Development and roles of vitelline cells in eggshell formation in Fasciola gigantica

Summary

In Fasciola gigantica, vitelline cells are the major contributors to the formation of the eggshell. The vitelline cells develop in vitelline follicles that are located in the posterior third of the adult parasite's body, in the areas lateral to the uterus and the testis. Mature vitelline cells are released and transported to the Mehlis' gland-ootype complex via a series of vitelline ducts. Based on ultrastructural features, the developing vitelline cells are classified into four stages: stem cell, protein-synthetic, carbohydrate-synthetic and mature cell stages. At the protein-synthetic stage, the eggshell globules are formed, whereas during the carbohydrate-synthetic stage glycogen particles and glycan vesicles are synthesized. The mature vitelline cells are detached from the nurse cells, and pass successively into the intrafollicular, interfollicular, longitudinal and transverse vitelline ducts, to be stored in the vitelline reservoir before being transported to the ootype via the median vitelline duct. At the same time, ova are transported from the ovary through the oviduct into the ootype lumen where each becomes surrounded by a number of vitelline cells. Vitelline cells secrete eggshell globules to surround a group of vitelline cells and an ovum in the ootype lumen, and these globules coalesce into the definitive eggshell. In the middle part of the uterus fertilization occurs, after which the eggshell is completely formed. Within the egg proper, vitelline cells break down, releasing glycogen and other products to nourish the developing embryo.     

Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan, Iran

Fascioliasis is an important human and animal disease caused by Fasciola hepatica and Fasciola gigantica. In Iran, the distribution of these two species overlaps in most areas, including the northern human endemic province of Gilan where both fasciolids are simultaneously found in individual cattle and buffaloes. A phenotypic study of fasciolid adult flukes from naturally infected bovines from Gilan was carried out by means of an exhaustive morphometric analysis using traditional microscopic measurements and an allometric model. The Iranian fasciolids were compared to F. hepatica and F. gigantica standard populations, i.e. from geographical areas where both species do not co-exist (Bolivia and Burkina Faso, respectively). Although morphometric values somewhat overlapped, there were clear differences in allometric growth. The allometric function was adjusted to 25 pairs of variables. Results obtained revealed that Iranian F. hepatica-like specimens are larger than the F. hepatica standard and Iranian F. gigantica-like specimens are longer and narrower than the F. gigantica standard, but with smaller body area. Measurements which permit a specific differentiation in allopatric populations (distance between ventral sucker and posterior end of the body; ratio between body length and body width) overlap in the specimens from Gilan, thus proving the presence of intermediate forms. When compared to the standard populations, the different Iranian fasciolid morphs show greater differences in F. gigantica-like specimens than in F. hepatica-like specimens. This study shows that simple, traditional microscopic measurements may be sufficient for the morphometric characterisation of fasciolids, even in areas where intermediate forms are present.     

大片吸虫成虫cDNA表达文库的构建及其表达序列标签初步分析

为发现潜在的抗大片吸虫病的候选疫苗分子,控制大片形吸虫病,本研究以大片吸虫成虫为材料,应用SMARTTcDNA文库构建技术,构建了以表达载体λTriplEx2为基础的大片吸虫成虫cDNA表达文库。经测定,库容量为1·08×106PFU/ml ,重组率为96·6 %,扩增后的文库滴度为2·41×109PFU/ml ,插入片段平均大小约为1 000 bp;经大肠杆菌BM25·8质粒化后,从文库中随机挑选40个重组克隆测序,获得32条有效ESTs ;经BLASTX和BLASTn程序检索和分析,发现有9条ESTs代表已知基因, 16条ESTs相似性较低或无匹配,列为新基因。9条已知基因代表了半胱氨酸蛋白酶、卵壳蛋白、钙连接蛋白等三类功能蛋白,其余新基因也暗示与信号传导、蛋白合成、免疫刺激等基因相关,具有潜在的研究价值[动物学报51 (5) : 879 -883 , 2005]。     

Identification of potential inhibitors of Fasciola gigantica thioredoxin1: computational screening,molecular dynamics simulation,and binding free energy studies

