RESUMO
OBJECTIVE: The Wnt signalling antagonist Dickkopf-1 (DKK1) inhibits osteoblast differentiation and function and has been described to play a central role in promoting bone loss, while blockade of DKK1 increases bone formation. We investigated the effects of DKK1 on periosteal new bone formation in two murine models of inflammatory arthritis, the antigen-induced arthritis (AIA) and K/BxN serum transfer arthritis (STA) models. METHOD: The flare variant of AIA was induced in wild-type mice and a blocking antibody to DKK1, control rat immunoglobulin G (IgG), or phosphate-buffered saline (PBS) was administered starting on day 14, a time at which inflammation and erosions are known to be established. Knees were assessed for histological inflammation and periosteal new bone formation was quantitated. In addition, STA was generated in transgenic (Tg) mice with osteoblast-specific overexpression of Dkk1 and littermate controls. New bone formation around the wrists of these mice was quantified by micro-computed tomography. RESULTS: Blockade of DKK1 in arthritic mice resulted in significantly more periosteal new bone formation compared to mice treated with control rat IgG or PBS. Conversely, in the setting of increased Dkk1 expression, arthritic Dkk1 Tg mice developed significantly less periosteal new bone than arthritic controls. CONCLUSION: DKK1 is a regulator of periosteal bone formation in inflammatory arthritis. Thus, regulation of DKK1 may be considered as a therapeutic approach in inflammatory diseases in which patients suffer from excessive periosteal bone formation, such as spondyloarthritis.
Assuntos
Osteogênese , Espondilartrite , Camundongos , Ratos , Animais , Osteogênese/fisiologia , Modelos Animais de Doenças , Microtomografia por Raio-X , Peptídeos e Proteínas de Sinalização Intercelular , Inflamação , Imunoglobulina GRESUMO
An understanding of insect olfaction allows for more specific alternative methods of pest control. We evaluated the responses of the western flower thrips (WFT, Frankliniella occidentalis) in a Y-olfactometer to estimate gas-phase concentrations of the aggregation pheromone neryl (S)-2-methylbutanoate and known kairomones such as methyl isonicotinate, (S)-(-)-verbenone, and p-anisaldehyde. The gas-phase concentrations of these compounds were obtained from the release rates measured in dynamic headspace cells. The compounds were collected from the headspace using dried solid-phase extraction (SPE) cartridges and analyzed with a triple quadrupole GC-MS/MS. We observed that the aggregation pheromone significantly attracted WFT females at doses of 10 and 100 µg, whereas methyl isonicotinate and p-anisaldehyde significantly attracted WFT females at the highest dose. Verbenone did not produce any significant results. A completely different picture was obtained when the gas-phase concentrations were considered. The minimal gas-phase concentrations of the pheromone required to attract WFT females was 0.027 ng/mL, at least 100 times lower than that of the other two compounds. The relevance and implications of our results are discussed in light of the insect's biology and pest management methods.
RESUMO
The recent natural product isolates spiroviolene and spirograterpene A are two relatively non-functionalized linear triquinane terpenes with a large number of structural homologies. Nevertheless, three significant areas of structural disparity exist based on their original assignments, one of which implies a key stereochemical divergence early in their respective biosyntheses. Herein, using two known bicyclic ketone intermediates, a core Pd-catalyzed Heck cyclization sequence, and several chemoselective transformations, we describe concise total syntheses of both natural product targets and propose that the structure of spiroviolene should be reassigned. As a result, these natural products possess greater homology than previously anticipated.
RESUMO
Despite the proven value in utilizing pyrone dienes to create molecular complexity via Diels-Alder reactions with varied dienophiles, few examples of effective catalytic, asymmetric variants of this process have been developed. Herein, we show that the use of Jørgensen-Hayashi-type catalysts can convert an array of α,ß-unsaturated aldehydes into chiral dienamines that can formally add in a Diels-Alder fashion to a number of electron-deficient pyrones of the coumalate-type to generate optically active [2.2.2]-bicyclic lactones. In most cases, the reactions proceed with good to excellent diastereo- and enantiocontrol (up to 99% ee). Models to explain that stereoselectivity, as well as several additional transformations of the resultant products, are also presented.
