A refined blood collection method for quantifying corticosterone

In many rodent laboratories, blood samples are collected from rats using the tail vein nick procedure and analyzed to quantify blood corticosterone levels as an indicator of stress. The standard method of corticosterone quantification often requires the collection of a relatively large volume of blood, followed by the extraction of the blood plasma. An alternative blood sampling method requires the collection of only a drop of blood on paper (the 'drop' method), minimizing handling of the animals, and does not require plasma extraction. The authors aimed to validate the drop method of blood sampling for use in corticosterone quantification. They induced stress in rats by cerebral ischemia, collected blood samples at various intervals using both the drop method and the plasma extraction method and then quantified corticosterone by radioimmunoassay. Corticosterone levels of the ischemic rats were compared with those of sham-operated rats and those of ischemic rats that had been given metyrapone, a glucocorticoid synthesis inhibitor, prior to vessel occlusion. Blood corticosterone levels in the samples obtained from the same animal using the two different methods were highly correlated for all rats. The authors further provide a regression model that can be used to predict plasma corticosterone values from those obtained from the drop blood samples. Quantification of corticosterone from only a small drop of blood has many practical and ethical advantages and should be considered as an alternative to standard methods.     

Faunal changes and geographic crypticism indicate the occurrence of a biogeographic transition zone along the southern East Pacific Rise

Aim Deep‐sea hydrothermal vents have now been reported along all active mid‐ocean ridges and back‐arc basins, but the boundaries of biogeographic entities remain questionable owing to methodological issues. Here we examine biogeographic patterns of the vent fauna along the East Pacific Rise (EPR) and determine the relative roles of regional and local factors on the distribution of biodiversity associated with mussel beds along a poorly explored zone, the southern EPR (SEPR). Location East Pacific Rise. Methods A species list of macrobenthic invertebrates along the EPR was compiled from the literature and supplemented with data recovered during the French research cruise BIOSPEEDO carried out in 2004 along the SEPR. Biogeographic patterns were assessed by combining the identification of morphological species with a molecular barcoding approach. A multivariate regression tree (MRT) analysis was performed to identify any geographic breaks, and an empirical distribution of species richness was compared with predictions provided by a mid‐domain effect model. Macrofaunal community structure associated with mussel beds along the SEPR was analysed in relation to environmental factors using cluster and canonical redundancy analyses. Results Sequencing of the cytochrome c oxidase subunit I gene revealed the occurrence of several cryptic species complexes along the EPR, with the equator separating the southern and northern clades. Furthermore, during the BIOSPEEDO cruise at least 10 still unnamed species were collected between 7°25′ S and 21°33′ S. The shift in community structure identified by MRT analysis was located south of 17°34′ S or south of 13°59′ S, depending on the data used, suggesting that the southern part of the SEPR (17°25′–21°33′ S) constitutes a biogeographic transition zone in the vent fauna along the EPR. At a regional scale, latitude combined with the type of venting was significantly correlated with the community structure associated with mussel beds. Main conclusions Together, the molecular data, in situ observations, and the distribution of species suggest that the high diversity of vent fauna species presently observed between 17°25′ S and 21°33′ S is probably a result of the overlap of several distinct biogeographic provinces. We argue that this area thus constitutes a biogeographic vent fauna transition zone along the EPR.     

Spindle assembly defects leading to the formation of a monopolar mitotic apparatus

Mitotic spindle formation in animal cells involves microtubule nucleation from two centrosomes that are positioned at opposite sides of the nucleus. Microtubules are captured by the kinetochores and stabilized. In addition, microtubules can be nucleated independently of the centrosome and stabilized by a gradient of Ran—GTP, surrounding the mitotic chromatin. Complex regulation ensures the formation of a bipolar apparatus, involving motor proteins and controlled polymerization and depolymerization of microtubule ends. The bipolar apparatus is, in turn, responsible for faithful chromosome segregation. During recent years, a variety of experiments has indicated that defects in specific motor proteins, centrosome proteins, kinases and other proteins can induce the assembly of aberrant spindles with a monopolar morphology or with poorly separated poles. Induction of monopolar spindles may be a useful strategy for cancer therapy, since ensuing aberrant mitotic exit will usually lead to cell death. In this review, we will discuss the various underlying molecular mechanisms that may be responsible for monopolar spindle formation.     

