Effects of daunorubicin on ganglioside expression and neuronal differentiation of mouse embryonic stem cells

Gangliosides are implicated in neuronal development processes. The regulation of ganglioside levels is closely related to the induction of neuronal cell differentiation. In this study, the relationship between ganglioside expression and neuronal cell development was investigated using an in vitro model of neural differentiation from mouse embryonic stem (mES) cells. Daunorubicin (DNR) was applied to induce the expression of gangliosides in embryoid body (EB) (4+). We observed an increase in expression of gangliosides in all stages of EBs by treatment of DNR (2microM). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GD3, GD1a, GT1b, and GQ1b increased in DNR-treated 7-day-old EB (4+) [EB (4+):7]. DNR treatment significantly increased the expression of gangliosides, especially GT1b and GQ1b in comparison to control cells. Interestingly, GQ1b co-localized with microtubule-associated protein 2 (MAP-2) expressing cells in DNR-treated EB (4+):7. The co-localization of GQ1b and MAP-2 was found in neurite-bearing cells in DNR-treated 15-day-old EB (4+) [EB (4+):15], whereas no significant expression of GQ1b and less neurite formation were observed in untreated control. Also, the expression of synaptophysin and NF200, both neuronal markers associated with neruites, was increased by DNR treatment. These results demonstrate that DNR increases expression of gangliosides, especially GQ1b, in differentiating neuronal cells. Further, neurite-bearing neuronal cell differentiation can be facilitated by DNR, possibly through the induction of gangliosides. Thus, the present data suggest that DNR is beneficial for facilitating neuronal differentiation from ES cells and among the gangliosides analyzed in the present study, GQ1b is mainly involved in neurite formation.     

Enzymatic fractionation of conjugated linoleic acid isomers by selective esterification

Attempt was done to prepare food supplements with high content of c9, t11-CLA or t10, c12-CLA. A free acid mixture containing CLA isomers was esterified with ethanol by enzyme catalysis. Novozyme 435 and Lipase AY30 were screened, and Lipase AY30 was employed to catalyze esterification reaction because of its high fractionation efficiency. Effect of reaction conditions on total esterification was investigated, and the optimal reaction conditions were: 140 U of lipase amount, reaction temperature at 50 °C, a pH of 6.5, and molar ratio of FFA–CLA to ethanol at 1:1. Based on the studies above, experiments of esterification and purification were done, and the best fractionation efficiency was obtained when the total esterification was 37%, and the corresponding purity and recovery of c9, t11-CLA were 75.50 and 49.85%, and that of t10, c12-CLA were 72.02 and 62.32%.     

Functional insights from structural genomics

Structural genomics efforts have produced structural information, either directly or by modeling, for thousands of proteins over the past few years. While many of these proteins have known functions, a large percentage of them have not been characterized at the functional level. The structural information has provided valuable functional insights on some of these proteins, through careful structural analyses, serendipity, and structure-guided functional screening. Some of the success stories based on structures solved at the Northeast Structural Genomics Consortium (NESG) are reported here. These include a novel methyl salicylate esterase with important role in plant innate immunity, a novel RNA methyltransferase (H. influenzae yggJ (HI0303)), a novel spermidine/spermine N-acetyltransferase (B. subtilis PaiA), a novel methyltransferase or AdoMet binding protein (A. fulgidus AF_0241), an ATP:cob(I)alamin adenosyltransferase (B. subtilis YvqK), a novel carboxysome pore (E. coli EutN), a proline racemase homolog with a disrupted active site (B. melitensis BME11586), an FMN-dependent enzyme (S. pneumoniae SP_1951), and a 12-stranded β-barrel with a novel fold (V. parahaemolyticus VPA1032).     

Improving freezing tolerance of transgenic tobacco expressing sucrose: sucrose 1-fructosyltransferase gene from Lactuca sativa

Sucrose: sucrose 1-fructosyltransferase (1-SST) cDNA from Lactuca sativa, coding the enzyme responsible for lower degree polymers fructan biosynthesis, was cloned by RT-PCR and RACE methods. The 1-SST cDNA under the control of CaMV 35S promoter was introduced into tobacco by Agrobacterium-mediated leaf disc transformation protocol. Fructan synthesis in vitro and carbohydrate analysis showed that sense transgenic tobacco plant displayed sucrose: sucrose 1-fructosyltransferse activity. After freezing stress, significant increases in electrolyte leakage and malondialdehyde were found in the wild type and anti-sense transgenic plants, while no apparent differences were observed in sense transgenic plants. Meanwhile, water soluble carbohydrate, fructan and fructose of sense transgenic plants remarkably increased, compared with those of wild type and anti-sense plants. No significant difference was detected in superoxide dismutase activity between transgenic and wild type plants. The above results demonstrated that the expression of 1-SST gene improved the freezing resistance of transgenic tobacco plants.     

