Identification of capsule, biofilm, lateral flagellum, and type IV pili in Vibrio mimicus strains.

Vibrio mimicus is a bacterium that causes gastroenteritis; it is closely related to Vibrio cholerae, and can cause acute diarrhea like cholera- or dysentery-type diarrhea. It is distributed worldwide. Factors associated with virulence (such as hemolysins, enterotoxins, proteases, phospholipases, aerobactin, and hemagglutinin) have been identified; however, its pathogenicity mechanism is still unknown. In pathogenic Vibrio species such as V. cholerae, Vibrio. parahaemolyticus and Vibrio vulnificus, capsule, biofilms, lateral flagellum, and type IV pili are structures described as essential for pathogenicity. These structures had not been described in V. mimicus until this work. We used 20 V. mimicus strains isolated from water (6), oyster (9), and fish (5) samples and we were able to identify the capsule, biofilm, lateral flagellum, and type IV pili through phenotypic tests, electron microscopy, PCR, and sequencing. In all tested strains, we observed and identified the presence of capsular exopolysaccharide, biofilm formation in an in vitro model, as well as swarming, multiple flagellation, and pili. In addition, we identified homologous genes to those described in other bacteria of the genus in which these structures have been found. Identification of these structures in V. mimicus is a contribution to the biology of this organism and can help to reveal its pathogenic behavior.

Effects of heavy metals on the oyster (Crassostrea virginica) at Mandinga Lagoon, Veracruz, Mexico.

The Mandinga Lagoon in the Mexican State of Veracruz is an important ecological zone that produces 32% of the oyster output in the state of Veracruz, the main oyster producer in Mexico. Samples of water, sediment, and oysters were collected in 2003 and 2004 to study heavy metal pollution. Metal concentrations were determined in water, soil, and oyster tissues from fresh and detoxified Crassostrea virginica, and histology samples were analyzed. Metal (Cr, Cd, and Pb) concentrations in water were within the Mexican legal limits. The recorded values in sediments corresponded to those not producing biological effects (ERL). In the tissues, the highest concentrations corresponded to Pb, above 5.84 microgg(-1) dry weight (d.w.); Cd was of 2.23 microgg(-1) d.w., and Cr above 6 microgg(-1) d.w. The metal levels detected in oysters exceeded the maximum permissible limits (MPL) for Cd and Pb, and oysters were unable to eliminate the concentrations of the bioaccumulated metals during the detoxification stage. The histopathological analysis revealed lesions in the digestive gland, edema, atrophy of epithelia in the digestive tubules, the presence of brown vesicles, hemocytic reaction, and necrosis. During detoxification, a higher number of epithelia were observed in the tubules, as well as an increase in brown vesicles and hemocytic reaction. Forty seven percent of oysters presented histopathological lesions related to metal concentrations. It is important to monitor metal concentrations, to detect the source of pollution, andto evaluate the effects on organisms to establish culture areas and adequate criteria for the exploitation of such an important fishery resource.

Effects of moderate and severe malnutrition in rats on splenic T lymphocyte subsets and activation assessed by flow cytometry.

Malnutrition is distributed widely throughout the world and is a particular problem in developing countries. Laboratory animals have been very useful in studying the effects of varying levels of malnutrition because non-nutritional factors that affect humans may be controlled. The objective of the present study was to determine the effects of moderate and severe malnutrition on lymphocyte proportions and activation markers of T cells in experimentally malnourished rats during lactation by flow cytometry. Lower absolute (total) and relative (%) numbers of CD3+ and CD4+ lymphocyte subpopulations were observed in moderately (second degree) and severely (third degree) malnourished rats compared with well-nourished rats (P < 0.05). Both groups of malnourished rats showed a significant decrease in the percentage of CD71+ cells at 24 h post-activation with phytohaemagglutinin (PHA). After 24 h activation of spleen cells with PHA, a lower percentage of CD25+ cells was observed in malnourished than well-nourished rats (P < 0.05). In conclusion, the results of this study indicated an altered expression of CD71 and CD25 during activation of T lymphocytes in malnourished rats and may partially explain increased susceptibility to infection associated with malnutrition. Moreover, these results demonstrated that moderate malnutrition affects the response of T lymphocytes as much as severe malnutrition.

