Yeast 14-3-3 proteins

14-3-3 proteins form a family of highly conserved proteins which are present in all eukaryotic organisms investigated, often in multiple isoforms, up to 13 in some plants. They interact with more than 200 different, mostly phosphorylated proteins. The molecular consequences of 14-3-3 binding are diverse: this binding may result in stabilization of the active or inactive phosphorylated form of the protein, to a conformational alteration leading to activation or inhibition, to a different subcellular localization, to the interaction with other proteins or to shielding of binding sites. The binding partners, and hence the 14-3-3 proteins, are involved in almost every cellular process and 14-3-3 proteins have been linked to several diseases, such as cancer, Alzheimer's disease, the neurological Miller-Dieker and spinocerebellar ataxia type 1 diseases and bovine spongiform encephalopathy (BSE). The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both have two genes encoding 14-3-3 proteins, BMH1 and BMH2 and rad24 and rad25, respectively. In these yeasts, 14-3-3 proteins are essential in most laboratory strains. As in higher eukaryotes, yeast 14-3-3 proteins bind to numerous proteins involved in a variety of cellular processes. Recent genome-wide studies on yeast strains with impaired 14-3-3 function support the participation of 14-3-3 proteins in numerous yeast cellular processes. Given the high evolutionary conservation of the 14-3-3 proteins, the experimental accessibility and relative simplicity of yeasts make them excellent model organisms for elucidating the function of the 14-3-3 protein family.     

TOR kinase pathway and 14-3-3 proteins regulate glucose-induced expression of HXT1, a yeast low-affinity glucose transporter

Expression of HXT1, a gene encoding a Saccharomyces cerevisiae low-affinity glucose transporter, is regulated by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are scarce. In this study we show that 14-3-3 proteins are involved in the regulation of the expression of HXT1 by glucose. We also demonstrate that 14-3-3 proteins, in complex with Reg1, a regulatory subunit of Glc7 protein phosphatase, interact physically with Grr1 (a component of the SCF-Grr1 ubiquitination complex), a key player in the process of HXT1 induction by glucose. In addition, we show that the TOR kinase pathway participates actively in the induction of HXT1 expression by glucose. Inhibition of the TOR kinase pathway by rapamycin treatment abolishes HXT1 glucose induction. A possible involvement of PP2A protein phosphatase complex, through the Cdc55 B-subunit, in the glucose induction of HXT1 is also discussed.     

The Candida albicans 14-3-3 gene, BMH1, is essential for growth.

The 14-3-3 proteins are a family of conserved small acidic proteins that have been implicated in playing major roles in a wide variety of signalling cascades. In Saccharomyces cerevisiae, the 14-3-3 genes (BMH1 and BMH2) are essential for normal pseudohyphal induction and normal bud cell development. The Bmh proteins function in the cAMP-dependent RAS/MAPK and rapamycin-sensitive signalling cascades. Deletion of only one BMH gene demonstrates no phenotypic differences under normal growth conditions. Strains deleted of both BMH1 and BMH2 are either non-viable or demonstrate sensitivity to environmental stresses. In Schizosaccharomyces pombe, the BMH homologues (RAD24 and RAD25) are essential for cell cycle control after DNA damage and deletion of both genes renders the cell inviable. The 14-3-3 gene in Candida albicans (BMH1) was identified using a novel adherence assay and differential display RT-PCR. Unlike other yeasts, C. albicans has only one 14-3-3 gene (BMH1). It was not possible to construct double knockouts by routine methods. These results suggested that the C. albicans BMH1 gene is essential. The essentiality of C. albicans BMH1 was confirmed by a PCR disruption technique. The C. albicans bmh1 Delta/BMH1 heterozygotes exhibit growth and morphogenetic defects. Therefore, the BMH1 gene in C. albicans (Accession No. AF038154) is an excellent candidate to improve our understanding of the coordinate regulation of cell cycle and morphogenesis.     

Potential weekly intake of artificial food colours by 3-14-year-old children in Brazil.

