Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

Volume capacity and contraction control of the seal spleen

Cabanac, Arnaud, Lars P. Folkow, and Arnoldus Schytte Blix.Volume capacity and contraction control of the seal spleen. J. Appl. Physiol. 82(6):1989-1994, 1997.Volume changes in the spleens of hooded seals(Cystophora cristata) and harp seals(Phoca groenlandica) were measuredplethysmographically in vitro in response to epinephrine,norepinephrine, isoprenaline, phentolamine, and acetylcholine. Dilated spleens contracted forcefullywithin 1-3 min of -adrenoceptor activation with 1.0-5.0µg epinephrine/kg body mass, whereas stimulation of -adrenoceptorsand cholinergic receptors had little effect. The mass of dilated hoodedseal spleens corresponded to 2-4%(n = 7) of body mass, with volume (V;ml) relating to body mass (M; kg) as follows: V = 12.0M + 910 (r² = 0.96, n = 4). Thus the spleen of a 250-kghooded seal maximally expels 3.9 liters, or 13%, of its estimatedtotal blood volume. Average hematocrit in splenic venous outflow fromdilated spleens was 90 ± 3% (n = 3) in hooded seals and 85% (n = 2) inharp seals. From these data we have estimated that the aerobic divinglimit of a 250-kg hooded seal increases only 105 s, at the most, if complete emptying of the spleen occurs during diving, while the corresponding estimate for a 112-kg harp seal is 80 s.

     

Mise en évidence d'un paléokarst turonien daté par des Bryozoaires dans le Vercors septentrional (France SE)

Discovery of Turonian-Senonian Bryozoans in thefilling of a karstic cavity in Vercors (E. Voigt in this publication) establishes the presence of an Upper Cretaceous paleokarst in this part of the northern subalpine ranges.     

Delayed insemination is successful with a new extender for storing fresh equine semen at 15 degrees C under aerobic conditions

Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage before artificial insemination. Milk is a biological fluid with a complex composition and contains components which are beneficial or harmful to spermatozoa. The aim of this study was to test the fertility of stallion semen after long-term storage using different milk diluents (INRA 82 or Kenney's diluent) vs one diluent chemically defined (INRA 96), which is composed of efficient milk components and optimized for sperm survival and storage temperature. The milk fraction used was that which best maintained spermatozoal survival based on motility measured in previous studies. Four breeding trials were conducted to determine the influence of combination of new diluent and storage conditions on fertility of the stallion. We compared the standard protocol of storing semen in a skim milk diluent (INRA 82 or Kenney's diluent) at 4 degrees C under anaerobic conditions with the experimental protocol which consisted of storing in a chemically defined, milk-free diluent (INRA 96), at 15 degrees C, under aerobic conditions. After 4 breeding trials, in which the semen was stored for 24 h under the 2 protocols, we obtained 57% (n = 178) and 40% (n = 173) of fertility per cycle using the experimental and the standard protocol respectively (p < 0.001). Another breeding trial was conducted to determine the influence of storage time on the fertility of spermatozoa. We have compared the fertility of semen inseminated immediately (68% of fertility per cycle, n = 50) vs the fertility of semen stored under the experimental protocol for 72 h before insemination (48% of fertility per cycle, n = 52). The experimental protocol improved sperm fertility compared to the standard protocol and seems to be a particular alternative for stallions with cold shock sensitive spermatozoa. Storing semen for 72 h under the experimental protocol seems to be useful in the field.     

Induction of complete segregation of cellular DNA and non-encapsidated viral genomes in herpes simplex virus type 1 infected HeLa cells as revealed by in situ hybridization

The intranuclear distribution of human Alu elements and herpes simplex virus type 1 (HSV-1) genomes was examined in HeLa cells by post-embedding in situ hybridization using in parallel appropriate biotinylated DNA probes. The bound probes were detected by direct immunogold labeling. In non-infected cells, human Alu elements detected by BLUR 8 were randomly scattered over the masses and strands of chromatin throughout the mucleus. The marked asynchrony of the HSV-1 cycle in individual HeLa cells of 17 h infected cultures allowed us to study the respective distributions of cell and viral DNA during the course of the infectious cycle. Labeling of human Alu elements revealed that cellular DNA had become confined to the border of infected nuclei without extension of cellular DNA fibers into the newly formed electron-translucent regions that occupied the centers of the infected nuclei. Labeling of HSV-1 DNA detected by a viral DNA probe revealed that non-encapsidated viral genomes were present exclusively within this centrally located viral region whereas encapsidated HSV-1 genomes, which were more widely distributed in the infected cell, were seen within the marginated host chromatin as well as the central viral region. Therefore, HSV-1 infection induces a regrouping of human Alu elements, that is, of host chromatin at the nuclear border. Non-encapsidated HSV-1 genomes and cellular DNA do not co-localize. Instead, they always constitute two adjacent compartments without spatial interrelationships.     

Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improves myocardial function and coronary perfusion in senescent hearts.

