西藏雅鲁藏布大峡谷国家级自然保护区金猫的色型类别与活动节律

2013-2018年间采用红外相机技术对西藏雅鲁藏布大峡谷国家级自然保护区的金猫(Catopuma temminckii)进行长期监测, 累计布设70个点位, 14,071个相机工作日。共收集金猫独立照片101张, 其中60张可识别色型, 占金猫独立照片总数的59.40%。共记录到4种不同色型的金猫个体, 拍摄频次比例为麻褐色型:红棕色型:黑色型:灰色型 = 46:7:4:3; 其中灰色型为我国野生金猫实体首次记录。不同色型的个体可同域分布, 而以雅鲁藏布大峡谷自然保护区为代表的藏东南地区是我国金猫色型变异最丰富的地区。基于红外相机照片对金猫的共性形态特征和不同色型的独特形态特征进行了详细的图文描述, 以期为该物种的深入研究提供详实的基础资料。活动节律的分析结果显示, 研究区内金猫以昼行性活动为主, 10:00-12:00为其最强的活动高峰。不同月份的日活动差异指数(daily-discrepancy index) α和昼行性指数(diurnal-nocturnal index) β均存在显著差异, 且活动强度具有明显的季节性变化, 不同季节的活动峰型存在显著差异。本研究为东喜马拉雅生物多样性热点区动物多样性本底的完善积累了基础资料, 也为后续的猫科动物色型多样性的形成机制研究提供了基础信息与参考。     

Expression and localization of recombinant human EDG-1 receptors in Pichia pastoris

The receptor for human endothelial differentiation gene-1 protein (EDG-1) was C-terminally tagged with green fluorescent protein and expressed in the methylotrophic yeast, Pichia pastoris. EDG-1 expression was driven by the highly inducible alcohol oxidase 1 promoter. Expression of EDG-1 recombinant protein was detected by Western blot analysis and confocal microscopy. The recombinant EDG-1 receptor protein was located in the plasma membrane. Radioligand binding assays demonstrated that the␣EDG-1 receptors expressed in Pichia pastoris␣have specific and saturation binding of ³²P-labeled sphingosine 1-phosphate.     

Identification and characterization of a novel (+)-γ-lactamase from <Emphasis Type="Italic">Microbacterium hydrocarbonoxydans</Emphasis>

2-Azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) is an important precursor of many carbocyclic nucleoside analogs and pharmaceuticals. (?)-γ-Lactam has attracted much attention because of its role as an intermediate of antiviral drugs such as abacavir and carbovir. (+)-γ-Lactamase can be used for the kinetic resolution of γ-lactam to obtain (?)-γ-lactam. In this study, a novel (+)-γ-lactamase (Mh33H4-5540) was discovered from the gene library of Microbacterium hydrocarbonoxydans based on a colorimetric high-throughput screening method and it could be used to enantioselectively catalyze the bioresolution of racemic γ-lactam with high enantiomeric excess (ee) (>99 %) and yield (>49 %). An unexpected finding was that Mh33H4-5540 was unrelated to other known γ-lactamases (5.7, 4.8, 7.2, and 5.4 % similarities in amino sequence with (+)-γ-lactamase from Comamonas acidovorans, Bradyrhizobium japonicum, Aeropyrum pernix, and Sulfolobus solfataricus, respectively) but rather related to isochorismatases. The homolog analysis of Mh33H4-5540 revealed that it was similar in structure with bacterial isochorismatases (an isochorismatase from Pseudomonas putida (PDB number 4H17) and a putative isochorismatase from Oleispira antarctica (PDB number 3LQY)). Thus, Mh33H4-5540 represented another type of (+)-γ-lactamase. Mh33H4-5540 was overexpressed in E. coli Rosetta (DE3), purified to homogeneity and functionally characterized. The enzyme displayed optimal activity at 25 °C and pH 8.0. The activity showed a 5.5-fold increase in the presence of 0.5 M Ni²⁺ or Co²⁺. Mh33H4-5540 displayed much higher (+)-γ-lactamase activity than any other biochemically characterized (+)-γ-lactamases. Overall, we discovered a novel (+)-γ-lactamase Mh33H4-5540 which displayed the highest activity. It could be a promising candidate of biocatalyst for industrial applications of highly valuable chiral pharmaceutical chemicals.     

西藏墨脱县孟加拉虎种群数量调查

孟加拉虎曾在西藏墨脱县各乡镇均有分布记录,但自2002年后该地区未见确认报道。2013—2018年间,我们利用红外相机技术、信息网络收集法和足迹鉴定法调查了墨脱县孟加拉虎的9个潜在分布区域。结果显示,墨脱县仅存1~3只孟加拉虎非定居个体,仅在旱季(每年10月至来年3月)游荡活动于背崩乡、墨脱镇雅鲁藏布江南岸区,以及格当乡金珠藏布南岸区。该区域内孟加拉虎的保护前景不容乐观,其原因是多方面的。本文提出如下建议以加强针对野生孟加拉虎的保护:(1)继续加强虎的基础性监测;(2)加强针对偷盗猎活动的野外巡护与执法管理;(3)加强对原始森林的保护;(4)改善原住民生计,加强宣传教育,以减少对自然资源的消耗;(5)恢复与建立景观廊道,改善栖息地连通性,以促进虎的自然扩散;(6)加强不同机构与参与方之间的合作研究和技术交流。     

Discovery of a Novel (+)-γ-Lactamase from Bradyrhizobium japonicum USDA 6 by Rational Genome Mining

A novel (+)-γ-lactamase used for the resolution of racemic γ-lactam from Bradyrhizobium japonicum USDA 6 was found as a result of sequence-structure guided genome mining. It consists of 409 amino acids, only 49% of which are identical to the amino acid sequences of the known (+)-γ-lactamase from Sulfolobus solfataricus. This is only the third (+)-γ-lactamase gene to be reported.     