RESUMO
G protein-coupled receptor 137b (GPR137b) is an orphan seven-pass transmembrane receptor of unknown function. In mouse, Gpr137b is highly expressed in osteoclasts in vivo and is upregulated during in vitro differentiation. To elucidate the role that GPR137b plays in osteoclasts, we tested the effect of GPR137b deficiency on osteoclast maturation and resorbing activity. We used CRISPR/Cas9 gene editing in mouse-derived ER-Hoxb8 immortalized myeloid progenitors to generate GPR137b-deficient osteoclast precursors. Decreasing Gpr137b in these precursors led to increased osteoclast differentiation and bone resorption activity. To explore the role of GPR137b during skeletal development, we generated zebrafish deficient for the ortholog gpr137ba. Gpr137ba-deficient zebrafish are viable and fertile and do not display overt morphological defects as adults. However, analysis of osteoclast function in gpr137ba-/- mutants demonstrated increased bone resorption. Micro-computed tomography evaluation of vertebral bone mass and morphology demonstrated that gpr137ba-deficiency altered the angle of the neural arch, a skeletal site with high osteoclast activity. Vital staining of gpr137ba-/- fish with calcein and alizarin red indicated that bone formation in the mutants is also increased, suggesting high bone turnover. These results identify GPR137b as a conserved negative regulator of osteoclast activity essential for normal resorption and patterning of the skeleton. Further, these data suggest that coordination of osteoclast and osteoblast activity is a conserved process among vertebrates and may have similar regulation.
Assuntos
Remodelação Óssea/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Sequência de Bases , Reabsorção Óssea/patologia , Osso e Ossos/patologia , Diferenciação Celular , Homeostase , Mutação com Perda de Função/genética , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , OsteogêneseRESUMO
BACKGROUND: Exit from mitosis requires inactivation of mitotic cyclin-dependent kinases (CDKs). A key mechanism of CDK inactivation is ubiquitin-mediated cyclin proteolysis, which is triggered by the late mitotic activation of a ubiquitin ligase known as the anaphase-promoting complex (APC). Activation of the APC requires its association with substoichiometric activating subunits termed Cdc20 and Hct1 (also known as Cdh1). Here, we explore the molecular function and regulation of the APC regulatory subunit Hct1 in Saccharomyces cerevisiae. RESULTS: Recombinant Hct1 activated the cyclin-ubiquitin ligase activity of APC isolated from multiple cell cycle stages. APC isolated from cells arrested in G1, or in late mitosis due to the cdc14-1 mutation, was more responsive to Hct1 than APC isolated from other stages. We found that Hct1 was phosphorylated in vivo at multiple CDK consensus sites during cell cycle stages when activity of the cyclin-dependent kinase Cdc28 is high and APC activity is low. Purified Hct1 was phosphorylated in vitro at these sites by purified Cdc28-cyclin complexes, and phosphorylation abolished the ability of Hct1 to activate the APC in vitro. The phosphatase Cdc14, which is known to be required for APC activation in vivo, was able to reverse the effects of Cdc28 by catalyzing Hct1 dephosphorylation and activation. CONCLUSIONS: We conclude that Hct1 phosphorylation is a key regulatory mechanism in the control of cyclin destruction. Phosphorylation of Hct1 provides a mechanism by which Cdc28 blocks its own inactivation during S phase and early mitosis. Following anaphase, dephosphorylation of Hct1 by Cdc14 may help initiate cyclin destruction.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Ligases/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Tirosina Fosfatases , Proteínas de Saccharomyces cerevisiae , Complexos Ubiquitina-Proteína Ligase , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdh1 , Ativação Enzimática , Mitose , Fosforilação , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: Following chromosome segregation in anaphase, ubiquitin-dependent degradation of mitotic cyclins contributes to the exit from mitosis. A key step in this process is catalyzed by a ubiquitin-protein ligase known as the anaphase-promoting complex (APC), the regulation of which is poorly understood. The Polo-related protein kinase Cdc5 in Saccharomyces cerevisiae might encode a regulator of the APC, because cdc5 mutant cells arrest with a late mitotic phenotype similar to that