Characterization and Quantification of Chitosan Extracted from Nacre of the Abalone Haliotis tuberculata and the Oyster Pinctada maxima

This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (η) of extraction was calculated (η= 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (η= 0.053%) was isolated rather than chitosan. Received February 16, 2000; accepted July 4, 2000.     

Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles

We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions.     

Myosin VI regulates actin dynamics and melanosome biogenesis

Myosin VI has been implicated in various steps of organelle dynamics. However, the molecular mechanism by which this myosin contributes to membrane traffic is poorly understood. Here, we report that myosin VI is associated with a lysosome-related organelle, the melanosome. Using an actin-based motility assay and video microscopy, we observed that myosin VI does not contribute to melanosome movements. Myosin VI expression regulates instead the organization of actin networks in the cytoplasm. Using a cell-free assay, we showed that myosin VI recruited actin at the surface of isolated melanosomes. Myosin VI is involved in the endocytic-recycling pathway, and this pathway contributes to the transport of a melanogenic enzyme to maturing melanosomes. We showed that depletion of myosin VI accumulated a melanogenic enzyme in enlarged melanosomes and increased their melanin content. We confirmed the requirement of myosin VI to regulate melanosome biogenesis by analysing the morphology of melanosomes in choroid cells from of the Snell's waltzer mice that do not express myosin VI. Together, our results provide new evidence that myosin VI regulates the organization of actin dynamics at the surface of a specialized organelle and unravel a novel function of this myosin in regulating the biogenesis of this organelle.     

Evaluation of a rapid immunochromatographic diagnostic test for the detection of rabies from brain material of European mammals

The surveillance of rabies relies on investigations conducted on dead suspected animals or animals showing clinical signs suggestive of rabies. An immunochromatographic method based on lateral flow principle has been evaluated against a collection of brain samples mainly of European mammals including bats. The performance of this new test has been compared to the conventional gold standard methods: the fluorescent Antibody Test (FAT) and the Rapid Tissue Culture Infection Test (RTCIT). This test enabled the detection of various rabies strains belonging to rabies species 1, 5, 6 and 7 and demonstrated an overall specificity of 100% and a sensitivity of more than 88% when compared to FAT and RTCIT. A total agreement between the Rapid Immunochromatographic Diagnostic Test and conventional technique results have been obtained for European bat samples.     

Restoring specific lactobacilli levels decreases inflammation and muscle atrophy markers in an acute leukemia mouse model

The gut microbiota has recently been proposed as a novel component in the regulation of host homeostasis and immunity. We have assessed for the first time the role of the gut microbiota in a mouse model of leukemia (transplantation of BaF3 cells containing ectopic expression of Bcr-Abl), characterized at the final stage by a loss of fat mass, muscle atrophy, anorexia and inflammation. The gut microbial 16S rDNA analysis, using PCR-Denaturating Gradient Gel Electrophoresis and quantitative PCR, reveals a dysbiosis and a selective modulation of Lactobacillus spp. (decrease of L. reuteri and L. johnsonii/gasseri in favor of L. murinus/animalis) in the BaF3 mice compared to the controls. The restoration of Lactobacillus species by oral supplementation with L. reuteri 100-23 and L. gasseri 311476 reduced the expression of atrophy markers (Atrogin-1, MuRF1, LC3, Cathepsin L) in the gastrocnemius and in the tibialis, a phenomenon correlated with a decrease of inflammatory cytokines (interleukin-6, monocyte chemoattractant protein-1, interleukin-4, granulocyte colony-stimulating factor, quantified by multiplex immuno-assay). These positive effects are strain- and/or species-specific since L. acidophilus NCFM supplementation does not impact on muscle atrophy markers and systemic inflammation. Altogether, these results suggest that the gut microbiota could constitute a novel therapeutic target in the management of leukemia-associated inflammation and related disorders in the muscle.     

Yohimbine-induced amygdala activation in pathological gamblers: a pilot study

Rationale and Objectives

There is evidence that drug addiction is associated with increased physiological and psychological responses to stress. In this pilot functional magnetic resonance imaging (fMRI) study we assessed whether a prototype behavioral addiction, pathological gambling (PG), is likewise associated with an enhanced response to stress.

