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1.
Mol Ecol Resour ; 11(5): 935-6, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21777398

RESUMO

This article documents the addition of 92 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anopheles minimus, An. sinensis, An. dirus, Calephelis mutica, Lutjanus kasmira, Murella muralis and Orchestia montagui. These loci were cross-tested on the following species: Calephelis arizonensi, Calephelis borealis, Calephelis nemesis, Calephelis virginiensis and Lutjanus bengalensis.


Assuntos
Anfípodes/genética , Anopheles/genética , Bases de Dados Genéticas , Gastrópodes/genética , Repetições de Microssatélites/genética , Mariposas/genética , Perciformes/genética , Animais , Marcadores Genéticos/genética
2.
Health Promot. Int ; 22(2): 92-101, Jun. 2007. ilus, tab
Artigo em Inglês | CidSaúde - Cidades saudáveis | ID: cid-56805

RESUMO

This article describes results from an investigation of the health impacts of community gardening, using Toronto, Ontario as a case study. According to community members and local service organizations, these gardens have a number of positive health benefits. However, few studies have explicitly focused on the health impacts of community gardens, and many of those did not ask community gardeners directly about their experiences in community gardening. This article sets out to fill this gap by describing the results of a community-based research project that collected data on the perceived health impacts of community gardening through participant observation, focus groups and in-depth interviews. Results suggest that community gardens were perceived by gardeners to provide numerous health benefits, including improved access to food, improved nutrition, increased physical activity and improved mental health. Community gardens were also seen to promote social health and community cohesion. These benefits were set against a backdrop of insecure land tenure and access, bureaucratic resistance, concerns about soil contamination and a lack of awareness and understanding by community members and decision-makers. Results also highlight the need for ongoing resources to support gardens in these many roles. (AU)


Assuntos
Estudos de Casos e Controles , Estudos de Casos Organizacionais , População Urbana , Qualidade de Vida , Canadá
3.
Health Promot. Int ; 22(2): 92-101, Jun. 2007. tab
Artigo em Inglês | CidSaúde - Cidades saudáveis | ID: cid-59570

RESUMO

This article describes results from an investigation of the health impacts of community gardening, using Toronto, Ontario as a case study. According to community members and local service organizations, these gardens have a number of positive health benefits. However, few studies have explicitly focused on the health impacts of community gardens, and many of those did not ask community gardeners directly about their experiences in community gardening. This article sets out to fill this gap by describing the results of a community-based research project that collected data on the perceived health impacts of community gardening through participant observation, focus groups and in-depth interviews. Results suggest that community gardens were perceived by gardeners to provide numerous health benefits, including improved access to food, improved nutrition, increased physical activity and improved mental health. Community gardens were also seen to promote social health and community cohesion. These benefits were set against a backdrop of insecure land tenure and access, bureaucratic resistance, concerns about soil contamination and a lack of awareness and understanding by community members and decision-makers. Results also highlight the need for ongoing resources to support gardens in these many roles. (AU)


Assuntos
Saúde da População Urbana , Promoção da Saúde , Entrevistas como Assunto , Ontário , Estudos de Casos Organizacionais
4.
J Biol Chem ; 276(29): 27731-9, 2001 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-11356840

RESUMO

The major glycosylphosphatidylinositols (GPIs) transferred to protein in mammals and trypanosomes contain three mannoses. In Saccharomyces cerevisiae, however, the GPI transferred to protein bears a fourth, alpha1,2-linked Man on the alpha1,2-Man that receives the phosphoethanolamine (EthN-P) moiety through which GPIs become linked to protein. We report that temperature-sensitive smp3 mutants accumulate a GPI containing three mannoses and that smp3 is epistatic to the gpi11, gpi13, and gaa1 mutations, which normally result in the accumulation of Man(4)-GPIs, including the presumed substrate for the yeast GPI transamidase. The Smp3 protein, which is encoded by an essential gene, is therefore required for addition of the fourth Man to yeast GPI precursors. The finding that smp3 prevents the formation of the Man(4)-GPI that accumulates when addition of EthN-P to Man-3 is blocked in a gpi13 mutant suggests that the presence of the fourth Man is important for transfer of EthN-P to Man-3 of yeast GPIs. The Man(3)-GPI that accumulates in smp3 is a mixture of two dominant isoforms, one bearing a single EthN-P side branch on Man-1, the other with EthN-P on Man-2, and these isoforms can be placed in separate arms of a branched GPI assembly pathway. Smp3-related proteins are encoded in the genomes of Schizosaccharomyces pombe, Candida albicans, Drosophila melanogaster, and Homo sapiens and form a subgroup of a family of proteins, the other groups of which are defined by the Pig-B(Gpi10) protein, which adds the third GPI mannose, and by the Alg9 and Alg12 proteins, which act in the dolichol pathway for N-glycosylation. Because Man(4)-containing GPI precursors are normally formed in yeast and Plasmodium falciparum, whereas addition of a fourth Man during assembly of mammalian GPIs is rare and not required for GPI transfer to protein, Smp3p-dependent addition of a fourth Man represents a target for antifungal and antimalarial drugs.


