Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure.

BACKGROUND: We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels. METHOD: Combined two-photon-excited fluorescence (2PEF) and second harmonic generation (SHG) imaging of large arteries reveals the architecture of elastin and collagen fibers in the vessel wall with remarkable specificity. RESULTS: We present elastin/collagen imaging in unstained rat vessels at both micrometer and whole vessel scales, and we characterize the optical properties of rat carotid artery and aorta walls. We apply this method to evidence deleterious effects of residual doses of a pesticide on the vessel wall. CONCLUSION: This study illustrates the potential of 2PEF/SHG microscopy for pharmacological studies in unlabeled arteries.     

Biochemical characterization of the L-plastin-actin interaction shows a resemblance with that of alpha-actinin and allows a distinction to be made between the two actin-binding domains of the molecule

Actin interaction with L-plastin, a plastin/fimbrins isoform of the alpha-actinin family of molecules, is poorly characterized, from the biochemical point of view. Besides, molecular modeling of the T-isoform has recently provided a complete model of interaction with filamentous actin [Volkmann, N., DeRosier, D., Matsudaira, P., and Hanein, D. (2001) J. Cell Biol. 153, 947-956]. In this study, we report that recombinant L-plastin binds actin in a manner that strongly resembles that of the alpha-actinin-actin interface. The similitudes concern the absence of specificity toward the actin isoform and the inhibition of the binding by phosphoinositides. Furthermore, the participation of actin peptides 112-125 and 360-372 in the interface together with an inhibition of the rate of pyrenyl F-actin depolymerization is in favor of a lateral binding of the plastin isoform along the filament axis and strenghtens the similitudes in the way L-plastin and alpha-actinin bind to actin. We have also investigated the functional aspect and the putative equivalence of the two actin-binding domains of L-plastin toward actin binding. We demonstrate for the first time that the two recombinant fragments, expressed as single domains, have different affinities for actin. We further analyzed the difference using chemical cross-linking and F-actin depolymerization experiments assayed by fluorescence and high-speed centrifugation. The results clearly demonstrate that the two actin-binding domains of plastin display different modes of interaction with the actin filament. We discuss these results in light of the model of actin interaction proposed for T-plastin.     

The founder mutation MSH2*1906G-->C is an important cause of hereditary nonpolyposis colorectal cancer in the Ashkenazi Jewish population

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in the mismatch-repair genes. We report here the identification and characterization of a founder mutation in MSH2 in the Ashkenazi Jewish population. We identified a nucleotide substitution, MSH2*1906G-->C, which results in a substitution of proline for alanine at codon 636 in the MSH2 protein. This allele was identified in 15 unrelated Ashkenazi Jewish families with HNPCC, most of which meet the Amsterdam criteria. Genotype analysis of 18 polymorphic loci within and flanking MSH2 suggested a single origin for the mutation. All colorectal cancers tested showed microsatellite instability and absence of MSH2 protein, by immunohistochemical analysis. In an analysis of a population-based incident series of 686 Ashkenazi Jews from Israel who have colorectal cancer, we identified 3 (0.44%) mutation carriers. Persons with a family history of colorectal or endometrial cancer were more likely to carry the mutation than were those without such a family history (P=.042), and those with colorectal cancer who carried the mutation were, on average, younger than affected individuals who did not carry it (P=.033). The mutation was not detected in either 566 unaffected Ashkenazi Jews from Israel or 1,022 control individuals from New York. In hospital-based series, the 1906C allele was identified in 5/463 Ashkenazi Jews with colorectal cancer, in 2/197 with endometrial cancer, and in 0/83 with ovarian cancer. When families identified by family history and in case series are included, 25 apparently unrelated Ashkenazi Jewish families have been found to harbor this mutation. Although this pathogenic mutation is not frequent in the Ashkenazi Jewish population (accounting for 2%-3% of colorectal cancer in those whose age at diagnosis is <60 years), it is highly penetrant and accounts for approximately one-third of HNPCC in Ashkenazi Jewish families that fulfill the Amsterdam criteria.     

Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence

To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.     

Starting to unravel the toxoglossan knot: molecular phylogeny of the "turrids" (Neogastropoda: Conoidea)

The superfamily Conoidea is one of the most speciose groups of marine mollusks, with estimates of about 340 recent valid genera and subgenera, and 4000 named living species. Previous classifications were based on shell and anatomical characters, and clades and phylogenetic relationships are far from well assessed. Based on a dataset of ca. 100 terminal taxa belonging to 57 genera, information provided by fragments of one mitochondrial (COI) and three nuclear (28S, 18S and H3) genes is used to infer the first molecular phylogeny of this group. Analyses are performed on each gene independently as well as for a data matrix where all genes are concatenated, using Maximum Likelihood, Maximum Parsimony and Bayesian approaches. Several well-supported clades are defined and are only partly identifiable to currently recognized families and subfamilies. The nested sampling used in our study allows a discussion of the classification at various taxonomical levels, and several genera, subfamilies and families are found polyphyletic.     

Sexual conflict in Gerris gillettei (Insecta: Hemiptera): intraspecific intersexual correlated morphology and experimental assessment of behaviour and fitness

The contemporary dynamics of sexually antagonistic coevolution caused by sexual conflicts have seldom been investigated at the intraspecific level. We characterized natural populations of Gerris gillettei and documented significant intersexual correlations for morphological traits previously related to sexual conflict in water striders. These results strongly indicate that sexually antagonistic coevolution contributed to population differentiation and resulted in different balances of armaments between the sexes within natural populations of this species. No-choice mating experiments further revealed that both male and male-female relative arms levels influence copulation duration. However, there were no asymmetries in reproductive behaviour and fitness between sympatric and allopatric mating pairs, suggesting that differentiation by sexual conflict was not sufficient to influence the outcome of mating interactions. Altogether, these results question the relative importance of female connexival spines vs. genitalia traits in mediating pre- and post-copulatory conflict in Gerris.     

Characterization of peptide aptamers targeting Bfl-1 anti-apoptotic protein

Bfl-1, an anti-apoptotic protein of the Bcl-2 family, has been identified as a potential therapeutic target for B-cell malignancies. We describe herein the first characterization of peptide aptamers selected against Bfl-1. We show that most of the Bfl-1 peptide aptamers do not interact with Bcl-2, Bcl-xL, or Mcl-1 in yeast and that some of them restore the pro-apoptotic activity of Bax in yeast in which Bax and Bfl-1 proteins are coexpressed. When expressed in mammalian cells, peptide aptamers interact with Bfl-1 and sensitize B-cell lines to apoptosis induced by chemotherapeutic agents. We further demonstrate that a nonconstrained peptide derived from one aptamer variable region reverses Bfl-1 anti-apoptotic activity in HeLa cells through disruption of Bax-Bfl-1 interaction. This peptide also promotes cell death in lymphoma B-cell lines expressing a high level of Bfl-1 and sensitizes these cells to drug-induced apoptosis. Taken together, these results further validate Bfl-1 as a therapeutic target for malignant B-cells and suggest that peptide aptamers may be a useful tool for guiding the identification of small compounds that target the anti-apoptotic Bfl-1 protein.