Fasciola gigantica is the causative organism of fascioliasis and is responsible for major economic losses in livestock production globally. F. gigantica thioredoxin1 (FgTrx1) is an important redox-active enzyme involved in maintaining the redox homeostasis in the cell. To identify a potential anti-fasciolid compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,740) against the FgTrx1 structure. The ligands were docked against FgTrx1 and 309 ligands were found to have better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 30 compounds were found to fit well for re-docking studies. After refinement by molecular docking and drug-likeness analysis, three potential inhibitors (ZINC15970091, ZINC9312362, and ZINC9312661) were identified. These three ligands were further subjected to molecular dynamics simulation (MDS) to compare the dynamics and stability of the protein structure after binding of the ligands. The binding free energy analyses were calculated to determine the intermolecular interactions. The results suggested that the two compounds had a binding free energy of –82.237, and –109.52 kJ.mol^?1 for compounds with IDs ZINC9312362 and ZINC9312661, respectively. These predicted compounds displayed considerable pharmacological and structural properties to be drug candidates. We concluded that these two compounds could be potential drug candidates to fight against F. gigantica parasites.     

Sequence heterogeneity in a repetitive dna element of Fasciola

A repetitive DNA sequence used as a specific probe for Fasciola hepatica infections in snails was examined in F. hepatica and Fasciola gigantica (36 individuals) specimens from five continents. The degree of intraspecific identity ranged from 79 to 99% in F. hepatica and from 93 to 99% in F. gigantica. The interspecific identity ranged from 81 to 100%, confirming the suggestion that the DNA probe sequence could be used worldwide as an epidemiological tool for the examination of the intermediate host snail in fasciolosis. Differentiation between F. hepatica and F. gigantica infections in snails was not possible using the probe.     

Analysis of thioredoxin peroxidase as a promising antigen for diagnosis of Fasciola gigantica infection: a preliminary study

Buffalo fasciolosis induced by Fasciola gigantica causes important economic losses in tropical areas of Asia. Detection of prepatent infection is essential to control this disease. Classical tools such as coprology, necroscopy or ELISA based on crude extracts from F. gigantica are poorly sensitive or specific. Purified antigens could be used to increase these parameters. Western blot analysis and mass spectrometry of a fraction of F. gigantica excretory-secretory products obtained by gel filtration showed that thioredoxin peroxidase could be a potential antigen for serodiagnosis: it was recognized from the 2nd week after infection, by all buffalo experimentally or naturally infected with F. gigantica but not by healthy animals.     

Fasciola gigantica: enzymes of the ornithine-proline-glutamate pathway--characterization of delta1-pyrroline-5-carboxylate dehydrogenase

Ornithine aminotransferase (OAT), proline oxidase (PO), Delta 1-pyrroline-5-carboxylate reductase (P5CR), and Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CD) were assessed in Fasciola gigantica. All enzymes are involved in the conversion of ornithine into glutamate and proline. High levels of P5CD suggest that the direction of the metabolic flow from ornithine is more toward glutamate than proline. F. gigantica P5CD1 and P5CD2 were separated from the majority of contaminating proteins in crude homogenate using a CM-cellulose column. A Sephacryl S-200 column was employed for P5CD2 to obtain pure enzyme with increased specific activity. The molecular mass of P5CD2 was estimated to be 50kDa using a Sephacryl S-200 column and SDS-PAGE. It migrated as a single band on SDS-PAGE, indicating a monomeric enzyme. P5CD2 had Km values of 1.44mM and 0.37mM for NAD and P5C, respectively. P5CD2 oxidized a number of aliphatic and aromatic aldehydes, where the aromatic compounds had higher affinity toward the enzyme. All amino acids examined had partial inhibitory effects on the enzyme. While 3mM AMP caused 31% activation of enzyme, 3mM ADP and ATP inhibited activity by 18% and 23%, respectively. Apart from Cu2+, the divalent cations that were studied caused partial inhibitory effects on the enzyme.     

Overexpression of OsJAC1, a Lectin Gene, Suppresses the Coleoptile and Stem Elongation in Rice

Lectin plays an Important role In defense signaling In plants, but its function In plant growth and development is not well known. Previously, we cloneds rice （Oryza sativa L.） gene OsJAC1 encoding s msnnose-blndlng Jscslln-relsted lectln, and found that OsJAC1 was Jssmonlc acid （JA） Inducible. Here we cloned the promoter of OsJAC1, and GUS activity was detected In young roots, coleoptlles, sheaths, leaves, nodes of stems, stems, rschlses, pistils, stsmens and lemmss of OsJAC1：：GUS trsnsgenlc rice, suggesting that OsJAC1 Is s constitutive expression gene In rice. Moreover, OsJAC1-overexpressed （Ubi：：OsJAC1） rice showed dwarfism with shorter coleptlles resulting from the failure of cell elongation of coleoptlles. In addition, compared with coleoptlles of wild-type plants, those of OsJAC1 overexpresslon rice were more sensitive to JA treatment. These data revealed that, besides Its roles in defense response, lectin plays an Important role in rice growth and development.     

