基因工程菌中通过辅酶再生提高酶催化的生物转化效率的研究进展,以期为构建辅酶再生型基因工程菌,以及实现这类工程菌自动化和一体化的发酵工艺提供借鉴。

Phosphatase of Regenerating Liver and Its Association with Tumors

Protein kinases and protein phosphatases play key roles in regulating functions of diverse proteins which control numerous essential events in eukaryotes, such as transcriptional regulation, apoptosis, cell cycle progression, protein degradation and protein trafficking. Protein kinases transfer a phosphate from ATP to a specific residue（s）, typically at serine, threonine, or tyrosine, in proteins, while phosphatases remove phosphoryl groups from proteins. Such a phosphorylation-dephosphorylation cycle can be regarded as a molecular ＂on-off＂ switch. It is estimated that the human genome contains over 2,000 kinases and 1,000 phosphatases. A very large number of protein kinases has been identified and studied in detail, and more and more, information concerning protein phosphatases has emerged recently.

Significance of regenerating islet-derived type Ⅳ gene expression in gastroenterological cancers

The regenerating islet-derived members （Reg）, a group of small secretory proteins, which are involved in cell proliferation or differentiation in digestive organs, are upregulated in several gastrointestinal cancers, functioning as trophic or antiapoptotic factors. Regenerat- ing islet-derived type Ⅳ （RegⅣ）, a member of the Reg gene family, has been reported to be overexpressed in gastroenterological cancers. RegIV overexpression in tumor cells has been associated with carcinogen- esis, cell growth, survival and resistance to apoptosis. Cancer tissue expressing RegIV is generally associated with more malignant characteristics than that with- out such expression, and RegⅣ is considered a novel prognostic factor as well as diagnostic marker in some gastroenterological cancers. We previously investigated the expression levels of RegⅣ mRNA of 202 surgical colorectal cancer specimens with quantitative real-time reverse-transcriptase polymerase chain reaction and reported that a higher level of RegⅣ gene expression was a significant independent predictor of colorec- tal cancer. The biologic functions of RegⅣ protein in cancer tissue, associated with carcinogenesis, anti- apoptosis and invasiveness, are being elucidated by molecular investigations using transfection techniques or neutralizing antibodies of RegIV, and the feasibility of antibody therapy targeting RegIV is being assessed. These studies may lead to novel therapeutic strate- gies for gastroenterological cancers expressing RegⅣ. This review article summarizes the current information related to biological functions as well as clinical impor- tance of RegⅣ gene to clarify the significance of Reg~ expression in gastroenterological cancers.

Omentum facilitates liver regeneration

AIM:To investigate the mechanism of liver regeneration induced by fusing the omentum to a small traumatic injury created in the liver. We studied three groups of rats. In one group the rats were omentectomized; in another group the omentum was left in situ and was not activated,and in the third group the omentum was activated by polydextran particles. METHODS:We pre-activated the omentum by injecting polydextran particles and then made a small wedge wound in the rat liver to allow the omentum to fuse to the wound. We monitored the regeneration of the liver by determining the ratio of liver weight/body weight,by histological evaluation (including immune staining for cytokeratin-19,an oval cell marker),and by testing for developmental gene activation using reverse transcription polymerase chain reaction (RT-PCR). RESULTS:There was no liver regeneration in the omentectomized rats,nor was there significant regeneration when the omentum was not activated,even though in this instance the omentum had fusedwith the liver. In contrast,the liver in the rats with the activated omentum expanded to a size 50% greater than the original,and there was histologically an interlying tissue between the wounded liver and the activated omentum in which bile ducts,containing cytokeratin-19 positive oval cells,extended from the wound edge. In this interlying tissue,oval cells were abundant and appeared to proliferate to form new liver tissue. In rats pre-treated with drugs that inhibited hepatocyte growth,liver proliferation was ongoing,indicating that regeneration of the liver was the result of oval cell expansion. CONCLUSION:Activated omentum facilitates liver regeneration following injury by a mechanism that depends largely on oval cell proliferation.

Busuishengxue ranules mediate their effects upon non-severe aplastic anemia via mitogen-activated protein kinase/extracellular signal-regulated kinase pathway

OBJECTIVE:To observe the clinical efficacy of Busuishengxue granules on non-severe aplastic anemia(NSAA)and investigate its effect on the mitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK)pathway.METHODS:Sixty NSAA patients were divided equally into two groups.Subjects in the experimental group were treated with Busuishengxue granules,and the control group with Zaizaoshengxue tablets.The treatment course was 6 months and cu-rative efficacy was compared between the two groups as well as with 10 healthy individuals.Flow cytometry(FCM)was used to detect the intracellular concentration of Ca2+([Ca2+]i).Western blotting was employed to detect the expression of enzymes in the MAPK/ERK pathway.RESULTS:The efficacy of Busuishengxue granules was significantly better than that of Zaizaoshengxue tablets(P<0.05).Before treatment,expression of JNK,phospho-ERK 1/2 and p-JNK was higher,and[Ca2+]i higher,than that of the control group(P<0.05).After treatment with Busuishengxue granules,expression of all enzymes related to signal transduction pathways in the blood cells of NSSA patients were altered to different degrees.CONCLUSION:Busuishengxue granules had a better effect with regard to improving symptom scores,increasing the number of blood leukocytes,and increasing hemoglobin levels than Zaizaoshengxue tablets,and they differed slightly in terms of increasing the number of platelets.

Acetylcholinesterase liquid crystal biosensor for identification of AChE inhibitors by a reactivator

An improved acetylcholinesterase liquid crystal(LC) biosensor has been developed for the identification of organophosphates(OPs) by using a reactivator. When the acetylcholinesterases(AChEs) inhibited by different kinds of OPs are reactived by a reactivator, the catalytic activity of AChEs can be recovered with different activation efficiency because of the different phosphorylation structures formed in the inhibited AChEs. Accordingly, the reactived AChEs can catalyze the hydrolysis of acetylthiocholine to generate thiocholine product in different degrees, which will result in different catalytic growth of AuNPs and further form distinct orientational response of LCs. Based on such a reactivation mechanism, the AChE LC biosensor with a simple, rapid and visual procedure achieves an obvious identification of three OPs pesticides, methamidophos, trichlorfon and paraoxon, by using a pralidoxime reactivator.