Introduction of large DNA inserts into the barley pathogenic fungus, <Emphasis Type="Italic">Ustilago hordei</Emphasis>, via recombined binary BAC vectors and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Genetic transformation of organisms with large genome fragments containing complete genes, with regulatory elements or clusters of genes, can contribute to the functional analysis of such genes. However, large inserts, such as those found on bacterial artificial chromosome (BAC) clones, are often not easy to transfer. We exploited an existing technique to convert BAC clones, containing genomic DNA fragments from the barley-covered smut fungus Ustilago hordei to binary BACs (BIBACs) to make them transferable by the Agrobacterium tumefaciens T-DNA transfer machinery. Genetic transformation of U. hordei with BAC clones using polyethylene glycol or electroporation is difficult. As a proof of concept, two BAC clones were successfully converted into BIBAC vectors and transferred by A. tumefaciens into U. hordei and U. maydis, the related corn smut fungi. Molecular analysis of the transformants showed that the T-DNA containing the BAC clones with their inserts was stably integrated into the U. hordei genome. A transformation frequency of approximately 10⁻⁴ was achieved both for U. hordei sporidia and protoplasts; the efficiencies were 25–30 times higher for U. maydis. The combination of in vivo recombineering technology for BAC clones and A. tumefaciens-mediated transformation of Ustilago species should pave the way for functional genomics studies.     

Deletion of the <Emphasis Type="Italic">cpku80</Emphasis> gene in the chestnut blight fungus, <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis>, enhances gene disruption efficiency

The chestnut blight fungus, Cryphonectria parasitica, and associated virulence-attenuating hypoviruses have emerged as an important model system for studying molecular mechanisms underlying fungal–plant pathogenic interactions. As more gene sequence information becomes available as a result of C. parasitica express sequence tags (ESTs) and ongoing whole genome sequencing projects, the development of an efficient gene disruption system has become an urgent need for functional genomics studies of this important forestry pathogen. Here, we report the cloning of the C. parasitica gene cpku80 that encodes a key component of the nonhomologous end joining DNA repair pathway and the construction of a corresponding deletion mutant strain. The cpku80 mutant was indistinguishable from the parental wild-type strain EP155 in colony morphology, ability to support hypovirus replication, conidiation and virulence. As predicted, the Δcpku80 strain did exhibit an increased sensitivity to the mutagen methyl methanesulfonate. A test with three selected genes resulted in a gene disruption efficiency of about 80% for the Δcpku80 strain, a significant increase over the 2–5% levels of homologous recombination generally observed for the wild-type strain EP155. This efficient homologous recombination system provides a powerful tool for large-scale analysis of gene functions in C. parasitica. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The authors Xiuwan Lan and Ziting Yao contributed equally to this work.     

Association of positional and functional candidate genes<Emphasis Type="Italic"> FGF1</Emphasis>,<Emphasis Type="Italic"> FBN2</Emphasis>, and<Emphasis Type="Italic"> LOX</Emphasis> on 5q31 with intracranial aneurysm

We previously performed a genome-wide linkage study of intracranial aneurysm (IA) and found positive evidence of linkage at chromosomes 5q22–31, 7q11, and 14q22. In the present study, we focus on 5q31, where three candidate genes, fibroblast growth factor 1 (FGF1), fibrillin 2 (FBN2), and lysyl oxidase gene (LOX) lie, and evaluate associations with IA. Genomic DNAs were obtained from 172 IA patients and 192 controls. Association analysis was performed with ten, five, and four single-nucleotide polymorphisms (SNPs) identified in FGF1, FBN2, and LOX, respectively. A difference in allelic frequency was observed for only the SNP at intron 4 in FGF1 (χ²=4.44, df=1, P=0.035). Although a haplotype association was observed with the combination of ten SNPs in FGF1 (χ²=16.04, df=1, P=0.00006), significant haplotype associations were not observed when haplotypes were constructed with the three, two, and four SNPs in FGF1 according to the linkage disequilibrium structure. No associations of FBN2 and LOX with IA were detected in the present study.     

