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2.
Insect Mol Biol ; 25(2): 163-70, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26748768

RESUMO

The Cry48Aa/Cry49Aa mosquitocidal toxin from Lysinibacillus sphaericus was uniquely composed of a three-domain (Cry) toxin and binary (Bin) toxin-like protein, with high toxicity against Culex spp. However, its mode of action against the target mosquitoes is still unknown. In this study, Cry48Aa, Cry49Aa and its N- and C-terminal truncated proteins were expressed and purified, and the binding affinities of the purified proteins with midgut brush-border membrane fractions (BBMFs) from Culex quin-quefasciatus larvae were performed. The results showed that both Cry48Aa and Cry49Aa have specific and high binding affinity to BBMFs, with dissociation constants of 9.5 ± 1.8 and 25.4 ± 3.8 nM, respectively. Competition assays demonstrated that Cry49Aa C-terminal derivatives were able to bind to the BBMFs, whereas Far-Western dot blot analysis revealed that its N-terminal constructs interacted with Cry48Aa. Nevertheless, larvicidal activity was almost lost when Cry49Aa truncated proteins, either individually or in pairs, combined with Cry48Aa. It is concluded that Cry49Aa is responsible for receptor binding and interaction with Cry48Aa and plays an important role in the mechanism of action of these two-component toxins.


Assuntos
Toxinas Bacterianas/farmacologia , Culex/efeitos dos fármacos , Larva/efeitos dos fármacos , Animais , Bacillus/química , Toxinas Bacterianas/química , Membrana Celular/efeitos dos fármacos , Culex/química , Sistema Digestório/química , Larva/química , Microvilosidades/efeitos dos fármacos
3.
Genet Mol Res ; 14(3): 10524-9, 2015 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-26400284

RESUMO

The aim of this study was to assess the role of the VEGF -2578C/A, +936C/T, and -460T/C gene polymorphisms in the development of osteosarcoma. A total of 182 patients with osteosarcoma and 182 age- and gender-matched healthy controls were enrolled into our study during January 2011 and December 2013. Genotype frequencies of the VEGF -2578C/A and -460T/C alleles in controls were found to be within the parameters of Hardy-Weinberg equilibrium, but the genotype frequencies of +936C/T alleles were not. By conditional regression analysis, we detected a statistically significantly increased risk of osteosarcoma in patients with the AA genotype (OR = 1.97; 95%CI = 1.02-3.83) and the CA+AA genotype (OR = 1.57; 95%CI = 1.01-2.44) of -2578C/A when compared with CC genotype. Therefore, our study showed that the AA and CA+AA genotypes of the VEGF -2578C/A polymorphism might modify the risk of osteosarcoma in a Chinese population.


Assuntos
Neoplasias Ósseas/genética , Predisposição Genética para Doença , Osteossarcoma/genética , Polimorfismo de Nucleotídeo Único , Fator A de Crescimento do Endotélio Vascular/genética , Adolescente , Adulto , Alelos , Povo Asiático , Neoplasias Ósseas/etnologia , Neoplasias Ósseas/patologia , Estudos de Casos e Controles , Criança , Feminino , Expressão Gênica , Frequência do Gene , Genótipo , Humanos , Masculino , Razão de Chances , Osteossarcoma/etnologia , Osteossarcoma/patologia , Análise de Regressão , Risco
4.
J Fish Biol ; 78(1): 150-65, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21235552

RESUMO

Gynogenesis was induced by using UV-irradiated spermatozoa of blunt snout bream Megalobrama amblycephala to activate eggs of common carp Cyprinus carpio. The maternal genome was then duplicated by cold shock in 0 to 4° C cold water to retain the second polar body. Two kinds of fry, normal fry and abnormal tortuous fry, were hatched. Their DNA content was measured by flow cytometry. The normal fry were identified as diploid, representing the successful gynogenesis in C. carpio whereas the abnormal tortuous fry were haploid. Ten microsatellite loci were used to study the genetic diversity among C. carpio, diploid gynogenetic C. carpio and unduplicated haploid tortuous fry. The results indicated that the genetic homozygosity of gynogenetic C. carpio was significantly higher than that of C. carpio. The genetic homozygosity of the haploid C. carpio was intermediate between that of gynogenetic C. carpio and C. carpio. It might be easier for the allogenetic DNA fragments to be integrated into the haploid genome than into diploid gynogenetic genome.


