RESUMO
South Africa's water resources are unequally distributed over space and time and an already stressed water resource situation will only be exacerbated by climate change if current predictions are correct. The potential for conflict over increasingly strained water resources in South Africa is thus very real. In order to deal with these complex problems, national legislation is demanding that water resource management be decentralized to the local level where active participation can take place in an integrated manner in accordance with the principles of Integrated Water Resource Management (IWRM). However, administrative and political boundaries rarely match those of catchments as, throughout South Africa, rivers have been employed extensively to delineate administrative and political boundaries at a number of spatial scales. The aim of this research is to determine if rivers act as dividing or uniting features in a socio-political landscape and whether topography will influence their role in this context. The Orange-Senqu River is used as a case study. This paper goes on to consider the implications of this for catchment management in South Africa. No study known to the authors has explored the effect of the river itself, and its topographic setting, on the drivers that foster either conflict or cooperation, and allow for participatory management. This study presents evidence that the topography of a catchment has the ability to aggravate or reduce the impact of the variables considered by water managers and thereby influence the role of a river as a dividing or uniting feature. South Africa's proposed form of decentralized water management will have to contend with the effects of different topographies on the way in which rivers are perceived and utilized.
Assuntos
Conservação dos Recursos Naturais , Geografia , Rios , Abastecimento de Água , Comunicação , Política , Apoio Social , África do SulRESUMO
The Ensembl project (http://www.ensembl.org) is a comprehensive genome information system featuring an integrated set of genome annotation, databases, and other information for chordate, selected model organism and disease vector genomes. As of release 51 (November 2008), Ensembl fully supports 45 species, and three additional species have preliminary support. New species in the past year include orangutan and six additional low coverage mammalian genomes. Major additions and improvements to Ensembl since our previous report include a major redesign of our website; generation of multiple genome alignments and ancestral sequences using the new Enredo-Pecan-Ortheus pipeline and development of our software infrastructure, particularly to support the Ensembl Genomes project (http://www.ensemblgenomes.org/).
Assuntos
Bases de Dados Genéticas , Genômica , Animais , Variação Genética , Humanos , Internet , Alinhamento de SequênciaRESUMO
The Ensembl project (http://www.ensembl.org) is a comprehensive genome information system featuring an integrated set of genome annotation, databases and other information for chordate and selected model organism and disease vector genomes. As of release 47 (October 2007), Ensembl fully supports 35 species, with preliminary support for six additional species. New species in the past year include platypus and horse. Major additions and improvements to Ensembl since our previous report include extensive support for functional genomics data in the form of a specialized functional genomics database, genome-wide maps of protein-DNA interactions and the Ensembl regulatory build; support for customization of the Ensembl web interface through the addition of user accounts and user groups; and increased support for genome resequencing. We have also introduced new comparative genomics-based data mining options and report on the continued development of our software infrastructure.
Assuntos
Bases de Dados Genéticas , Genômica , Animais , Gráficos por Computador , Humanos , Internet , Camundongos , Elementos Reguladores de Transcrição , Software , Interface Usuário-ComputadorRESUMO
The Ensembl (http://www.ensembl.org/) project provides a comprehensive and integrated source of annotation of chordate genome sequences. Over the past year the number of genomes available from Ensembl has increased from 15 to 33, with the addition of sites for the mammalian genomes of elephant, rabbit, armadillo, tenrec, platypus, pig, cat, bush baby, common shrew, microbat and european hedgehog; the fish genomes of stickleback and medaka and the second example of the genomes of the sea squirt (Ciona savignyi) and the mosquito (Aedes aegypti). Some of the major features added during the year include the first complete gene sets for genomes with low-sequence coverage, the introduction of new strain variation data and the introduction of new orthology/paralog annotations based on gene trees.
Assuntos
Bases de Dados de Ácidos Nucleicos , Genômica , Animais , Sequência de Bases , Bases de Dados de Ácidos Nucleicos/normas , Variação Genética , Genoma Humano , Humanos , Internet , Camundongos , Proteínas/genética , Padrões de Referência , Alinhamento de Sequência , Integração de Sistemas , Interface Usuário-ComputadorRESUMO
The Ensembl (http://www.ensembl.org/) project provides a comprehensive and integrated source of annotation of large genome sequences. Over the last year the number of genomes available from the Ensembl site has increased from 4 to 19, with the addition of the mammalian genomes of Rhesus macaque and Opossum, the chordate genome of Ciona intestinalis and the import and integration of the yeast genome. The year has also seen extensive improvements to both data analysis and presentation, with the introduction of a redesigned website, the addition of RNA gene and regulatory annotation and substantial improvements to the integration of human genome variation data.
