Presence and spatio-temporal habitat characterization of Dermatemys mawii (Testudines: Dermatemydidae) in the Grijalva-Usumacinta watershed, Tabasco, Mexico

The Central American River Turtle (Dermatemys mawii) is an endangered species that has been poorly studied. There are no reports on their population status, habitat condition, and the species distribution area is still unknown. This study analyzes the seasonal and spatial variations of their habitat and the presence/absence of D. mawii in three rivers within the Pantanos de Centla Biosphere Reserve (Tabasco, Mexico). For habitat characterization, natural segmentation of rivers was used and three sites per segment were identified, 9 in each rivers (Grijalva and Usumacinta) and 6 in Tabasquillo. Additionally, the evaluation of 11 environmental variables such as water hydrological, physicochemical characteristics and riparian and hydrophytic vegetation were carried out during two different seasons (dry and rainy). The presence/absence of species was assessed with eight fike nets that were set per segment, with a capture effort of 384 hours per trap. The capture per unit effort (CPUE) was used as an indicator of relative abundance. The results indicated spatio-temporal variations in habitat characteristics and the presence of environmental gradients. The principal components analysis (PCA) applied allowed us to determine that the first three components explained 67.8% of the environmental variability. The species presence was confirmed in all rivers, however significant differences exists in their relative abundance: the highest was registered in the Tabasquillo River where the species was present in both seasons and in all segments. Of the 11 environmental variables analyzed, the gradient, shelter and depth were the most indicative of species presence. The obtained results evidenced the importance of riparian vegetation as habitat for Dermatemys. This represents the first approach towards an action plan for a species and its habitat protection within the Pantanos de Centla Biosphere Reserve.     

Genomic relationships among diploid and polyploid species of the genus Eryngium L. using genomic in situ hybridization

Eryngium L. (Umbelliferae) is a large genus including more than 250 species worldwide. The large morphological variability in this genus makes it difficult to delimit the species or to establish phylogenetic relationships. The occurrence of different ploidy levels within the genus might indicate a hybrid origin of the polyploid species. In the present study, the chromosome number and karyotype of E. regnellii are reportedfor the first time and the ploidy level of a population of E. paniculatum is confirmed. We compare the genomes of the diploids E. horridum and E. eburneum, the tetraploids E. megapotamicum and E. regnellii, and the hexaploids E. pandanifolium (as a representative of the whole pandanifolium complex) and E. paniculatum using genomic in situ hybridization (GISH). Although it was not possible to identify the parental species of the polyploid taxa analyzed, the GISH technique allowed us to postulate some hypotheses about their origin. Eryngium horridum and E. eburneum do not seem to be the direct progenitors of the polyploids analyzed. On the other hand, it seems that other diploid species unrelated to E. horridum and E. eburneum are involved in their origin. Our results are consistent with morphological and phylogenetic studies, indicating a close relationship between the species of the series Latifolia.     

Dietary omega-3 fatty acids attenuate cellular damage after a hippocampal ischemic insult in adult rats

The role of omega-3 polyunsaturated fatty acids (3PUFAs) on brain function is increasingly demonstrated. Here, the effect of dietary deprivation of essential 3PUFAs on some parameters related to neuroprotection was investigated. Rats were fed with two different diets: omega-3 diet and omega-3-deprived diet. To assess the influence of 3PUFAs on brain responses to ischemic insult, hippocampal slices were subjected to an oxygen and glucose deprivation (OGD) model of in vitro ischemia. The omega-3-deprived group showed higher cell damage and stronger decrease in the [³H]glutamate uptake after OGD. Moreover, omega-3 deprivation influenced antiapoptotic cell response after OGD, affecting GSK-3beta and ERK1/2, but not Akt, phosphorylation. Taken together, these results suggest that 3PUFAs are important for cell protection after ischemia and also seem to play an important role in the activation of antiapoptotic signaling pathways.     

