Mutational analysis of<Emphasis Type="Italic"> BRAF</Emphasis> in gallbladder carcinomas in association with<Emphasis Type="Italic"> K-ras</Emphasis> and<Emphasis Type="Italic"> p53</Emphasis> mutations and microsatellite instability

Background Little is known about the genetic changes involved in the pathogenesis of gallbladder cancer. The aim of this study was to examine the presence of mutations in exon 15 of the B-raf gene to investigate its role in gallbladder carcinogenesis.Materials and methods We examined the mutational status in exon 15 of B-raf gene in 21 gallbladder carcinoma specimens and investigated its association with the presence of K-ras and p53 alterations, microsatellite instability and the clinicopathological features of tumors.Results B-raf mutations were observed in 7 of 21 (33%) gallbladder carcinomas examined, and all were located at the hot spot codon 599 of exon 15. K-ras and B-raf mutations were never in the same specimens.Conclusions B-raf gene mutations seem to be a quite common event in gallbladder carcinomas, implying that B-raf may play an important role in the pathogenesis of this tumor.     

Phylogenetic analysis of<Emphasis Type="Italic"> Theileria</Emphasis> species transmitted by<Emphasis Type="Italic"> Haemaphysalis qinghaiensis</Emphasis>

The phylogenetic relationships between six isolates of Theileria spp. infective to small ruminants, and two isolates of Theileria spp. infective to yak, all transmitted by Haemaphysalis qinghaiensis, together with the Theileria orientalis/sergenti/buffeli group and T. sinensis, were analyzed using the 18S ssrRNA gene sequence. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was used either for direct sequencing or was ligated to the PCR II vector for sequencing. The length of the 18S ssrRNA gene of all Theileria spp. involved in this study was around 1,740 bp. Two phylogenetic trees were inferred based on the 18S ssrRNA gene sequence of the Chinese isolates only, and Chinese isolates and other species of Theileria available in GenBank. In the first tree, the Theileria sp. infective to yaks was found to be T. sinensis. The Theileria sp. infective to small ruminants was found to be composed of two separate species of Theileria. Theileria sp. from Qinghai, Madang, Ningxian and Lintan, which was identical to the unidentified Theileria sp. described previously, is designated Theileria sp (China 1). The Theileria sp. from Longde, Zhangjiachuan and Lintan, which has not been described previously, is designated Theileria sp. (China 2) in order to avoid confusion. In the second tree, Theileria sp. (China 1) was closely related to benign Theileria, such as T. buffeli and T. sergenti, while Theileria sp. (China 2) was separated from other Theileria spp. The results indicate that H. qinghaiensis transmit at least three species of Theileria, two which are infective to sheep and goats, but not yak and one which is infective to yaks and cattle, but not to sheep and goats.     

Immunological comparison of 124 isolates of<Emphasis Type="Italic"> Toxoplasma gondii</Emphasis>

We tested 124 isolates of Toxoplasma gondii, as determined morphologically and by their ability to elicit antibodies in the dye test with the RH strain of Toxoplasma in mice. They were compared for their capacity to immunize CF-1 mice against isolate T-1, and T-1 immune mice for their capacity to resist each of the 123 other isolates. Of the 125 isolates, 52 had been isolated in the continental USA, 33 in Central America, 15 in Europe, 9 in Hawaii, five in Japan, two in Taiwan, five in Australia, one in Indonesia, one in Tunisia, and one was of unknown origin. Complete cross-immunity was found. This suggests that only one immunotype of Toxoplasma is prevalent in the United States, and perhaps all over the earth. Vaccines are likely to immunize against most or all Toxoplasma isolates.     

