Roles of phospholipase D in phorbol myristate acetate‐stimulated neutrophil respiratory burst

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10‐fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA‐stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA‐stimulated respiratory burst. Using 1‐butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA‐stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA‐stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA‐stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA‐stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2‐dioctanoyl‐sn‐glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA‐stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA‐stimulated respiratory burst.     

Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes

The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K⁺ current and decreased the inward basal I Ca significantly. The reversal potential of both carbachol-and antibody-induced extra currents were close to –80 mV, being in proximity to the calculated E_k of –90 mV. A -adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward I_ca and outward I_k simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated I_Ca but not the isoprenaline-stimulated I_k. The antibody- or carbachol-induced outward K⁺ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated I_ca were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.     

Purification and characterization of 2,6-dichloro-p-hydroquinone chlorohydrolase from Flavobacterium sp. strain ATCC 39723.

The biochemistry of pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723 has been studied, and two enzymes responsible for the conversion of PCP to 2,6-dichloro-p-hydroquinone (2,6-DiCH) have previously been purified and characterized. In this study, enzymatic activities consuming 2,6-DiCH were identified from the cell extracts of strain ATCC 39723. The enzyme was purified to apparent homogeneity by a purification scheme consisting of seven steps. Gel filtration chromatography showed a native molecular weight of about 40,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 42,500 Da. The purified enzyme converted 2,6-DiCH to 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene), which was easily oxidized by molecular oxygen and hard to detect. The end product, 6-chlorohydroxyquinol, was detected only in the presence of a reductase and NADH in the reaction mixture. The enzyme dechlorinated 2,6-DiCH but not 2,5-DiCH. The enzyme required Fe2+ for activity and was severely inhibited by metal chelating agents. The optimal conditions for activity were pH 7.0 and 40 degrees C. The Kcat for 2,6-DiCH was 35 microM, and the kcat was 0.011 s-1.     

Purification and characterization of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp.

Tetrachloro-p-hydroquinone (TeCH) is the first intermediate in pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723. We previously purified a PCP hydroxylase that oxidized PCP to TeCH. Subsequently, we identified the reductive dehalogenation of TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone in a cell extract with the reduced form of glutathione as the reducing agent under anaerobic conditions. Here we report the purification of a TeCH reductive dehalogenase that reductively dehalogenated TeCH to trichlorohydroquinone and then to dichlorohydroquinone. The enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, and phenyl-agarose, anion-exchange, and gel filtration column chromatographies. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the protein has a molecular weight of about 30,000; nondenaturing polyacrylamide gel electrophoresis analysis suggests that the native enzyme exists as a dimer. The enzyme used glutathione but not NADPH, NADH, dithiothreitol, or ascorbic acid as the reducing agent. The optimal pH was close to neutral.     

一株兔源A型多杀性巴氏杆菌的分离鉴定和全基因组测序及分析

【背景】多杀性巴氏杆菌可导致猪肺疫、牛出血性败血症和兔出血性败血症等多种疾病,严重威胁多种动物畜牧养殖业的健康发展。【目的】重庆某兔场送检一批病死兔,为研究其病原和治疗方法,对病原进行了微生物分离和全基因组测序分析。【方法】从2022年重庆某兔场送检兔病料中进行细菌分离纯化、生化试验、16S rRNA基因鉴定、荚膜血清型分型、药敏试验和毒力基因检测,同时通过全基因组测序结果进行毒力、耐药基因注释和遗传进化等分子生物学信息分析。【结果】该菌为兔源A:ST74多杀性巴氏杆菌,命名为LXSS001,基因组序列上传到NCBI数据库(登录号为CP119523.1),药敏试验显示该菌对四环素、杆菌肽、复方新诺明和磺胺异恶唑耐药,对头孢噻肟、头孢哌酮和丁胺卡那等药物敏感。全基因组长度为2 480 671 bp,并注释到了58个毒力基因和9类药物的靶向抗药基因。通过联合建树表明其与3480株一致性最高。【结论】本研究完成了一株A型多杀性巴氏杆菌的分离鉴定和全基因组测序,并揭示了其与国内外其他分离株的进化关系,为多杀性巴氏杆菌的后续研究提供了参考依据。     

Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells

The recent COVID-19 pandemic poses a global health emergency. Cellular entry of the causative agent SARS-CoV-2 is mediated by its spike protein interacting with cellular receptor-human angiotensin converting enzyme 2 (ACE2). Here, by using lentivirus based pseudotypes bearing spike protein, we demonstrated that entry of SARS-CoV-2 into host cells was dependent on clathrin-mediated endocytosis, and phosphoinositides played essential roles during this process. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 were not required for efficient virus entry. Finally, we showed that the current predominant Delta variant, although with high infectivity and high syncytium formation, also entered cells through clathrin-mediated endocytosis. These results provide new insights into SARS-CoV-2 cellular entry and present proof of principle that targeting viral entry could be an effective way to treat different variant infections.     

