松材线虫携带一株荧光假单胞菌分泌毒素的初步研究

本文研究了离体情况下松材线虫携带的致病菌一株荧光假单胞菌(Pseudomonas fluorescens GcM5-1A)在LB、NB和PD三种培养基中的毒性, 以及产生的毒素对黑松(Pinus thunbergii)切根苗和悬浮细胞的效应。结果显示, 菌体在LB和NB 培养液的毒性较高, 其中 LB培养液的毒性最高, 且培养液的pH值为7时比pH值为5时毒性高, 而该菌在PD培养基中几乎不产毒。细菌培养液经硫酸铵分级沉淀, 得到了主要含有50 kDa蛋白的蛋白组分, 该蛋白组分对黑松悬浮细胞和切根苗均有较高的毒性, 并能改变黑松悬浮细胞细胞膜的透性, 导致胞内可溶性糖和游离氨基酸外渗。     

荧光假单胞菌鞭毛蛋白对黑松细胞的致死作用

采用细胞免疫荧光分析、细胞染色观察、电导率测定以及基因组DNA电泳分析技术,研究了松材线虫携带的荧光假单胞菌分泌的鞭毛蛋白对黑松愈伤组织细胞的作用及其致死方式。结果表明,鞭毛蛋白与黑松细胞之间存在直接的相互作用;鞭毛蛋白处理的细胞,细胞膜皱缩变形、细胞质浓缩、细胞核解体,形成若干小核,细胞质中的RNA降解;处理细胞的着色力增强,细胞培养液的电导率增加,说明鞭毛蛋白可增加处理细胞的细胞膜透性;基因组DNA的电泳分析证实,处理细胞的DNA发生了断裂,但无明显的梯子形成,推测鞭毛蛋白对黑松细胞的致死方式为非典型的细胞凋亡。     

松材线虫的天然毒素研究进展

概述了松材线虫病的现行防治措施及存在问题,介绍了植物源和真菌源天然毒素毒杀松材线虫的研究现状及研究中遇到的问题,指出天然毒素在未来松材线虫病生物防治中的研究方向。     

沼泽红假单胞菌对微囊藻毒素的降解

以铜绿微囊藻为材料,通过固相萃取-高效液相色谱方法(SPE-HPLC),研究了沼泽红假单胞菌对微囊藻毒素MC-LR的降解作用。结果表明:沼泽红假单胞菌在厌氧、光照强度2000lx、35℃、pH7.0、乙酸钠为碳源、菌液初始浓度OD680为0.325和初始MC-LR为3mg.L-1时,6d降解率为36.5%,12d达到最高,降解率为78.7%。此降解条件和蓝藻水华爆发的环境条件基本一致,因此该菌株在水华爆发季节消除水中的微囊藻毒素方面具有应用潜力。     

荧光假单胞菌的条件致病性

作者从血、尿标本中分离出12株荧光假单胞菌,在研究生物学性状的基础上探讨了本菌的条件致病性问题,结果显示,凡能使机体免疫力下降的各种原因均可导致本菌感染。治疗本菌感染,必须进行药敏试验,选择有效的抗生素。     

丁香假单胞菌环式脂肽毒素的生理和分子生物学研究

综合评述了近１０年来在丁香假单胞菌脂肽毒素生理和分子生物学研究上的发现。这些毒素依肽部ＡＡ数目可分两组。丁香假单胞霉素组（ｓｙｒｉｎｇｏｍｙｃｕｎｓ）已报告４个成员,肽部有９个ＡＡ;丁香假单胞肽毒素组有２个成员,肽部分别有２２个和２５个ＡＡ。肽部Ｃ－端羧基与分子内羟基氨基酸残基（ＡＡ）的羟基酯化闭合成环,再由羟基脂肪酸酰化。两组毒素都诱导植物电解质渗漏、人和动物红血球溶解,其机制在于在细胞膜上形成     

一株红假单胞菌分离及对幼鳖作用的初步研究

从露天养鳖池中分离到红假单胞菌CZ-2菌株。该菌具有生理需求简单、培养条件粗放、生长迅速的特点。在黑暗,好氧和光照/厌氧条件下均可良好生长,最适条件下培养6d,生物量可以达到1．57g/L。在温室幼鳖的饲料中添加CZ-2菌株培养液后幼鳖没有出现直接死亡,发病率也较对照池低50％左右。试验表明CZ-2菌株培养液对幼鳖生长有促进作用,比对照提高了8．9％。该文对CZ-2菌株培养特性及对幼鳖作用作了探讨。     

假单胞菌降解微囊藻毒素的效能及酶作用机理

以从福州市某富营养化水库底泥中筛选出的假单胞菌M-6为研究对象,分别考察接种量、温度、供氧量(转速)、pH值、微囊藻毒素MCLR初始浓度等条件因素对假单胞菌M-6降解MCLR的影响,并探讨假单胞菌M-6降解MCLR的分子生物学机理.结果表明,当接种量为10%时,菌体的降解效果较好,6d的降解率可达90%;降解反应的最适pH为7.0,最佳温度为30℃,摇床转速控制在150 r/min;MCLR初始浓度过高都将影响假单胞菌M-6对碳源和氮源的吸收和利用,最佳浓度为15.7 mg/L.SDS-PAGE电泳分析表明,假单胞菌M-6降解藻毒素过程中,主要有3种酶参与反应,且这3种酶都是细胞内本身所含有的组织酶.     

