重组hPRL-1的表达纯化及其理化和酶学性质研究

目的:表达纯化hPRL-1重组蛋白,分析其理化性质及酶学特性。方法:热激法将重组pET15b质粒转化入E.coli BL21中,IPTG诱导表达出His-tagged hPRL-1蛋白。使用Ni-NTA亲和层析法结合Mono Q离子交换层析法纯化。用SDS-PAGE法和Western Blot法进行表达情况的定性定量分析,并使用HPLC法鉴定蛋白纯度,计算出蛋白分子量,圆盘等电聚焦电泳分析重组蛋白等电点。比较分析以pNPP、4-MUP和DiFMUP为底物时的酶促反应动力学。同时以pNPP为底物测定酶的最适pH值;以4-MUP为底物测定酶的最适温度,分析探讨缓冲液离子强度与蛋白酪氨酸酶通用抑制剂钒酸钠对酶活力的影响。结果:以亲和层析和离子交换层析结合,可以纯化得到纯度约为95%的蛋白。测得蛋白分子量为24.54kD,等电点为9.11。以pNPP、4-MUP和DiFMUP为底物时Km分别为3720μmol/L,130μmol/L和50μmol/L。酶的最适pH值为7.6,最适温度为34℃。结论:纯化所得蛋白为目的蛋白hPRL-1;两步纯化相结合可以得到纯度较高的蛋白;三种底物特异性依次为DiFMUP>4-MUP>pNPP。     

肠内营养对乳腺癌术后化疗患者肠道菌群及生活质量的影响

目的研究肠内营养对乳腺癌术后化疗患者肠道菌群、生活质量、营养指标水平的影响。方法选取2012年7月至2018年1月于我院进行乳腺癌术后化疗的91例女性乳腺癌患者为研究对象,根据是否进行肠内营养将患者分为肠内营养组和对照组,观察化疗前后2组患者肠道菌群、生活质量、营养指标水平的变化及不良反应发生情况。结果治疗前2组患者肠道菌群、生活质量、营养指标水平差异无统计学意义(均P0.05)。治疗后,肠内营养组患者肠道双歧杆菌数量为(6.7±0.5)lg CFU/g、乳杆菌数量为(8.5±0.4)lg CFU/g,均显著高于治疗前,同时显著高于对照组(均P0.05)。治疗后肠内营养组患者出现反酸、腹胀、便秘等胃肠道不良反应的比例与对照组比较差异无统计学意义(均P0.05)。治疗后两组患者总体健康、认知功能、情绪功能、角色功能、社会功能、活力、躯体疼痛、躯体功能评分显著高于治疗前(均P0.05),且肠内营养组患者生活质量各指标的评分显著高于对照组(均P0.05)。治疗后肠内营养组患者血清总蛋白(TB)、血清清蛋白(ALB)、血清前清蛋白(PA)、转铁蛋白(TF)水平均显著高于对照组(均P0.05)。结论肠内营养可预防乳腺癌术后化疗患者肠道菌群的紊乱及营养不良的发生,安全有效,有助于提高乳腺癌患者的生活质量。     

不同温度、气体微环境对红托竹荪干品储藏品质的影响

本研究针对红托竹荪干品在储藏过程中易发生褐变、降低商品性问题,探究了不同储藏条件(温度、气体微环境)对红托竹荪干品储藏品质的影响。以红托竹荪干品为原材料,考察了在气体微环境(空气、N₂、CO₂和脱氧)和不同储藏温度(5、25和45 ℃)下红托竹荪干品储藏品质的动态变化。在60 d的储藏期内,所有样品的褐变指数、剪切力、多酚氧化酶、过氧化氢酶、总酚、还原糖和5-羟甲基糠醛含量均增加,游离氨基酸、白度值、复水比均降低。与25、45 ℃相比,以上指标在5 ℃条件下均表现最优,5 ℃储藏条件下呈味氨基酸和挥发性成分指标更接近于0 d;在不同气体微环境比较下,CO₂储藏环境下干品品质保持最好,通过综合评分得出5 ℃低温结合CO₂充气条件下干品品质最优,其次为N₂结合5 ℃低温。结合经济成本,5 ℃低温结合CO₂或N₂充气可以作为红托竹荪干品延长货架期的推荐储藏技术。     

ERRATUM: New Body Mass Estimates for Omomys carteri,a Middle Eocene Primate From North America. Am J Phys Anthropol 109:41–52.

Payseur BA, Covert HA, Vinyard CJ, Dagosto M. 1999. New Body Mass Estimates for Omomys carteri, a Middle Eocene Primate From North America. Am J Phys Anthropol 109:41–52. This article included an incomplete Table 2. The final two columns, showing “Intercept” and “SEE” data were omitted. The complete Table 2, with these two columns included, is provided below.     