An autophagy gene, <Emphasis Type="Italic">MgATG5</Emphasis>, is required for cell differentiation and pathogenesis in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Autophagy is a conserved degradation pathway that is involved in the maintenance of normal cell differentiation and development. The Saccharomyces cerevisiae ATG5 gene is an important component of the autophagy process. In this study, we identified MgATG5 as an autophagy-related gene in Magnaporthe oryzae that is homologous to ATG5. Using targeted gene replacement, an Mgatg5∆ mutant was generated and fungal autophagy was blocked. Cytological analysis revealed that the mutant had poor fungal morphogenic development, including a shortened aerial hyphae lifespan, decreased conidiation and perithecia formation, delayed conidial germination and appressorial formation, postponement of conidial cytoplasm transfer during appressorium formation, and reduction in formation of the penetration peg. Turnover of endogenous matter in the Mgatg5∆ mutant was also affected, as demonstrated by defects in the formation of conidial lipid droplets, and in the degradation of conidial glycogen deposits during appressorium formation. Lipid droplets and glycogen are necessary to generate adequate turgor in appressoria for invading the host surface. As a result of the decreased appressorium turgor and differentiation in the penetration peg, Mgatg5∆ pathogenicity was deficient in two host plants tested. The developmental and pathogenic phenotypes were restored by the introduction of an intact copy of MgATG5 into Mgatg5∆, demonstrating that the MgATG5 deletion was responsible for the cellular defects. Taken together, these findings suggest that autophagy promotes cell differentiation through turnover of endogenous matter during fungal development, and is thus essential for the pathogenicity of the rice blast fungus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J.-P. Lu and X.-H. Liu contributed equally to this work and are regarded joint first authors.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the antitumor clavaric acid-producing basidiomycete <Emphasis Type="Italic">Hypholoma sublateritium</Emphasis>

The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (Pgpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.     

Characterization of <Emphasis Type="Italic">Taenia solium</Emphasis> cysticerci microsomal glutathione <Emphasis Type="Italic">S</Emphasis>-transferase activity

Glutathione S-transferase activity has been shown to be associated with the microsomal fraction of Taenia solium. Electron microscopy and subcellular enzyme markers indicate the purity of the microsomal fraction that contains the glutathione S-transferase activity. T. solium microsomes were solubilized under conditions used to solubilize integral microsomal proteins. This procedure proved necessary to obtain enzymatic activity. To characterize this parasite enzyme activity, several substrates and inhibitors were used. The optimum activity for microsomal glutathione S-transferase was found to be pH 6.6, with a specific enzyme activity of 0.9, 0.1, 0.067, 0.03, and 0.05 μmol min⁻¹ mg⁻¹ with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 2,4-hexadienal, and trans-2-nonenal, respectively. No activity of glutathione peroxidase was observed. T. solium microsomes had an _app K _{m (GSH)} = 0.161 μM, _app K _{m (CDNB)} = 14.5 μM, and _app V _max of 0.15 and 27.9 μmol min⁻¹ mg⁻¹ for GSH and CDNB, respectively. T. solium microsomes were inhibited by several glutathione S-transferase enzyme inhibitors, and it was possible to establish a simple inhibition system as well as corresponding K _i ’s for each inhibitor. These results indicate that the T. solium microsomal glutathione S-transferase is different from the parasite cytoplasmic enzymes that catalyze similar reactions.     

In vitro and in vivo antiparasitic activity of Azadirachtin against <Emphasis Type="Italic">Argulus</Emphasis> spp. in <Emphasis Type="Italic">Carassius auratus</Emphasis> (Linn. 1758)

Argulus is one of the most common and predominant ectoparasites which cause serious parasitic disease and is a potent carrier of viruses and bacteria in the ornamental fish industry. In recent years, organic (herbs)-based medicines are widely used to cure the disease, and neem (Sarbaroganibarini) medicine is very popular and effective throughout the world. The present study was conducted to find the effects of Azadirachtin against Argulus spp. on Carassius auratus under in vitro and in vivo conditions. The 96-h median lethal concentration (LC₅₀) for Azadirachtin EC 25% against Carassius auratus was found to be 82.115 mg L⁻¹. The antiparasitic activity test under in vitro and in vivo was evaluated at 1 (T1), 5 (T2), 10 (T3), 15 (T4) and 20 mg L⁻¹ (T5) to treat Argulus for 3 h and 72 h, respectively. In vitro effect of Azadirachtin solution led to 100% mortality of Argulus at 20 and 15 mg L⁻¹ for 2.5 and 3 h, respectively. Whereas, under in vivo test, the 100% antiparasitic efficacy of Azadirachtin solution was found at 15 and 20 mg L⁻¹ for 72 and 48 h, respectively. The EC₅₀ for 48 h was 20 mg L⁻¹, and thus, therapeutic index is 4.10. The results provided evidence that Azadirachtin can be used as a potential agent for controlling Argulus.     

