光敏核不育水稻41 kDa和61 kDa蛋白质的发现、N-端序列分析及其雄性不育性状关系的探讨

光敏核不育水稻农垦58S是石明松于1973年在晚粳农垦58的大田中发现的雄性不育突变体，它在长日照下雄性不育可被用于与恢复系杂交生产杂种，而在短日照下雄性可育能用于自交繁殖，它的恢复系来源广泛。基于这些特性，育种学家用光敏核不育水稻建立的二系杂交水稻制种技术有很大的应用潜力。近十几年来，育种学家用农垦58S作基因供体转育了许多新的不育系，研究结果表明育成的粳型不育系均为光敏不育系，但在育成的籼型不育系中，绝大多数丧失光敏核不育特性，变成温敏不育系。目前因不知光敏核不育的分子遗传机制，尚不能解释这些问题。 本文用双向电泳技术分析了农垦58S和农垦58苗期和育性转换光敏感期叶绿体蛋白质的差异，在农垦58S中发现三个蛋白质(Pl，P2和P3)，其中Pl和P2在苗期和光敏感期叶片内均存在，P3仅在光敏感期的叶片中存在，它们不受长日照或短日照处理的影响。农垦58没有这三个蛋白质。 用制备型双向电泳纯化后，得到SDS - PAGE和IEF纯的Pl和P2。经SDS-PAGE和IEF测定，Pl的等电点是6.2，分子量是41 kDa;P2的等电点是5.8，分子量是61 kDa。现称Pl为P41，P2为P61。氨基酸序列分析和同源性检索发现P41与水稻叶绿体ATP合成酶p亚基和酵母转录因子CAD1有同源性，此外，P41的N-端序列中有一个与蛋白激酶催化核心中的多功能motif Y-G-X-G-X- (P/T)-G-V相似的序列;P61的14个氨基酸长的N-端序列与水稻叶绿体ATP合成酶β亚基的一致。P41和P61 N-端前12个氨基酸的序列也完全一致。 PCR扩增和Southern杂交分析没有发现农垦58S和农垦58之间ATP合成酶β亚基基因(atpB)的多态性。Nothern杂交分析表明农垦58S中仅有一种、与农垦58 atpB mRNA分子量相同的atpB转录产物，但它的atpB mRNA丰度明显低于农垦58的。没有检测到突变的atpB和其它形式的atpB转录产物。 分析P41和P61在其它水稻材料中的分布特点发现它们在粳型光敏不育系7001S、5088S、31301S、C407S和1647S，籼型光敏不育系W7415S和W9451S以及温（光）敏不育系培矮64S中存在，而在对照材料三系水稻马协A、珍汕97A、马协B、珍汕97B和明恢63以及常规粳稻C94153中不存在。根据这些不育系的系谱和它们与农垦58S之间基因的等位性研究结果，讨论了P41和P61与光敏核不育性的可能联系。

Isolation and preliminary characterization of a 22-kDa protein with trypsin inhibitory activity from toad Bufo andrewsi skin

A novel trypsin inhibitor termed BATI was purified to homogeneity from the skin extracts of toad Bufo andrewsi by successive ion-exchange, gel-filtration and reverse-phase chromatography. BATI is basic single chain glycoprotein, with apparent molecular weight of 22 kDa in SDS-PAGE. BATI is a thermal stable competitive inhibitor and effectively inhibits trypsin's catalytic activity on peptide substrate with the inhibitor constant (K-i) value of 14 nM and shows no inhibitory effect on chymotrypsin, thrombin and elastase. The N-terminal sequence of BATI is EKDSITD, which shows no similarity with other known trypsin inhibitors. (c) 2005 Elsevier Ltd. All rights reserved.

Cloning and characterization of cytochrome c oxidase subunit I (COXI) in Gobiocypris rarus

In study of gene expression profile in cloned embryos which derived from D. rerio embryonic nuclei and G. rarus enucleated eggs, cytochrome c oxidase subunit I (COXI) of G. rarus, exhibiting difference at expression level between cloned embryos and zebrafish embryo, was cloned. Its full cDNA length is 1654 bp and contains a 1551 bp open reading frame, encoding a 5.64 kDa protein of 516 amino acids. The alignment result shows that mitochondrion tRNA(ser) is co-transcripted with COXI, which just was the 3'-UTR of COXI. Molecular phylogenic analysis based on COXI indicates G. rarus should belong to Gobioninae, which was not in agreement with previous study according to morphological taxonomy. Comparison of DNA with cDNA shows that RNA editing phenomenon does not occur in the COXI of G. rarus.

Evaluation of Clonorchis sinensis recombinant 7-kilodalton antigen for serodiagnosis of clonorchiasis

The diagnostic applicability of the Clonorchis sinensis recombinant 7-kDa protein was evaluated. In enzyme-linked immunosorbent assays and immunoblots, the protein showed high sensitivities (81.3 and 71.9%, respectively) and specificities (92.6 and 89.7%, respectively) for sera obtained from various helminthic infections. Some paragonimiasis sera showed cross-reactions. The antigen might be valuable in the serodiagnosis of human clonorchiasis.