Assuntos
Bases de Dados de Ácidos Nucleicos , Genômica , Animais , Sequência de Bases , Variação Genética , Genoma Humano , Humanos , Internet , Camundongos , Proteínas/genética , RNA/genética , Ratos , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Interface Usuário-ComputadorRESUMO
The Ensembl (http://www.ensembl.org/) project provides a comprehensive and integrated source of annotation of large genome sequences. Over the last year the number of genomes available from the Ensembl site has increased by 7 to 16, with the addition of the six vertebrate genomes of chimpanzee, dog, cow, chicken, tetraodon and frog and the insect genome of honeybee. The majority have been annotated automatically using the Ensembl gene build system, showing its flexibility to reliably annotate a wide variety of genomes. With the increased number of vertebrate genomes, the comparative analysis provided to users has been greatly improved, with new website interfaces allowing annotation of different genomes to be directly compared. The Ensembl software system is being increasingly widely reused in different projects showing the benefits of a completely open approach to software development and distribution.
Assuntos
Bases de Dados de Ácidos Nucleicos , Genômica , Animais , Sequência de Bases , Bovinos , Cães , Humanos , Internet , Camundongos , Ratos , Alinhamento de Sequência , Software , Interface Usuário-ComputadorRESUMO
The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organize biology around the sequences of large genomes. It is a comprehensive and integrated source of annotation of large genome sequences, available via interactive website, web services or flat files. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. The facilities of the system range from sequence analysis to data storage and visualization and installations exist around the world both in companies and at academic sites. With a total of nine genome sequences available from Ensembl and more genomes to follow, recent developments have focused mainly on closer integration between genomes and external data.
Assuntos
Biologia Computacional , Bases de Dados Genéticas , Genoma , Genômica , Animais , Humanos , Armazenamento e Recuperação da Informação , Internet , SoftwareRESUMO
Improved molecular understanding of the pathogenesis of type 2 diabetes is essential if current therapeutic and preventative options are to be extended. To identify diabetes-susceptibility genes, we have completed a primary (418-marker, 9-cM) autosomal-genome scan of 743 sib pairs (573 pedigrees) with type 2 diabetes who are from the Diabetes UK Warren 2 repository. Nonparametric linkage analysis of the entire data set identified seven regions showing evidence for linkage, with allele-sharing LOD scores > or =1.18 (P< or =.01). The strongest evidence was seen on chromosomes 8p21-22 (near D8S258 [LOD score 2.55]) and 10q23.3 (near D10S1765 [LOD score 1.99]), both coinciding with regions identified in previous scans in European subjects. This was also true of two lesser regions identified, on chromosomes 5q13 (D5S647 [LOD score 1.22] and 5q32 (D5S436 [LOD score 1.22]). Loci on 7p15.3 (LOD score 1.31) and 8q24.2 (LOD score 1.41) are novel. The final region showing evidence for linkage, on chromosome 1q24-25 (near D1S218 [LOD score 1.50]), colocalizes with evidence for linkage to diabetes found in Utah, French, and Pima families and in the GK rat. After dense-map genotyping (mean marker spacing 4.4 cM), evidence for linkage to this region increased to a LOD score of 1.98. Conditional analyses revealed nominally significant interactions between this locus and the regions on chromosomes 10q23.3 (P=.01) and 5q32 (P=.02). These data, derived from one of the largest genome scans undertaken in this condition, confirm that individual susceptibility-gene effects for type 2 diabetes are likely to be modest in size. Taken with genome scans in other populations, they provide both replication of previous evidence indicating the presence of a diabetes-susceptibility locus on chromosome 1q24-25 and support for the existence of additional loci on chromosomes 5, 8, and 10. These data should accelerate positional cloning efforts in these regions of interest.
