超高压对植物乳杆菌能量代谢影响的研究

以植物乳杆菌ATCC8014为试材,研究超高压对其能量代谢的影响。建立了用氯化碘硝基四唑紫测定ATCC8014的INT代谢还原活性的比色法。用比色法测定了超高压对ATCC8014的INT代谢还原活性与葡萄糖利用的影响。试验结果表明,150~250MPa作用15min在MRS琼脂培养基上随着压力的增大菌落数显著降低,INT代谢还原活性降低显著,葡萄糖的利用变化不明显;超过300MPa后,葡萄糖的利用才显著降低;400MPa处理15min,尽管在MRS琼脂培养基上菌落数低于检测限,INT代谢还原活性为0%,而葡萄糖的利用能力仍为对照组的56.1%,超高压作用下ATCC8014的灭活与INT代谢还原活性的降低的相关性较好。说明ATCC8014的细胞膜上参与葡萄糖的吸收和运输的酶、糖酵解的酶与调节系统比三羧酸循环的酶与调节系统较耐压。三羧酸循环比糖酵解对超高压敏感,三羧酸循环的抑制是超高压灭活其的重要原因,这为了探讨超高压杀灭植物乳杆菌的机制提供了一定的理论依据。     

第十八讲 叶绿体代谢物运转与光合产物的调节

内容一、引言二、叶绿体代谢物的运转 1.叶绿体被膜对物质的透性 (1)“Pi运转器” (2)“双羧酸运转器” 2.ATP及还原力的运转 (1)3PGA—DHAP穿梭运转 (2)Mal—OAA穿梭运转     

还原型谷胱甘肽(GSH)的功能与应用

<正> GSH是由谷氨酸、半胱氨酸和甘氨酸组成的含巯基的三肽,广泛存在于生物体内。它参与维持细胞的正常氧化还原状态。对于需要巯基的酶有保护与恢复活性的功能,从而促进糖、脂肪与蛋白质代谢。它是多种酶的辅基与辅酶,参与三羧酸循环与糖代谢,使机体获得能量。谷胱甘肽过氧化酶     

微生物发酵生产C_4二元羧酸研究进展

C4二元羧酸(苹果酸、延胡索酸和琥珀酸等)是TCA循环中重要的中间代谢产物,广泛用于食品、医药和化工等行业。由于市场潜在需求量巨大,微生物发酵法生产C4二元羧酸备受关注。从自然界筛选的微生物可以大量积累C4二元羧酸,但存在诸多问题。而利用模式微生物生产C4二元羧酸具有很多优势,已成为国内外的研究热点。从三个方面综述了C4二元羧酸的研究进展:利用自然筛选及改造的微生物生产C4二元羧酸,代谢工程改造模式微生物生产C4二元羧酸,C4二元羧酸的发酵生产的代谢调控。最后,提出了微生物发酵法生产C4二元羧酸今后要解决的问题和发展方向。     

盐胁迫下小麦苗叶片吡咯-5-羧酸还原酶活性和游离脯氨酸积累

受NaCl、KCl或MsCl_2胁迫的小麦幼苗,当外部溶液的渗透势由—160 kPa下降到—900 kPa时,叶片吡咯—5—羧酸还原酶(PSC)活性增高;渗透势由—900 kPa降低到—1500kPa,酶活性下降;胁迫 1d和 2d的幼苗,还原酶活性显著增加;3～6d酶活性无大变化。游离脯氨酸含量随溶液渗透势下降和培养时间的延长而提高。胁迫解除后酶活性和脯氨酸含量均降低。受NcCl胁迫义在ABA影响下的幼苗,P5C还原酶活性和脯氨酸含量高于仅受NaCl胁迫的幼苗。     

疟疾媒介冈比亚按蚊杀虫剂代谢抗性解毒酶芯片

代谢抗性是蚊虫对各类杀虫剂产生抗性的一个重要而普遍的机制,但这一通路所涉及的基因仍不清楚。英国利物浦热带医学院媒介研究组构建了疟疾媒介冈比亚按蚊与代谢抗性相关的基因芯片,是从230个可能与杀虫剂代谢有关的基因选出的单一片段,包括细胞色素P450s、GSTs、羧酸酯酶、氧化还原基因,以及与P450氧化代谢复合体相关的基因和对照基因。     