The Potential Weekly Intake (PWI) of artificial food colours by 3-14-year-old children living in the District of Bar?o Geraldo, Campinas, S?o Paulo, Brazil, was estimated on the basis of average consumption data of artificially coloured food and analytically determined colour concentration in foodstuffs ingested. Coloured food consumption data were obtained through dietary recall interviews and collection of the packages and/or labels of the coloured foods consumed during a two-week period. Colours found in the individual types of foods detected through the consumption survey were identified and determined by methods that included wool dyeing and polyamide column extractions, ascending paper chromatography and spectrophotometry. The results showed that all artificial colours used in the composition of 83 commercial food products, including jellies, juices, soft drinks, syrups and 57 different candies, were permitted for use in food in Brazil the year the survey was conducted (1986), in amounts below those prescribed by law. Statistical analysis performed to compare the PWI for different population groups demonstrated that young male children, especially from lower social classes, were most exposed to artificial colours. Comparison of the estimated potential intakes with the toxicologically Acceptable Daily Intake (ADI) showed that consumption of Amaranth, Sunset Yellow, Indigotine and Tartrazine by all children in the study represented approximately 24%, 3%, 0.05% and 0.4%, of the actual ADI values, respectively.     

啤酒废酵母泥综合利用的研究

研究了利用啤酒废酵母泥制备酵母抽提物和β-葡聚糖的相关工艺条件。结果表明,自溶-酶联法是制备啤酒酵母抽提物的理想方法,用该法制备啤酒酵母抽提物的抽提率达68.6%、蛋白质利用率达87.3%,抽提物中蛋白质、游离氨基酸态氮和复合核苷酸的含量分别达10.6%、4.2%和3.9%,远高于普通自溶工艺的技术指标;将自溶后的酵母残渣进一步制备成β-1,3-葡聚糖,通过正交试验获得了其优化的工艺条件:酵母浓度10%、碱浓度2%、反应温度80℃、反应时间4 h、碱处理次数4次。在优化的工艺条件下,β-1,3-葡聚糖得率达26.8%、成品中杂蛋白含量仅0.4%、β-葡聚糖分子质量为158ku。实现了啤酒废酵母泥的综合利用和高值化。     

增强食品风味的酵母抽提物

<正> 在近几个世纪中,水解蛋白质原料在食品配料中扮演着重要的角色,为食品提供更佳的风味。现时,市场上的蛋白质水解物主要有水解植物蛋白(HVP)、酱油和自溶酵母抽提物三大类;全球的生产量估计为:水解植物蛋白约12万吨、酱油60余万吨、酵母抽提物约5万吨。就目前市场的情况来看,酵母抽提物的应用正有上升的趋势。     

耐酸高产清酒酵母ZJ-14发酵条件研究

以优质粳米为原料,在单因素实验基础上,采用正交试验法,研究耐酸性清酒酵母ZJ-14的发酵规律,为优质清酒生产提供理论基础.结果表明,料水比为1.5:1、酒母量为20%、米曲量为50%、乳酸量为8‰的条件下,测得清酒的酒精度为17.1%vol.     

营养调味品——酵母抽提物的研究

本文以啤酒厂废酵母泥（酵母成活率＞90％）为原料，采用现代生物技术，借助酵母体内多种酶系，在温和的条件下促使酵母细胞自溶，将自溶产物离心分离、喷雾干燥而制成一种粉状酵母抽提物。     

Quantitative and qualitative analysis of lipids in genetically modified potato tubers with varying rates of 14-3-3 protein synthesis

In six transgenic lines of potatoes with the varying rates of 14-3-3 protein synthesis as well as in control cultivar Desiree the content and composition of the lipids extracted from the mature tubers from three years field trials (1998-2000) were analyzed. The transgenic lines J2 and J1 are both overexpressing gene encoding 14-3-3 protein. The J2 exhibited an overexpression of the protein 14-3-3 derived from pumpkin (Cucurbita pepo) cDNA and in J1 the 14-3-3 overexpression resulted from modifying of ADP-ribosylation factor synthesis. In the remaining lines, synthesis of the protein 14-3-3 was modified by the antisense technology. In tubers from 1998, the content of total lipids was within the range of 0.45-0.88% of tuber dry matter. The highest amount of fat was in tubers of line J2 (69% more than in the control). The content of lipids in tubers from subsequent years ranged from 0.36 to 0.63% of dry matter. Consistently the highest amount of fat was in tubers of line J2, however, the increase was very slight (8.6% more than in the control). The fractionation of lipids into polar and nonpolar fractions showed that all transgenic lines from field trials 1998 and 2000 contained more nonpolar lipids than the control (up to 270% in line J2). The percentage of nonpolar fractions in fats of tubers from all transgenes harvested in 1999 were similar, but they were higher than in tubers from the previous years, and they amounted to 44.4-49.1%. Chromatographic separation of methyl esters of fatty acids demonstrated that cis-alpha-linoleic acid was the main fatty acid present in potato tubers. This acid composed the biggest part of all lipids in G2 line. In the nonpolar fraction of lipids, palmitic acid followed by cis-alpha-linoleic acid showed the highest amounts.     