Natural infections of<Emphasis Type="Italic"> Omphiscola glabra</Emphasis> (Lymnaeidae) with<Emphasis Type="Italic"> Fasciola hepatica</Emphasis> in central France

As larval forms of Fasciola hepatica have periodically been detected in Omphiscola glabra after their collection from watercress beds or from meadows since 1995, field investigations in 37 populations of O. glabra were carried from 1996 to 2002. This was done in order to determine the changes in prevalences and intensities of these natural infections with F. hepatica in relation to the type of snail habitat and the year of snail collection. Snails infected with F. hepatica were found in all samples made in swampy meadows and roadside ditches in all years. In fenced pools and walled gardens, snail infections were only found from 1998 and 1999 onwards, respectively. In the four types of habitats, the prevalences of F. hepatica infections increased slightly over time (0.8–2.1% for snails sampled in swampy meadows, for example) but this increase varied with the habitat. The mean shell heights of infected snails (6.2–7.8 mm) were similar whatever the type of habitat. The numbers of cercariae-containing rediae counted in snails sampled in swampy meadows, roadside ditches, and fenced pools significantly increased over time. Significant numerical variation between these redial burdens was also observed in relation to snail habitat. As the larval development of F. hepatica is facilitated by the presence of another trematode larval form (Paramphistomum daubneyi), the finding of some naturally infected O. glabra in watering places known to have no contact with domestic or wild large mammals might be explained by the development of P. daubneyi in small mammals such as lagomorphs. However, a progressive adaptation of F. hepatica miracidia to O. glabra over time, which would permit the infection of snails at sizes larger than 2 mm, could not be excluded.     

A novel polyketide biosynthesis gene cluster is involved in fruiting body morphogenesis in the filamentous fungi <Emphasis Type="Italic">Sordaria macrospora</Emphasis> and <Emphasis Type="Italic">Neurospora crassa</Emphasis>

During fungal fruiting body development, hyphae aggregate to form multicellular structures that protect and disperse the sexual spores. Analysis of microarray data revealed a gene cluster strongly upregulated during fruiting body development in the ascomycete Sordaria macrospora. Real time PCR analysis showed that the genes from the orthologous cluster in Neurospora crassa are also upregulated during development. The cluster encodes putative polyketide biosynthesis enzymes, including a reducing polyketide synthase. Analysis of knockout strains of a predicted dehydrogenase gene from the cluster showed that mutants in N. crassa and S. macrospora are delayed in fruiting body formation. In addition to the upregulated cluster, the N. crassa genome comprises another cluster containing a polyketide synthase gene, and five additional reducing polyketide synthase (rpks) genes that are not part of clusters. To study the role of these genes in sexual development, expression of the predicted rpks genes in S. macrospora (five genes) and N. crassa (six genes) was analyzed; all but one are upregulated during sexual development. Analysis of knockout strains for the N. crassa rpks genes showed that one of them is essential for fruiting body formation. These data indicate that polyketides produced by RPKSs are involved in sexual development in filamentous ascomycetes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The laccase gene family in<Emphasis Type="Italic"> Coprinopsis cinerea</Emphasis> (<Emphasis Type="Italic">Coprinus cinereus</Emphasis>)

In this study, we isolated and sequenced eight non-allelic laccase genes from Coprinopsis cinerea (Coprinus cinereus) homokaryon AmutBmut. These eight genes represent the largest laccase gene family identified so far in a single haploid fungal genome. We analyzed the phylogenetic relationships between these genes by intron positions, amino acid sequence conservation and similarities in promoter sequences. All deduced protein products have the laccase signature sequences L1–L4, the typical conserved cysteine and the ten histidine residues which are ligands in the two laccase copper-binding centers, T1 and T2/T3. Proteins Lcc2 and Lcc3 of Coprinopsis cinerea are most similar to the acidic, membrane-associated laccase CLAC2 from Coprinellus congregatus implicated in neutralization of acidic medium. All other laccases from the saprophyte Coprinopsis cinerea, including the well described enzyme Lcc1, form a cluster separate from these three enzymes and from various laccases of wood-rotting and plant-pathogenic basidiomycetes.Communicated by U. Kück     

Isolation and characterisation of the mating-type (<Emphasis Type="Italic">MAT</Emphasis>) locus from<Emphasis Type="Italic"> Rhynchosporium secalis</Emphasis>

