Understanding resistant germplasm‐induced virulence variation through analysis of proteomics and suppression subtractive hybridization in a maize pathogen Curvularia lunata

Curvularia lunata is an important pathogen causing Curvularia leaf spot in maize. Significant pathogenic variation has been found in C. lunata. To better understand the mechanism of this phenomenon, we consecutively put the selective pressures of resistant maize population on C. lunata strain WS18 (low virulence) artificially. As a result, the virulence of this strain was significantly enhanced. Using 2DE, 12 up‐regulated and four down‐regulated proteins were identified in virulence‐increased strain compared to WS18. Our analysis revealed that melanin synthesis‐related proteins (Brn1, Brn2, and scytalone dehydratase) and stress tolerance‐related proteins (HSP 70) directly involved in the potential virulence growth as crucial markers or factors in C. lunata. To validate 2DE results and screen differential genes at mRNA level, we constructed a subtracted cDNA library (tester: virulence‐increased strain; driver: WS18). A total of 188 unigenes were obtained this way, of which 14 were indicators for the evolution of pathogen virulence. Brn1 and hsp genes exhibited similar expression patterns corresponding to proteins detected by 2DE. Overall, our results indicated that differential proteins or genes, being involved with melanin synthesis or tolerance response to stress, could be considered as hallmarks of virulence increase in C. lunata.     

Multiple near-identical genotypes of Schistosoma japonicum can occur in snails and have implications for population-genetic analyses

We genotyped (using 16 or 17 microsatellite loci) numerous adult Schistosoma japonicum raised in rabbits exposed to pooled cercariae from small numbers of naturally infected snails from several localities in China. As expected, duplicate multi-locus genotypes (MLGs) were found among these worms. Additionally, many more MLGs, often near-identical, were found than snails used as sources of cercariae. Explanations for these results include (i) genotyping errors, (ii) development within each infected snail of multiple sibling miracidia and (iii) somatic mutation producing genetically varied cercariae from a single miracidium. To control for genotyping errors we re-analysed samples from many individual worms, including repeating the initial PCR. Explanations invoking the development of multiple sibling miracidia within a single snail are not likely to be correct because almost all duplicate MLGs fell within same-sex clusters in a principal coordinates analysis. We would expect both sexes to be represented in a multi-miracidium infection. In addition, we exposed several snails to infection by a single miracidium. One such snail, via an experimentally infected mouse, yielded 48 adult worms. The presence of at least nine near-identical MLGs among these worms was confirmed by re-genotyping. We regard somatic mutation as the most likely explanation for our results. The implications of multiple MLGs for population-genetic studies in S. japonicum are discussed.     

Proteomic analysis of the pinworm Syphacia muris (Nematoda: Oxyuridae), a parasite of laboratory rats

Syphacia muris (Nematoda: Oxyuridae) is a ubiquitous nematode that commonly infects rats in the laboratory which can interfere in the development of biological assays. The somatic extract of S. muris adults collected from infected rats was investigated using a proteomic approach. A shot-gun liquid chromatography/tandem mass spectrometry procedure was used. We used the MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems) for the database search. A total of 359 proteins were accurately identified from the worms. The largest protein families consisted of metabolic enzymes and those involved in the nucleic metabolism and cell cycle. Proteins of transmembrane receptors and those involved in protein metabolism, chaperones, structural and motor, signalling and calcium-binding proteins also were identified in the proteome of S. muris. Proteome array of S. muris may contribute to further elucidation of biological system of S. muris as well as host-parasite relationships.     

