基于工程化毕赤酵母一锅法合成硫酸软骨素A北大核心CSCD

硫酸软骨素(chondroitin sulfate,CS)是一种线性多糖,广泛应用于医疗和保健等领域。相比于传统动物组织提取法,微生物合成硫酸软骨素具有可控、易规模化放大等优势。为实现硫酸软骨素A(CSA)的高效合成,本研究首先通过整合软骨素合酶编码基因kfoC、kfoA以及UDP-葡萄糖脱氢酶编码基因tuaD至毕赤酵母GS115基因组中,构建了以甘油为唯一碳源发酵生产软骨素的毕赤酵母工程菌株。通过进一步优化软骨素合成途径,软骨素分批补料发酵水平达到2.6 g/L。在进一步整合表达软骨素-4-O-磺基转移酶的基础上,本研究通过向生产软骨素毕赤酵母工程菌株破碎液中添加3′-磷酸腺苷-5′-磷酰硫酸和软骨素-4-O-磺基转移酶,成功建立了CSA的一锅法生物合成体系。通过优化,最终实现0-40%不同磺酸化水平CSA的可控合成。本研究中CSA的一锅法生物合成体系操作简便、易放大,更适用于工业化大规模生产。本研究结果也为肝素等其他糖胺聚糖的合成提供了思路。     

The p53/miRNAs/Ccna2 pathway serves as a novel regulator of cellular senescence: Complement of the canonical p53/p21 pathway

Aging is a multifactorial process characterized by the progressive deterioration of physiological functions. Among the multiple molecular mechanisms, microRNAs (miRNAs) have increasingly been implicated in the regulation of Aging process. However, the contribution of miRNAs to physiological Aging and the underlying mechanisms remain elusive. We herein performed high‐throughput analysis using miRNA and mRNA microarray in the physiological Aging mouse, attempted to deepen into the understanding of the effects of miRNAs on Aging process at the “network” level. The data showed that various p53 responsive miRNAs, including miR‐124, miR‐34a and miR‐29a/b/c, were up‐regulated in Aging mouse compared with that in Young mouse. Further investigation unraveled that similar as miR‐34a and miR‐29, miR‐124 significantly promoted cellular senescence. As expected, mRNA microarray and gene co‐expression network analysis unveiled that the most down‐regulated mRNAs were enriched in the regulatory pathways of cell proliferation. Fascinatingly, among these down‐regulated mRNAs, Ccna2 stood out as a common target of several p53 responsive miRNAs (miR‐124 and miR‐29), which functioned as the antagonist of p21 in cell cycle regulation. Silencing of Ccna2 remarkably triggered the cellular senescence, while Ccna2 overexpression delayed cellular senescence and significantly reversed the senescence‐induction effect of miR‐124 and miR‐29. Moreover, these p53 responsive miRNAs were significantly up‐regulated during the senescence process of p21‐deficient cells; overexpression of p53 responsive miRNAs or knockdown of Ccna2 evidently accelerated the cellular senescence in the absence of p21. Taken together, our data suggested that the p53/miRNAs/Ccna2 pathway might serve as a novel senescence modulator independent of p53/p21 pathway.     

Advanced Enzymology,Expression Profile and Immune Response of Clonorchis sinensis Hexokinase Show Its Application Potential for Prevention and Control of Clonorchiasis

BackgroundApproximately 35 million people are infected with Clonorchis sinensis (C. sinensis) globally, of whom 15 million are in China. Glycolytic enzymes are recognized as crucial molecules for trematode survival and have been targeted for vaccine and drug development. Hexokinase of C. sinensis (CsHK), as the first key regulatory enzyme of the glycolytic pathway, was investigated in the current study.Conclusions/SignificanceDue to differences in putative spatial structure and enzymology between CsHK and HK from the host, its extensive distribution in adult worms, and its expression profile as a component of excretory/secretory products, together with its good immunogenicity and immunoreactivity, as a key glycolytic enzyme, CsHK shows potential as a vaccine and as a promising drug target for Clonorchiasis.     