Effect of perfluorooctane sulfonate on viability, maturation and gap junctional intercellular communication of porcine oocytes in vitro.

Perfluorooctane sulfonate (PFOS) is a broadly used man-made surfactant whose long half-life has led to bioaccumulation. This perfluorinated compound is ubiquitous in human body fluids. PFOS concentrations as high as 26µM in plasma have been reported in occupationally exposed populations, and high levels of PFOS in human follicular fluid have been associated with subfertility. However, the effect of PFOS on the maturation of oocytes in mammals has not been reported to date. The aim of this study was to determine the effects of PFOS during oocyte maturation. Results indicate that PFOS inhibits oocyte viability (Lethal Concentration50=32µM) and maturation (inhibition of maturation50=22µM) at physiologically relevant concentrations. In order to evaluate the mechanisms of oocyte maturation inhibition by PFOS, gap junctional intercellular communication (GJIC) between oocytes and granulosa cells was assessed. GJIC between granulosa cells and the oocyte was significantly affected during the first 8h of maturation. However, the inhibitory effect of PFOS on GJIC was not due to an alteration on the expression of connexin genes Cx43, Cx45 and Cx60. These findings suggest that occupationally exposed populations could be at risk, and that PFOS might affect oocyte maturation by interfering the GJIC in the cumulus-oocyte complexes during the first hours of maturation.

Antimicrobial resistance and characterization of Helicobacter pylori strains isolated from Mexican adults with clinical outcome.

Eradication of Helicobacter pylori infection in Mexico is of great importance due to the elevated seroprevalence, however, there is yet very little information about antibiotic resistance rates in H. pylori isolates in our country. We analyzed susceptibility to three antimicrobials used in therapy of 49 H. pylori strains isolated from patients with active chronic gastritis, active chronic gastritis with lymphoid follicles, intestinal metaplasia and gastric cancer. All isolated strains were susceptible to amoxicillin, 28 (58%) were resistant to metronidazole and 2 (4%) were resistant to both clarithromycin and metronidazole. Sequence analysis of the 23S rRNA of the two clarithromycin-resistant strains showed the A2142G mutation in one and A2143G and T2182C mutations in the other. Metronidazole resistance was associated with cagA negative strains with a frequency of 82% (9/11). No significant correlation was found between vacA s/m alleles and metronidazole resistance.

Mouse models for the study of postnatal cardiac hypertrophy.

The main objective of this study was to create a postnatal model for cardiac hypertrophy (CH), in order to explain the mechanisms that are present in childhood cardiac hypertrophy. Five days after implantation, intraperitoneal (IP) isoproterenol (ISO) was injected for 7 days to pregnant female mice. The fetuses were obtained at 15, 17 and 19 dpc from both groups, also newborns (NB), neonates (7-15 days) and young adults (6 weeks of age). Histopathological exams were done on the hearts. Immunohistochemistry and western blot demonstrated GATA4 and PCNA protein expression, qPCR real time the mRNA of adrenergic receptors (α-AR and ß-AR), alpha and beta myosins (α-MHC, ß-MHC) and GATA4. After the administration of ISO, there was no change in the number of offsprings. We observed significant structural changes in the size of the offspring hearts. Morphometric analysis revealed an increase in the size of the left ventricular wall and interventricular septum (IVS). Histopathological analysis demonstrated loss of cellular compaction and presence of left ventricular small fibrous foci after birth. Adrenergic receptors might be responsible for changing a physiological into a pathological hypertrophy. However GATA4 seemed to be the determining factor in the pathology. A new animal model was established for the study of pathologic CH in early postnatal stages.

Moderate and severe malnutrition alters proliferation of spleen cells in rats.

