Santino's growth chamber: A chamber for studies with Aphelenchoides besseyi on plants.

The soybean green stem and foliar retention syndrome (GFSR) [1], caused by Aphelenchoides besseyi, is reported in Brazilian fields located at the States of Mato Grosso, Pará, Amapá, Tocantins, and Maranhão, which correspond to warm climates with well-defined rainfall patterns when the air humidity is high during several consecutive days. Studies showed that the infection of plants by A. besseyi occurs in regions with a high frequency of rains and under temperatures higher than 28 °C, when the nematode founds adequate conditions to migrate from soil to shoot parts of plants to initiate its parasitism [2]. One of the challenges was the difficulty in simulating the natural conditions for the disease development, which needs luminosity, high temperatures, and moisture. These conditions can be reproduced using modern growth chambers, but these equipment are onerous and scarce in most Brazilian research centers. So, for the studies with A. besseyi and different plant hosts, it is difficult to reproduce the environmental conditions similar to those found in the field where this nematode is reported, especially under the operational and economic points of view. Considering these environmental conditions and the necessity in conducting studies under controlled environments with this pathossystem aiming a detailed investigation about the symptoms and the nematode parasitism, but also to isolate the effects due to exclusively the nematode parasitism instead of other effects that occur under field conditions, especially in crops like soybean, common bean, and cotton [1], [2], [3], [4], the objective of this project was to develop a growth chamber for the cultivation of these plants under controlled environmental, simulating the necessary conditions for the GFSR development. For this, we used an environmental chamber [5] as the base to our project, where we could find the important aspects that need to be adjusted to suit our purpose.•We developed a plant growth chamber to be used under greenhouse conditions for the studies with Aphelenchoides besseyi and different host plants.•The soybean green stem and foliar retention syndrome (GFSR) needs specific environmental conditions of humidity and temperature for the development of the characteristic symptoms and for the nematode multiplication in the parasitized plants.•This method simulates adequately the environmental conditions found in the field, since the chamber is installed inside a greenhouse, assuring the reliable observation of the plant behavior in relation to the pathogen and allowing the conduction of experiments of this nature in regions different from those where the disease naturally occurs.

TNFα deficiency results in increased IL-1ß in an early onset of spontaneous murine colitis.

Inflammatory bowel disease (Crohn's disease (CD) and ulcerative colitis (UC)) is a multifactorial disease resulting from immune dysregulation in the gut. The underlying colitis is characterized by high levels of inflammatory cytokines, including TNFα. Biological intervention for IBD patients using anti-TNFα antibodies is often an effective therapeutic solution. However, TNFα neutralization fails to induce remission in a subgroup of IBD patients, primarily in UC patients. There is a dearth of suitable animal models representing TNFα non-responders. Here we have combined one of the best UC models currently available, namely Winnie and the TNFαKO mouse to generate a TNFα-deficient Winnie to study early onset colitis. The induced TNFα deficiency with underlying colitis does not influence general health (viability and body weight) or clinical parameters (colon weight, colon length and histological colitis) when compared with the Winnie genotype alone. The molecular characterization resulted in identification of Il1ß as the major elevated cytokine during early phases of colitis. Further, in vitro functional assay using bone marrow-derived dendritic cells confirmed IL-1ß as the major cytokine released in the absence of TNFα. This study has generated a successful model of colitis that remains TNFα non-responsive and has demonstrated that IL-1ß expression is a major pathway for the progression of colitis in this system. These data also suggest that IL-1ß can be a potential target for clinical intervention of UC patients who fail to respond to TNFα neutralization.

[Polymorphism of KPI-A genes from plants of the subgenus Potatoe (sect. Petota, Estolonifera and Lycopersicum) and subgenus Solanum].

Kunitz-type proteinase inhibitor proteins of group A (KPI-A) are involved in the protection of potato plants from pathogens and pests. Although sequences of large number of the KPI-A genes from different species of cultivated potato (Solanum tuberosum subsp. tuberosum) and a few genes from tomato (Solanum lycopersicum) are known to date, information about the allelic diversity of these genes in other species of the genus Solanum is lacking. In our work, the consensus sequences of the KPI-A genes were established in two species of subgenus Potatoe sect. Petota (Solanum tuberosum subsp. andigenum--5 genes and Solanum stoloniferum--2 genes) and in the subgenus Solanum (Solanum nigrum--5 genes) by amplification, cloning, sequencing and subsequent analysis. The determined sequences of KPI-A genes were 97-100% identical to known sequences of the cultivated potato of sect. Petota (cultivated potato Solanum tuberosum subsp. tuberosum) and sect. Etuberosum (S. palustre). The interspecific variability of these genes did not exceed the intraspecific variability for all studied species except Solanum lycopersicum. The distribution of highly variable and conserved sequences in the mature protein-encoding regions was uniform for all investigated KPI-A genes. However, our attempts to amplify the homologous genes using the same primers and the genomes of Solanum dulcamarum, Solanum lycopersicum and Mandragora officinarum resulted in no product formation. Phylogenetic analysis of KPI-A diversity showed that the sequences of the S. lycopersicum form independent cluster, whereas KPI-A of S. nigrum and species of sect. Etuberosum and sect. Petota are closely related and do not form species-specific subclasters. Although Solanum nigrum is resistant to all known races of economically one of the most important diseases of solanaceous plants oomycete Phytophthora infestans aminoacid sequences encoding by KPI-A genes from its genome have nearly or absolutely no differences to the same from genomes of cultivated potatoes involved by P. infestans.

Single-molecule mechanical unfolding of amyloidogenic beta2-microglobulin: the force-spectroscopy approach.

The recombinant production of a novel chimeric polyprotein is described. The new protein contains either wild-type beta(2)-microglobulin (beta(2)m) or its truncated variant (DeltaN6 beta(2)m) (see picture). Structural characterization is achieved by means of single-molecule force spectroscopy studies of specific beta(2)m regions which could be involved in amyloidogenesis.

[Cloning of polygalacturonase inhibitor protein genes from Solanum brevidens Fill].

New data were obtained for the Solanum brevidens Fill. nucleotide sequences coding for polygalacturonase inhibitor proteins (PGIPs), which are involved in plant defense against phytopathogenic fungi. Highly degenerate primers directed to the conserved regions of the known PGIP genes of tomato, kiwi, apple, carrot, and grape were used to clone four pgip genes and one pseudogene from the genome of S. brevidens, a species that is closely related to cultivated potato, forms no tubers, is highly resistant to phytopathogens, and is often employed in potato breeding. The sequenced part of the coding region of the new genes is 924 bp and codes for a protein of 308 amino acid residues (without the leader peptide). The genes were designated as pgipSbr1(1), pgipSbr1 (2). pgipSbr2, pgipSbr3, and pgipSbr4. The amino acid sequences of the S. brevidens PGIPs have 90.9-99.4% identity to each other and 94% identity to PGIP of Lycopersicon esculentum Mill., another member of the family Solanaceae. The amino acid residues differing between S. brevidens PGIPs were assumed to determine the selectivity of interactions with particular polyglucuronases of phytopathogenic fungi.

Assessment of trichothecene chemotypes of Fusarium culmorum occurring in Europe.

Fusarium trichothecenes are a group of fungal toxic metabolites whose synthesis requires the action of gene products from three different genetic loci. We evaluated, both chemically and by PCR assays, 55 isolates of Fusarium culmorum from eight European countries and different host plants for their ability to produce trichothecenes. Specific sequences in the Tri6-Tri5 intergenic region were associated with deoxynivalenol production. Sequences in the Tri3 gene were also associated with deoxynivalenol production and specific primer sets were selected from these sequences to identify 3-acetyl-deoxynivalenol or 15-acetyl-deoxynivalenol chemotypes. Specific sequences in the Tri5 and Tri7 genes were associated with the nivalenol chemotype but not with the deoxynivalenol chemotype. Two chemotypes were identified by chemical analysis and confirmed by PCR. Strains of the nivalenol chemotype produced nivalenol (up to 260 microg g(-1)) and 4-acetyl-nivalenol (up to 60 microg g(-1)), strains with the 3-acetyl-deoxynivalenol chemotype produced deoxynivalenol (up to 1700 microg g(-1)) and 3-acetyl-deoxynivalenol (up to 600 microg g(-1)). Three strains of F. culmorum from France, previously reported as 15-acetyl-deoxynivalenol producers, had the 3-acetyl-deoxynivalenol chemotype. The results are consistent with data from other European countries on the occurrence of the nivalenol and 3-acetyl-deoxynivalenol chemotypes and provide support for the hypothesis that European isolates of F. culmorum producing deoxynivalenol belong only to the 3-acetyl-deoxynivalenol chemotype. The production of trichothecenes from F. culmorum isolates from walnut (3-acetyl-deoxynivalenol chemotype) and leek (nivalenol chemotype) is reported for the first time.

Advances on plant products with potential to control toxigenic fungi: a review.

In recent years, public pressure to reduce the use of synthetic fungicides in agriculture has increased. Concerns have been raised about both the environmental impact and the potential health risk related to the use of these compounds. Therefore, considerable efforts have been made towards the development of alternative crop protectants. The European Commission has been actively encouraging the development and commercial implementation of new compounds known as 'green chemicals'. In this context, an increase in the knowledge of plant defence responses to toxigenic fungi, which is covered in this review, will help to discover new plant products with antifungal activity and to design new strategies to improve plant resistance to these pathogens.

Molecular cloning of Kunitz-type proteinase inhibitor group B genes from potato.

Eighteen clones representing copies of four Kunitz-type proteinase inhibitor group B genes (PKPI-B) obtained by polymerase chain reaction cloning of potato (Solanum tuberosum L. cv. Istrinskii) genomic DNA were sequenced and analyzed. Three new genes were found and named PKPI-B1, PKPI-B2, and PKPI-B10: these were represented by five, two, and seven clones, respectively. The remaining four clones corresponded to the formerly characterized PKPI-B9 gene. These data show that at least four PKPI-B encoding genes are harbored in the genome of potato cv. Istrinskii. Their analysis suggests that variability of PKPI-B encoding genes in potato is limited and could be explained by cross-hybridization events in the ancestor forms rather than by random mutagenesis.

Liquid phase SPR imaging experiments for biosensors applications.

Surface plasmon resonance (SPR) has recently gained attention as a label-free method for the detection of biological molecules binding onto functionalised surfaces. It is one of the most sensitive detection method for monitor variations in the thickness and refractive index in ultra-thin films. Here, the adsorption processes of oligonucleotides onto gold substrates have been investigated in aqueous buffer solution using SPR imaging measurements. The hybridization of a thiol-modified, single stranded oligonucleotide anchored to a gold surface via thiol group, with its complementary sequence has been observed and characterised monitoring the hybridization process by SPR equipment. In situ investigation of smallest changes in SPR imaging measurements dynamically performed in liquid phase in the presence of DNA complementary probes was performed. Infrared spectroscopy and scanning electron microscopy characterisation of the functionalised gold surfaces of the biosensor were compared with the images obtained by SPR experimental apparatus.

Purification and characterisation of a beta-glucosidase abundantly expressed in ripe sweet cherry (Prunus avium L.) fruit.

A beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was purified to homogeneity from ripe fruits of sweet cherry (Prunus avium L.) by ammonium sulphate precipitation, ion exchange and size exclusion chromatography. The enzyme is a monomer with a molecular mass of approximately 68 kDa and an acidic isoelectric point. N-terminal sequence analysis indicated that sweet cherry beta-glucosidase is related to other plant cyanogenic beta-glucosidases. Substrate specificity studies revealed that the enzyme is able to attack and hydrolyse several synthetic substrates and total cell walls purified from ripe fruit. Biochemical and immunolocalisation studies showed that sweet cherry beta-glucosidases are mainly localised in the cytosol and in the apoplast, at the unripe stage of ripening; in ripe fruit it is also associated with cell wall.

Molecular cloning and biochemical characterization of a lipoxygenase in almond (Prunus dulcis) seed.

We have characterized an almond (Prunus dulcis) lipoxygenase (LOX) that is expressed early in seed development. The presence of an active lipoxygenase was confirmed by western blot analysis and by measuring the enzymatic activity in microsomal and soluble protein samples purified from almond seeds at this stage of development. The almond lipoxygenase, which had a pH optimum around 6, was identified as a 9-LOX on the basis of the isomers of linoleic acid hydroperoxides produced in the enzymatic reaction. A genomic clone containing a complete lipoxygenase gene was isolated from an almond DNA library. The 6776-bp sequence reported includes an open reading frame of 4667 bp encoding a putative polypeptide of 862 amino acids with a calculated molecular mass of 98.0 kDa and a predicted pI of 5.61. Almond seed lipoxygenase shows 71% identity with an Arabidopsis LOX1 gene and is closely related to tomato fruit and potato tuber lipoxygenases. The sequence of the active site was consistent with the isolated gene encoding a 9-LOX.

Recombinant lipoxygenases as biocatalysts for natural flavour production.

We identified a lipoxygenase expressed early during almond seed development. Biochemical and molecular characterisation showed that the enzyme produces almost exclusively 9-hydroperoxides which have been demonstrated to be important factors for the production of characteristic aromas in several fruits. An almond LOX cDNA was identified by RT-PCR using RNA extracted from immature almond seeds. Sequence analysis revealed that the identified gene is closely related to tomato fruit and potato tuber lipoxygenases. The isolated cDNA was cloned into pET24a and the expression of recombinant protein was induced in E. coli. The presence of an active LOX was confirmed in cells containing the recombinant vector. HPLC analysis of the reaction products of recombinant almond LOX confirmed that the isolated cDNA encodes a 9-LOX.