Expression and purification of the transcription factor StMsn2 from Setosphaeria turcica in Escherichia coli

Background: In Saccharomyces cerevisiae, Msn2, which acts as a key transcription factor downstream the MAPKHOG cascade pathway, also regulates the expression of genes related to stress responses. However, little is known about the regulation mechanisms of the transcription factor in Setosphaeria turcica. Results: In this study, a zinc finger DNA-binding protein, designated as StMSN2, was cloned from S. turcica. Sequencing results showed that StMSN2 had a 1752 bp open reading frame (ORF), which was interrupted by an intron (135 bp) and encoded a putative 538-amino acid protein. Phylogenetic analysis further revealed that StMsn2 was more closely related to Msn2 of Aspergillus parasiticus. StMSN2 was cloned into the pET-28a vector with His (Histidine) tags and induced with 1 mM IPTG (isopropyl-ß-D-thiogalactoside) at 37°C. The recombinant His-tagged StMsn2 was purified, and a band of size approximately 58.8 kDa was obtained. The high specificity of the polyclonal antibody Msn2-2 was detected with the StMsn2 protein from S. turcica and prokaryotic expression system, respectively. Conclusions: A new gene, named StMSN2, with 1617 bp ORF was cloned from S. turcica and characterized using bioinformatics methods. StMsn2 was expressed and purified in a prokaryotic system. A polyclonal antibody, named Msn2-2, against StMsn2 with high specificity was identified.

Cyclohelminthols Y1-Y4 Metabolites Possessing Two Spirocyclopropanes in Their Structure.

Cyclohelminthols Y1-Y4 (1-4) were isolated from the culture broth of Helminthosporium velutinum yone96. These compounds are diastereomers to each other featuring 3-azabicyclo[3.1.0]hexane-6-spirocyclopentane linked with a cyclopentanespirocyclopropane framework. Their planar structures were established via the comparison of their spectra with the simpler analogue cyclohelminthol X as well as analysis of their HMBC spectra. Although the proton-deficient core frameworks of 1-4 prevented us from obtaining configurational information via conventional NMR analysis, their total structures involving the relative and absolute configurations were established using density functional theory (DFT)-based molecular modeling calculations. The present study demonstrates the effectiveness of the comparison between the theoretical and experimental δ13C values for stereochemical analysis by focusing on the carbons that show relatively large δ13C deviations among the isomers. The G-ring of these molecules most likely originates from the cyclopropanation of the C6C7 double bond with the carbene equivalent 6 derived from cyclohelminthol IV (7), which was isolated from the same producer fungus. Preliminary biological experiments revealed the potent cytotoxicity of the (6 S)-isomers against COLO201 cells, whereas the (6 R)-isomers exhibited weak activity. The antifungal assay with Cochiobolus miyabeanus showed a slightly different profile.

Cyclohelminthol X, a Hexa-Substituted Spirocyclopropane from Helminthosporium velutinum yone96: Structural Elucidation, Electronic Circular Dichroism Analysis, and Biological Properties.

Helminthosporium velutinum yone96 produces cyclohelminthol X (1), a unique hexa-substituted spirocyclopropane. Although its molecular formula and NMR spectral data resemble those of AD0157, being isolated from marine fungus Paraconiothyrium sp. HL-78-gCHSP3-B005, our detailed analyses disclosed a totally different structure. Chemical shift calculations and electronic circular dichroism spectral calculations were quite helpful to establish the structure, when those were performed based on density functional theory. The carbon framework of cyclohelminthols I-IV is found at the C1-C8 propenylcyclopentene substructure of 1. Thus, 1 is assumed to be biosynthesized by cyclopropanation between an oxidized form of cyclohelminthol IV and a succinic anhydride derivative 4. Cytotoxicity for two cancer cell lines and proteasome inhibition efficiency are measured.

Two novel antimicrobial defensins from rice identified by gene coexpression network analyses.