Assuntos
Cromossomos Humanos Par 1 , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Mapeamento Cromossômico , Feminino , Testes Genéticos , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reino Unido/epidemiologia , População Branca/genéticaRESUMO
Chromosome 8 abnormalities are seen in 80% of patients with T-cell prolymphocytic leukemia (T-PLL). The abnormalities described are idic(8)(p11),t(8;8)(p11;q12),+8, and 8p+ with the involvement of 8p. To localize 8p11-p12 breakpoints in T-PLL, metaphases from seven cases were karyotyped. Those with idic(8)(p11) and add(8)(p11) were probed with a panel of contiguous YACs derived from 8p11-p12 using fluorescence in situ hybridization (FISH). Analysis of FISH results showed that 8p11-p12 breakpoints cluster into two regions. The first region is telomeric to YAC 899e2, which contains the fibroblast growth factor receptor-1 gene (FGFR1) and appears to cluster within a 1.5-MB YAC 807a2. The second region is more centromeric with breakpoints on either side of YAC 806e9, flanked by YAC 940f10 distally and YAC 910d7 proximally, the latter containing the MOZ gene. These findings showed that a segment of 8p was still present in the isodicentric, but the pattern of clustering does not seem to correspond to a breakpoint affecting a single gene. The clustering regions are likely to be hot spots for recombination and result in idic(8)(p11) and 8p+. These changes point to the pathogenesis of T-PLL involving deletion of a gene sequence on 8p and/or gain of a copy of 8q.
Assuntos
Cromossomos Humanos Par 8 , Leucemia Prolinfocítica/genética , Leucemia de Células T/genética , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Humanos , Hibridização in Situ FluorescenteRESUMO
Chromosome 1 abnormalities are the most commonly detected aberrations in many cancers including malignant melanomas. Specific breakpoints are reported for malignant melanomas throughout the chromosome but especially at 1p36 and at several sites throughout 1p22-q21. In addition, partial deletions and loss of heterozygosity have been found on 1p indicating the possible location of tumor suppressor genes. Here we have characterized the involvement of chromosome 1 in a series of seven malignant melanoma cell lines. Initial chromosome painting studies revealed that six of the cell lines had chromosome 1 rearrangements. Deletions involving 1p10-32, 1q11-44, and 1q25-44 were observed. The other rearrangement breakpoints included three in the 1q10-p11 region with the rest at 1p36, 1p34, 1p32, 1p31, 1p12-13, 1q21, and 1q23. The breaks at 1q10-p11 were investigated further using an alpha-satellite 1 centromere probe and yeast artificial chromosomes (YACs) from the region. Two of the 1q10-p11 breaks mapped in the centromeric region, while the others mapped to variable sites. This suggests that the role of these rearrangements in the pathogenesis of melanomas does not involve the alteration of specific oncogenes in the breakpoint region. During the YAC mapping a previously undetected, small (<1 Mbp) del(1)(p10p11) was identified. This deletion lies within minimal overlapping deleted regions reported in head and neck as well as breast carcinomas and it could therefore facilitate the isolation of a carcinoma-associated tumor suppressor gene.
Assuntos
Aberrações Cromossômicas/genética , Cromossomos Humanos Par 1/genética , Melanoma/genética , Transtornos Cromossômicos , Cromossomos Artificiais de Levedura/genética , Mapeamento de Sequências Contíguas , Humanos , Hibridização in Situ Fluorescente , Células Tumorais CultivadasRESUMO
Tandem repeats of a novel, putative, zinc-binding motif (MYM) have been described within the products of two, highly homologous genes: ZNF198/RAMP/FIM and ZNF261/DXS6673E. ZNF198, mapping to 13q11-q12, was recently shown to fuse to the fibroblast growth factor receptor 1 gene in the t(8;13)(p11;q11-q12) rearrangement associated with a stem cell leukemia/lymphoma syndrome. ZNF261 at Xq13.1 is disrupted by a t(X;13)(q13.1;q32) rearrangement in a mentally retarded patient and is a candidate gene for nonspecific X-linked mental retardation. Here we have cloned another member of this family, designated ZNF258, and mapped it to chromosome band 14q12. In addition, ZNF262/KIAA0425 was identified as a further member of the family and mapped to 1p32-p34. The predicted protein products of ZNF258 and ZNF262 maintain the repeats of the MYM motif. Isolation of these new members will facilitate the functional characterization of the MYM family and motif.
