RESUMO
The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F1F0ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development.
Assuntos
Transporte de Elétrons/efeitos dos fármacos , Lepidópteros/enzimologia , Lepidópteros/crescimento & desenvolvimento , Metamorfose Biológica , Metoprene/farmacologia , Mitocôndrias/enzimologia , Zea mays/parasitologia , Animais , Corpora Allata/metabolismo , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Hormônios Juvenis/metabolismo , Hormônios Juvenis/farmacologia , Larva , Lepidópteros/efeitos dos fármacos , Metamorfose Biológica/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Pupa/efeitos dos fármacos , Pupa/enzimologia , Pupa/crescimento & desenvolvimentoRESUMO
Balance between reactive oxygen species (ROS) and the antioxidant (AO) defense mechanisms is vital for organism survival. Insects serve as an ideal model to elucidate oxidative stress responses as they are prone to different kinds of stress during their life cycle. The present study demonstrates the modulation of AO enzyme gene expression in the insect pest, Achaea janata (castor semilooper), when subjected to different oxidative stress stimuli. Antioxidant enzymes' (catalase (Cat), superoxide dismutase (Sod), glutathione-S-transferase (GST) and glutathione peroxidase (Gpx)) partial coding sequences were cloned and characterized from larval whole body. Tissue expression studies reveal a unique pattern of AO genes in the larval tissues with maximum expression in the gut and fat body. Ontogeny profile depicts differential expression pattern through the larval developmental stages for each AO gene studied. Using quantitative RT-PCR, the expression pattern of these genes was monitored during sugar-induced (d-galactose feeding), infection-induced (Gram positive, Gram negative and non-pathogenic bacteria) and pesticide-induced oxidative stress (Bt Cry toxin). d-Galactose feeding differentially modulates the expression of AO genes in the larval gut and fat body. Immune challenge with Escherichia coli induces robust upregulation of AO genes when compared to Bacillus coagulans and Bacillus cereus in the larval fat body and gut. Cry toxin feeding predominantly induced GST upregulation in the gut. The current study suggests that though there are multiple ways of generation of oxidative stress in the insect, the organism tailors its response by insult- and tissue-specific recruitment of the antioxidant players and their differential regulation for each inducer.
Assuntos
Mariposas/fisiologia , Estresse Oxidativo , Sequência de Aminoácidos , Animais , Antioxidantes/metabolismo , Catalase/genética , Catalase/metabolismo , Clonagem Molecular , Escherichia coli/imunologia , Regulação da Expressão Gênica , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Mariposas/genética , Mariposas/imunologia , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Environmental estrogens are major cause of endocrine disruption in vertebrates, including aquatic organisms. Teleosts are valuable and popular models for studying the effects of endocrine disrupting chemicals (EDCs) in the environment. In the present study, we investigated the changes caused by exposure to the synthetic estrogens 17α-ethynylestradiol (EE2 ) and diethylstilbesterol (DES) during early stages of growth and sex differentiation of air-breathing catfish, Clarias gariepinus, at the morphological, histological, and molecular levels. Catfish hatchlings, 0 day post hatch (dph) were exposed continuously to sublethal doses of EE2 (50 ng/L) and DES (10 ng/L) until 50 dph and subsequently monitored for ovarian structural changes and alteration in the gene expression of steroidogenic enzymes till adulthood. Treated fish exhibited morphological deformities such as spinal curvature, stunted growth, and yolk-sac fluid retention. In addition to ovarian atrophy, DES-treated fish showed either rudimentary or malformed ovaries. Detailed histological studies revealed precocious oocyte development as well as follicular atresia. Further, transcript levels of various steroidogenic enzyme and transcription factor genes were altered in response to EE2 and DES. Activity of the rate-limiting enzyme of estrogen biosynthesis, aromatase, in the ovary as well as the brain of treated fish was in accordance with transcript level changes. These developmental and molecular effects imparted by EE2 and DES during early life stages of catfish could demonstrate the deleterious effects of estrogen exposure and provide reliable markers for estrogenic EDCs exposure in the environment.
