Effect of a partial trisomy in the mouse upon cellular and nuclear RNA

In a previous paper (Rake and Edwards, 1987) it was shown that the majority of the chromatin from trisomic mouse cells has nucleosomes with a smaller repeat length of DNA than the nucleosome repeat length of normal cells. Here it is shown that the RNA content of the total cell and of the nuclei is the same in all tissues studied, in both normal and trisomic cells. However, the amount per unit time or rate of RNA synthesis is depressed in the trisomic liver and brain nuclei. The depression of RNA synthesis could not be specified to the small trisomic section of the chromatin but instead must reflect the overall nuclear activity. These results, along with those of Devlinet al. (1988), indicate that the trisomic condition alters a substantial part of nuclear organization and activity, not just the small trisomic part.     

Comparison among the isozyme profiles associated with ethrel treatments of leaves, and with senescence and plum pox virus infection in Chenopodium foetidum

Changes in the isozyme profiles of peroxidases (POX, EC 1.11.1.7), glutamate oxaloacetate transaminase (GOT, EC 2.6.1.1) and esterases (EST, EC 3.1.1.1) have been studies in leaves of Chenopodium foetidum S. treated with ethrel. Different systems for administration of the ethrel led to different responses. In intact plants, treatment of the complete surface of leaves or local administration by pricking the leaves induced senescence and wilting as well as quick changes, characteristic of ageing, in the isozyme patterns of the treated leaves. Isolated leaves treated with ethrel in vitro also showed a senescence response, but this was followed by a necrosis that displayed an isozyme pattern highly similar to that of necrotic lesions induced by plum pox virus (PPV) infection. An accelerated senescence process seems to be involved in the induction of changes in the isozyme patterns of expression during the hypersensitive response of Chenopodium foetidum to PPV infection, and ethylene could participate in this process. However, other factors may also be required for necrotization.     

Functional characterization of the C-terminal domain of mouse capping enzyme

Mouse capping enzyme (Mce1) consists of two functional domains: the amino-terminal triphosphatase domain and the carboxyl-terminal guanylyltransferase (GTase) domain. The bifunctional Mce1 gene encodes 597 a.a. with a molecular weight approximately 68 kDa. Mce1 cDNA is located on chromosome 4A4 approximately 4A5 and is composed of 17 exons. To functionally characterize the C-terminus of Mce1, we generated four truncated proteins with 12, 30, 37, or 60 a.a. deletions from the C-terminus of either the wild type (Mce1) or the isolated GTase domain (211-597), respectively. Plasmid shuffling experiment with Saccharomyces cerevisiae GTase subunit gene CEG1 null mutant demonstrated that deletion mutants 211-567 and 211-585 were able to support cell viability in the presence of 5-fluoroorotic acid, whereas 211-537 and 211-560 were not. Consistent with the yeast genetic study, both 211-567 and 211-585 had significant GTase activity in vitro, while 211-537 and 211-560 that were only detected in the insoluble fraction in the bacterial expression system, were completely inactive. Overall, both in vivo and in vitro studies indicate that the functional domain of Mce1 is between a.a. 211 and 567, and the heptapeptide sequence between 561 and 567 may play an important role in the enzyme activity.     

Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin,Immune, and Male Reproductive Systems

UBE2W ubiquitinates N termini of proteins rather than internal lysine residues, showing a preference for substrates with intrinsically disordered N termini. The in vivo functions of this intriguing E2, however, remain unknown. We generated Ube2w germ line KO mice that proved to be susceptible to early postnatal lethality without obvious developmental abnormalities. Although the basis of early death is uncertain, several organ systems manifest changes in Ube2w KO mice. Newborn Ube2w KO mice often show altered epidermal maturation with reduced expression of differentiation markers. Mirroring higher UBE2W expression levels in testis and thymus, Ube2w KO mice showed a disproportionate decrease in weight of these two organs (∼50%), suggesting a functional role for UBE2W in the immune and male reproductive systems. Indeed, Ube2w KO mice displayed sustained neutrophilia accompanied by increased G-CSF signaling and testicular vacuolation associated with decreased fertility. Proteomic analysis of a vulnerable organ, presymptomatic testis, showed a preferential accumulation of disordered proteins in the absence of UBE2W, consistent with the view that UBE2W preferentially targets disordered polypeptides. These mice further allowed us to establish that UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm and that the absence of UBE2W does not alter cell viability in response to various stressors. Our results establish that UBE2W is an important, albeit not essential, protein for early postnatal survival and normal functioning of multiple organ systems.     