CD1d degradation in Chlamydia trachomatis-infected epithelial cells is the result of both cellular and chlamydial proteasomal activity

Chlamydia trachomatis is an obligate intracellular pathogen that can persist in the urogenital tract. Mechanisms by which C. trachomatis evades clearance by host innate immune responses are poorly described. CD1d is MHC-like, is expressed by epithelial cells, and can signal innate immune responses by NK and NKT cells. Here we demonstrate that C. trachomatis infection down-regulates surface-expressed CD1d in human penile urethral epithelial cells through proteasomal degradation. A chlamydial proteasome-like activity factor (CPAF) interacts with the CD1d heavy chain, and CPAF-associated CD1d heavy chain is then ubiquitinated and directed along two distinct proteolytic pathways. The degradation of immature glycosylated CD1d was blocked by the proteasome inhibitor lactacystin but not by MG132, indicating that degradation was not via the conventional proteasome. In contrast, the degradation of non-glycosylated CD1d was blocked by lactacystin and MG132, consistent with conventional cellular cytosolic degradation of N-linked glycoproteins. Immunofluorescent microscopy confirmed the interruption of CD1d trafficking to the cell surface, and the dislocation of CD1d heavy chains into both the cellular cytosol and the chlamydial inclusion along with cytosolic CPAF. C. trachomatis targeted CD1d toward two distinct proteolytic pathways. Decreased CD1d surface expression may help C. trachomatis evade detection by innate immune cells and may promote C. trachomatis persistence.     

谷氨酸脱羧酶与Ⅰ型糖尿病发病机制

谷氨酸脱羧酶（glutamic acid decarboxylase,GAD）与Ⅰ型糖尿病（type 1 diabetes,T1DM）的发病有很大关系,被认为是Ⅰ型糖尿病发病的自身免疫启动靶抗原。谷氨酸脱羧酶对于Ⅰ型糖尿病的预测、诊断、预防和治疗都有很大应用价值。该文阐述了谷氨酸脱羧酶的最新研究进展,以及谷氨酸脱羧酶与Ⅰ型糖尿病自身免疫发病机制的联系。     

Assay of L-3-hydroxyacyl-coenzyme A dehydrogenase with substrates of different chain lengths

A method for assaying L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which permits rate measurements with L-3-hydroxyacyl-CoA substrates of various chain lengths at physiological pH is described. The method is based on a coupled assay system in which 3-ketoacyl-CoA compounds formed by the dehydrogenase are cleaved by 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in the presence of CoASH. The advantages of this assay method are its irreversibility and elimination of product inhibition. The assay procedure was used to determine the kinetic parameters (Km, Vmax) of pig heart L-3-hydroxyacyl-CoA dehydrogenase with several substrates of various chain lengths. The data obtained show the enzyme to be most active with medium-chain substrates whereas Km values for medium-chain and long-chain substrates are almost equal but much lower than those previously reported.     

气孔下腔间的水汽扩散

本文把气孔及其下腔看作截面为椭圆形的柱形区域,提出一个水汽从气孔下腔内所有细胞表面扩散到气孔外端的三维扩散模型。根据 Fick 定律和质量守恒定律建立了支配该模型的水汽扩散方程。用有限差分法,借助于计算机求得水汽从气孔下腔的所有细胞表面扩散到气孔内端所遇到的阻力及其近似表达式。并从理论上对该阻力的倒数——导度随气孔面积而变化的方式做了分析和解释。通过将本模型求得的气孔下腔阻力计算公式与 Brown 等以及 Cooke 的公式比较,发现在气孔开度变化相当大的范围内用后面两公式计算的阻力偏大0.5—1倍左右。此外,计算结果还表明:在气孔下腔水散失总量中,腔内表皮细胞表面上的水散失量占86—96%,而保卫细胞表面上的水散失量又占后者的88—93%,副卫细胞表面上的水散失量仅7—12%。