Assuntos
Dietilestilbestrol/toxicidade , Disruptores Endócrinos/toxicidade , Estrogênios/biossíntese , Etinilestradiol/toxicidade , Ovário/efeitos dos fármacos , Animais , Aromatase/genética , Peixes-Gato/metabolismo , Feminino , Expressão Gênica , Ovário/metabolismo , Ovário/patologiaRESUMO
Cytochrome P450 aromatase (cyp19) catalyzes the conversion of androgens into estrogens. Teleosts have distinct, ovarian specific (cyp19a1a) and brain specific (cyp19a1b) cyp19 genes. Previous studies in teleosts demonstrated regulation of cyp19a1a expression by the NR5A nuclear receptor subfamily as well as a fork head transcription factor, FOXL2. In the present study, we investigated the involvement of fushi tarazu factor 1, FTZ-F1, a NR5A subfamily member, and FOXL2 in the regulation of cyp19a1b expression in brain of the air-breathing catfish, Clarias gariepinus. Based on the synchronous expression pattern of cyp19a1b, FTZ-F1 and FOXL2 in the brain, we isolated the 5' upstream region of cyp19a1b to analyse regulatory motifs. Promoter motif analysis revealed FTZ-F1/NR5A1 and FOXL2 binding nucleotide sequences. Transient transfection studies showed that FTZ-F1 and FOXL2 together enhanced the transcriptional activity of cyp19a1b gene in mammalian cell lines. Mutation in either of their putative binding sites within the cyp19a1b promoter abolished this effect. Electrophoretic gel mobility shift experiments indicated that FTZ-F1 and FOXL2 proteins bind to the synthesized radio-labelled oligomers used as probes and mobility shifted upon addition of their respective antibodies. Chromatin immunoprecipitation assay confirmed the binding of both these transcription factors to their corresponding cis-acting elements in the upstream region of cyp19a1b. To our knowledge, this study is the first report on the transcriptional regulation of cyp19a1b by FTZ-F1 and FOXL2 in a teleost fish.
Assuntos
Aromatase/genética , Encéfalo/metabolismo , Peixes-Gato/genética , Fatores de Transcrição Forkhead/genética , Regiões Promotoras Genéticas/genética , Fator Esteroidogênico 1/genética , Animais , Aromatase/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Peixes-Gato/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Células HEK293 , Humanos , Dados de Sequência Molecular , Motivos de Nucleotídeos , Fator Esteroidogênico 1/metabolismo , Ativação Transcricional , Regulação para CimaRESUMO
Juvenile hormone (JH) and 20-hydroxyecdysone (20E), co-ordinately orchestrate insect growth and development. The process of silk synthesis and secretion in lepidopteran insects is known to be under hormonal control. However, the role of JH in this process has not been demonstrated hitherto. The present study is aimed to elucidate the role of JH in H-fibroin regulation in Corcyra cephalonica, a serious lepidopteran pest. Reiterated amino acid stretches and the large molecular weight of H-fibroin render its cloning and characterization cumbersome. To address this, a commercially synthesized short amino acid peptide conjugated with a carrier protein was used to generate antibodies against the N-terminal region of H-fibroin. ELISA and immunoblot experiments demonstrated the sensitivity and specificity of antibody. Further, immunohistochemical analyses revealed the antibody's cross-reactivity with H-fibroins of C. cephalonica and Bombyx mori in the silk gland lumen. Quantitative RT-PCR and Western blot analysis demonstrated the tissue-specificity and developmental expression of H-fibroin. Hormonal studies revealed that JH alone does not alter the expression of H-fibroin. However, in the presence 20E, JH reverses the declined expression caused by 20E administration to normal levels. This study provides molecular evidence for the regulation of H-fibroin by the cumulative action of JH and 20E.
