Biosynthesis of Depsipeptide Mycotoxins in Fusarium

The cyclic hexadepsipeptide enniatin is known as a phytopathogenic compound from Fusaria causing necrosis and wilt. The molecule consists of three alternating residues each of a branched chain amino acid and D-hydroxyisovaleric acid (D-Hiv). Enniatins are synthesized by a 347kDa multienzyme (enniatin synthetase) via a thiol template mechanism. The corresponding gene esyn1 has an open reading frame of 9393 nucleotides and harbours two modules, one responsible for D-hydroxy acid activation and one for L-amino acid activation with an integrated N-methyltransferase domain. Such methyltransferases build an homologous group among N-methyl peptide synthetases. Enniatins are synthesized by step-wise condensation of dipeptidol building blocks in an iterative manner resembling fatty acid synthesis. A key enzyme in enniatin biosynthesis is the NADPH-dependent D-2-hydroxyisovalerate dehydrogenase, that supplies enniatin synthetase with D-Hiv. Enniatins contribute to the wilt toxic character of Fusaria. Virulence was significantly reduced in F. avenaceum after disruption of the esyn1 gene.     

Carrier rate of the c.235G>C mutation in the bovine isoleucyl-tRNA synthetase (IARS) gene of Japanese Black cows at Kagoshima prefecture, Japan,and analysis of the metabolic profiling and reproductive performance of heterozygous cows

Bovine isoleucyl-tRNA synthetase (IARS) disorder, a major cause of weak calf syndrome, is caused by a homozygous missense (c.235G>C) mutation in the bovine IARS gene of Japanese Black (JB) cattle, which was identified in 2013. However, the extent to which the carrier rate has changed at Kagoshima prefecture, Japan, and whether the carrier status is associated with any clinical or reproductive problems, have yet to be ascertained. In this study, using a real-time polymerase chain reaction-based genotyping assay, we determined the carrier rate in a regional JB cow population at Kagoshima prefecture. Comparative analyses were performed on the metabolic profile test (MPT) results and reproductive performance data obtained for heterozygous carrier and homozygous wild-type cows. In 2009 and 2018, DNA samples were collected from 130 and 462 clinically healthy JB cows, respectively, in Kagoshima prefecture. MPT results and reproductive performance data were evaluated for 62 cows, comprising four heterozygous carriers and 58 wild-type cows. Genotyping revealed that the carrier rate was 6.9% in 2009 and 1.5% in 2018, the difference of which was statistically significant (P<0.005). There were no statistically significant differences between the carrier and wild-type cows with respect to either MPT results or reproductive performance, indicating that the carrier cows have necessary IARS activity to maintain minimal health and reproductive potential.     

玉米果穗发育的生理特性研究

为了探讨玉米优良自交系选育中出现短苞叶品种的原因，从生理角度分析了两玉米自交系品种穗轴与苞叶生长发育变化趋势。结果表明：四个时期中139号苞叶硝酸还原酶活性非常低，对照1号的稍高一些。139号苞叶谷氨酰胺合成酶活性小于1号，且1号的酶活性变化较平缓。139号雌穗轴硝酸还原酶活性变化剧烈，授粉后15d达高峰，明显大于1号。139号雌穗轴谷氨酰胺合成酶活性前三时期始终大于1号，最后一时期二者几乎相等，但1号的酶活性变化平缓。就上述结果从氮代谢方面分析了139号苞叶与穗轴发育失调的原因。     

Effect of smoking on GSH and γ-GCS in lungs of rats and the protective role of N-acetylcysteine

AIM: To investigate the effects of cigarette smoking on the GSH and γ-GCS in SD rats, and the protective role of N-acetylcysteine (NAC). METHODS: SD rats were exposed to cigarette smoke, and GSH concentrations in BALF were measured by GSH-specific DTNB-GSH reductase recycling assay. Expression of γ-GCS protein in lung tissue was determined by immunohistochemistry. RESULTS: Exposure to cigarette smoking increased GSH content in BALF and the expression of γ-GCS protein in the lung tissue of SD rats. CONCLUSION: Cigarette smoking affects intracellular GSH concentration and the expression of γ-GCS protein in lungs of SD rats. NAC attenuates the inflammatory reaction resulting from cigarette smoking.     