Gene deletion and allelic replacement in the filamentous fungus <Emphasis Type="Italic">Podospora anserina</Emphasis>

Gene replacement via homologous recombination is a fundamental tool for the analysis of gene function. However, this event is rare in organisms like the filamentous fungus Podospora anserina. We show here that deletion of the PaKu70 gene is an efficient strategy for improving gene manipulation in this organism. By using the ΔPaKu70 strain, it is now possible (1) to produce deletion mutants with an efficiency of 100%, (2) to achieve allelic exchange by introducing a mutated allele associated with a selection cassette at the locus, (3) to introduce a mutation in a gene without co-insertion of a selectable marker and without any modification of the target locus.     

Biological control of <Emphasis Type="Italic">Fasciola hepatica</Emphasis> eggs with the <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> fungus after passing through the cattle gastrointestinal tract

Fasciolosis is a disease caused by Fasciola hepatica responsible for causing significant losses in livestock. This study aimed to evaluate the Pochonia chlamydosporia fungus (isolate VC1) on F. hepatica eggs after passing through the cattle gastrointestinal tract. For this evaluation, 1 g pellet was given in sodium alginate matrix per kilogram live weight containing 25% of fungal mycelium from isolate VC1 per animal. Twelve animals were used, six treated and six untreated (control). Some stool samples were collected from the groups of treated and control animals, at the times of 12, 18, 24, 48, 72, and 96 h after the pellets' administration. Then, from each stool sample of treated and control groups, 2 g was placed in a Petri dish of 9 cm in diameter, containing 2% water–agar and 1,000 eggs of F. hepatica. It was observed that the fungus was effective in preying upon the eggs in the samples recovered at all of the schedules starting at 12 h. Furthermore, differences were observed (p < 0.01) in the destruction of eggs in the Petri dishes in the treated group compared with the control group. The ovicidal effect was observed after 7 days of interaction. The ovicidal P. chlamydosporia fungus was effective in destroying F. hepatica eggs; therefore, it is suggested that this fungus could be employed as agent for the control of helminth eggs.     

<Emphasis Type="Italic">In vitro</Emphasis> effects of thyroliberin on structural state of plasma membranes in mouse brain and liver

The spin probe method was employed to study in vitro the effect of regulatory peptide thyroliberin on structural state of surface (0.8 nm) and deep (2 nm) lipid layers of the plasma membranes in mouse liver and brain. Thyroliberin in a concentration range of 10⁻³–10⁻¹⁸ M enhanced structural order of surface lipids, the maximum effect was observed at 10⁻⁹–10⁻¹⁰ M. The dose-effect dependencies for microviscosity of deep lipids were nonlinear and had 3 extrema at 10⁻⁴–10⁻⁷ M, 10⁻⁹ M, and 10⁻¹⁴–10⁻¹⁶ M. The greatest changes in lipid microviscosity produced by 10⁻⁹ M thyroliberin are explained by lipid-receptor interaction. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 8, pp. 151–154, August, 2007     

Occurrence of cystacanths of <Emphasis Type="Italic">Plagiorhynchus cylindraceus</Emphasis> (Acanthocephala) in the terrestrial isopods <Emphasis Type="Italic">Trachelipus squamuliger</Emphasis> and <Emphasis Type="Italic">Armadillidium vulgare</Emphasis> (Oniscidea) in Bulgaria

In total, 2097 individuals of Trachelipus squamuliger and 20 individuals of Armadillidium vulgare from four habitats (three woodland sites and one pasture) in the region of Stara Zagora, Bulgaria, were examined for the presence of cystacanths of Plagiorhynchus cylindraceus, a common acanthocephalan parasite of passerine birds. In T. squamuliger from woodland habitats, cystacanths were found with prevalence 4.0–9.3%, intensity 1–5 (mean 1.22–1.57) and mean abundance 0.057–0.113. No significant differences were observed between infections in males and females of T. squamuliger. None of the T. squamuliger individuals from the pasture examined was infected. Out of 48 infected females of T. squamuliger, only one had developed eggs (in agreement with previous studies revealing the negative effect of the cystacanths on the development of female gonads of woodlice). One individual of A. vulgare was infected with a single cystacanth. The occurrence of P. cylindraceus in T. squamuliger is a new host record.     