OBJECTIVES: Previous studies have shown alterations in bone marrow cell proliferation in malnourished rats, during lactation. The objective of this study was to determine in vivo effects of moderate and severe malnutrition on spleen cell proliferation in 21-day-old rat pups. MATERIALS AND METHODS: Spleen cell proliferation was determined following administration of bromodeoxyuridine (BrdUrd) over a time course of 2, 4, 6 and 8 h. Incorporation of BrdUrd was detected using FITC-conjugated anti-BrdUrd monoclonal antibodies and total DNA content was detected and evaluated using propidium iodide using flow cytometry. RESULTS: Proportions of cells in S and G2 /M were reduced in the rats with moderate (MN2(nd) ) and severe (MN3(rd) ) malnutrition. BrdUrd incorporation was lower in both groups of malnourished rat. In cells of MN2nd individuals, length of G1 became shorter, while length of S-phase increased. In contrast, fraction of cells in proliferation was significantly lower in both groups of malnourished rat, with MN3rd group having lowest percentage of cell population growth. In this study, severe malnutrition did not significantly affect duration of phases of the cell cycle, although fractions of proliferating cells were dramatically reduced. CONCLUSION: Moderate malnutrition increased time of cells in DNA synthesis and time of total cell cycle and severe malnutrition reduced growth fraction of spleen cells in malnourished rats during lactation.

Normal development of the muscular region of the interventricular septum. II. The importance of myocardial proliferation.

In a first paper, we concluded that the muscular region of the interventricular septum is developed by the trabecular branches and showed evidence that the developing interventricular septum elongates in a direction opposite to that of atria. Nevertheless, to date the literature is lacking precise information on the importance of myocardial proliferation not only in this process but also in the morphogenesis of the ventricular cavities. The aim of this study was to determine the spatial and temporal distribution of high-intensity foci of cycling myocytes in the ventricular region of the heart of chicken embryos during cardiac septation. Histological studies, detection of the proliferating cell nuclear antigen by light and confocal microscopy and flow cytometric analysis were carried out. The results corroborate that the developing interventricular septum grows in a direction opposite to that of atria. A remoulding mechanism that results in fenestrated trabecular sheets and trabecular branching is discussed. Our findings allowed us to summarize the normal morphogenesis of the muscular region of the interventricular septum in a way that is different from that suggested by other researchers.

Reference map of soluble proteins from Streptococcus thermophilus by two-dimensional electrophoresis.

Streptococcus thermophilus is a lactic acid bacterium widely used for the production of fermented dairy products. The two-dimensional electrophoresis (2-DE) protein profile was obtained from three independent analyses of 2-DE gels of soluble proteins of the strain PB18. About 270 spots were detected by silver staining and the average molecular weight and isoelectric point of each protein spot were calculated to be 41 600 and 5.2, respectively. Twelve proteins were purified by chromatographic techniques because their concentration was too low for direct sequencing from blots. Eleven were located in the PB18 2-DE profile after silver staining. These preliminary results contribute to the setting up of a two-dimensional image (or reference map) of the proteins from S. thermophilus in order to identify and compare strains of various origin or to follow metabolic process such as stress. Bidimensional autoradiographs of two strains (PB18 and ST105) of S. thermophilus grown in exponential phase at 42 degrees C with [35S]methionine were compared with an image analysis system. Among the eleven located proteins in the 2-DE silver-stained profile, nine were found in PB18 and eight in ST105 autoradiographs. One protein was specific to PB18. The eight proteins could play the role of internal 2-D PAGE markers of p/ and Mr for S. thermophilus.

Expression of lectin receptors on the membrane surface of sperm of fertile and subfertile boars by flow cytometry.

