Optically-Active compounds via biocatalytic methods

Different approaches to improve the enantioselectivity of biocatalytic systems are presented. These methodologies have been successfully applied to the preparation of optically active dihydropyridine, calcium antagonists, and cis-3-hydroxy-6-acetoxy-cyclohex-1-ene, a valuable chiral building block for natural products synthesis. The phenomenon of enantioselective inhibition is described and several new heterocyclic amines have been shown to be effective enantioselective inhibitors in the Candida lipase system.     

Immunogold labelling of cryosectioned pea chloroplasts and initial localization of the proteins associated with the protein import machinery

The electron-microscopic technique for immunogold labelling of thawed cryosectioned material (K.T. Tokuyasu, 1989, Histochem J 21: 163–171) has been adapted for use with isolated chloroplasts. Percoll-purified pea (Pisum Sativum L. cv Feltham First) chloroplasts were fixed in a buffered glutaraldehyde solution and then infiltrated with a buffered solution of 10% polyvinylpyrrolidone in 2.07 M sucrose prior to freezing in liquid nitrogen and sectioning in an ultracryomicrotome. Sections were thawed, immunolabelled, and stained with ammonium molybdate in methyl cellulose on Formvar/carbon-coated Cu or Cu/Pd electron-microscope grids. Cryosectioning gave excellent structural preservation and retained antigenicity. The effectiveness of this technique in localizing proteins to their specific chloroplast compartment was assayed using antibodies raised against: (i) the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a stromal protein, (ii) the chloroplast ATP synthase (CF₁), a peripheral thylakoid protein, and (iii) different envelope membrane proteins. Antibodies raised against three members of the chloroplasticouterenvelopeprotein (OEP) import machinery, a 34-kDa protein (OEP34 or IAP34), the channel-forming 75-kDa protein (OEP75 or IAP75), and the 86-kDa precursor protein receptor (OEP86 or IAP86) were tested for their localization. The previous localization of OEP86, OEP75 and OEP34 to the outer envelope by biochemical methods was confirmed by our immuno electronmicroscopic analysis. Additionally, a constituent of the chloroplastic inner envelope protein (IEP) import machinery IEP 110 (IAP 100) was clearly localized to this membrane. Therefore, cryosectioning and immunogold labelling of intact chloroplasts provides a method for studying the localization of chloroplast proteins, especially those residing in the inner and outer envelope membranes.Abbreviations FCS fetal calf serum - IAP import intermediate associated protein - IEP inner envelope protein - OEP outer envelope protein (numbers signifying the relative molecular mass in kilodaltons) - PBS phosphate buffered saline - PVP polyvinyl pyrrolidone - Rubisco ribulose-1,5-biophosphate carboxylase/oxygenase     

Potential of adipose derived stem cell preparations in neurological repair and regeneration

Stem cell therapy is a promising treatment for neurogenerative disease as well as inflammatory and immune mediated diseases.Decades of preclinical research has ...     

Molecular characterization of an ecdysteroid inducible carboxylesterase with GQSCG motif in the corn borer, Sesamia nonagrioides

We obtained a full-length cDNA encoding a carboxylesterase in Sesamia nonagrioides. The complete cDNA sequence is comprised of 1838 bp with an open reading frame encoding 576 amino acid residues with predicted molecular mass of 64.24 kDa. The deduced amino acid sequence showed high identity to JHE-Related of Trichoplusia ni (65% amino acid identity) and 49-46% amino acid identity to JHEs of other lepidopterans and contained all five functional motifs of insect JHEs. The gene has been termed as SnJHE-Related (SnJHER) to denote its similarity to other insect JHE genes and the occurrence of an unusual cysteine residue immediately adjacent to the catalytic serine, instead of the conventional alanine residue. Phylogenetic analyses localised SnJHER together with TnJHER in a branch of the lepidopteran's JHEs group, with other carboxylesterases (COEs) occuring in separated groups. The JH analog methoprene did not affect the expression of SnJHER in contrast to other insect JHEs. Additionally, ecdysteroid analogs induced SnJHER gene expression. The SnJHER mRNA levels were higher in long-day non-diapausing larvae than in short-day diapausing ones. In the fifth instar of non-diapausing and ninth instar of diapausing larvae, the SnJHER mRNAs reached higher expression levels on the days close to each larval molt. In the last (sixth) non-diapausing larval instar, SnJHER mRNA levels peaked in the intermolt period but were lower than during the fifth instar.     

