PubMed Health⌕ Search

Biomedical subjects

E Monte

Publications and source records attributed to E Monte.

At least 19 recordsLinked to original sources
PubMed2006

Cloning and characterization of the erg1 gene of Trichoderma harzianum: effect of the erg1 silencing on ergosterol biosynthesis and resistance to terbinafine.

Trichoderma species are commonly used as biocontrol agents of different plant-pathogenic fungi. Terpene compounds are involved in the biocontrol process due to their antifungal properties (e.g., ergokonins and viridins) but additionally their structural function in the cell membranes (ergosterol) is essential. We report here the characterization of the T. harzianum erg1 gene, encoding a squalene epoxidase, a key enzyme in the biosynthesis of triterpene derivatives such as ergosterol. In T. harzianum the partial silencing of the erg1 gene gave rise to transformants with a higher level of sensitivity to terbinafine, an antifungal compound that acts specifically over the squalene epoxidase activity. In addition, these silenced transformants produced lower levels of ergosterol than the wild type strain. Finally, the silencing of the erg1 gene resulted in an increase in the expression level of the erg7 gene that encodes the oxidosqualene lanosterol-cyclase, another enzyme of the terpene biosynthesis pathway.

Antifungal Agents↗
PubMed2006

ThPTR2, a di/tri-peptide transporter gene from Trichoderma harzianum.

The generation of a wide ESTs library and database from Trichoderma harzianum CECT 2413 was the base for identifying the gene ThPTR2, coding for a PTR family di/tri-peptide transporter. The deduced protein sequence of the ThPTR2 gene showed the conserved motifs and also the 12 transmembrane domains typical of the PTR transporters. The highest level of ThPTR2 expression was found when the fungus was grown in chitin as sole carbon source. We also found that ThPTR2 expression was increased when Trichoderma interacted directly in solid medium with the plant-pathogenic fungus Botrytis cinerea, showing that ThPTR2 is involved in the mycoparasitic process. Additionally, its expression was triggered by nitrogen starvation and a higher level of expression was also found when Trichoderma was grown in secondary nitrogen sources like allantoin, yeast extract, and urea. However, no difference was found when Trichoderma was grown in presence or absence of glucose as carbon source. Strain T34-15, a transformant that overexpressed the ThPTR2 gene, showed about a 2-fold increase in the uptake of the dipeptide Leu-Leu. Additionally, two transformants from the strain Trichoderma longibrachiatum T52 that overexpressed ThPTR2 were also studied, confirming the role of this gene in peptide transport. Other homologous genes to ThPTR2 were identified in other Trichoderma strains. ThPTR2 is the first experimentally confirmed PTR family transporter gene from filamentous fungi.

Amino Acid Motifs↗
PubMed2006

Detection of peptaibols and partial cloning of a putative peptaibol synthetase gene from T. harzianum CECT 2413.

The characterization of 11- and 18-residue peptaibols (peptides synthesized by peptide synthetases) at Trichoderma harzianum CECT 2413 (a filamentous fungus) was performed. Using a heterologous probe from tex1, the only peptaibol synthetase cloned and characterized so far in Trichoderma species, was cloned; a region that comprised 11676 bp of a second peptide synthetase gene detected in these strain (called salps2) and sequenced. The deduced sequence of Salps2 (3891 amino acids) contained three complete and a fourth incomplete module of a peptide synthetase, in which the typical adenylation, thiolation and condensation domains were found, but also an additional dehydrogenase/reductase domain in the C-terminus of the last module. Based on sequence similarity and analysis of its modular structure, it is proposed that Salps2 is a peptaibol synthetase. Additionally, analysis of =4.4-kb sequence downstream of salps2 was done and the signature sequences of Salps2 were identified and compared with those of available sequences of the other Trichoderma peptaibol synthetases.

Cloning, Molecular↗
PubMed2005

Detection of putative peptide synthetase genes in Trichoderma species: application of this method to the cloning of a gene from T. harzianum CECT 2413.