Vascular endothelial cell activation by adult Dirofilaria immitis antigens

Dirofilaria immitis is the causal agent of cardiopulmonary dirofilariosis (heartworm disease). Adult worms lodge in the pulmonary arteries and right ventricle, thus vascular endothelium is exposed to high concentrations of Dirofilaria antigenic products. Heartworm disease habitually develops as a chronic foreseeable pathology. Moreover, the simultaneous death of many adult worms, naturally or induced by a filaricide treatment, can cause acute thromboembolisms and endarteritis. To better understand the effects of the massive death of D. immitis adult worms on the blood vessel endothelium, we cultured vascular endothelial cells in the presence or absence of an antigenic extract of D. immitis adult worms (DiSA). The parasite products increased the expression of enzymes and the synthesis of eicosanoids related to inflammation, such as COX-2, 5-LO, PGE₂ and LTB₄. The expression of ICAM-1 and PECAM-1 adhesion molecules and endothelial and inducible Nitric Oxide Synthases (eNOS and iNOS) was also increased in cultures treated with DiSA. Nevertheless, DiSA decreased endothelial permeability and does not alter both proliferation and apoptosis. These results suggest that the somatic extract of D. immitis adult worms stimulate inflammatory mechanisms in endothelial cells, without altering their basic physiologic processes.     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung     

Gymnophallid digenean Parvatrema duboisi uses Manila clam as the first and second intermediate host

To identify the metacercariae of a gymnophallid trematode in the Manila clam Ruditapes philippinarum from the Ariake Sea, experimental infection and molecular analysis were conducted. Based on the morphology of adult worms obtained from experimentally infected mice, the parasite was identified as Parvatrema duboisi. Comparison of internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) sequences for metacercariae and sporocysts from Manila clams and adult worms collected from wild wigeon Anas penelope showed minor differences ranging from 0 to 0.8%. These data strongly suggest that in the Ariake Sea, the parasite has a lifecycle using the Manila clam as the first and second intermediate hosts and wigeon as the definitive host.     

Differential proteomic analysis of developmental stages of <Emphasis Type="Italic">Acca sellowiana</Emphasis> somatic embryos

Feijoa (Acca sellowiana, Myrtaceae), a native fruit species from southern Brazil and northern Uruguay, is considered to constitute a reference system for somatic embryogenesis in woody dicots. This in vitro regenerative pathway is an efficient micropropagation method, and a suitable model system for studies in plant developmental physiology. This study attempts to detect and identify proteins that are expressed during the different developmental stages of somatic embryos of A. sellowiana. Using high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE), a high degree of similarity between protein profiles of the assayed somatic embryos was observed. Of the 74 different protein spots extracted for analysis, 60 were identified by means of 2-DE/MALDI-TOF/MS. Twelve proteins were expressed in all the assayed stages. Ten proteins were expressed in the initial stages and 22 proteins were expressed in the mature developmental stages of somatic embryos. Only one protein was expressed exclusively in the torpedo stage, whereas four were expressed in the pre-cotyledonary, and none in the cotyledonary stage. The proteins identified were involved in the synthesis of phenylalanine ammonia-lyase, a conspicuous polyphenol present in the induction of feijoa embryogenic cultures. The presence of essential proteins of nitrogen metabolism, such as the cytosolic glutamine synthetase protein, was also observed. The physiological implications of these findings are discussed.     

Green synthesis of gold nanoparticles with Zingiber officinale extract: Characterization and blood compatibility

Biosynthesis of gold nanoparticles with small size and biostability is very important and used in various biomedical applications. There are lot of reports for the synthesis of gold nanoparticles by the addition of reducing agent and stabilizing agent. In the present study we have synthesized gold nanoparticles, with a particle size ranging from 5 to 15 nm, using Zingiber officinale extract which acts both as reducing and stabilizing agent. Z. officinale extract is reported to be a more potent anti-platelet agent than aspirin. Therefore, green synthesis of gold nanoparticles with Z. officinale extract, as an alternative to chemical synthesis, is beneficial from its biological and medical applications point of view, because of its good blood biocompatibility and physiological stability. The formation and size distribution of gold nanoparticles were confirmed by dynamic light scattering (DLS), UV–vis spectrophotometer and transmission electron microscopy (TEM). Gold nanoparticles synthesized using citrate and Z. officinale extract demonstrated very low protein adsorption. Both nanoparticles were non platelet activating and non complement activating on contact with whole human blood. They also did not aggregate other blood cells, however, nanoparticles synthesised with Z. officinale extract was highly stable at physiological condition compared to citrate capped nanoparticles, which aggregated. Thus the usage of nanoparticles, synthesized with Z. officinale extract, as vectors for the applications in drug delivery, gene delivery or as biosensors, where a direct contact with blood occurs is justified.     