The mating-type (MAT) genes from Rhynchosporium secalis were isolated using PCR-based methods. Characterisation of the MAT idiomorphs suggests that R. secalis is closely related to the discomycetes Pyrenopeziza brassicae and Tapesia yallundae in terms of sequence and MAT locus gene composition. The MAT1-2 idiomorph contains a single gene encoding a protein with a high-mobility group (HMG) DNA-binding domain. The MAT1-1 idiomorph contains two genes, one encoding a protein with a HMG domain and the other encoding an alpha box domain. A second, previously undescribed, intron was identified within the P. brassicae MAT1-2-1 gene. Two introns were also present in the corresponding gene in R. secalis and this showed the similarity between these genes at the discomycete MAT1-2 locus. Using PCR, we identified isolates of both mating types from barley crops in different parts of the UK and showed that the composition of the MAT idiomorphs is conserved in these isolates. These findings support the hypothesis that R. secalis is a heterothallic discomycete which has an as yet unidentified teleomorph.Communicated by J. Heitman     

Genetic analysis of coenzyme A biosynthesis in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: identification of a conditional mutation in the pantothenate kinase gene <Emphasis Type="Italic">CAB1</Emphasis>

Coenzyme A (CoA) is a ubiquitous cofactor required for numerous enzymatic carbon group transfer reactions. CoA biosynthesis requires contributions from various amino acids with pantothenate as an important intermediate which can be imported from the medium or synthesized de novo. Investigating function and expression of structural genes involved in CoA biosynthesis of the yeast Saccharomyces cerevisiae, we show that deletion of ECM31 and PAN6 results in mutants requiring pantothenate while loss of PAN5 (related to panE from E. coli) still allows prototrophic growth. A temperature-sensitive mutant defective for fatty acid synthase activity could be functionally complemented by a gene significantly similar to eukaryotic pantothenate kinases (YDR531W). Enzymatic studies and heterologous complementation of this mutation by bacterial and mammalian genes showed that YDR531W encodes a genuine pantothenate kinase (new gene designation: CAB1, “coenzyme A biosynthesis”). A G351S missense mutation within CAB1 was identified to cause the conditional phenotype of the mutant initially studied. Similar to CAB1, genes YIL083C, YKL088W, YGR277C and YDR106C responsible for late CoA biosynthesis turned out as essential. Null mutants could be complemented by their bacterial counterparts coaBC, coaD and coaE, respectively. Comparative expression analyses showed that some CoA biosynthetic genes are weakly de-repressed with ethanol as a carbon source compared with glucose.     

Large-scale association analysis of <Emphasis Type="Italic">TNF</Emphasis>/<Emphasis Type="Italic">LTA</Emphasis> gene region polymorphisms in type 2 diabetes

Background  

The TNF/LTA locus has been a long-standing T2D candidate gene. Several studies have examined association of TNF/LTA SNPs with T2D but the majority have been small-scale and produced no convincing evidence of association. The purpose of this study is to examine T2D association of tag SNPs in the TNF/LTA region capturing the majority of common variation in a large-scale sample set of UK/Irish origin.     

The efficacy of inhibitors involved in spermidine metabolism in<Emphasis Type="Italic"> Plasmodium falciparum</Emphasis>,<Emphasis Type="Italic"> Anopheles stephensi</Emphasis> and<Emphasis Type="Italic"> Trypanosoma evansi</Emphasis>

