Components and proteolytic processing sites of arylsulfatase B from human placenta.

Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.     

D type cyclins associate with multiple protein kinases and the DNA replication and repair factor PCNA.

Human cyclin D1 has been associated with a wide variety of proliferative diseases but its biochemical role is unknown. In diploid fibroblasts we find that cyclin D1 is complexed with many other cellular proteins. Among them are protein kinase catalytic subunits CDK2, CDK4 (previously called PSK-J3), and CDK5 (also called PSSALRE). In addition, polypeptides of 21 kd and 36 kd are identified in association with cyclin D1. We show that the 36 kd protein is the proliferating cell nuclear antigen, PCNA. Cyclin D3 also associates with multiple protein kinases, p21 and PCNA. It is proposed that there exists a quaternary complex of D cyclin, CDK, PCNA, and p21 and that many combinatorial variations (cyclin D1, D3, CDK2, 4, and 5) may assemble in vivo. These findings link a human putative G1 cyclin that is associated with oncogenesis with a well-characterized DNA replication and repair factor.     

Expression of glucoamylase gene usingSUC2 promoter inSaccharomyces cerevisiae

Summary The cloning of glucoamylase geneSTA using theSUC2 promoter intoSaccharomyces cerevisiae was performed. The signal sequence ofSTA gene was used for the secretion of glucoamylase protein. The plasmid constructed in this way was named YEpSUCSTA and its expression was identified. The expression of YEpSUCSTA was repressed in the presence of glucose in growth medium, but derepressed when glucose became depleted. YEpSUCSTA showed the similar efficiency of glucoamylase secretion as YEpSTA-F which has the entireSTA gene. Glucoamylase activity in starch-glucose medium was largely increased because cell mass and plasmid stability were high in biosynthesis phase compared to extracellular glucoamylase activities in media which starch or glucose was the only carbon source.     

Reactive oxygen species alter contractile properties of pulmonary arterial smooth muscle

Reactive oxygen species alter pulmonary arterial vascular tone and cause changes in pulmonary vascular resistance. The objective of this investigation was to determine direct effects of oxygen radicals on the contractile properties of pulmonary arterial smooth muscle. Isolated pulmonary arterial rings from Sprague-Dawley rats were placed in tissue baths containing Earle's balanced salt solution (gassed with 95% O2 - 5% CO2, 37 degrees C, pH 7.4). Vessels were contracted with 80 mM KCl to establish maximum active force production (Po). All other responses were normalized as percentages of Po for comparative purposes. Reactive oxygen metabolites were generated enzymatically with either the xanthine oxidase (XO) reaction or the glucose oxidase (GO) reaction, or hydrogen peroxide (H2O2) was added directly to the muscle bath. Exposure to XO, GO, or to H2O2 resulted in a contractile response that was sustained during the 30-min exposure period. The muscle fully relaxed following removal of the reactive oxygen species. Resting tension remained unchanged throughout the experimental period, suggesting no functional change in membrane potential. The contractile response was dose dependent and was not prevented by either cyclooxygenase or lipoxygenase inhibition, or by removal of the endothelium. Pretreatment of vessels with superoxide dismutase (SOD) partially blocked the XO-induced contraction, while mannitol or deferoxamine had no effect on the response to XO. However, pretreatment with catalase (CAT) completely blocked the XO-induced contraction. These data suggest that superoxide ions and hydrogen peroxide are the major causative agents. Following O2-radical exposure, vessels showed a decrease in contractile responsiveness to 80 mM KCl (recovery response), suggesting damage to the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

福氏痢疾菌膜成分与细菌抗原性和毒力的关系

应用免疫转移技术,用从感染福氏痢疾菌的病人获取的恢复期血清分析了福氏痢疾菌膜成分与细菌抗原性和毒力的关一。发现福氏痢疾菌的膜蛋白67kD和63kD均含有两种成分,一种和膜蛋白60kD都可能是保护性抗原,而另一种与膜蛋白78kD和35kD一样与福氏痢疾菌的毒力有关。采用微细胞同位素掺入示踪显示这些与病人恢复期血清反应的膜蛋白由质粒编码。     

Entrapment of microbial cells and organelles with hydrophilic urethane prepolymers

Summary Microbial cells and cellular organelles were immobilized by mixing aqueous suspensions of the biocatalysts with water-miscible urethane prepolymers. Thus immobilized preparations of acetone-dried cells of Arthrobacter simplex and thawed cells of Nocardia rhodocrous showed appreciable {ie351-1} activities in the transformation of hydrocortisone into prednisolone and 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione, respectively. The activities of catalase and alcohol oxidase were observed in the immobilized peroxisomes (microbodies) of a methanol-grown yeast Kloeckera sp. No. 2201. Yeast mitochondria entrapped with the prepolymer showed adenylate kinase activity. These results indicate the usefulness of the urethane prepolymers as convenient materials for entrapment of not only enzymes, but also organelles and microbial cells.     

基于高分辨率遥感影像的北亚热带森林生物量反演

以北亚热带湖北省太子山林场为研究对象,基于高空间分辨率GF-2与SPOT-6卫星影像,提取不同窗口大小下的纹理信息与光谱信息,利用随机森林回归算法,并结合野外实测106块样地的生物量数据,建立不同影像下的太子山林场森林生物量反演模型。结果显示：（1） GF-2和SPOT-6虽然空间分辨率有差异,但是从其不同波段反射率的相关系数（0.75、0.78、0.73、0.61）发现,两种影像的波段反射率具有较高的相关性,说明两者的辐射性能相近;（2）通过分析不同纹理特征对生物量模型的影响,发现均值和对比度纹理参数对生物量反演具有很好的效果。（3）高分辨率的遥感数据在生物量反演中具有较好的表现,且GF-2生物量模型精度（R²=0.88,RMSE=27.11 Mg/hm²）与SPOT-6生物量模型的精度（R²=0.89,RMSE=23.93 Mg/hm²）相近。（4）两种影像对不同森林类型的生物量预测值不存在显著差异,都适合对不同林分类型的生物量进行预测。     

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.     

高产青霉素酰化酶巨大芽胞杆菌的诱变选育及产酶条件优化

【目的】选育高产青霉素G酰化酶(PGA)工业菌株。【方法】采用LiCl-紫外线复合诱变以及常压室温等离子体(ARTP)诱变技术对巨大芽胞杆菌(Bacillus megaterium) ATCC 14945进行处理。处理菌体涂平板后,将长出的菌落接种到液体培养基中,向培养6 h后的二代菌液中添加终浓度为0.1%的苯乙酸,28 °C、250 r/min条件下诱导培养40 h。对离心后获得的上清(粗酶液)采用NIPAB法测定PGA酶活力。以PGA酶活力最高的菌株为材料,对苯乙酸最佳添加量和最佳诱导时间进行优化,采用NIPAB法测定PGA酶活力。采用SDS-PAGE检测诱变前后巨大芽胞杆菌粗酶液中PGA的蛋白特性。【结果】从诱变菌落中筛选到PGA酶活力为39.60 U/mL的菌株12-4,酶活力比出发菌株提高了8.5倍。该菌株在液体培养6 h后添加终浓度为0.2%的苯乙酸,继续培养50 h后,PGA酶活力可达78.45 U/mL,比出发菌株提高了16.8倍。诱变前后菌株培养液中的PGA蛋白均具α、β亚基;诱变后菌株PGA α亚基的量没有明显变化,β亚基的量明显增多;α、β亚基之间的蛋白条带明显增多。【结论】采用诱变技术可提高巨大芽胞杆菌PGA活性,获得的诱变菌株12-4及培养条件对PGA工业化生产具有重要价值。