Assuntos
Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos/genética , Clonagem Molecular , Primers do DNA/genética , Feminino , Expressão Gênica , Humanos , Leucemia/genética , Linfoma/genética , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Gravidez , Sequências Repetitivas de Aminoácidos , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Identification of the t(X;18)(p11.2;q11.2) and the fusion gene products, SYT-SSX1 and SYT-SSX2, associated with a high proportion of synovial sarcomas, has been shown to be a useful diagnostic aid. This study demonstrates the application of dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the derivative X chromosome and also the position of the breakpoint on chromosome X at either the SSX1 or the SSX2 gene. This used region specific markers from chromosomes X and 18 and an optimized protocol involving microwave exposure. Novel and rapid scoring criteria were validated which circumvented potential problems of nuclear truncation and defining cell boundaries. This involved blind analysis of two negative sarcoma samples and three synovial sarcomas in which corresponding frozen material had been previously shown to have the translocation involving different SSX genes. Six new cases diagnosed as synovial sarcoma were also analysed; two monophasic and two biphasic case were deduced to have a breakpoint in the SSX1 gene, one monophasic case an SSX2 breakpoint, and one case did not show rearrangement of the region. The ability to analyse formalin-fixed, paraffin-embedded samples in this way has practical implications for aiding the diagnosis of difficult cases, recently ascribed prognostic relevance, and allows further retrospective studies to be carried out. The methodology is also applicable to the identification of other tumour specific translocations in paraffin-embedded material.
Assuntos
Cromossomos Humanos Par 18 , Proteínas de Neoplasias/genética , Proteínas Repressoras/genética , Sarcoma Sinovial/diagnóstico , Translocação Genética , Cromossomo X , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Prognóstico , Sarcoma Sinovial/genéticaRESUMO
The t(8;13)(p11;q12) is the most common translocation associated with the 8p11 myeloproliferative syndrome and results in an identical mRNA fusion between ZNF198 at 13q12 and FGFR1 at 8p11 in all cases thus far reported. ZNF198 is a widely expressed gene that is predicted to encode a 1377-amino-acid protein with five Zn finger-related motifs known as MYM domains. To determine the genomic DNA structure of ZNF198, we employed bubble PCR from PAC clones with a panel of gene-specific primers. Sequencing of these products revealed that ZNF198 consists of 26 exons with the initiation codon located in exon 4. The t(8;13) results in a consistent mRNA fusion of ZNF198 exon 17 to FGFR1 exon 9. Notable features of the structure of ZNF198 include three noncanonical GC donor splice sites and the presence of an alternatively spliced intron within exon 4. Amplification of genomic DNA from six t(8;13) patients with primers to ZNF198 exon 17 and FGFR1 exon 9 yielded patient-specific products ranging in size from 500 bp to 2.5 kb, indicating that the positions of the breakpoints in the t(8;13) are tightly clustered. The positions of the six t(8;13) breakpoints were determined and found to be distributed across ZNF198 intron 17 and FGFR1 intron 8 with no apparent subclustering. No consistent sequence motifs, repeats, or topoisomerase II cleavage sites were found at or near the breakpoints. It remains unclear why the t(8;13) translocation breakpoints occur within such small genomic regions, and it is possible that strict ZNF198-FGFR1 coding requirements restrict the positions of the breakpoints.
Assuntos
Proteínas de Transporte , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 8 , Proteínas de Ligação a DNA/genética , Transtornos Mieloproliferativos/genética , Translocação Genética , Sequência de Aminoácidos , Mapeamento Cromossômico , Éxons , Humanos , Íntrons , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome , Fatores de TranscriçãoRESUMO
The ZNF198- FGFR1 fusion gene arises as a result of the t(8;13)(p11;q12) in the 8p11 myeloproliferative syndrome. To determine the transforming properties of this chimeric protein we transfected ZNF198-FGFR1 into the interleukin (IL)-3 dependent cell line Ba/F3. Growth factor independent subclones were obtained in which ZNF198-FGFR1, STAT1, and STAT5 were constitutively tyrosine phosphorylated, as determined by immunoprecipitation and Western blot analysis. To test the hypothesis that constitutive activation of ZNF198-FGFR1 tyrosine kinase activity is a result of self-association of the fusion protein, we in vitro transcribed and translated ZNF198-FGFR1 and a derivative construct, ZNF198- FGFR1deltaC-myc, in which the C-terminal FGFR1 epitope was replaced by a c-myc tag. As expected, an anti-FGFR1 antibody immunoprecipitated ZNF198-FGFR1 but not ZNF198-FGFRdeltaC-myc. However when both products were translated together, both were coimmunoprecipitated by anti-FGFR1 antisera. Similar results were obtained by using an anti-myc antibody and demonstrated a physical interaction between the two proteins. Analysis of COS-7 cells transfected with ZNF198-FGFR1 demonstrated that the fusion gene, in contrast to normal FGFR1, is located in the cytoplasm. We conclude that ZNF198-FGFR1 is a cytoplasmic protein that self-associates and has constitutive transformation activity. These data suggest that ZNF198-FGFR1 plays a primary role in the pathogenesis of the t(8;13) myeloproliferative syndrome and is the first report to implicate STAT proteins in FGFR1-mediated signaling.