鸟氨酸脱羧酶结构、功能及表达调控研究进展

多胺对细胞生长、存活以及增殖具有重要的调控作用,体内多胺水平变化与疾病和衰老的关系密切。鸟氨酸脱羧酶(Ornithine decarboxylase,ODC)是多胺生物合成途径中的第一个限速酶,能催化细胞内鸟氨酸脱羧转化成腐胺。因此,ODC可通过对细胞内多胺水平的调节来参与调控细胞增殖过程。近年来,对ODC功能的研究取得了一定的进展,特别是在ODC调控多胺水平、细胞增殖、癌症以及ODC表达调控等方面研究较多。文章对ODC结构、功能及其表达调控的研究进行了评述,并对今后ODC的深入研究进行了展望。     

Low-protein diets supplemented with glutamic acid or aspartic acid ameliorate intestinal damage in weaned piglets challenged with hydrogen peroxide

Glutamic acid (Glu) and aspartic acid (Asp) are acidic amino acids with regulatory roles in nutrition, energy metabolism, and oxidative stress. This study aimed to evaluate the effects of low-protein diets supplemented with Glu and Asp on the intestinal barrier function and energy metabolism in weaned piglets challenged with hydrogen peroxide (H₂O₂). Forty piglets were randomly divided into 5 groups: NC, PC, PGA, PG, and PA (n = 8 for each group). Pigs in the NC and PC groups were fed a low-protein diet, while pigs in the PGA, PG, or PA groups were fed the low-protein diet supplemented with 2.0% Glu +1.0% Asp, 2.0% Glu, or 1.0% Asp, respectively. On day 8 and 11, pigs in the NC group were intraperitoneally injected with saline (1 mL/kg BW), while pigs in the other groups were intraperitoneally administered 10% H₂O₂ (1 mL/kg BW). On day 14, all pigs were sacrificed to collect jejunum and ileum following the blood sample collection in the morning. Notably, low-protein diets supplemented with Glu or Asp ameliorated the intestinal oxidative stress response in H₂O₂-challenged piglets by decreasing intestinal expression of genes (P < 0.05) (e.g., manganese superoxide dismutase [MnSOD], glutathione peroxidase [Gpx]-1, and Gpx-4) encoding oxidative stress-associated proteins, reducing the serum concentration of diamine oxidase (P < 0.05), and inhibiting apoptosis of the intestinal epithelium. Glu and Asp supplementation attenuated the upregulated expression of energy metabolism-associated genes (such as hexokinase and carnitine palmitoyltransferase-1) and the H₂O₂-induced activation of acetyl-coenzyme A carboxylase (ACC) in the jejunum and adenosine monophosphate-activated protein kinase–acetyl-ACC signaling in the ileum. Dietary Glu and Asp also ameliorated intestinal barrier damage as indicated by restored intestinal histology and morphology. In conclusion, low-protein diets supplemented with Glu and Asp protected against oxidative stress-induced intestinal dysfunction in piglets, suggesting that this approach could be used as a nutritional regulatory protectant against oxidative stress.     

Colostral growth factors. Possible role in bovine udder epithelial cell regeneration.

Growth of the secretory epithelium during prepartum time, and for a short period after calving, is under hormonal control by estrogen, progesterone and prolactin. The mechanism(s) by which these hormones act is not known but colostrum and milk have been shown to contain different growth promoting substances. In an attempt to unravel these relationships the effect of bovine colostrum on cellular proliferation in vitro have been characterized. Colostral thermostable factors not present in milk nor associated with fat, potently induce the proliferation associated enzyme, ornithine decarboxylase, in fibroblast cell lines. However, mammary epithelial cells appear to proliferate in response to different colostral heat sensitive factor(s) that await further characterization.     

单根阴离子交换柱法提取L-谷氨酰胺工艺研究

[目的]研究不同影响因素及工艺条件对提取L-谷氨酰胺的影响,为谷氨酰胺的工业化生产提供一种新型分离工艺。[方法]利用单根阴离子交换柱,采用静态吸附试验和动态分离试验研究从酶法合成液中提取谷氨酰胺的各种条件。[结果]模拟液离交分析的最适条件为：树脂201x4,进样流速为3．0BV／h,进样pH值6．5,谷氨酸浓度1．O％。酶反应液处理的工艺条件为：脱色温度40℃,pH值6．5,10．0g/L粉末状活性炭进行二次脱色,15．0mmol／L的Ca2＋添加量来去除样液中PO4^3＋,离交分离条件同模拟液。Gin收率为66．6％、Glu去除率为95．O％。[结论]该研究得到的处理方法简单易行,Gin收率和Glu去除率高,在工业生产中有较好的应用前景。     