Analysis of expression of the binary toxin genes from Bacillus sphaericus in Anabaena and the potential in mosquito control

Anabaena strains expressing the binary toxin genes of Bacillus sphaericus produce high larvicidal activity with living cells. Western blot analysis showed that the 51-kDa and 42-kDa toxin proteins were stable in Anabaena. When a DNA fragment upstream of the 51-kDa protein gene was deleted, the toxicity was reduced by over a hundred-fold, whereas deletions at the coding regions showed that the cooperation of the two proteins expressed in Anabaena is essential for the larvicidal activity. Outdoor tests showed that the genetically altered Anabaena could keep containers with natural water from being inhabited by Culex larvae for over 2 months.

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the photosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.

Bioorganic/inorganic hybrid composition of sponge spicules: Matrix of the giant spicules and of the comitalia of the deep sea hexactinellid Monorhaphis

The giant basal spicules of the siliceous sponges Monorhaphis chuni and Monorhaphis intermedia (Hexactinellida) represent the largest biosilica structures on earth (up to 3 m long). Here we describe the construction (lamellar organization) of these spicules and of the comitalia and highlight their organic matrix in order to understand their mechanical properties. The spicules display three distinct regions built of biosilica: (i) the outer lamellar zone (radius: >300 mu m), (ii) the bulky axial cylinder (radius: <75 mu m), and (iii) the central axial canal (diameter: <2 mu m) with its organic axial filament. The spicules are loosely covered with a collagen net which is regularly perforated by 7-10 mu m large holes; the net can be silicified. The silica layers forming the lamellar zone are approximate to 5 mu m thick; the central axial cylinder appears to be composed of almost solid silica which becomes porous after etching with hydrofluoric acid (HF). Dissolution of a complete spicule discloses its complex structure with distinct lamellae in the outer zone (lamellar coating) and a more resistant central part (axial barrel). Rapidly after the release of the organic coating from the lamellar zone the protein layers disintegrate to form irregular clumps/aggregates. In contrast, the proteinaceous axial barrel, hidden in the siliceous axial cylinder, is set up by rope-like filaments. Biochemical analysis revealed that the (dominant) molecule of the lamellar coating is a 27-kDa protein which displays catalytic, proteolytic activity. High resolution electron microscopic analysis showed that this protein is arranged within the lamellae and stabilizes these surfaces by palisade-like pillars. The mechanical behavior of the spicules was analyzed by a 3-point bending assay, coupled with scanning electron microscopy. The load-extension curve of the spicule shows a biphasic breakage/cracking pattern. The outer lamellar zone cracks in several distinct steps showing high resistance in concert with comparably low elasticity, while the axial cylinder breaks with high elasticity and lower stiffness. The complex bioorganic/inorganic hybrid composition and structure of the Monorhaphis spicules might provide the blueprint for the synthesis of bio-inspired material, with unusual mechanical properties (strength, stiffness) without losing the exceptional properties of optical transmission. (C) 2007 Elsevier Inc. All rights reserved.

Identification of a silicatein(-related) protease in the giant spicules of the deep-sea hexactinellid Monorhaphis chuni

Silicateins, members of the cathepsin L family, are enzymes that have been shown to be involved in the biosynthesis/condensation of biosilica in spicules from Demospongiae (phylum Porifera), e. g. Tethya aurantium and Suberites domuncula. The class Hexactinellida also forms spicules from this inorganic material. This class of sponges includes species that form the largest biogenic silica structures on earth. The giant basal spicules from the hexactinellids Monorhaphis chuni and Monorhaphis intermedia can reach lengths of up to 3 m and diameters of 10 mm. The giant spicules as well as the tauactines consist of a biosilica shell that surrounds the axial canal, which harbours the axial filament, in regular concentric, lamellar layers, suggesting an appositional growth of the spicules. The lamellae contain 27 kDa proteins, which undergo post-translational modification (phosphorylation), while total spicule extracts contain additional 70 kDa proteins. The 27 kDa proteins cross-reacted with anti-silicatein antibodies. The extracts of spicules from the hexactinellid Monorhaphis displayed proteolytic activity like the silicateins from the demosponge S. domuncula. Since the proteolytic activity in spicule extracts from both classes of sponge could be sensitively inhibited by E-64 (a specific cysteine proteinase inhibitor), we used a labelled E-64 sample as a probe to identify the protein that bound to this inhibitor on a blot. The experiments revealed that the labelled E-64 selectively recognized the 27 kDa protein. Our data strongly suggest that silicatein(-related) molecules are also present in Hexactinellida. These new results are considered to also be of impact for applied biotechnological studies.