Assuntos
Fibroínas/metabolismo , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lepidópteros/crescimento & desenvolvimento , Lepidópteros/metabolismo , Metoprene/metabolismo , Animais , Western Blotting , Ecdisterona/metabolismo , Fibroínas/genética , Proteínas de Insetos/genética , Hormônios Juvenis/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Background: Staphylococcal mastitis is a major cause of concern to the dairy industry in India and several countries worldwide. Though Staphylococcus aureus is the major cause, coagulase negative staphylococcal species (CoNS) are being increasingly reported in recent years. Aims: To investigate the incidence of coagulase negative staphylococcal species in bovine mastitis. Methods: Isolation of staphylococci was carried out from 237 milk samples of cows and She buffaloes with clinical and subclinical mastitis from different regions of Andhra Pradesh and Karnataka. CoNS isolates were identified by tube coagulase test using fresh rabbit plasma and coagulase gene PCR. We employed the biochemical test scheme published elsewhere previously for identification of the CoNS isolates up to species and subspecies levels. Seven representative isolates were identified by 16S rDNA sequencing to check the accuracy of biochemical test based identification. Results: The CoNS constitute the majority of the staphylococcal isolates from mastitis (80/125, 64%) in this region. Using biochemical test scheme, the CoNS isolates from bovine mastitis were identified as S. cohnii sub sp. cohnii, S. simulans, S. capitis sub sp. capitis, S. cohnii sub sp. xylosus, and S. lugdunensis. The CoNS species S. schleiferi, S. haemolyticus, S. sciuri, S. xylosus, S. chromogenes, and Macrococcus epidermidis were identified by 16S rDNA sequencing. Conclusion: The 16S rDNA sequencing is the appropriate method for the identification of CoNS species. This study highlighted coagulase negative staphylococcal species as possible etiological agents of mastitis.
RESUMO
The malaria parasite has an extraordinary ability to evade the immune system due to which the development of a malaria vaccine is a challenging task. Extensive research on malarial infection in the human host particularly during the liver stage has resulted in the discovery of potential candidate vaccines including RTS,S/AS01 and R21. However, complete elimination of malaria would require a holistic multi-component approach. In line with this, under the World Health Organization's PATH Malaria Vaccine Initiative (MVI), the research focus has shifted towards the sexual stages of malaria in the mosquito host. Last two decades of scientific research obtained seminal information regarding the sexual/mosquito stages of the malaria. This updated and comprehensive review would provide the basis for consolidated understanding of cellular, biochemical, molecular and immunological aspects of parasite transmission right from the sexual stage commitment in the human host to the sporozoite delivery back into subsequent vertebrate host by the female Anopheles mosquito.
Assuntos
Anopheles/parasitologia , Vacinas Antimaláricas/uso terapêutico , Malária Falciparum , Plasmodium falciparum/metabolismo , Animais , Feminino , Humanos , Malária Falciparum/metabolismo , Malária Falciparum/prevenção & controle , MasculinoRESUMO
Development of insect resistance to biopesticides is a current and pertinent global issue. Earlier, it was established that lepidopteran larvae can recover from Bt intoxication via a midgut regenerative response and subsequently generate resistance. Molecular aspects of restoration of the midgut integrity following toxin exposure are emerging recently. In the present study, we bring out the pivotal role of gut arylphorin in mediating the midgut regenerative response following sublethal Bt exposure in Achaea janata. Bt-induced midgut damage was characterized by microscopic analysis using differential interference contrast (DIC) and immunofluorescence (IF). Extensive disruption of brush-border membrane, associated with the underlying cytoskeletal alterations including F-actin, α-actin and ß-tubulin was observed. Single-photon fluorescence microscopy combined with fluorescence lifetime imaging (FLIM) established the metabolic state associated with enhanced stem cell proliferation and migration from the basal side towards the luminal side following the damage. In-silico analysis revealed the phylogenetic relationship of gut arylphorin with closely related insect species and indicated the presence of two different subunits. Transient RNAi knockdown of the arylphorin resulted in diminished expression of mitotic Cyclin B mRNA levels. Human monoclonal Cyclin B antibody cross-reactivity with the Cyclin B located in the stem cells further validate the role of arylphorin as the mitogenic factor responsible for stem cell proliferation and epithelial regeneration. An in-depth understanding of resistance mechanisms will aid in the design of new strategies for the long-term usage and efficacy of Bt technology against pest control.