Synergistic Effects of the Lactobacillus acidophilus Surface Layer and Nisin on Bacterial Growth

We have previously described a murein hydrolase activity for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356. Here we show that, in combination with nisin, this S-layer acts synergistically to inhibit the growth of pathogenic Gram-negative Salmonella enterica and potential pathogenic Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus. In addition, bacteriolytic effects were observed for the Gram-positive species tested. We postulate that the S-layer enhances the access of nisin into the cell membrane by enabling it to cross the cell wall, while nisin provides the sudden ion-nonspecific dissipation of the proton motive force required to enhance the S-layer murein hydrolase activity.Natural preservatives active against Gram-positive and Gram-negative pathogens are highly desirable for the food industry and consumers. Nisin, a small peptide bacteriocin produced by Lactococcus lactis subsp. lactis, is the only bacteriocin that has approved GRAS (generally recognized as safe) status for certain applications (Generally Regarded as Safe; US 21CFR170.30-Food Additives) by the Food and Drug Administration (FDA) for use in food products (3, 19). The antimicrobial mechanism of nisin has been extensively studied and is well documented (1, 5, 8, 9, 20). Nisin is not active against Gram-negative bacteria compared to Gram-positive bacteria, due to its inability to penetrate the external membrane, which prevents its access to the inner membrane. However, the combination of nisin with the chelating agent lactoferrin acts synergistically to inhibit the growth of Escherichia coli O157:H7 (13). Nisin has been used as a food preservative for over 30 years, is active in the nanomolar range, and has no known toxicity to humans (5, 19), which has placed it in the unique position of worldwide acceptance as a powerful and safe food additive in control of food spoilage and certain food pathogens.We have previously described a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against cell wall preparations of Salmonella enterica serovar Newport (17); however, we failed to observe any effect on whole cells from Gram-positive bacteria such as Bacillus cereus.We now wonder if the purified S-layer protein having this endopeptidase activity may act synergistically with nisin, allowing reduction of the levels of the bacteriocin and control of bacterial growth. For this purpose, the antibacterial activity of S-layer from Lactobacillus acidophilus ATCC 4356, either alone or in combination with nisin, was analyzed using three models of food-borne pathogenic bacteria: Salmonella enterica serovar Newport (10), Staphylococcus aureus ATCC 6538 (used as a representative of Gram-positive bacteria in standard medium tests; laboratory collection), and Bacillus cereus 6A1 (Bacillus Genetic Stock Center [BGSC]). The S-layer proteins were extracted as previously described (17) and conserved as a 1-mg/ml suspension in sterile distilled water at −20°C until use. Purity was determined by SDS-PAGE as a unique band and detected by Western blotting.Nisin was used as its commercial product, 2.5% (wt/wt) (Sigma), and was dissolved in 0.02 N HCl to a concentration of 16,000 IU/ml, equivalent to 0.4 mg/ml pure nisin. The MIC that inhibited growth was determined twice by microtiter broth dilution, and we measured the optical density at 600 nm (OD₆₀₀) reached after 16 h of growth. Bacillus cereus and Salmonella enterica were grown in LB (yeast extract, 5 g/liter; peptone, 10 g/liter; NaCl, 10 g/liter; pH 7.0), while Staphylococcus aureus was grown in BHI (brain heart infusion; Biokar, France) and incubated at 37°C. The MIC was 595 IU/ml for Bacillus cereus, 298 IU/ml for Staphylococcus aureus, and over 5,000 IU/ml for Salmonella enterica serovar Newport. For Salmonella enterica, nisin solution was prepared to a concentration of 160,000 IU/ml, equivalent to 4 mg/ml pure nisin, in order to avoid a pH modification once it was added to the growth medium.In a first approach, growth curves of the Gram-negative Salmonella enterica serovar Newport in the presence of nisin, S-layer, or both were performed. Nisin at half of its MIC showed no inhibition. The S-layer alone was inhibitory, but the addition of both nisin and S-layer decreased growth (for growth rate and maximal OD reached, see Fig. ​Fig.1).1). In view of these findings, we decided to evaluate the effects of the combination (nisin and S-layer) in Gram-positive bacteria. Bacillus cereus and Staphylococcus aureus, two food-borne pathogen models, were chosen. No effect was observed when the S-layer (10 μg/ml) was added alone, and nisin added alone, at a subinhibitory concentration of 250 IU/ml for B.cereus and 150 IU/ml for S. aureus, partially delayed growth. In contrast, the combination of both the S-layer (10 μg/ml) and nisin at a subinhibitory concentration inhibited growth of both B. cereus and S. aureus cultures (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Effect of S-layer, nisin, or both on the growth of S. enterica, S. aureus, and B. cereus. To determine the effect of nisin and S-layer protein separately or in combination, bacterial cells were first cultured at 37°C for 16 h with agitation. These cultures were diluted in fresh medium (0.5 ml into 10 ml) and distributed in aliquots, each containing either the S-layer, nisin, or both, and OD₆₀₀ was monitored every hour. Three or more independent experiments were performed for each bacterium. Nisin was added at the following concentrations: 2,500 IU/ml for Salmonella enterica, 250 IU/ml for Bacillus cereus, and 150 IU/ml for Staphylococcus aureus; the concentration of S-layer was 10 μg/ml.To determine the mode of action of the combination of S-layer and nisin, lysis was monitored by OD₆₀₀ decrease of exponential cultures of B. cereus and S. aureus when exposed to nisin or nisin plus S-layer and viable counts were determined by serial decimal dilutions on LB agar plates. A bacteriolytic effect was observed. As shown in Fig. ​Fig.2A,2A, B. cereus cultures lysed instantly in the presence of both compounds as observed from the decrease in OD and confirmed by the viable count determinations. However, S. aureus showed a 2-h lag period before lysis was observed (Fig. ​(Fig.2B).2B). This delay may be attributed to the typical cell aggregation profile characteristic of Staphylococcus cultures, which might hide the target for the S-layer and/or nisin. In fact, after this period cells disaggregated as visualized by microscopic observation (data not shown) and both compounds led to cell lysis. In addition, when viable counts were performed, a rapid decline in viability was observed even before the OD decrease. The reduction in viability after a 6-h treatment with both nisin and S-layer was 5 and 4 logs for B. cereus and S. aureus, respectively.Open in a separate windowFIG. 2.Type of effect of the combination of nisin and S-layer. Exponentially growing bacteria were incubated in the presence of either nisin or nisin and S-layer. (A) Effect on Bacillus cereus with nisin (250 IU/ml) or nisin and S-layer (250 IU/ml and 10 μg/ml, respectively). (B) Effect on Staphylococcus aureus with nisin (150 IU/ml) or nisin and S-layer (150 IU/ml and 10 μg/ml, respectively). Dotted line, cfu/ml; unbroken line, OD at 600 nm.Concerning the Gram-negative Salmonella enterica, a peculiar behavior was observed: while whole cells suspended in buffer were lysed by the sole presence of S-layer (17), no lytic effect was observed when exponentially growing cultures were treated even with both S-layer and nisin (data not shown). However, the S-layer protein remains intact after incubation with whole cells as observed by Western blotting after 8 h of incubation (Fig. ​(Fig.3).3). We suspect that actively dividing cells of this Gram-negative pathogen may release an inhibitor either of the S-layer activity or of nisin. Further experiments should be performed in order to evaluate these hypotheses.Open in a separate windowFIG. 3.S-layer integrity during incubation with Salmonella. Viable cells from Salmonella enterica were washed once with phosphate-buffered saline buffer, resuspended at an optical density at 600 nm of 1 mixed with S-layer protein (10 μg/ml), and incubated at 37°C with constant rotation to keep the contents in suspension at the indicated times. At different times S-layer was analyzed by Western blotting. Electrotransfer to parablot polyvinylidene difluoride was according to the manufacturer''s instructions (Macherey-Nagel, Germany). Polyclonal antibody anti-S-layer was used at 1:1,000.To check if the synergic effect of S-layer on nisin is indeed due to its murein hydrolase activity, a mild condition of heat inactivation (60°C for 2 h) was assayed with the S-layer. This treatment inactivates the endopeptidase activity as seen by zymogram analysis (Fig. ​(Fig.4,4, zymogram, lane 3) but conserves the protein without dramatic changes, since no differences in migration or antibody detection were observed (compare lanes 3 and 1 in Fig. ​Fig.4).4). The addition of mild-heat-inactivated S-layer protein and nisin to S. aureus cultures was unable to inhibit growth (Table ​(Table1).1). In contrast, while nisin and S-layer separately inhibited growth to 22% or 0%, respectively, the addition of both inhibited dramatically the growth (89%) and growth rate (90%), representing an 8-log reduction in viable cell counts, pointing to the benefit of this synergetic effect (Table ​(Table11).Open in a separate windowFIG. 4.S-layer treatments. S-layer preparations were submitted to different treatments and analyzed by PAGE, zymography, and Western immunoblotting as previously described (16). Lane 1, S-layer; lane 2, pretreatment with nisin of L. acidophilus cultures before S-layer preparation; lane 3, heat-inactivated S-layer (2 h at 60°C); lane 4, S-layer preparation incubated with nisin.