A novel zinc-carboxypeptidase SURO-1 regulates cuticle formation and body morphogenesis in Caenorhabditis elegans

Cuticle formation and molting are critical for the development of Caenorhabditis elegans. To understand cuticle formation more clearly, we screened for suppressors in transgenic worms that expressed dominant ROL-6 collagen proteins. The suro-1 mutant, which is mild dumpy, exhibited a different ROL-6::GFP localization pattern compared to other Dpy mutants. We identified mutations in three suro-1 mutants, and found that suro-1 (ORF R11A5.7) encodes a putative zinc-carboxypeptidase homologue. The expression of this enzyme in the hypodermis and the genetic interactions between this enzyme and other collagen-modifying enzyme mutants suggest a regulatory role in collagen processing and cuticle organization for this novel carboxypeptidase. These findings aid our understanding of cuticle formation during worm development.     

In vivo analysis of <Emphasis Type="Italic">Drosophila</Emphasis> SU(<Emphasis Type="Italic">Z</Emphasis>)12 function

Polycomb group (PcG) proteins are required to maintain a stable repression of the homeotic genes during Drosophila development. Mutants in the PcG gene Supressor of zeste 12 (Su(z)12) exhibit strong homeotic transformations caused by widespread misexpression of several homeotic genes in embryos and larvae. Su(z)12 has also been suggested to be involved in position effect variegation and in regulation of the white gene expression in combination with zeste. To elucidate whether SU(Z)12 has any such direct functions we investigated the binding pattern to polytene chromosomes and compared the localization to other proteins. We found that SU(Z)12 binds to about 90 specific eukaryotic sites, however, not the white locus. We also find staining at the chromocenter and the nucleolus. The binding along chromosome arms is mostly in interbands and these sites correlate precisely with those of Enhancer-of-zeste and other components of the PRC2 silencing complex. This implies that SU(Z)12 mainly exists in complex with PRC2. Comparisons with other PcG protein-binding patterns reveal extensive overlap. However, SU(Z)12 binding sites and histone 3 trimethylated lysine 27 residues (3meK27 H3) do not correlate that well. Still, we show that Su(z)12 is essential for tri-methylation of the lysine 27 residue of histone H3 in vivo, and that overexpression of SU(Z)12 in somatic clones results in higher levels of histone methylation, indicating that SU(Z)12 is rate limiting for the enzymatic activity of PRC2. In addition, we analyzed the binding pattern of Heterochromatin Protein 1 (HP1) and found that SU(Z)12 and HP1 do not co-localize.     

Eumycetoma caused by Curvularia lunata in a dog

Curvularia lunata was cultured from black granules found in granulomatous tumefactions excised from the subcutis of a three year old Medium Schnauzer dog. Draining sinuses were present in some of the tumefactions. Accordingly the diagnosis of eumycotic mycetoma was made. This diagnosis was confirmed by histopathological examination. During the four years following the first surgical intervention, several more similar tumefactions were excised on three different occasions. The dog died of chronic renal failure at the age of 8 years. There was no bone involvement or visceral diffusion of the fungus. The granules were examined by scanning electron microscopy. Immunoglobulins in the dog's serum, assessed by a qualitative test, proved to be equal to immunoglobulins in the serum of a control dog. Precipitating antibodies against C. lunata were not found. The dog was treated for 150 days with itraconazole. In spite of good initial results, recurrence of the fungal lesions were observed after the treatment's interruption. Further treatment with itraconazole for 45 days proved ineffective. No side effects of the drug were observed. This is, to the best of our knowledge, the first case in which C. lunata is identified as the causative agent of an animal eumycetoma.     