Fasciola gigantica fatty acid binding protein (FABP) as a prophylactic agent against Schistosoma mansoni infection in CD1 mice

Although schistosomicidal drugs and other control measures exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study, native fatty acid binding protein (FABP) from Fasciola gigantica was purified from the adult worm's crude extract by saturation with ammonium sulphate followed by separation on DEAE-Sephadex A-50 anion exchange chromatography and gel filtration using Sephacryl HR-100, respectively. CD1 mice were immunized with the purified, native F. gigantica FABP in Freund's adjuvant and challenged subcutaneously with 120 Schistosoma mansoni cercariae. Immunization of CD1 mice with F. gigantica FABP has induced heterologous protection against S. mansoni, evidenced by the significant reduction in mean worm burden (72.3%), liver and intestinal egg counts (81.3% and 80.8%, respectively), and hepatic granuloma counts (42%). Also, it elicited mixed IgG(1)/IgG(2b) immune responses with predominant IgG1 isotype, suggesting that native F. gigantica FABP is mediated by a mixed Th1/Th2 response. However, it failed to induce any significant differences in the oogram pattern or in the mean granuloma diameter. This indicated that native F. gigantica FABP could be a promising vaccine candidate against S. mansoni infection.     

Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp. strain DM 86-1

Bacteria that produced NAD⁺-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among l-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an isoelectric point of 5.8 and a relative molecular weight (M _r) of approximately 330,000. The enzyme is composed of eight identical subunits with an M _r of approximately 41,000. The apparent K _m values for l-phenylalanine and NAD⁺ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55° C for 10 min. Received: 30 July 1997 / Accepted: 4 November 1997     

定位于染色体9p21-22的一个新基因UBAP1的克隆测序及在鼻咽癌中的表达分析

 在染色体 9p2 1 2 2鼻咽癌杂合性丢失 (lossofheterozygosity,LOH)高频区 ,应用EST介导的定位 侯选克隆策略 ,用RT PCR及Northern杂交检测了 2 2个表达序列标记 (expressedsequencetag ,EST)在鼻咽癌细胞株HNE1和原代培养的正常鼻咽上皮细胞中的表达差异 ,并对其中一个在鼻咽癌细胞株HNE1中表达下调的EST检测了在鼻咽癌活检组织中的表达 .用生物信息学方法获得其全长cDNA序列 ,GenBank登录号AF2 2 2 0 4 3.该基因cDNA全长 2 70 1bp ,其开放阅读框 (openreadingframe ,ORF)编码一个含 50 2个氨基酸、分子量为 55kD的碱性蛋白质 ,在蛋白羧基端含有 2个连续的重要UBA功能域 (ubiquitinassociateddomain) ,属于遍在蛋白相关蛋白家族的一个新成员 ,经国际人类基因命名委员会同意 ,将其命名为UBAP1 (ubiquitinassociatedprotein 1 ) .Northern表达分析显示UBAP1在所检测的人组织中广泛表达 ,但在人的心脏、骨骼肌及肝脏中的表达较强 .UBAP1基因在63 2 % ( 1 2 1 9)的鼻咽癌活检组织中表达下调 .UBAP1基因作为一个遍在蛋白相关蛋白家族的新成员 ,结合其在 9p的重要定位信息 ,有必要进一步研究其表达下调参与鼻咽癌发生发展的可能机制 .     

