Role of proline residues in the expression and function of the human noradrenaline transporter

The aim was to investigate the roles of proline residues in extracellular loop 2 (P172, P183, P188 and P209) and transmembrane domains 2, 5, 11 and 12 (P108, P270, P526, P551, P552 and P570) in determining noradrenaline transporter (NET) expression and function. Mutants of human NET with these residues mutated to alanine were pharmacologically characterized. Mutation of P108, P270 and P526 disrupted cell surface expression, from [H-3]nisoxetine binding and confocal microscopy data. Mutations of P526, P551 and P570 reduced transporter turnover (V-max of [H-3]noradrenaline uptake/B-max of [H-3]nisoxetine binding) by 1.5-1.7-fold compared with wild-type NET, so these residues might be involved in conformational changes associated with substrate translocation. Conversely, mutations of P172, P183, P188 and P209 increased V-max/B-max by 2-3-fold compared with wild-type, indicating that the presence of these proline residues limits turnover of the NET. The mutations had few effects on apparent affinities of substrates or affinities of inhibitors, except decreases in inhibitor affinities after mutations of the P270 and P570 residues, and increases after mutation of the P526 residue. Hence, proline residues in extracellular loop 2 and in transmembrane domains have a range of roles in determining expression and function of the NET.

Molecular crowding effects of linear polymers in protein solutions

Measurement of protein-polymer second virial coefficients (B-AP) by sedimentation equilibrium studies of carbonic anhydrase and cytochrome c in the presence of dextrans (T10-T80) has revealed an inverse dependence of B-AP upon dextran molecular mass that conforms well with the behaviour predicted for the excluded-volume interaction between a spherical protein solute A and a random-flight representation of the polymeric cosolute P. That model of the protein-polymer interaction is also shown to provide a reasonable description of published gel chromatographic and equilibrium dialysis data on the effect of polymer molecular mass on BAP for human serum albumin in the presence of polyethylene glycols, a contrary finding from analysis of albumin solubility measurements being rejected on theoretical grounds. Inverse dependence upon polymer chainlength is also the predicted excluded-volume effect on the strength of several types of macromolecular equilibria-protein isomerization, protein dimerization, and 1 : 1 complex formation between dissimilar protein reactants. It is therefore concluded that published experimental observations of the reverse dependence, preferential reaction enhancement within DNA replication complexes by larger polyethylene glycols, must reflect the consequences of cosolute chemical interactions that outweigh those of thermodynamic nonideality arising from excluded-volume effects. (c) 2005 Elsevier B.V. All rights reserved.

Community paediatrics: Soft science or firm foundations?

Community paediatrics strives to integrate the biology of health into the social and psychological worlds within which children grow and develop. Consumer demand for limited community paediatric clinical services is increasing and medico-legal pressures escalate professional and personal concern. Meanwhile, the profession, through training and professional support, has struggled to keep up. Research into community paediatrics and its integration into policy and clinical practice remains limited, raising the perception that it is a 'soft' science. Our viewpoint is that necessary progress in this field requires leadership, apprenticeship and research. We argue that to build firm foundations for the future requires structures to enable clinical specialisation and continuing professional development in this area.

A novel venom peptide from an endoparasitoid wasp is required for expression of polydnavirus genes in host hemocytes

Maternal factors introduced into host insects by endoparasitoid wasps are usually essential for successful parasitism. This includes polydnaviruses (PDVs) that are produced in the reproductive organ of female hymenopteran endoparasitoids and are injected, together with venom proteins, into the host hemocoel at oviposition. Inside the host, PDVs enter various tissue cells and hemocytes where viral genes are expressed, leading to developmental and physiological alterations in the host, including the suppression of the host immune system. Although several studies have shown that some PDVs are only effective when accompanied by venom proteins, there is no report of an active venom ingredient(s) facilitating PDV infection and/or gene expression. In this study, we describe a novel peptide (Vn1.5) isolated from Cotesia rubecula venom that is required for the expression of C. rubecula bracoviruses (CrBVs) in host hemocytes (Pieris rapae), although it is not essential for CrBV entry into host cells. The peptide consists of 14 amino acids with a molecular mass of 1598 Da. In the absence of Vn1.5 or total venom proteins, CrBV genes are not expressed in host cells and did not cause inactivation of host hemocytes.

Novel protein domains and motifs in the marine planctomycete Rhodopirellula baltica

The planctomycetes are a phylum of bacteria that have a unique cell compartmentalisation and yeast-like budding cell division and peptidoglycan-less proteinaceous cell walls. We wished to further our understanding of these unique organisms at the molecular level by searching for conserved amino acid sequence motifs and domains in the proteins encoded by Rhodopirellula baltica. Using BLAST and single-linkage clustering, we have discovered several new protein domains and sequence motifs in this planctomycete. R. baltica has multiple members of the newly discovered GEFGR protein family and the ASPIC C-terminal domain family, whilst most other organisms for which whole genome sequence is available have no more than one. Many of the domains and motifs appear to be restricted to the planctomycetes. It is possible that these protein domains and motifs may have been lost or replaced in other phyla, or they may have undergone multiple duplication events in the planctomycete lineage. One of the novel motifs probably represents a novel N-terminal export signal peptide. With their unique cell biology, it may be that the planctomycete cell compartmentalisation plan in particular needs special membrane transport mechanisms. The discovery of these new domains and motifs, many of which are associated with secretion and cell-surface functions, will help to stimulate experimental work and thus enhance further understanding of this fascinating group of organisms. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G(4)SG(4)) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I < 0.1 M, 30% glycerol, 1 mM CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR- H, IC50 &SIM; 1 μM). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.

Biology and phenology of the eriophyid mite, Floracarus perrepae, on its native host in Australia, Old World climbing fern, Lygodium microphyllum

The biology and phenology of the eriophyid mite, Floracarus perrepae Knihinicki and Boczek,a potential biological control agent of Lygodium microphyllum (Cav.) R. Br., was studied in its native range - Queensland, Australia. F. perrepae forms leaf roll galls oil tile subpinnae of L. microphyllum. It has a simple biology, with females and males produced throughout the year. Tile Population was female biased at 10.5 to 1. The immature development time was 8.9 &PLUSMN; 0.1 and 7.0 &PLUSMN; 0.1 days; adult longevity was 30.6 &PLUSMN; 1.6 and 19.4 &PLUSMN; 1.2 days and mean fecundity per female was 54.5 &PLUSMN; 3.2 and 38.5 &PLUSMN; 1.6 eggs at 21 and 26 &DEG; C, all respectively. Field studies showed that tile mite was active year round, with populations peaking when temperatures were cool and soil moisture levels were highest. Two species of predatory mites, Tarsonemus sp. and a species of Tydeidae, along with the pathogen Hirsutella thompsonii, had significant effects oil all life stages of F. perrepae. Despite high levels of predators and the pathogen, F. perrepae caused consistent damage to L. microphyllum at all the field sites over the entire 2 years of the study.

Functional promoter analysis using an approach based on an in vitro evolution strategy

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the Goldfinger isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter Junction. The selective-shotgun approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional linker-scanning deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the, prospect of identifying interacting promoter regions.

Opsins: Evolution in waiting

Complete vertebrate genome sequencing has revealed a remarkable stability and uniformity in the protein-coding gene set, which at first glance might suggest that gene duplication events are relatively rare. This may be a red herring, or at least a red cichlid, as the Lake Malawi cichlid fishes show rapid and extensive duplication and diversification of their retinal cone photoreceptor opsin genes.