虾青素提高血管性痴呆小鼠学习记忆能力

目的 探讨腹腔给药虾青素对于血管性痴呆(VaD)小鼠学习记忆能力的影响及其作用机制.方法 采用双侧颈总动脉夹闭法建立小鼠VaD模型后随机分成假手术组、模型组、虾青素低剂量组及虾青素高剂量组,分别给予相应形式的药物治疗.Morris水迷宫观察各组小鼠的学习记忆及空间探索能力,尼氏染色、免疫组织化学染色、Western b...     

虾青素抑制氧化应激改善陈旧红细胞保存质量

目的:观察虾青素能否抑制陈旧悬浮红细胞内的氧化应激,改善红细胞保存质量。方法:采集志愿者血液,制备去白悬浮红细胞,将其随机分为4组,对照组加入DMSO,另外3组悬浮红细胞的保存液内加入抗氧化剂虾青素使其终浓度分别为5、10和20μmol/L,悬浮红细胞于2℃~6℃内保存。保存至28 d、42 d采用倒置荧光显微镜观察悬浮红细胞内活性氧族的表达状况,采用荧光酶标仪测定红细胞内活性氧族的含量,采用硫代巴比妥酸比色法测定红细胞内丙二醛含量,扫描电镜观察红细胞的超微结构,采用化学比色法测定三磷酸腺苷含量,采用紫外测试法测定2,3-二磷酸甘油酸含量。结果:与各自对照组相比,加虾青素的三组储存28 d和42 d悬浮红细胞内活性氧族和丙二醛含量降低,三磷酸腺苷和2,3-二磷酸甘油酸水平升高,改善了红细胞的形态。结论:虾青素可以通过降低储存悬浮红细胞内的氧化应激水平改善红细胞保存质量。     

甲醛致小鼠海马结构毒性及虾青素对其的影响

目的探讨虾青素对甲醛小鼠海马结构中SYN、BDNF蛋白的表达及学习记忆功能的影响。方法将40只雄性昆明系小鼠随机分为4组:CON组(空白对照组)、FA组(染毒甲醛组)、AST组(单纯虾青素组)和FA+AST组(甲醛虾青素组);利用Morris水迷宫测试小鼠学习记忆功能变化情况,Western blot检测BDNF和SYN两种蛋白在海马结构中的表达情况。结果甲醛可明显降低小鼠学习记忆功能并出现相应的行为学改变,而虾青素可以改善小鼠的学习记忆功能,上调染毒甲醛小鼠海马结构中SYN、BDNF的表达水平。虾青素强效的抗氧化性可以逆转甲醛染毒所产生的神经毒性。结论虾青素改善甲醛小鼠学习记忆功能可能与其上调小鼠海马结构中SYN、BDNF表达水平相关。     

虾青素对叔丁基过氧化氢诱导软骨细胞损伤的保护作用

背景:关节软骨损伤是一种临床上常见的骨科疾病,探索其损伤修复机制有助于改善临床治疗策略。虾青素对关节软骨具有保护作用,但相关分子机制尚不明确。目的:评价虾青素对叔丁基过氧化氢诱导的C28/I2细胞损伤的保护作用,并探讨环状RNA circHP1BP3如何参与该作用。方法:采用叔丁基过氧化氢诱导软骨细胞C28/I2损伤,通过细胞活力、凋亡、炎症反应、氧化应激和胞外基质降解情况评价虾青素对叔丁基过氧化氢诱导C28/I2细胞损伤的保护作用。通过实时荧光定量PCR检测叔丁基过氧化氢和虾青素处理对环状RNA表达的影响,并对差异表达的环状RNA进行特性评价;通过转染过表达质粒或小干扰RNA,研究circHP1BP3在叔丁基过氧化氢诱导的C28/I2细胞损伤和虾青素介导的细胞损伤保护中的作用。结果与结论:(1)叔丁基过氧化氢可降低C28/I2细胞活性,增加凋亡率、炎症反应、氧化应激和胞外基质降解,诱导细胞损伤;虾青素处理对叔丁基过氧化氢诱导的C28/I2细胞损伤起保护作用;(2)叔丁基过氧化氢可下调C28/I2细胞中circHP1BP3的表达,虾青素处理可显著上调叔丁基过氧化氢诱导的细胞中circH...     

虾青素调控氧自由基/JNK信号通路介导脊髓损伤小鼠神经凋亡的研究

目的 探讨虾青素(Astaxanthin,AST)调控氧自由基/JNK信号通路对脊髓损伤处神经细胞的保护作用.方法 采用高空坠物法的方法制备脊髓损伤小鼠模型.将造模成功的小鼠随机分为SCI组与虾青素组(灌胃给予25 mg/kg的AST),另取正常C57BL/6小鼠作为假手术组.评价各组小鼠后肢功能;检测各组小鼠脊髓含水...     