Metabolism of [U-14C; 2,3-3H]-L-valine by the isolated perfused goat udder.

Two lactating mammary glands excised from 2 goats were perfused for several hours in the presence of [U-14C; 2,3-3H]-L-valine and received adequate quantities of glucose, acetate and amino acids. In the synthesized milk 96 and 89% respectively of the casein valine was derived from free plasma valine. Valine was extensively catabolized by mammary tissue, resulting in a considerable 14CO2 production and in the incorporation of 14C into milk citric acid and to a lesser extent into casein aspartic acid and glutamic acid. About 30% of the valine molecules which were taken up by the mammary gland were oxidized to CO2 and 70% were incorporated in casein as valine residues. About 10% of the plasma valine molecules were reversibly transaminated during one passage through the udder. An important amount of radioactivity of plasma was present in unknown metabolites. Only 7% of this activity was localized in isobutyrate. The radioactivity of total milk fat was very low. Mainly iso-14:0, iso-16:0 and 15:0 were labelled.     

Yeast GGA proteins interact with GTP-bound Arf and facilitate transport through the Golgi

ARF proteins regulate the formation of transport vesicles at many steps of the secretory and endocytic pathways. A recently identified family of ARF effectors, named GGAs, appears to regulate membrane traffic exiting the trans-Golgi network in mammalian cells (Boman et al., 2000). We have identified two GGA homologues in the yeast S. cerevisiae. These previously uncharacterized open reading frames, YDR358w and YHR108w, have been named GGA1 and GGA2, respectively. Using the two-hybrid assay and GST-affinity chromatography, we show that Gga1p and Gga2p interact with Arf1p and Arf2p in a GTP-dependent manner, suggesting that both are functional homologues of the human GGA proteins. The Arf-binding domain resides in the amino-terminal half of Gga1p (amino acids 170-330), and the carboxy-terminal 100 amino acids resemble the gamma-adaptin 'ear domain'. Gene deletion experiments indicate that GGA1 and GGA2 are not essential genes, as single and double knockouts are viable at both 30 degrees C and 37 degrees C. However, cells lacking GGA1 and GGA2 exhibit defects in invertase processing and CPY sorting, but not endocytosis. We conclude that yeast Gga proteins are effectors of Arf in yeast that facilitate traffic through the late Golgi.     

优良香梨酒酵母的选育

介绍了从新疆库尔勒香梨果皮上和库尔勒香梨果园土壤中分离选育优良香梨酵母菌的方法及控制要点，并据生理生化特性初步鉴定为酿酒酵母。     

破壁方法对红球菌11-3胞内几丁质脱乙酰酶释放的影响

红球菌11-3是一株高产几丁质脱乙酰酶(CDA)的菌株,几丁质在该酶的催化作用下可转化为壳聚糖,该酶在壳聚糖的生产中具有重要作用。红球菌11-3菌株所产CDA为胞内酶,成为催化反应的一大障碍。为了提高CDA的释放率,本研究首先利用不同的物理方法(反复冻融、超声、球磨、匀浆和液氮研磨)、化学方法(表面活性剂处理、氯仿处理)和生物学方法(溶菌酶处理)对红球菌11-3进行破壁处理,测定CDA酶活力和释放率,并通过扫描电子显微镜观察细胞形态变化。结果表明,不同方法的破壁效果存在很大差异,其中,液氮研磨法破壁效果最佳,菌体表面出现细密的孔洞,CDA释放率为45.13%,总酶活力损失率为2.03%;匀浆处理法次之,CDA释放率为16.00%,总酶活力损失率为9.18%。利用匀浆和液氮研磨联合处理红球菌11-3细胞,细胞表面产生更多、更大的孔洞,CDA释放率高达86.17%,总酶活力损失率为9.11%,上清液中CDA酶活力为480.2 U/mL,较液氮研磨法相比提高了1.48倍。结论:匀浆和液氮研磨联合处理可有效破坏红球菌11-3细胞壁,提高胞内CDA释放效率。本研究结果对红球菌11-3内CDA应用于壳聚糖的生产具有参考作用。