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Substâncias de Crescimento/metabolismo , Proteínas do Leite , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transativadores/metabolismo , Tirosina/metabolismo , Animais , Western Blotting , Células COS , Linhagem Celular , Citoplasma/metabolismo , Imunofluorescência , Humanos , Interleucina-3/metabolismo , Camundongos , Transtornos Mieloproliferativos/genética , Fosforilação , Testes de Precipitina , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Fator de Transcrição STAT1 , Fator de Transcrição STAT5 , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
A recently described atypical myeloproliferative disorder is invariably associated with reciprocal translocations involving 8p11-12. The most common rearrangement is a t(8;13)(p11;q11-12). Here we determine that this translocation results in the fusion of the fibroblast growth factor receptor 1 gene (FGFR1), a member of the receptor tyrosine kinase family at 8p11, to a novel gene at 13q11-12 designated RAMP . The predicted RAMP protein exhibits strong homology to the product of a recently cloned candidate gene for X-linked mental retardation, DXS6673E . We also provide the first report of a novel, putative metal-binding motif, present as five tandem repeats in both RAMP and DXS6673E. RT-PCR detected only one of the two possible fusion transcripts, encoding a product in which the N-terminal 641 amino acids of RAMP become joined to the tyrosine kinase domain of FGFR1. Receptor tyrosine kinases are not commonly involved in the formation of tumour-specific fusion proteins. However, the previous reports of involvement of receptor tyrosine kinases in fusion proteins in non-Hodgkin's lymphoma, chronic myelomonocytic leukaemia and papillary thyroid carcinoma described similar rearrangements. By analogy with these, we propose that the RAMP-FGFR1 fusion product will contribute to progression of this myeloproliferative disorder by constitutive activation of tyrosine kinase function.
Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 8 , Proteínas de Ligação a DNA , Transtornos Mieloproliferativos/genética , Receptores Proteína Tirosina Quinases , Receptores de Fatores de Crescimento de Fibroblastos/genética , Translocação Genética , Sequência de Aminoácidos , Fusão Gênica Artificial , Sequência de Bases , Sítios de Ligação , Southern Blotting , Fatores de Crescimento de Fibroblastos/genética , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Fatores de Transcrição , Transcrição GênicaRESUMO
Fourteen cases of an atypical myeloproliferative disorder associated with consistent translocations involving 8p11-12 have previously been described. A t(8;13)(p11;q11-12) was the most common, but variant t(8;9)(p11-12;q32-34) and t(6;8)(q27;p12) were also reported. Here we have used a series of yeast artificial chromosomes (YACs) derived from the 8p11 and 13q11-12 regions to analyse one of the t(8;13) cases by fluorescence in situ hybridization (FISH). YACs flanking the 13q11-12 breakpoint and spanning the 8p11 breakpoint have been isolated. These YACs will facilitate characterization of the genes involved in this rearrangement.
Assuntos
Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 8/genética , Transtornos Mieloproliferativos/genética , Translocação Genética , Cromossomos Artificiais de Levedura , Humanos , Cariotipagem , Metáfase , Hibridização de Ácido NucleicoRESUMO
We demonstrate that the cytogenetically defined translocation t(X;1)(p11.2;p34) observed in papillary renal cell carcinomas results in the fusion of the splicing factor gene PSF located at 1p34 to the TFE3 helix-loop-helix transcription factor gene at Xp11.2. In addition we define an X chromosome inversion inv(X)(p11.2;q12) that results in the fusion of the NonO (p54nrb) gene to TFE3. NonO (p54nrb), the human homologue of the Drosophila gene NonAdiss which controls the male courtship song, is closely related to PSF and also believed to be involved in RNA splicing. In each case the rearrangement results in the fusion of almost the entire splicing factor protein to the TFE3 DNA-binding domain. These observations suggest the possibility of intriguing links between the processes of RNA splicing, DNA transcription and oncogenesis.