Assuntos
Proteínas Fúngicas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Manose/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/genética , Glicosilfosfatidilinositóis/química , Manosiltransferases/metabolismo , Mutagênese , Ligação Proteica
5.
Mol Biol Cell ; 11(5): 1611-30, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10793139

RESUMO

Glycosylphosphatidylinositols (GPIs) are critical for membrane anchoring and intracellular transport of certain secretory proteins. GPIs have a conserved trimannosyl core bearing a phosphoethanolamine (EthN-P) moiety on the third mannose (Man-3) through which the glycolipid is linked to protein, but diverse GPI precursors with EthN-Ps on Man-1 and Man-2 have also been described. We report on two essential yeast genes whose products are required late in GPI assembly. GPI11 (YDR302w) encodes a homologue of human Pig-Fp, a protein implicated in the addition of EthN-P to Man-3. PIG-F complements the gpi11 deletion, but the rescued haploids are temperature sensitive. Abolition of Gpi11p or Pig-Fp function in GPI11 disruptants blocks GPI anchoring and formation of complete GPI precursors and leads to accumulation of two GPIs whose glycan head groups contain four mannoses but differ in the positioning and number of side chains, probably EthN-Ps. The less polar GPI bears EthN-P on Man-2, whereas the more polar lipid has EthN-P on Man-3. The latter finding indicates that Gpi11p is not required for adding EthN-P to Man-3. Gpi13p (YLL031cp), a member of a family of phosphoryltransferases, is a candidate for the enzyme responsible for adding EthN-P to Man-3. Depletion of Gpi13p in a Gpi11p-defective strain prevents formation of the GPI bearing EthN-P on Man-3, and Gpi13p-deficient strains accumulate a Man(4)-GPI isoform that bears EthN-P on Man-1. We further show that the lipid accumulation phenotype of Gpi11p-deficient cells resembles that of cells lacking Gpi7p, a sequence homologue of Gpi13p known to add EthN-P to Man-2 of a late-stage GPI precursor. This result suggests that in yeast a Gpi11p-deficiency can affect EthN-P addition to Man-2 by Gpi7p, in contrast to the Pig-Fp defect in mammalian cells, which prevents EthN-P addition to Man-3. Because Gpi11p and Pig-Fp affect EthN-P transfer to Man-2 and Man-3, respectively, these proteins may act in partnership with the GPI-EthN-P transferases, although their involvement in a given EthN-P transfer reaction varies between species. Possible roles for Gpi11p in the supply of the EthN-P donor are discussed. Because Gpi11p- and Gpi13p-deficient cells accumulate isoforms of Man(4)-GPIs with EthN-P on Man-2 and on Man-1, respectively, and because the GPIs that accumulate in Gpi11p-defective strains are likely to have been generated independently of one another, we propose that the yeast GPI assembly pathway is branched.


Assuntos
Etanolaminas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Sequência de Carboidratos , Genes Letais , Teste de Complementação Genética , Lipídeos/química , Manose/metabolismo , Proteínas de Membrana , Dados de Sequência Molecular , Mutação , Polissacarídeos/química , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
6.
Biophys J ; 75(3): 1372-83, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9726938