香菇乳清酸核苷-5'-单磷酸脱羧酶编码基因le-pyrG的克隆

通过巢式简并PCR和基因组步行技术从香菇(Lentinula edodes)单核体中克隆到乳清酸核苷-5′-单磷酸脱羧酶(orotidine-5'-monophosphate decarboxylase,OMPDC)的编码基因le-pyrG,长度为946 bp.经生物信息学分析,香菇le-pyrG基因含有2个长度分别为67 bp和51 bp的内含子,该基因可以编码1个含有275个氨基酸残基的蛋白质序列,该序列经比对分析显示与裂褶菌(Schizophyllum commne)和灰盖鬼伞(Coprinus cinereus) OMPDC序列的相同性分别为73%和70%,相似性分别为84% 和83%.这是首次报道从栽培食用真菌中克隆出乳清酸核苷-5′-单磷酸脱羧酶编码基因的全序列.     

茶树中精氨酸脱羧酶基因的cDNA克隆及序列分析

通过SMARTRACE PCR技术获得了茶树中精氨酸脱羧酶(ADC)的cDNA全长序列,并登录GenBank(登录号为JQ653274)。ADC基因cDNA全长为2 988 bp,开放阅读框(ORF)全长为2 163 bp,共编码720个氨基酸,编码的蛋白质的分子量为77.430 kDa,理论等电点为5.373,其序列中不存在卷曲螺旋结构。ADC蛋白为亲水性非分泌不稳定蛋白,不跨膜运动,无信号肽序列存在,共有41个可能的磷酸化位点,存在于叶绿体基质中,是细胞质蛋白。精氨酸脱羧酶的基因cDNA全长序列的获得,对于进一步研究精氨酸在茶树氮代谢中的作用及茶氨酸代谢途径提供基础。     

Determining potential glutamine synthetase and glutamate dehydrogenase activity in soil

Ammonium (NH₄⁺), an important nitrogen (N) source for microorganisms, is assimilated via two major pathways. One route is catalyzed by glutamate dehydrogenase (GDH), while the other mechanism involves two enzymes, glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS/GOGAT enzyme system requires more energy to operate, but has a much higher affinity for NH₄⁺ than GDH. We describe procedures to determine potential GS and GDH activity in soil samples. GS and GDH are intracellular enzymes. We used chloroform fumigation to make cell membranes permeable for substrates and products of the enzymes. Fumigation for 4 h increased GS activity almost ten-fold compared to the unfumigated control. Under optimized assay conditions, GS activity increased linearly for at least 80 min, indicating that the substrates were not limiting. In contrast to what was found for GS activity, direct addition of substrates to the soil to assay GDH activity did not result in a linear increase in GDH activity over time. A linear response for 3 h, however, resulted when the soil samples were first extracted with buffer solution and the reagents were added after centrifugation. The differences between the assays explain why fumigation for 3 d prior to the assay increased GDH activity by only 60%. In a microcosm study with glucose and NH₄⁺ addition, the activity of the two enzymes depended on the carbon (C) to N ratio of the amendment. With increasing C to N ratios from 5 to 120, GS activity doubled, while C to N ratios higher than 120 did not further increase GS activity. In contrast, GDH activity decreased by 13% with increasing C to N ratios from 5 to 200. The GDH to GS activity ratio in soil may therefore yield valuable information about the availability of N relative to C at a specific time.     

洋常春藤甲羟戊酸焦磷酸脱羧酶基因 HhMVD 的克隆与表达 分析

甲羟戊酸焦磷酸脱羧酶（Mevalonate diphosphate decarboxylase,MVD）是三萜皂苷生物合成途径中的关键酶, 为研究其在洋常春藤中的基因功能,通过 RT-PCR 和 RACE 技术从洋常春藤叶片中克隆获得 HhMVD 基因的 cDNA 全 长序列,并通过 RT-qPCR 技术分析其表达规律。结果表明：RACE 克隆获得的 HhMVD 基因 cDNA 序列全长 1 799 bp, 包含一个完整开放阅读框（ORF）1 263 bp、5′非编码区（5′UTR）192 bp、3′非编码区（3′UTR）344 bp;该基因编码 420 个氨基酸,分子质量 46.6 ku,理论等电点为 6.57,不含跨膜区,属于非分泌型蛋白;HhMVD 蛋白具有 GHMP 激 酶 N 端结构域,属于甲羟戊酸激酶系列,与刺五加、人参、三七等同科植物亲缘关系较近。RT-qPCR 结果表明,洋常 春藤中 HhMVD 基因的时空表达相对稳定,但与常春藤皂苷含量差异变化之间存在一定的负相关性。洋常春藤 HhMVD 基因的成功克隆及表达分析研究,为深入探讨其基因功能、阐明其在常春藤皂苷生物合成途径中的作用提供理论依据。