EGCG Enhances Cisplatin Sensitivity by Regulating Expression of the Copper and Cisplatin Influx Transporter CTR1 in Ovary Cancer

Cisplatin is one of the first-line platinum-based chemotherapeutic agents for treatment of many types of cancer, including ovary cancer. CTR1 (copper transporter 1), a transmembrane solute carrier transporter, has previously been shown to increase the cellular uptake and sensitivity of cisplatin. It is hypothesized that increased CTR1 expression would enhance the sensitivity of cancer cells to cisplatin (cDDP). The present study demonstrates for the first time that (-)-epigallocatechin-3-gallate (EGCG), a major polyphenol from green tea, can enhance CTR1 mRNA and protein expression in ovarian cancer cells and xenograft mice. EGCG inhibits the rapid degradation of CTR1 induced by cDDP. The combination of EGCG and cDDP increases the accumulation of cDDP and DNA-Pt adducts, and subsequently enhances the sensitivity of ovarian cancer SKOV3 and OVCAR3 cells to the chemotherapeutic agent. In the OVCAR3 ovarian cancer xenograft nude mice model, the combination of the lower concentration of cDDP and EGCG strongly repressed the tumor growth and exhibited protective effect on the nephrotoxicity induced by cisplatin. Overall, these findings uncover a novel chemotherapy mechanism of EGCG as an adjuvant for the treatment of ovarian cancer.     

羊毛防毡缩用蛋白酶的化学修饰

为减少防毡缩整理中蛋白酶对羊毛纤维主体结构的破坏作用,分别研究了戊二醛、微生物谷氨酰胺转氨酶(MTG)和水溶性碳二亚胺(EDC)对蛋白酶Savinase 16L的化学修饰,以期达到增大蛋白酶分子量,从而将水解作用限制在纤维表面的目的。主要通过体积排阻色谱、SDS-PAGE谱图以及荧光光谱研究修饰酶的分子量和结构变化。结果表明,戊二醛不能对蛋白酶分子进行有效修饰;MTG会被蛋白酶水解,无法催化酶分子间发生共价交联;而碳二亚胺既可以使蛋白酶分子间发生交联,又能将含有伯胺基的大分子修饰剂偶联到酶分子上。     

花生体细胞的器官发生和植株再生

以已萌发的花生种子不同部位为材料,比较了完整的胚芽、主芽,侧芽,胚轴及子叶等培养力的差异,得出了如下结果: 1.在外植体培养过程中,花生的胚轴具有强烈的再生能力;其它部位培养力大小的顺序依次是:完整的胚芽>主芽>侧芽>子叶>叶片。2.通过试验证明,花生外植体培养的最佳激素组合是NAA 0.6毫克/升+BA 0.9毫克/升。     

草地生态系统中土壤氮素矿化影响因素的研究进展

氮素是各种植物生长和发育所需的大量营养元素之一,也是牧草从土壤吸收最多的矿质元素．土壤中的氮大部分以有机态形式存在,而植物可以直接吸收利用的是无机态氮．这些有机态氮在土壤动物和微生物的作用下。由难以被植物直接吸收利用的有机态转化为可被植物直接吸收利用的无机态的过程就是土壤氮的矿化．氮素矿化受多种因子的影响,这些因子可以归结为生物因子和非生物因子．生物因子包括：土壤动物、土壤微生物和植物种类．土壤动物可以促进土壤有机质的矿化;土壤微生物种类、结构及功能与氮的分解、矿化有密切的关系;不同的植物种类对土壤氮素的矿化作用是不相同的,一般来说。有豆科植物生长的土壤比其它种类土氮素矿化的作用大．非生物因素一般可以分为环境因子和人类活动干扰．环境因子中土壤温度和含水量对土壤氮素矿化的影响是国内外众多科学家研究的方向．尽管如此,在此方面的研究还没有取得一致意见,仍然需要进行这方面的研究,而在其他诸如：不同的土壤质地与土壤类型方面,研究报道的结论也很不一致,草地生态系统中人类活动对土壤氮素矿化的影响主要包括,不同强度的放牧,割草以及施肥、火烧强度等．非生物因子对氮素矿化的影响非常直接和明显,尤其是人类活动．本文综述了近年来影响草地生态系统土壤氮素矿化有关因素的一些进展．     