Defensins form an antimicrobial peptides (AMP) family, and have been widely studied in various plants because of their considerable inhibitory functions. However, their roles in rice (Oryza sativa L.) have not been characterized, even though rice is one of the most important staple crops that is susceptible to damaging infections. Additionally, a previous study identified 598 rice genes encoding cysteine-rich peptides, suggesting there are several uncharacterized AMPs in rice. We performed in silico gene expression and coexpression network analyses of all genes encoding defensin and defensin-like peptides, and determined that OsDEF7 and OsDEF8 are coexpressed with pathogen-responsive genes. Recombinant OsDEF7 and OsDEF8 could form homodimers. They inhibited the growth of the bacteria Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Erwinia carotovora subsp. atroseptica with minimum inhibitory concentration (MIC) ranging from 0.6 to 63µg/mL. However, these OsDEFs are weakly active against the phytopathogenic fungi Helminthosporium oryzae and Fusarium oxysporum f.sp. cubense. This study describes a useful method for identifying potential plant AMPs with biological activities.

[Cross-reactivity in allergic fungal sinusitis. Case report]. / Reactividad cruzada en sinusitis alérgica fúngica. Reporte de caso.

BACKGROUND: The presence of allergic mucin in allergic fungal sinusitis (AFS) is a manifestation that identifies it as a hypersensitivity process. AFS has a phenomenon of cross-reactivity to IgE-bound proteins having at least two shared epitopes. CLINICAL REPORT: A 13-year-old male with nasal obstructive symptoms of three years of evolution. An obstructive mass was identified in the sinuses through physical examination and CT. In endoscopic surgery, the left nostril polyp was identified with the macroscopic appearance of allergic mucin; the polyp was resected. Final histopathological examination using periodic acid-Schiff and Grocott's methenamine silver staining indicated Aspergillus. Two weeks after surgery, percutaneous tests showed sensitization to Alternaria, Helminthosporium sativum, and Deramatophagoides farianae with negativity to Aspergillus fumigatus. CONCLUSIONS: The absence of significant titers of specific IgE antibodies to Aspergillus fumigatus was the evidence that the hypersensitivity response was triggered by a pathogen other than that isolated in histopathological study, which coupled with positive tests for other fungi may be explained by the cross-reactivity phenomenon in a phenomenon of likely hypersensitivity.

Antecedentes: La presencia de mucina alérgica en la rinosinusitis alérgica fúngica (RAF) es una manifestación que la identifica como un proceso de hipersensibilidad. En la RAF existe un fenómeno de reactividad cruzada entre proteínas unidas a IgE que tienen al menos dos epítopes compartidos. Caso clínico: Varón de 13 años de edad con síntomas obstructivos nasales de tres años de evolución. Por exploración física y tomografía se identificó masa obstructiva en los senos paranasales. En la cirugía endoscópica, en la fosa nasal izquierda se identificó pólipo con aspecto macroscópico de mucina alérgica; el pólipo fue resecado. El examen histopatológico final mediante tinciones con ácido peryódico de Schiff y metenamina plata de Grocott indicó Aspergillus. Dos semanas después de la cirugía, las pruebas percutáneas mostraron sensibilización a Alternaria alternata, Helminthosporium sativum y Deramatophagoides farianae, con negatividad a Aspergillus fumigatus. Conclusiones: La ausencia de títulos significativos de anticuerpos IgE específicos para Aspergillus fumigatus constituyó la evidencia de que la respuesta de hipersensibilidad fue desencadenada por un patógeno distinto del aislado en el estudio histopatológico, que aunada a las pruebas positivas para otros hongos puede explicarse por el fenómeno de reactividad cruzada en un probable fenómeno de hipersensibilidad.

Purification and characterization of an antifungal peptide with potent antifungal activity but devoid of antiproliferative and HIV reverse transcriptase activities from Legumi secchi beans.

