Targeting of the human adrenoleukodystrophy protein to the peroxisomal membrane by an internal region containing a highly conserved motif

In this study we addressed the targeting requirements of peroxisomal ABC transporters, in particular the human adrenoleukodystrophy protein. This membrane protein is defective or missing in X-linked adrenoleukodystrophy, a neurodegenerative disorder predominantly presenting in childhood. Using adrenoleukodystrophy protein deletion constructs and green fluorescent protein fusion constructs we identified the amino acid regions 1-110 and 67-164 to be sufficient for peroxisomal targeting. However, the minimal region shared by these constructs (amino acids 67-110) is not sufficient for peroxisomal targeting by itself. Additionally, the NH2-terminal 66 amino acids enhance targeting efficiency. Green fluorescent protein-labeled fragments of human peroxisomal membrane protein 69 and Saccharomyces cerevisiae Pxa1 corresponding to the amino acid 67-164 adrenoleukodystrophy protein region were also directed to the mammalian peroxisome. The required region contains a 14-amino-acid motif (71-84) conserved between the adrenoleukodystrophy protein and human peroxisomal membrane protein 69 and yeast Pxa1. Omission or truncation of this motif in the adrenoleukodystrophy protein abolished peroxisomal targeting. The single amino acid substitution L78F resulted in a significant reduction of targeting efficiency. The in-frame deletion of three amino acids (del78-80LLR) within the proposed targeting motif in two patients suffering from X-linked adrenoleukodystrophy resulted in the mislocalization of a green fluorescent protein fusion protein to nucleus, cytosol and mitochondria. Our data define the targeting region of human adrenoleukodystrophy protein containing a highly conserved 14-amino-acid motif.     

Screening of natural products extracts for the presence of phosphodiesterase inhibitors using liquid chromatography coupled online to parallel biochemical detection and chemical characterization

The ability to rapidly identify active compounds in a complex mixture (e.g., natural products extract) is still one of the major problems in natural products screening programs. An elegant way to overcome this problem is to separate the complex mixture by gradient liquid chromatography followed by online biochemical detection parallel with chemical characterization, referred to as high-resolution screening (HRS). To find and identify phosphodiesterase (PDE) inhibitors in natural products extracts using the HRS technology, the authors developed a continuous-flow PDE enzymatic assay. The suitability of the continuous-flow PDE enzymatic assay for natural products screening was demonstrated. After optimization of the continuous-flow PDE assay, the limit of detection for 3-isobutyl-1-methyl-xanthine (IBMX) was 1 muM, with a dynamic range from 1 to 100 muM IBMX. The applicability of the HRS technology for the detection of PDE inhibitors in natural products extracts was demonstrated by the analysis of a plant extract spiked with 2 naturally occurring PDE inhibitors. The plant extract was analyzed with 2 assay lines in parallel, enabling background fluorescence correction of the sample. The simultaneous quantification of the active compounds using evaporative light-scattering detection allowed the estimation of the IC(50) value of the active compounds directly in the crude extract.     

Formation of alkaloids from Penicillium species fungi during growth on wheat kernels

The ability to produce alkaloids has been studied in 13 strains belonging to 10 species of the genus Penicillium. Most of these strains produce identical ranges of alkaloids when grown on wheat grain and synthetic Abe medium. They are roquefortine, 3,12-dihydroroquefortine, and glandicolines A and B in strain P. chrysogenum VKM F-1987; fumigaclavines A and B, festuclavine, and pyroclasine in P. commune VKM F-308, F-3491, and KBP4; agroclavine 1 and epoxyagroclavine 1 in P. fellutanum VKM F-1073; fellutanine A in P. fellutanum F-3020; roquefortine, 3,12-dihydroroquefortine, meleagrin, and glandicolines A and B in P. glandicola VKM F-743; aurantioclavine in P. nalgiovense VKM F-229; isofumigaclavines A and B, festuclavine, roquefortine, and 3,12-dihydroroquefortine in P. roquefortii VKM F-2389; roquefortine, 3,12-dihydroroquefortine, and meleagrin in P. vitale VKM F-3624; roquefortine and oxaline in P. vulpinum VKM F-256; and alpha-cyclopiazonic acid and rugulovasine B in P. viridicatum C-47. No alkaloids were found in P. rugulosum VKM F-352 grown on wheat grain. A simple method is proposed for isolating alkaloids from affected grains.     

