A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay—the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg²⁺ and is not enhanced by other divalent metal ions (Zn²⁺ and Mn²⁺), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors.     

Characterization of a human SWI2/SNF2 like protein hINO80: demonstration of catalytic and DNA binding activity

The proteins belonging to SWI2/SNF2 family of DNA dependent ATPases are important members of the chromatin remodeling complexes that are implicated in epigenetic control of gene expression. We have identified a human gene with a putative DNA binding domain, which belongs to the INO80 subfamily of SWI2/SNF2 proteins. Here we report the cloning, expression, and functional activity of the domains from hINO80 gene both in terms of the DNA dependent ATPase as well as DNA binding activity. A differential expression of the various domains within this gene is detected in human tissues while a ubiquitous expression is detected in mice. The intranuclear localization is demonstrated using antibodies directed against the DBINO domain of hINO80.     

皱纹盘鲍消化腺细胞类型和分泌产物

对皱纹盘鲍消化腺进行了组织学、组织化学、超微结构及酶活性测定等研究。消化腺由消化细胞和嗜碱性细胞组成。消化细胞能内吞腺管腔内的外源性物质,细胞内充满大量与异噬功能有关的囊泡。消化细胞具有内吞和细胞内消化、分泌、贮存等功能。嗜碱性细胞含有发达的粗面内质网以及许多含铁的折光小体,具有分泌和贮存金属离子的功能。消化腺还呈现多种水解酶活性。     

The potential role of ubiquitin c-terminal hydrolases in oncogenesis

Deubiquitinating enzymes (DUBs), capable of removing ubiquitin (Ub) from protein substrates, are involved in numerous biological processes. The ubiquitin C-terminal hydrolases (UCHs) subfamily of DUBs consists of four members: UCH-L1, UCH-L3, UCH37 and BRCA1-associated protein-1 (BAP1). UCH-L1 possesses deubiquitinating activity and dimerization-dependent ubiquitin ligase activity, and functions as a mono-ubiquitin stabilizer; UCH-L3 does both deubiquitinating and deneddylating activity, except dimerization or ligase activity, and unlike UCH-L1, can interact with Lys48-linked Ub dimers to protect it from degradation and in the meanwhile to inhibit its hydrolase activity; UCH37 is responsible for the deubiquitinating activity in the 19S proteasome regulatory complex, and as indicated by the recent study, UCH37 is also associated with the human Ino80 chromatin-remodeling complex (hINO80) in the nucleus and can be activated via transient association of 19S regulatory particle- or proteasome-bound hRpn13 with hINO80; BAP1, binding to the wild-type BRCA1 RING finger domain, is regarded as a tumor suppressor, but for such suppressing activity, as demonstrated otherwise, both deubiquitinating activity and nucleus localization are required. There is growing evidence that UCH enzymes and human malignancies are closely correlated. Previous studies have shown that UCH enzymes play a crucial role in some signalings and cell-cycle regulation. In this review, we provided an insight into the relation between UCH enzymes and oncogenesis.     

A Preliminary Genetic Linkage Map of the Pacific Abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F₁ family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (α = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.     

A fitness-based interferential genetics approach using hypertoxic/inactive gene alleles as references

Genetics, genomics, and biochemistry have all been of immense help in characterizing macromolecular cell entities and their interactions. Still, obtaining an overall picture of the functioning of even a simple unicellular species has remained a challenging task. One possible way to obtain a comprehensive picture has been described: by capitalizing on the observation that the overexpression on a multicopy plasmid of apparently any wild-type gene in yeast can lead to some negative effect on cell fitness (referring to the concept of “gene toxicity”), the FIG (fitness-based interferential genetics) approach was devised for selecting normal genes that are in antagonistic (and potentially also agonistic) relationship with a particular gene used as a reference. Herein, we take a complementary approach to FIG, by first selecting a “hypertoxic” allele of the reference gene—which easily provides the general possibility of obtaining gene products with the remarkable property of being inactive without altering their macromolecular interactivity—and then looking for the genes that interact functionally with this reference. Thus, FIG and the present approach (Trap-FIG), both taking advantage of the negative effects on cell fitness induced by various quantitative modulations in cellular networks, could potentially pave the way for the emergence of efficient in situ biochemistry.     

咖啡因加热休克诱导皱纹盘鲍多倍体的研究

报道了用咖啡因加热休克抑制受精卵的第一极体的释放诱导皱纹盘鲍（Ｈａｌｉｏｔｉｓ ｄｉｓｃｕｓ ｈａｎｎａｉ Ｉｎｏ）多倍体的研究结果。皱纹盘鲍的卵在２１℃海水中受精,受精后１０分钟开始处理,药物浓度分别为２．５ｍｍｏｌ／Ｌ、５ｍｍｏｌ／Ｌ、１０ｍｍｏｌ／Ｌ,热休克温度分别为２４℃、２６℃、２８℃,处理的持续时间为１０分钟至３０分钟,共分５个时间段。结果表明,药物浓度５ｍｍｏｌ／Ｌ和１０ｍｍｏｌ／Ｌ     

皱纹盘鲍的个体能量收支

对皱纹盘鲍的呼吸、摄食、生长及能量收支等实验研究表明, 鲍的耗氧率与壳长、体重、温度及昼夜变化有关, 耗氧率与壳长、体重均呈幂函数关系, 一天中16~4时(夜间)耗氧率高于4~16时(白天)且在18~20时达峰值.同温度下鲍日摄食率与体重呈幂指数关系, 日(相对)摄食率随温度升高而增加, 而日相对摄食率、日相对生长率均随壳长、体重增加呈下降趋势.鲍在14~20℃内对海带的总转化效率为53%.鲍软体部、海带及鲍粪便干品的比能值分别为19.2、8.57和7.23kJ·g^-1.14~20℃皱纹盘鲍摄入能量的34.6~48.6%为粪能, 22.0~38.2%的能量用于自身代谢, 5.6~28.2%用于贝体软体部的生长.