Studies suggest that carbohydrates are important in different stages of fertilization. Plasma membrane changes accompanying in vitro capacitation and acrosome reaction (AR), such as removal or appearance of specific glycoproteins, have been studied using lectins that bind specifically to carbohydrate residues. In specialized artificial insemination farms and semen production centers, identification of boars with decreased fertilization ability (subfertility) is a newborn necessity. This investigation is a sequential study to determine the kinetics of surface carbohydrates turnover during in vitro capacitation and AR in fertile and subfertile boar sperm. Flow cytometry determinations of the binding of three FITC-labeled lectins were assessed. WGA binding was significantly lower in fresh, capacitated, and acrosome-reacted sperm of subfertile boars than in fertile boars. Con-A binding was not significantly different in fresh sperm of fertile and subfertile boars. However. Con-A labeling in capacitated, and acrosome-reacted sperm differed significantly in both groups. UEA binding increased only in capacitated sperm of subfertile boars. These findings could be used as indicators of capacitation and AR and may also be a good indicator of sperm fertilizing ability in boars.

Flow cytometric analysis of spontaneous and dexamethasone-induced apoptosis in thymocytes from severely malnourished rats.

Severe malnutrition is widely distributed throughout the world, showing a high prevalence in developing countries. Experimental animal models have been useful to study the effects of malnutrition at different levels and ages. Apoptosis is a well recognised process of cell death occurring under several physiological and pathological conditions. It represents the principal mechanism involved in cell selection in the thymus. Thymocyte apoptosis induction by dexamethasone is one of the best characterised experimental models of programmed cell death. The aim of the present study was to determine whether severe malnutrition increased spontaneous and/or dexamethasone-induced apoptosis in vivo in thymocytes of experimentally malnourished rats during lactation. Thymocytes were obtained from malnourished rats at weaning (21d of age). Apoptosis frequency was estimated by the terminal transferase-mediated dUTP nick end labelling assay. Spontaneous apoptosis was 1.9 (sd 1.0) % in well nourished rats in contrast to 13.3 (sd 3.8) % in malnourished animals; this is seven times greater (P<0.001). Interestingly, the frequency of dexamethasone-induced apoptosis was similar in both groups of animals (47.9 (sd 10.1) % in well nourished rats and 53.8 (sd 8.0) % in malnourished rats). The results obtained in the present study indicate that malnutrition is associated with a significant increase of spontaneously apoptotic cells. In addition, the data showed that the fraction of thymocytes susceptible to dexamethasone-induced apoptosis was similar in well nourished and malnourished animals. The greater levels of spontaneously apoptotic cells associated with malnutrition could be related to alterations of the microenvironment of the thymus and/or to an obstruction of early thymocyte maturation.

Effects of heavy metals on the oyster (Crassostrea virginica) at Mandinga Lagoon, Veracruz, Mexico

The Mandinga Lagoon in the Mexican State of Veracruz is an important ecological zone that produces 32% of the oyster output in the state of Veracruz, the main oyster producer in Mexico. Samples of water, sediment, and oysters were collected in 2003 and 2004 to study heavy metal pollution. Metal concentrations were determined in water, soil, and oyster tissues from fresh and detoxified Crassostrea virginica, and histology samples were analyzed. Metal (Cr, Cd, and Pb) concentrations in water were within the Mexican legal limits. The recorded values in sediments corresponded to those not producing biological effects (ERL). In the tissues, the highest concentrations corresponded to Pb, above 5.84 μgg-1 dry weight (d.w.); Cd was of 2.23 μgg-1 d.w., and Cr above 6 μgg-1 d.w. The metal levels detected in oysters exceeded the maximum permissible limits (MPL) for Cd and Pb, and oysters were unable to eliminate the concentrations of the bioaccumulated metals during the detoxification stage. The histopathological analysis revealed lesions in the digestive gland, edema, atrophy of epithelia in the digestive tubules, the presence of brown vesicles, hemocytic reaction, and necrosis. During detoxification, a higher number of epithelia were observed in the tubules, as well as an increase in brown vesicles and hemocytic reaction. Forty seven percent of oysters presented histopathological lesions related to metal concentrations. It is important to monitor metal concentrations, to detect the source of pollution, andto evaluate the effects on organisms to establish culture areas and adequate criteria for the exploitation of such an important fishery resource. Rev. Biol. Trop. 57 (4): 955-962. Epub 2009 December 01.