表达核糖核酸酶基因的雄性不育油菜的获得

从细菌Ｂａｃｉｌｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ染色体ＤＮＡ中克隆了ＲＮａｓｅ（ｂａｒｎａｓｅ）基因,构建了ＴＡ－２９基因５＇调控区（－１３００－＋３）与ｂａｒｎａｓｅ基因的嵌合基因,通过农杆菌介导的遗传转化,获得了“双低”甘蓝型油菜“中双８２１”的转基因植株。转化植株与末转化植株在高度、生长速度、花器形态、花色等方面基本相同,但转化植株花丝短小、花药干瘪、没有花粉;自花授粉或以其为父本进行的异花授粉均不能结实,表现为完全的雄性不育。花药的横向解剖结构表明：转基因油菜雄性不育与绒毡层细胞的破坏有关     

Functional Connectivity of the Caudal Anterior Cingulate Cortex Is Decreased in Autism

The anterior cingulate cortex (ACC) is frequently reported to have functionally distinct sub-regions that play key roles in different intrinsic networks. However, the contribution of the ACC, which is connected to several cortical areas and the limbic system, to autism is not clearly understood, although it may be involved in dysfunctions across several distinct but related functional domains. By comparing resting-state fMRI data from persons with autism and healthy controls, we sought to identify the abnormalities in the functional connectivity (FC) of ACC sub-regions in autism. The analyses found autism-related reductions in FC between the left caudal ACC and the right rolandic operculum, insula, postcentral gyrus, superior temporal gyrus, and the middle temporal gyrus. The FC (z-scores) between the left caudal ACC and the right insula was negatively correlated with the Stereotyped Behaviors and Restricted Interests scores of the autism group. These findings suggest that the caudal ACC is recruited selectively in the pathomechanism of autism.     

+]-Huperzine A treatment protects against N-methyl-D-aspartate-induced seizure/status epilepticus in rats

The toxicity of organophosphorous (OP) nerve agents is attributed to their irreversible inhibition of acetylcholinesterase (AChE), which leads to excessive accumulation of acetylcholine (ACh) and is followed by the release of excitatory amino acids (EAA). EAAs sustain seizure activity and induce neuropathology due to over-stimulation of N-methyl-d-aspartate (NMDA) receptors. Huperzine A (Hup A), a blood-brain barrier permeable selective reversible inhibitor of AChE, has been shown to reduce EAA-induced cell death by interfering with glutamate receptor-gated ion channels in primary neuronal cultures. Although [-]-Hup A, the natural isomer, inhibits AChE approximately 38-fold more potently than [+]-Hup A, both [-]- and [+]-Hup A block the NMDA channel similarly. Here, we evaluated the protective efficacy of [+]-Hup A for NMDA-induced seizure in a rat model. Rats implanted with radiotelemetry probes to record electroencephalography (EEG), electrocardiography (ECG), body temperature, and physical activity were administered various doses of [+]-Hup A (intramuscularly) and treated with 20mug/kg NMDA (intracerebroventricular) 20-30min later. For post-exposure, rats were treated with [+]-Hup A (3mg/kg, intramuscularly) 1min after NMDA (20mug/kg). Our data showed that pre- and post-exposure, [+]-Hup A (3mg/kg) protects animals against NMDA-induced seizures. Also, NMDA-administered animals showed increased survival following [+]-Hup A treatment. [+]-Hup A has no visible effect on EEG, heart-rate, body temperature, or physical activity, indicating a reduced risk of side effects, toxicity, or associated pathology. Our results suggest that [+]-Hup A protects against seizure and status epilepticus (SE) by blocking NMDA-induced excitotoxicity in vivo. We propose that [+]-Hup A, or a unique combination of [+]- and [-]-Hup A, may prove to be effective for pre- and post-exposure treatment of lethal doses of OP-induced neurotoxicity.