Assuntos
Toxinas de Bacillus thuringiensis/toxicidade , Proteínas de Insetos/metabolismo , Intestinos , Mariposas/metabolismo , Animais , Bacillus thuringiensisRESUMO
The biologically active ecdysteroid hormone, 20-hydroxyecdysone (20E), regulates various processes like molting, metamorphosis and reproduction in insects. However, its role in expression of silk genes is obscure. The silk core in insects is generally constituted of a complex of three proteins namely, H-chain fibroin (H-fibroin), L-chain fibroin (L-fibroin) and P25. In the present study, we report full-length cDNA cloning and characterization of L-fibroin and P25 genes from rice moth, Corcyra cephalonica. Northern analyses demonstrated 1.1 and 1kb transcripts of L-fibroin and P25 respectively. The tissue expression pattern shows the presence of these transcripts specifically in the salivary gland. These two genes are developmentally regulated at transcriptional level and their maximum expression is observed during the late-last instar larval stage. Semi-quantitative and real-time PCR studies revealed that 20E regulates the expression of these genes in a dose-dependant manner. This study further shows that physiological dose of 20E does not alter the normal expression of these two genes, whereas treatments with higher doses cause a significant decline in the expression. This study clearly suggests the role of 20E in the regulation of L-fibroin and P25 at molecular level.
Assuntos
Ecdisterona/metabolismo , Fibroínas , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Mariposas , Animais , Northern Blotting , Southern Blotting , Clonagem Molecular , Fibroínas/genética , Fibroínas/metabolismo , Imunoensaio , Larva , Dados de Sequência Molecular , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
Larvae of most lepidopteran insect species are known to be voracious feeders and important agricultural pests throughout the world. Achaea janata larvae cause serious damage to Ricinus communis (Castor) in India resulting in significant economic losses. Microbial insecticides based on crystalline (Cry) toxins of Bacillus thuringiensis (Bt) have been effective against the pest. Excessive and indiscriminate use of Bt-based biopesticides could be counter-productive and allow susceptible larvae to eventually develop resistance. Further, lack of adequate genome and transcriptome information for the pest limit our ability to determine the molecular mechanisms of altered physiological responses in Bt-exposed susceptible and tolerant insect strains. In order to facilitate biological, biochemical and molecular research of the pest species that would enable more efficient biocontrol, we report the midgut de novo transcriptome assembly and clustering of susceptible Cry toxin-exposed and Cry toxin tolerant Achaea janata larvae with appropriate age-matched and starvation controls.
Assuntos
Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Larva/genética , Mariposas/genética , Transcriptoma , Animais , Toxinas de Bacillus thuringiensis , Índia , Controle de Insetos , Inseticidas , RNA-SeqRESUMO
India is the major producer and exporter of castor oil in the world. Castor semilooper, Achaea janata is one of the main castor crop pests, which causes serious economic loss of crop, hence management and control of the pest are important. Currently, Bacillus thuringiensis (Bt) based biopesticides are being used for their control. However, the insects are known to develop resistance not only against chemical pesticides but also to Bt based biopesticides. In the present study, de novo transcriptome analysis was conducted to monitor the expression pattern of larval midgut genes in Achaea janata exposed to sublethal dose of Bt formulation. A total of 34,612 and 41,109 transcripts were identified in control and toxin-exposed larval midgut samples out of which 18,836 in control and 21,046 in toxin-exposed samples are annotated. Microarray data analysis employed to monitor the gene expression upon Cry toxin exposure revealed that 375 genes were upregulated and 579 genes were downregulated during all the time points (12-60â¯h) of toxin exposure. The differentially expressed transcripts include i.e. Cry toxin receptors, gut proteases, arylphorin, REPATs, detoxification enzymes and aquaporins. Validation of microarray data was performed by real-time quantitative PCR using few randomly selected genes and the results obtained were in corroboration. This is the first study on transcriptome data from the castor semilooper and the results would provide valuable resources for the characterization of Bt toxin response in the pest.