Methods

We induced stress by injecting yohimbine (0.2–0.3 mg/kg, IV), an alpha-2 adrenoceptor antagonist that elicits stress-like physiological and psychological effects in humans and in laboratory animals, to four subjects with PG and to five non-gamblers mentally healthy control subjects. Their fMRI brain responses were assessed along with subjective stress and gambling urges ratings.

Results

Voxelwise analyses of data sets from individual subjects, utilizing generalized linear model approach, revealed significant left amygdala activation in response to yohimbine across all PG subjects. This amygdala effect was not observed in the five control individuals. Yohimbine elicited subjective stress ratings in both groups with greater (albeit not statically significantly) average response in the PG subjects. On the other hand, yohimbine did not induce urges to gamble.

Conclusions

The present data support the hypothesis of brain sensitization to pharmacologically-induced stress in PG.     

Early functional deficit and microglial disturbances in a mouse model of amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by selective motoneurons degeneration. There is today no clear-cut pathogenesis sequence nor any treatment. However growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. To better characterize the time course of pathological events in an animal model that recapitulates human ALS symptoms, we investigated functional and cellular characteristics of hSOD1^G93A mice.

Methods and Findings

We have evaluated locomotor function of hSOD1^G93A mice through dynamic walking patterns and spontaneous motor activity analysis. We detected early functional deficits that redefine symptoms onset at 60 days of age, i.e. 20 days earlier than previously described. Moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. To tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. We show that (1) decrease in neuromuscular junction''s number correlates with motor impairment, (2) astrocytes number is not altered at pre- and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and (3) microglia modifications precede disease onset. At pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge.

Conclusions

In conclusion, precise motor analysis updates the onset of the disease in hSOD1^G93A mice and allows locomotor monitoring until the end stage of the disease. Early functional deficits coincide with alterations of neuromuscular junctions. Importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. This finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets.     

Brain phenotype of transgenic mice overexpressing cystathionine β-synthase

Background

The cystathionine β-synthase (CBS) gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS) cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA) metabolism, a pathway important for several brain physiological processes.

Methodology/Principal Findings

Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1) expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.

Conclusion/Significance

We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.     

Exosomal Lipids Impact Notch Signaling and Induce Death of Human Pancreatic Tumoral SOJ-6 Cells

Exosomes are of increasing interest as alternative mode of cell-to-cell communication. We previously reported that exosomes secreted by human SOJ-6 pancreatic tumor cells induce (glyco)protein ligand-independent cell death and inhibit Notch-1 pathway, this latter being particularly active during carcinogenesis and in cancer stem cells. Therefore, we asked whether exosomal lipids were key-elements for cell death and hypothesized that cholesterol-rich membrane microdomains were privileged sites of exosome interactions with tumor cells. To address these questions and based on the lipid composition of exosomes from SOJ-6 cells (Ristorcelli et al. (2008) FASEB J. 22; 3358–3369) enriched in cholesterol and sphingomyelin (lipids forming liquid-ordered phase, Lo) and depleted in phospholipids (lipids forming liquid-disordered phase, Ld), we designed Synthetic Exosome-Like Nanoparticles (SELN) with ratios Lo/Ld from 3.0 to 6.0 framing that of SOJ-6 cell exosomes. SELN decreased tumor cell survival, the higher the Lo/Ld ratio, the lower the cell survival. This decreased survival was due to activation of cell death with inhibition of Notch pathway. FRET analyses indicated fusions/exchanges of SELN with cell membranes. Fluorescent SELN co-localized with the ganglioside GM1 then with Rab5A, markers of lipid microdomains and of early endosomes, respectively. These interactions occurred at lipid microdomains of plasma and/or endosome membranes where the Notch-1 pathway matures. We thus demonstrated a major role for lipids in interactions between SELN and tumor cells, and in the ensued cell death. To our knowledge this is the first report on such effects of lipidic nanoparticles on tumor cell behavior. This may have implications in tumor progression.