BDNF gene is a risk factor for schizophrenia in a Scottish population.

Schizophrenia is a severe psychiatric disease with a strong genetic component. Brain-derived neurotrophic factor (BDNF) has been implicated in the pathogenesis of schizophrenia and bipolar (BP) disorders. The present study has examined two polymorphisms in linkage disequilibrium in the BDNF gene, which have been variously reported as associated with schizophrenia and BP. In our study, 321 probands with a primary diagnosis of schizophrenia or schizoaffective disorder, and 263 with a diagnosis of bipolar affective disorder, were examined together with 350 controls drawn from the same geographical region of Scotland. The val66met single-nucleotide polymorphism (SNP) showed significant (P = 0.005) association for valine (allele G) with schizophrenia but not bipolar disorder. Haplotype analysis of val/met SNP and a dinucleotide repeat polymorphism in the putative promoter region revealed highly significant (P < 1 x 10(-8)) under-representation of the methionine or met-1 haplotype in the schizophrenic but not the BP population. We conclude that, although the val66met polymorphism has been reported to alter gene function, the risk may depend upon the haplotypic background on which the val/met variant is carried.

The VirR response regulator from Clostridium perfringens binds independently to two imperfect direct repeats located upstream of the pfoA promoter.

Regulation of toxin production in the gram-positive anaerobe Clostridium perfringens occurs at the level of transcription and involves a two-component signal transduction system. The sensor histidine kinase is encoded by the virS gene, while its cognate response regulator is encoded by the virR gene. We have constructed a VirR expression plasmid in Escherichia coli and purified the resultant His-tagged VirR protein. Gel mobility shift assays demonstrated that VirR binds to the region upstream of the pfoA gene, which encodes perfringolysin O, but not to regions located upstream of the VirR-regulated plc, colA, and pfoR genes, which encode alpha-toxin, collagenase, and a putative pfoA regulator, respectively. The VirR binding site was shown by DNase I footprinting to be a 52-bp core sequence situated immediately upstream of the pfoA promoter. When this region was deleted, VirR was no longer able to bind to the pfoA promoter. The binding site was further localized to two imperfect direct repeats (CCCAGTTNTNCAC) by site-directed mutagenesis. Binding and protection analysis of these mutants indicated that VirR had the ability to bind independently to the two repeated sequences. Based on these observations it is postulated that the VirR positively regulates the synthesis of perfringolysin O by binding directly to a region located immediately upstream of the pfoA promoter and activating transcription.

Matrix adhesion characteristics of corneal myofibroblasts.

PURPOSE: To investigate the adhesion characteristics of corneal myofibroblasts in a cell culture model. METHODS: Immunocytochemistry, immunoprecipitation, western blot analysis, and attachment assays were used to evaluate matrix adhesion characteristics of myofibroblasts. RESULTS: Myofibroblasts, defined by their expression of the smooth muscle isoform of alpha-actin, were evaluated and compared with fibroblasts. Myofibroblasts had larger vinculin-containing focal adhesions and expressed more of the classic fibronectin receptor (FNR) alpha5beta1 per cell. However, myofibroblasts had less surface expression of the higher molecular weight alpha4 subunit of another FNR, alpha4beta1, than did fibroblasts. Myofibroblasts adhered more avidly in an integrin-dependent manner to fibronectin than did fibroblasts. The attachment to fibronectin was actin-dependent for both phenotypes, but the myofibroblasts' adhesion was more resistant to disruption by cytochalasin than were fibroblasts'. In addition to the previously described expression of a 135-kDa classic cadherin, myofibroblasts also expressed a 115-kDa mesenchymal cadherin, cadherin-11. CONCLUSIONS: Differentiation of corneal fibroblasts into myofibroblasts is associated with characteristics that would indicate that the latter have a special role in wound closure. The increase in focal and cell adhesion molecules that accompanies smooth muscle-specific actin expression provides the basis for the myofibroblasts' enhanced cell-fibronectin and cell-cell adhesion.

Complete nucleotide sequence of the 27-kilobase virulence related locus (vrl) of Dichelobacter nodosus: evidence for extrachromosomal origin.

The vrl locus is preferentially associated with virulent isolates of the ovine footrot pathogen, Dichelobacter nodosus. The complete nucleotide sequence of this 27.1-kb region has now been determined. The data reveal that the locus has a G+C content much higher than the rest of the D. nodosus chromosome and contains 22 open reading frames (ORFs) encoding products including a putative adenine-specific methylase, two potential DEAH ATP-dependent helicases, and two products with sequence similarity to a bacteriophage resistance system. These ORFs are all in the same orientation, and most are either overlapping or separated by only a few nucleotides, suggesting that they comprise an operon and are translationally coupled. Expression vector studies have led to the identification of proteins that correspond to many of these ORFs. These data, in combination with evidence of insertion of vrl into the 3' end of an ssrA gene, are consistent with the hypothesis that the vrl locus was derived from the insertion of a bacteriophage or plasmid into the D. nodosus genome.

Identification of integrins in cultured corneal fibroblasts and in isolated keratocytes.

PURPOSE: The integrins are a family of transmembrane glycoproteins that function in attachment of cells to one another and to the extracellular matrix. When cell--cell and cell--matrix interactions are altered, the population of integrins may change. In particular, removing cells from their normal environment may be used as a model of wounding. The current study reports the identification of the integrins expressed at the cell surface of noncultured keratocytes and of cultured corneal fibroblasts, which are derived from keratocytes grown in primary culture. METHODS: For integrin identification, the surface proteins of keratocytes and cultured corneal fibroblasts were labeled with biotin, and the integrins were immunoprecipitated using anti-integrin antibodies. Attachment assays determined (1) the extracellular matrix preference of the cultured corneal fibroblasts and (2) the effects of function-perturbing antibodies against the fibronectin receptor (alpha 5 beta 1) or against other beta 1-containing integrins. RESULTS: The integrins of noncultured keratocytes were present as heterodimeric alpha, beta surface proteins that were immunoprecipitated by anti-beta 1, anti-alpha v, anti-alpha 6, anti-alpha 3, anti-alpha 1, and anti-beta 3. Furthermore, when the keratocytes were placed in culture, the integrin pattern changed. The classic fibronectin receptor, alpha 5 beta 1, is then expressed along with additional integrins that bind to fibronectin. Using attachment assays, we determined that the cultured corneal fibroblasts prefer fibronectin to collagen, vitronectin, or laminin as extracellular matrix substrate. In addition, function-perturbing antibodies against the fibronectin receptor (alpha 5 beta 1) or against beta 1 inhibit attachment of cultured corneal fibroblasts to fibronectin. CONCLUSIONS: Receptors for fibronectin and other extracellular matrix molecules are expressed at the cell surface in cultured corneal fibroblasts, and are in position to play a significant functional role as seen in attachment to extracellular matrix.