TNF controls the infiltration of dendritic cells into the site of <Emphasis Type="Italic">Leishmania major</Emphasis> infection

TNF-negative C57BL/6 (B6.TNF^−/−) mice are highly susceptible to Leishmania (L.) major infection and succumb rapidly to fatal leishmaniasis. A T helper type 1 (Th1) cell-mediated immune response is central for protective anti-leishmanial immunity. Therefore, the observed susceptibility of B6.TNF^−/− mice to L. major parasites could be caused by a deficiency in mounting a Th1 response. Analysis of infected footpads revealed, that B6.TNF^−/− mice exhibited a substantially diminished formation of DCs at the site of infection. Furthermore, Th1 cytokines such as IFN-γ were reduced in footpads of infected B6.TNF^−/− mice. Cutaneous reconstitution of B6.TNF^−/− mice with either bone marrow derived DCs (BM-DCs) or recombinant TNF simultaneous to infection resulted in an increased expression of cytokines such as IFN-γ and in an enhanced presence of Leishmania-antigen in skin draining lymph nodes. In addition, the individual time of survival was doubled. In conclusion we demonstrate that the expression of dermal TNF is necessary to provide an environment that initiates a local inflammatory response, but is not sufficient to induce protective immunity.     

Effect of <Emphasis Type="Italic">n</Emphasis>-tyrosol on blood viscosity and platelet aggregation

Experiments on rats showed that n-tyrosol limited the increase in blood viscosity during thermal exposure at a shear rate of 5–300 sec⁻¹ and inhibited ADP-induced platelet aggregation. The effects of n-tyrosol are comparable to that of pentoxyphylline. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 1, pp. 67–69, January, 2007     

Energetics of karate (<Emphasis Type="Italic">kata</Emphasis> and <Emphasis Type="Italic">kumite</Emphasis> techniques) in top-level athletes

Breath-by-breath O₂ uptake ( [(V)\dot]_\textO2 \dot{V}_{{{\text{O}}_{2} }} , L min⁻¹) and blood lactate concentration were measured before, during exercise, and recovery in six kata and six kumite karate Word Champions performing a simulated competition. [(V)\dot]_{\textO 2 \textmax} , \dot{V}_{{{\text{O}}_{{ 2 {\text{max}}}} }} , maximal anaerobic alactic, and lactic power were also assessed. The total energy cost ( V_{\textO 2 \textTOT} , V_{{{\text{O}}_{{ 2 {\text{TOT}}}} }} , mL kg⁻¹ above resting) of each simulated competition was calculated and subdivided into aerobic, lactic, and alactic fractions. Results showed that (a) no differences between kata and kumite groups in [(V)\dot]_{\textO 2 \textmax} , \dot{V}_{{{\text{O}}_{{ 2 {\text{max}}}} }} , height of vertical jump, and Wingate test were found; (b) V_{\textO 2 \textTOT} V_{{{\text{O}}_{{ 2 {\text{TOT}}}} }} were 87.8 ± 6.6 and 82.3 ± 12.3 mL kg⁻¹ in kata male and female with a performance time of 138 ± 4 and 158 ± 14 s, respectively; 189.0 ± 14.6 mL kg⁻¹ in kumite male and 155.8 ± 38.4 mL kg⁻¹ in kumite female with a predetermined performance time of 240 ± 0 and 180 ± 0 s, respectively; (c) the metabolic power was significantly higher in kumite than in kata athletes (p ≤ 0.05 in both gender); (d) aerobic and anaerobic alactic sources, in percentage of the total, were significantly different between gender and disciplines (p < 0.05), while the lactic source was similar; (e) HR ranged between 174 and 187 b min⁻¹ during simulated competition. In conclusion, kumite appears to require a much higher metabolic power than kata, being the energy source with the aerobic contribution predominant.     

The combination of <Emphasis Type="Italic">ApoCIII</Emphasis>, <Emphasis Type="Italic">hepatic lipase</Emphasis> and <Emphasis Type="Italic">hormono sensitive lipase</Emphasis> gene polymorphisms suggests an association with susceptibility to gestational hypertension