Analysis of the behaviour of<Emphasis Type="Italic"> Dictyostelium discoideum</Emphasis> in immobilised state by means of continuous cultivation

The social amoeba Dictyostelium discoideum is amenable to cultivation in the immobilised state most simply by colonisation of porous supports. An analysis of the growth behaviour of D. discoideum in the immobilised state is reported. For this purpose, D. discoideum was cultivated in continuously operated reactors in a suspension culture (homogeneous system) and immobilised on a porous support (heterogeneous system). Thus, it is possible to compare homogeneous and heterogeneous systems under steady-state conditions. Immobilisation was achieved by the colonisation of porous glass beads (SIRAN). Simple models are applied in order to describe the growth behaviour of fractions of both the cells in free suspension and the cells inside the porous carrier. This analysis shows that D. discoideum inside the pores grows at a rate of only about 10% compared with that in free solution. The consequence of this behaviour is discussed in terms of reactor performance.     

Detection of in situ labeled cell surface proteins by mass spectrometry: application to the membrane subproteome of human mammary epithelial cells

Characterization of the surface exposed membrane subproteome of human mammary epithelial cells (strain 184 A1L5) implemented lysine specific in situ labeling of the proteins using sulfosuccinimidyl-6-(biotinamido)hexanoate, followed by enrichment of the biotinylated, tryptically digested peptides, and then liquid chromatography-tandem mass spectrometry analysis of the labeled peptides. Probing the membrane subproteome in this manner yielded unambiguous identification of proteins situated on the cell surface. The method reported can be adapted to include stable isotope labeling of proteins for quantitation of changes occurring on the cell surface in response to specific perturbations.     

Genetic analysis of two distinct reproductive strategies in sexual and asexual field populations of an endoparasitic wasp,Venturia canescens

Asexual (thelytokous) females of the parasitoid Venturia canescens, which develop inside another insect, exhibit evolutionarily stable mixtures of life-history strategies, allowing two genetically distinct wasp lines to coexist sympatrically on the same host resources. Since the two asexual lines differ in a virus-like particle protein-coding gene (VLP1), the question is whether the VLP1 gene is genetically associated with the phenotype. The recent isolation of facultative sexual (arrhenotokous) and asexual V. canescens strains from the same location in Southern France has enabled an investigation of the genetic basis for the observed phenotypic differences, by comparing the two asexual lines with the corresponding homozygous VLP1 genotypes in arrhenotokous strains. This analysis showed similar patterns of morphological and functional differences exist in the ovaries of the two asexual VLP1 lines and in the two homozygous VLP1 genotypes from the field, suggesting that the VLP1 gene alteration either causes the ovarian phenotype or is genetically closely linked to the putative gene. However, the VLP1-gene may not be the only gene contributing to the phenotypic effects observed in the asexual lines. Although the two VLP1-alleles segregate with the relative differences in the ovary distribution of eggs, the absolute egg numbers differ in the corresponding asexual and sexual genotypes. This suggests that an additional unlinked gene may be involved in the transfer of eggs from the ovarioles into the oviduct.     

Detection of the rodent tapeworm Rodentolepis (=Hymenolepis) microstoma in humans. A new zoonosis?

A longitudinal survey of gastro-intestinal parasites was conducted over a 3-year period in remote communities in the north-west of Western Australia where, based on diagnosis by microscopy of faecal samples, Rodentolepis (=Hymenolepis) nana was found to be the most common enteric parasite. In the present study, using molecular tools, we describe the unexpected discovery, of a mixed infection with a second hymenolepidid species, Rodentolepis (=Hymenolepis) microstoma in four of the surveyed individuals. In the absence of any reliable earlier reports we believe this is to be the first instance of the detection of R. microstoma from human hosts. The development of a diagnostic restriction fragment polymorphism has enabled the study of R. microstoma in human populations and will greatly facilitate a more thorough understanding of the epidemiology of this parasite in the future.