Assuntos
Bacillus thuringiensis , Agentes de Controle Biológico/toxicidade , Mariposas/efeitos dos fármacos , Mariposas/genética , Transcriptoma/efeitos dos fármacos , Animais , Bacillus thuringiensis/química , Agentes de Controle Biológico/química , Regulação da Expressão Gênica/efeitos dos fármacos , Genes de Insetos/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/genéticaRESUMO
AIM: The aim of this study was to characterize beta-lactamase antimicrobial resistance in Klebsiella and Enterobacter species isolated from healthy and diarrheic dogs in Andhra Pradesh. MATERIALS AND METHODS: A total of 136 rectal swabs were collected from healthy (92) and diarrheic (44) dogs, bacteriological cultured for Klebsiella and Enterobacter growth and screened for beta-lactamase antimicrobial resistance phenotypically by disc diffusion method and genotypically by polymerase chain reaction targeting blaTEM, blaSHV, blaOXA, blaCTX-M Group 1, 2, blaAmpC, blaACC, and blaMOX genes. RESULTS: A total of 33 Klebsiella and 29 Enterobacter isolates were recovered. Phenotypic beta-lactamase resistance was detected in 66.6% and 25% of Klebsiella and Enterobacter isolates, respectively, from healthy dogs and 66.6% and 60% of Klebsiella and Enterobacter isolates, respectively, from diarrheic dogs. Overall, incidence of extended-spectrum beta-lactamase (ESBL) phenotype was found to be 21.2% (7/33) in Klebsiella isolates, whereas none of the Enterobacter isolates exhibited ESBL phenotype. Predominant beta-lactamase genes detected in Klebsiella species include blaSHV (84.8%), followed by blaTEM (33.3%), blaCTX-M Group 1 (15.1%), and blaOXA (6.1%) gene. Predominant beta-lactamase genes detected in Enterobacter species include blaSHV (48.2%), followed by blaTEM (24.1%), blaAmpC (13.7%), and blaOXA (10.3%) gene. CONCLUSION: The present study highlighted alarming beta-lactamase resistance in Klebsiella and Enterobacter species of canine origin in India with due emphasis as indicators of antimicrobial resistance.
RESUMO
The lack of homogeneity in field application of Bacillus thuringiensis formulation often results in ingestion of sub-lethal doses of the biopesticide by a fraction of pest population and there by promotes the toxin tolerance and resistance in long term. Gut regeneration seems to be one of the possible mechanism by which this is accomplished. However, the existing information is primarily derived from in vitro studies using mid-gut cell cultures. Present study illustrates cellular and molecular changes in mid-gut epithelium of a Bt-susceptible polyphagous insect pest castor semilooper, Achaea janata in response to a Cry toxin formulation. The present report showed that prolonged exposure to sub-lethal doses of Cry toxin formulation has deleterious effect on larval growth and development. Histological analysis of mid-gut tissue exhibits epithelial cell degeneration, which is due to necrotic form of cell death followed by regeneration through enhanced proliferation of mid-gut stem cells. Cell death is demonstrated by confocal microscopy, flow-cytometry, and DNA fragmentation analysis. Cell proliferation in control vs. toxin-exposed larvae is evaluated by bromodeoxyuridine (BrdU) labeling and toluidine blue staining. Intriguingly, in situ mRNA analysis detected the presence of arylphorin transcripts in larval mid-gut epithelial cells. Quantitative PCR analysis further demonstrates altered expression of arylphorin gene in toxin-exposed larvae when compared with the control. The coincidence of enhanced mid-gut cell proliferation coincides with the elevated arylphorin expression upon Cry intoxication suggests that it might play a role in the regeneration of mid-gut epithelial cells.