基于rDNA序列的酵母整合载体的构建及应用

目的构建一个基于rDNA序列的酵母整合载体。方法通过PCR扩增得到酿酒酵母rDNA序列;通过常规分子生物学技术构建以rDNA序列为整合位点的酵母整合载体。为了确证该载体的效能,将青蒿紫穗槐-4,11-二烯合酶基因克隆到该载体上,构建了含紫穗槐-4,11-二烯合酶基因的整合型酿酒酵母工程菌。提取该工程酵母的基因组,通过紫穗槐-4,11-二烯合酶基因的特异引物进行扩增,确认紫穗槐-4,11-二烯合酶基因是否已整合到酵母基因组上。发酵酵母工程菌,对发酵产物进行GC—MS检测,确认是否产生紫穗槐-4,11-二烯。结果构建了1个酵母整合载体,具有如下特点：①能将外源基因表达盒序列整合到酿酒酵母rDNA序列上,增加目的基因的拷贝数;②选择标记能反复应用,方便获得多拷贝的整合形式;③整合片段不需要酶切线性化,节省了内切酶,而且简化了操作程序。酵母基因组PCR结果表明,紫穗槐-4,11-二烯合酶基因已经整合到了酵母基因组上。以朱栾倍半萜为标准品,对该酿酒酵母工程菌的代谢产物进行GC—MS检测,发现其能产生紫穗槐-4,11-二烯,产量达到（91．33±7．57）mg／L朱栾倍半萜当量。结论初步成功构建了一个基于rDNA序列的酵母整合载体。     

虾青素通过抑制活性氧簇/NLRP3炎症小体减轻对比剂引起的大鼠急性肾损伤

目的:研究虾青素(AST)减轻大鼠对比剂急性肾损伤(CI-AKI)的作用并探讨其潜在机制.方法:SD雄性大鼠随机分为5组:空白对照(control)组、虾青素对照(AST)组、模型(CM)组、虾青素治疗(AST+CM)组和N-乙酰半胱氨酸治疗(NAC+CM)组,每组8只.建立大鼠CI-AKI模型72 h后收集血清和肾脏...     

虾青素通过上调PINK1/parkin通路增强线粒体自噬并减轻大鼠对比剂急性肾损伤

目的：探讨虾青素（astaxanthin,AST）减轻大鼠对比剂急性肾损伤（contrast-induced acute kidney injury,CI-AKI）的作用机制。方法：雄性SD大鼠随机分为5组：假手术（sham）组、造模组（CI-AKI组）、AST治疗组（AST+CI-AKI组）、自噬抑制剂3-甲基腺嘌呤（3-methyladenine,3-MA）干预组（3-MA+CI-AKI组）和AST联合自噬抑制剂组（AST+3-MA+CI-AKI组）,每组10只。建立大鼠CI-AKI模型后72 h收集血清和肾脏组织,检测大鼠血清肌酐（serum creatinine,SCr）和血尿素氮（blood urea nitrogen,BUN）水平,检测肾脏组织总超氧化物歧化酶（total superoxide dismutase,T-SOD）和丙二醛（malondialdehyde,MDA）水平;HE染色观察肾脏结构;活性氧簇（reactive oxygen species,ROS）染色观察肾组织冰冻切片ROS水平;Western blot检测线粒体自噬及细胞自噬相关蛋白PTEN诱导假定激...     

虾青素对缺氧缺血性脑损伤新生大鼠模型的神经保护作用

背景：研究发现虾青素有良好的神经保护作用,但是对于其在新生儿缺氧缺血性损伤中的治疗作用,目前尚无相关报道。目的：构建缺氧缺血性脑损伤新生大鼠模型,观察虾青素对其产生的神经保护作用及作用的途径。方法：从98只7 d龄的SD乳鼠中随机取30只作为假手术组,其余大鼠结扎左颈总动脉2 h后,置于体积分数92%的特种标准气体、8%的氧气缺氧舱2 h建立缺血缺氧性脑损伤模型。假手术组仅分离颈总动脉,不予缺血缺氧处理。将造模成功的大鼠随机分为脑缺血缺氧组和虾青素治疗组,各30只。虾青素治疗组大鼠在脑缺血缺氧模型建成后立即通过腹腔注射80 mg/kg虾青素。结果与结论：与假手术组相比,脑缺血缺氧组大鼠缺血损伤区顶叶皮质中p-Akt、p-GSK3β、cleaved-caspase3蛋白的表达水平显著增加,Bcl-2蛋白的表达水平显著减少(P < 0.05);与脑缺血缺氧组相比,虾青素治疗可以显著减少凋亡相关蛋白cleaved-caspase3蛋白的表达水平(P < 0.05),显著上调Bcl-2蛋白的表达水平(P < 0.05),明显减少凋亡细胞的数量(P < 0.05)。提示虾青素可以显著改善新生大鼠缺氧缺血性脑损伤的预后及作用途径与上调Akt/GSK3β信号通路相关。中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程     

