高尿酸血症动物模型的实验研究

[目的]制备稳定持续、简便易行的高尿酸血症动物模型。[方法]采用单纯氧嗪酸钾、氧嗪酸钾+次黄嘌呤、腺嘌呤+乙胺丁醇、单纯酵母膏、酵母膏+腺嘌呤、酵母膏+腺嘌呤+氧嗪酸钾不同药物与不同浓度进行实验,检测模型动物血清尿酸值。[结果]单纯氧嗪酸钾、氧嗪酸钾+次黄嘌呤模型组尿酸值升高,与空白组比较有差异,但模型不稳定,重复性差,个体差异较大。腺嘌呤+乙胺丁醇、单纯酵母膏、酵母膏+腺嘌呤模型组尿酸值与空白组比较无差异。酵母膏+腺嘌呤+氧嗪酸钾模型组尿酸值升高,与空白组比较有显著性差异(P0.01),且模型稳定,个体差异小。[结论]采用酵母膏+腺嘌呤+氧嗪酸钾复制高尿酸血症动物模型最佳。     

Anti-Candida activity of the water-soluble lectin from Moringa oleifera seeds (WSMoL)

This work reports the effects of the water-soluble lectin from Moringa oleifera seeds (WSMoL) on growth and survival of Candida species. In addition, cellular alterations linked to the antifungal effect were investigated. The minimal inhibitory (MIC) and fungicidal (MFC) concentrations were determined and 24-h growth curves in absence and presence of lectin were established. Flow cytometry was used to evaluate the induction of apoptosis/necrosis, alterations in mitochondrial membrane potential (ΔΨm), and occurrence of lysosomal damage. WSMoL inhibited the growth of C. albicans, C. glabrata, C. krusei and C. parapsilosis with MIC of 20 μg/mL. The lowest MFC (20 μg/mL) was detected for C. glabrata and the highest (80 μg/mL) for C. albicans and C. parapsilosis. The inhibitory effect started from the ninth to nineteenth hour of incubation depending on the fungal species. Incubation with the lectin at the MIC for 24 h increased the number of cells undergoing apoptosis and necrosis. Hyperpolarization of the mitochondrial membrane was detected after 12-h treatment, followed by reduction of ΔΨm or depolarization after 24 h. No lysosomal damage was detected in treated cells. In conclusion, WSMoL is a fungistatic and fungicide agent against Candida with differential effects depending on the species.     

Pbp1 mediates the aberrant expression of genes involved in growth defect of ccr4∆ and pop2∆ mutants in yeast Saccharomyces cerevisiae

CCR4 and POP2 genes encode the catalytic subunit of the Ccr4-Not complex involved in shortening mRNA poly(A) tail in Saccharomyces cerevisiae. The ccr4Δ and pop2∆ mutants exhibit pleiotropic phenotypes such as slow and temperature-sensitive growth, aberrant expression of glucose repression genes and abnormal cell wall synthesis. We previously found that the growth defect of the ccr4Δ and pop2∆ mutants is suppressed by deletion of the PBP1 gene, which encodes poly(A)-binding protein (Pab1)-binding protein 1. In this study, we investigated the functional relationship between Ccr4/Pop2 and Pbp1 by measuring changes in gene expression in ccr4Δ and pop2∆ single mutants and ccr4Δ pbp1∆ and pop2∆ pbp1∆ double mutants. We found that expression of HSP12, HSP26, PIR3, FUS1 and GPH1 was increased in ccr4Δ and pop2∆ single mutants. The pbp1∆ mutation not only restored the growth defect but also reduced the increased expression of those genes found in the ccr4Δ and pop2∆ mutants. Over-expression of PBP1 in the ccr4Δ mutant further increased the expression of HSP12, HSP26, PIR3 and FUS1 and exacerbated the cell growth. These results suggest that the aberrant expression of a subset of genes, which is facilitated by Pbp1, contributes to the pleiotropic phenotypes of the ccr4Δ and pop2∆ mutants.     

A Homozygous Mutation in LYRM7/MZM1L Associated with Early Onset Encephalopathy,Lactic Acidosis,and Severe Reduction of Mitochondrial Complex III Activity

Mutations in nuclear genes associated with defective complex III (cIII) of the mitochondrial respiratory chain are rare, having been found in only two cIII assembly factors and, as private changes in single families, three cIII structural subunits. Recently, human LYRM7/MZM1L, the ortholog of yeast MZM1, has been identified as a new assembly factor for cIII. In a baby patient with early onset, severe encephalopathy, lactic acidosis and profound, isolated cIII deficiency in skeletal muscle, we identified a disease‐segregating homozygous mutation (c.73G>A) in LYRM7/MZM1L, predicting a drastic change in a highly conserved amino‐acid residue (p.Asp25Asn). In a mzm1Δ yeast strain, the expression of a mzm1^D25N mutant allele caused temperature‐sensitive respiratory growth defect, decreased oxygen consumption, impaired maturation/stabilization of the Rieske Fe–S protein, and reduced complex III activity and amount. LYRM7/MZM1L is a novel disease gene, causing cIII‐defective, early onset, severe mitochondrial encephalopathy.     