The Dutch N-cascade in the European perspective

The Netherlands is "well known" for its nitrogen problems; it has one of the highest reactive nitrogen (Nr) emission densities in the world. It is a small country at the delta of several large European rivers. Ever since the industrial revolution, there has been a growing excess of nutrients and related emissions into the atmosphere (ammonia, nitrogen oxides and nitrous oxide) and into groundwater and surface water (nitrate), leading to a large range of cascading environmental impacts. Vehicular traffic, sewage and animal husbandry are the main sources of oxidized and reduced forms of Nr. This paper provides an overview of the origin and fate of nitrogen in the Netherlands, the various reported impacts of nitrogen, the Dutch and European policies to reduce nitrogen emissions and related impacts. In addition, ways are presented to go forward to potentially solve the problems in a European perspective. Solutions include the improvement of nitrogen efficiencies in different systems, technological options and education.     

Inhibition of myostatin in adult mice increases skeletal muscle mass and strength

A human therapeutic that specifically modulates skeletal muscle growth would potentially provide a benefit for a variety of conditions including sarcopenia, cachexia, and muscular dystrophy. Myostatin, a member of the TGF-beta family of growth factors, is a known negative regulator of muscle mass, as mice lacking the myostatin gene have increased muscle mass. Thus, an inhibitor of myostatin may be useful therapeutically as an anabolic agent for muscle. However, since myostatin is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. In order to test the hypothesis that myostatin regulates muscle mass in adults, we generated an inhibitory antibody to myostatin and administered it to adult mice. Here we show that mice treated pharmacologically with an antibody to myostatin have increased skeletal muscle mass and increased grip strength. These data show for the first time that myostatin acts postnatally as a negative regulator of skeletal muscle growth and suggest that myostatin inhibitors could provide a therapeutic benefit in diseases for which muscle mass is limiting.     

黑素皮质素受体-4的研究进展

黑素皮质素受体 4 (MC4R)是人类中枢神经系统中参与调节肥胖症发生的重要因素 ,可调节动物的体重和采食量。自MC4R基因克隆以来 ,学者们对MC4R的结构 ,生理功能 ,调控 ,作用机制及其基因突变与体重的关系等方面进行了大量的研究。     

Structural requirements for catalysis and membrane targeting of mammalian enzymes with neutral sphingomyelinase and lysophospholipid phospholipase C activities. Analysis by chemical modification and site-directed mutagenesis

The sequence similarity with bacterial neutral sphingomyelinase resulted in the isolation of putative mammalian counterparts and, subsequently, identification of similar molecules in a number of other eukaryotic organisms. Based on sequence similarities and previous characterization of the mammalian enzymes, we have chemically modified specific residues and performed site-directed mutagenesis in order to identify critical catalytic residues and determinants for membrane localization. Modification of histidine residues and the substrate protection experiments demonstrated the presence of reactive histidine residues within the active site. Site directed mutagenesis suggested an essential role in catalysis for two histidine residues (His-136 and His-272), which are conserved in all sequences. Mutations of two additional histidines (His-138 and His-151), conserved only in eukaryotes, resulted in reduced neutral sphingomyelinase activity. In addition to sphingomyelin, the enzyme also hydrolyzed lysophosphatidylcholine. Exposure to an oxidizing environment or modification of cysteine residues using several specific compounds also inactivated the enzyme. Site-directed mutagenesis of eight cysteine residues and gel-shift analysis demonstrated that these residues did not participate in the catalytic reaction and suggested the involvement of cysteines in the formation/breakage of disulfide bonds, which could underlie the reversible inactivation by the oxidizing compounds. Cellular localization studies of a series of deletion mutants, expressed as green fluorescent protein fusion proteins, demonstrated that the transmembrane region contains determinants for the endoplasmic reticulum localization.     

Use of a crude extract or purified antigen from first‐instar cattle grubs,Hypoderma lineatum,for the detection of anti‐Hypoderma antibodies in free‐ranging cervids from southern Spain

During the 2003–2005 hunting seasons, a total of 120 Cervidae, including 39 red deer (Cervus elaphus hispanicus) and 81 fallow deer (Dama dama), were examined for subcutaneous myiasis. Animals were shot from January to June in southern Spain. Specific antibodies against Hypodermatinae (Diptera: Oestridae) were detected by indirect enzyme‐linked immunosorbent assay (iELISA) using a crude larval extract (CLE) and a purified antigen [hypodermin C (HC)] obtained from first instars of Hypoderma lineatum (De Villers) (Diptera: Oestridae). Hypoderma actaeon Brauer was the only species detected in this study, which represents the first confirmation of this species in fallow deer from Spain. The overall prevalence of animals presenting subcutaneous larvae (14.2%) was considerably lower than the prevalences determined by iELISA with CLE (43.3%) and HC (40.0%). Red deer showed a higher prevalence of Hypoderma than fallow deer. The concordance between larval examination during the hunting season and iELISA using both antigens was low, whereas the concordance between the CLE and HC ELISAs was good. Larval antigens obtained from H. lineatum constitute a good tool for the diagnosis of H. actaeon in Cervidae, especially when the hunting season does not coincide with the maximum presence of larvae on the back.