Comparative study of Saccharomyces cerevisiae LPS

LPS of yeast strains producing human epidermal growth factor were studied. Experiments demonstrated the absence of essential differences in the characteristics of these LPS and LPS of nonrecombinant Saccharomyces cerevisiae strains, which allowed us to develop a universal complex technology of simultaneous preparation of heterologous proteins and highly active immunomodulating LPS.     

狂犬病病毒糖蛋白在酿酒酵母中的分泌表达

目的使狂犬病病毒糖蛋白在酿酒酵母中获得分泌表达,为制备口服狂犬病疫苗奠定基础。方法克隆狂犬病病毒糖蛋白基因,与菊粉酶信号肽序列连接为融合基因InG,该融合基因克隆至pYes2载体Gal1启动子下游,构建成诱导分泌表达载体pYes2-InU-G,该重组载体转化酿酒酵母筛选阳性克隆,阳性重组子经半乳糖诱导12h后,收集上清进行SDS-PAGE电泳和Western-blot鉴定。结果 RT-PCR和Western-blot鉴定显示狂犬病病毒糖蛋白已获得分泌表达,并且具有良好的抗原性。结论 CVS24病毒株的糖蛋白在酿酒酵母中获得分泌表达且具有良好的抗原性,为进一步研究重组酵母能否在消化道中存活并继续进行分泌表达提供了研究条件。     

红色荧光蛋白在酿酒酵母中的表达特性研究

目的研究红色荧光蛋白编码基因Dsred在酿酒酵母菌中的快速克隆与表达。方法根据已发表基因序列设计引物,采用连续重叠PCR方法快速克隆获得全长Dsred基因,将其与pMD-18T载体连接后进行测序鉴定。通过In-fusion方法将鉴定正确的pMD-Dsred重组载体与酿酒酵母表达载体pYeDP60进行连接,测序后利用LiAc方法将鉴定正确的pYeDP60-Dsred重组表达载体转化至酿酒酵母菌W303-1B中,PCR扩增筛选阳性克隆,获得的W303B[pYeDP60-Dsred]工程菌经诱导培养后进行SDS-PAGE电泳分析和绿光激发荧光成像检测。将工程菌分别接种至YPD、YPG、SCG和SCD培养基,培养48、72、96、120和144h后分别测定吸光度(A600)值。取诱导表达后的工程菌(菌液组)进行离心(菌体组)、离心后加甘油(高渗组)处理,分别置于–70、–20、4、28和37℃条件培养,荧光显微镜观察其表达特性。结果连续重叠PCR扩增和测序鉴定结果表明,扩增获得的Dsred基因长为678bp,其序列与已发表的基因序列完全一致;Dsred基因已成功插入pYeDP60-Dsred重组表达载体,且受酵母诱导型启动子GAL10-CYC1调控表达。PCR扩增筛选和SDS-PAGE分析显示,pYeDP60-Dsred重组表达载体已成功导入酿酒酵母菌中,诱导表达产物相对分子质量与预期相符,且诱导后工程菌可在绿光激发下发出红色荧光。4种培养基内工程菌的菌体生长情况无明显差别,重组Dsred红色荧光蛋白表达不会抑制酿酒酵母菌体生长。荧光显微镜观察显示,工程菌经不同处理后,高渗组红色荧光蛋白成熟时间最短,菌液组时间最长;红色荧光蛋白在37℃条件下培养时成熟最早,但降解速度较快。结论成功构建了pYeDP60-Dsred重组酿酒酵母表达载体,实现了Dsred基因在酿酒酵母菌体内的异源表达。红色荧光蛋白表达对酿酒酵母菌体生长无明显影响,且离心保留菌体和加入甘油等缺水高渗处理都有利于红色荧光蛋白的成熟。     

Cysteine uptake by Saccharomyces cerevisiae is accomplished by multiple permeases