observed in cells with defective cyclin destruction. RESULTS: We investigated the role of Cdc5 in the regulation of mitotic cyclin degradation. In cdc5-1 mutant cells, we observed a defect in the destruction of cyclins and a reduction in the cyclin-ubiquitin ligase activity of the APC. Overexpression of CDC5 resulted in increased APC activity and mitotic cyclin destruction in asynchronous cells or in cells arrested in metaphase. CDC5 mutation or overexpression did not affect the degradation of the APC substrate Pds 1, which is normally degraded at the metaphase-to-anaphase transition. Cyclin-specific APC activity in cells overexpressing CDC5 was reduced in the absence of the APC regulatory proteins Hct 1 and Cdc20. In G1, Cdc5 itself was degraded by an APC-dependent and Hct1-dependent mechanism. CONCLUSIONS: We conclude that Cdc5 is a positive regulator of cyclin-specific APC activity in late mitosis. Degradation of Cdc5 in G1 might provide a feedback mechanism by which the APC destroys its activator at the onset of the next cell cycle.
Assuntos
Ciclina B , Ciclinas/metabolismo , Proteínas Fúngicas/fisiologia , Proteínas Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/fisiologia , Proteína da Polipose Adenomatosa do Colo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/fisiologia , Proteínas Cdc20 , Proteínas Cdh1 , Proteínas de Ciclo Celular/fisiologia , Ciclinas/fisiologia , Proteínas do Citoesqueleto/fisiologia , Proteínas Fúngicas/metabolismo , Fase G1/fisiologia , Mutação/genética , Mutação/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Quinases/biossíntese , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Saccharomyces cerevisiae/genética , Securina , Fuso Acromático/metabolismoRESUMO
Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase-cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15, cdc5, cdc14, dbf2, and tem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, and SIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1.
Assuntos
Ciclo Celular/fisiologia , Ciclina B , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Complexos Ubiquitina-Proteína Ligase , Ciclossomo-Complexo Promotor de Anáfase , Ciclo Celular/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina , Ciclinas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genótipo , Peptídeos e Proteínas de Sinalização Intracelular , Mitose , Modelos Biológicos , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/citologia , Supressão Genética , Ubiquitina-Proteína LigasesRESUMO
Esophageal squamous cell carcinoma is a form of cancer occurring most commonly in males, particularly those living in some areas of Asia, Africa, and western Europe. In some of these tumors, a sequence alteration has been identified in the coding region of the TP53 gene which is known to inactivate the tumor suppressor function of its product. Using a GC clamp (i.e., a GC rich domain) denaturing gradient gel electrophoresis assay we have been able to identify sequence modifications in 27 of the 32 tumor samples analyzed (84%). Most of the mutations occur in exon 6, a region of the gene which has not previously been reported as being a hot spot for the mutations of other cancers. Twelve of the mutations reported here have not been described in other types of tumors and these consist mostly of frameshift or splice mutations. The distribution of mutations [transitions (45%), transversions (34%), and frameshift (21%)] suggests that the etiological contribution of genotoxic factors might be complex and might associate different exogenous and endogenous mutagen exposures.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Genes p53 , Adulto , Idoso , Sequência de Bases , Dano ao DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , MutaçãoRESUMO
A patient with a testicular interstitial cell tumor and gynecomastia is reported. The testicular tumor was capable of aromatizing 8.3% testosterone. Spermatic venous samples taken from the tumor-bearing side had marked elevations of 17 beta-estradiol (E2), progesterone, and 17-hydroxyprogesterone (170 HP). The progesterone to 17OHP and 17OHP to androstene-dione ratios suggested an enzymatic block of 17 alpha-hydroxylase and 17-20 lyase possibly secondary to locally produced E2. The E2 produced by the tumor appeared to suppress gonadotropin secretion. The plasma testosterone and gonadotropin levels rose within 7 days after the removal of tumor, and the gynecomastia began to decrease.