RESUMO

We report a simple new nuclear magnetic resonance (NMR) spectroscopic method to investigate order and dynamics in phospholipids in which inter-proton pair order parameters are derived by using high resolution 13C cross-polarization/magic angle spinning (CP/MAS) NMR combined with 1H dipolar echo preparation. The resulting two-dimensional NMR spectra permit determination of the motionally averaged interpair second moment for protons attached to each resolved 13C site, from which the corresponding interpair order parameters can be deducted. A spin-lock mixing pulse before cross-polarization enables the detection of spin diffusion amongst the different regions of the lipid molecules. The method was applied to a variety of model membrane systems, including 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/sterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sterol model membranes. The results agree well with previous studies using specifically deuterium labeled or predeuterated phospholipid molecules. It was also found that efficient spin diffusion takes place within the phospholipid acyl chains, and between the glycerol backbone and choline headgroup of these molecules. The experiment was also applied to biosynthetically 13C-labeled ergosterol incorporated into phosphatidylcholine bilayers. These results indicate highly restricted motions of both the sterol nucleus and the aliphatic side chain, and efficient spin exchange between these structurally dissimilar regions of the sterol molecule. Finally, studies were carried out in the lamellar liquid crystalline (L alpha) and inverted hexagonal (HII) phases of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). These results indicated that phosphatidylethanolamine lamellar phases are more ordered than the equivalent phases of phosphatidylcholines. In the HII (inverted hexagonal) phase, despite the increased translational freedom, there is highly constrained packing of the lipid molecules, particularly in the acyl chain region.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Lipídeos de Membrana/química , Fosfolipídeos/química , Fenômenos Biofísicos , Biofísica , Isótopos de Carbono , Colesterol/química , Dimiristoilfosfatidilcolina/química , Ergosterol/química , Lanosterol/química , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Prótons , Termodinâmica
7.
Biochem J ; 334 ( Pt 3): 609-16, 1998 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-9729469

RESUMO

Glycosylphosphatidylinositol (GPI) represents an important anchoring molecule for cell surface proteins. The first step in its synthesis is the transfer of N-acetylglucosamine (GlcNAc) from UDP to phosphatidylinositol (PI). The products of three mammalian genes, PIG-A, PIG-C and PIG-H, have previously been shown to be involved in the putative enzymic complex. Here we report the cloning of human and mouse cDNAs encoding a fourth participant in the GlcNAc transfer reaction which are homologues of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Gpi1 proteins. To provide evidence for their function, these proteins were expressed in GPI1-disrupted yeast strains. In Sacch. cerevisiae, where GPI1 disruption results in a temperature-sensitive phenotype and abolishes in vitro GlcNAc-PI synthesis, restoration of growth could be demonstrated in a temperature-dependent manner. In addition, in vitro GlcNAc-PI synthetic activity was again detectable. In Schiz. pombe, gpi1+ disruption is lethal. Using random spore analysis, we were able to show that the mammalian GPI1 homologues can rescue haploids harbouring the lethal gpi1+::his7+ allele. Our data demonstrate that the genes identified are indeed involved in the first step of GPI biosynthesis, and allow conclusions about a specific function for Gpi1p in stabilizing the enzymic complex. The finding that, despite a low degree of identity, the mammalian Gpi1 proteins are able to participate in the yeast GlcNAc-PI synthetic machinery as heterologous components further demonstrates that GPI biosynthesis has been highly conserved throughout evolution.


Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glicosilfosfatidilinositóis/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Sequência de Bases , Membrana Celular/metabolismo , Clonagem Molecular , Primers do DNA/genética , Proteínas Fúngicas/química , Expressão Gênica , Humanos , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
8.
EMBO J ; 17(4): 877-85, 1998 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-9463366

RESUMO

Biosynthesis of glycosylphosphatidylinositol (GPI) is initiated by transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol (PI). This chemically simple step is genetically complex because three genes are required in both mammals and yeast. Mammalian PIG-A and PIG-C are homologous to yeast GPI3 and GPI2, respectively; however, mammalian PIG-H is not homologous to yeast GPI1. Here, we report cloning of a human homolog of GPI1 (hGPI1) and demonstrate that four mammalian gene products form a protein complex in the endoplasmic reticulum membrane. PIG-L, which is involved in the second step in GPI synthesis, GlcNAc-PI de-N-acetylation, did not associate with the isolated complex. The protein complex had GPI-GlcNAc transferase (GPI-GnT) activity in vitro, but did not mediate the second reaction. Bovine PI was utilized approximately 100-fold more efficiently than soybean PI as a substrate, and lyso PI was a very inefficient substrate. These results suggest that GPI-GnT recognizes the fatty acyl chains of PI. The unusually complex organization of GPI-GnT may be relevant to selective usage of PI and/or regulation.


