高半胱氨酸膳食对小鼠生长和免疫力的影响

探讨了高半胱氨酸膳食对小鼠生长及对外来抗原刺激产生抗体的免疫力的影响。实验取小白鼠32只,随机分为高半胱氮酸组(H)和对照组(C)各16只。H组每天给予含半胱氨酸质量分数为3％的饲料,分别在1、4、8周对其腹腔注射牛血清白蛋白,在第9周处死动物,取血测量血清抗体效价。结果显示实验组生长缓慢,血清抗体效价显著高于对照组,表明高半胱氨酸膳食可提高小鼠血清抗体效价,但高含量的半胱氨酸对小鼠生长有抑制作用。     

温度对不同类型早籼稻灌浆期间直链淀粉、蛋白质积累的影响

高温条件下早籼稻籽粒直链淀粉积累速率快,持续时间短;蛋白质含量高;灌浆前期谷草转氨酶和谷丙转氨酶活性高,开花11d后迅速下降．适温条件下籽粒直链淀粉积累速率慢,持续时间长;蛋白质含量低;谷草转氨酶和谷丙转氨酶活性变化缓慢,3个不同类型品种中适合作米粉的湘早籼33号直链淀粉含量最高,饲料稻湘早籼24号蛋白质含量最高．高温有助于湘早籼33号直链淀粉含量和湘早籼24号蛋白质含量的提高。     

Type IIE and IIF Restriction Endonucleases Interacting with Two Recognition Sites on DNA

Recent studies have shown that restriction endonucleases (REs), which are broadly used in genetic engineering and molecular biology, vary not only in nucleotide sequence of the recognition site, but also in the mechanism of their interaction with DNA. This review focuses on type IIF and IIE REs, which require simultaneous interaction with two nucleotide sequences for efficient DNA cleavage. Crystal structures of these REs and their complexes with DNA, stepwise interactions with DNA, catalytic mechanisms of DNA hydrolysis, and DNA looping are considered. Type IIE REs have provided an example of a new type of DNA–protein recognition: two copies of one recognition sequence interact specifically with two different amino acid sequences and two different structural motifs of one polypeptide chain.     

Ⅰ型蛋白磷酸酶研究进展

Ⅰ型蛋白磷酸酶(PP1)属丝/苏氨酸磷酸酶的一种,在生物体中广泛存在,参与调节多种重要的生理功能,包括转录、翻译、代谢、细胞生长及分化等.PP1分子结构表面的3个凹槽及β 12-β 13 Loop环结构,它在底物与抑制剂的结合方面起决定作用.近期研究发现,Loop环结构除了是抑制剂的结合部位之外,对整个酶分子的结构和性质都起重要作用.功能研究也证明PP1还参与HIV-1转录过程的调节,并且与老年性痴呆等多种疾病密切相关.主要对PP1的组织分布、分子结构、酶学特性、催化机制以及生物学功能等方面进行了相应的综述.     

Ambrosiozyma kamigamensis sp. nov. and A. neoplatypodis sp. nov., two new ascomycetous yeasts from ambrosia beetle galleries

Two new yeast species of the genus Ambrosiozyma are described on the basis of comparison of nucleotide sequences of large subunit of ribosomal DNA D1/D2 region. Ambrosiozyma kamigamensis and Ambrosiozyma neoplatypodis differ from Ambrosiozyma ambrosiae by 17 nucleotides (3.0%) and 16 nucleotides (2.8%), respectively, out of 565. The two species differ from each other by 13 nucleotides. Ambrosiozyma kamigamensis was isolated from galleries of the ambrosia beetle, Platypus quercivorus, in specimens of Quercus laurifolia and Castanopsis cuspidata located in the southern part of Kyoto, Japan. Ambrosiozyma neoplatypodis was isolated from similar material, but only in Q. laurifolia. Ambrosiozyma kamigamensis can be distinguished from the other Ambrosiozyma species by the inability to assimilate erythritol, whereas A. neoplatypodis can be distinguished by the ability to assimilate both L: -arabinose and nitrate. The type strains of A. kamigamensis and A. neoplatypodis are JCM 14990(T) (=CBS 10899(T)) and JCM 14992(T) (=CBS 10900(T)), respectively. This is the first report of new Ambrosiozyma species since the genus was proposed.     