A monomeric 9.4-kDa peptide with antifungal activity was isolated from seeds of Phaseolus vulgaris cv Legumi secchi by using a protocol that involved affinity chromatography on Blue-Sepharose, ion exchange chromatography on Q-Sepharose, and gel filtration on Superdex 75. It was adsorbed on Blue-Sepharose and unadsorbed on Q-Sepharose. Its N-terminal sequence resembled those of other leguminous defensins. It impeded mycelial growth in the fungi Helminthosporium maydis, Rhizoctonia solani, Mycosphaerella arachidicola, and Fusarium oxysporum with an IC(50) value of 9.5, 3.5, 1, and 9.2 µM, respectively, but there was no effect on Valsa mali. SYTOX Green uptake by R. solani indicated that the antifungal peptide induced fungal membrane permeabilization. In contrast to the majority of previously reported defensins/defensin-like peptides, Legumi secchi antifungal peptide did not reduce the viability of MCF-7 breast cancer cells and HepG2 hepatoma cells or inhibit HIV-1 reverse transcriptase, indicating a dissociation between antifungal, antiproliferative and HIV-1 reverse transcriptase inhibitory activities.

Acaconin, a chitinase-like antifungal protein with cytotoxic and anti-HIV-1 reverse transcriptase activities from Acacia confusa seeds.

From the seeds of Acacia confusa, a chitinase-like antifungal protein designated as acaconin that demonstrated antifungal activity toward Rhizoctonia solani with an IC50 of 30±4 µM was isolated. Acaconin demonstrated an N-terminal sequence with pronounced similarity to chitinases and a molecular mass of 32 kDa. It was isolated by chromatography on Q-Sepharose, SP-Sepharose and Superdex 75 and was not bound by either ion exchanger. Acaconin was devoid of chitinase activity. The antifungal activity against Rhizoctonia solani was completely preserved from pH 4 to 10 and from 0°C to 70°C. Congo Red staining at the tips of R. solani hyphae indicated inhibition of fungal growth. However, there was no antifungal activity toward Mycosphaerella arachidicola, Fusarium oxysporum, Helminthosporium maydis, and Valsa mali. Acaconin inhibited proliferation of breast cancer MCF-7 cells with an IC50 of 128±9 µM but did not affect hepatoma HepG2 cells. Its IC50 value toward HIV-1 reverse transcriptase was 10±2.3 µM. The unique features of acaconin include relatively high stability when exposed to changes in ambient pH and temperature, specific antifungal and antitumor actions, potent HIV-reverse transcriptase inhibitory activity, and lack of binding by strongly cationic and anionic exchangers.

[The purification and characterization of a novel lipid transfer protein from caryopsis of barnyard grass (Echinochloa crusgalli)].

A novel lipid transfer protein called Ec-LTP was isolated from resting caryopsis of weed barnyard grass Echinochloa crusgalli (L.) Beauv.; its molecular weight, amino acid content and N-terminal amino acid sequence were determined. Ec-LTP was a 9150 Da protein, containing eight cysteine residues, which formed four disulfide bonds. The isolated protein could significantly inhibit the development of pathogenic fungi Phytophthora infestans and Helminthosporium sativum, causing the late blight of potato and tomato and the root rot of herbs, respectively.

Anti-neuroblastoma activity of Helminthosporium carbonum (HC)-toxin is superior to that of other differentiating compounds in vitro.

Treatment of high-risk neuroblastoma (NB) is difficult. Novel therapeutics improving survival rates are urgently required. We have previously shown that the histone deacetylase inhibitor (HDACI) Helminthosporium carbonum (HC)-toxin induces differentiation of neuroblastoma (NB) cells. Here, we show that HC-toxin inhibits the growth of both established NB cell lines and primary cultures with and without amplified MYCN stronger than retinoids (RAs) and other HDACIs (MS-275, n-butyric acid, suberoylanilide hydroxamic acid, trichostatin A, valproic acid). Nanomolar dosages suppress E2F-1, N-myc, Skp2, Mad2 and survivin proteins, found at high levels in high-risk NBs, more efficiently than both RAs and other HDACIs. The level of hypophosphorylated active retinoblastoma (RB) tumor suppressor protein is increased most effectively. HC-toxin's epoxy group is essential for inhibiting HDACs and promoting anti-NB activity. Without this functional group, those cellular effects are not observed. In conclusion, the anti-NB activity of HC-toxin is superior to that of RAs and that of all other HDACIs tested.