Assuntos
Glicosilfosfatidilinositóis/biossíntese , Proteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Sequência de Bases , Ceramidas/farmacologia , Clonagem Molecular , DNA Complementar/isolamento & purificação , Ativação Enzimática/efeitos dos fármacos , Glutationa Transferase/fisiologia , Glicosilfosfatidilinositóis/metabolismo , Hexosiltransferases , Humanos , Substâncias Macromoleculares , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fosfolipídeos/farmacologia , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transfecção
9.
Proc Natl Acad Sci U S A ; 94(15): 7873-8, 1997 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-9223280

RESUMO

Dolichol phosphate mannose (Dol-P-Man), formed upon transfer of Man from GDPMan to Dol-P, is a mannosyl donor in pathways leading to N-glycosylation, glycosyl phosphatidylinositol membrane anchoring, and O-mannosylation of protein. Dol-P-Man synthase is an essential protein in Saccharomyces cerevisiae. We have cloned cDNAs encoding human and Schizosaccharomyces pombe proteins that resemble S. cerevisiae Dol-P-Man synthase. Disruption of the gene for the S. pombe Dol-P-Man synthase homolog, dpm1(+), is lethal. The known Dol-P-Man synthase sequences can be divided into two classes. One contains the S. cerevisiae, Ustilago maydis, and Trypanosoma brucei enzymes, which have a COOH-terminal hydrophobic domain, and the other contains the human, S. pombe, and Caenorhabditis synthases, which lack a hydrophobic COOH-terminal domain. The two classes of synthase are functionally equivalent, because S. cerevisiae DPM1 and its human counterpart both complement the lethal null mutation in S. pombe dpm1(+). The findings that Dol-P-Man synthase is essential in yeast and that the Ustilago and Trypanosoma synthases are in a different class from the human enzyme raise the possibility that Dol-P-Man synthase could be exploited as a target for inhibitors of pathogenic eukaryotic microbes.


Assuntos
Manosiltransferases/metabolismo , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Sequência de Aminoácidos , Animais , Genes Fúngicos , Genes Letais , Teste de Complementação Genética , Humanos , Manosiltransferases/genética , Dados de Sequência Molecular , Mutação , Ratos , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/enzimologia , Homologia de Sequência de Aminoácidos
10.
Glycobiology ; 5(6): 603-10, 1995 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8563148

RESUMO

The gene encoding a beta-galactosidase from Xanthomonas manihotis was cloned into Escherichia coli. The gene resides on a 2.4 kb DNA fragment which was isolated from a partial Sau3A library in the cloning vector pUC19 using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as the selection. The enzyme produced by the clone has a specificity for beta 1-3- > beta 1-4-linked galactose. The nucleotide sequence of the gene was determined. The deduced protein sequence contained 597 amino acids yielding a monomeric molecular mass of 66 kDa. The cloned beta-galactosidase showed no similarity to any known prokaryotic beta-galactosidase. However, extensive similarity was observed with eukaryotic beta-galactosidases from animals, plants and fungi. The strongest similarity was with the beta-galactosidases found in the human and mouse lysosomes (42 and 41% identity, respectively). Alignment of the X.manihotis and eukaryotic beta-galactosidase sequences revealed seven highly conserved domains common to each protein. Additionally, Domain 1 in X.manihotis showed similarity to regions within catalytic domains from seven xylanases and cellulases belonging to family 10 of glucosyl hydrolases. A region spanning Domain 2 showed similarity to the catalytic domain of endo beta 1-3 glucanases from tobacco and barley.


Assuntos
Xanthomonas/genética , beta-Galactosidase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Xanthomonas/enzimologia , beta-Galactosidase/metabolismo
11.
Gene ; 155(1): 19-25, 1995 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-7698663

RESUMO

NaeI, a type-II restriction-modification (R-M) system from the bacterium Nocardia aerocolonigenes, recognizes the sequence 5'-GCCGGC. The NaeI DNA methyltransferase (MTase)-encoding gene, naeIM, had been cloned previously in Escherichia coli [Van Cott and Wilson, Gene 74 (1988) 55-59]. However, none of these clones expressed detectable levels of the restriction endonuclease (ENase). The absence of the intact ENase-encoding gene (naeIR) within the isolated MTase clones was confirmed by recloning the MTase clones into Streptomyces lividans. The complete NaeI system was finally cloned using E. coli AP1-200 [Piekarowicz et al., Nucleic Acids Res. 19 (1991) 1831-1835] and less stringent MTase-selection conditions. The naeIR gene was expressed first by cloning into S. lividans, and later by cloning under control of a regulated promoter in an E. coli strain preprotected by the heterologous MspI MTase (M.MspI). The DNA sequence of the NaeI R-M system has been determined, analyzed and compared to previously sequenced R-M systems.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Genes Bacterianos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Desoxirribonucleases de Sítio Específico do Tipo II/biossíntese , Escherichia coli/genética , Dados de Sequência Molecular , Nocardia/genética , Proteínas Recombinantes/biossíntese , Homologia de Sequência de Aminoácidos , Streptomyces/genética
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