Raw cow milk bacterial population shifts attributable to refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.     

Description of two new species of Glypthelmins Stafford, 1905 (Digenea: Macroderoididae) in Rana spp. from Mexico, based on morphology and mtDNA and rDNA sequences

Glypthelmins Stafford, 1905 includes 29 putative species commonly found in the intestine and liver of anurans from all over the world but mainly in the Americas. Partial sequences of the cytochrome c oxidase subunit 1 ( cox 1), ribosomal internal transcribed spacer region 2 (ITS2) and the large subunit 28S rDNA gene were obtained and analysed using pairwise distance matrices and parsimony methods in order to characterise the interrelationships between 14 isolates of four nominal species of Glypthelmins recognised on morphological grounds. The highest intra-specific sequence divergence occurred in the cox 1 (18.53%) sequence, followed by that of the ITS2 (5.44%) and 28S (4.63%). Genetic variability was detected between the three isolates originally identified as G. facioi Brenes et al., 1959 from two localities in Mexico and one locality in Costa Rica. Sequence divergence exhibited among these isolates ranged from 10.70 to 11.22%, from 0.48 to 0.97% and from 1.33 to 1.88% for cox 1, ITS2 and 28S, respectively. Phylogenetic analysis combining all three data-sets generated a single most parsimonious tree. The three isolates of G. facioi form a clade, with an isolate collected from frogs in Veracruz State as the sister group to an isolate from Tabasco State + G. facioi from Costa Rica. The information derived from pairwise distance of independent data-sets plus the phylogenetic information indicate that each of the two isolates from Mexico, identified a priori as G. facioi, represent separate species. A re-examination of specimens was carried out and a re-evaluation made of the morphological characters to find reliable differences that had been overlooked. As a consequence, G. brownorumae n. sp. from Tabasco and G. tuxtlasensis n. sp. from Veracruz are described based on molecular and morphological differences.     

A unigene catalogue of 5700 expressed genes in cassava

Two economically important characters, starch content and cassava bacterial blight resistance, were targeted to generate a large collection of cassava ESTs. Two libraries were constructed from cassava root tissues of varieties with high and low starch contents. Other libraries were constructed from plant tissues challenged by the pathogen Xanthomonas axonopodis pv.manihotis. We report here the single pass sequencing of 11 954 cDNA clones from the 5’ ends, including 111 from the 3’ ends. Cluster analysis permitted the identification of a unigene set of 5700 sequences. Sequence analyses permitted the assignment of a putative functional category for 37% of sequences whereas ~ 16% sequences did not show any significant similarity with other proteins present in the database and therefore can be considered as cassava specific genes. A group of genes belonging to a large multigene family was identified. We characterize a set of genes detected only in infected libraries putatively involved in the defense response to pathogen infection. By comparing two libraries obtained from cultivars contrasting in their starch content a group of genes associated to starch biosynthesis and differentially expressed was identified. This is the first large cassava EST resource developed today and publicly available thus making a significant contribution to genomic knowledge of cassava.     

Effects of a chromogranin-derived peptide (CgA 47-66) in the writhing nociceptive response induced by acetic acid in rats

Chromogranin A (CgA) is an acidic protein identified within a large variety of endocrine cells. Colocalized with catecholamines in chromaffin cells, CgA is a prohormone precursor of small biologically active peptides. Vasostatin (CgA 1-76) is the most conserved fragment of CgA and chromogranin A 47-66 peptide (CgA 47-66) possesses potent antimicrobial activities. The aim of this study was to test the hypothesis that CgA 47-66 may be involved in mechanisms modulating nociception. Thus, we used acetic acid (AA) which produces a delayed inflammatory response and episodes of abdominal writhing, a marker of pain, when injected intraperitoneally (i.p.) to rats. Administration (i.p.) of CgA 47-66 induced specific opposite dose-dependent effects depending on concentration. That is, CgA 47-66 below 0.5 mg/kg produced antinociceptive effects, whereas at 2 mg/kg it produced a marked pronociceptive effect. The latter effect was blocked by diltiazem and indomethacin. CgA 47-66-induced antinociceptive effects on AA-induced responses were reversed when the corticotropin-releasing factor (CRF) antagonist alpha-helical CRF 9-41 was i.p. injected to animals prior to AA and CgA 47-66 administration. The administration of i.p. calcitonin gene-related peptide (CGRP) or substance P (SP) evoked dose-dependent abdominal writhing; this effect was abolished when CgA 47-66 was injected. The present data suggest, for the first time, that a fragment of CgA, CgA 47-66, possesses potent antinociceptive effects at low doses. Although the mechanism triggered by this peptide is unknown, CRF receptors are likely to be involved.     

Production of highly 13C-labeled polyphenols in Vitis vinifera cell bioreactor cultures

The use of Vitis vinifera cells grown in a 2 l-stirred tank bioreactor for producing isotopically 13C-labeled phenolic substances is presented. Several culture parameters were optimized to achieve characteristics of growth and polyphenol metabolism similar to that recorded in shake flasks. Administration of [1-13C]L-phenylalanine (3 mM) to grape cell suspension cultures led to the production of 13C-labeled stilbenes (trans- and cis-piceids), catechins (catechin and epicatechin) and anthocyanins (delphinidin-, cyanidin-, petunidin-, peonidin- and malvidin-3-O-beta-glucosides). Incorporation of [1-13C]L-phenylalanine into polyphenols was measured by means of 13C satellites in the proton NMR spectrum and EA-IRMS. The enrichment of labeling obtained for all the compounds (between 40 and 65%) is sufficient to investigate their absorption and metabolism in humans.     

