Bacterial diversity in a deep-subsurface clay environment.

The presence of bacteria in a deep clay sediment was analyzed in a 20-m-long core horizontally drilled from a mine gallery at a depth of 224 m in the Boom clay formation (Mol, Belgium). This clay deposit is the result of a marine sedimentary process that occurred 35 million years ago. Bacterial activities were estimated by measuring respiration on [14C]glucose. Using the same samples, universal primers for the genes coding for eubacterial 16S rRNA were used to amplify extracted DNA. PCR products were then cloned, sequenced, and analyzed by molecular phylogeny. Our data showed a decrease in bacterial densities as a function of distance from the gallery, with few bacteria detectable by culture at more than 80 cm from the gallery wall. PCR experiments showed the presence of bacteria in all samples, and phylogenetic analyses were then used to tentatively identify these organisms. Because of low bacterial densities in deep clay samples, direct counts and enumeration of viable bacteria on diverse culture media remained negative. All experiments, both cultures and PCR, demonstrated the difficulty of analyzing samples that contain only a few poorly active bacteria as it is difficult to avoid a small contamination by active bacteria during sampling. Since the porosity of the Boom clay formation is less than the expected size of bacteria, it is possible that some of the bacteria present in this 35-million-year-old deep clay deposit derive from cells initially trapped during the sedimentation process.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Bioconversion of substituted naphthalenes to the corresponding salicylic acids

The Pseudomonas fluorescens N3 was isolated from soil for its ability to utilize naphthalene as a carbon source. The strain transforms 2,3-dimethyl-, 2-methoxy-, 1- and 2-ethylnaphthalenes to the corresponding salicylic acids competitively with chemical synthesis. The identification of 2-hydroxy-2-carboxy-7-ethylchromane by biotransformation of 2-ethylnaphthalene, contributes to elucidating the steps involved in the catabolic pathways of naphthalenes to salicylaldehydes. Correspondence to: F. Pelizzoni     

Multiple DNA Variant Association Analysis: Application to the Insulin Gene Region in Type 1 Diabetes

Association and linkage studies have shown that at least one of the genetic factors involved in susceptibility to insulin-dependent diabetes mellitus (IDDM) is contained within a 4.1-kb region of the insulin gene. Sequence analysis has led to the identification of 10 DNA variants in this region that are associated with increased risk for IDDM. These variants are in strong linkage disequilibrium with each other, and previous studies have failed to distinguish between the variant(s) that cause increased susceptibility to IDDM and others that are associated with the disease because of linkage disequilibrium. To address this problem, we have undertaken a large population study of French diabetics and controls and have analyzed genotype patterns for several of the variant sites simultaneously. This has led to the identification of a subset consisting of four variants (−2733AC, −23HphI, −365VNTR, and +1140AC), at least one of which appears to be directly implicated in disease susceptibility. The multiple-DNA-variant association-analysis approach that is applied here to the problem of identifying potential susceptibility variants in IDDM is likely to be important in studies of many other multifactorial diseases.     

The making of the minibody: An engineered β-protein for the display of conformationally constrained peptides

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue β-protein aimed at retaining some desirable features of immunogloblin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of main chain conformation of the same regions, based on the ‘canonical structures’ model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical syntyhesis and expression in E. coli and charactgerized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low soubility. We were able to overcome this problem both by further; mutagenesis of the framework and by addition of a solublizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.     

Effect of arachidonic acid on [3H]d-aspartate outflow in the rat hippocampus

The aim of this study was to investigate the effect of arachidonic acid on [³H]d-aspartate outflow in rat hippocampus synaptosomes and slices. Arachidonic acid 1) increased basal outflow of [³H]d-aspartate in both synaptosomes and slices, and 2) increased K⁺-evoked overflow in slices but not in synaptosomes. The latter effect was dependent (at least in part) on arachidonic acid metabolism, most likely mediated by lipo-oxygenase metabolites and free radical production. It was prevented by nordihydroguaiaretic acid but not by indomethacin, and was significantly reduced by free radical scavengers (superoxide-desmutase and catalase). This effect was dependent upon stimulation since it could not be observed after a continuous perfusion of arachidonic acid in the absence of stimulation. Furthermore, it was long-lasting since a 30 min perfusion of arachidonic acid was sufficient to exert a significant effect on a stimulation following termination of the application.     

High-resolution solution structure of two members of a conformationally homogeneous combinatorial peptide library based on the classical zinc-finger motif

Summary We describe the high-resolution structure by NMR of two peptides that belong to a combinatorial library based on the zinc-finger motif. The library represents, to the best of our knowledge, the first example of a conformationally homogeneous peptide library and was obtained by introducing random residues in five positions of the -helical portion of a 26-residue consensus peptide (CP1) belonging to the Cys²-Hys² zinc-finger family. The result was shown to be a highly homogeneous -helical library (Bianchi et al., 1995). The structures of the parent compound (CP1) and of a representative member (CP1m) that was selected by screening the library with a monoclonal antibody are compared in detail as an example of the very high stability of the zinc-finger scaffold upon sequence variability. The two peptides exhibit an extremely high degree of structural similarity. The use of this type of conformationally constrained combinatorial library might represent a step forward in the design of peptidomimetics, as it considerably accelerates the process of the identification of the spatial relationship among the pharmacophoric groups.Abbreviations t-Bu tert-butyloxycarbonyl - Fmoc 9-fluorenylmethoxycarbonyl