Cloning and Characterization of an Agglutinin Gene from Arisaema lobatum

A novel agglutinin gene was cloned from Arisaema lobatum using SMART RACE-PCR technology. The full-length cDNA of Arisaema lobatum agglutinin (ala) was 1078 bp and contained a 774 bp open reading frame encoding a lectin precursor (proproprotein) of 258 amino acid residues with a 23 aa signal peptide. ALA contained three mannose-binding sites (QXDXNXVXY) with two-conserved domains of 45% identity, ALA-DOM1 and ALA-DOM2. The three-dimensional structure of ALA was very similar to that of GNA (Galanthus nivalis agglutinin). ALA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families, such as Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. Genomic sequence of ala was also cloned using genomic walker technology, and it was found to contain three putative TATA boxes and eight possible CAAT boxes in the 5′-flanking region. No intron was found within the region of genomic sequence. Southern blot analysis indicated that the ala belonged to a multi-copy gene family. Expression pattern analysis revealed that the ala preferentially expressed in the tissues with the higher expression being found in spadix, bud, leaf, spathe and tuber. The cloning of the ala gene not only provides a basis for further investigation of its structure, expression and regulation mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.     

Hydrogen sulfide may improve the hippocampal damage induced by recurrent febrile seizures in rats

The aim of the present study was to investigate the possible role of hydrogen sulfide (H(2)S) in the pathogenesis of recurrent febrile seizures (FS) in rats. On a rat model of recurrent FS, the ultrastructure of hippocampal neurons, the plasma level of H(2)S, the expressions of cystathionine b-synthase (CBS) and c-fos, and the development of mossy fiber sprouting (MFS) in hippocampus were examined after treatment with NaHS, a donor of H(2)S, or hydroxylamine (HA), an inhibitor of CBS. We found that the plasma level of H(2)S increased significantly, the expressions of CBS and c-fos increased markedly, and MFS was evident in hippocampus in FS group. NaHS alleviated the neuronal damage of recurrent FS rats, decreased the expression of c-fos, and inhibited MFS obviously. HA aggravated the neuronal damage of recurrent FS rats, further increased the expression of c-fos, and enhanced the mossy fiber outgrowth. The results showed that endogenous H(2)S system was involved in the development of FS. Exogenous H(2)S may exert beneficial effect on the pathogenesis of FS-related brain damage.     

Mapping peptides correlated with transmission of intrasteric inhibition and allosteric activation in human cystathionine beta-synthase

Cystathionine beta-synthase plays a key role in the intracellular disposal of homocysteine and is the single most common locus of mutations associated with homocystinuria. Elevated levels of homocysteine are correlated with heart disease, Alzheimer's and Parkinson's diseases, and neural tube defects. Cystathionine beta-synthase is modular and subjected to complex regulation, but insights into the structural basis of this regulation are lacking. We have employed hydrogen exchange mass spectrometry to map peptides whose motions are correlated with transmission of intrasteric inhibition and allosteric activation. The mass spectrometric data provide an excellent correlation between kinetically and conformationally distinguishable states of the enzyme. We also demonstrate that a pathogenic regulatory domain mutant, D444N, is conformationally locked in one of two states sampled by the wild type enzyme. Our hydrogen exchange data identify surfaces that are potentially involved in the juxtaposition of the regulatory and catalytic domains and form the basis of a docked structural model for the full-length enzyme.     

Rabphilin regulates SNARE-dependent re-priming of synaptic vesicles for fusion

Synaptic vesicle fusion is catalyzed by assembly of synaptic SNARE complexes, and is regulated by the synaptic vesicle GTP-binding protein Rab3 that binds to RIM and to rabphilin. RIM is a known physiological regulator of fusion, but the role of rabphilin remains obscure. We now show that rabphilin regulates recovery of synaptic vesicles from use-dependent depression, probably by a direct interaction with the SNARE protein SNAP-25. Deletion of rabphilin dramatically accelerates recovery of depressed synaptic responses; this phenotype is rescued by viral expression of wild-type rabphilin, but not of mutant rabphilin lacking the second rabphilin C2 domain that binds to SNAP-25. Moreover, deletion of rabphilin also increases the size of synaptic responses in synapses lacking the vesicular SNARE protein synaptobrevin in which synaptic responses are severely depressed. Our data suggest that binding of rabphilin to SNAP-25 regulates exocytosis of synaptic vesicles after the readily releasable pool has either been physiologically exhausted by use-dependent depression, or has been artificially depleted by deletion of synaptobrevin.     

香鱼补体成分C9基因的克隆、序列分析及表达

补体成分C9是构成膜攻击复合体引起靶细胞溶解破坏的重要组成成分。该文测定了香鱼C9(aC9)基因的cDNA全序列,序列全长2125个核苷酸,编码一个由592个氨基酸组成、相对分子质量为6.56×104的前体蛋白,N端22个氨基酸为信号肽序列。序列分析表明,aC9与虹鳟C9的氨基酸同源性最高,达56.8%,与其它鱼类C9的同源性介于40.9%~53.8%之间。aC9在健康香鱼肝、脾、肠、鳃和肌肉有表达,其中在肝内的表达量最高。实时荧光定量PCR的结果显示,鳗利斯顿氏菌侵染4h后,肝中aC9mRNA表达量显著上调,并随着时间的推移在16h时达到峰值。Westernblotting分析的结果显示,鳗利斯顿氏菌侵染后香鱼血清中的aC9蛋白随着时间的推移呈显著上调。以上结果表明,香鱼肝组织C9基因表达变化与鳗利斯顿氏菌的侵染密切相关,揭示了C9在鱼类抗细菌免疫反应中具有重要的作用。     

梅氏新贝尼登虫热休克蛋白60基因的克隆、序列分析及应激表达分析

热休克蛋白60(HSP60)是一种维持线粒体蛋白正常结构和功能的分子伴侣。该文克隆了梅氏新贝尼登虫HSP60基因cDNA序列(NmHSP60),并采用荧光定量RT-PCR和Western blot研究其在不同温度和盐度下的表达变化。以25℃为参照,18℃时,NmHSP60在虫卵和成虫中均表达下调;而32℃时,在成虫中表达上调。以盐度24为参照,盐度18时,NmHSP60在成虫中表达下调;盐度30时,在虫卵中表达上调。该结果揭示NmHSP60可能在梅氏新贝尼登虫应对环境应激中起重要作用。