Assuntos
Carpas/genética , Repetições de Microssatélites , Ploidias , Animais , Carpas/anatomia & histologia , DNA/análise , Feminino , Masculino , Ovário/anatomia & histologia , Partenogênese
5.
J Appl Microbiol ; 105(1): 151-7, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18266703

RESUMO

AIMS: To improve ultraviolet (UV) resistance of Bacillus thuringiensis for increasing the duration of the Bt product applied in the field, a genetically engineered strain Bt TD841 that produced both melanin and Cry1A protein was constructed, and its UV resistance was evaluated in the laboratory. METHODS AND RESULTS: Melanin quantitative analysis revealed that the recombinant strain Bt TD841 could synthesize 0.15 mg melanin ml(-1) sporulated culture. Atomic force microscopy confirmed the production of diamond crystal and SDS-PAGE results showed the expression of the 130 kDa Cry1A protein. Bioassay results demonstrated that the LC(50) value of Bt TD841 was 3.69 microl ml(-1) against Helicoverpa armigera and the UV resistance of this recombinant was enhanced 9.7-fold compared to its parental strain Bt HC42 after 4-h UV irradiation. CONCLUSION: Expression of the mel gene can significantly increase UV resistance of B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on genetically engineered Bt strain with co-expression of melanin and the insecticidal crystal proteins gene, and the results may offer a practical solution for improving the photoprotection of Bt products in field application.


Assuntos
Bacillus thuringiensis/genética , Microbiologia Industrial/métodos , Melaninas/genética , Raios Ultravioleta , Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/efeitos da radiação , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Cristalização , Endotoxinas/genética , Escherichia coli/genética , Expressão Gênica , Engenharia Genética , Proteínas Hemolisinas/genética , Melaninas/metabolismo , Organismos Geneticamente Modificados , Controle Biológico de Vetores/métodos
6.
Arch Microbiol ; 187(6): 425-31, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17216168

RESUMO

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44-56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 x 10(-6) CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.


Assuntos
Bacillus cereus/genética , Conjugação Genética , Transferência Genética Horizontal , Lepidópteros/microbiologia , Plasmídeos/genética , Animais , Bacillus cereus/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Larva/microbiologia , Lepidópteros/classificação , Lepidópteros/crescimento & desenvolvimento
7.
J Appl Microbiol ; 93(3): 374-9, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12174034

RESUMO

AIMS: To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase-producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. METHODS AND RESULTS: The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirty-eight strains produced different levels of chitinase at pH 7.0, and so did 17 strains at pH 10.0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml(-1) in liquid medium. SDS-PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase-producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2.35-fold. CONCLUSION: This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide.


Assuntos
Bacillus thuringiensis/classificação , Bacillus thuringiensis/enzimologia , Quitinases/metabolismo , Controle Biológico de Vetores , Spodoptera/crescimento & desenvolvimento , Animais , Bioensaio , Larva/crescimento & desenvolvimento , Sorotipagem
8.
Oncogene ; 18(25): 3766-72, 1999 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-10391685

RESUMO

The HTLV-I oncoprotein Tax is required for high level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Tax stimulates HTLV-I transcription through high affinity binding to the KIX domain of CBP, a pleiotropic coactivator. Several cellular proteins, including c-jun, also bind to KIX and utilize CBP as a coactivator. To test whether Tax binding to KIX may disable cellular CBP function, we examined the potential interplay between Tax and c-jun for binding to KIX. We show that Tax represses the transcription function of c-jun in vivo and demonstrate that both transcription factors bind to an overlapping minimal region of KIX in vitro. c-jun binding to KIX is displaced by Tax, indicating that their binding is mutually exclusive and providing a molecular basis for the observed repression. The competition between Tax and cellular transcription factors for CBP represents a novel pathway for HTLV-I dependent deregulation of gene expression, and may have significant implications for cellular homeostasis and transformation in the HTLV-I infected T-cell.