In the present study, we have tested the effect of different polyamine inhibitors of the spermidine metabolizing enzymes deoxyhypusine synthase and homospermidine synthase in different chloroquine resistant Plasmodium falciparum strains, in the mosquito Anopheles stephensi (Diptera: Culicidae) and in a Trypanosoma evansi clone I from strain STIB 806 K China. Recent experiments have shown that agmatine is a growth inhibitor of the malaria parasite P. falciparum (Kaiser et al. 2001) in vitro. A comparison of agmatine efficacy with the new antimalarials artemisinin, triclosan and conventional chloroquine showed similar or even better results on the basis of growth inhibition and the reduction of developmental forms. However, no effect of triclosan or agmatine was observed at the ribonucleic acid level. In a second set of experiments, we tested the effect of 1,7-diaminoheptane and agmatine on oocyst formation in A. stephensi after infection with Plasmodium yoelii. Agmatine had an antisporozoite effect since 1,000 M led to a 59.5% inhibition of oocysts. A much weaker inhibitor of oocyst formation was 1,7-diaminoheptane. The most effective in in vitro inhibition of T. evansi was dicyclohexylamine, an inhibitor of spermidine biosynthesis with an IC₅₀ value of 47.44 M and the deoxyhypusine inhibitor 1,7-diaminoheptane with an IC₅₀ value of 47.80 M. However, both drugs were ineffective in in vivo experiments in a Trypanosoma mouse model. Two different spermidine analogues, 1,8-diaminooctane and 1,3-diaminopropane with IC₅₀ values of 171 M and 181.37 M, respectively, were moderate inhibitors in vitro and ineffective in vivo.     

<Emphasis Type="Italic">Fasciola hepatica</Emphasis> and<Emphasis Type="Italic"> Paramphistomum daubneyi</Emphasis>: vertical distribution of metacercariae on plants under natural conditions

Four experiments on the metacercariae of Fasciola hepatica and Paramphistomum daubneyi were carried out under natural conditions in order to study their vertical location on submerged plants and to determine whether simultaneous cercarial shedding of both digenea causes changes in the distributions of the metacercariae. These experiments were performed in experimental boxes, each containing six tufts of rushes. Most metacercariae (73.0%) of F. hepatica were found along the walls of boxes, while 81.5% of P. daubneyi metacercariae were found on rush stems. In the case of snails infected by either of the two digenea, 80.1% of F. hepatica cercariae encysted on submerged parts of rushes and of the box walls near the water surface (to a depth of 1 cm), whereas 73.0% of P. daubneyi metacercariae were found in the lower sections (from –4 to –7 cm). If snails dually infected with P. daubneyi and F. hepatica were used, the vertical distributions of the metacercariae were significantly different from those found for snails infected by either of the two digenea. If snails having 42-day old infections with F. hepatica and other snails with 70-day old infections with P. daubneyi were simultaneously introduced into the boxes, the frequency of F. hepatica cysts was significantly lower in the section located under the water surface (29% only), while the frequencies of metacercariae in the lower sections (from –1 to –5 cm) were increased. Some significant changes were also observed for the metacercariae of P. daubneyi. The disturbance noted in the vertical distributions of F. hepatica and P. daubneyi metacercariae suggest that the encystment of F. hepatica cercariae can be disturbed by the simultaneous encystment of P. daubneyi cercariae, or conversely.     

Malo-ethanolic fermentation in<Emphasis Type="Italic"> Saccharomyces</Emphasis> and<Emphasis Type="Italic"> Schizosaccharomyces</Emphasis>

Yeast species are divided into the K(+) or K(–) groups, based on their ability or inability to metabolise tricarboxylic acid (TCA) cycle intermediates as sole carbon or energy source. The K(–) group of yeasts includes strains of Saccharomyces, Schizosaccharomyces pombe and Zygosaccharomyces bailii, which is capable of utilising TCA cycle intermediates only in the presence of glucose or other assimilable carbon sources. Although grouped together, these yeasts have significant differences in their abilities to degrade malic acid. Typically, strains of Saccharomyces are regarded as inefficient metabolisers of extracellular malic acid, whereas strains of Sch. pombe and Z. bailii can effectively degrade high concentrations of malic acid. The ability of a yeast strain to degrade extracellular malic acid is dependent on both the efficient transport of the dicarboxylic acid and the efficacy of the intracellular malic enzyme. The malic enzyme converts malic acid into pyruvic acid, which is further metabolised to ethanol and carbon dioxide under fermentative conditions via the so-called malo-ethanolic (ME) pathway. This review focuses on the enzymes involved in the ME pathway in Sch. pombe and Saccharomyces species, with specific emphasis on the malate transporter and the intracellular malic enzyme.Communicated by S. Hohmann     