Regions in theZ5 mating gene ofSchizophyllum commune involved in Y-Z binding and recognition

The A mating locus of the woodrotting fungusSchizophyllum commune encodes two multiallelic genes,Y andZ, which regulate the A-pathway of development. TheY alleles contain a homeobox, suggesting that the Y proteins may be DNA-binding regulatory proteins. During mating, development is induced when Y from one mating partner interacts with Z from the other mating partner; self combinations of Y and Z are inactive. Two-hybrid analyses indicate that nonself combinations of Y and Z form heteromultimers and self combinations do not. To understand Y-Z binding and self- nonself recognition further we used mutagenesis and chimeras to identify regions in one allele ofZ(Z5) that are involved in these processes. Here we report the results, which broadly define regions in Z5 that are essential for activity, Y-Z binding and Z5 allelic specificity.The sequence reported in this paper has been deposited in the Genbank database under accession number U22049     

小叶石楠果实中低极性化学成分GC-MS分析

采用溶剂提取法从小叶石楠果实中提取低极性化学成分,并利用气相色谱—质谱仪对果实中的低极性化学成分进行分离和鉴定,同时用面积归一法测定各成分的相对百分含量。结果表明:已确认了25种成分,占果实中低极性化学成分的96.04%,其主要成分为亚麻酸甲酯(13.11%)、邻苯二甲酸二辛醇酯(10.13%)、角鲨烯(9.19%)、维生素E(8.67%)、十九烷(8.03%)。所鉴定的化合物多为该种植物中首次发现,为小叶石楠的进一步开发利用提供了科学依据。     

盐角草内生真菌分离鉴定及抑制水产腐败细菌菌株的筛选

盐角草是一种耐盐植物,有重要的食用、药用价值.为研究盐角草的内生真菌及其活性次生代谢产物的多样性,该文对采集于广西北部湾沿海盐角草的内生真菌进行分离纯化,采用RAPD对内生真菌多样性进行分析,采用ITS基因序列对内生真菌进行鉴定,并对内生真菌提取物抑制等3种水产腐败细菌的活性进行筛选.结果表明:(1)从北部湾盐角草植物...     

Differential cytotoxicity and mycotoxin content among isolates of Fusarium moniliforme

Curvularia lunata was found causing (disseminated phaeohyphomycosis among a group of Nezara viridula (Insecta:Heteroptera) parasitizing vegetable crop Vigna unguiculata. Dark lesions were seen on pronotum and abdominal sterna. Experimental lesions were produced by applying 0.1 ml of 6.2 × 10⁸cfu/ml^–1 on abdominal sterna. Histopathology revealed that almost all internal organs and tissues showed extensive damage. It is interesting to note that C. lunata exhibited predeliction for chitinous tissues and elicited cellular immune response by granulocytes (phagocytosis). This is the first report of phaeohyphomycosis of an insect, extending the disease to invertebrates.A promising research career of one of the authors (Mrs. Vinita Dubey) was cut short by untimely death. This paper is dedicated to her memory.     

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying theNeo ^r selectable marker and theEscherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G₁ progeny at a frequency of 2.7%.Neo ^r andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G₂ progeny.     

Effect of polymer differences on fungal penetration of hydrogel lenses

Summary Two chemically distinct types of hydrogel lenses, vifilcon A and bufilcon A, each with a water content of 55%, were challenged in a balanced salts solution withAspergillus fumigatus, Cladosporium cladosporioides, Curvularia lunata andFusarium solani. The lenses were cleaned, disinfected and stained after varying periods of incubation and examined with light microscopy and scanning electron microscopy. For three of the four fungi, the bufilcon A lens was more susceptible to fungal attack than the vifilcon A lens.Curv. lunata produced the greatest number of penetration pegs within 72 h for both lens types. Etching of lens surfaces was observed withC. cladosporioides. In general, the susceptibility of a hydrogel lens to penetration with a fungus appeared to vary with the species of fungus and the chemical composition of the lens.     

Optimization of culture conditions for the production of rifamycin oxidase by Curvularia lunata

Maximum activity (8.9 IU/ml) of rifamycin oxidase in Curvularia lunata, grown in shake-flask culture at 28°C and pH 6.5, was after 96 h. Nearly all the glucose was used in 72 h. An initial culture pH of 6.5 and 28°C were optimum for the growth and enzyme production. Among various carbon and organic nitrogen sources, carboxymethylcellulose and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when yeast extract was also added to the medium. The optimum medium for the production of rifamycin oxidase contained 10 g each of yeast extract, peptone and carboxymethylcellulose/l and 0.04% (NH₄)₂SO₄.The author is with the Biochemical Engineering Research and Process Development Centre, Institute of Microbial Technology, Post Box 1304, Sector 39-A, Chandigarh 160 014, India