Reproductive toxicity of the hydroethanolic extract of the flowers of Acmella oleracea and spilanthol in zebrafish: In vivo and in silico evaluation.

Hydroethanolic preparations of Acmella oleracea is used in the north of Brazil as a female aphrodisiac. Thus, the objective of this study was to evaluate the action of the hydroethanolic extract of Acmella oleracea (EHFAo) flowers (21.873 and 44.457 mg/kg) and spilanthol (3 mg/kg) administered orally on reproductive performance and effects on the embryonic development of zebrafish F1 generation. It was observed that in the groups in which males and females received EHFAo and spilanthol, the spawning was interrupted, whereas in the groups in which only the females were treated, spawning occurred during the 21 days. Thus, in the histopathological evaluation of the gonads, it was possible to observe that the percentage of mature cells in the spermatozoa and females was significantly reduced. Only the embryo groups in which parental generation was treated with EHFAo showed lethal and teratogenic effects. On the other hand, the parental groups treated with the spilanthol presented only the lethality. Spilanthol and some metabolites showed good oral availability and important toxicological properties. Thus, it is suggested that the treatment of parental generation of zebrafish with EHFAo and spilanthol caused severe changes in the gonads and on fertility. However, on the embryo, the most striking effects in the development were recorded in the groups in which the parental generation was treated with the EHFAo, while the spilanthol influenced the lethality of the embryos.

Antifungal, anti-oomycete and phytotoxic effects of volatile organic compounds from the endophytic fungus Xylaria sp. strain PB3f3 isolated from Haematoxylon brasiletto.

AIMS: To determine the antifungal, anti-oomycete and phytotoxic activity; and chemical composition of the volatile organic compounds (VOCs) produced by endophytic fungus Xylaria sp. PB3f3 isolated from Haematoxylon brasiletto Karst. METHODS AND RESULTS: Bioactivity and chemical composition of the VOCs from Xylaria sp. PB3f3 were established by using simple and multiple antagonism bioassays, and gas chromatography/mass spectrometry, respectively. The results showed that Xylaria sp. PB3f3 inhibited the growth of the oomycetes Pythium aphanidermatum (78·3%), Phytophthora capsici (48·3%), and the fungi Alternaria solani (24·5%) and Fusarium oxysporum (24·2%), in multiple antagonism bioassays. Volatile organic compounds, produced at 20 and 30 days of fungal growth, inhibited root elongation on Amaranthus hypochondriacus (27·6%) and on Solanum lycopersicum (53·2%). Forty VOCs were identified at 10, 20 and 30 days in Xylaria sp. PB3f3 cultures. The compounds with the highest fibre affinity were: 3-methyl-1-butanol and thujopsene, at 10 days of fungal growth; an unidentified amine and 2-methyl-1-butanol at 20 days; and 2-methyl-1-propanol at 30 days. In the gas phase assay method 2-methyl-1-propanol and 2-methyl-1-butanol showed significant inhibitory effects on root elongation and germination of Am. hypochondriacus and S. lycopersicum. CONCLUSIONS: Xylaria sp. PB3f3 and its VOCs showed significant phytotoxic effects on root elongation and germination of Am. hypochondriacus and S. lycopersicum. SIGNIFICANCE AND IMPACT OF THE STUDY: The genus Xylaria produces a great variety of secondary metabolites, but, up date, there are no reports of the identification of bioactive volatile compounds. Thus, Xylaria sp. PB3f3 and its VOCs are a possible candidate for the biological control of weeds.

Comparison of STAT5 mRNA levels in GH-treated male and female rats analysed by a solution hybridization assay.

In this study we describe the development of a RNA:RNA solution hybridization-RNase protection assay to quantify STAT5 mRNA in total RNA extracts from rat tissues. The assay is sensitive and reproducible. We quantified STAT5 mRNA levels in liver and thymus lymphocytes from male and female control rats and from rats treated with a single dose of recombinant human growth hormone (rhGH). No significant sex differences in the expression pattern were observed in both studied tissues, but STAT5 mRNA levels were significantly (P< 0.05) higher in liver than in thymus lymphocytes. STAT5 mRNA levels were significantly (P< 0.05) increased by a pulse of GH given to either male or female normal rats, suggesting a regulation of STAT5 gene expression in the studied tissues. In conclusion, quantitative solution hybridization-RNase protection assay of STAT5 mRNA provides a tool to further advance the study of the regulatory mechanisms involved in STAT5 gene expression.

Characterisation of the carbohydrate components of Taenia solium metacestode glycoprotein antigens.