Histone deacetylase inhibitor Helminthosporium carbonum (HC)-toxin suppresses the malignant phenotype of neuroblastoma cells.

The survival rate of children with advanced neuroblastoma (NB) is dismal despite intensive multimodal therapy. The limited efficacy and the frequent and serious side effects of currently used therapeutic regimens necessitate the development of new, less toxic treatment strategies. This study shows that the histone deacetylase inhibitor Helminthosporium carbonum (HC)-toxin suppresses the malignant phenotype of both established NB cell lines and primary NB cells with and without amplified MYCN at dosages lower than 20 nM. HC-toxin induces cell cycle arrest and apoptosis as well as neuronal differentiation and diminishes both colony formation and invasive growth. These cellular changes are accompanied by the transcriptional repression of cell cycle regulators of the retinoblastoma (RB) tumor suppressor network found at high levels in NBs with poor prognosis, like E2F-1 and its targets Skp2, N-myc, Mad2 and survivin. The levels of the hypophosphorylated active form of RB, and of cyclin-dependent kinase inhibitors including p15(INK4b), p16(INK4a), p21(cip1/waf-1) and p27(kip1) are increased. In conclusion, nanomolar doses of the HDACI HC-toxin cause a shift to a differentiated and benign phenotype of NB cells that is associated with an activation of the RB tumor suppressor network.

Identification of Bipolaris bicolor and Bipolaris sorokiniana on wheat seeds (Triticum aestivum L) in Brazil

Diseases caused in wheat by Helminthosporium spp. have led to considerable yield and production losses. Different species in this genus are associated with wheat seeds. In Brazil, spot blotch in wheat is caused by Bipolaris sorokiniana (Sacc) Schoem, and another fungus Bipolaris bicolor (Mitra) Shoem that has been also isolated from wheat seeds. The current study was undertaken to identify the most frequent fungus species that normaly infects wheat seeds and compared them with B. sorokiniana. The fungus Bipolaris bicolor (Mitra) Shoem., isolated from wheat seeds cultivar IAPAR, was identified by taxonomic methods and compared with the fungus Bipolaris sorokiniana (Sacc.) Shoem., in relation to growth characteristics on the seeds, as well as to growth characteristics in PDA and morphology of the structures. Type of colony observed on the seeds is important for the differentiation between the fungus species. Bipolaris sorokiniana presented black colonies, which were well-adherent to the seeds, whereas B. bicolor presented grayish, aerial, cotton-like colonies. The size of the conidia also differed in length and width, and B. bicolor presented the smallest dimensions. In relation to septa, B. bicolor conidia presented deep ones, with dark color bases, but seldom presented dark apex. Bipolaris sorokiniana presented homogenous color.

Doenças causadas por Helminthosporium spp. em trigo, causam consideráveis perdas na produção. Diferentes espécies do gênero do fungo podem ser encontradas em sementes. No caso do Brasil, a mancha foliar do trigo tem sido causada por Bipolaris sorokiniana (Sacc) Schoem, entretanto, outro fungo como Bipolaris bicolor (Mitra) Shoem tem sido isolado de sementes do trigo. O objetivo do presente trabalho foi identificar as espécies de fungo que normalmente infectam sementes de trigo e comparar com a mais comum B. sorokiniana. O fungo Bipolaris bicolor (Mitra) Shoem., isolado de sementes de trigo var. IAPAR, foi identificado por métodos taxonômicos e comparado com o fungo Bipolaris sorokiniana (Sacc.) Shoem. em relação ao crescimento sobre as sementes bem como às características culturais no meio BDA e morfologia das estruturas. O tipo de colônia observada sobre as sementes é importante para diferenciar as espécies entre si. O fungo B. sorokiniana apresentou colônias de cor preta sendo bem aderidas às sementes, enquanto B. bicolor apresentou colônias aéreas acinzentadas e com aspecto cotonoso. O tamanho dos conídios também diferenciaram no comprimento e largura onde os valores menores corresponderam a B. bicolor. Em relação aos septos, os conídios de B. bicolor apresentavam septo "profundo", com coloração escura na base e, raras vezes no ápice. Os conídios de B. sorokiniana apresentaram coloração homogênea.