RESUMO
Transmission-blocking vaccines (TBV) interrupt malaria parasite transmission and hence form an important component for malaria eradication. Mosquito midgut exopeptidases such as aminopeptidase N & carboxypeptidase B have demonstrated TBV potential. In the present study, we cloned and characterized carboxypeptidase A (CPA) from the midgut of an important malarial vector, Anopheles stephensi. ClustalW amino acid alignment and in silico 3-dimensional structure analysis of CPA predicted the presence of active sites involved in zinc and substrate binding that are conserved among all the known mosquito species. Real-time PCR analysis demonstrated that CPA is predominantly expressed in the midgut throughout the mosquito life cycle and that this gene is significantly elevated in P. berghei-infected mosquitoes compared to uninfected blood-fed controls. The high midgut CPA activity correlated with the prominent mRNA levels observed. Peptide-based anti-CPA antibodies were raised that cross-reacted specifically to â¼48kDa and â¼37kDa bands, which correspond to zymogen and active forms of CPA. Further, the addition of CPA-directed antibodies to P. berghei-containing blood meal significantly reduced the mosquito infection rate in the test group compared to control and blocked the parasite development in the midgut. These results support further development of A. stephensi CPA as a candidate TBV.
Assuntos
Anopheles/enzimologia , Carboxipeptidases A/genética , Clonagem Molecular , Trato Gastrointestinal/enzimologia , Insetos Vetores/enzimologia , Vacinas Antimaláricas , Plasmodium berghei , Animais , Anopheles/anatomia & histologia , Anopheles/genética , Anopheles/parasitologia , Carboxipeptidases A/química , Carboxipeptidases A/imunologia , Carboxipeptidases A/metabolismo , Feminino , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/parasitologia , Regulação Enzimológica da Expressão Gênica , Humanos , Insetos Vetores/anatomia & histologia , Insetos Vetores/parasitologia , Malária/parasitologia , Malária/prevenção & controle , Malária/transmissão , Masculino , Plasmodium berghei/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Arylphorin hexamerins are one of the major insect storage proteins involved in diverse functions during metamorphosis. However, their regulation during development is not elucidated so far. In the present study, we documented 20-hydroxyecdysone (20E)-mediated regulation of arylphorin expression in the fat body of the stored grain pest, Corcyra cephalonica. Based on the differential developmental expression and 20E-induced transcriptional as well as translational level alterations of arylphorin, we isolated the 5' upstream region of the gene to analyze regulatory motifs. Promoter motif analysis revealed the presence of ecdysone response element (ERE). Transient transfection studies showed the functionality of the ERE. Enzyme mobility shift experiments with radiolabelled, cold and mutated probes indicate ERE-nuclear factor binding. This study is the first to report transcriptional regulation of arylphorins by 20E in lepdopteran insect species.