Some of the secondary metabolites produced by Trichoderma, such as the peptaibols and other antibiotics, have a peptide structure and in their biosynthesis are involved proteins belonging to the Non-Ribosomal Peptide Synthetase family. In the present work, a PCR-mediated strategy was used to clone a region corresponding to an adenylation domain of a peptide synthetase (PS) gene from 10 different strains of Trichoderma. In addition, and using the fragment isolated by PCR from T. harzianum CECT 2413 as a probe, a fragment of 19.0 kb corresponding to a PS-encoding gene named salps1, including a 1.5 kb fragment of the promoter, was cloned and sequenced. The cloned region of salps1 contains four complete, and a fifth incomplete, modules, in which are found the adenylation, thiolation and condensation domains, but also an additional epimerization domain at the C-terminal end of the first module. The analysis of the Salps1 protein sequence, taking into consideration published data, suggests that it is neither a peptaibol synthetase nor a protein involved in siderophore biosynthesis. The presence of two breaks in the open reading frame and the expression of this gene under nitrogen starvation conditions suggest that salps1 could be a pseudogene.

Amino Acid Sequence↗
PubMed2005

Specific PCR assays for the detection and quantification of DNA from the biocontrol strain Trichoderma harzianum 2413 in soil.

Strain identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of sequence-characterized amplified region (SCAR) markers to detect genomic DNA from Trichoderma harzianum 2413 from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 27 isolates of Trichoderma spp. and amplified a 990-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum 2413, using an annealing temperature of 68 degrees C. These fragments showed no significant homology to any sequence deposited in the databases. The primer pair, BR1 and BR2, was designed to the 1.5-kb fragment amplified from T. harzianum 2413, generating a SCAR marker. To test the specificity of these primers, experiments were conducted using the DNA from 27 Trichoderma spp. strains and 22 field soil samples obtained from four different countries. PCR results showed that BR1 and BR2 amplified an 837-bp fragment unique to T. harzianum 2413. Assays in which total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma spp. strains, indicated that the BR1 and BR2 primers could specifically detect T. harzianum 2413 in a pool of mixed DNA. No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 837-bp SCAR marker of T. harzianum 2413 could be used in real-time PCR experiments, new primers (Q2413f and Q2413r) conjugated with a TaqMan fluorogenic probe were designed. Real-time PCR assays were applied using DNA from sterile and nonsterile soil samples inoculated with a known quantity of spores of Trichoderma spp. strains.

DNA↗
PubMed2004

Adsorption of methotrexate and calcium leucovorin onto cholestyramine in vitro.

Methotrexate (MTX), an antimetabolite of folic acid, is a drug widely used in the treatment of different types of cancer. When high doses are administered, it is necessary to interrupt its action by administering calcium leucovorin (CaL). The main pathway of MTX and CaL elimination in humans occurs through the kidney, but about 10% is excreted in the faeces via the bile. Drugs, foods and sorbents in intestinal lumen modify MTX and CaL reabsorption. Individual and simultaneous studies on the adsorption of MTX and CaL from aqueous phosphate buffer by cholestyramine were carried out in order to calculate the adsorption process of MTX and CaL to cholestyramine, and to characterize the influence of CaL in the adsorption of MTX to cholestyramine and vice versa. The Langmuir binding isotherms determined in buffer solutions at pH 6 indicated a greater (12.58%) adsorption capacity of cholestyramine (1.43 mmol of drug/g of resin) than at pH 7 (1.25 mmol of drug/g of cholestyramine). The affinity constant of MTX to cholestyramine was a 45.27% higher (6.67 mM(-1)) than the affinity constant of CaL to the resin (3.65 mM(-1)). Results from simultaneous assays indicate that a displacement of the MTX bound to cholestyramine by CaL is not foreseeable. The results suggest that cholestyramine may be a potentially useful adjunctive therapy in the treatment of an overdose of MTX. Consequently, cholestyramine may be of clinical value in patients who develop early renal function impairment whilst undergoing MTX therapy.

Adsorption↗
PubMed2002

[ROC curve analysis of factors predictive of response to treatment with interferon plus ribavirin in patients with chronic hepatitis C relapse after previous interferon treatment].

This study aimed to identify the factors predictive of response before the initiation of treatment and throughout the treatment period in patients with chronic hepatitis C relapse after treatment with interferon-a who were retreated with a standard regimen of interferon-a plus ribavirine and followed up for 40 months. Forty-four patients (40 with genotype 1, four without genotype 1) were included in the study. Four patients (genotype 1) were excluded because of adverse effects. The rate of maintained response was 55% (50% genotype 1, 100% non-genotype 1). The stage of histological damage (>2), glutamic-pyruvic transaminase (GPT) concentration (< or = 26 UI/l) and the association between the GPT concentration and the detection of the RNA-HCV in the first and third treatment months were the variables with an area under the ROC curve and a confidence interval >0.5. The probability of predicting a maintained response (negative predictive value) if the stage of histological lesion was <2 was 62.9%, while the positive predictive value was 100%. During the treatment, the disappearance of the RNA-HCV together with GPT values < or =26 in the first treatment month were the best predictive values. In this case, the negative predictive value was 78.3% and the positive predictive value was 76.5% (OR: 11.7, 2.6-52.2). Furthermore, the GPT value with the best predictive value (<26 UI/l) was a more effective predictor of the response to treatment than the normal value of the GPT. Finally, the GPT values >26 UI/l and the detection of RNA/HCV in the first or third treatment month were certain predictors of the absence of response but with low sensitivity (10-12%). It was concluded that is possible to predict the response to the combined treatment with an acceptable level of confidence, although not unequivocally. Ninety percent of the patients would be candidates for maintaining treatment for at least 6-12 months, while approximately 10% could undergo early interruption of treatment due to the absence of response.

Adult↗
PubMed2001

Gibberellins signal nuclear import of PHOR1, a photoperiod-responsive protein with homology to Drosophila armadillo.

S. tuberosum ssp. andigena potato plants require short days (SD) for tuberization. We have isolated PHOR1 (photoperiod-responsive 1), which shows upregulated expression in induced leaves (SD). PHOR1 encodes an arm repeat protein with homology to the Drosophila segment polarity protein armadillo. Antisense inhibition of PHOR1 produces a semidwarf phenotype similar to that of GA-deficient plants, and the antisense lines show reduced GA responsiveness combined with a higher endogenous GA content than wild-type plants. Feedback regulation of GA biosynthetic genes is also altered in these lines. Conversely, transgenic lines overexpressing PHOR1 show an enhanced response to GA. GA application induces rapid migration of PHOR1-GFP protein to the nucleus. Thus, PHOR1 appears to be a general component of GA signaling pathways that relocalizes to the nucleus in the presence of GA.

Active Transport, Cell Nucleus↗
PubMed2001

Development of a strain-specific SCAR marker for the detection of Trichoderma atroviride 11, a biological control agent against soilborne fungal plant pathogens.

The genus Trichoderma includes biocontrol agents (BCAs) effective against soilborne plant pathogenic fungi. Several potentially useful strains for biological control are difficult to distinguish from other strains of Trichoderma found in the field. So, there is a need to find ways to monitor these strains when applied to natural pathosystems. We have used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum and T. longibrachiatum previously selected as BCAs, and to obtain fingerprinting patterns. Analysis of these polymorphisms revealed four distinct groups, in agreement with previous studies. Some of the RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identify the strain T. atroviride 11, showing that DNA markers may be successfully used for identification purposes. This SCAR (sequence-characterised amplified region) marker can clearly distinguish strain 11 from other closely related Trichoderma strains.

Genetic Markers↗
PubMed2001

An antifungal exo-alpha-1,3-glucanase (AGN13.1) from the biocontrol fungus Trichoderma harzianum.

Trichoderma harzianum secretes alpha-1,3-glucanases when it is grown on polysaccharides, fungal cell walls, or autoclaved mycelium as a carbon source (simulated antagonistic conditions). We have purified and characterized one of these enzymes, named AGN13.1. The enzyme was monomeric and slightly basic. AGN13.1 was an exo-type alpha-1,3-glucanase and showed lytic and antifungal activity against fungal plant pathogens. Northern and Western analyses indicated that AGN13.1 is induced by conditions that simulated antagonism. We propose that AGN13.1 contributes to the antagonistic response of T. harzianum.

Antibiosis↗
PubMed2000

Separation and identification of volatile components in the fermentation broth of Trichoderma atroviride by solid-phase extraction and gas chromatography-mass spectrometry.

A preseparated fermentation broth of Trichoderma atroviride strain 11 is analyzed by gas chromatography followed by mass-spectral detection using a Finnigan MAT GCQ apparatus. After preseparation in a C18 and a silica gel column, nineteen pyrone and dioxolane derivatives and two aliphatic esters are obtained, respectively. Among these, the four dioxolane derivatives have not been identified previously. The main component is found to be 5,5'-dimethyl-2H-pyran-2-on. The relative standard deviation for the determination of the retention time and the peak area (measured in ion counts) is 0.1% and 4.5%, respectively.

Culture Media↗
PubMed2000

Molecular characterization and identification of biocontrol isolates of Trichoderma spp.

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as "Trichoderma harzianum" Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.

DNA, Mitochondrial↗
PubMed1999

Chloride and ethyl ester morpholine thiourea derivatives and their Ni(II) complexes. Crystal and molecular structures of the thiourea derivative L-leucine methyl ester and its complexes with Cu(II) and Pt(II). Growth of the pathogenic fungus Botrytis cinerea.

We have synthesized a series of ligands (1, 3, 4, 6 and 7) and some of their complexes with Ni(II), Cu(II) and Pt(II) (2, 5, 8 and 9). These compounds were studied and characterized by elemental analysis, IR and UV-Vis spectra, conductivity measurements in solution, FAB+/MS, 1H and 13C NMR, ESR, etc. Compound 7 crystallized in the orthorhombic space group P2(1)2(1)2(1), with Z = 4. Unit cell parameters were as follows: a = 21.307(2) A, alpha = 90 degrees, b = 12.498(1) A, beta = 90 degrees, c = 7.7232(4) A, gamma = 90 degrees. For seven of these compounds, the antifungal activity of a major pathogen responsible for important crop damage was studied. In general, inhibition by the ligands was higher than that of the complexes. When the thiourea was linked to some diethyl groups, the compounds showed higher antifungal activity than the morpholine groups. Compound 3 achieved total inhibition (100%).

Antifungal Agents↗
PubMed1999

Leaf C40.4: a carotenoid-associated protein involved in the modulation of photosynthetic efficiency?

Comparative analysis by differential RNA display (DDRT-PCR) of the expression patterns of potato plants induced and non-induced to tuberize, led to the isolation of a cDNA clone, C40.4, that is strongly upregulated in the leaves of tuberizing plants. Leaf expression of this transcript was shown to be light-dependent, with increased levels of mRNA and protein being detected during the light hours. Sequence analysis revealed near complete identity to potato CDSP34, a thylakoid protein induced by drought stress, and strong homology to the carotenoid-associated proteins fibrillin, ChrB and PAP from pepper fruit, and CHRC from cucumber flowers. By using an antibody against pepper fibrillin, we were able to demonstrate a chloroplastic location of the C40.4 protein in association with the thylakoid membranes. Two-dimensional analysis of thylakoid pigment-protein complexes showed a specific association of the protein with the photosystem II (PSII) multi-subunit complex. Antisense plants with reduced levels of accumulation of C40.4 showed a stunted growth and decreased tuber yield and exhibited reduced values of non-photochemical quenching of chlorophyll a fluorescence. Altogether, these results indicate a preferential association of the C40.4 protein with the light harvesting complex of photosystem II (LHCII) antenna pigment complexes, and suggest a functional role of this protein in photosynthesis, by modulating photosynthetic efficiency and dissipation of excess absorbed light energy within the antenna complex.

Amino Acid Sequence↗
PubMed1999

A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis.

Although Arabidopsis has numerous well documented advantages for genetic and molecular analyses, its small size can be a limitation for biochemical and immunochemical assays requiring protein extraction. We have developed a rapid method to extract total protein from small amounts of Arabidopsis tissue that can be used for quantitative immunoblot analysis. The procedure involves direct extraction of tissue into SDS-containing buffer under conditions permitting immediate protein quantification in the extract, using commercially available kits without prior fractionation. This approach provides maximal extraction and quantitative recovery of total cellular protein, together with accurate evaluation of target protein levels as a proportion of the total. We have examined the utility and sensitivity of the procedure using monoclonal antibodies to phytochromes A and C (phyA and phyC), which are high- and low-abundance members, respectively, of the phytochrome family in Arabidopsis. Both phytochromes could be rapidly and readily quantified in the tissues examined, with phyC being detectable in extracts representing as few as five dark-grown seedlings, two light-grown seedlings, or half a single leaf from 3-week-old adult plants. The data indicate that the procedure may have broad utility for the detection and quantitative analysis of many proteins, including those of low abundance, in a variety of applications in Arabidopsis. In one such application, we used transgenic Arabidopsis phyC-overexpressor seedlings to demonstrate that the procedure can be used to detect transgene-encoded protein early at the segregating T2 generation, thereby offering the capacity for accelerated screening and selection of lines engineered to overexpress target proteins.

Arabidopsis↗
PubMed1999

Characterization of beet necrotic yellow vein furovirus from Spanish sugar beets.

Rhizomania is a viral disease, caused by beet necrotic yellow vein furovirus (BNYVV), which was detected in Spanish sugar beets in 1988, it being focused on the Castilla y León region. BNYVV has five RNA fragments with specific functions, and the different composition and proportion of RNA in the virions allow their separation and the characterization of their activities during the development of the disease. Thirty-six samples of sugar beet rootlets and frozen pulps from three different sugar beet zones of Castilla y León were analyzed by DAS-ELISA and Immunocapture-Reverse Transcription-Polymerase Chain Reaction (IC-RT-PCR) using specific primers. The identity of the cDNA products was confirmed by nested-PCR and restriction fragment length polymorphism (RFLP). The uniformity of the patterns obtained by RFLP analyses with nine endonucleases showed the existence of a unique strain of BNYVV in 80,000 Ha of crop surface which could be explained by a recent arrival of the rhizomania disease to this region. The isolates studied were more similar to type A, which has been previously described in BNYVV, but a non-expected cleavage site for this molecular group was observed with endonuclease HincII on the RNA-2 IC-RT-PCR product (nt 2133-3293) in the thirty-six Spanish samples and also in a North American strain taken as reference. The use of frozen pulps obtained as a previous step to the industrial extraction of sugar avoids problems due to erratic distribution of the virus in the roots, provides repetitive results for a particular sample, and facilitates epidemiological and distributional studies on rhizomania disease.

Antibodies, Viral↗
PubMed1997

Cloning and expression analysis of a gene that shows developmental regulation upon tuberization in potato.

Differential screening of a potato leaf cDNA library with cDNA probes made from tuberizing and non-tuberizing Solanum demissum plants led to the identification of a clone that is upregulated in leaves and other tissues upon tuberization. This clone was also shown to have a high level of expression in green tomato fruit, its expression falling off as the fruit turns red. No sucrose or hormonal regulation of the expression of this clone was observed and it did not respond to wounding or heat stress. Clone 32B is 532 bp long and contains an open reading frame encoding a small protein of 98 amino acids. The deduced protein sequence has a putative signal peptide for ER transport and a 10 amino acid domain in the C-terminal region of the protein, both of which are also found in the cotton LEA5, Arabidopsis Di21 and the mungbean Arg2 proteins.

Amino Acid Sequence↗