松毛虫赤眼蜂和螟黄赤眼蜂羧酸酯酶性质比较研究

该文对松毛虫赤眼蜂Trichogramma dendrolimi和螟黄赤眼蜂T.Chilonis羧酸酯酶的毒理学和生物化学性质进行了初步研究。由时间进程曲线确定了赤眼蜂羧酸酯酶活性测定中的最适反应时间为40min。对两种赤眼蜂单头羧酸酯酶活性的测定表明,松毛虫赤眼蜂羧酸酯酶活性主要分布在0—1OD/(mg·min)之间,而螟黄赤眼蜂羧酸酯酶活性主要分布在1-4OD/(mg·min)之间。比较两种赤眼蜂羧酸酯酶米氏常数(K_m)值,螟黄赤眼蜂羧酸酯酶对底物的亲和力比松毛虫赤眼蜂羧酸酯酶对底物的亲和力高。对氧磷对螟黄赤眼蜂和松毛虫赤眼蜂羧酸酯酶的抑制作用没有明显差异,而对氧磷对松毛虫赤眼蜂羧酸酯酶的抑制作用明显强于磷酸三苯酯(TPP)。该文还将棉铃虫Helicoverpa armigera (Hubner)羧酸酯酶部分性质与两种赤眼蜂作了比较。     

生物催化立体选择性氧化还原中存在问题及其发展策略

以立体选择性氧化还原酶或其全细胞催化的不对称氧化还原反应已经成为转化光学活性手性醇及其他手性化合物的有效手段。然而,生物催化氧化还原反应体系存在着催化活性与专一性、反应体系与催化稳定性等生物催化剂所固有的局限性问题,而且,生物氧化还原反应必需辅酶及其再生问题也是限制该转化途径产业化应用的一个重要因素。围绕上述生物催化立体选择性氧化还原中存在的关键问题,现代分子生物技术及反应工程的不断突破和发展为改善生物催化立体选择性氧化还原在催化剂本身和反应工程方面的局限性提供了有效的发展策略,为其进一步大规模产业应用提供了发展基础。     

羧酸酯酶A2在大肠杆菌中的表达及特征分析

本研究采用RT-PCR法从抗性蚊虫(Culex quinquefasciatus)中克隆了羧酸酯酶A2的全长cDNA,对其进行了序列测定,并构建了融合表达质粒pET-ESTA2。转化大肠杆菌BL21后,在异丙基硫代牛乳糖苷(IPTG)的诱导下,使羧酸酯酶A2在大肠杆菌内得到表达。表达产物经亲和层析纯化获得了1条带的重组蛋白。与报道的从蚊虫体内纯化的羧酸酯酶相比,从表达产物中纯化的羧酸酯酶Km与其一致,但从表达产物纯化的羧酸酯酶的Vm比蚊虫中纯化的羧酸酯酶的Vm高。表明用亲和层析纯化的羧酸酯酶A2比从蚊虫中提取的纯度高。羧酸酯酶A2表达纯化及特征分析为其应用奠定了基础。     

从三羧酸循环代谢异常中探寻癌症进展与治疗新思路

三羧酸循环是有氧生物获得生命活动所需能量的主要途径。整个三羧酸循环的过程需要多种酶的协同作用,产生多种中间代谢产物,以此来维持细胞稳定的生存环境。在众多参与三羧酸循环的酶中,当其中某一种或多种酶的活性发生改变时,就会有新的代谢产物生成,从而改变细胞的命运。该文以三羧酸循环中异柠檬酸脱氢酶、琥珀酸脱氢酶和延胡索酸酶的突变所引起的一系列致癌代谢物的积累为切入点,就三羧酸循环中间酶突变引起的表观遗传学改变与肿瘤发生发展的关系进行综述,以期从三羧酸循环代谢异常中,探寻癌症进展与治疗的新思路。     

Carotenoids,versatile components of oxygenic photosynthesis

Carotenoids (CARs) are a group of pigments that perform several important physiological functions in all kingdoms of living organisms. CARs serve as protective agents, which are essential structural components of photosynthetic complexes and membranes, and they play an important role in the light harvesting mechanism of photosynthesizing plants and cyanobacteria. The protection against reactive oxygen species, realized by quenching of singlet oxygen and the excited states of photosensitizing molecules, as well as by the scavenging of free radicals, is one of the main biological functions of CARs. X-ray crystallographic localization of CARs revealed that they are present at functionally and structurally important sites of both the PSI and PSII reaction centers. Characterization of a CAR-less cyanobacterial mutant revealed that while the absence of CARs prevents the formation of PSII complexes, it does not abolish the assembly and function of PSI. CAR molecules assist in the formation of protein subunits of the photosynthetic complexes by gluing together their protein components. In addition to their aforementioned indispensable functions, CARs have a substantial role in the formation and maintenance of proper cellular architecture, and potentially also in the protection of the translational machinery under stress conditions.     