Biochemical characterization of new strains of<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis> and<Emphasis Type="Italic"> T. rangeli</Emphasis> isolates from Peru and Mexico

Seven trypanosome stocks isolated have been characterized by lectin agglutination, isoenzyme analysis, and the end products excreted. The stocks were isolated from different geographic areas—one from Mexico (TM5), and six from Peru, four of these isolated from different species of triatoma (TP504, TP702, TP704 and TP706), the other two isolated from the salivary glands of Rhodnius ecuadorensis (TRa605 and TRa606). Additionally, one strain of Trypanosoma cruzi isolated from a human case (strain TC-Maracay) and one strain of T. rangeli (TRa, Cajamarca-Peru strain), characterized and maintained in our laboratory, were used as reference strains. According to statistical study, the stocks were grouped into three clusters: (1) cluster I included the reference strain of T. cruzi (TC-Maracay); (2) cluster II was subdivided into two groups—subcluster IIA for the Mexican isolate (TM5) and subcluster IIB for the Peruvian ones, isolated from the salivary glands of Rhodnius ecuadorensis (TRa 605 and TRa 606) and the reference strain T. rangeli (TRa); these two new isolates were classified as T. rangeli; and (3) cluster III for the rest of the Peruvian isolates, which should be considered at least as a different strain from the T. cruzi strain Maracay. We show that the identification of T. cruzi and T. rangeli in mixed infections is readily achieved by biochemical methods. These findings identified three clusters of Mexican and Peruvian stocks that correlate with geographic origin, although assignment to a T. cruzi linage was not possible.     

Synteny between <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> and <Emphasis Type="Italic">Hordeum vulgare</Emphasis> as revealed by FISH

We investigated by fluorescence in situ hybridization (FISH) the synteny between Brachypodium distachyon with a small genome (1C = 320 Mb) and barley with a large genome (1C = 5,100 Mb) at the chromosome level. Reciprocal genomic in situ hybridization (GISH) between B. distachyon and barley labeled mainly 45S ribosomal DNA loci, indicating that most high copy DNA is weakly conserved between both grasses. Of 13 BAC clones with inserts from different B. distachyon chromosomes, only two belonging to chromosome 1 yielded hybridization signals on a barley metaphase chromosome (on 7HS and 7HL, respectively), confirming synteny between both chromosomes. FISH experiments to characterize the synteny of single-copy loci were performed. Two of four Brachypodium sylvaticum BACs spanning a 223-kb interval homologous to the region of barley that harbors a gibberellic-acid-insensitive semi-dwarfing gene, sdw3, hybridized specifically to a central position of B. distachyon chromosome 1 short arm but not to the homologous region of the barley genome. Repeat-free sequences PCR amplified from four non-overlapping barley BACs linked to the core of Sdw3 region yielded signals at distinct positions in the middle of barley chromosome arm 2HS. Together, these results (1) confirmed the synteny between B. distachyon chromosome 1 and barley chromosomes 2H and 7H at the cytological level, (2) indicated mid-arm position for the Sdw3 locus genetically mapped at the centromere of barley chromosome 2H, and (3) proved that the sdw3 core interval of <100 kb in B. distachyon corresponds to a megabase-sized syntenic region in barley.     

