细胞胞吐的分子基础

近年来对神经末梢突触小泡膜蛋白的蛋白质化学,药理学研究及酵母细胞蛋白运输及分泌的遗传学的研究,发现了胞吐蛋白复合体的存在。ＳＮＡＲＥ假说的提出使得对神经递质释放,内外分泌细胞分泌机制的研究有了突飞猛进的进展。     

In vitro metabolism of 3-t-butyl-4-hydroxyanisole and its irreversible binding to proteins

A method for the preparation of methyl-labelled 3-t-butyl-4-hydroxyanisole (BHA) is described. Metabolism of [14C]BHA using four different enzyme systems (liver microsomes + NADPH; liver microsomes + cumene hydroperoxide (CHP); sheep seminal vesicle (SSV) microsomes (as a source of prostaglandin synthetase) + arachidonic acid (AA); horseradish peroxidase (HRP) + hydrogen peroxide) was investigated. In all systems, BHA was oxidized to a variety of products including formaldehyde, a dimer di-BHA, polar and water soluble metabolites as well as a reactive intermediate(s) that binds irreversibly to proteins. With liver microsomes and NADPH, phenobarbital (PB) induction gave increased yields of all products while 3-methylcholanthrene (MC) induction specifically increased protein binding but decreased other metabolite formation. BHA addition effectively discharged the activated oxygen complex of cytochrome P-450 (liver microsomes) as well as Comp. I and Comp. II of HRP suggesting that it is a good one electron peroxidase donor. BHA addition also increased the net rate of NADPH oxidation in the presence of liver microsomes suggesting uncoupling. It is proposed that in all system investigated BHA is oxidized predominantly via a one electron oxidation process to yield first the BHA free radical which then dimerizes, forms more products or binds to proteins.     

Cryopreservation of murine testicular Leydig cells by modified solid surface vitrification with supplementation of antioxidants

Reports on the vitrification of somatic cells are scarce. Here, we show that Leydig cells (murine cell line TM3) could be successfully vitrified by both open vitrification [plastic straw (PS) and plastic vials (PV)] and closed ultravitrification [microdrop (MD) and solid surface vitrification (SSV)], after protocol optimization. However, open ultravitrification resulted in better post-warming viability than closed systems of vitrification with highest success obtained in modified SSV (84.8 ± 1.86%; p < 0.05). Leydig cells vitrified-warmed by modified SSV also showed superior (p < 0.05) cell growth, mitochondrial activity and cytoplasmic esterase enzyme activity, than MD, PS and PV, respectively. It was also observed that vitrified-warmed cells had higher level of ROS activity than non-vitrified control cells (41.6 ± 4.0 vs. 16.7 ± 1.0; p < 0.05). Treatment of cells with glutathione (GSH) or 2-mercaptoethanol (2-ME) (0, 10, 50, 100 μM) significantly (p < 0.05) reduced the ROS activity but had no significant (p > 0.05) effect on post-warm viability. Nevertheless, antioxidant-treated cells had improved mitochondrial activity, cytoplasmic esterase activity and cell growth during in vitro culture (p < 0.05). In conclusion, our results suggest that modified SSV offers a viable method for vitrifying single cell suspension of Leydig cells. To the best of our knowledge, this is the first report on cryopreservation of Leydig cells by vitrification.     

A specific prostaglandin I2 synthetase inhibitor, 3-hydroperoxy-3-methyl-2-phenyl-3H-indole.

Inhibitory effects of 3-hydroperoxy-3-methyl-2-phenyl-3H-indole(HPI) on prostaglandin endoperoxide synthase(EC 1.14.99.1) and prostaglandin I₂(PGI₂) synthetase were compared with those of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, namely, 15-hydroperoxyarachidonic acid(15-HPAA) and tranylcypromine (TCP). Sheep seminal vesicle microsomes were used as a source of prostaglandin endoperoxide synthase and bovine aortic microsomes as that of PGI₂ synthetase. 15-HPAA and HPI inhibited PGI₂ synthetase with IC₅₀s of 5 × 10^?7 and 3.5 × 10^?6 M, respectively, whereas neither compound had effect on prostaglandin endoperoxide synthase at the concentration inhibiting PGI₂ synthetase by 90%. TCP was a weak(IC₅₀ = 5 × 10^?4M) PGI₂ synthetase inhibitor with low specificity.     

Surface resistance to SSVs and SIRVs in pilin deletions of Sulfolobus islandicus

Characterizing the molecular interactions of viruses in natural microbial populations offers insights into virus–host dynamics in complex ecosystems. We identify the resistance of Sulfolobus islandicus to Sulfolobus spindle-shaped virus (SSV9) conferred by chromosomal deletions of pilin genes, pilA1 and pilA2 that are individually able to complement resistance. Mutants with deletions of both pilA1 and pilA2 or the prepilin peptidase, PibD, show the reduction in the number of pilins observed in TEM and reduced surface adherence but still adsorb SSV9. The proteinaceous outer S-layer proteins, SlaA and SlaB, are not required for adsorption nor infection demonstrating that the S-layer is not the primary receptor for SSV9 surface binding. Strains lacking both pilins are resistant to a broad panel of SSVs as well as a panel of unrelated S. islandicus rod-shaped viruses (SIRVs). Unlike SSV9, we show that pilA1 or pilA2 is required for SIRV8 adsorption. In sequenced Sulfolobus strains from around the globe, one copy of each pilA1 and pilA2 is maintained and show codon-level diversification, demonstrating their importance in nature. By characterizing the molecular interactions at the initiation of infection between S. islandicus and two different types of viruses we hope to increase the understanding of virus–host interactions in the archaeal domain.     

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.     

SSV1-encoded site-specific recombination system in Sulfolobus shibatae

Summary We present evidence for the existence of a conservative site-specific recombination system in Archaea by demonstrating integrative recombination of Sulfolobus shibatae virus SSV1 DNA with the host chromosome, catalysed by the SSVI-encoded integrase in vitro. The putative int gene of SSV1 was expressed in Escherichia coli yielding a protein of about 39 kDa. This protein alone efficiently recombined linear DNA substrates containing chromosomal (attA) and viral (attP) attachment sites; recombination with either negatively or positively supercoiled SSV1 DNA was less efficient. Intermolecular attA × attA and attP × attP recombination was also promoted by the SSV integrase. The invariant 44 by common attachment core present in all att sites contained sufficient information to allow recombination, whilst the flanking sequences effected the efficiency. These features clearly distinguish the SSV1 — encoded site — specific recombination system from others and make it suitable for the study of regulatory mechanisms of SSV1 genome — host chromosome interaction and investigations of the evolution of the recombination machinery.