Peptide mapping of β-casein by capillary zone electrophoresis

A method to the study of -casein proteolysis by aspartic proteinases is developed. The 3% trichloroacetic acid-soluble peptides of -casein digested with cardosin A were separated by capillary zone electrophoresis under different experimental conditions in an uncoated fused silica capillary. The best separation was at pH 3.01, 30 kV and 30 °C.     

Capillary electrophoresis of caseino-peptides: influence of applied voltage and column temperature

The peptides formed upon action of purified cardoon rennet on the Ala₁₈₉-Phe₁₉₀-Leu₁₉₁-Leu₁₉₂-Tyr₁93 -casein fragment were separated by capillary electrophoresis in an uncoated fused silica capillary. There was a linear correlation between electrophoretic mobility and Z/M^2/3 (Z, net charge; M, molecular mass) under all experimental conditions tested; under the optimal condicitions, 25 kV and 40 °C, the correlation coefficient was 0.994. The reported method is fast (migration times less than 7 min) and may be used to study the action of aspartic proteinases on the C-terminal domain of -casein and thus to help elucidate their effect on cheese quality.     

Genetic diversity and differentiation in Portuguese cattle breeds using microsatellites

Genotype data from 30 microsatellites were used to assess genetic diversity and relationships among 10 native Portuguese cattle breeds, American Charolais and the Brazilian Caracú. Hardy–Weinberg equilibrium was observed for all loci/population combinations except for five loci in Brava de Lide and one locus in Alentejana that exhibited heterozygote deficiency. Estimates of average observed and expected heterozygosities, total number of alleles (TNA) per breed and mean number of alleles (MNA) per locus/population were obtained. A total of 390 alleles were detected. TNA among Iberian cattle ranged from 170 to 237 and MNA ranged from 5.67 to 8.07. The highest observed heterozygosities were found in the Caracú, Maronesa, Garvonesa and Arouquesa and the lowest in Brava de Lide and Mirandesa. Estimation of population subdivision using Wright's F_ST index showed that the average proportion of genetic variation explained by breed differences was 9%. Neighbour‐joining phylogenetic trees based on D_A distances showed that the genetic relationships of present‐day Portuguese native breeds are consistent with historical origins in the Brown Concave (Arouquesa, Mirandesa, Marinhoa) and Red Convex (Mertolenga, Alentejana, Garvonesa, Minhota) evolutionary groups. The Iberian Black Orthoide group, represented by Brava de Lide and Maronesa, and the Barrosã breed appeared to be more closely related to the Brown Concave group but may represent a separate lineage. The Caracú breed was not found to be closely associated with any of the native Portuguese breeds.     

Pulmonary aspergillosis outbreak in Rhea americana in Southern Brazil

Commercial raising of rheas is currently in expansion in the south of Brazil, and many diseases previously restricted to other avian species are currently emerging on rhea farms, especially as a result of careless management of these animals. The objective of the present article is to report a pulmonary aspergillosis outbreak that occurred in great rhea (Rhea americana) in the south of Brazil. About 50 birds aged 30 to 60 days died suddenly and one of them was submitted to autopsy which revealed the presence of white caseous nodules 0.5 mm in diameter occupying 95% of the lung area. One lung was sent to the Federal University of Santa Maria for histopathological and mycological analyses. Histopathological analysis revealed multifocal areas with necrosis and inflammatory infiltrates and the presence of fungal hyphae, giant cells and fibrous tissue proliferation at the periphery. Aspergillus fumigatus was recovered as pure culture from all culture media. This appears to be the first report of aspergillosis among great rhea in Brazil and the second in the world.     

Relationships in subtribe Diocleinae (Leguminosae; Papilionoideae) inferred from internal transcribed spacer sequences from nuclear ribosomal DNA

The complete sequences of nuclear ribosomal DNA (nrDNA) internal transcribed spacer regions (ITS/5.8S) were determined for species belonging to six genera from the subtribe Diocleinae as well as for the anomalous genera Calopogonium and Pachyrhizus. Phylogenetic trees constructed by distance matrix, maximum parsimony and maximum likelihood methods showed that Calopogonium and Pachyrhizus were outside the clade Diocleinae (Canavalia, Camptosema, Cratylia, Dioclea, Cymbosema, and Galactia). This finding supports previous morphological, phytochemical, and molecular evidence that Calopogonium and Pachyrhizus do not belong to the subtribe Diocleinae. Within the true Diocleinae clade, the clustering of genera and species were congruent with morphology-based classifications, suggesting that ITS/5.8S sequences can provide enough informative sites to allow resolution below the genus level. This is the first evidence of the phylogeny of subtribe Diocleinae based on nuclear DNA sequences.     

Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus

An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml(-1) in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter(-1) was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml(-1). These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter(-1) when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml(-1) (i.e., 360 mg liter(-1)) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter(-1). In this case, maximal activities were 3,900 and 4,660 nkat ml(-1), respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.     

Simultaneous production of triploid and haploid eggs by triploid Squalius alburnoides (Teleostei: Cyprinidae)

The hybrid minnow Squalius alburnoides comprises diploid and polyploid forms with altered modes of reproduction. In the present paper, we report a cross where a triploid female generated both large, triploid and small, haploid eggs simultaneously, which were fertilized with S. pyrenaicus sperm. Although the large eggs were rarer (15%), they originated offspring with higher survivorship, so that tetraploids were dominant among the surviving siblings. The cross yielded apparently all female progeny. Inheritance patterns were inferred using four microsatellite markers and NORs (Nucleolus Organizer Regions) phenotypes, and suggested that haploid eggs were probably produced by an atypical hybridogenesis, in which the elimination of the unmatched genome permitted random segregation and recombination between the homospecific genomes, while the triploid eggs were clonal. The present results suggest that the occurrence of triploid unreduced eggs may be a new route for the natural tetraploidization in the complex.     

A light in multidrug resistance: Photodynamic treatment of multidrug-resistant tumors

The major drawback of cancer chemotherapy is the development of multidrug-resistant (MDR) tumor cells, which are cross-resistant to a broad range of structurally and functionally unrelated agents, making it difficult to treat these tumors. In the last decade, a number of authors have studied the effects of photodynamic therapy (PDT), a combination of visible light with photosensitizing agents, on MDR cells. The results, although still inconclusive, have raised the possibility of treating MDR tumors by PDT. This review examines the growing literature concerning the responses of MDR cells to PDT, while stressing the need for the development of new photosensitizers that possess the necessary characteristics for the photodynamic treatment of this class of tumor.