TABLE 1.

Nature of the synergic combination^a

Strains nodulating Lupinus albus on different continents belong to several new chromosomal and symbiotic lineages within Bradyrhizobium

In this work we analysed different chromosomal and symbiotic markers in rhizobial strains nodulating Lupinus albus (white lupin) in several continents. Collectively the analysis of their rrs and atpD genes, and 16S-23S intergenic spacers (ITS), showed that they belong to at least four chromosomal lineages within the genus Bradyrhizobium. Most isolates from the Canary Islands (near to the African continent) grouped with some strains isolated on mainland Spain and were identified as Bradyrhizobium canariense. These strains are divided into two ITS subgroups coincident with those previously described from isolates nodulating Ornithopus. The remaining strains isolated on mainland Spain grouped with most isolates from Chile (American continent) forming a new lineage related to Bradyrhizobium japonicum. The strains BLUT2 and ISLU207 isolated from the Canary Islands and Chile, respectively, formed two new lineages phylogenetically close to different species of Bradyrhizobium depending on the marker analyzed. The analysis of the nodC gene showed that all strains nodulating L. albus belong to the biovar genistearum; nevertheless they form four different nodC lineages of which lineage C is at present exclusively formed by L. albus endosymbionts isolated from different continents.     

Ursodeoxycholic acid modulates the ubiquitin-proteasome degradation pathway of p53

p53/Mdm-2 interaction is a prime target of ursodeoxycholic acid (UDCA) for regulating apoptosis in primary rat hepatocytes. Here, we further explored the role of UDCA in downregulating p53 by Mdm-2. UDCA reduced the stability of p53 by decreasing protein half-life. Although proteasomal activity was slightly increased with UDCA, the effect was also observed for other bile acids. More importantly, immunoprecipitation assays revealed that UDCA promoted p53 ubiquitination, therefore leading to increased p53 degradation. In this regard, proteasome inhibition after UDCA pre-treatment resulted in accumulation of ubiquitinated p53, which in turn was prevented in cells overexpressing a mutated form of p53 that does not undergo Mdm-2 ubiquitination. The involvement of Mdm-2 in UDCA-mediated response was further confirmed by siRNA-mediated gene silencing experiments. Finally, the protective effect of UDCA against p53-induced apoptosis was abolished after inhibition of proteasome activity and prevention of p53 ubiquitination by Mdm-2. These findings suggest that UDCA protects cells from p53-mediated apoptosis by promoting its degradation via the Mdm-2-ubiquitin-proteasome pathway.     

Hypoglycemic activity of leaf organic extracts from Smallanthus sonchifolius: Constituents of the most active fractions

The aim of the present study was to determine the in vivo hypoglycemic activity of five organic extracts and enhydrin obtained from yacon leaves. The main constituents of the most active fraction were identified. Five organic extracts and pure crystalline enhydrin were administered to normoglycemic, transiently hyperglycemic and streptozotocin (STZ)-diabetic rats. The fasting and post-prandial blood glucose, and serum insulin levels were estimated and an oral glucose tolerance test (OGTT) was performed for the evaluation of hypoglycemic activity and dose optimization of each extract.We found that the methanol, butanol and chloroform extracts showed effective hypoglycemic activity at minimum doses of 50, 10 and 20 mg/kg body weight, respectively, and were selected for further experiments. Oral administration of a single-dose of each extract produced a slight lowering effect in the fasting blood glucose level of normal healthy rats, whereas each extract tempered significantly the hyperglycemic peak after food ingestion. Daily administration of each extract for 8 weeks produced an effective glycemic control in diabetic animals with an increase in the plasma insulin level. Phytochemical analysis of the most active fraction, the butanol extract, showed that caffeic, chlorogenic and three dicaffeoilquinic acids were significant components. Additionally, enhydrin, the major sesquiterpene lactone of yacon leaves, was also effective to reduce post-prandial glucose and useful in the treatment of diabetic animals (minimum dose: 0.8 mg/kg body weight).The results presented here strongly support the notion that the phenolic compounds above as well as enhydrin are important hypoglycemic principles of yacon leaves that could ameliorate the diabetic state.