衰老相关蛋白prelamin A与MT-2A的相互作用

目的分析衰老相关蛋白prelamin A是否与MT-2A相互作用,以及MT-2A与衰老的关系。方法采用酵母双杂交方法从人骨骼肌文库中筛选prelamin A的相互作用蛋白,酵母一对一回复性杂交和荧光共定位验证MT-2A与prelamin A是否相互作用。RT-PCR分析MT-2A在正常和早老细胞中的表达。结果通过酵母双杂交方法从人骨骼肌文库中筛选到prelamin A的候选相互作用蛋白MT-2A,一对一回复性验证也证明两者能在酵母细胞中相互作用。构建含绿色荧光蛋白的MT-2A融合表达载体转染HEK-293细胞,激光共聚焦显微观察发现MT-2A能与带红色荧光蛋白的prelamin A蛋白在核膜共定位。研究还发现MT-2A的表达在早老细胞中显著下调(P<0.01)。结论 MT-2A作为一个新的prelamin A结合蛋白,可能在细胞早老过程中具有重要的作用。     

Production of melatonin by Saccharomyces strains under growth and fermentation conditions

Abstract: Melatonin is a bioactive compound that is present in wine because it is contained in vinification grapes and synthesized by yeast during alcoholic fermentation. The purpose of this study was to determine the capacity of various Saccharomyces strains to form melatonin during its growth and alcoholic fermentation. A selection of yeasts including six S. cerevisiae (Lalvin CLOS, Lalvin ICV‐D254, Enoferm QA23 Viniferm ARM, Viniferm RVA, and Viniferm TTA), one S. uvarum (Lalvin S6U) and one S. cerevisiae var. bayanus (Uvaferm BC) were tested to determine whether they produce melatonin in yeast extract peptose dextrose and synthetic must media in a variety of conditions. Two S. cerevisiae strains (ARM, and QA23), the S. uvarum and the S. cerevisiae var. bayanus, synthesized melatonin. The conditions in which they did so, however, were different: the QA23 strain produced melatonin best in a medium with a low concentration of reducing sugars and Lalvin S6U and Uvaferm BC required a synthetic must under fermentation conditions. Melatonin synthesis largely depended on the growth phase of the yeasts and the concentration of tryptophan, reducing sugars and the growth medium. These results indicate that melatonin may have a role as a yeast growth signal molecule.     

新生儿5微克重组乙型肝炎疫苗（酿酒酵母）无应答者不同剂次和剂量再免疫血清学效果研究

目的评价新生儿接种5微克（μg）重组乙型肝炎（乙肝）疫苗（酿酒酵母）[Hepatitis B Vaccine（HepB）Made by Recombinant Deoxyribonucleic Acid Techniques in Saccharomyces Cerevisiae Yeast,HepB-SCY]后无应答者,使用不同剂次、剂量HepB再免疫后的血清学效果。方法抽取上海、山东、江苏、广西等省（自治区、直辖市）完成5μg HepBSCY全程免疫后的7~12月龄婴儿8945人,将其中的无应答者随机分为2组,分别采用5μg HepB-SCY和10μg HepB（汉逊酵母）（Hansenula Polymorpha Yeast,HPY）,按0、1、6个月程序进行再免疫,于再免疫第1、3剂后各1个月采血检测抗乙肝病毒表面抗原抗体（Antibody to Hepatitis B Virus Surface Antigen Anti-HBs）。结果 5μg HepBSCY组再免疫第1、3剂后,Anti-HBs阳转率分别为78.95%（60/76）和91.67%（66/72）,差异有统计学意义（χ2=4.73,P=0.03）。10μg HepB-HPY组再免疫第1、3剂后,Anti-HBs阳转率分别为92.65%（63/68）和100.00%（64/64）,差异无统计学意义[费希尔（Fisher）精确检验法,P=0.06]。5μg HepB-SCY组再免疫第1、3剂后,Anti-HBs的几何平均浓度（Geometric Mean Concentration,GMC）分别为45.79毫国际单位/毫升（mIU/ml）和193.83mIU/ml,差异有统计学意义（t=3.55,P=0.001）。10μg HepB-HPY组再免疫第1、3剂后,GMC分别上升为107.71 mIU/ml和302.54mIU/ml,差异有统计学意义（t=3.52,P=0.001）。再免疫第1剂后,10μg HepB-HPY组Anti-HBs的GMC高于5μg HepB-SCY组,差异有统计学意义（t=-2.10,P=0.037）。结论对5μg HepB-SCY初次免疫后无应答婴儿使用5μg HepB-SCY再免疫3剂,或10μg HepB-HPY再免疫1剂,能获得较理想的免疫应答;再免疫3剂比1剂可获得更高的抗体水平。     

Chemical genetic analysis of FTY720‐ and Ca2+‐sensitive mutants reveals a functional connection between FTY720 and membrane trafficking

FTY720, a sphingosine‐1‐phosphate (S1P) analog, is used as an immune modulator to treat multiple sclerosis. Accumulating evidence has suggested the mode of action of FTY720 independent of an S1P modulator. In fission yeast, FTY720 induces an increase in intracellular Ca²⁺ and ROS levels. We have previously identified 49 genes of which deletion causes FTY720 sensitivity. Here, we characterized the FTY720‐sensitive mutants in terms of their relevance to the Ca²⁺ homeostasis and identified the 16 F TY720‐ and C a²⁺‐s ensitive mutants (fcs mutants). Most of the FTY720‐sensitive mutants showed elevated Ca²⁺ levels and exhibited Ca²⁺ dysregulation by FTY720 treatment. One of the functional categories among the genes whose deletion renders cells susceptible to FTY720 and Ca²⁺ include the Golgi/endosomal membrane trafficking. Notably, FTY720, but not phosphorylated FTY720 incapable of inducing Ca²⁺ increase, inhibited the secretion of acid phosphatase in the wild‐type cells. Importantly, secretory defects of the Golgi/endosomal trafficking mutants, Vps45, or Ryh1 deletion, were further exacerbated by FTY720. Our fcs mutant screen also identified the adenylyl cyclase‐associated protein Cap1 and a Rictor homolog Ste20, whose deletion markedly exacerbated FTY720‐sensitive secretory impairment. Collectively, our data may suggest a synergistic impact of FTY720 combined with secretion perturbation on proliferation and Ca²⁺ homeostasis.