Bioprospecting for fungi with potential pathogenic activity on leaves of Agave tequilana Weber var. Azul

Thirty different fungal strains were isolated from A. tequilana leaves showing disease symptoms such as wilt and curled leaves, black, red and chlorotic spots. Ten genera were identified and confirmed by using the LSU D1/D2 rDNA and ITS1‐5.8S‐ITS2 regions, mainly of the Ascomycota phylum, where the Lasiodiploidia and Neoscytalidium genera were the more (46.6%) abundant. The other genera identified were Cladosporium, Cytospora, Epicoccum, Flavodon, Lasiodiplodia, Myrmaecium, Neoscytalidium, Penicillium, Peniophora, Purpureocillium, Trametes and Fusarium. Five strains of Lasiodiplodia and one of Fusarium were selected based on their representativeness and pathogenic potential on Agaves. Pathogenic potential was analysed by both, an infection assay, evidenced as necrosis, and by pectinolytic activity. Specifically, necrosis infection assay was conducted by puncture (wounded) infection and by direct mycelium contact. In general, Lasiodiplodia strains exhibited different pathogenic profiles according to their necrosis percentages, regardless of the infection method used. Fusarium strain analysed also showed a high necrosis infection (> 99%). Pectinolytic activity used as an indirect measurement of pathogenesis presented a high Fusarium extract activity (peaking at 23.9 U). Lasiodiplodia strains exhibited up 6 times more enzymatic activity (peaking at 143.5) than Fusarium strain analysed. In addition, Agave leaf extracts used totally or partially as carbon source during fungal induction culture may induce different pathogenic activities in these strains. In general, the two pathogenicity assays implemented evidenced differences in the pathogenicity profile of these analysed strains.     

Fusarium Species from the Fusarium fujikuroi Species Complex Involved in Mixed Infections of Maize in Northern Sinaloa,Mexico

Fusarium species belonging to the Fusarium fujikuroi species complex (FFSC) are associated with maize in northern Mexico and cause Fusarium ear and root rot. In order to assess the diversity of FFSC fungal species involved in this destructive disease in Sinaloa, Mexico, a collection of 108 fungal isolates was obtained from maize plants in 2007–2011. DNA sequence analysis of the calmodulin and elongation factor 1α genes identified four species: Fusarium verticillioides, F. nygamai, F. andiyazi and F. thapsinum (comprising 79, 23, 4 and 2 isolates, respectively). Differential distribution of Fusarium species in maize organs was observed, that is F. verticillioides was the most frequently isolated species from maize seeds, while F. nygamai predominated on maize roots. Mixed infections with F. verticillioides/F. thapsinum and F. verticillioides/F. nygamai were detected in maize seeds and roots, respectively. Pathogenicity assay demonstrated the ability of the four species to infect maize seedlings and induce different levels of disease severity, reflecting variation in aggressiveness, plant height and root biomass. Isolates of F. verticillioides and F. nygamai were the most aggressive. These species were able to colonize all root tissues, from the epidermis to the vascular vessels, while infection by F. andiyazi and F. thapsinum was restricted to the epidermis and adjacent cortical cells. This is the first report of F. nygamai, F. andiyazi and F. thapsinum infecting maize in Mexico and co‐infecting with F. verticillioides. Mixed infections should be taken into consideration due to the production and/or accumulation of diverse mycotoxins in maize grain.     

Black shank resistant tobacco by silencing of glutathione S-transferase

A glutathione S-transferase gene was amplified from cDNA of Nicotiana tabacum roots infected with Phytophthora parasitica var. nicotianae. The gene was cloned in sense and anti-sense orientation to an RNAi vector for induced gene silencing, and reduced expression of the gene was detected by RT-PCR. A statistically significant increase in resistance of N. tabacum to infection following gene silencing was found for glutathione S-transferase-silenced plants compared with control plants. Some defense genes were up-regulated in glutathione S-transferase-silenced plants during the interaction with the pathogen. This is the first evidence of the role of glutathione S-transferase as negative regulator of defense response.     

Polyisoprenylated benzophenones in cuban propolis; biological activity of nemorosone

The Copey tree (Clusia rosea) has a large distribution in Cuba and its floral resin is a rich source of polyisoprenylated benzophenones. To determine the presence of these natural products, we carried out a study by HPLC of 21 propolis samples produced by honey bees (Apis mellifera) from different provinces of Cuba. Nemorosone resulted to be the most abundant polyisoprenylated benzophenone and the mixture of xanthochymol and guttiferone E was also observed, but in minor proportion. We studied the biological activity of the pure natural product nemorosone and its methyl derivatives. We found that nemorosone has cytotoxic activity against epitheloid carcinoma (HeLa), epidermoid carcinoma (Hep-2), prostate cancer (PC-3) and central nervous system cancer (U251). It also exhibited antioxidant capacity. Methylated nemorosone exhibited less biological activity than the natural product.     

Distribution of conserved and specific epitopes on the VP8 subunit of rotavirus VP4.

Three cDNA clones comprising the VP8 subunit of the VP4 of human rotavirus strain KU (VP7 serotype G1; VP4 serotype P1A) G1 were constructed. The corresponding encoded peptides were designated according to their locations in the VP8 subunit as A (amino acids 1 to 102), B (amino acids 84 to 180), and C (amino acids 150 to 246 plus amino acids 247 to 251 from VP5). In addition, cDNA clones encoding peptide B of the VP8 subunit of the VP4 gene from human rotavirus strains DS-1 (G2; P1B) and 1076 (G2; P2) were also constructed. These DNA fragments were inserted into plasmid pGEMEX-1 and expressed in Escherichia coli. Western immunoblot analysis using antisera to rotavirus strains KU (P1A), Wa (P1A), DS-1 (P1B), 1076 (P2), and M37 (P2) demonstrated that peptides A and C cross-reacted with heterotypic human rotavirus VP4 antisera, suggesting that these two peptides represent conserved epitopes in the VP8 subunit. In contrast, peptide B appears to be involved in the VP4 serotype and subtype specificities, because it reacted only with the corresponding serotype- and subtype-specific antiserum. Antiserum raised against peptide A, B, or C of strain KU contained a lower level of neutralizing activity than did that induced by the entire VP8 subunit. In addition, the serotype-specific neutralizing activity of anti-KU VP8 serum was ablated after adsorption with the KU VP8 protein but not with a mixture of peptides A, B, and C of strain KU, suggesting that most of the serotype-specific epitopes in the VP8 subunit are conformational and are dependent on the entire amino acid sequence of VP8.     

Epitope mapping of anti-human transferrin monoclonal antibodies: potential uses for transferrin-transferrin receptor interaction studies

Human transferrin (hTf) is an 80 kDa glycoprotein involved in iron transport from the absorption sites to the sites of storage and utilization. Additionally, transferrin also plays a relevant role as a bacteriostatic agent preventing uncontrolled bacterial growth in the host. In this work we describe a well-characterized Mabs panel in terms of precise epitope localization and estimate affinity for the two major hTf isoforms. We found at least four antigenic regions in the hTf molecule, narrowed down the interacting antigen residues within three of such regions, and located them on a molecular model of hTf. Two of the antigenic regions partially overlap with previously described transferrin-binding sites for both human receptor (antigenic region I: containing amino acid residues from Asp-69 to Asn-76 at the N-lobe) and bacterial receptors from two pathogenic species (antigenic region III: amino acid residues from Leu-665 to Ser-672 at the C-lobe). Hence, such monoclonal antibodies (Mabs) could be used as an additional tool for conformational studies and/or the characterization of the interaction between hTf and both types of receptor molecules.     

Effect of ethanol in preovulatory periods on LH, FSH, prolactin and ovulation in rats

The effect of ethanol (4 g/kg) as well as the role of serotoninergic neurons on the rate of ovulation and plasma LH, FSH and prolactin secretion have been studied in rats at preovulatory periods (18th hour of diestrus). It has been found that administration of ethanol in preovulatory periods decreased the number of ovules per rat (p less than 0.001), the number of ovulating rats and LH levels (p less than 0.001). These effects were accompanied by an increase in prolactin concentration (0.05 greater than p greater than 0.02), which was followed by a diffuse luteinization in the ovarian tissue. These results showed that ethanol had an effect of central depression in preovulatory periods. These effects could be mediated through the hypothalamic releasing factors. Under previous serotonin depletion with p-chlorophenylalanine (PCPA: 300 mg/kg), ethanol caused similar effects on LH and FSH levels as compared with the control group with PCPA. However, prolactin concentration was not increased. These results showed that serotoninergic neurons could be mediated in changes caused by ethanol on prolactin secretion, but do not affect directly in changes caused on LH and FSH secretion.     

The probability of matching genetic markers in different samples

Hypervariable DNA polymorphisms in humans have been introduced in forensic science for the exclusion of innocent persons, and possibily for the identification of guilty ones, through mismatches and matches of DNA patterns in incriminating samples. Under the assumption of random mating and linkage equilibrium, it is observed that the probability of mismatch, then of exclusion of innocent persons, is very high. The probability of a match on the contrary may be very low, particularly when several hypervariable DNA polymorphisms are used for the DNA pattern. When a match is observed, and the probability of match is calculated, and it is lower than one in five billions, this might be considered incriminating by a judge. It is concluded that an innocent person has all advantages in submitting to the DNA fingerprinting test.     

Extracellular HIV-1 Nef protein modulates lytic activity and proliferation of human CD8+ T lymphocytes

The effect of extracellular HIV Nef (exNef) protein on the induction of lytic activity and proliferation of CD8+T lymphocytes from 18 donors was studied. At 10 ng/ml, exNef-induced a 2- to 8-fold enhancement of basal lytic activity in cells from all donors in an allogeneic induction assay, whereas it was ineffective at 100ng/ml. The extent of enhancement was inversely correlated with the basal level of lytic activity without exNef. Only in combination with PHA did both exNef concentrations stimulate proliferation, and in a manner inversely related to the effect of PHA alone. Thus, concentrations of exNef commonly found in sera of HIV-infected patients were found to modulate the induction of lytic activity and proliferation of CD8+ T lymphocytes in vitro, to an extent strongly dependent on the quite variable responsiveness of each donor. These findings point to Nef as a potential agent for modulating CD8+ T cell function in pathogenesis and therapy.