青海可可西里盆地雅西措组介形类动物群及其地层意义

青海可可西里盆地是青藏高原腹地最大的沉积盆地。然而对于该盆地新生代地层的沉积时代目前仍然存在争议。可可西里盆地南侧雁石坪地区雅西措组中含丰富的介形类化石,该介形类动物群对青藏高原腹地新生代生物地层对比具有重要意义。文中通过对可可西里盆地南侧雁石坪地区通天河剖面进行系统的采样和室内分析,共获得652枚介形类壳体,共鉴定识别出14属48种,自下而上建立了2个介形类组合带,即Austrocypris cf. posticaudata-Candoniella albicans-Leucocythere tropis组合带和Ilyocypris errabundis- Darwinula stenimpudica组合带。通过与柴达木盆地、伦坡拉盆地、江汉盆地以及渤海湾沿岸等地区的介形类生物地层进行区域对比研究,推测可可西里盆地通天河剖面雅西措组的沉积时代为晚始新世—早渐新世。     

Asp271 is critical for substrate interaction with the surface binding site in β-agarase a from Zobellia galactanivorans

In the marine environment agar degradation is assured by bacteria that contain large agarolytic systems with enzymes acting in various endo- and exo-modes. Agarase A (AgaA) is an endo-glycoside hydrolase of family 16 considered to initiate degradation of agarose. Agaro-oligosaccharide binding at a unique surface binding site (SBS) in AgaA from Zobellia galactanivorans was investigated by computational methods in conjunction with a structure/sequence guided approach of site-directed mutagenesis probed by surface plasmon resonance binding analysis of agaro-oligosaccharides of DP 4-10. The crystal structure has shown that agaro-octaose interacts via H-bonds and aromatic stacking along 7 subsites (L through R) of the SBS in the inactive catalytic nucleophile mutant AgaA-E147S. D271 is centrally located in the extended SBS where it forms H-bonds to galactose and 3,6-anhydrogalactose residues of agaro-octaose at subsites O and P. We propose D271 is a key residue in ligand binding to the SBS. Thus AgaA-E147S/D271A gave slightly decreasing K_D values from 625 ± 118 to 468 ± 13 μM for agaro-hexaose, -octaose, and -decaose, which represent 3- to 4-fold reduced affinity compared with AgaA-E147S. Molecular dynamics simulations and interaction analyses of AgaA-E147S/D271A indicated disruption of an extended H-bond network supporting that D271 is critical for the functional SBS. Notably, neither AgaA-E147S/W87A nor AgaA-E147S/W277A, designed to eliminate stacking with galactose residues at subsites O and Q, respectively, were produced in soluble form. W87 and W277 may thus control correct folding and structural integrity of AgaA.     

A novel variant of Thermotoga neapolitana beta-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation

Alkyl glycosides are surfactants with good biodegradability and low toxicity, attractive to produce by an enzymatic method to get a well-defined product. In this paper, we report a novel thermostable variant of a family 3 beta-glucosidase to be an efficient catalyst in alkyl-glucoside forming reactions using transglycosylation with hexanol or octanol as the acceptor molecule. The enzyme has an apparent optimum for hydrolysis at 90 degrees C, which coincides with its unfolding temperature. The total activity is lower at lower temperature (60 degrees C), but the ratio of alcoholysis/hydrolysis is slightly more favourable. This ratio is however more heavily influenced by the water content and the pH. Optimal reaction conditions for hexyl glucoside synthesis from p-nitrophenyl-beta-glucopyranoside were a water/hexanol two-phase system containing 16% (v/v) water, pH 5.8, and a temperature of 60 degrees C. Under these conditions, the total initial reaction rate was 153 micromol min(-1)mg(-1) and the alcoholysis/hydrolysis ratio was 5.1. Comparing with alcoholysis/hydrolysis ratios of other beta-glycosidases, TnBgl3B can be considered to be a very promising catalyst for alkyl glucoside production.     

An improved method to identify BAC clones using pooled overgos

Hybridization using overgo probes is an established approach for screening arrayed bacterial artificial chromosome (BAC) libraries. We have improved the use of overgos by increasing the yield of positive clones using reduced levels of radioisotopes and enzyme. The strategy involves labeling with all four radiolabeled nucleotides in a hot pulse followed by a cold nucleotide chase and then extending the exposure time to compensate for reduced specific activity of the probes. The resulting cost savings and reduced human exposure to radiation make the use of highly pooled overgo probes a more attractive approach for screening of BAC libraries from organisms with large genomes.     

光质对韭菜碳氮代谢、生长和品质的影响

以‘平韭2号’为试材,以白光(W)处理为对照(CK),研究白+红(WR)、白+蓝(WB)、白+绿(WG)、白+紫(WP)4种不同光质处理对韭菜碳氮代谢、生长和品质的影响.结果表明: WR处理韭菜的光合速率(P_n)显著高于CK,而WB、WG和WP与CK无显著差异,但RuBP羧化酶(RuBPCase)活性均显著高于CK.各处理相比,总糖含量以WR最高,WP其次,WB和WG最低.WR下韭菜蔗糖磷酸合成酶(SPS)活性显著高于CK,其余三者均显著低于CK,其中以WB最低,但蔗糖合成酶(SS)和淀粉酶(AMS)活性均以WB最高,WR最低,WG和WP的SS活性与CK无差异,但AMS活性显著低于CK.可见,增加红、紫光比例可促进韭菜碳的同化和转化,加速糖的积累.总氮、蛋白氮含量、硝酸还原酶(NR)、谷氨酰胺酶(GS)和谷氨酸合成酶(GOGAT)活性以WB最高,显著高于CK,但WB下谷氨酸脱氢酶(GDH)活性最低,显著低于CK;WR的蛋白氮和非蛋白氮含量、NR和GS活性最低,显著低于CK,GOGAT和GDH活性显著高于CK,且GDH在所有处理中最高;WG的全氮、非蛋白氮含量及GDH活性均显著低于CK,但蛋白氮含量、NR和GOGAT活性显著高于CK,WP的氮含量及相关酶活性呈现出与WG相同的趋势,且GS活性亦同样高于CK.说明增加蓝光、紫光和绿光可使韭菜氮代谢增强,而增加红光对蛋白质的合成有一定的抑制作用.红光和紫光下韭菜生长较好,而蓝光则抑制其加粗生长,叶片较薄,单株生长量较低.紫光下韭菜的粗纤维含量最低,营养品质最优,因此,紫光对韭菜的生长最有利.     