Virtual screening of carboxylic acid reductases for biocatalytic synthesis of 6-aminocaproic acid and 1,6-hexamethylenediamine

The key precursors for nylon synthesis, that is, 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD), are produced from petroleum-based feedstocks. A sustainable biocatalytic alternative method from bio-based adipic acid has been demonstrated recently. However, the low efficiency and specificity of carboxylic acid reductases (CARs) used in the process hampers its further application. Herein, we describe a highly accurate protein structure prediction-based virtual screening method for the discovery of new CARs, which relies on near attack conformation frequency and the Rosetta Energy Score. Through virtual screening and functional detection, five new CARs were selected, each with a broad substrate scope and the highest activities toward various di- and ω-aminated carboxylic acids. Compared with the reported CARs, KiCAR was highly specific with regard to adipic acid without detectable activity to 6-ACA, indicating a potential for 6-ACA biosynthesis. In addition, MabCAR3 had a lower K_m with regard to 6-ACA than the previously validated CAR MAB4714, resulting in twice conversion in the enzymatic cascade synthesis of HMD. The present work highlights the use of structure-based virtual screening for the rapid discovery of pertinent new biocatalysts.     

Enhanced Cytotoxicity of Natural Killer Cells following the Acquisition of Chimeric Antigen Receptors through Trogocytosis

Natural killer (NK) cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs), which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL) cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.     

T cells redirected to interleukin-13Rα2 with interleukin-13 mutein–chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Rα1

Background aimsOutcomes for patients with glioblastoma remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL)13Rα2, human epidermal growth factor receptor 2, epidermal growth factor variant III or erythropoietin-producing hepatocellular carcinoma A2 has shown promise for the treatment of glioma in preclinical models. On the basis of IL13Rα2 immunotoxins that contain IL13 molecules with one or two amino acid substitutions (IL13 muteins) to confer specificity to IL13Rα2, investigators have constructed CARS with IL13 muteins as antigen-binding domains. Whereas the specificity of IL13 muteins in the context of immunotoxins is well characterized, limited information is available for CAR T cells.MethodsWe constructed four second-generation CARs with IL13 muteins with one or two amino acid substitutions, and evaluated the effector function of IL13-mutein CAR T cells in vitro and in vivo.ResultsT cells expressing all four CARs recognized IL13Rα1 or IL13Rα2 recombinant protein in contrast to control protein (IL4R) as judged by interferon-γ production. IL13 protein produced significantly more IL2, indicating that IL13 mutein–CAR T cells have a higher affinity to IL13Rα2 than to IL13Rα1. In cytotoxicity assays, CAR T cells killed IL13Rα1- and/or IL13Rα2-positive cells in contrast to IL13Rα1- and IL13Rα2-negative controls. Although we observed no significant differences between IL13 mutein–CAR T cells in vitro, only T cells expressing IL13 mutein–CARs with an E13K amino acid substitution had anti-tumor activity in vivo that resulted in a survival advantage of treated animals.ConclusionsOur study highlights that the specificity/avidity of ligands is context-dependent and that evaluating CAR T cells in preclinical animal model is critical to assess their potential benefit.     

Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.     

Bispecific chimeric antigen receptors targeting the CD4 binding site and high-mannose Glycans of gp120 optimized for anti–human immunodeficiency virus potency and breadth with minimal immunogenicity

Background aims

Chimeric antigen receptors (CARs) offer great potential toward a functional cure of human immunodeficiency virus (HIV) infection. To achieve the necessary long-term virus suppression, we believe that CARs must be designed for optimal potency and anti-HIV specificity, and also for minimal probability of virus escape and CAR immunogenicity. CARs containing antibody-based motifs are problematic in the latter regard due to epitope mutation and anti-idiotypic immune responses against the variable regions.

From humble beginnings to success in the clinic: Chimeric antigen receptor-modified T-cells and implications for immunotherapy

In the past 50 years, disease burden has steadily shifted from infectious disease to cancer. Standard chemotherapy has long been the mainstay of cancer medical management, and despite vast efforts towards more targeted and personalized drug therapy, many cancers remain refractory to treatment, with high rates of relapse and poor prognosis. Recent dramatic immunotherapy clinical trials have demonstrated that engineering T-cells with chimeric antigen receptors (CARs) to target CD19 can lead to complete remission in relapsed or refractory B-cell malignancies, generating a great deal of enthusiasm in the field. Here we provide a comprehensive overview of the history of adoptive T-cell therapy, including CARs, in solid tumors as well as hematologic malignancies. CAR therapy has the potential to fundamentally transform cancer treatment with specific and even personalized targeting of tissue- and tumor-specific antigens. However, before CARs become standard first-line treatment modalities, critical issues regarding efficacy, combinatorial regimens, and mechanisms of treatment failure and toxicity will need to be addressed.     

Construction and functional characterization of a fully human anti-CD19 chimeric antigen receptor (huCAR)-expressing primary human T cells

Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti-CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti-mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti-mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti-CD19 CAR T cells—thereby reducing the risk of tumor relapse—but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second-generation fully human anti-CD19 CAR (or huCAR19) containing a fully human single-chain variable fragment (ScFv) fused with a CD8a hinge, a 4-1BB transmembrane domain and intracellular T cell signaling domains of 4-1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19⁺ target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen-independent tonic signaling in the absence of cognate antigen. Considering our results, our anti-CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse-derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice.