Pectinolytic enzymes secreted by yeasts from tropical fruits

Three hundred yeasts isolated from tropical fruits were screened in relation to secretion of pectinases. Twenty-one isolates were able to produce polygalacturonase and among them seven isolates could secrete pectin lyase. None of the isolates was able to secrete pectin methylesterase. The pectinolytic yeasts identified belonged to six different genera. Kluyveromyces wickerhamii isolated from the fruit mangaba (Hancornia speciosa) secreted the highest amount of polygalacturonase, followed by K. marxianus and Stephanoascus smithiae. The yeast Debaryomyces hansenii produced the greatest decrease in viscosity while only 3% of the glycosidic linkages were hydrolysed, indicating that the enzyme secreted was an endo-polygalacturonase. The hydrolysis of pectin by polygalacturonase secreted by S. smithiae suggested an exo-splitting mechanism. The other yeast species studied showed low polygalacturonase activity.     

Proteome analysis for the identification of in vivo estrogen-regulated proteins in bone

Estrogen deficiency results in a reduced bone mass, which can be prevented by treatment with estrogens. This study used a proteomic approach for the first time to obtain a global perspective of estrogens' effects on whole-bone proteins. Bone proteome profiles were examined in three groups of mice: (1) sham-operated with normal ovarian functions, (2) ovariectomised and (3) ovariectomised with estrogen replacement therapy. Bone proteins extracted from the humerus were separated by 2-DE and visualised by CBB colloidal staining. Spot detection and quantification was done by image analysis. Differentially expressed proteins were identified by MS and database search, using peptide mass fingerprint and peptide sequence analysis. Differential expression analysis in the three experimental groups showed significant changes for 14 proteins. These included proteins related to bone metabolism, cytoskeleton components and energy metabolic pathways. Our data suggest that some proteins related to cytoskeleton and to energy pathways, such as tropomyosins, aconitase 2 and enolase beta, might be new molecular targets responsive to the effects of estrogen. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the pleiotropic effects of estrogens on bone.     

Alpha1- and alpha2-adrenoreceptor antagonist profiles of 1- and 2-[omega-(4-arylpiperazin-1-yl)alkyl]-1,2,3-benzotriazoles

A series of pharmacologically interesting 1- and 2-[omega-(4-arylpiperazin-1-yl)alkyl]-1,2,3-benzotriazoles, compounds 1-27, were synthesized (Scheme) and subjected to various biological studies to identify structure-activity relationships (SAR). The new compounds were found to exhibit good non-selective binding affinity towards the alpha1-adrenoreceptor (Table 1). In several cases, high functional antagonism was observed towards the alpha1A-, alpha1B-, and alpha1D-adrenoreceptor subtypes (Table 2). The selectivity for these three subtypes was comparable with or superior to that displayed by the standard drug prazosin. The most-common selectivity rank order was alpha1D > alpha1B > alpha1A, followed by alpha1B > alpha1D > alpha1A. In functional experiments, antagonism towards the alpha2-adrenoreceptor was generally low; however, a few compounds were endowed with significant antagonist properties (pA2 values of up to 7.87).     

Antioxidant defences in cybrids harboring mtDNA mutations associated with Leber's hereditary optic neuropathy

Oxidative stress and imbalance between free radical generation and detoxification may play a pivotal role in the pathogenesis of Leber's hereditary optic neuropathy (LHON). Mitochondria, carrying the homoplasmic 11778/ND4, 3460/ND1 and 14484/ND6 mtDNA point mutations associated with LHON, were used to generate osteosarcoma-derived cybrids. Enhanced mitochondrial production of reactive oxygen species has recently been demonstrated in these cybrids [Beretta S, Mattavelli L, Sala G, Tremolizzo L, Schapira AHV, Martinuzzi A, Carelli V & Ferrarese C (2004) Brain 127, 2183-2192]. The aim of this study was to characterize the antioxidant defences of these LHON-affected cells. The activities of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutases (SOD) and catalase, and the amounts of glutathione (GSH) and oxidized glutathione (GSSG) were measured in cybrids cultured both in glucose-rich medium and galactose-rich medium. The latter is known to cause oxidative stress and to trigger apoptotic death in these cells. In spite of reduced SOD activities in all LHON cybrids, and of low GPx and GR activities in cells with the most severe 3460/ND1 and 11778/ND4 mutations, GSH and GSSG content were not significantly modified in LHON cybrids cultured in glucose medium. In contrast, in galactose, GSSG concentrations increased significantly in all cells, indicating severe oxidative stress, whereas GR and MnSOD activities further decreased in all LHON cybrids. These data suggest that, in cells carrying LHON mutations, there is a decrease in antioxidant defences, which is especially evident in cells with mutations associated with the most severe clinical phenotype. This is magnified by stressful conditions such as exposure to galactose.     

Synthesis, antimicrobial activity and molecular modeling studies of halogenated 4-[1H-imidazol-1-yl(phenyl)methyl]-1,5-diphenyl-1H-pyrazoles

During the course of our studies in the azole antifungals area, we synthesized a number of 1,5-disubstituted 4-[1H-imidazol-1-yl(phenyl)methyl]-1H-pyrazoles, analogues of bifonazole. 1,5-Diphenyl-1H-pyrazole 3 showed weak antimycotic and antibacterial activities in vitro against Candida albicans, Cryptococcus neoformans and Staphylococcus aureus. In order to increase these properties, given that the halo substitution was found to be capable of enhancing antifungal effects, we prepared a series of fluoro and chloro derivatives of 3. The microbiological evaluation carried out on newly synthesized compounds included in vitro assays for antifungal, antibacterial and antimycobacterial activities. Among the tested compounds, some dichloro and trichloro-derivatives showed interesting antimicrobial properties. In particular, compounds 10j,k,l produced inhibitory effects against pathogen representatives of yeast (C. albicans, C. neoformans) and Gram positive bacteria (S. aureus) similar or superior to those of bifonazole. In addition, their activity against Mycobacterium tuberculosis was superior to that of clotrimazole and econazole, which were used as reference drugs. The replacement, in these compounds, of chlorine with fluorine atoms led to inactive derivatives. Docking studies were carried out on the most active compounds, in order to rationalize the pharmacological results.