Interaction of transmembrane helices in ATP synthase subunit a in solution as revealed by spin label difference NMR

Subunit a in the membrane traversing F₀ sector of Escherichia coli ATP synthase is known to fold with five transmembrane helices (TMHs) with residue 218 in TMH IV packing close to residue 248 in TMH V. In this study, we have introduced a spin label probe at Cys residues substituted at positions 222 or 223 and measured the effects on the Trp ?NH indole NMR signals of the seven Trp residues in the protein. The protein was purified and NMR experiments were carried out in a chloroform-methanol-H₂O (4:4:1) solvent mixture. The spin label at positions 222 or 223 proved to broaden the signals of W231, W232, W235 and W241 located at the periplasmic ends of TMH IV and TMH V and the connecting loop between these helices. The broadening of W241 would require that the loop residues fold back on themselves in a hairpin-like structure much like it is predicted to fold in the native membrane. Placement of the spin label probe at several other positions also proved to have broadening effects on some of these Trp residues and provided additional constraints on folding of TMH IV and TMH V. The effects of the 223 probes on backbone amide resonances of subunit a were also measured by an HNCO experiment and the results are consistent with the two helices folding back on themselves in this solvent mixture. When Cys and Trp were substituted at residues 206 and 254 at the cytoplasmic ends of TMHs IV and V respectively, the W254 resonance was not broadened by the spin label at position 206. We conclude that the helices fold back on themselves in this solvent system and then pack at an angle such that the cytoplasmic ends of the polypeptide backbone are significantly displaced from each other.     

Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles

Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca²⁺- and Mg²⁺-ATPase activities of CF1 deficient in its inhibitory ε subunit (CF1-ε) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca²⁺-ATPase activity by CTAB is irreversible. The Ca²⁺-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg²⁺-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-ε or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-ε from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the α/β heterohexamer.     

Defective assembly of a hybrid vacuolar H-ATPase containing the mouse testis-specific E1 isoform and yeast subunits

Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane V_o and catalytic V₁ sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 °C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098-18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 °C. Corresponding to the reversible defect of the hybrid V-ATPase, the V_o subunit a epitope was exposed to the corresponding antibody at 37 °C, but became inaccessible at 30 °C. However, the V₁ sector was still associated with V_o at 37 °C, as shown immunochemically. The control yeast V-ATPase was active at 37 °C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V₁ from V_o in yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.     

Prefibrillar transthyretin oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture

Recent studies suggest that soluble, oligomeric species, which are intermediates in the fibril formation process in amyloid disease, might be the key species in amyloid pathogenesis. Soluble oligomers of human wild type transthyretin (TTR) were produced to elucidate oligomer properties. Employing ThT fluorescence, time-resolved fluorescence anisotropy of pyrene-labeled TTR, chemical cross-linking, and electron microscopy we demonstrated that early formed soluble oligomers (within minutes) from A-state TTR comprised on the average 20-30 TTR monomers. When administered to neuroblastoma cells these early oligomers proved highly cytotoxic and induced apoptosis after 48 h of incubation. More mature fibrils (>24 h of fibrillation) were non-toxic. Surprisingly, we also found that native tetrameric TTR, when purified and stored under cold conditions (4 °C) was highly cytotoxic. The effect could be partially restored by increasing the temperature of the protein. The cytotoxic effects of native tetrameric TTR likely stems from a hitherto unexplored low temperature induced rearrangement of the tetramer conformation that possibly is related to the conformation of misfolded TTR in amyloigogenic oligomers.