PRRT2 mutations cause benign familial infantile epilepsy and infantile convulsions with choreoathetosis syndrome

Benign familial infantile epilepsy (BFIE) is a self-limited seizure disorder that occurs in infancy and has autosomal-dominant inheritance. We have identified heterozygous mutations in PRRT2, which encodes proline-rich transmembrane protein 2, in 14 of 17 families (82%) affected by BFIE, indicating that PRRT2 mutations are the most frequent cause of this disorder. We also report PRRT2 mutations in five of six (83%) families affected by infantile convulsions and choreoathetosis (ICCA) syndrome, a familial syndrome in which infantile seizures and an adolescent-onset movement disorder, paroxysmal kinesigenic choreoathetosis (PKC), co-occur. These findings show that mutations in PRRT2 cause both epilepsy and a movement disorder. Furthermore, PRRT2 mutations elicit pleiotropy in terms of both age of expression (infancy versus later childhood) and anatomical substrate (cortex versus basal ganglia).     

Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP‐dependent biogenesis pathway

Mitochondria are separated from the remainder of the eukaryotic cell by the mitochondrial outer membrane (MOM). The MOM plays an important role in different transport processes like lipid trafficking and protein import. In yeast, the ER–mitochondria encounter structure (ERMES) has a central, but poorly defined role in both activities. To understand the functions of the ERMES, we searched for suppressors of the deficiency of one of its components, Mdm10, and identified a novel mitochondrial protein that we named Mdm10 complementing protein 3 (Mcp3). Mcp3 partially rescues a variety of ERMES‐related phenotypes. We further demonstrate that Mcp3 is an integral protein of the MOM that follows a unique import pathway. It is recognized initially by the import receptor Tom70 and then crosses the MOM via the translocase of the outer membrane. Mcp3 is next relayed to the TIM23 translocase at the inner membrane, gets processed by the inner membrane peptidase (IMP) and finally integrates into the MOM. Hence, Mcp3 follows a novel biogenesis route where a MOM protein is processed by a peptidase of the inner membrane.     

Swiss people's attitudes towards field margins for biodiversity conservation

Arable land in Switzerland harbours low biodiversity and lacks permanent species-rich structures. To remedy this situation, improved field margins (IFMs) will be introduced as a new ecological compensation type in the Swiss Lowlands. IFMs are extensively managed, sown species- and flower-rich vegetation strips which provide both habitats for a wide range of species and valuable structures for the ecological network. However, the success of ecological compensation measures depends strongly on their acceptance by farmers and the general public. In summer 2004, we investigated in a case study the attitudes of 108 Swiss people to IFMs directly in the field. Study participants were asked to rate the attractiveness of IFMs of different species richness and composition that were presented to them, to explain their rating and to estimate the number of species present. In addition, they were asked to imagine a field margin of their particular liking, to describe it, and to state their opinion on several aspects of IFMs. Study participants responded very positively to species-rich vegetation. The more species-rich an IFM was perceived to be, the more it appealed to them. Species richness and general diversity were named as the main reasons for a positive rating. Study participants strongly approved the establishment of improved field margins. The positive rating and high acceptance of IFMs in this study indicate that they may be a successful new tool for biodiversity enhancement in intensively used agricultural landscapes.     

Discovery and SAR of hydrazide antagonists of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor type 1 (PAC1-R)

Potent small molecule antagonists for the PAC(1)-R have been discovered. Previously known antagonists for the PAC(1)-R were slightly truncated peptide ligands. The hydrazides reported here are the first small molecule antagonists ever reported for this class B GPCR.     

On-chip PCR amplification of very long templates using immobilized primers on glassy surfaces

In this paper we describe a novel method for visualizing very long DNA fragments (for example >6 kb) which are difficult to spot with commonly used arrayers or capillary samplers with very small nanoliter volumes, using directly bound primers on "on-chip" polymerase chain reaction (PCR). We have used the genomes of the M13 bacteriophage (7.2 kb) the human mitochondrion (16.5 kb) as examples of long DNA templates to test the PCR and were able to elicit robust reactivity. Over 75% of the immobilized primers could be elongated to their fullest extent. In addition we were able to elicit the PCR reaction with double stranded templates in which one primer was immobilized and the other suspended in the reaction solution. These synthesized PCR products were visualized by either confocal microarray scanning or fluorescence microscopy using Cy5-dye fluorescence of the modified free primer, or the fluorescence of intercalating dyes.