Human neurocysticercosis is caused by Taenia solium metacestodes. It usually affects the central nervous system of humans and can be confused with other brain pathologies. The Lens culinaris-binding glycoproteins from this parasite have been shown to be ideal targets for the development of a highly specific immunoassay for the diagnosis of neurocysticercosis. In the present study we characterised the carbohydrates associated with five antigenic glycoproteins of T. solium metacestodes in the range of 12-28 kilodaltons. Lectin-affinities and enzymatic deglycosylations suggested that each of the five antigens contain various glycoforms of asparagine-linked carbohydrates of the hybrid, complex and probably high mannose type. These carbohydrates accounted for at least 30-66% of the apparent molecular mass of the glycoconjugates. In contrast, there was no evidence for the presence of O-linked carbohydrates. Lectin affinity patterns suggested that the sugars are short and truncated in their biosynthetic route, and that some contain terminal galactose moieties. Elucidating the precise structure of the carbohydrates and establishing their role in antigenicity will be essential to design strategies to produce them in large and reproducible amounts for the development of improved immunoassays.

Use of the 27-kilodalton recombinant protein from Paracoccidioides brasiliensis in serodiagnosis of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it is usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods. Although the latter are effective, two circumstances, cross-reactions with other mycotic agents and antigen preparation that is marked by extreme variability among lots, hinder proper standardization of the procedures. To circumvent this lack of reproducibility, molecular biology tools were used to produce a recombinant 27-kDa-molecular-mass antigen from this fungus; a sizable quantity of this antigen was obtained through fermentation of Escherichia coli DH5alpha, which is capable of expressing the fungal protein. The latter was purified by the Prep-Cell System (Bio-Rad); the recovery rate of the pure protein was approximately 6%. A battery of 160 human serum samples, consisting of 64 specimens taken at the time of diagnosis from patients with PCM representing the various clinical forms plus 15 serum specimens each from patients with histoplasmosis and aspergillosis, 10 each from patients with cryptococcosis and tuberculosis, 6 from patients with coccidioidomycosis, and 40 from healthy subjects, were all tested by an indirect enzyme-linked immunosorbent assay with the purified 27-kDa recombinant protein. The latter was used at a concentration of 1.0 microgram/well; there were three serum dilutions (1:1,000, 1:2,000, and 1:4,000). The experiment was repeated at least twice. The average sensitivity for both experiments was 73.4%; in comparison with the healthy subjects, the specificity for PCM patients was 87.5% while for patients with other mycoses, it was 58.7%. Important cross-reactions with sera from patients with aspergillosis and histoplasmosis were detected. The positive predictive value of the test was 90.4%. These results indicate that it is possible to employ recombinant antigenic proteins for the immunologic diagnosis of PCM and, by so doing, achieve high coverage rates. Furthermore, antigen reproducibility can now be ensured, thus facilitating inter- and intralaboratory standardization.

Stress proteins in fungal diseases.

Heat shock proteins (hsps) are ubiquitous families of proteins, found in all organisms studied so far. They are highly conserved across the species barrier and serve fundamental functions in cell physiology. The term 'heat shock' was adopted because of the early observation of the heat-inducible nature of these proteins, although, as it is now realized that they can be induced by a variety of stressful stimuli, it is probably more appropriate to call them 'stress proteins'. The nomenclature of many hsps, for example hsp90, hsp70 and hsp60, reflects the approximate molecular mass of hsps within each of these families. For many bacterial and parasitic infections, hsps were first recognized as immunodominant antigens on immunoblots of extracts from the organism probed with immune sera, or in T-cell proliferation assays. They have now been identified in a range of fungal pathogens, again often linked to an immune response. In this symposium, we review the association of hsps with humoral immunity to candidosis and aspergillosis, cellular immunity to histoplasmosis, and the identification of hsp70 in another dimorphic fungus, Paracoccidioides brasiliensis. Finally, the crucial role of the membrane in setting the temperature of the heat shock response in yeasts is discussed.

Development of a novel antigen detection test for histoplasmosis.

Histoplasmosis is an important systemic fungal infection, particularly among immunocompromised individuals living or travelling in areas of endemicity, who, without antifungal therapy, may develop a progressive disseminated fatal infection. For such patients, the detection of antibody responses by immunodiffusion or complement fixation test is of limited use. In contrast, the detection of Histoplasma capsulatum circulating antigens may provide a more practical approach to the rapid diagnosis of the disease. Accordingly, an inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of a 69- to 70-kDa H. capsulatum-specific determinant and incorporating a species-specific murine monoclonal antibody was developed. With sera from patients with different forms of the disease (n = 35), the overall sensitivity of the test was found to be 71.4%, while the specificity was found to be 98% with normal human sera from areas of endemicity (n = 44) and 85.4% with sera from patients with other chronic fungal or bacterial infections (n = 48). This novel, highly specific ELISA provides a significant addition to the existing diagnostic tests for the detection of histoplasmosis.

Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis.

A gene encoding a 27-kDa antigenic protein from Paracoccidioides brasiliensis was cloned, sequenced, and characterized. A cDNA library of the mycelial phase was produced and packed in Uni-Zap-XR vector, lambda Zap II synthesis kit (Stratagene, La Jolla, Ca). The screening of the library was carried out using a pool of sera from paracoccidioidomycosis patients that had proven reactive in serological testing. Among 44,000 immuno-screened clones from the library, 2 were positive (clones 2 and 3). The former was not characterized further. The latter has a 1-kb DNA insert with an open reading frame encoding a protein of 259 amino acids with a predicted molecular mass of 28.6 kDa (27 kDa by SDS-PAGE). This protein corresponds to a 25-kDa protein in antigenic preparations of P. brasiliensis as determined by Western blot analysis. Comparison of the transcribed sequence with different gene banks failed to reveal a high degree of homology with other proteins. The cloned DNA fragment was easily expressed in Escherichia coli without the need of induction by isopropyl-beta-D-thiogalactopyranoside. These findings suggest that the gene encodes a P. brasiliensis-specific protein.

Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis.

We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis.

In search of the natural habitat of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis (Pb) is the dimorphic fungus responsible for paracoccidioidomycosis (PCM), one of the most important systemic mycosis in Latin America where the disease is geographically restricted. The natural habitat of the causative agent remains undetermined. We are planning to use PCR-based technology in order to amplify specific DNA fragments. The high sensitivity of this technique may allow us to detect the natural habitat of Pb. In this study, we prepared a cDNA library from which we cloned a protein of approximately 27 kDa MW. When this recombinant antigenic protein was tested by the immunoblot technique, it was able to recognize antibodies in the sera of 91% of the PCM patients studied. No cross reactions were observed with sera from patients with other systemic mycoses. Presently we are sequencing and characterizing this clone, in order to design specific primers for amplification of Pb DNA.

Characterization of the yeast acidic ribosomal phosphoproteins using monoclonal antibodies. Proteins L44/L45 and L44' have different functional roles.

In order to characterize the acidic ribosomal proteins immunologically and functionally, a battery of monoclonal antibodies specific for L44, L44' and L45, the three acidic proteins detected in Saccharomyces cerevisiae, were obtained. Eight monoclonal antibodies were obtained specific for L45, three for L44' and one for L44. In addition, two mAbs recognizing only the phosphorylated forms of the three proteins were obtained. The specific immunogenic determinants are located in the middle region of the protein structure and are differently exposed in the ribosomal surface. The common determinants are present in the carboxyl end of the three proteins. An estimation of the acidic proteins by ELISA indicated that, in contrast to L44 and L45, L44' is practically absent from the cell supernatant; this suggests that protein L44' does not intervene in the exchange that has been shown to take place between the acidic proteins in the ribosome and in the cytoplasmic pool. It has also been found that, while IgGs specific for L44 and L45 do not inhibit the ribosome activity, the anti-L44' effectively blocks the polymerizing activity of the particles. These results show for the first time that the different eukaryotic acidic ribosomal proteins play a different functional role.

Ribosomal protein interactions in yeast. Protein L15 forms a complex with the acidic proteins.

Protein L15 from Saccharomyces cerevisiae ribosomes has been shown to interact in solution with acidic ribosomal proteins L44, L44' and L45 by different methods. Thus, the presence of the acidic proteins changes the elution characteristics of protein L15 from CM-cellulose and DEAE-cellulose columns and from reverse-phase HPLC columns. Moreover, immunoprecipitation using anti-L15 specific monoclonal antibodies coprecipitates the acidic proteins, too. Conversely, antibodies raised against the acidic proteins immunoprecipitate protein L15. This coprecipitation seems to be specific since it does not involve other ribosomal proteins present in the sample. Similarly, plastic-adsorbed antibodies specific for one of the components in the L15--acidic-protein complex are able to retain the other component of the complex but cannot bind unrelated proteins. Moreover, protein L15 can be chemically cross-linked to the acidic proteins in solution. These results indicate that protein L15 might be equivalent to bacterial ribosomal protein L10 in forming a complex with the acidic proteins. Since, on the other hand, protein L15 has been shown to be immunologically related to bacterial protein L11 [Juan Vidales et al. (1983) Eur. J. Biochem. 136, 276-281] and to interact with the same region of the large ribosomal RNA as does protein L11 [El-Baradi et al. (1987) J. Mol. Biol. 195, 909-917], these results suggest strongly that protein L15 plays the same role in the yeast ribosome as proteins L10 and L11 do in the bacterial particles.