Assuntos
Ecdisterona/farmacologia , Proteínas de Insetos/genética , Mariposas/crescimento & desenvolvimento , Oryza/parasitologia , Animais , Sítios de Ligação , Corpo Adiposo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Mariposas/genética , Mariposas/metabolismo , Elementos de RespostaRESUMO
In the present study, a riboflavin-binding hexamerin (RbHex) was cloned and characterized from the larval fat body of Corcyra cephalonica. The complete cDNA (2121bp) encodes a 706-amino acid protein with a molecular mass ~82kDa. Expression of RbHex 82 was predominant in fat body among larval tissues. Further, it is prominently expressed during the last instar larval development. Homology modeling and docking studies predicted riboflavin binding site of the hexamerin. Spectrofluorimetric analysis further confirmed riboflavin release from the hexamerin fraction. Quantitative RT-PCR studies demonstrated hormonal regulation of RbHex 82. 20-Hydroxyecdysone (20HE) had a stimulatory effect on its transcription whereas JH alone did not show any effect. However, JH in the presence of 20HE maintains the RbHex 82 expression which indicates the JH's role as a status quo factor. This study is the first to report the characterization of riboflavin-binding hexamerin in a lepidopteran pest. Further, the possibility of RbHex 82 as a pest control target is discussed.
Assuntos
Corpo Adiposo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/metabolismo , Mariposas/fisiologia , Riboflavina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Ecdisterona/farmacologia , Corpo Adiposo/efeitos dos fármacos , Corpo Adiposo/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Índia , Proteínas de Insetos/agonistas , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Metoprene/farmacologia , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Fases de Leitura Aberta , Especificidade de Órgãos , Filogenia , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Riboflavina/química , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
Pyrethroids including allethrin are the most common commercial household insecticides. The detrimental effects caused by pyrethroids on humans are gaining considerable attention. The present study was aimed to elucidate the effects of allethrin on the human corneal epithelial (HCE) cells. Allethrin inhibited the proliferation of HCE cells in a dose-dependent manner. In the presence of allethrin, cells showed membrane blebbing and nuclear fragmentation along with significant decrease in mitochondrial membrane potential resulting in increased cytochrome c (Cyt c) release into the cytosol. Further, flow cytometry analysis demonstrated a marked increase in sub G0-G1 cells, characteristic of apoptosis. Increased expression of pro-apoptotic protein, Bax, a simultaneous decrease of anti-apoptotic protein, Bcl-2, and activation of Caspase 3 was evident in the treated cells. In addition, extracellular matrix digesting metalloproteinase 9 (MMP-9) was also stimulated. Furthermore, significant increase in the gene expression of inflammatory cytokines, TNF-α and IL-1ß was observed. Taken together, these findings suggest that allethrin (IC50≈85µM) is toxic to HCE cells causing death through mitochondrial pathway.
Assuntos
Aletrinas/toxicidade , Células Epiteliais/efeitos dos fármacos , Epitélio Corneano/citologia , Inseticidas/toxicidade , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , Interleucina-1beta/genética , Metaloproteinase 9 da Matriz/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Bacillus thuringiensis (Bt) crystal proteins (Cry) bind to aminopeptidase N (APN) receptors on insect midgut membrane leading to pore formation and subsequent death. However, evolution of insect resistance to Bt toxins threatens their long-term application. Therefore, search for new targets which could function as Cry toxin receptors is an immediate mandate. In the present study, two full-length APN cDNAs were cloned from Malpighian tubule and salivary gland tissues of the moth, Achaea janata. Both these APNs showed 99% and 32% sequence homology with fat body and midgut APNs respectively. Tissue distribution analysis revealed the presence of two different APN isoforms, one predominant in non-gut visceral tissues while the other exclusively expressed in the midgut. Immunofluorescence and western blot analyses showed cross-reactivity in Malpighian tubule and salivary gland when probed with anti-fat body APN antiserum. These results clearly indicated the presence of non-gut (AjAPN1) and gut-specific (AjAPN4) isoforms in this moth. The expression of both the isoforms steadily increased during the larval development. Hormonal studies indicated regulation of the APN genes by the morphogenetic hormones, 20-hydroxyecdyone and juvenile hormone. Further, in vitro ligand-blotting studies demonstrated binding of Cry toxins to APNs in Malpighian tubule and salivary gland indicating their potential as alternate targets.