Integrated control of Boophilus microplus ticks in Cuba based on vaccination with the anti-tick vaccine Gavac TM

Boophilus microplus has developed resistance against a range of chemical acaricides which has stimulated the development of alternative methods such as vaccination against ticks. In Cuba, the Bm86-based recombinant vaccine Gavac^TM has been successfully used in a number of controlled laboratory and field trials in cattle against B. microplus. In this paper, we have evaluated Gavac^TM in a large scale field trial wherein 588,573 dairy cattle were vaccinated with the aim to reduce the number of acaricidal treatments. It was found that the number of acaricidal treatments could be reduced by 87% over a period of 8 years (1995–2003). Prior to the introduction of the vaccine, 54 clinical cases of babesiosis and six fatal cases were reported per 1000 animals. Six years later, the incidence of babesiosis was reduced to 1.9 cases per 1000 cattle and mortality reduced to 0.18 per 1000. The national consumption of acaricides in Cuba could be reduced by 82% after the implementation of the integrated anti-B. microplus control program.     

What is genetic quality?

Mate choice is favored by indirect selection if choosy females mate with males of high genetic quality. We believe, however, that testing hypotheses about indirect selection has been constrained by how we conceptualize and therefore empirically measure male genetic quality. Here, we argue that genetic quality is the breeding value of an individual for total fitness. We can therefore learn little about genetic quality from measures of only a few fitness components. We explain breeding value for total fitness, drawing on concepts from life-history theory and quantitative genetics, and suggest how approaches incorporating these insights might result in empirical progress.     

Effects induced by mono- and divalent cations on protein regions responsible for thermal adaptation in β-glycosidase from<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>

The perturbation induced by mono- and divalent cations on the thermophilicity and thermostability of Solfolobus solfataricus -glycosidase, a hyperthermophilic tetrameric enzyme, has been investigated by spectroscopic and computational simulation methods to ascertain the Hofmeister effects on two strategic protein regions identified previously. Specifically, (1) an extra segment (83–124), present only in the sequence of hyperthermophilic glycosidases and recognized as an important thermostability determinant for the enzyme structure; and (2) a restricted area of the subunit interface responsible for the quaternary structure maintenance. Mono- and divalent cations inhibit to a different extent the -glycosidase activity, whose kinetic constants show an apparent competitive inhibition of the catalytic process that reflects the Hofmeister order. The thermostability is also affected by the nature and charge of the cations, reaching maximal effects for the case of Mg²⁺. Fourier transform infrared spectroscopy has revealed very small changes in the protein secondary structure in the presence of the investigated cations at 20 °C, while large effects on the protein melting temperatures are observed. Computational analysis of the enzyme structure has identified negative patches on the accessible surface of the two identified regions. Following the Hofmeister series, cations weaken the existing electrostatic network that links the extra segment to the remaining protein matrix. In particular, the perturbing action of cations could involve the ionic pair interactions E107–R245 and E109–R185, thus leading to a local destructuring of the extra segment as a possible starting event for thermal destabilization. A detailed investigation of the electrostatic network at the A–C intermolecular interface of Sgly after energy minimization suggests that cations could cause a strong attenuation of the ion pair interactions E474–K72 and D473–R402, with consequent partial dissociation of the tetrameric structure.Abbreviations Amide I amide I band in a ²H₂O medium - EM energy minimization - ONPG o-nitrophenyl--d-galactopyranoside - Sgly Escherichia coli expressed Sulfolobus solfataricus -glycosidase     

Effects of corticosterone on muscle mitochondria identifying different sensitivity to glucocorticoids in Lewis and Fischer rats

Previous studies in rat have demonstrated decreased number of mitochondria and uncoupling of oxidative phosphorylation after administration of glucocorticoids but at supraphysiological doses and using synthetic glucocorticoids. To analyze the relationships between corticosterone levels (the natural glucocorticoid in rat) and muscle mitochondrial metabolism, Lewis and Fischer 344 rats were bilaterally adrenalectomized and implanted with different corticosterone pellets (0, 12, 50, 100, and 200 mg of corticosterone). Rats bearing a corticosterone pellet delivering corticosterone at concentrations in the range of chronic stress-induced levels presented a lower amount of functional muscle mitochondria with a decrease in cytochrome c oxidase and citrate synthase activities and a depletion of mitochondrial DNA. Moreover, a strain difference in tissue sensitivity to corticosterone was depicted both in end-organ sensitive to glucocorticoids (body, thymus, and adrenal weights) and in muscle mitochondrial metabolism (Lewis > Fischer). Interestingly, this strain difference was also observed in the absence of corticosterone, with a deleterious effect on muscle mitochondrial metabolism in Fischer rats, whereas no effects were observed in Lewis rats. We therefore postulate that corticosterone is necessary for muscle mitochondrial metabolism exerting its effects in Fischer rats with an inverted U curve, whereby too little (only Fischer) or too much (Fischer and Lewis) corticosterone is deleterious to muscle mitochondrial metabolism. In conclusion, we propose a general model of coordinate regulation of mitochondrial energetic metabolism by glucocorticoids.     

Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis

Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and immunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.