Dyslipidemia and insulin resistance contribute to the endothelial cell dysfunction in hypertensive disorders of pregnancy (HDP) and increase the long-term risk of cardiovascular disease (CVD). The genes linking susceptibility to gestational hypertension (GH) and/or preeclampsia (PE) to the long-term risk of CVD are still unknown. We evaluated the potential association between 14 polymorphisms from six genes involved in lipid metabolism and insulin action and the risk of HDP: namely the lipoprotein lipase (LPL), hepatic lipase (LIPC), hormone sensitive lipase (LIPE), cholesteryl ester transfer protein (CETP), ApoCIII and ApoE gene polymorphisms. Overall, 169 women with HDP [proteinuria (PE) and gestational hypertension without proteinuria (GH)] and 169 controls matched for age and year of delivery were genotyped. Homozygosity of the −514T allele of the −514C > T polymorphism (LIPC gene) decreased the risk of GH (OR = 0.17, CI₉₅: 0.02–0.76), while there were more −60G carriers of the −60C > G LIPE gene polymorphism (OR = 3.51, CI₉₅:1.02–12.10) among GH cases, but not in PE cases. The common ApoCIII two-locus −482CC/3238CC genotype was lower in women with GH compared with controls (OR = 0.53, CI₉₅: 0.3–0.9). The combined frequency of at-risk genotypes was higher in cases of GH compared with controls [one at-risk genotype: OR = 3.38 (95% CI: 0.48–41.8); two or more at-risk genotypes: OR = 7.14 (95% CI: 1.21–92.3, P = 0.01)], suggesting a gene-dose effect. We conclude that the combined effect of LIPC, LIPE and ApoCIII gene polymorphisms may increase the likelihood of GH, but seemingly not of PE.     

<Emphasis Type="Italic">Sarcocystis</Emphasis> sp. from the herring gull (<Emphasis Type="Italic">Larus argentatus</Emphasis>) identity to <Emphasis Type="Italic">Sarcocystis wobeseri</Emphasis> based on cyst morphology and DNA results

Having studied 11 herring gulls (Larus argentatus) Sarcocystis cysts were found in neck and leg muscles of 4 birds. One type of sarcocysts (cyst type I) that have a thin (∼1.0 μm), smooth, or slightly wavy cyst wall without clearly visible protrusions and small (6.0–8.0 μm) lancet- or banana-shaped cystozoites was identified by the light microscopy. Sarcocysts extracted from one herring gull were used for electron microscopy and DNA analysis. Ultrastructurally, Sarcocystis sp. from the herring gull had the same tissue cyst wall type-1 as S. calchasi, S. columbae, and S. wobeseri parasitizing in birds. According to first internal transcribed spacer (ITS-1) region, 18S rRNA and 28S rRNA gene sequences, Sarcocystis sp. from the herring gull belongs to S. wobeseri. Nevertheless, without evidences of cross-transmission experiment sarcocysts extracted from herring gull at present time are named as S. wobeseri-like.     

Antileishmanial activity of crude extract and coumarin from <Emphasis Type="Italic">Calophyllum brasiliense</Emphasis> leaves against <Emphasis Type="Italic">Leishmania amazonensis</Emphasis>

Infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which show renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro leishmanicidal activity of the (−) mammea A/BB. The compound (−) mammea A/BB is a coumarin-type mammea purified from a dichloromethane crude extract of leaves of Calophyllum brasiliense Cambess (Clusiaceae). The isolated compound was characterized using spectral analyses by UV, infrared, nuclear magnetic resonance of ¹H, ¹³C, distortionless enhancement by polarization transfer, correlation spectroscopy, heteronuclear multiple bond correlation, and heteronuclear multiple quantum coherence. The compound (−) mammea A/BB showed significant activity against promastigote and amastigote forms of L. amazonensis, with IC₅₀ (50% inhibition concentration of cell growth) at a concentration of 3.0 and 0.88 μg/ml and IC₉₀ (90% inhibition concentration of cell growth) of 5.0 and 2.3 μg/ml, respectively. The coumarin (−) mammea A/BB showed no cytotoxicity against J774G8 macrophages in culture, when it was tested at high concentrations that inhibited promastigote forms. Electron microscopy studies revealed considerable ultrastructural changes when promastigote forms of L. amazonensis were treated with 3.0 μg/ml of the coumarin (−) mammea A/BB for 72 h. We observed significant changes such as mitochondrial swelling with concentric membranes in the mitochondrial matrix and intense exocytic activity in the region of the flagellar pocket. Other alterations included the appearance of binucleate cells and multiple cytoplasmic vacuolization. These results showed that (−) mammea A/BB is a potent growth inhibitor of L. amazonensis and caused important changes in the parasite’s ultrastructure. This study provided new perspectives on the development of novel drugs with leishmanicidal activity obtained from natural products.