Assuntos
Estradiol/biossíntese , Ginecomastia/etiologia , Tumor de Células de Leydig/metabolismo , Neoplasias Testiculares/metabolismo , Adulto , Androstenodiona/sangue , Castração , Hormônio Foliculoestimulante/sangue , Humanos , Hidroxiprogesteronas/sangue , Tumor de Células de Leydig/complicações , Tumor de Células de Leydig/patologia , Tumor de Células de Leydig/cirurgia , Hormônio Luteinizante/sangue , Masculino , Progesterona/sangue , Neoplasias Testiculares/complicações , Testosterona/sangueRESUMO
The hormonal profile of estrogen-secreting Leydig cell tumors was studied in four patients. Plasma testosterone (T) and estradiol (E2) levels varied from day-to-day whereas the T/E2 ratios were decreased (22:85, normal 170 to 440). hCG administration induced a higher estrogen response in the patients than in normal men. The finding in the spermatic venous blood of the tumor-bearing testis of a particular biochemical profile, including a low T/E2 ratio (12:27), associated with high progesterone/17-hydroxyprogesterone ratios (0.13:0.26) and high 17-hydroxyprogesterone-androstenedione ratios (26:44), allowed localization of a small testicular tumor when no testicular abnormality was found clinically. Also, the E2 level was moderately elevated in the spermatic vein of one patient compared with normal men. Spermatic venous blood also was obtained after hCG administration in two patients. Increased estrogen and reduced T responses were found in the tumoral testis in comparison with the contralateral testis. In conclusion, the hormone content of spermatic venous effluent from testes containing an interstitial cell tumor is abnormal in several respects and such abnormalities allow detection of the tumor when it is not recognizable clinically.
Assuntos
Ginecomastia/sangue , Hormônios/sangue , Tumor de Células de Leydig/sangue , Neoplasias Testiculares/sangue , Adulto , Androgênios/sangue , Gonadotropina Coriônica , Estradiol/sangue , Estrogênios/sangue , Hormônio Foliculoestimulante/sangue , Ginecomastia/etiologia , Humanos , Tumor de Células de Leydig/complicações , Hormônio Luteinizante/sangue , Masculino , Progestinas/sangue , Neoplasias Testiculares/complicações , Testosterona/sangueRESUMO
The apparent success in vector control observed between 1950 and 1970 was followed by worldwide resistance to organosynthetic insecticides wherever they were used intensively. Insect resistance to one or more categories of insecticides has limited the effectiveness of these compounds, and their non-selective mode of action adversely affects non-target organisms. This scenario highlights the need for selective agents in integrated vector control programs. This article gives an overview of the main fundamental and applied research topics on entomopathogenic bacteria in relation to their role in vector control.
Assuntos
Bacillus thuringiensis/patogenicidade , Toxinas Bacterianas/farmacologia , Culicidae/microbiologia , Insetos Vetores/microbiologia , Controle Biológico de Vetores/métodos , Animais , Culicidae/crescimento & desenvolvimento , Insetos Vetores/efeitos dos fármacosRESUMO
Midguts from Anopheles gambiae fourth instars were dissected and processed for immuno-light microscopy. Cloned insecticidal crystal proteins (ICPs) from Bacillus thuringiensis var. israelensis (Bti) were individually expressed in crystal-negative strains of Bacillus thuringiensis. Tissue sections of A. gambiae were incubated in vitro with each solubilized and trypsin-activated ICP. Immunodetection of CryIVA, CryIVB, CryIVD and CytA toxins on sections was performed using purified rabbit IgG directed against Bti ICPs, in combination with an anti-rabbit IgG/peroxidase. CryIVA, CryIVB, CryIVD and CytA toxins were detected on the apical brush border of midgut cells, in the gastric caecae and posterior stomach. CytA was also detected, to a lesser extent, on microvilli of anterior stomach cells.
