Mutagenesis of D80-82 and G83 residues in West Nile Virus NS2B: Effects on NS2B-NS3 activity and viral replication

Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D⁸⁰DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D⁸⁰DDG in NS2B on protease activity and viral replication, the negatively charged region D⁸⁰DD and the conserved residue G83 of NS2B were mutated (D⁸⁰DD/E⁸⁰EE, D⁸⁰DD/K⁸⁰KK, D⁸⁰DD/A⁸⁰AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D⁸⁰DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D⁸⁰DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.     

Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.     

Caffeic acid phenethyl ester modulates aflatoxin B1‐induced hepatotoxicity in rats

Aflatoxin B1 (AFB1) is the most potent of the mycotoxins and is widely observed in nutrition abnormalities. There are some studies suggesting oxidative stress‐induced toxic changes on liver related to AFB1 toxicity. The aim of the present study was to evaluate whether antioxidant caffeic acid phenethyl ester (CAPE) relieves oxidative stress in AFB1‐induced liver injury in rat. Twenty‐four male rats were equally divided into three groups. The first group was used as a control. The second group received three doses of AFB1. The three doses of CAPE were given to constitute the third group with doses of AFB1. After 10 days of experiment, liver and serum samples were taken from all animals. Serum gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), glutathione s‐transferase (GST), nitric oxide (NO) and sulfhydryl values were higher in the AFB1 group than in control, whereas serum GGT, ALP, GST and NO values were decreased by in the AFB1 + CAPE group than in AFB1 group. Liver GST, total oxidant capacity, sulfhydryl, apoptosis index and ischemia‐modified albumin values were higher in the AFB1 group than in control, whereas the GST activity and apoptosis index were lower in the AFB1 + CAPE group than in the AFB1 group. There were histopathological degeneration and apoptosis in hepatocytes of AFB1 group. The findings were totally recovered by CAPE administration. In conclusion, we observed that AFB1 caused oxidative and nitrosative hepatoxicity to hepatocytes in the rat. However, CAPE induced protective effects on the AFB1‐induced hepatoxicity by modulating free radical production, biochemical values and histopathological alterations. Copyright © 2013 John Wiley & Sons, Ltd.     

以胞质分裂阻滞法研究女性年龄与体细胞中 21号染色体分离异常的关系

21三体是人类最常见的先天性疾病,约95%患儿的超数21号染色体来源于母亲,且随母龄增加患儿出生率也随之增加。本文首次运用胞质分裂阻滞法(CB法)对不同年龄女性体细胞进行荧光原位杂交,对其21号染色体的分离情况进行分析。结果表明,随年龄的增加,女性体细胞中21号染色体不分离也随之增加,但21号染色体丢失无年龄效应, 且21号染色体不分离频率远高于其丢失。该结果表明,女性体细胞中21号染色体不分离与生殖细胞同样存在年龄效应。 Abstract　　Trisomy 21 is the most common genetic desease in human. More than 90% is derived from mother. With the advancing of mother's age, the frequency of trisomy 21 is increasing. We detected the relation between chromosome 21 missegregation and age in cytokinesis-blocked female lymphocytes by　in situ　hybridization with chromosome 21 specfic probe. We have found that the age effect also exists in female somatic cells as in gamets.     

A Deletion in the α2B‐Adrenergic Receptor Gene and Autonomic Nervous Function in Central Obesity

Objective: We investigated the impact of a three‐amino acid deletion (12Glu9) polymorphism in the α_2B‐adrenergic receptor gene on autonomic nervous function. The short form (Glu⁹/Glu⁹) of the polymorphism has previously been associated with a reduced basal metabolic rate in obese subjects. Because autonomic nervous function participates in the regulation of energy metabolism, there could be a link between this polymorphism and autonomic nervous function. Research Methods and Procedures: Data of a 10‐year follow‐up study with 126 nondiabetic control subjects and 84 type 2 diabetic patients were used to determine the effects of the 12Glu9 polymorphism on autonomic nervous function. A deep breathing test and an orthostatic test were used to investigate parasympathetic and sympathetic autonomic nervous function. In addition, cardiovascular autonomic function was studied using power spectral analysis of heart rate variability. Results: No significant differences were found in the frequency of the 12Glu9 deletion polymorphism between nondiabetic and diabetic subjects. The nondiabetic men with the Glu⁹/Glu⁹ genotype, especially those with abdominal obesity, had significantly lower total and low‐frequency power values in the power spectral analysis when compared with other men. Furthermore, in a longitudinal analysis of 10 years, the decrease in parasympathetic function was greater in nondiabetic men with the Glu⁹/Glu⁹ genotype than in the men with the Glu⁹/Glu¹² or Glu¹²/Glu¹² genotypes. Discussion: The results of the present study suggest that the 12Glu9 polymorphism of the α_2B‐adrenergic receptor gene modulates autonomic nervous function in Finnish nondiabetic men. In the nondiabetic men with the Glu⁹/Glu⁹ genotype, the general autonomic tone is depressed, and vagal activity especially becomes impaired with time. Furthermore, this association is accentuated by central obesity.     

Biosynthesis of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 using liquid acid protein residue of soybean as culture medium

Vitamin B12 deficiency still persists, mainly caused by low intake of animal food products affecting vegetarians, vegans, and populations of underdeveloped countries. In this study, we investigate the biosynthesis of vitamin B12 by potential probiotic bacterium using an agroindustry residue, the liquid acid protein residue of soybean (LAPRS), as a low-cost, animal derivate-free alternative culture medium. Cultures of Propionibacterium freudenreichii subsp. shermanii ATCC 13673 growing in LAPRS for vitamin B12 biosynthesis were studied using the Plackett–Burman experimental approach, followed by a central composite design 2² to optimize the concentration of significant variables. We also performed a proteolytic treatment of LAPRS and evaluated the optimized–hydrolyzed medium influence on the microbial growth and metabolism in shaker flask and bioreactor experiments. In this all-plant source medium, P. freudenreichii subsp. shermanii produced high concentrations of cells and high amounts of vitamin B12 (0.6 mg/g cells) after process optimization. These results suggest the possibility of producing vitamin B12 by a potential probiotic bacterium in a very cheap, animal derivate-free medium to address the needs of specific population groups, at the same time reducing the production costs of this essential vitamin.