Fungi allergens produced by solid-state fermentation process: optimization and allergen characterization.

Allergenic extracts were produced from Drechslera (Helminthosporium) monoceras biomass cultured by solid-state fermentation using wheat bran as the substrate. The main fermentation variables were selected by statistical design, and the optimized biomass yield (1.43 mg/[g of dry substrate d]) was obtained at pH 9.5 and 45.8% moisture. The allergenic extracts were produced from crude extract by protein precipitation and polyphenol removal. Proteins in the range of 16-160 kDa were identified in the extracts. Their reactions in patients were characterized by in vivo cutaneous tests (positive in 40% of the atopic patients) and by dot-blotting assays.

Enhancement of NGF- and cholera toxin-induced neurite outgrowth by butyrate in PC12 cells.

It has been shown that sodium butyrate (NaBu) does not elicit neurite outgrowth of PC12, one of the most widely used cell lines as a model of neuronal differentiation. In this study, the effects of NaBu on nerve growth factor (NGF)- and cholera toxin-induced neurite outgrowth in PC12 cells were examined. NaBu dose-dependently enhanced neurite formation induced by both agents. The maximum responses obtained at 0.5 mM NaBu were nearly twice those of the inducers alone. Propionate and valerate were also effective, but acetate and caproate were ineffective. Among the butyrate analogs with a moiety of three to five carbon atoms tested, isobutyrate, isovalerate, vinylacetate and 3-chloropropionate enhanced neurite outgrowth promoted by both inducers. However, neither alpha-, beta-, and gamma-aminobutyrates nor alpha-, beta-, and gamma-hydroxybutyrates were effective. All of the effective short-chain fatty acids and their analogs increased the level of histone acetylation, while ineffective ones did not. Furthermore, Helminthosporium carbonum toxin (HC toxin), a structurally dissimilar inhibitor of histone deacetylase, mimicked the effect of butyrate. These results suggest that NaBu enhances neurite outgrowth induced by NGF and cholera toxin in PC12 cells through a mechanism involving an increase in the level of histone acetylation.

[Protein and peptide factors from Bacillus sp.739 inhibit the growth of phytopathogenic fungi]. / Belkovye i peptidnye factory Bacillus sp.739--ingibitory rosta fitopatogennykh gribov.

Thermolabile peptides inhibiting the growth of Helminthosporium sativum, a facultative phytopathogen, have been isolated from the low-molecular-weight fraction of extracellular metabolites of the strain Bacillus sp. 739. Paper chromatography of the fraction, followed by bioautography, revealed the presence of three components exhibiting antifungal activity. These components were separated by gel chromatography on Toyopearl HW-40. SDS-PAGE (the Laemmli procedure) demonstrated that only one component was a protein (MW, approximately 14 kDa). The other two substances were polypeptides with molecular weights less than 6 kDa each. The protein factor inhibited the growth of H. sativum with a minimum effective concentration of 0.1 to 0.2 mg/ml.

Rare sesquiterpenes from the algicolous fungus Drechslera dematioidea.

From the inner tissue of the marine red alga Liagora viscida the fungus Drechslera dematioidea was isolated. After mass cultivation, the fungus was investigated for its secondary metabolite content, and 10 new sesquiterpenoids [isosativenetriol (1), drechslerines A (2) and B (3), 9-hydroxyhelminthosporol (5), drechslerines C-G (6-10), and sativene epoxide (12)] were isolated. Compounds 8 and 10 exhibited antiplasmodial activity against Plasmodium falciparum strains K1 and NF54. The known compounds helminthosporol (4), cis-sativenediol (11), isocochlioquinone A (14), isocochlioquinone C (15), and cochlioquinone B (16) were also isolated. All structures were elucidated using spectroscopic methods, mainly 1D and 2D NMR and MS.