The levels of 2′, 5′ oligoadenylate synthetase,interleukin 2 and interleukin 12 in hepatitis B virus infection

AIM: In order to study the effect of endogenous interferon system and Th1 response modes on hepatitis B virus infection, the 2′, 5′ oligoadenylate synthetase (2-5OAS), IL-2 and IL-12 were selected as the research parameters. METHODS: The activity of 2-5OAS in peripheral blood mononeuclear cells was determined by sensitive radioenzymatic assay. IL-2 and IL-12 were determined by ELISA. RESULTS: Compared to normal control, the 2-5OAS, IL-2 or IL-12 were not significantly changed (P>0.05) in the asymptomatic HBsAg carricer group. The 2-5OAS, IL-2 and IL-12 were significantly up-regulated (P<0.01) in the group of acute hepatitis, but in the groups of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, the 2-5OAS, IL-2, IL-12 were significantly down-regulated (P<0.05). Moreover, with the progression of patient′s conditions and with the complications of liver cirrhosis and hepatocellular carcinoma, the 2-5OAS, IL-2 and IL-12 decreased progressively, the 2-5OAS, IL-2, IL-12 were the lowest in guoups of liver cirrhosis and hepatocellular carcinoma (vs each groups of chronic hepatitis, P<0.05). CONCLUSION: The endogenous interferon system and Th1 response are significantly alterable in the different period of hepatitis B virus infection and among the different clinical types. The cellular immunity plays an important role in recovery from HBV infection.     

蜡样芽孢杆菌致吐毒素的毒性作用与生物合成研究进展

蜡样芽孢杆菌是一类兼性厌氧的革兰氏阳性杆菌,可以产生芽孢抵抗不良环境,并广泛存在于土壤、水、空气和多种食物中。致病性蜡样芽孢杆菌是常见的食源性条件致病菌之一,其引发的食物中毒主要是由其产生的毒素导致的。致吐毒素cereulide是致病性蜡样芽孢杆菌产生的重要毒素之一,是一种小分子亲脂性十二环肽,结构性质十分稳定。Cereulide能够引起恶心、呕吐等轻微的食物中毒症状,也可导致如肝性脑病、急性肝脏衰竭等严重致死的疾病。当前对于cereulide的毒性作用机制研究局限于其刺激传入迷走神经引起呕吐症状,以及作为钾离子载体,诱导线粒体膜电位丧失,并最终导致细胞死亡,而对于其导致的严重肝脏和脑部病变的毒性作用机制研究仍十分不足。Cereulide是由cereulide合成酶基因簇(ces)编码,非核糖体肽合成酶(nonribosomal peptide-synthetase, NRPS)系统控制合成的。Cereulide由两个羟基酸和两个氨基酸残基[-D-HIC-D-Ala-L-HIV-L-Val-]经3次迭代组成三聚体缩酚酞,其结构特殊并有很强的代表性,但因NRPS合成系统的灵活性会产生许多变体,因此cereulide的毒性与其生物合成过程息息相关。文章在现有文献报道和研究数据的基础上,总结并提出了cereulide的生物合成机理：首先,cereulide合成基因簇的CesA和CesB结构域分别识别D-α-酮羧酸、L-丙氨酸、L-α-酮异戊酸和L-缬氨酸,通过共价结合形成cereulide的主要合成单元二肽;其次,依照上述过程重复合成四肽;再通过重复反应合成第二个四肽,两个四肽通过酯化形成八肽;再次重复上述反应,形成三元络合的产物肽;最后,由于ces-NRPS的硫酯酶结构域活性中心表面结构阻止外部水分子进入,并诱导内部亲核攻击反应,最终释放出环状cereulide。目前,由产cereulide的蜡样芽孢杆菌引起的食物中毒风险被低估。并且,本团队前期研究发现部分芽孢杆菌微生态制剂中混有产cereulide的蜡样芽孢杆菌菌株。因此,产cereulide蜡样芽孢杆菌的存在对食品安全和公共健康均构成了潜在风险。文章综述了cereulide的毒性作用及机制,为进一步研发cereulide防控措施提供科学依据;总结并提出了cereulide的生物合成机理,强调了催化酮酸形成酯的酮还原酶域(KR),及形成重复单元和环肽的硫酯酶域(TE)在其合成中的重要作用,为阐明类似结构的非核糖体肽合成提供新的思路。     