Assuntos
Regulação Viral da Expressão Gênica , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/genética , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Sítios de Ligação , Ligação Competitiva , Proteína de Ligação a CREB , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Genes pX , Humanos , Células Jurkat , Proteínas Nucleares/química , Fosforilação , Mutação Puntual , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/química , Transfecção
9.
J Cell Biol ; 146(1): 71-84, 1999 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-10402461

RESUMO

Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi- enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged-GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the beta-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.


Assuntos
Brefeldina A/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/metabolismo , Proteínas/metabolismo , Fatores de Ribosilação do ADP , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Células CHO , Divisão Celular/efeitos dos fármacos , Clonagem Molecular , Proteína Coatomer , Cricetinae , Citosol/metabolismo , Citosol/ultraestrutura , Resistência a Medicamentos/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Fatores de Troca do Nucleotídeo Guanina , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Magnésio/farmacologia , Masculino , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas/química , Proteínas/genética , Ratos , Ratos Sprague-Dawley
10.
J Mol Biol ; 281(3): 395-400, 1998 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-9698555

RESUMO

The oncoprotein Tax, encoded by the human T-cell leukemia virus type I (HTLV-I), is required for high-level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Recent evidence suggests that Tax stimulates HTLV-I transcription through recruitment of the cellular coactivator protein CBP to the HTLV-I promoter, promoting high-level viral replication via the transcriptional activation properties associated with CBP. Tax directly contacts the KIX domain of CBP to stably anchor the coactivator to nucleoprotein complexes at the promoter. Here, we identify KIX amino acid residues 588 to 683 as the minimal region sufficient for interaction with Tax. This region is similar to the minimal KIX amino acid residues necessary for strong interaction with phosphorylated CREB, and is composed of a structural domain that forms an extensive hydrophobic core. We further show that a double point mutation in KIX differentially affects the binding of Tax and phosphorylated CREB, suggesting that these transcription factors may recognize unique amino acid residues within the KIX domain. These observations suggest that Tax directly contacts the hydrophobic core of KIX, and provides a structural framework to further define the molecular interactions between Tax and CBP.


Assuntos
Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Proteína de Ligação a CREB , Humanos , Proteínas Nucleares/genética , Fosforilação , Mutação Puntual , Ligação Proteica , Proteínas Recombinantes de Fusão , Deleção de Sequência , Transativadores/genética , Ativação Transcricional/fisiologia
11.
J Biol Chem ; 270(39): 22836-41, 1995 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-7559416

RESUMO

Recent in vitro work with Golgi-enriched membranes showed that 3'-azidothymidine-5'-monophosphate (AZTMP), the primary intracellular metabolite of 3'-azidothymidine (AZT), is a potent inhibitor of glycosylation reactions (Hall et al. (1994) J. Biol. Chem. 269, 14355-14358) and predicted that AZT treatment of whole cells should cause similar inhibition. In this report, we verify this prediction by showing that treatment of K562 cells with AZT inhibits lipid and protein glycosylation. AZT treatment dramatically alters the pattern of glycosphingolipid biosynthesis, nearly abolishing ganglioside synthesis at clinically relevant concentrations (1-5 microM), and suppresses the incorporation of both sialic acid and galactose into proteins. Control experiments demonstrate that these changes do not result from nonspecific effects on either the secretory apparatus or protein synthesis. On the other hand, studies using isolated nuclei as a model system for chromosomal DNA replication show that AZTTP is a very weak inhibitor of DNA synthesis. These observations strongly suggest that the myelosuppressive effects of AZT in vivo are due to inhibition of protein and/or lipid glycosylation and not to effects on chromosomal DNA replication.