Pathway of Programmed Cell Death and Oxidative Stress Induced by β-Hydroxybutyrate in Dairy Cow Abomasum Smooth Muscle Cells and in Mouse Gastric Smooth Muscle

The administration of exogenous β-hydroxybutyrate (β-HB), as well as fasting and caloric restriction, is a condition associated with β-HB abundance and decreased appetite in animals. Increased β-HB and decreased appetite exist simultaneously in some diseases, such as bovine left displaced abomasums (LDA) and human chronic gastritis. However, the effects of β-HB on stomach injuries have not been explored. To elucidate the possible effects of exogenous β-HB on the stomach, mice were injected intraperitoneally with β-HB, and bovine abomasum smooth muscle cells (BSMCs) were treated with different concentrations of β-HB. We found that β-HB induced BSMCs endoplasmic reticulum- and mitochondria-mediated apoptotic cell death. β-HB promoted Bax expression and caspase-12, -9, and -3 activation while blocking Bcl-2 expression. β-HB also promoted AIF, EndoG release and p53 expression. β-HB acted on key molecules in the apoptotic cell death pathway and increased p38 and c-June NH2-terminal kinase phosphorylation while inhibiting ERK phosphorylation and PCNA expression. β-HB upregulated P27 and P21 mRNA levels while downregulating cyclin and CDK mRNA levels, arresting the cell cycle. These results suggest that BSMCs treated with β-HB can induce oxidative stress, which can be prevented by intracellular calcium chelators BAPTA/AM but not antioxidant NAC. Additionally, these results suggest that β-HB causes ROS generation through a Ca²⁺-dependent mechanism and that intracellular Ca²⁺ levels play a critical role in β-HB -induced apoptotic cell death. The impact of β-HB on programmed cell death and oxidative stress in vivo was confirmed in murine experiments. For the first time, we show oxidative stress effects of β-HB on smooth muscle. We propose that β-HB is a possible cause of some stomach diseases, including bovine LDA.     

Protective effects of exogenous β-hydroxybutyrate on paraquat toxicity in rat kidney

In this study, we demonstrated the protective effects of β-hydroxybutyrate (β-HB) against paraquat (PQ)-induced kidney injury and elucidated the underlying molecular mechanisms. By histological examination and renal dysfunction specific markers (serum BUN and creatinine) assay, β-HB could protect the PQ-induced kidney injury in rat. PQ-induced kidney injury is associated with oxidative stress, which was measured by increased lipid peroxidation (MDA) and decreased intracellular anti-oxidative abilities (SOD, CAT and GSH). β-HB pretreatment significantly attenuated that. Caspase-mediated apoptosis pathway contributed importantly to PQ toxicity, as revealed by the activation of caspase-9/-3, cleavage of PARP, and regulation of Bcl-2 and Bax, which were also effectively blocked by β-HB. Moreover, treatment of PQ strongly decreased the nuclear Nrf2 levels. However, pre-treatment with β-HB effectively suppressed this action of PQ. This may imply the important role of β-HB on Nrf2 pathway. Taken together, this study provides a novel finding that β-HB has a renoprotective ability against paraquat-induced kidney injury.     

Functional expression of cystic fibrosis transmembrane conductance regulator in rat oviduct epithelium

The aim of this study was to investigate the functional expression of cystic fibrosis transmembrane conductance regulator (CFTR) with electrophysiological and molecular technique in rat oviduct epithelium. In whole-cell patch clamp, oviduct epithelial cells responded to 100 microM 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) with a rise in inward current in Gap-free mode, which was inhibited successively by 5 microM CFTR(inh)-172, a CFTR specific inhibitor, and 1 mM diphenylamine-2-carboxylate (DPC), the Cl- channel blocker. The cAMP-activated current exhibited a linear current-voltage (I-V) relationship and time- and voltage-independent characteristics. The reversal potentials of the cAMP-activated currents in symmetrical Cl- solutions were close to the Cl- equilibrium, 0.5+/-0.2 mV (n=4). When Cl- concentration in the bath solution was changed from 140 mM to 70 mM and a pipette solution containing 140 mM Cl- was used, the reversal potential shifted to a value close to the new equilibrium for Cl-, 20+/-0.6 mV (n=4), as compared with the theoretic value of 18.7 mV. In addition, mRNA expression of CFTR was also detected in rat oviduct epithelium. Western blot analysis showed that CFTR protein is found in the oviduct throughout the cycle with maximal expression at estrus, and immunofluorescence and immunohistochemistry analysis revealed that CFTR is located at the apical membrane of the epithelial cells. These results showed that the cAMP-activated Cl- current in the oviduct epithelium was characteristic of CFTR, which provided direct evidence for the functional expression of CFTR in the rat oviduct epithelium. CFTR may play a role in modulating fluid transport in the oviduct.