Direct observation of adsorption sites of protein impurities and their effects on step advancement of protein crystals

We measured noninvasively step velocities of elementary two-dimensional (2D) islands on {110} faces of tetragonal lysozyme crystals, under various supersaturations, by laser confocal microscopy combined with differential interference contrast microscopy. We studied the correlation between the effects of protein impurities on the growth of elementary steps and their adsorption sites on a crystal surface, using three kinds of proteins: fluorescent-labeled lysozyme (F-lysozyme), covalently bonded dimers of lysozyme (dimer), and a 18 kDa polypeptide (18 kDa). These three protein impurities suppressed the advancement of the steps. However, they exhibited different supersaturation dependencies of the suppression of the step velocities. To clarify the cause of this difference, we observed in situ the adsorption sites of individual molecules of F-lysozyme and fluorescent-labeled dimer (F-dimer) on the crystal surface by single-molecule visualization. We found that F-lysozyme adsorbed preferentially on steps (i.e., kinks), whereas F-dimer adsorbed randomly on terraces. Taking into account the different adsorption sites of F-lysozyme and F-dimer, we could successfully explain the different effects of the impurities on the step velocities. These observations strongly suggest that 18 kDa also adsorbs randomly on terraces. Seikagaku lysozyme exhibited a complex effect that could not alone be explained by the two major impurities (dimer and 18 kDa) present in Seikagaku lysozyme, indicating that trace amounts of other impurities significantly affect the step advancement.

Molecular evolution of CXCR1, a G protein-coupled receptor involved in signal transduction of neutrophils

Human neutrophils are a type of white blood cell, which forms an early line of defense against bacterial infections. Neutrophils are highly responsive to the chemokine, interleukin-8 (IL-8) due to the abundant distribution of CXCR1, one of the IL-8 receptors on the neutrophil cell surface. As a member of the GPCR family, CXCR1 plays a crucial role in the IL-8 signal transduction pathway in neutrophils. We sequenced the complete coding region of the CXCR1 gene in worldwide human populations and five representative nonhuman primate species. Our results indicate accelerated protein evolution in the human lineage, which was likely caused by Darwinian positive selection. The sliding window analysis and the codon-based neutrality test identified signatures of positive selection at the N-terminal ligand/receptor recognition domain of human CXCR1.

Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVl

Stejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (alpha), 20 kDa (beta(1)) and 22 kDa (beta(2)), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that beta(1) and beta(2) subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between 1 and 2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the U. and beta, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against UNA inhibited the aggregation response to stejnulxin, indicating that activation of alpha(IIb)beta(3) and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbalpha or alpha(2)beta(1) as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.

A novel heme-containing protein with anti-HIV-1 activity from skin secretions of Bufo andrewsi

A novel protein, named BAS-AH, was purified and characterized from the skin of the toad Bufo andrewsi. BAS-AH is a single chain protein and the apparent molecular weight is about 63 kDa as judged by SDS-PAGE. BAS-AH was determined to bind heme (0.89 mol heme/mol protein) as determined by pyridine haemochrome analysis. Fifty percentage cytotoxic concentration (CC50) of BAS-AH on C8166 cells was 9.5 mu M. However, at concentrations that showed little effect oil cell viability, BAS-AH displayed dose dependent inhibition oil HIV-1 infection and replication. The antiviral selectivity indexes corresponding to the measurements of syncytium formation and HIV-1 p24 (CC50/EC50) were 14.4 and 11.4, respectively, corresponding to the . BAS-AH also showed an inhibitory effect on the activity of recombinant HIV-1 reverse transcriptase (IC50 = 1.32 mu M). The N-terminal sequence of BAS-AH was determined to be NAKXKADVIGKISILLGQDNLSNIVAM, which exhibited little identity with other known anti-HIV-1 proteins. BAS-AH is devoid of antibacterial, protcolytic, trypsin inhibitory activity, (L)-amino acid oxidase activity and catalase activity. (c) 2005 Elsevier Ltd. All rights reserved.

Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent mole

A new protein structure of P-II class snake venom metalloproteinases: it comprises metalloproteinase and disintegrin domains

A new metalloproteinase-disintegrin, named Jerdonitin, was purified from Trimeresurus jerdonii venom with a molecular weight of 36 kDa on SDS-PAGE. It dose-dependently inhibited ADP-induced human platelet aggregation with IC50 of 120 nM. cDNA cloning and sequencing revealed that Jerdonitin belonged to the class II of snake venom metalloproteinases (SVMPs) (P-II class). Different from other P-II class SVMPs, metalloproteinase and disintegrin domains of its natural protein were not separated, confirmed by internal peptide sequencing. Compared to other P-II class SVMPs, Jerdonitin has two additional cysteines (Cys219 and Cys238) located in the spacer domain and disintegrin domain, respectively. They probably form a disulfide bond and therefore the metalloproteinase and disintegrin domains cannot be separated by posttranslationally processing. In summary, comparison of the amino acid sequences of Jerdonitin with those of other P-II class SVMPs by sequence alignment and phylogenetic analysis, in conjunction with natural protein structure data, suggested that it was a new type of P-II class SVMPs. (C) 2003 Elsevier Inc. All rights reserved.

Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells.