Regional genetic variation in the major sperm protein genes of Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea)

Onchocerca volvulus and Mansonella ozzardi are two human filarial parasites present in South and Central America. In the Brazilian Amazonia they are found in sympatry, and the lack of clear morphological diagnostic characters in the microfilariae hinders their identification. The major sperm protein (MSP) gene of both species has been sequenced and characterised to determine its potential as a molecular diagnostic character. The length of the MSP gene is different in each species, and this could be used to detect and differentiate them by running the polymerase chain reaction (PCR) product in an agarose gel. Two major gene groups were identified in O. volvulus with a genetic distance of 6% between them. In M. ozzardi only one major group of genes was observed. The high similarity between the protein amino acid sequence of both filarial species confirms that the MSP has been highly conserved through nematode evolution.     

细胞运动、细胞迁移与细胞骨架研究进展

细胞定向运动与细胞骨架的动态循环密切相关。运动细胞在其伪足前沿依靠细胞骨架的不断聚合推动细胞膜的前进,在基部靠近细胞体部位通过细胞骨架的不断解聚收缩拖拉细胞体向前运动,细胞骨架的聚合与解聚通过伪足与支撑表面的吸附与解吸附而偶连。肌动蛋白组成的微丝骨架是大多数运动细胞的主要成分。外界刺激引起微丝细胞骨架动态变化的信号通路已逐步明了。线虫精子细胞的运动行为与阿米巴变形运动相似,但是在线虫精子细胞中没有肌动蛋白,而是以精子主要蛋白为基础形成细胞骨架驱动精子细胞的运动。与肌动蛋白不同,精子主要蛋白没有分子极性、ATP结合位点和马达蛋白。通过比较研究以上两种运动体系将有助于在分子水平上进一步阐明细胞运动的机理。     

Major surface protein 1a effects tick infection and transmission of Anaplasma marginale.

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

Neither a germ line-specific nor several somatically expressed genes are lost or rearranged during embryonic chromatin diminution in the nematode Ascaris lumbricoides var. suum

Ascaris lumbricoides var. suum is a parasitic nematode of pigs. Its embryos undergo chromatin diminution between the third and fifth cleavages, resulting in the loss of about 30% of the DNA from all somatic precursor cells while the germ line DNA stays intact. Most of the eliminated DNA has been shown to be satellite sequences. Theodor Boveri [(1910) In "Festschrift fur R. Hertwig, III," Vol. 3, pp. 131-214, Fischer] proposed that functions essential only to the germ line might be lost from the soma. We have examined this proposal by cloning a gene encoding the major sperm protein (MSP) using a cloned MSP gene from Caenorhabditis elegans as a probe. The MSP appears to be expressed only in the testis of Ascaris, as it is in Caenorhabditis. Actin and alpha tubulin were also cloned to serve as somatically expressed gene controls. By probing Southern blots of somatic and germ line DNA with these cloned genes, it was found that none of them was lost or rearranged during chromatin diminution. Thus at least one germ line-specific gene is neither lost nor rearranged during chromatin diminution. We also found that the two nematode species differ widely in their numbers of both MSP and actin genes. Caenorhabditis has greater than 30 MSP genes, but Ascaris has no more than three; whereas Ascaris has many more actin genes than Caenorhabditis.     

Transformation: how do nematode sperm become activated and crawl?

Nematode sperm undergo a drastic physiological change during spermiogenesis (sperm activation). Unlike mammalian flagellated sperm, nematode sperm are amoeboid cells and their motility is driven by the dynamics of a cytoskeleton composed of major sperm protein (MSP) rather than actin found in other crawling cells. This review focuses on sperm from Caenorhabditis elegans and Ascaris suum to address the roles of external and internal factors that trigger sperm activation and power sperm motility. Nematode sperm can be activated in vitro by several factors, including Pronase and ionophores, and in vivo through the TRY-5 and SPE-8 pathways. Moreover, protease and protease inhibitors are crucial regulators of sperm maturation. MSP-based sperm motility involves a coupled process of protrusion and retraction, both of which have been reconstituted in vitro. Sperm motility is mediated by phosphorylation signals, as illustrated by identification of several key components (MPOP, MFPs and MPAK) in Ascaris and the characterization of GSP-3/4 in C. elegans.     

恶性疟裂殖子表面蛋白1合成基因在毕赤酵母中的表达

恶性疟原虫裂殖子表面蛋白1是当今疟疾疫苗主要的候选抗原。由于天然MSP1基因AT含量异常高(为74%),使得克隆全长天然基因无法实现。本文已全合成了msp1基因(4940bp),解决了该天然基因在异源系统中不稳定的问题。为制备大量msp1重组蛋白进行疫苗有效性试验,本研究建立了msp1基因在毕赤酵母中的表达,将合成的msp1基因克隆到毕赤酵母胞内表达载体pPIC3.5,构建了重组质粒pPIC3.5/msp1,用电击转化毕赤酵母得到重组转化子,经PCR证实msp1基因已整合于毕赤酵母染色体中。含有重组表达质粒的毕赤酵母菌经甲醇诱导后表达出全长msp1重组蛋白。表达产物能与识别MSP1分子二硫键依赖构象表位的特异性单抗发生很强的反应,表明msp1重组蛋白至少在该表位构象上与天然蛋白一致。从毕赤酵母中分离得到大量msp1为开展该蛋白的结构与功能,特别是测定其疟疾保护性免疫提供可能。     

Trichinella zimbabwensis n.sp. (Nematoda), a new non-encapsulated species from crocodiles (Crocodylus niloticus) in Zimbabwe also infecting mammals

Since 1995, Trichinella larvae have been detected in 39.5% of farmed crocodiles (Crocodylus niloticus) in Zimbabwe. Morphological, biological, biochemical and molecular studies carried out on one isolate from a farmed crocodile in 2001 support the conclusion that this parasite belongs to a new species, which has been named Trichinella zimbabwensis n.sp. This species, whose larvae are non-encapsulated in host muscles, infects both reptiles and mammals. The morphology of adults and larvae is similar to that of Trichinella papuae. Adults of T. zimbabwensis cross in both directions with adults of T. papuae (i.e. male of T. zimbabwensis per female of T. papuae and male of T. papuae per female of T. zimbabwensis), producing F1 offspring which produce very few and less viable F2 larvae. Muscle larvae of T. zimbabwensis, like those of T. papuae, do not infect birds. Three allozymes (of a total of 10) are diagnostic between T. zimbabwensis and T. papuae, and five are diagnostic between T. zimbabwensis and Trichinella pseudospiralis, the third non-encapsulated species. The percentage of the pairwise alignment identity between T. zimbabwensis and the other Trichinella species for the cytochrome oxidase subunit I gene, the large subunit ribosomal-DNA (mt-lsrDNA) gene and the expansion segment five, shows that T. zimbabwensis is more similar to the two non-encapsulated species T. papuae (91% for cytochrome oxidase I; 96% for mt-lsrDNA; and 88% for expansion segment five) and T. pseudospiralis (88% for cytochrome oxidase I; 90% for mt-lsrDNA; and 66–73% for expansion segment five) than to any of the encapsulated species (85–86% for cytochrome oxidase I; 88–89% for mt-lsrDNA; and 71–79% for expansion segment five). This is the first non-encapsulated species discovered in Africa. The finding of a new Trichinella species that infects both reptiles and mammals suggests that the origin of Trichinella parasites dates back further than previously believed and can contribute to understanding the phylogeny and the epidemiology of the genus Trichinella.     

Species relationships in Bulnesia as shown by seed protein electrophoresis

The seed proteins of seven species of Bulnesia were studied by polyacrylamide electrophoresis. Some of the bands are characteristic and constant “markers” of each species; these allow the unequivocal identification of their electrophoregrams. In total 84 different bands were identified. These were treated numerically by cluster analysis. There were no constant differences between geographic races of B. arborea from Colombia and Venezuela. The electrophoregram of B. carrapo shows differences with that of B. arborea giving support to the idea that both taxa are separate allopatric species. The species pair B. foliosa-B. schickendantzii present the most similar electrophoregrams; this determines a short taxonomic distance between them in the phenogram. The Prim network shows the supposedly more primitive species (B. arborea, B. carrapo and B. bonariensis) well separated from the more advanced group (B. schickendantzii, B. foliosa and B. retama). B. sarmientoi, however, appears as rather distant and unrelated from all other taxa. In general, the results from protein electrophoresis agree with results from a previous numerical study based on 43 morphological characters.     

Cloning and modeling of CD8 beta in the amphibian ambystoma Mexicanum. Evolutionary conserved structures for interactions with major histocompatibility complex (MHC) class I molecules

Bovine anaplasmosis is a rickettsial disease of world-wide economic importance caused by Anaplasma marginale. Several major surface proteins with conserved gene sequences have been examined as potential candidates for vaccines and/or diagnostic assays. Major surface protein 1 (MSP1) is composed of polypeptides MSP1a and MSP1b. MSP1a is expressed from the single copy gene msp1 alpha and MSP1b is expressed by members of the msp1 beta multigene family. In order to determine if the msp1 genes are conserved, primers specific for msp1 alpha, msp1 beta(1), and msp1 beta(2) genes were synthesized and used to amplify msp1 sequences of A. marginale from tick cell cultures, from cattle during acute and chronic infections and from salivary glands of Dermacentor variabilis. Protein sequences of MSP1a, MSP1b(1) and MSP1b(2) were conserved during the life cycle of the parasite. No amino acid changes were observed in MSP1a. However, small variations were observed in the MSP1b(1) and MSP1b(2) protein sequences, which could be attributed to recombination, selection for sub-populations of A. marginale in the vertebrate host and/or PCR errors. Several isolate-specific sequences were also observed. Based on the information obtained in this study, the MSP1 protein appears to be fairly well conserved and a potential vaccine candidate.     

Major Sperm Protein Genes from Globodera rostochiensis

Three genes in the major sperm protein (MSP) gene family from the potato cyst nematode Globodera rostochiensis were cloned and sequenced. In contrast to the absence of introns in Caenorhabditis elegans MSP genes, these genes in G. rostochiensis contained a 57 nucleotide intron, with normal exon-intron boundaries, in the same relative location as the intron in Onchocerca volvulus. The MSP genes of G. rostochiensis had putative CAAT, TATA, and polyadenylation signals. The predicted G. rostochiensis MSP gene product is 126 amino acids long, one residue shorter than the products in the other species. The comparison of MSP amino acid sequences from four diverse nematode species suggests that O. volvulus, Ascaris suum, and C. elegans may be more closely related to each other than they are to G. rostochiensis.     

Cytosolic Ca2+ as a multifunctional modulator is required for spermiogenesis in Ascaris suum

The dynamic polar polymers actin filaments and microtu-bules are usually employed to provide the structural ba-sis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the abil-ity to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly under-stood. Here we show that Ca²⁺ oscillations induced by the Ca²⁺ release from intracellular Ca²⁺ store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suumsperm activation. The chelation of cytosolic Ca²⁺ suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing ex-ogenous Ca²⁺ into sperm cells. Ca²⁺ promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca²⁺ promotes MSP disassembly by activating Ca²⁺/calmodulin-depend-ent serine/threonine protein phosphatase calcineurin. In addition, Ca²⁺/camodulin activity is required for the fusion of sperm-specific membranous organelle with the plasma membrane, a regulated exocytosis required for sperm mo-tility. Thus, Ca²⁺ plays multifunctional roles during sperm activation in Ascaris suum.     

Actin and major sperm protein in spermatids and spermatozoa of the parasitic nematode Heligmosomoides polygyrus

Nematode spermatozoa are amoeboid cells. In Caernorhabditis elegans and Ascaris suum, previous studies have reported that sperm motility does not involve actin, but, instead, requires a specific cytoskeletal protein, name y major-sperm-protein (MSP). In Heligmosomoides polygyrus, a species with large and elongate spermatids and spermatozoa, cell organelles are easily identified even with light microscopy. Electrophoresis of Heligmosomoides sperm proteins indicates that the main protein band has a molecular weight of about 15 kDa, as MSP in other nematodes, and is specifically labelled by an anti-MSP antibody raised against C. elegans MSP. A minor band at 43 kDa was specifically labelled by an anti-actin antibody. Reaction of anti-actin and anti-MSP antibodies is specific to, and restricted to, their respective targets. Actin and MSP localisation, studied by indirect immunofluorescence in male germ cells of Heligmosomoides polygyrus, are similar: spermatids show rows of dots, corresponding to the fibrous bodies, around an unlabelled central longitudinal core; spermatozoa are labelled strictly in an anterior crescent-shaped cap, at the opposite pole to the nucleus, which contains fibres of the MSP cytoskeleton. Phalloidin labelling shows that F-actin is present in spermatids, but absent in spermatozoa. Tropomyosin shows a distinct pattern in spermatids, but is located in the MSP and actin-containing cap in spermatozoa. It is hypothesized that actin plays a role in the shaping of the cell and in the arrangement of its organelles during nematode spermiogenesis, when MSP is present, in an inactive state, in the fibrous bodies. The concentration of actin and tropomyosin in the anterior cap is not compatible with previous theories about the MSP cytoskeleton which is supposed to act in the absence of actin. © 1996 Wiley-Liss, Inc.     

Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli

A major immunodominant surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis 381 has been purified and its amino-terminal amino acid sequence has been determined. Using oligonucleotide probes corresponding to the sequence, we identified a recombinant plasmid clone carrying a single 4.2-kb BamHI fragment from pUC19 libraries of P. gingivalis. The BamHI fragment transferred to the bacteriophage T7 RNA polymerase/promoter expression vector system produced a slightly larger (77-kDa) protein, a precursor form, immunoreactive to the antibody against the 75-kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 75-kDa protein. Genomic Southern analysis revealed a single copy of the 75-kDa protein gene per genome among all P. gingivalis strains tested, and that no homologous genes are present in other black-pigmented Bacteroides species. These observations suggest that the 75-kDa protein gene may be useful as a specific DNA probe to classify or to detect this organism.     

斑鳢肝脏解偶联蛋白2cDNA核心片断的克隆及分析

解偶联蛋白2(uncouplingprotein 2,UCP2)可使氧化磷酸化解偶联。本研究首次成功从斑鳢(Channa maculata)肝脏通过简并引物克隆获得UCP2基因cDNA核心序列,该片段长502bp,编码167个氨基酸残基。使用vector NTI suite 6.0软件进行氨基酸同源性序列比较分析表明,斑鳢UCP2与真鲷、鲤鱼、草鱼、斑马鱼UCP2同源性高达91%、73%、72%、71%,与人、大鼠、小鼠UCP2同源性较高为70%、71%、70%。UCP2编码区在鱼类、哺乳类中均具有较高保守性,提示着脊椎动物UCP2可能在线粒体有氧呼吸代谢过程中承担某种最基本的生命功能。     

2.6 A resolution crystal structure of helices of the motile major sperm protein (MSP) of Caenorhabditis elegans

The amoeboid locomotion of nematode sperm is mediated by the assembly dynamics of the major sperm protein (MSP). MSP forms fibrous networks based on a hierarchy of macromolecular assemblies: helical subfilaments are built from MSP dimers; filaments are formed from two subfilaments coiling round one another; and filaments themselves supercoil to produce bundles. To provide a structural context for understanding the role of these macromolecular assemblies in cell locomotion, we have determined the 2.6 A resolution structure of crystals of Caenorhabditis elegans MSP that are constructed from helices of MSP chains that are analogous to the subfilaments from which filaments are constructed. Comparison with the crystal structures of dimers and helical assemblies of Ascaris suum MSP has identified five conserved interaction interfaces that suggest how subfilaments interact in filaments and how filaments can form bundles. The interfaces frequently involve the loop containing residues 78-85, which is divergent between MSP homologues, and the loop containing residues 98-103, which is highly conserved.     

A novel gene encoding a ras-like GTP-binding protein from Trypanosoma brucei: an evolutionary ancestor of the ras and rap genes of higher eukaryotes?

The ras superfamily of GTP binding proteins encompasses a wide range of family members, related by conserved amino-acid motifs, and act as molecular binary switches that play key roles in cellular processes. Gene duplication and divergence has been postulated as the mechanism by which such family members have evolved their specific functions. We have cloned and sequenced a ras-like gene, tbrlp, from the primitive eukaryote Trypanosoma brucei. The gene encodes a protein of 227 amino acids and contains the six conserved subdomains that designate it as a ras/rap subfamily member. However, the presence of key diagnostic residues characteristic of both the ras and rap families of GTP confuse the familial classification of this gene. Phylogenetic analysis of the GTP binding domain places its origins at the divergence point of the ras/rap families and suggests that tbrlp is an ancestral gene to the ras/rap genes of higher eukaryotes.     

A Plasmodium vivax vaccine candidate displays limited allele polymorphism, which does not restrict recognition by antibodies.

BACKGROUND: The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)) has been suggested as candidate for part of a subunit vaccine against malaria. A major concern in vaccine development is the polymorphism observed in different plasmodial strains. The present study examined the extension and immunological relevance of the allelic polymorphism of the MSP1(19) from Plasmodium vivax, a major human malaria parasite. MATERIALS AND METHODS: We cloned and sequenced 88 gene fragments representing the MSP1(19) from 28 Brazilian isolates of P. vivax. Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera to bacterial and yeast recombinant proteins representing the two allelic forms of P. vivax MSP1(19) described thus far. RESULTS: We observed that DNA sequences encoding MSP1(19) were not as variable as the equivalent region of other species of Plasmodium, being conserved among Brazilian isolates of P. vivax. Also, we found that antibodies are directed mainly to conserved epitopes present in both allelic forms of the protein. CONCLUSIONS: Our findings suggest that the use of MSP1(19) as part of a subunit vaccine against P. vivax might be greatly facilitated by the limited genetic polymorphism and predominant recognition of conserved epitopes by antibodies.     

Inter-specific sequence conservation and intra-individual sequence variation in a spider silk gene

Currently, studies on major ampullate spidroin 1 (MaSp1) genes of non-orb weaving spiders are few, and it is not clear whether genes of these organisms exhibit the same characteristics as those of orb-weavers. In addition, many studies have proposed that MaSp1 might be a single gene with allelic variants, but supporting evidence is still lacking. In this study, we compared partial DNA and amino acid sequences of MaSp1 cloned from different spider guilds. We also cloned partial MaSp1 sequences from genomic DNA and cDNA of the same individuals of spiders using the same primer combination to see if different molecular forms existed. In the repetitive region of partial MaSp1 sequences obtained, GGX, GA and poly-A motifs were present in all Araneomorphae and Mygalomorpae species examined. An extreme similarity in MaSp1 non-repetitive portions was found in sequences of ecribellate, cribellate and Mygalomorphae web-builders and such a result suggested that this sequence might exhibit an important function. A comparison of sequences amplified from the same individual showed that substitutions in amino acids occurred in both repetitive and non-repetitive regions, with a much higher variation in the former. These results suggest that the MaSp1 of Araneomorphae spiders exhibits several forms in an individual spider and it might be either a multiple gene or a single gene with a multiple exon/intron organization.