Outcomes after discontinuing immunotherapy for allergic fungal sinusitis.

Although the treatment of allergic fungal sinusitis with specific immunotherapy after surgical intervention has proved successful, the question of what happens when such injections are discontinued remains unanswered. In this initial, admittedly small series, no recurrence has been noted in follow-up of 7 to 17 months.

Assembly of the Hv190S totivirus capsid is independent of posttranslational modification of the capsid protein.

The genome of Helminthosporium victoriae 190S totivirus (Hv190SV) consists of two large overlapping open reading frames (ORFs), encoding a capsid protein (CP) and an RNA-dependent RNA polymerase. The capsid of Hv190SV, even though encoded by a single gene, contains three closely related capsid polypeptides: p88, p83, and p78. p88 and p83 are phosphorylated, whereas p78, which is derived from p88 via proteolytic processing at the C terminus, is nonphosphorylated. In this study we expressed the CP ORF in bacteria and determined that a single product comigrating with virion p88 was generated. Evidence from in vivo phosphorylation studies indicated that the bacterially expressed p88 was unmodified, and thus autophosphorylation was ruled out. Enzymatic-dephosphorylation experiments using 32P-labeled p88 as a substrate demonstrated that the phosphorylated and nonphosphorylated forms of p88 could not be differentiated based on their mobilities in SDS gels and suggested that the two forms occur in purified virions. We also showed that the unmodified p88 is competent for assembly into virus-like particles, indicating that neither phosphorylation nor proteolytic processing of CP is required for capsid assembly. Posttranslational modification of CP, however, is proposed to play an important role in the life cycle of Hv190SV, including regulation of transcription/replication and/or packaging/release from virions of the viral (+) strand RNA transcript.

Immunotherapy for allergic fungal sinusitis: three years' experience.

Since August 1994, we have treated patients with histologically proven allergic fungal sinusitis with surgery followed by immunotherapy, employing fungal and nonfungal antigens to which hypersensitivity has been demonstrated. Our results continue to be encouraging. Not only have we encountered no indication that fungal immunotherapy has worsened these patients' condition or caused a recurrence of disease, we have confirmed dramatic improvement in these patients compared with the generally accepted course of this disease. Of 11 patients who have received immunotherapy for 1 to 3 years (mean 28 months), none has required regular or frequent treatment with a single brief course of systemic steroids, and only three are receiving topical nasal steroids. No repeat surgeries for recurrent allergic fungal sinusitis have been required in the treatment group. This combination of surgery and immunotherapy has continued to prove beneficial, and we urge others to consider this approach to therapy.

KM-01, a brassinolide inhibitor, its production, isolation and structure from two fungi Drechslera avenae and Pycnoporus coccineus.

A new brassinolide inhibitor, named KM-01, was isolated from the culture filtrates of two fungal species, Drechslera avenae and Pycnoporus coccineus, and the structure with absolute stereochemistry was elucidated as the fatty acid ester of an eremophilane sesquiterpene, bipolaroxin, based on spectroscopic analysis, chemical degradation, and synthesis of the fatty acid.

Prevalence of mold-specific immunoglobulins in a Midwestern allergy practice.

Mold allergy surveys are an important part of the correct identification and treatment of mold allergies. This study included 100 patients who were referred to a Midwestern allergy clinic for the evaluation of rhinitis, suspected to be of allergic origin. An in vitro screening test for allergen-specific IgE (ImmunoCAP) comprised of 10 allergens, including Candida, Aspergillus, Helminthosporium, and Alternaria, was used. To assess the seasonal distribution of mold allergies, we randomly selected 8 patients out of the 100 from each season during which the clinical contact occurred, and we tested them for 14 varieties of mold. The overall incidence of mold allergy in atopic subjects was 44%. The most common molds were (in descending order of frequency) Alternaria, Helminthosporium, Aspergillus, Candida, and Curvularia. Mold allergy was diagnosed most frequently in the winter; the second highest period was the fall. Population surveys of IgE antibody sensitization by in vitro techniques can provide useful information about fungal allergy.