云南含笑花粉萌发研究

应用离体培养和人工授粉的方法对云南含笑花粉的萌发进行了研究。栽培和野生云南含笑花粉的萌发率有很大的差异,分别为20％和90％。首次报道了云南含笑的花粉粒在离体培养基上萌发2条花粉管的现象。云南含笑花粉在灰岩含笑花柱头上的萌发率和萌发时间与离体培养基上的相同,表明灰岩含笑的柱头对云南含笑的花粉没有排异现象。     

Copper-induced germline apoptosis in Caenorhabditis elegans: The independent roles of DNA damage response signaling and the dependent roles of MAPK cascades

Excess copper is toxic to life. Copper has been shown to induce apoptosis in various cell lines and tissues. However, due to the lack of appropriate gene knockout animal models, data concerning the underlying pathways of copper-induced apoptosis are insufficient, especially with regards to in vivo systems. The nematode Caenorhabditis elegans is a good model to study basic biological processes, including stress responses and apoptosis. In the present study, we investigated copper-induced germline apoptosis in the C. elegans strains carrying mutated alleles of homologs to known mammalian genes that are involved in apoptosis regulation. We show here that exposing C. elegans to copper causes dose- and time-dependent germline apoptosis. The knockout of checkpoint genes hus-1, clk-2, the Bcl-2 homolog ced-9, and the BH3-only domain egl-1 did not prevent cells of the germline from copper-induced apoptosis. The loss-of-function of the tumor suppressor gene, p53/cep-1, caused a significant increase in germline apoptosis with exposure to copper, and the depletion of p53 antagonist ABL1 significantly enhanced apoptosis. The knockout of the caspase gene ced-3 and the Apaf-1 homolog ced-4 abrogated both copper-induced and physiological germline apoptosis. Germline apoptosis stopped increase in the strains lin-45(ku51), mek-2(n1989), mpk-1(ku1) under copper stresses, respectively. Copper-induced apoptosis was blocked in the loss-of-function alleles of both JNK and p38 MAPK cascades excepting pmk-3, one of the three p38 MAPK components. Together, the results of this study suggest that caspase and Apaf-1 are required for copper-induced germline apoptosis while DNA damage response genes are not essential, and that the Raf-MEK-ERK, ASK1/2-MKK7-JNK, ASK1/2-MKK3/6-p38 signaling pathways are indispensable in mediating this apoptotic response.     

昆虫飞行速度和持续时间监测仪硬件设计

本文设计了利用微机记录昆虫飞行状态的实验装置,这种装置由两部分组成,一部分是设计和制作一个飞行磨供昆虫飞行,另一部分是制作光电传感器和微机检测系统,记录信号并把信号进行识别、分析、归类,送往计算机显示和打印。介绍了其工作原理和使用方法。     

Revisit on the evolutionary relationship between alternative splicing and gene duplication

Gene duplications and alternative splicing (AS) isoforms are two widespread types of genetic variations that can facilitate diversification of protein function. A number of studies claimed that after gene duplication, two AS isoforms with differential functions can be 'fixed', respectively, in each of the duplicate copies. This simple 'functional-sharing' hypothesis was recently challenged by Roux and Robinson-Rechavi (2011). Instead, they proposed a more sophisticated hypothesis, invoking that less alternative splicing genes tend to be duplicated more frequently, and single-copy genes are younger than duplicate genes, or the 'duplicability-age' hypothesis for short. In this letter, we show that all these genome-wide analyses of AS isoforms actually did not provide clear-cut evidence to nullify the basic idea of functional-sharing hypothesis. After updating our understanding of genome-wide alternative splicing, duplicability and CNV (copy number variation), we argue that the foundation of the duplicability-age hypothesis remains to be justified carefully. Finally, we suggest that a better approach to resolving this controversy is the correspondence analysis of indels (insertions and deletions) between duplicate genes to the genomic exon-intron structure, which can be used to experimentally test the effect of functional-sharing hypothesis.