Assuntos
Bacillus thuringiensis/metabolismo , Toxinas Bacterianas/metabolismo , Antígenos CD13/metabolismo , Túbulos de Malpighi/enzimologia , Mariposas/enzimologia , Glândulas Salivares/enzimologia , Sequência de Aminoácidos , Animais , Antígenos CD13/química , Antígenos CD13/genética , Clonagem Molecular , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Larva/crescimento & desenvolvimento , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Especificidade de Órgãos , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
20-Hydroxyecdysone (20E) controls molting, metamorphosis and reproduction of insects. It binds to a heterodimeric complex of ecdysone receptor (EcR) and ultraspiracle (USP), and regulates the transcription of genes containing ecdysone response elements (EcREs). However, the 20E regulation of silk fibroin genes is largely unexplored. In most lepidopteran larvae, the silk fibroin primarily consists of a large protein, heavy chain fibroin (H-fibroin) that is associated with two small proteins, L-chain fibroin and P25. In the present study, we demonstrate that 20E regulates the expression of H-fibroin gene in Corcyra cephalonica, in a dose-dependent manner during the last instar larval development. Semi-quantitative and real-time PCR studies reveal that physiological doses of 20E do not alter the normal expression, whereas higher doses cause a significant decline in the expression. Luciferase activity assays and gel shift experiments further confirm the presence of a functional EcRE in the upstream region of H-fibroin which regulates the ecdysteroid dependent transcriptional activity of fibroin gene through EcR. In vitro treatment with 20E mimicking insecticides, RH-5849 and RH-5992 decreases the expression of H-fibroin in isolated salivary glands. Insects fed with similar concentrations of these insecticides, metamorphose abnormally. Differences are also observed in the ultrastructure of the silk fibers of control and insecticide fed insects providing additional insight into the disruptive effects of these non-steroidal ecdysteroid agonists.
Assuntos
Ecdisterona/farmacologia , Fibroínas/metabolismo , Larva/metabolismo , Mariposas/metabolismo , Sequência de Aminoácidos , Animais , Fibroínas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hidrazinas/farmacologia , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Mariposas/crescimento & desenvolvimento , Alinhamento de SequênciaRESUMO
BACKGROUND: Curcumin (diferuloylmethane) shows significant activity across a wide spectrum of conditions, but its usefulness is rather limited because of its low bioavailability. Use of nanoparticle formulations to enhance curcumin bioavailability is an emerging area of research. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, curcumin-loaded apotransferrin nanoparticles (nano-curcumin) prepared by sol-oil chemistry and were characterized by electron and atomic force microscopy. Confocal studies and fluorimetric analysis revealed that these particles enter T cells through transferrin-mediated endocytosis. Nano-curcumin releases significant quantities of drug gradually over a fairly long period, â¼50% of curcumin still remaining at 6 h of time. In contrast, intracellular soluble curcumin (sol-curcumin) reaches a maximum at 2 h followed by its complete elimination by 4 h. While sol-curcumin (GI(50)â=â15.6 µM) is twice more toxic than nano-curcumin (GI(50)â=â32.5 µM), nano-curcumin (IC(50)<1.75 µM) shows a higher anti-HIV activity compared to sol-curcumin (IC(50)â=â5.1 µM). Studies in vitro showed that nano-curcumin prominently inhibited the HIV-1 induced expression of Topo II α, IL-1ß and COX-2, an effect not seen with sol-curcumin. Nano-curcumin did not affect the expression of Topoisomerase II ß and TNF α. This point out that nano-curcumin affects the HIV-1 induced inflammatory responses through pathways downstream or independent of TNF α. Furthermore, nano-curcumin completely blocks the synthesis of viral cDNA in the gag region suggesting that the nano-curcumin mediated inhibition of HIV-1 replication is targeted to viral cDNA synthesis. CONCLUSION: Curcumin-loaded apotransferrin nanoparticles are highly efficacious inhibitors of HIV-1 replication in vitro and promise a high potential for clinical usefulness.