Vertebrate palaeohistology: Past and future

Vertebrate palaeohistology has been considered for a long time as a modest subdivision of Palaeontology. Starting in the 1930s and 1940s, comparative bone tissue histology and palaeohistology progressively demonstrated the multiple correlations between bone tissue distribution and numerous biological variables, such as ontogenetic origin, growth, size, shape, biomechanics, physiology, and ecology. During the last three decades, Palaeohistology has focussed on deciphering the numerous, complex causes explaining the patterns and processes of Vertebrate evolution. Palaeohistology is a powerful tool, in connection with Biology, for the reconstruction of fossil Vertebrates as living organisms.     

Effects of disturbance intensity and frequency on bacterial community composition and function

Disturbances influence community structure and ecosystem functioning. Bacteria are key players in ecosystems and it is therefore crucial to understand the effect of disturbances on bacterial communities and how they respond to them, both compositionally and functionally. The main aim of this study was to test the effect of differences in disturbance strength on bacterial communities. For this, we implemented two independent short-term experiments with dialysis bags containing natural bacterial communities, which were transplanted between ambient and 'disturbed' incubation tanks, manipulating either the intensity or the frequency of a salinity disturbance. We followed changes in community composition by terminal restriction fragment analysis (T-RFLP) and measured various community functions (bacterial production, carbon substrate utilization profiles and rates) directly after and after a short period of recovery under ambient conditions. Increases in disturbance strength resulted in gradually stronger changes in bacterial community composition and functions. In the disturbance intensity experiment, the sensitivity to the disturbance and the ability of recovery differed between different functions. In the disturbance frequency experiment, effects on the different functions were more consistent and recovery was not observed. Moreover, in case of the intensity experiment, there was also a time lag in the responses of community composition and functions, with functional responses being faster than compositional ones. To summarize, our study shows that disturbance strength has the potential to change the functional performance and composition of bacterial communities. It further highlights that the overall effects, rates of recovery and the degree of congruence in the response patterns of community composition and functioning along disturbance gradients depend on the type of function and the character of the disturbance.     

Postnatal gender-dependent maturation of cellular cysteine uptake

Background. In view of the functional capacity of glutathione synthesis in premature infants, and because the availability of cysteine is one the rate limiting steps in glutathione synthesis, we hypothesized that the low glutathione levels in premature infants may be due to immaturity of the active cellular uptake of cysteine.

Objective. To document in cells from newborn infants the effect of maturity and gender on cysteine uptake and consequently on glutathione levels.

Methods. Incorporation of L-[35S] cysteine was measured in leukocytes from cord blood and from tracheal aspirates (TAC) of newborn infants of varying (gestational as well as postnatal) ages and gender. Cysteine uptake was correlated with glutathione in TAC.

Results. The maturity of newborn girls positively influences cysteine uptake, which is responsible for 78% of the variation in their glutathione content. However, in newborn boys, gestational and postnatal ages did not influence the cysteine uptake.

Discussion. Cysteine uptake appears to be the limiting step explaining the reported gender-related differences in glutathione as well as the low levels of this central antioxidant found in premature infants. The immature cysteine uptake found in cells from premature infants raises questions about the bioavailability of this conditionally essential amino acid in regimens of parenteral nutrition for human neonates.     

An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons

Summary A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll^® gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the -glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed.     

Importance of data structure in comparing two dimension reduction methods for classification of microarray gene expression data

Background  

With the advance of microarray technology, several methods for gene classification and prognosis have been already designed. However, under various denominations, some of these methods have similar approaches. This study evaluates the influence of gene expression variance structure on the performance of methods that describe the relationship between gene expression levels and a given phenotype through projection of data onto discriminant axes.     

Diversity surveys and evolutionary relationships of aoxB genes in aerobic arsenite-oxidizing bacteria

A new primer set was designed to specifically amplify ca. 1,100 bp of aoxB genes encoding the As(III) oxidase catalytic subunit from taxonomically diverse aerobic As(III)-oxidizing bacteria. Comparative analysis of AoxB protein sequences showed variable conservation levels and highlighted the conservation of essential amino acids and structural motifs. AoxB phylogeny of pure strains showed well-discriminated taxonomic groups and was similar to 16S rRNA phylogeny. Alphaproteobacteria-, Betaproteobacteria-, and Gammaproteobacteria-related sequences were retrieved from environmental surveys, demonstrating their prevalence in mesophilic As-contaminated soils. Our study underlines the usefulness of the aoxB gene as a functional marker of aerobic As(III) oxidizers.