2型猪链球菌葡萄糖胺-6-磷酸脱氨酶基因的克隆表达及酶活性测定

目的:克隆表达2型猪链球菌葡萄糖胺-6-磷酸脱氨酶(NagB)编码基因,并测定其酶反应体系活性。方法:根据2型猪链球菌05ZYH33基因组序列,全合成nagB基因(ssu05_0195),并将其克隆至pET32a载体,在大肠杆菌中表达;利用Ni亲和层析柱纯化表达产物,获得纯化的NagB蛋白,Western印迹鉴定后测定其酶反应体系活性。结果:在大肠杆菌中高效表达了nagB基因,重组NagB相对分子质量约为56×10~3;在25℃、pH9.5、底物浓度为15 mmol/L、反应40 min时,NagB酶促反应体系表现出最大活性;在最适条件下,2型猪链球菌中NagB酶促反应体系的体外活性为3.73 U/mL,酶比活为12.43 U/mg。结论:2型猪链球菌05ZYH33中含有编码NagB的nagB基因,在原核系统中表达的NagB蛋白具有酶学活性。     

Triazole‐Pyridine Dicarbonitrile Targets Phosphodiesterase 4 to Induce Cytotoxicity in Lung Carcinoma Cells

Phosphodiesterase 4 (PDE4) is a key enzyme involved in the hydrolysis of cyclic adenosine monophosphate (cAMP) and widely expressed in several types of cancers. The inhibition of PDE4 results in an increased concentration of intracellular cAMP levels that imparts the anti‐inflammatory response in the target cells. In the present report, two series of triazolo‐pyridine dicarbonitriles and substituted dihydropyridine dicarbonitriles were synthesized using green protocol (TBAB in refluxed water). We next evaluated the title compounds for their cytotoxicity towards lung cancer (A549) cells and identified 7′‐[4‐(methylsulfonyl)phenyl]‐5′‐oxo‐1′,5′‐dihydrospiro[cyclohexane‐1,2′‐[1,2,4]triazolo[1,5‐a]pyridine]‐6′,8′‐dicarbonitrile ( 5h ) and 7′‐(1‐methyl‐1H‐imidazol‐2‐yl)‐5′‐oxo‐1′,5′‐dihydrospiro[cyclohexane‐1,2′‐[1,2,4]triazolo[1,5‐a]pyridine]‐6′,8′‐dicarbonitrile ( 5j ) as lead analogs with the IC₅₀ values of 15.2 and 24.1 μm , respectively. Furthermore, all the new compounds were tested for PDE4 inhibitory activity and 5j showed relatively good inhibitory activity towards PDE4 with inhibition of 50.9 % at 10 μm . In silico analysis demonstrated the favorable interaction of the title compounds with the target enzyme. Taken together, the present study introduces a new scaffold for the development of novel PDE4 inhibitors to fight against inflammatory diseases.     

不同剂量白藜芦醇对糖尿病性白内障大鼠晶状体抗氧化酶活力的影响

目的:探究不同剂量白藜芦醇对糖尿病性白内障大鼠晶状体抗氧化酶活力的影响。方法:75只5周龄健康SPF级雄性SD大鼠按照随机数字表法分为正常对照组、模型组,白藜芦醇低剂量组,白藜芦醇中剂量组和白藜芦醇高剂量组,每组各15只。五组大鼠均给予常规适应性喂养,模型组和白藜芦醇低、中、高剂量组大鼠采用链脲佐菌素(STZ)以60 mg/kg的给药剂量制作糖尿病大鼠模型,成模后白藜芦醇低剂量组按20 mg/kg、白藜芦醇中剂量组按50 mg/kg、白藜芦醇高剂量组按100 mg/kg的给药剂量每日给予白藜芦醇灌胃。观察12周后5组大鼠晶状体的混浊程度,检测血糖、体重后处死大鼠,检测晶状体内超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)以及过氧化氢酶(CAT)的活性改变。结果:随着白藜芦醇剂量的升高,白藜芦醇低、中、高剂量组大鼠的血糖逐渐降低而体重逐渐升高,且组间比较均具有统计学差异(P0.05)。三组不同剂量白藜芦醇干预组大鼠晶状体的浑浊程度均低于模型组,且白藜芦醇高剂量组大鼠晶状体的浑浊程度最低,差异均具有统计学意义(P0.05)。白藜芦醇低、中、高剂量组大鼠SOD、GSH-PX和CAT酶活力与模型组大鼠相比均明显升高,而与正常对照组相比均明显降低(均P0.05)。随着白藜芦醇剂量的升高,白藜芦醇低、中、高剂量组大鼠SOD、GSH-PX和CAT酶活力逐渐升高,且组间比较均具有统计学差异(P0.05)。结论:高剂量白藜芦醇可更为明显地降低血糖浓度,提高晶状体SOD、GSH-Px及CAT酶活力,改善糖尿病性白内障晶状体的浑浊程度。