Sequence analysis of the equine SLC26A2 gene locus on chromosome 14q15-->q21

The solute carrier family 26, member 2 (SLC26A2) gene belongs to a family of multifunctional anion exchangers. Mutations in the human SLC26A2 gene are associated with autosomal recessively inherited chondrodysplasias. Hence, we postulate that the equine SLC26A2 could be a candidate gene for conformational traits in horses. An equine BAC clone harboring the SLC26A2 gene was isolated. The complete 142,625 bp insert sequence of this clone was determined by transposon sequencing. Together with the SLC26A2 gene the BAC clone contains four genes, i.e. the macrophage colony stimulating factor 1 receptor precursor (CSF1R), KIAA0194 protein gene similar to the SMF protein (KIAA0194), a tigger transposable element derived 14 (TIGD14), the 3'-5'-cyclic GMP phosphodiesterase alpha-chain (EC 3.1.4.35) and one unidentified open reading frame. The equine SLC26A2 gene encompassing 6,152 bp consists of two exons. The complete open reading frame of 2,211 bp encodes a protein of 736 amino acids. A comparison of the amino acid sequence with other mammalian orthologs revealed homologies with identity in a range between 80% and 88%. By contrast, the equine SLC26A2 protein lacks five C-terminal amino acids. Four single nucleotide polymorphisms (SNP) were identified (three synonymous and one non-synonymous variant Ser210Leu) in the coding region by comparative sequencing of 50 DNA samples representing the German Riding horse. Allele frequencies and distribution were further evaluated in a variety of different breeds: Arabians (for all four SNPs), Old Kladrub Horses, Draught Horses (including Westphalian Draught Horses, Rheinish Westphalian Draught Horses, Saxon-Thuringia Coldbloods, Altmarker Coldbloods), American Saddlebreds, Miniature Horses, Australian Riding Ponies, Appaloosa, Morgan Horses, and Lipizzaner for C629T (Ser210Leu) alone. No animal carrying the homozygous genotype TT has been detected. The overall frequency of the newly described variant T is low (between 2% and 6%). Simulation studies on the protein conformation predict structural protein changes mediated by the SNP.     

Phosphoinositides determine specificity of the guanine-nucleotide exchange activity of cytohesin-1 for ADP-ribosylation factors derived from a mammalian expression system.

ADP-ribosylation factors (ARFs) are small Ras-like GTPases which play important roles in intracellular vesicle transport and in the remodeling of the actin cytoskeleton. Guanine nucleotide exchange factors (GEFs) for ARFs have recently been identified. One of them, cytohesin-1, a 47-kDa cytoplasmic protein acts as an inside-out signaling molecule and regulates binding of the beta2 integrin leukocyte function antigen 1 (LFA-1) to its ligand intercellular adhesion molecule 1 (ICAM-1). In this study, we address the regulation of the GEF activity of cytohesin-1 by phosphoinositides, using mammalian expression of functional ARF-Ig chimeras. The fusion proteins, which can be quantitatively immunoprecipitated on protein A-Sepharose, target to the expected intracellular compartments, and they are readily induced to bind GTP in vitro. We show that both ARF1-Ig and ARF6-Ig chimeras are activated in vitro by cytohesin-1. However, GEF activity towards ARF6 is strongly suppressed by phosphatidylinositol-(3,4,5)-trisphosphate (PtdInsP3). In contrast, cytohesin-1-dependent GTP binding of ARF1 is significantly enhanced by PtdInsP3. We conclude that the membrane phospholipid PtdInsP3 determines the specificity of the GEF activity of cytohesin-1.     

DNA cloning and sequence analysis of chicken AFLP

Amplified fragment length polymorphisms (AFLP) have been shown to be useful for linkage mapping in chickens and other domestic animals. It is often desirable to convert AFLP bands to sequence-tagged site (STS) markers, in particular, so that AFLP-based linkage information can be integrated with recombinant DNA clone-based maps. Sixteen chicken AFLP bands were excised from gels, re-amplified, cloned and analysed. All inserts proved to be EcoRI-TaqI fragments, which suggests that unlabelled TaqI-TaqI AFLP fragments do not amplify well, and therefore do not significantly contaminate AFLP bands. For eight of the AFLP, the cloned fragment was used to probe blots of AFLP reaction fingerprints, confirming that the predominant DNA clone indeed contained the polymorphic fragment. Flanking regions of selected AFLP fragments were isolated using Vectorette cloning. The results obtained suggest that the these chicken AFLP most commonly arise from sequence polymorphism at or near the TaqI site.     

Identification of acidic heterocycle-substituted 1H-pyrazolo[3,4-b]pyridines as soluble guanylate cyclase stimulators

Novel guanylate cyclase stimulators are disclosed. Design, synthesis, SAR, and pharmacological profile of the compounds are discussed.     

Enzymatic lysis of yeast cell walls

Lysis of yeast cell walls using zymolase and lysozyme was studied. During coupled zymolase–lysozyme treatment, nearly three times more reducing sugars were released from the yeast cells compared to controls. Enzyme treatment followed by extraction at pH 9 resulted in a yield of more than 80% of the total nitrogen of the yeast cell. Protein degradation occurred during enzyme treatment. The precipitation of proteins was significantly increased by succinylation after enzyme treatment. This also reduced the nucleic acid content of the yeast proteins to less than 2% and enhanced the extractability of nitrogenous material.     

Exudation: an expanding technique for continuous production and release of secondary metabolites from plant cell suspension and hairy root cultures

This review addresses methods of obtaining secondary metabolites from plant cell suspension and hairy root cultures and their exudates, particularly the physiological mechanisms of secondary metabolites release and trafficking. The efficiency for product recovery of metabolites can be increased by various methods, based on the principle of continuous product release into the cultivation medium. The most common methods for metabolite recovery are elicitation, influencing membrane permeability, and in situ product removal. The biosynthetic pathways can be influenced by cultivation conditions, transformation, or application of elicitors. The membrane permeability can be altered through the application of chemical or physical treatments. Product removal can be greatly increased through a two-phase system and the introduction of absorbents into the cultivation medium. In this review, we describe some improved approaches that have proven useful in these efforts.