Two Cyclic Dipeptides from Pseudomonas fluorescens GcM5-1A Carried by the Pine Wood Nematode and Their Toxicities to Japanese Black Pine Suspension Cells and Seedlings in vitro

Pseudomonas fluorescens GcM5-1A, isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, was cultured in Luria Broth medium (LB). The clarified culture was extracted with ethyl acetate, and two dipeptides were purified from the extract. The chemical structures of 1 and 2 were identified as cyclo(-Pro-Val-)and cyclo(-Pro-Tyr-), respectively, by MS, ¹H NMR, ¹³C NMR,¹H-¹H COSY, 1H -¹³C COSY spectra. Bioassay results showed that the two compounds were toxic to both suspension cells and seedlings of Pinus thunbergii, which may offer some clues to research the mechanism of pine wilt disease caused by PWN.     

荧光假单胞菌天冬氨酸转氨酶的基因克隆及其在大肠杆菌中的表达

采用克隆基因测序技术,从荧光假单胞菌GcM5-1A基因组文库中筛选到了天冬氨酸转氨酶的编码基因aspC。通过聚合酶链式反应（PCR）扩增目的基因,插入pET-15b构建重组表达质粒pET-15bAAT,转化E.coli BL21(DE3),IPTG诱导天冬氨酸转氨酶在大肠杆菌中高效表达,利用亲和层析法初步分离纯化了重组蛋白。生物活性分析表明,纯化的重组天门冬氨酸转氨酶具有氨基转移活性。     

LncRNA OIP5-AS1 regulates radioresistance by targeting DYRK1A through miR-369-3p in colorectal cancer cells

Object

This study aimed to investigate the role of lncRNA OIP5-AS1 in regulating radioresistance of colorectal cancer (CRC) cells.

MicroRNA-340-5p increases telomere length by targeting telomere protein POT1 to improve Alzheimer's disease in mice

Alzheimer′s disease (AD) is a chronic neurodegenerative disorder which is the primary cause of dementia in the elderly. Telomere attrition has been proposed as a hallmark of aging. Our study aimed to explore the mechanism of the protection of telomere 1 (POT1) in regulating telomere length and affecting cellular senescence in AD. The AD mouse model was established by d -galactose and aluminum chloride, and the water maze test and dark avoidance test were used to detect the behaviors of mice and confirm the success of AD mouse model. AD cell model was established with HT22 cells induced by Aβ₄₂ oligomers. POT1 expression in the AD model was detected by quantitative real-time polymerase chain reaction. Cellular telomere length in hippocampal tissue was analyzed by telomere restriction fragment. Localization of intracellular POT1, telomerase, and telomeres was analyzed by immunofluorescence and fluorescence in situ hybridization. Dual-luciferase assay was used to validate the targeted binding relationship between microRNA-340-5p (miR-340-5p) and POT1. After inhibiting POT1 expression, the symptoms of AD in mice were improved. Aβ_1–42 deposition was reduced, whereas telomere length and telomerase activity was increased. Dual-luciferase assay verified the binding relationship between miR-340-5p and POT1. An increase in miR-340-5p expression could alleviate cellular senescence and AD symptoms. miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms. This study made a conclusion that miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms in mice.     

Silencing circ-USP1 protects the renal tubular from kidney injury induced by hypoxia via modulating miR-194-5p/DNMT3A axis in acute renal allografts

Recent studies indicate that circular RNAs are involved in dysregulation of kidney injury. Nevertheless, the underlying mechanisms remain largely unclear. Therefore, this study sought to investigate the role of circ-USP1 in the pathogenesis of early renal allografts. Thirty-two male C57BL/6J mice aged between 6 and 8 weeks were divided into the sham and allograft groups. Thereafter, the association between miR-194-5p, circ-USP1 and DNMT3A was confirmed using a combination of bioinformatics and the luciferase reporter gene assay. Additionally, the expression of circ-USP1, miR-194-5p and DNMT3A mRNA was detected through qPCR. Afterwards, the Western blot assay was performed to examine the expression of DNMT3A protein. Finally, the TUNEL assay was conducted to determine the rate of apoptosis in DNMT3A cells. The expression of circ-USP1 increased, while that of miR-194-5p decreased in renal allografts. Additionally, silencing circ-USP1 reduced kidney injuries caused by renal allografts in mice. Moreover, miR-194-5p was a target for circ-USP1, and DNMT3A was a target of miR-194-5p. Finally, it was shown that silencing circ-USP1 reduced DNMT3A expression in the kidney of mice that received renal allografts. Circ-USP1 functions as a competing endogenous RNA for miR-194-5p. This occurs in order to regulate DNMT3A expression in kidney injury induced by hypoxia in acute renal allografts.     

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.