Expression of PGK-A in the Australian brush-tailed possum,Trichosurus vulpecula (Kerr), consistent with paternal X inactivation

An extensive survey of erythrocytes of marsupials other than kangaroos for electrophoretic variation in X-linked enzymes revealed two rare PGK-A phenotypes in the phalangerid Trichosurus vulpecula and one in Trichosurus caninus. Four putatively heterozygous females expressed only the variant allelic isozyme in some tissues but expressed a trace of the normal isozyme in others. A putatively hemizygous male expressed only the variant isozyme in all tissues. The phenotypic patterns were consistent with those observed in kangaroos known to exhibit partial or complete paternal X inactivation in cells of females. Two of the T. vulpecula were a mother and her female pouch young, further suggesting that paternal X inactivation occurs in T. vulpecula. This peculiar mechanism of dosage compensation may not be restricted to kangaroos.This work was supported by grants to D. W. C. from the Australian Research Grants Committee and Macquarie University, J. L. V. was the recipient of a Fulbright-Hays Award from the Australian-American Educational Foundation and a postgraduate fellowship from General Motors-Holden.     

Introduction of sense and antisense cDNA for branching enzyme in the amylose-free potato mutant leads to physico-chemical changes in the starch

One isoform of the branching enzyme (BE; EC 2.4.1.18) of potato (Solarium tuberosum L.) is known and catalyses the formation of α-1,6 bonds in a glucan chain, resulting in the branched starch component amylopectin. Constructs containing the antisense or sense-orientated distal 1.5-kb part of a cDNA for potato BE were used to transform the amylose-free (amf) mutant of potato, the starch of which stains red with iodine. The expression of the endogenous BE gene was inhibited either largely or fully as judged by the decrease or absence of the BE mRNA and protein. This resulted in a low percentage of starch granules with a small blue core and large red outer layer. There was no effect on the amylose content, degree of branching or λ_max of the iodine-stained starch. However, when the physico-chemical properties of the different starch suspensions were assessed, differences were observed, which although small indicated that starch in the transformants was different from that of theamf mutant.     

Enzyme activities in Pennisetum seedlings germinated in the presence of abscisic and gibberellic acids

Effects of abscisic acid (ABA) and gibberellic acid (GA₃), alone and in combination, on growth and activity of alanine aminotransferase (GPT), aspartate aminotransferase (GOT), and glutamate dehydrogenase (GLDH) were studied in aerial parts of Pennisetum typhoides seedlings. ABA inhibited growth and activity of GLDH, but stimulated the activity of GPT and weakly that of GOT. GA₃, on the other hand, did not affect the activity of any of the enzymes tested, but in combination with ABA tended to antagonise the efrect of the latter.     

Effects of metal cations and other chemicals upon the in vitro activity of two enzymes in the blood plasma of the white sucker, Catostomus commersoni (Lacépède)

Relative changes in the activity of glutamic oxalacetic transaminase (GOT, l-aspartate-2-oxoglutarate aminotransferase) and lactic dehydrogenase (LDH, l-lactate-NAD oxidoreductase) in blood plasma from white suckers were determined after incubation with 49 compounds, principally inorganic chlorides, at concentrations of the ions up to 2 mg/ml in the reaction mixture. A sequence of inhibitory effect was arranged for each enzyme. Dose-response curves were qualitatively similar for most of the chemicals. GOT was most sensitive to silver and mercury, and LDH to palladium and mercury. Both enzymes are highly inhibited by metals which are highly toxic to aquatic animals. Correlations were studied between the inhibitory effect and certain physicochemical properties of chemicals, the best being found between the inhibition of GOT and the equilibrium constants of metal sulfides.     

水稻sbe1启动子驱动的反义sbe-GUS融合基因在转基因水稻中的表达

为研究水稻基因启动子对外源基因在转基因水稻中表达的影响,构建了由sbe1启动子引导的反义sbe-GUS融合基因。经农杆菌介导,将不同的融合基因导入水稻中,定量测定转基因水稻植株不同组织中的GUS酶活力。结果表明,sbe1启动子可驱动反义sbe-GUS融合基因在转基因水稻植株的胚乳中高效表达,而在颖壳、胚和茎叶等组织中的表达活性较弱。证实sbe1启动子在驱动外源基因的表达上表现有明显的组织特异性。     

The distribution and structure of cells in the tracheal epithelium of the mouse

Summary The tracheal epithelium of the mouse is a single layer of columnar cells resting on a basement membrane. Many of the cell types resemble those of other species. However, goblet cells are rare and ciliated cells occur only in scattered patches. Submucosal glands are absent from all but the highest reaches of the airway.The major proportion of the epithelial cells are non-ciliated. These usually project into the lumen of the trachea. Large amounts of smooth endoplasmic reticulum and many secretory vesicles occur within the cytoplasm. Secretory activity of these cells may be either apocrine or merocrine and these cells may transform into other cell types.It is suggested that these non-ciliated cells are Clara cells and that the mouse tracheal epithelium may make a useful model for the study of this type of cell.     

嗜酸氧化亚铁硫杆菌亚铁氧化酶基因分子多态性研究

嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans, A.f）属于化能自养、革兰氏阴性细菌,好氧嗜酸,以氧化亚铁离子或还原态硫化物（S0&S2-）获得能量生长。在其Fe2+氧化系统中,亚铁氧化酶起重要作用。对不同硫化矿区分离得到的5株A.f进行生长动力学和对亚铁氧化活性的对比研究,结果显示不同菌株之间存在表型差异。提取A.f菌株的基因组DNA,设计引物,对亚铁氧化酶基因进行PCR扩增,同时对扩增产物直接进行测序,并同GenBank的参考序列进行比对分析,发现序列相似性在99%～97%之间,分析编码区域发现在第187～195位可能存在一个高变异区：在第187～189位,菌株YTW编码的氨基酸Thr→Pro;而在第193～195位,菌株TK是Met→Asn; 菌株BY则是Met→Ile。另外在位点第219位,所有菌株都是T→C,但编码的氨基酸未发生变化,属于同义密码子。对于编码区上游序列,比对后没有差异;而对于编码区下游序列,经比对发现存在一些差异位点。     

Effect of quinolinic acid on the distribution of hepatic metabolites

The intracellular distribution of hepatic metabolites in normal and quinolinic acid (QA)-treated rats has been calculated. QA, an inhibitor of gluconeogenesis, raises the total cell content of malate, aspartate, α-ketoglutarate and citrate. The calculated mitochondrial content of all four metabolites was raised, as was the mitochondrial/cytosolic gradient, and the cytosolic content of oxaloacetate and α-ketoglutarate decreased. The fall of cytosolic oxaloacetate in QA-treated rats suggests a control at PEPCK by substrate limitation. It is suggested that QA may have an action on the translocation of polycarboxylate anions across the mi tochondrial membrane.     

Structure of UBE2Z Enzyme Provides Functional Insight into Specificity in the FAT10 Protein Conjugation Machinery

FAT10 conjugation, a post-translational modification analogous to ubiquitination, specifically requires UBA6 and UBE2Z as its activating (E1) and conjugating (E2) enzymes. Interestingly, these enzymes can also function in ubiquitination. We have determined the crystal structure of UBE2Z and report how the different domains of this E2 enzyme are organized. We further combine our structural data with mutational analyses to understand how specificity is achieved in the FAT10 conjugation pathway. We show that specificity toward UBA6 and UBE2Z lies within the C-terminal CYCI tetrapeptide in FAT10. We also demonstrate that this motif slows down transfer rates for FAT10 from UBA6 onto UBE2Z.     

不同浓度外源乙酸钠对耐乙酸大肠埃希菌DA19代谢和关键酶酶活的影响

为研究外源乙酸钠对大肠埃希菌DA19生长代谢的影响,将该菌株在氮源限制基本培养基及添加不同浓度乙酸钠的氮源限制基本培养基中连续培养,测定稳态时生长代谢参数和胞内关键酶酶活。与MN培养基相比,葡萄糖比消耗速率和延胡索酸比生成速率随外源乙酸钠质量浓度增加而逐渐下降,丙酮酸比生成速率则随外源乙酸钠质量浓度增加而明显增加,而乙酸比生成速率则明显降低(除9 g/L乙酸钠外)。磷酸果糖激酶、异柠檬酸脱氢酶、异柠檬酸裂解酶、苹果酸脱氢酶、磷酸烯醇式丙酮酸羧化酶和乙酸激酶酶活随外源乙酸钠质量浓度增加而呈先下降后上升的趋势,而6-磷酸葡萄糖脱氢酶则随着外源乙酸钠质量浓度增加而逐渐降低。为了应对外源乙酸钠压力,大肠埃希菌DA19的生长代谢和中心代谢途径酶活都发生了明显改变。     

老黄酶OYE家族的蛋白质工程的研究进展 *

老黄酶OYE家族酶是一类广泛分布的能够催化烯烃化合物不对称还原的酶类,其能够用于多种手性化合物的制备.分析了OYE家族酶的系统分类及催化反应类型,针对目前该类酶在应用过程中出现的稳定性差,活性低及底物特异性强等问题,综述了蛋白质工程方法对该类酶进行改造的研究进展,为深入研究该家族酶的催化机制及进一步改造提供参考,同时为进一步拓展OYE酶的工业化应用奠定基础.     

Generation of a novel germline stem cell line expressing a germline‐specific reporter in the mouse

Germline stem (GS) cells are stem cell lines derived from postnatal male germline cells. Remarkably, GS cells can form functional spermatozoa when transplanted into infertile host mouse testes, indicating that GS cells have spermatogonial stem cell (SSC) activity. As GS cells are the only type with SSC activity, they are most suitable for in vitro studies on male germ cell differentiation. However, GS cells can deviate from the germ cell state to become other types of cells, depending on culture conditions. Therefore, it is desirable to have a monitor system to ensure that GS cells are kept at the germ cell state in culture. Here, we established GS cell lines from neonatal testes of transgenic mice that express the fluorescent protein, Venus, whose gene expression is driven by the promoter of Mvh (mouse Vasa homolog), a gene highly specific to mammalian germ cells. This novel cell line has genuine GS cell properties equivalent to existing GS lines, including the ability to generate viable offspring. This Mvh–Venus GS cell line, to our knowledge, is the first one expressing a germ cell‐specific reporter. This valuable resource should provide new opportunities for studies on male germ cell differentiation. genesis 51:498–505. © 2013 Wiley Periodicals, Inc.