María Iranzo

Involvement of transglutaminase in the formation of covalent cross-links in the cell wall ofCandida albicans

Activity of the enzyme glutaminyl-peptide-γ-glutamylyl-transferase (EC 2.3.2.13; transglutaminase), which forms the interpeptidic cross-link N∈-(γ-glutamic)-lysine, was demonstrated in cell-free extracts obtained from both the yeast like and mycelial forms ofCandida albicans. Higher levels of enzymatic activity were observed in the cell wall fraction, whereas the cytosol contained only trace amounts of activity. Cystamine, a highly specific inhibitor of the enzyme, was used to analyze a possible role of transglutaminase in the organization of the cell wall structure of the fungus. Cystamine delayed protoplast regeneration and inhibited the yeast-to-mycelium transition and the incorporation of proteins into the cell wall. The incorporation of covalently bound high-molecular-weight proteins into the wall was sensitive to cystamine. Proteic epitopes recognized by two monoclonal antibodies, one of which is specific for the mycelial walls of the fungus, were also sensitive to cystamine. These data suggest that transglutaminase may be involved in the formation of covalent bonds between different cell wall proteins during the final assembly of the mature cell wall.

Bassianolone: an antimicrobial precursor of cephalosporolides E and F from the entomoparasitic fungus Beauveria bassiana.

We have established the chemical structure of (+)-bassianolone (3), the antimicrobial compound precursor of cephalosporolides E and F, and that of the furan metabolite 4 from the entomopathogenic fungus Beauveria bassiana.

Structural organization of the components of the cell wall from Candida albicans.

The organization of the components of the cell wall from Candida albicans was studied by means of sequential treatment with hot SDS, anhydrous ethylenediamine (EDA) and lytic enzymes, followed by chemical and microscopic analyses of the different separated fractions. The EDA-insoluble fraction retained the original morphology of the wall, which was destroyed by beta-glucanase, but not by chitinase treatments. Staining with fluorescent lectins revealed distinct distributions of mannoproteins, glucans and chitin in the wall. Amino acid analysis of SDS-extracted walls, and the EDA-soluble and -resistant fractions gave similar results, with seven amino acids making up about 70% of the total protein weight. Treatment of the EDA-insoluble fraction with Zymolyase or chitinase released fragments of variable size whose susceptibility to these and other hydrolases suggests that they are made of glucan, chitin and mannan oligomers associated with proteins. Treatment of the Zymolyase-insoluble residue with chitinase released a series of low-molecular-mass oligomers made of neutral sugars, GlcNAc and amino acids, mainly lysine. It is suggested that they represent fragments of the core making up the scaffold of the cell wall of the fungus.

The use of trypsin to solubilize wall proteins from Candida albicans led to the identification of chitinase 2 as an enzyme covalently linked to the yeast wall structure

The use of trypsin to break proteins covalently linked to the yeast walls of Candida albicans released approx. 50% of the proteins, but also glucose and N-acetylglucosamine. Analysis by affinity chromatography indicated that glucose and/or N-acetylglucosamine formed part of the same supramolecular complexes with mannoproteins. These complexes would represent a new type of cell wall structuration in which beta-1,6 glucan and chitin are linked to proteins. An internal peptide from a 50-kDa protein released by trypsin was sequenced, showing 100% identity with chitinase 2 protein and 92% with chitinase 3. The electrophoretic mobility of the chitinase 2 protein was changed by treatment with EndoH or beta-elimination, indicating that the enzyme was both N- and O-mannosylated.

Possible Roles of Mannoproteins in the Construction of Candida Albicans Cell Wall

The shape of Candida albicans cells depends on their cell walls and some of their mannoproteins may act as modulators of the final molecular architecture. If that were the case, the wall mannoproteins might form part of what could be called a “morphogenetic code”.

Study of supramolecular structures released from the cell wall of Candida albicans by ethylenediamine treatment

Candida albicans cell wall components were analyzed by ethylenediamine (EDA) treatment. Based on their different solubility properties, the cell wall components produced three fractions (A, B, and C). Fractions B (EDA-soluble, water-insoluble) and C (EDA-insoluble) contained glucan, chitin, and protein in different proportions. After zymolyase (mainly a β-glucanase complex) or chitinase treatment of fractions B and C, more polysaccharides and proteins were solubilized by a second EDA treatment, suggesting that the solubility of the polymers in EDA depends on the degree of polymer interactions. Western blot analysis using two monoclonal antibodies (1B12 and 4C12) revealed electrophoretic patterns that were similar in mycelial and yeast morphologies, except that in material obtained from mycelial walls, an additional band was detected with MAb 1B12. Fluorescence microscopy of cell wall fractions treated with FITC-labeled Con-A, Calcofluor white, and FITC-labeled agglutinin showed that glucan and mannoproteins are uniformly distributed in fractions B and C, while chitin is restricted to distinct patches. Transmission electron microscopy demonstrated that fraction C maintained the original shape of the cells, with an irregular thickness generally wider than the walls. When fraction C was treated with chitinase, the morphology was still present and was maintained by an external glucan layer, with an internal expanded fibrillar material covering the entire cellular lumen. Degradation of the glucan skeleton of fraction C with zymolyase resulted in the loss of the morphology.

Effects of inspiratory muscle training and yoga breathing exercises on respiratory muscle function in institutionalized frail older adults: a randomized controlled trial.

Background:In older adults, respiratory function may be seriously compromised when a marked decrease of respiratory muscle (RM) strength coexists with comorbidity and activity limitation. Respiratory muscle training has been widely studied and recommended as a treatment option for people who are unable to participate in whole-body exercise training (WBET); however, the effects of inspiratory muscle training and yoga breathing exercises on RM function remain unknown, specifically in impaired older adults. Purpose:To evaluate the effects of inspiratory threshold training (ITT) and yoga respiratory training (YRT) on RM function in institutionalized frail older adults. Methods:Eighty-one residents (90% women; mean age, 85 years), who were unable to perform WBET (inability to independently walk more than 10 m), were randomly assigned to a control group or one of the 2 experimental groups (ITT or YRT). Experimental groups performed a supervised interval-based training protocol, either through threshold inspiratory muscle training device or yoga breathing exercises, which lasted 6 weeks (5 days per week). Outcome measures were collected at 4 time points (pretraining, intermediate, posttraining, and follow-up) and included the maximum respiratory pressures (maximum inspiratory pressure [MIP] and maximum expiratory pressure [MEP]) and the maximum voluntary ventilation (MVV). Results:Seventy-one residents completed the study: control (n = 24); ITT (n = 23); YRT (n = 24). The treatment on had a significant effect on MIP YRT (F6,204 = 6.755, P < .001, &eegr;2 = 0.166), MEP (F6,204 = 4.257, P < .001, &eegr;2 = 0.111), and MVV (F6,204 = 5.322, P < .001, &eegr;2 = 0.135). Analyses showed that the YRT group had a greater increase of RM strength (MIP and MEP) and endurance (MVV) than control and/or ITT groups. Conclusion:Yoga respiratory training appears to be an effective and well-tolerated exercise regimen in frail older adults and may therefore be a useful alternative to ITT or no training, to improve RM function in older population, when WBET is not possible.

Maximal respiratory pressure reference values for Navajo children ages 6–14

Since anthropometric variables are critical to the creation of pulmonary nomograms for FVC, FEV1, and other volumes and capacities, it is logical that anthropometric variables also influence the values of the maximal respiratory pressures (MRPs). Since nomograms are race‐specific, it is important that tribe‐specific tables of normal maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP) be developed. To date normal tables for MRPs do not exist for Navajo children.

The SRD2 gene is involved in Saccharomyces cerevisiae morphogenesis

Saccharomyces cerevisiae presents two alternative vegetative forms of growth, switching between yeast forms to pseudohyphal forms depending on the specific environmental conditions. To identify genes involved in cell wall morphogenesis, a haploid S. cerevisiae monomorphic mutant, W27, which exhibits pseudohyphal growth in the absence of the normal external signals that induce the formation of filamentous forms, was characterized. S. cerevisiae W27 did not demonstrate agar-invasive growth, a characteristic of most filamentous strains. The mutant wall had no obvious alterations with respect to mannan and glucan content, but had three times more chitin than the parental strain. This produced an increase in the amount of proteins linked covalently to chitin. The same protein species, however, were released from the cell walls of the mutant and the parental strain. The W27 mutation was complemented with a genomic library and the SRD2/ECM23 gene was identified as the complementing ORF. Transformation of S. cerevisiae W27 with the Ycplac33 vector carrying the SRD2 gene produced the original phenotype. These results suggest that the SRD2 gene acts as a negative regulator of pseudohyphal growth.

Reaggregation and binding of cell wall proteins from Candida albicans to structural polysaccharides

Urea or hot sodium dodecyl sulphate extracted a significant amount of the same proteins from the matrix of the cell wall of the yeast form and mycelial cells of Candida albicans. Gel filtration analysis of the urea-extracted proteins revealed that they occurred in the form of large complexes which were unaffected by up to 8 M urea. Among them, proteins en route to becoming covalently associated within the wall scaffold were identified by their reaction with specific antibodies. When urea was removed by dialysis, some of these proteins specifically reassociated into large aggregates which bound strongly with ConA, whereas others remained soluble in smaller associated products. The ability of some of these proteins to bind to the insoluble wall polysaccharides was also assessed. No self-assembling proteins were able to bind to glucans and/or chitin. Specificity of the binding to polysaccharides made of beta-bound glucosyl or N-acetylglucosaminyl residues was determined by the competitive effect of several disaccharides. Whereas laminaribiose and diacetylchitobiose were strong inhibitors of protein binding to both glucan and chitin, lactose, maltose and sucrose were ineffective.