Assuntos
Glicoesfingolipídeos/biossíntese , Inibidores da Transcriptase Reversa/farmacologia , Zidovudina/farmacologia , Configuração de Carboidratos , Sequência de Carboidratos , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Galactose/metabolismo , Glicoesfingolipídeos/química , Glicosilação/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Hexosaminas/metabolismo , Humanos , Cinética , Leucemia Eritroblástica Aguda , Dados de Sequência Molecular , Técnica de Diluição de Radioisótopos , Relação Estrutura-Atividade , Trítio , Células Tumorais Cultivadas , Zidovudina/análogos & derivados , Zidovudina/metabolismo
12.
J Cell Biol ; 126(1): 65-75, 1994 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8027187

RESUMO

22 CHOBFY (BFY) cell lines were isolated at a frequency 2-30 x 10(-7) from mutagenized populations on the basis of their ability to grow in the presence of 1 microgram/ml brefeldin A (BFA). Four of the five mutant lines tested are genetically stable and none of the mutant lines characterized degrade this drug. Immunofluorescence studies reveal that whereas early endosomes and the Golgi complex have nearly identical BFA sensitivities in the parent CHO line, the relative sensitivities of these two organelles were dramatically altered in all six mutant lines tested. Four cell lines maintain normal Golgi appearance at a BFA concentration as high as 10 micrograms/ml. Mutant lines show wide variation in the level of resistance to growth inhibition by BFA, but none of the mutant lines characterized grow above 2 micrograms/ml BFA. This specific growth inhibition is observed under conditions where Golgi morphology and function remain unaffected, suggesting that some factor(s) unrelated to Golgi function remains sensitive to BFA in BFY mutant lines. These observations provide strong evidence for the presence of multiple, organelle-specific targets for BFA. Cell-free measurements with membrane extracts establish that resistance to BFA in BFY-1 cells involves a membrane-associated factor distinct from ARFs and coatomers. This collection of mutant lines may prove valuable for the identification of intracellular target(s) for BFA and/or of effectors that interact upstream or downstream with these targets, thereby uncovering the cascade which regulates assembly of organelle-specific coats.


Assuntos
Células CHO/fisiologia , Compartimento Celular , Ciclopentanos/farmacologia , Glicoproteínas de Membrana , Mutação , Organelas/efeitos dos fármacos , Fatores de Ribosilação do ADP , Animais , Brefeldina A , Divisão Celular/efeitos dos fármacos , Proteína Coatomer , Cricetinae , Ciclopentanos/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos/genética , Proteínas de Ligação ao GTP/análise , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/análise , Modelos Biológicos , Organelas/ultraestrutura , Fenótipo , Seleção Genética , Proteínas do Envelope Viral/metabolismo
13.
J Biol Chem ; 269(20): 14355-8, 1994 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-8182037

RESUMO

3'-Azido-3'-deoxythymidine (AZT) is one of the primary chemotherapeutic agents used in the treatment of human immunodeficiency virus (HIV) infection. Unfortunately, AZT therapy is accompanied by severe side effects. Using Golgi-enriched membrane fractions, we have determined that 3'-azido-3'-deoxythymidine monophosphate, the primary AZT metabolite in treated cells, potently inhibits protein glycosylation. This inhibition results from direct competition with several pyrimidine-sugars for transport into Golgi membranes. This potential mechanism of cytotoxicity does not involve 3'-azido-3'-deoxythymidine triphosphate, the AZT metabolite most likely responsible for its antiviral effects; thus, it may be possible to develop novel therapeutic strategies that prevent inhibition of glycosylation without affecting the anti-HIV properties of AZT.


Assuntos
Proteínas de Membrana/metabolismo , Açúcares de Nucleosídeo Difosfato/metabolismo , Zidovudina/toxicidade , Animais , Células CHO , Cricetinae , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Didesoxinucleotídeos , Glicosilação/efeitos dos fármacos , Complexo de Golgi/metabolismo , HIV/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Humanos , Membranas Intracelulares/metabolismo , Cinética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Nucleotídeos de Timina/toxicidade , Uridina Difosfato Galactose/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , Zidovudina/análogos & derivados , Zidovudina/metabolismo , Zidovudina/uso terapêutico
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