Modulation of the Acetylcholine System in the Superior Cervical Ganglion of Rat: Effects of GABA and Hypoglossal Nerve Implantation After In Vivo GABA Treatment

gamma-Aminobutyric acid (GABA) was applied to the superior cervical ganglion (SCG) of CFY rats in vitro and in vivo, with or without implantation of a hypoglossal nerve, to evaluate the effects of these experimental interventions on the acetylcholine (ACh) system, which mainly serves the synaptic transmission of the preganglionic input. Long-lasting GABA microinfusion into the SCG in vivo apparently resulted in a "functional denervation." This treatment reduced the acetylcholinesterase (AChE; EC 3.1.1.7) activity by 30% (p less than 0.01) and transiently increased the number of nicotinic acetylcholine receptors, but had no significant effect on the choline acetyltransferase (acetyl-coenzyme A:choline-O-acetyltransferase; EC 2.3.1.6) activity, the ACh level, or the number of muscarinic acetylcholine receptors. The relative amounts of the different molecular forms of AChE did not change under these conditions. In vivo GABA application to the SCG with a hypoglossal nerve implanted in the presence of intact preganglionic afferent synapses exerted a significant modulatory effect on the AChE activity and its molecular forms. The "hyperinnervation" of the ganglia led to increases in the AChE activity (to 142.5%, p less than 0.01) and the 16S molecular form (to 200%, p less than 0.01). It is concluded that in vivo GABA microinfusion and GABA treatment in the presence of additional cholinergic synapses has a modulatory effect on the elements of the ACh system in the SCG of CFY rats.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

Human alpha-galactosidase A: characterization of the N-linked oligosaccharides on the intracellular and secreted glycoforms overexpressed by Chinese hamster ovary cells

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi- , 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.      

Dependence of Ethanolic Fermentation, Cytoplasmic pH Regulation, and Viability on the Activity of Alcohol Dehydrogenase in Hypoxic Maize Root Tips

We examined the role of alcohol dehydrogenase (ADH) in the metabolism and survival of hypoxic maize (Zea mays L.) root tips. The dependence of the rate of ethanolic fermentation, cytoplasmic pH, and viability on the activity of ADH in maize root tips during extreme hypoxia was determined. Maize lines with ADH activities differing over about a 200-fold range were studied. Effects of genetic background were controlled by comparing pairs of F4 progeny of crosses between mutant (low ADH activity) and reference inbred lines. The capacity of hypoxic root tips to perform ethanolic fermentation exhibited a dependence on ADH activity only at activities found in Adh 1 nulls. The ability of maize root tips to withstand prolonged and extreme hypoxia was like-wise independent of ADH activity, except at the lowest activities. Root tips that exhibited lower tolerance of hypoxia had more acidic cytoplasm during extreme hypoxia. We conclude that the activity of ADH in normal maize root tips does not limit the capacity for energy production via fermentation, and does not determine viability under extreme hypoxia. The significance of the induction of ADH activity in plants by hypoxia is discussed.     

Flow-fluorometric diagnosis of euploid and aneuploid human lymphocytes.

In this paper the potential of flow-fluorometric DNA determination as a convenient and economical alternative to conventional cytogenetics for the diagnosis of aneuploidy in human lymphocytes is explored. By comparing euploid and aneuploid samples, we found that the fluorescence signals emitted from propidium iodide (PI) stained cells are linearly proportional to DNA content. Variation in DNA content between euploid individuals of a given sex was sufficiently low to permit diagnosis of aneuploidy involving chromosomes with greater than 1.8% of the total diploid DNA (e.g., X, 8, 9, and 13). Interindividual DNA content variation with flow fluorometry was too substantial, however, to confidently diagnose trisomy 21. Fluorescent stains which exclude (variant) simple sequence DNA might overcome this limitation.     

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Synthesis and neuroprotective activity of analogues of glycyl-L-prolyl-L-glutamic acid (GPE) modified at the alpha-carboxylic acid

The synthesis of nine GPE* analogues, wherein the alpha-carboxylic acid group of glutamic acid has been modified, is described by coupling readily accessible N-benzyloxycarbonyl-glycyl-L-proline 2 with various analogues of glutamic acid. Pharmacological evaluation of the novel compounds was undertaken to further understand the role of the glutamate residue on the observed neuroprotective properties of the endogenous tripeptide GPE.     

Evolution of the Sry genes

Existing DNA sequence data on the Sry gene, the mammalian sex- determining locus in the Y chromosome, were analyzed for primates, rodents, and bovids. In all three taxonomic groups, the terminal sequences evolved faster than the HMG (high mobility group) boxes, and this applies both to synonymous (Ks) and nonsynonymous (Ka) nucleotide substitutions. Similar intragenic correlation between synonymous and nonsynonymous substitution rates was not found either in other mammalian genes that contain a conservative box (Sox, Msx) or in the MADS-box genes of plants. The rate of nonsynonymous substitutions exceeds significantly that of synonymous substitutions in the terminal Sry sequences of apes. We did not find good support for the hypothesis that the high evolutionary rate of Sry would be associated with a promiscuous mating system.      

Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach

Background  

Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event.