Role of ACSL3 expression in suppressing prostate cancer cell metastasis

AIM:To analyze the difference of long-chain acyl-CoA synthetase 3 (ACSL3) expression between normal prostate epithelial cells and prostate cancer cells.METHODS:ACLS3 mRNA expression between normal prostate epithelial cells and prostate cancer cells was compared using RT-PCR. Meanwhile, ACSL3 gene was amplified from prostate cancer cells, and the eukaryotic expression plasmid pCDNA3.1(+)-Flag-ACLS3 and lentivirus Lenti-ACLS3 were constructed. After transfection of ACSL3-plasmid and lentivirus into the prostate cancer cells, ACSL3 expressive was detected by RT-PCR and Western blotting, and then Matrigel invasion assay was performed to investigate the alteration of the invasive ability of the prostate cancer cells with over-expression of ACSL3. RESULTS:A significant difference of ACSL3 mRNA level between normal prostate epithelial cells and prostate cancer cells was observed. ACSL3 was highly expressed in localized prostate cancer cells compared to metastatic prostate cancer cells, while ACSL3 expression was higher in androgen-dependent prostate cancer cells than that in androgen-independent prostate cancer cells. Furthermore, the eukaryotic expression plasmid and lentivirus containing ACLS3 gene were successfully constructed. The prostate cancer cell line which stably over-expressed ACLS3 was established. Up-regulation of ACSL3 inhibited the invasive ability of prostate cancer cells. CONCLUSION:ACSL3 plays an antagonistic role in invasiveness of prostate cancer.     

Functional analysis of the venom allergen-like protein gene from pine wood nematode Bursaphelenchus xylophilus using a baculovirus expression system

The pine wood nematode, Bursaphelenchus xylophilus, infects pine trees, leading to fatal pine wilt disease. Here, recombinant venom allergen-like protein (VAP) was obtained by expressing Bx-vap-1 in insect cells. Three-year-old Pinus massoniana were inoculated with recombinant VAP, simulating B. xylophilus esophageal gland secretions. Recombinant VAP up-regulated α-pinene synthase gene expression, the trees showed disease symptoms 15 d after inoculation and the xylem pith revealed brown tissue discoloration, indicating that recombinant VAP could damage P. massoniana cells. Recombinant VAP did not, however, lead to cavitation, indicating that the VAP secreted from B. xylophilus acts as a defense response elicitor.     

氮素不同形态配比对菠菜体内游离氨基酸含量和相关酶活性的影响

采用溶液培养试验,研究了氮素不同形态配比对菠菜茎叶中游离氨基酸含量及3种主要氮代谢酶活性的影响。结果表明:1)随着营养液中铵硝比(NH4+-N/NO3--N)的降低,菠菜茎叶中游离氨基酸的总量呈下降趋势。在全硝营养下(NH4+-N/NO3--N=0∶100)下,菠菜茎叶中游离氨基酸的总量只有全铵营养(NH4+-N/NO3--N=100∶0)的34.4%。2)在全铵营养下,菠菜茎叶中游离氨基酸的主要组分是谷氨酰胺、精氨酸和谷氨酸,三者占游离氨基酸总量的百分比依次为39.8%、20.2%和8.9%;在全硝营养下,菠菜茎叶中游离氨基酸以谷氨酸、天冬氨酸和丝氨酸为主,三者占游离氨基酸总量的百分比分别为30.3%1、8.6%和8.5%。3)提高营养液中硝态氮的比例,可以显着提高菠菜茎叶中硝酸还原酶(NR)的活性,同时降低了谷氨酸脱氢酶(GDH)的活性,谷氨酰胺合成酶(GS)活性则呈现先升后降的抛物线状变化规律。4)菠菜茎叶中NR活性与谷胺酰胺含量之间存在着显著负相关关系(r=-0.968)。     

谷氨酰胺合成酶植物基因工程应用研究进展

简要概述了通过植物遗传操作分别增强细胞质型谷氨酰胺合成酶(GS1)和叶绿体型谷氨酰胺合成酶(GS2)在植物叶片叶肉细胞的细胞质和叶绿体中的表达水平以提高植物氮素利用率的应用研究进展.