GABAA Receptor Complex in an Experimental Model of Hepatic Encephalopathy: Evidence for Elevated Levels of an Endogenous Benzodiazepine Receptor Ligand

The involvement of the gamma-aminobutyric acidA (GABAA) receptor complex in the pathogenesis of hepatic encephalopathy was examined in thioacetamide-treated rats with fulminant hepatic failure. Partially purified extracts from encephalopathic rat brain were approximately three times more potent in inhibiting [3H]Ro 15-1788 binding to benzodiazepine receptors than identically prepared extracts from control rats. High levels of inhibitory activity were also found in extracts of plasma, heart, and liver from thioacetamide-treated rats. The inhibition of [3H]Ro 15-1788 binding by brain extracts appeared to be competitive and reversible and was unaffected by treatment with either proteolytic enzymes or boiling. Further, GABA significantly enhanced the potency of these extracts in inhibiting [3H]flunitrazepam binding. In contrast, no differences were found in radioligand binding to the constituent recognition sites of the GABAA receptor complex in well-washed brain membranes prepared from control and encephalopathic animals. These findings suggest that the recognition-site qualities of the constituent proteins of the GABAA receptor complex are unchanged in an experimental model of hepatic encephalopathy. However, significant elevations in the level of a substance or substances with neurochemical properties characteristic of a benzodiazepine receptor agonist may contribute to the electrophysiological and behavioral manifestations of hepatic encephalopathy.     

The function of a ribosomal frameshifting signal from human immunodeficiency virus-1 in Escherichia coli

A 15-17 nucleotide sequence from the gag-pol ribosome frameshift site of HIV-1 directs analogous ribosomal frameshifting in Escherichia coli. Limitation for leucine, which is encoded precisely at the frameshift site, dramatically increased the frequency of leftward frameshifting. Limitation for phenylaianine or arginine, which are encoded just before and just after the frameshift, did not significantly affect frameshifting. Protein sequence analysis demonstrated the occurrence of two closeiy related frameshift mechanisms. In the first, ribosomes appear to bind leucyl-tRNA at the frameshift site and then slip leftward. This is the 'simultaneous slippage’mechanism. In the second, ribosomes appear to slip before binding amlnoacyl-tRNA, and then bind phenylaianyl-tRNA, which is encoded in the left-shifted reading frame. This mechanism is identicai to the‘overlapping reading’we have demonstrated at other bacterial frameshift sites. The HIV-1 sequence is prone to frame-shifting by both mechanisms in E. coli.     

Identification and characterization of a basic cell surface-located protein from Lactobacillus fermentum BR11.

Extraction of Lactobacillus fermentum BR11 cells with 5 M LiCl yielded a preparation containing a single predominant polypeptide with an apparent molecular mass of 32 kDa. A clone encoding an immunoreactive 32-kDa polypeptide was isolated from a pUC18 library of L. fermentum BR11 DNA by screening with an antiserum raised against whole cells of L. fermentum BR11. Sequence determination of the insert in the clone revealed a complete 795-bp open reading frame (ORF) that defines a 28,625-Da polypeptide (BspA). N-terminal sequencing of the LiCl-extracted polypeptide from L. fermentum BR11 confirmed that it is the same as the cloned BspA. BspA was found to have a sequence similar to those of family III of the bacterial solute-binding proteins. The sequences of two ORFs upstream of bspA are consistent with bspA being located in an operon encoding an ATP-binding cassette-type uptake system. Unusually, BspA contains no lipoprotein cleavage and attachment motif (LXXC), despite its origin in a gram-positive bacterium. Biotin labelling and trypsin digestion of whole cells indicated that this polypeptide is exposed on the cell surface. The isoelectric point as predicted from the putative mature sequence is 10.59. It was consequently hypothesized that the positively charged BspA is anchored by electrostatic interaction with acidic groups on the cell surface. It was shown that BspA could be selectively removed from the surface by extraction with an acidic buffer, thus supporting this hypothesis.     

Inhibition of [3H]Diazepam and [3H]3-Carboethoxy-β-Carboline Binding by Irazepine: Evidence for Multiple "Domains" of the Benzodiazepine Receptor

The binding of [3H]diazepam and [3H]3-carboethoxy-beta-carboline was examined in rat brain synaptosomal membranes treated with irazepine, an alkylating benzodiazepine. Under incubation conditions that resulted in a 25-33% reduction in the Bmax of [3H]diazepam binding, only modest (less than 8.5%) reductions in the Bmax of [3H]3-carboethoxy-beta-carboline were observed. The differential effects of irazepine on the binding of these two compounds may be explained by the presence of multiple areas or "domains" on the benzodiazepine receptor.     

Discovery and development of exome‐based,co‐dominant single nucleotide polymorphism markers in hexaploid wheat (Triticum aestivum L.)

Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)‐based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP‐based wheat markers have been made available via the use of next‐generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial‐scale breeding programmes. To identify exome‐based co‐dominant SNP‐based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re‐sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95 266 putative single nucleotide polymorphisms, of which 10 251 were classified as being putatively co‐dominant. Validation of a subset of these putative co‐dominant markers confirmed that 96% were true polymorphisms and 65% were co‐dominant SNP assays. The new co‐dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web‐based database to facilitate their use on genotyping programmes worldwide.     

Patterns of bee diversity in mosaic agricultural landscapes of central Uganda: implication of pollination services conservation for food security

Little is known about bee communities and pollination services conservation strategies in sub-Sahara Africa. A study was conducted at 26 different sites with varying local landscape characteristics in farmlands of central Uganda in 2006. Bees were sampled using coloured pantraps, handnet and line transect counts. Overall 80,883 bee individuals from 6 families and 652 species were encountered. The bee fauna was characterized by a lower diversity of Melittidae and Andrenidae and a high diversity of Apidae, Megachilidae and Halictidae. Megachile and Lasioglossum were the two most species-rich genera. The most abundant species was Apis mellifera adansonii Linnaeus (23 % of total individuals) followed by Hypotrigona gribodoi Magretti (19 %), Meliponula ferruginea Lepeletier (13 %), Lasioglossum ugandicum Cockerell (7 %), Apis mellifera scutellata Latreille (6 %), Allodapula acutigera Cockerell (6 %), Ceratina rufigastra Cockerell (5 %), Ceratina tanganyicensis Strand (5 %), Braunsapis angolensis Cockerell (5 %), Megachile rufipes Fabricius (5 %), Meliponula bocandei Spinola (5 %) and Seladonia jucundus Smith (5 %). The mean number of species recorded per study site per day ranged between 14 and 49, whereas the abundance ranged between 188 and 1,859 individuals. Study sites in areas with intense land-use had species-poor bee communities compared to sites with medium to low land-use intensities. Study sites with riparian forest fragments and wetlands, or with forest fallows in their vicinity had significantly (P < 0.05) higher species richness and diversity than sites dominated by small-scale monoculture/polyculture fields or sites dominated by either simple or complex traditional agroforestry systems. An ordination analysis also revealed that bee communities were significantly (P < 0.01) influenced by the presence of semi-natural habitats (woodlands, fallows) and forest fragments in the surrounding of fields. Thus, natural and semi-natural habitats are of great value for afrotropical farmland bee communities. There is a need to put in place strategies and policies for semi-natural and forest fragments preservation for spatio-temporal stability of pollination services in rural landscapes. Farmers are recommended to increase on-farm trees cover to safeguard and enhance pollination function and services in fields. Mimicking natural vegetation through promoting establishment of forest plantations and village community forestry in rural landscapes is also critical for conserving pollination services.