Assuntos
Anopheles/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas , Endotoxinas/metabolismo , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Sistema Digestório/metabolismo , Endotoxinas/genética , Expressão Gênica , Proteínas Hemolisinas , Larva/metabolismo , Microvilosidades/metabolismo , Proteínas Recombinantes/metabolismo , Solubilidade , Tripsina/farmacologiaRESUMO
Fourth instar larvae of Anopheles gambiae were intoxicated with doses of purified crystals from Bacillus thuringiensis serovar israelensis (Bti) corresponding to 50-fold the LC50 after 24 h. Midguts were dissected after various contact times, then processed for immuno-light and -electron microscopy. Immunodetection on thin sections was performed using affinity-purified rabbit IgG against Bti crystal CryIVD or CytA polypeptides, in combination with anti-rabbit IgG/peroxidase. Both polypeptides were detected by optical and electron microscopy after 15 min of contact with Bti crystals on the apical brush border of midgut cells, but only in the gastric caeca and posterior stomach. No specific signal was detected in the other parts of the midgut, i.e. the cardia cells and the anterior stomach. These results confirm that mosquito midgut cells are the primary target for the toxins and that binding to specific receptors on the apical microvilli membrane is the initial step of delta-endotoxin action.
Assuntos
Anopheles/microbiologia , Bacillus thuringiensis/isolamento & purificação , Toxinas Bacterianas/isolamento & purificação , Mucosa Intestinal/microbiologia , Animais , Bacillus thuringiensis/imunologia , Toxinas Bacterianas/imunologia , Mucosa Gástrica/ultraestrutura , Técnicas In Vitro , Mucosa Intestinal/ultraestrutura , Microscopia Eletrônica , Microvilosidades/ultraestruturaRESUMO
Mosquitocidal proteins of 42 and 51 kDa from Bacillus sphaericus were produced in acrystallogenic B. sphaericus and B. subtilis strains. In both species, transformants containing each protein expressed individually were non-toxic for mosquito larvae and produced small inclusions which did not have a crystalline ultrastructure. When both components of the binary toxin were expressed together, toxicity was restored: oval and round amorphous inclusions were produced in B. subtilis, and typical native-type crystals were synthesized in B. sphaericus. In B. subtilis, native-type crystals were produced only when the two components of the binary toxin were synthesized as a 93-kDa fusion protein.
Assuntos
Bacillus subtilis/ultraestrutura , Bacillus/ultraestrutura , Toxinas Bacterianas/análise , Proteínas Recombinantes de Fusão/ultraestrutura , Cristalização , Técnicas In Vitro , Transformação BacterianaRESUMO
Sporulation of Clostridium bifermentans serovar malaysia, which has a larvicidal activity towards mosquitoes, was examined by electron microscopy. Parasporal inclusion bodies lacking a crystalline structure were first detected at t5 (5 h after the end of exponentional growth). Also, the presence of "brush-bottle"-like appendages appearing first at t5 was noted; these remained attached to the spores when released after sporangium lysis. Larvicidal activity assayed on Anopheles stephensi larvae appeared at t0 and increased rapidly to a maximum between t5 and t8. However, a decrease in bacterial toxicity occurred with sporangium lysis.
Assuntos
Clostridium/fisiologia , Animais , Divisão Celular/fisiologia , Clostridium/citologia , Clostridium/crescimento & desenvolvimento , Culicidae , Técnicas In Vitro , Microscopia Eletrônica , Esporos Bacterianos/citologia , Esporos Bacterianos/crescimento & desenvolvimentoRESUMO
Culex pipiens larval midgut is the primary target of the binary toxin (Bin) present in parasporal inclusions of Bacillus sphaericus. Cpm1, a 60-kDa protein purified from brush border membranes, has been proposed as the receptor of the Bin toxin in the midgut epithelial cells of mosquitoes. We have cloned and characterized the corresponding cDNA from midgut of Culex pipiens larvae. The open reading frame predicted a 580 amino-acid protein with a putative signal peptide at the N-terminus and a putative GPI-anchoring signal at the C-terminus. The amino acid sequence of the cloned Cpm1 exhibited 39-43% identities with insect maltases (alpha-glucosidases and alpha-amylases). Recombinant Cpm1 expressed in E. coli specifically bound to the Bin toxin and had a significant alpha-glucosidase activity but no alpha-amylase activity. These results support the view that Cpm1 is an alpha-glucosidase expressed in Culex midgut where it constitutes the receptor for the Bin toxin. To date, this is the first component involved in the mosquitocidal activity of the Bacillus sphaericus Bin toxin to be characterized. Its identification provides a key step to elucidate the mode of action of the Bin toxin and the mechanisms of resistance developed against it by some mosquito strains.
Assuntos
Bacillus/metabolismo , Culex/genética , Proteínas de Membrana/genética , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting/métodos , Clonagem Molecular , Culex/metabolismo , DNA Complementar , Sistema Digestório , Expressão Gênica , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de Aminoácidos , alfa-GlucosidasesRESUMO
The binary toxin (Bin) from Bacillus sphaericus crystals specifically binds to soluble midgut brush border membrane proteins from Culex pipiens larvae. A single 60 kDa midgut membrane protein is identified as the binding protein. This protein is anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor, and is partially released by phosphatidylinositol specific-phospholipase C (PI-PLC). Fractionation of soluble proteins by anion exchange chromatography indicates that the binding protein does not co-elute with leucine aminopeptidase activity. After partial purification, the sequences of internal amino acid fragments of the 60 kDa protein were determined. The peptide sequences were compared with data in GenBank, and showed a very high degree of similarity with enzymes belonging to the alpha-amylase family. Further enzymatic investigation showed that the receptor of the Bin toxin in C. pipiens larval midgut may be an alpha-glucosidase.
Assuntos
Bacillus , Toxinas Bacterianas/metabolismo , Culex/enzimologia , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , alfa-Glucosidases/metabolismo , Animais , Ácidos Cólicos , Cromatografia por Troca Iônica , Detergentes , Proteínas de Membrana/genética , Microvilosidades/enzimologia , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Receptores de Superfície Celular/genética , Solubilidade , Fosfolipases Tipo C/metabolismo , alfa-Glucosidases/genéticaRESUMO
The toxicity of Clostridium bifermentans serovar malaysia to mosquito larvae is due to protein toxins, belonging to a novel class of insecticidal toxins. Toxic extracts contains three major proteins of 66, 18 and 16 kDa. The 18-kDa and 16-kDa proteins are probably involved in toxicity. They are synthesised during sporulation, concomitant with activity. They are absent from non-toxic strains of C. bifermentans and are present at very low levels in non-toxic C. bifermentans serovar malaysia cultures produced at 42 degrees C. The 66-kDa protein is present throughout the growth phases of C. bifermentans serovar malaysia, and an immunologically related 66-kDa protein is present in non-toxic C. bifermentans strains.
Assuntos
Anopheles/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Clostridium/metabolismo , Animais , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/química , Clostridium/fisiologia , Larva/efeitos dos fármacos , Dose Letal Mediana , Esporos , TemperaturaRESUMO
The 42- and 51-kDa protein genes of Bacillus sphaericus 1593 have been subcloned independently downstream from the cytA gene promoter of Bacillus thuringiensis serovar israelensis and introduced into a non-mosquitocidal strain of Bacillus thuringiensis. Consequently, each protein was overproduced and accumulated as inclusion bodies which were purified. For the first time, the 42-kDa protein inclusions alone were found to be toxic to Culex pipiens larvae (LC50 at 48 h 300 ng ml-1); in contrast, the 51-kDa protein inclusions were not. Moreover, a synergistic effect between these two components was observed.