Effect of Hypoxia on Caspase-3, -8, and -9 Activity and Expression in the Cerebral Cortex of Newborn Piglets

Caspases play an important role in programmed cell death. Caspase-3 is a key executioner of apoptosis, whose activation is mediated by the initiator caspases, caspase-8 and caspase-9. The present study tested the hypothesis that cerebral hypoxia results in increased activation and expression of caspases-3, -8, and -9 in the cytosolic fraction of the cerebral cortex of newborn piglets. To test this hypothesis the activity and expression of caspases-3, -8, and -9 were determined in newborn piglets divided into normoxic and hypoxic groups. Caspase activity was determined spectrofluorometrically using enzyme specific substrates. The expression of caspase protein was assessed by Western blot analysis using enzyme specific antibody. Caspases-3, -8, and -9 activity and expression was significantly higher in the hypoxic group than in the normoxic group. These results demonstrate that hypoxia induces activation and increased expression of both the initiator caspases and the executioner caspase in the cerebral cortex of newborn piglets. We conclude that hypoxia results in stimulation of both the pathways of caspase-3 activation.     

Identification of tetratricopeptide repeat domain 9, a hormonally regulated protein

Tetratricopeptide repeat domain 9 (TTC9) mRNA was drastically up-regulated by progesterone in progesterone receptor-transfected breast cancer cells MDA-MB-231. This up-regulation is coupled with progesterone-mediated growth inhibition and induction of focal adhesion. We have generated mouse polyclonal antibody against a predicted 222 aa TTC9 protein and identified a 25 kDa TTC9 protein that is widely expressed in human tissues, with the highest expression in the brain. Immunostaining and cell fractionation studies revealed that TTC9 is predominantly localized to the endoplasmic reticulum. The level of TTC9 protein in MCF-7 cells is regulated by various factors and chemical reagents including estrogen, progesterone, growth factors, ICI182,780, and p38 kinase inhibitor SB203580. Growth factor-induced TTC9 protein expression was inhibited by estrogen and abolished by ERK inhibitor PD98059. Though the function of TTC9 is not yet clear, the susceptibility of its protein level to biological and chemical agents suggests that TTC9 is a biologically significant protein.     

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.     

Boundaries and physical characterization of a new domain shared between mammalian 53BP1 and yeast Rad9 checkpoint proteins

Eukaryotic cells have evolved DNA damage checkpoints in response to genome damage. They delay the cell cycle and activate repair mechanisms. The kinases at the heart of these pathways and the accessory proteins, which localize to DNA lesions and regulate kinase activation, are conserved from yeast to mammals. For Saccharomyces cerevisiae Rad9, a key adaptor protein in DNA damage checkpoint pathways, no clear human ortholog has yet been described in mammals. Rad9, however, shares localized homology with both human BRCA1 and 53BP1 since they all contain tandem C-terminal BRCT (BRCA1 C-terminal) motifs. 53BP1 is also a key mediator in DNA damage signaling required for cell cycle arrest, which has just been reported to possess a tandem Tudor repeat upstream of the BRCT motifs. Here we show that the major globular domain upstream of yeast Rad9 BRCT domains is structurally extremely similar to the Tudor domains recently resolved for 53BP1 and SMN. By expressing several fragments encompassing the Tudor-related motif and characterizing them using various physical methods, we isolated the independently folded unit for yeast Rad9. As in 53BP1, the domain corresponds to the SMN Tudor motif plus the contiguous HCA predicted structure region at the C terminus. These domains may help to further elucidate the structural and functional features of these two proteins and improve knowledge of the proteins involved in DNA damage.     

橡胶蛋白——种与胶乳凝固有关的具有抗真菌活性的植物凝集素

 橡胶蛋白(Hevein)是一种存在于巴西橡胶树(Hevea brasiliensis)乳管细胞特化的液泡——黄色体(lutoid)内、与几丁质结合的小分子(4.7kD)单链蛋白质,在橡胶粒子的凝絮和胶乳的凝固过程中表现出极为重要的作用,是一类具有抗真菌活性的植物凝集素。     

Comparison of a Reversed Passive Latex Agglutination and a Polymerase Chain Reaction for Identification of Cholera Toxin Producing Vibrio cholerae O1

Production of cholera toxin (CT) in AKI medium and conservation of CT gene (ctx) of 49 strains of Vibrio cholerae O1 were compared by reversed passive latex agglutination (RPLA) and polymerase chain reaction (PCR). The production of CT agreed with conservation of the ctx in 48 out of the 49 strains. Ten strains were positive, and 38 strains were negative by both methods. Only one strain was negative in RPLA and positive in PCR. This suggested that the combination of AKI-SW and RPLA is comparable to PCR to identify CT-producing V. cholerae O1.