Cardiovascular ageing is associated with an increase in cardiac susceptibility to ischaemia and reperfusion and production of reactive oxygen species has been suspected to be responsible for this age-associated particular vulnerability. To determine whether administration of antioxidant treatment could afford some protection against ischaemia and reperfusion during aging, isolated perfused hearts from adult and senescent rats were submitted to normoxia (180 min), prolonged low-flow ischaemia (15% of initial coronary flow;180 min) or low-flow ischaemia/reperfusion (45 min/30 min), without or with antioxidant enzymes (superoxide dismutase+catalase; 50IU/ml). Contractile function and coronary perfusion were measured and protein oxidation was quantitated in left ventricle after normoxia, ischaemia and ischaemia/reperfusion. Protein oxidation was higher in senescent than in adult hearts after ischaemia-reperfusion, in contrast to prolonged ischaemia. During prolonged ischaemia, antioxidant treatment prevented coronary vasoconstriction at both ages and delayed contractile dysfunction in senescent hearts but did not limit protein oxidation. During reperfusion, antioxidant treatment prevented coronary vasoconstriction and protein oxidation at both ages and considerably improved recovery of contractile function in senescent hearts. In conclusion, antioxidant treatment fully protects the senescent heart against ischaemia/reperfusion but not against prolonged ischaemia injury, indicating that oxidative stress plays a central role in the age-associated vulnerability to ischaemia-reperfusion.     

POLYPHASIC STUDY OF ANTARCTIC CYANOBACTERIAL STRAINS1

We isolated 59 strains of cyanobacteria from the benthic microbial mats of 23 Antarctic lakes, from five locations in two regions, in order to characterize their morphological and genotypic diversity. On the basis of their morphology, the cyanobacteria were assigned to 12 species that included four Antarctic endemic taxa. Sequences of the ribosomal RNA gene were determined for 56 strains. In general, the strains closely related at the 16S rRNA gene level belonged to the same morphospecies. Nevertheless, divergences were observed concerning the diversity in terms of species richness, novelty, and geographical distribution. For the 56 strains, 21 operational taxonomic units (OTUs, defined as groups of partial 16S rRNA gene sequences with more than 97.5% similarity) were found, including nine novel and three exclusively Antarctic OTUs. Sequences of Petalonema cf. involvens and Chondrocystis sp. were determined for the first time. The internally transcribed spacer (ITS) between the 16S and the 23S rRNA genes was sequenced for 33 strains, and similar groupings were observed with the 16S rRNA gene and the ITS, even when the strains were derived from different lakes and regions. In addition, 48 strains were screened for antimicrobial and cytotoxic activities, and 17 strains were bioactive against the gram‐positive Staphylococcus aureus, or the fungi Aspergillus fumigatus and Cryptococcus neoformans. The bioactivities were not in coincidence with the phylogenetic relationships, but rather were specific to certain strains.     

Modeling spatial expansion of invasive alien species: relative contributions of environmental and anthropogenic factors to the spreading of the harlequin ladybird in France

Species distribution models (SDM) have often been used to predict the potential ranges of introduced species and prioritize management strategies. However, this approach assumes equilibrium between occurrences and environmental gradients, an assumption which is violated during the invasion process, where many suitable sites are empty because the species has not yet reached them. Here we considered the invasive ladybird Harmonia axyridis as a case study to show the benefits of using a dynamic colonization–extinction model that does not assume equilibrium. We used a multi‐year occupancy model incorporating environmental, anthropogenic and neighborhood effects, to identify factors that explained spreading variation of this species in France from 2004, when only a few occupied sites were detected, to 2011. We found that anthropogenic factors (urbanization, agriculture, vineyards, and presence/absence of highways) explained more variation in the diffusion process than environmental factors (winter and summer temperatures, wind‐speed, and rainfall). The surface of urbanization was the major anthropogenic factor increasing the probability of colonization. The average summer temperature was the main environmental factor affecting colonization, with a negative effect when high or low. The neighborhood effect revealed that colonization was mostly influenced by contributions coming from a radius of 24 km around the focal cell. The contribution of neighborhood decreases over time, suggesting that H. axyridis is reaching its equilibrium in France. This is confirmed by the small discrepancy observed between the performance of our approach and a SDM approach when predicting a single year occupancy pattern at the end of the study period. Our approach has the advantage of explicitly modelling the state of the biological system during the spatial expansion and identifying colonization constraints. This allows managers to explore the effect of different actions on the system at key moments of the invasion process, hence providing a powerful approach to prioritize management strategies.     

The genetic basis of color‐related local adaptation in a ring‐like colonization around the Mediterranean

Uncovering the genetic basis of phenotypic variation and the population history under which it established is key to understand the trajectories along which local adaptation evolves. Here, we investigated the genetic basis and evolutionary history of a clinal plumage color polymorphism in European barn owls (Tyto alba). Our results suggest that barn owls colonized the Western Palearctic in a ring‐like manner around the Mediterranean and meet in secondary contact in Greece. Rufous coloration appears to be linked to a recently evolved nonsynonymous‐derived variant of the melanocortin 1 receptor (MC1R) gene, which according to quantitative genetic analyses evolved under local adaptation during or following the colonization of Central Europe. Admixture patterns and linkage disequilibrium between the neutral genetic background and color found exclusively within the secondary contact zone suggest limited introgression at secondary contact. These results from a system reminiscent of ring species provide a striking example of how local adaptation can evolve from derived genetic variation.