Assuntos
Fusão Gênica Artificial , Carcinoma Papilar/genética , Carcinoma de Células Renais/genética , Proteínas de Ligação a DNA/genética , Neoplasias Renais/genética , Proteínas Associadas à Matriz Nuclear , Proteínas Nucleares/genética , Splicing de RNA , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Southern Blotting , Carcinoma Papilar/metabolismo , Carcinoma de Células Renais/metabolismo , Inversão Cromossômica , Cromossomos Humanos Par 1 , Feminino , Sequências Hélice-Alça-Hélice/genética , Humanos , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fatores de Transcrição de Octâmero , Fator de Processamento Associado a PTB , Reação em Cadeia da Polimerase , Transcrição Gênica , Translocação Genética , Células Tumorais Cultivadas , Cromossomo XRESUMO
The spontaneous insertion of Bacillus thuringiensis Cry delta-endotoxins into planar lipid bilayers to form discrete channels in the absence of receptors is the subject of conflicting reports in the literature. Because these proteins are synthesized as protoxins requiring proteolytic activation for conversion to the active form, differences in the in-vitro protocol used for this activation could be responsible for the contradictory results. To investigate this, CrylA(c) toxin was activated by different procedures, and its ability to release glucose entrapped within liposomes and to form channels in planar lipid bilayers assessed. The toxin preparations exhibited widely differing activities on the lipid membranes; SDS-PAGE and immunoblot analysis suggested that variations in the protein profile of the activated samples could be responsible. These findings raise important practical considerations for further in-vitro studies into the mechanism of action of these toxins.
Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas , Endotoxinas/química , Canais Iônicos/química , Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis , Eletrofisiologia , Endopeptidases/metabolismo , Proteínas Hemolisinas , Bicamadas Lipídicas , Lipossomos , Precursores de Proteínas/química , Relação Estrutura-AtividadeRESUMO
Information on the molecular determinants of receptor recognition, membrane insertion and toxin pore-formation was sought by making 42 single and multiple substitutions of residues 312-314 (GYY), 367-370 (YRRP) and 438-441 (SGFS) in the Bacillus thuringiensis insecticidal CrylAc delta-endotoxin by site-directed mutagenesis. These three regions correspond to three putative surface-exposed loops (loops 1, 2 and 3, respectively) in domain II of the delta-endotoxin, forming the molecular apex of the structure. All except mutants GFY (loop 1), YKRA, SRRA, YRKA (loop 2) and TGFS (loop 3) expressed delta-endotoxin protein at wild-type levels which was stable upon activation by Pieris brassicae gut extract or trypsin. Toxicity assays for all the fully stable mutants using Manduca sexta larvae showed that G312, Y367, R368, R369, S438 and G439 are important for activity. Wild-type toxin was then labelled in vivo with [35S]methionine and heterologous competition binding assays were carried out for all the mutants using brush border membrane vesicles prepared from Manduca sexta midgut. Most and least conservative mutations of G439 and least conservative substitutions of Y367, R368 and R369 reduced the ability of the toxin to bind competitively. The most conservative mutation, S441T, gave significantly increased binding. These results suggested that these four residues play a role in the initial receptor binding step in the toxin mechanism. As no significant effect on binding affinity was observed in relatively non-toxic mutants in which residues G312 and S438 were mutated, we suggest that these residues are involved in the subsequent steps of membrane insertion and pore-formation.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas , Endotoxinas/genética , Endotoxinas/toxicidade , Proteínas de Insetos , Sequência de Aminoácidos , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Membrana Celular/metabolismo , DNA Bacteriano/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas , Larva/efeitos dos fármacos , Manduca/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Receptores de Superfície Celular/metabolismoRESUMO
The effects of air, water, and air plus water head cooling on thermoregulatory responses and human operator performance were studied in nonacclimatized, heat-exposed men. Forty chamber exposures (46 degrees C, 30 mm Hg water vapor pressure) were conducted under noncooled and the aforementioned subconditions of head cooling. Five subjects, exposed for 80 min, were monitored for mean skin and rectal temperatures, heart rate, sweat loss, and compensatory tracking performance. A modified Air Force helmet shell provided facial air ventilation (24 degrees C) at 8 cfm. Eight interconnected neoprene modules fastened beneath a helmet linear provided water cooling (20 degrees C at 0.9 l/min). Tracking performances was unchanged across conditions. Elevation of rectal temperature and heart rate, sweat loss, and Physiological Index of Strain were significantly reduced by each condition of head cooling. Air is as effective as water as a cooling agent. Air ventilation acts synergistically with water cooling in reducing physiological strain. Relative merits of each approach to head cooling, in an operational context, are discussed.