Immunogold detection of co-localized neuropeptides: methodological aspects.

Whatever the protocol used, electron microscopic immunogold detection still suffers from a lack of sensitivity. In rat supraoptico-posthypophyseal neurons, unlabeled secretory granules are always detectable after electron microscopic immunocytochemistry, and their real status remains questionable. To improve the sensitivity of this approach, we assessed a protocol to visualize either one or the other of co-localized neuropeptides, i.e., vasopressin or galanin, after two successive rounds of immunogold with the same primary antibody performed on both faces of the grid. The use of different-sized gold particles enabled us to visualize the respective contribution of each face of the section to the final labeling. Our results showed a moderate but significant increase in both the proportion of labeled granules and the labeling intensity. Although limited, this improvement of immunogold detection strengthens the relevance of quantitative studies at the electron microscopic level, likely to reveal fine variations of the neuron peptidergic content. However, this enhancement depended on the peptide studied. The present data confirmed a progressive decrease of vasopressin immunoreactivity, already suggested by the single-staining procedure, all along the hypothalamo-posthypophyseal tract. In contrast, labeling intensity for galanin remained steady. Finally, our double-face labeling supported a preferential routing of galanin-containing secretory granules towards dendrites.     

Inactivation of the Spirochete recA Gene Results in a Mutant with Low Viability and Irregular Nucleoid Morphology

Recently, we have shown the first evidence for allelic exchange in Leptospira spp. By using the same methodology, the cloned recA of Leptospira biflexa was interrupted by a kanamycin resistance cassette, and the mutated allele was then introduced into the L. biflexa chromosome by homologous recombination. The recA double-crossover mutant showed poor growth in liquid media and was considerably more sensitive to DNA-damaging agents such as mitomycin C and UV light than the wild-type strain. The efficiency of plating of the recA mutant was about 10% of that of the parent strain. In addition, microscopic observation of the L. biflexa recA mutant showed cells that are more elongated than those of the wild-type strain. Fluorescent microscopy of stained cells of the L. biflexa wild-type strain revealed that chromosomal DNA is distributed throughout most of the length of the cell. In contrast, the recA mutant showed aberrant nucleoid morphologies, i.e., DNA is condensed at the midcell. Our data indicate that L. biflexa RecA plays a major role in ensuring cell viability via mechanisms such as DNA repair and, indirectly, active chromosome partitioning.     

Cytogenetic evolution of human ovarian cell lines associated with chemoresistance and loss of tumorigenicity.

In order to identify genomic changes associated with a resistant phenotype acquisition, we used comparative genomic hybridization (CGH) to compare a human ovarian cell line, Igrov1, and four derived subcell lines, resistant to vincristine and presenting a reversion of malignant properties. Multicolor FISH (Multiplex-FISH and Spectral Karyotype) and conventional FISH are also used to elucidate the karyotype of parental cell line. The drug-resistant subcell lines displayed many chromosomal abnormalities suggesting the implication of different pathways leading to a multidrug resistance phenotype. However, these cell lines shared two common rearrangements: an unbalanced translocation der(8)t(8;13)(p22;q?) and a deletion of the 11p. These chromosomal imbalances could reflected the acquisition of the chemoresistance (der(8)) or the loss of tumorigenicity properties (del(11p)).     

Hyperthermia assists survival of astrocytes from oxidative-mediated necrotic cell death.

In response to many stresses and pathologic states, including different models of nervous system injury, cells synthesize a variety of proteins, most notably the inducible 72 kDa heat shock protein 70 (Hsp70), which plays important roles in maintaining cellular integrity and viability. We report here that cultured astrocytes from rat diencephalon express high levels of Hsp70 upon exposure to elevated temperatures, and are less vulnerable to a subsequent oxidative stress. Complex oxidative stress was induced by exposure of astrocytes to an aqueous extract of tobacco smoke. This resulted in both glutathione and ATP depletion, along with cell death that proceeded through a necrotic pathway. Pretreatment of cultures with the glutathione replenishing agent, N-acetyl-L-cysteine, prevented glutathione and ATP loss as well as necrotic cell death. Thermal stress also protected astrocytes from necrotic cell death but without affecting glutathione or ATP levels. We propose that heat shock protects astrocytes from necrosis induced by oxidative stress, probably as a result of Hsp70 synthesis, through an antioxidant-ATP independent mechanism. As Hsp70 may transfer from glial to neuronal cells, its synthesis by astrocytes may represent an important survival mechanism by which astrocytes protect neurons against oxidative-mediated cell death.     

Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model

The human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa , and identified mutants with attenuated virulence towards Dictyostelium . These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa .     

Complete genome sequence of Bacteroides helcogenes type strain (P 36-108)

Bacteroides helcogenes Benno et al. 1983 is of interest because of its isolated phylogenetic location and, although it has been found in pig feces and is known to be pathogenic for pigs, occurrence of this bacterium is rare and it does not cause significant damage in intensive animal husbandry. The genome of B. helcogenes P 36-108(T) is already the fifth completed and published type strain genome from the genus Bacteroides in the family Bacteroidaceae. The 3,998,906 bp long genome with its 3,353 protein-coding and 83 RNA genes consists of one circular chromosome and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Early specialization for voice and emotion processing in the infant brain

Human voices play a fundamental role in social communication, and areas of the adult "social brain" show specialization for processing voices and their emotional content (superior temporal sulcus, inferior prefrontal cortex, premotor cortical regions, amygdala, and insula). However, it is unclear when this specialization develops. Functional magnetic resonance (fMRI) studies suggest that the infant temporal cortex does not differentiate speech from music or backward speech, but a prior study with functional near-infrared spectroscopy revealed preferential activation for human voices in 7-month-olds, in a more posterior location of the temporal cortex than in adults. However, the brain networks involved in processing nonspeech human vocalizations in early development are still unknown. To address this issue, in the present fMRI study, 3- to 7-month-olds were presented with adult nonspeech vocalizations (emotionally neutral, emotionally positive, and emotionally negative) and nonvocal environmental sounds. Infants displayed significant differential activation in the anterior portion of the temporal cortex, similarly to adults. Moreover, sad vocalizations modulated the activity of brain regions involved in processing affective stimuli such as the orbitofrontal cortex and insula. These results suggest remarkably early functional specialization for processing human voice and negative emotions.     

Altered White Matter Architecture in BDNF Met Carriers

Brain-derived neurotrophic factor (BDNF) modulates the pruning of synaptically silent axonal arbors. The Met allele of the BDNF gene is associated with a reduction in the neurotrophin''s activity-dependent release. We used diffusion-weighted imaging to construct structural brain networks for 36 healthy subjects with known BDNF genotypes. Through permutation testing we discovered clear differences in connection strength between subjects carrying the Met allele and those homozygotic for the Val allele. We trained a Gaussian process classifier capable of identifying the subjects'' allelic group with 86% accuracy and high predictive value. In Met carriers structural connectivity was greatly increased throughout the forebrain, particularly in connections corresponding to the anterior and superior corona radiata as well as corticothalamic and corticospinal projections from the sensorimotor, premotor, and prefrontal portions of the internal capsule. Interhemispheric connectivity was also increased via the corpus callosum and anterior commissure, and extremely high connectivity values were found between inferior medial frontal polar regions via the anterior forceps. We propose that the decreased availability of BDNF leads to deficits in axonal maintenance in carriers of the Met allele, and that this produces mesoscale changes in white matter architecture.