Efficacy of female <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> with entomopathogenic fungus <Emphasis Type="Italic">Fusarium pallidoroseum</Emphasis>

This study was conducted to isolate and identify natural entomopathogenic fungi from female Culex quinquefasciatus and to test their adulticidal activity. Field-collected female C. quinquefasciatus died early and were placed on a Saboraud’s dextrose agar plates for growth and isolation of natural entomopathogenic fungi. The plates were maintained in an incubator at 24 ± 2°C for 3 days. Four fungal species were isolated in two genera namely, Aspergillus and Fusarium. The identified fungal species were A. niger, A. flavus, A. nidulans var acristatus (ITCC-6327.04), and F. pallidoroseum (ITCC-6324.06). Adult bioassays were carried out using spore-impregnated paper in WHO-holding tubes. F. pallidoroseum was found to be more effective than the others. Exposure of C. quinquefasciatus to spores of A. flavus and A. niger for 4 h caused 5.53% and 5.51% mortality in the mosquitoes within a week, respectively. All the female C. quinquefasciatus were killed within 4 days of exposure to F. pallidoroseum at a concentration of 1.11 × 10¹⁰ conidia per m². Significant difference of longevity was observed between the F. pallidoroseum-treated C. quinquefasciatus and control mosquitoes. The LT₅₀ of F. pallidoroseum was 2.08 days for 4 h exposure to C. quinquefasciatus. Results of the present study confirm that F. pallidoroseum is one of the alternative biological control agents of adult mosquitoes.     

NADH-reductive stress in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> induces the expression of the minor isoform of glyceraldehyde-3-phosphate dehydrogenase (<Emphasis Type="Italic">TDH1</Emphasis>)

A strain of Saccharomyces cerevisiae lacking the GPD2 gene, encoding one of the glycerol-3-phosphate dehydrogenases, grows slowly under anaerobic conditions, due to reductive stress caused by the accumulation of cytoplasmic NADH. We used 2D-PAGE to study the effect on global protein expression of reductive stress in the anaerobically grown gpd2 strain. The most striking response was a strongly elevated expression of Tdh1p, the minor isoform of glyceraldehyde-3-phosphate dehydrogenase. This increased expression could be reversed by the addition of acetoin, a NADH-specific redox sink, which furthermore largely restored anaerobic growth of the gpd2 strain. Additional deletion of the TDH1 gene (but not of TDH2 or TDH3) improved anaerobic growth of the gpd2 strain. We therefore propose that TDH1 has properties not displayed by the other TDH isogenes and that its expression is regulated by reductive stress caused by an excess of cytoplasmic NADH.H. Valadi and Å. Valadi contributed equally to this work     

Genetic features of DNA of <Emphasis Type="Italic">Borrelia miyamotoi</Emphasis> transmitted by <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>

16S rRNA, p66 and glpQ gene fragments in Borrelia miyamotoi transmitted by Ixodes persulcatus are determined and analyzed. Specific nucleotide sequences of every locus have been shown to be identical. The highest homology level for nucleotide sequences of three loci has been shown for the sequences of strains isolated earlier in Japan. An analysis of the P66 protein amino-acid sequence has shown that the locus corresponding to the surface-exposed domain differs considerably from both Borrelia hermsii, a typical member of tick-borne relapsing fever and Borrelia lonestari, the closest related species. Three genetic variants of Borrelia miyamotoi P66 protein is characterized not only by amino-acid substitutions, but also by deletions.     

Subcellular localization of an extracellular serine protease in<Emphasis Type="Italic"> Leishmania</Emphasis> (<Emphasis Type="Italic">Leishmania</Emphasis>)<Emphasis Type="Italic"> amazonensis</Emphasis>

Extracellular proteolytic activity was detected in a Leishmania (L.) amazonensis culture supernatant and a 56-kDa protein was purified using (NH₄)₂SO₄ precipitation followed by affinity chromatography on aprotinin–agarose. A rabbit serum obtained against the 56-kDa extracellular serine protease was used in order to analyze its location in L. (L.) amazonensis parasites. Immunocytochemistry studies revealed that the enzyme is mainly found in the flagellar pocket and cytoplasmic vesicles of promastigote forms, whereas in amastigotes, it is located in electron-dense structures resembling megasomes. These results indicate that the 56-kDa serine protease is released into the extracellular environment through the flagellar pocket; and its intracellular location suggests either a correlated enzymatic activity or intracellular trafficking.