Uptake by Saccharomyces cerevisiae of the sulphur-containing amino acid L-cysteine was found to be non-saturable under various conditions, and uptake kinetics suggested the existence of two or more transport systems in addition to the general amino-acid permease, Gap1p. Overexpression studies identified BAP2, BAP3, AGP1 and GNP1 as genes encoding transporters of cysteine. Uptake studies with disruption mutants confirmed this, and identified two additional genes for transporters of cysteine, TAT1 and TAT2, both very homologous to BAP2, BAP3, AGP1 and GNP1. While Gap1p and Agp1p appear to be the main cysteine transporters on the non-repressing nitrogen source proline, Bap2p, Bap3p, Tat1p, Tat2p, Agp1p and Gnp1p are all important for cysteine uptake on ammonium-based medium. Furthermore, whereas Bap2p, Bap3p, Tat1p and Tat2p seem most important under amino acid-rich conditions, Agp1p contributes significantly when only ammonium is present, and Gnp1p only contributes under the latter condition. Received: 25 January / 15 March 1999     

Molecular and biochemical analysis of Saccharomyces cerevisiae cox1 mutants

We report on the molecular and biochemical analysis of a set of 13 respiratory deficient mutants of Saccharomyces cerevisiae which are specifically altered in COX1, the gene encoding the subunit Cox1p of cytochrome c oxidase. DNA sequence analysis shows that three are due to frameshift mutations, two to nonsense mutations, and eight to missense mutations. All, except the missense mutant S157L, have impaired electron transfer and respiratory activity. Analysis of the mitochondrial translation products shows that when Cox1p is absent, Cox2p and Cox3p are still synthesized. In the missense mutants, the steady state levels in the mitochondrial membranes of the three mitochondrially encoded subunits Cox1p, Cox2p and Cox3p and the nuclear-encoded subunit Cox4p are reduced. In the frameshift and nonsense mutants, Cox1p is absent and Cox2p, Cox3p and Cox4p are considerably decreased or undetectable. A comparison of the steady state levels of Cox1p through Cox4p in the COX1, COX2, COX3 and COX4 mutants shows the interdependance of the accumulation of these four subunits in the mitochondrial membranes. Received: 20 January / 23 June 1998     

Reciprocal translocation at duplicated RPL2 loci might cause speciation of Saccharomyces bayanus and Saccharomyces cerevisiae

By a genomic comparison of two sibling yeasts, Saccharomyces bayanus and S. cerevisiae, we previously demonstrated that chromosomes II and IV of S. cerevisiae were rearranged into chromosomes 12 and 14 of S. bayanus or vice versa. In the present study we have delimited the translocation break sites in chromosomes II and IV by Southern hybridization using DNA fragments of S. cerevisiae cosmid clones as probes. The results suggest that the reciprocal translocation of chromosomes II and IV had occurred at duplicated RPL2 loci. Furthermore, the translocation sites in S. bayanus were confirmed by the cloning and sequence analysis of the regions flanking RPL2 loci. Several genes in the regions flanking the RPL2 loci were present in the order expected for a translocation at these loci between the two species. These results indicated that the reciprocal translocation between chromosomes II and IV was generated by homologous recombination at duplicated RPL2 loci on the two chromosomes. Therefore, we propose that duplicated genes or duplicated regions play an important role in altering genomic organization during the speciation of S. bayanus and S. cerevisiae. Received: 20 October 1997 / 2 March 1998     

Endopolygalacturonase genes and enzymes from Saccharomyces cerevisiae and Kluyveromyces marxianus

The gene encoding endopolygalacturonase (EC 3.2.1.15) has been cloned, sequenced and expressed from three strains of Saccharomyces cerevisiae (including non-secretors) and three strains of Kluyveromyces marxianus. Both control and coding regions showed small differences within each species, one including loss of a potential glycosylation site. Two non-secreting S. cerevisiae strains (FY1679 and var. uvarum) had non-transcribed copies of functional genes. Maximum enzyme activity was achieved with the S. cerevisiae FY1679 gene in an expressing vector, with an enzyme activity of 51 μmol of reducing sugar released from polygalacturonic acid μg protein⁻¹ min⁻¹, the highest so far reported for a yeast. Received: 19 May 2000 / Accepted: 6 August 2000     

6-Azauracil inhibition of GTP biosynthesis in Saccharomyces cerevisiae

Summary The addition of 6-azauracil to the growth medium causes a strong reduction of the GTP level in the nucleotide pool of Saccharomyces cerevisiae. In-vitro experiments show a strong inhibition of IMP dehydrogenase activity by 6-azaUMP explaining the preceeding effect. PPR2 mutants, previously characterized by an increased sensitivity to 6-azauracil compared to the wildtype, are specifically susceptible to the lowering of the GTP pool, and are able to grow in presence of 6-azauracil when guanine is added to the medium.