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1.
Arch Insect Biochem Physiol ; 103(1): e21626, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31562754

RESUMO

Insects can produce various antimicrobial peptides (AMPs) upon immune stimulation. One class of AMPs are characterized by their high proline content in certain fragments. They are generally called proline-rich antimicrobial peptides (PrAMPs). We previously reported the characterization of Spodoptera litura lebocin-1 (SlLeb-1), a PrAMP proprotein. Preliminary studies with synthetic polypeptides showed that among the four deductive active fragments, the C-terminal fragment SlLeb-1 (124-158) showed strong antibacterial activities. Here, we further characterized the antibacterial and antifungal activities of 124-158 and its four subfragments: 124-155, 124-149, 127-158, and 135-158. Only 124-158 and 127-158 could agglutinate bacteria, while 124-158 and four subfragments all could agglutinate Beauveria bassiana spores. Confocal microscopy showed that fluorescent peptides were located on the microbial surface. Fragment 135-158 lost activity completely against Escherichia coli and Staphylococcus aureus, and partially against Bacillus subtilis. Only 124-149 showed low activity against Serratia marcescens. Negative staining, transmission, and scanning electron microscopy of 124-158 treated bacteria showed different morphologies. Flow cytometry analysis of S. aureus showed that 124-158 and four subfragments changed bacterial subpopulations and caused an increase of DNA content. These results indicate that active fragments of SlLeb-1 may have diverse antimicrobial effects against different microbes. This study may provide an insight into the development of novel antimicrobial agents.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Insetos/farmacologia , Spodoptera/química , Animais , Peptídeos Catiônicos Antimicrobianos/química , Bacillus subtilis/efeitos dos fármacos , Beauveria/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Insetos/química , Serratia marcescens/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos
2.
Int J Mol Sci ; 20(19)2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31590392

RESUMO

The American cockroach (Periplaneta americana) is a medicinal insect. Its extract is used clinically to promote wound healing and tissue regeneration, but the effective medicinal components and mechanisms are not yet clear. It has been reported that human thymosin beta 4 (Tß4) may accelerate skin wound healing, however, the role of P. americana thymosin (Pa-THYs) is still poorly understood. In the present study, we identify and analyze the DNA sequences of Pa-THYs by bioinformatics analysis. Then we clone, express, and purify the Pa-THYs proteins and evaluate the activity of recombinant Pa-THYs proteins by cell migration and proliferation assays in NIH/3T3 cells. To elucidate the role of Pa-THYs in wound healing, a mouse model is established, and we evaluate wound contraction, histopathological parameters, and the expressions of several key growth factors after Pa-THYs treatment. Our results showed that three THY variants were formed by skipping splicing of exons. Pa-THYs could promote fibroblast migration, but have no effect on fibroblast proliferation. In wound repair, Pa-THYs proteins could effectively promote wound healing through stimulating dermal tissue regeneration, angiogenesis, and collagen deposition. On the molecular mechanism, Pa-THYs also stimulated the expression of several key growth factors to promote wound healing. The data suggest that Pa-THYs could be a potential drug for promoting wound repair.


Assuntos
Baratas/genética , Proteínas de Insetos/farmacologia , Timosina/farmacologia , Cicatrização/efeitos dos fármacos , Células 3T3 , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Baratas/metabolismo , Proteínas de Insetos/genética , Masculino , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Timosina/genética
3.
Int J Biol Macromol ; 141: 1072-1087, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31520705

RESUMO

Infections with HCV and HBV are serious worldwide health problems. Here, we report the anti-HCV and -HBV proficiency of Apis mellifera major royal-jelly protein (MRJP) 2 and its isoform X1. The efficiency of these proteins was evaluated in vitro and their safety was examined in vivo in comparison with Sofosbuvir (SOF) drug. Various in-silico methodologies were achieved for better understanding the antiviral mechanism of these MRJPs. Results proved their precluding ability to the viral receptors, CD81 and scavenger receptor class B type I (SR-B1). In addition, they targeted HCV-NS3/NS4A protease, HCV-NS5B polymerase, and HBV-polymerase (DNA-dependent DNA polymerase, and reverse transcriptase). Co-treatment with these proteins exerted different efficiencies toward CD81 and SR-B1 (synergistic), HBV-enzymes (antagonistic), and HCV-enzymes (either additive or synergistic). The studied proteins maximized their antiviral effect by their safety and superior potency to SOF. Collectively, these outcomes will shed the light on MRJP2 and its isoform X1 as two promising safe-inhibitors for both HCV and HBV.


Assuntos
Abelhas , Hepacivirus/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Segurança , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Antivirais/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Hepacivirus/fisiologia , Vírus da Hepatite B/fisiologia , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Proteínas de Insetos/toxicidade , Masculino , Simulação de Acoplamento Molecular , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Isoformas de Proteínas/toxicidade , Ratos , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
4.
Insect Biochem Mol Biol ; 113: 103215, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31449847

RESUMO

In this study, two novel antibacterial peptide genes, termed lugensin A and B were identified and characterized from a rice sap-sucking hemipteran insect pest, the brown planthopper, Nilaparvata lugens. Lugensin gene expression was significantly induced by Gram-negative and Gram-positive bacterial stains under the regulation of a signal receptor, the long peptidoglycan recognition protein (PGRP-LC) in the IMD pathway. Knockdown of PGRP-LC by RNAi eliminated bacterium induced Lugensin gene expression. Lugensins had the apparent antibacterial activities against Escherichia coli K12, Bacillus subtilis and the rice bacterial brown stripe pathogen Acidovorax avenae subsp. avenae (Aaa) strain RS-1. Lugensins inhibited bacterial proliferation by disrupting the integrity of the bacterial membranes. Scanning electron microscopy revealed abnormal membrane morphology of the recombinant Lugensin-treated bacteria. Lugensins induced complete cell disruption of E. coli K12 and B. subtilis strains while formed the holes on the cell surface of Aaa RS-1 strain. Immunofluorescence showed that Lugensins localized in the cell membrane of E. coli K12 while accumulated in the cytosol of B. subtilis. Differently, Lugensins remained in both the cell membrane and the cytosol of Aaa RS-1 strain, suggesting different action modes of Lugensins to different microbes. This is the first report of the novel antibacterial peptides found in the rice sap-sucking hemipteran insect species.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Regulação da Expressão Gênica , Hemípteros/genética , Proteínas de Insetos/genética , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Comamonadaceae/efeitos dos fármacos , Escherichia coli K12/efeitos dos fármacos , Feminino , Hemípteros/crescimento & desenvolvimento , Hemípteros/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Masculino , Ninfa/genética , Ninfa/metabolismo , Oócitos/metabolismo , Interferência de RNA
5.
Insect Biochem Mol Biol ; 111: 103181, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31265906

RESUMO

Although dengue is the most prevalent arthropod-borne viral disease in humans, no effective medication or vaccine is presently available. Previous studies suggested that mosquito salivary proteins influence infection by the dengue virus (DENV) in the mammalian host. However, the effects of salivary proteins on DENV replication within the Aedes aegypti mosquito remain largely unknown. In this study, we investigated the effect of a specific salivary protein (named AaSG34) on DENV serotype 2 (DENV2) replication and transmission. We showed that transcripts of AaSG34 were upregulated in the salivary glands of Aedes aegypti mosquitoes after a meal of blood infected with DENV2. Transcripts of the dengue viral genome and envelop protein in the salivary glands were significantly diminished after an infectious blood meal when AaSG34 was silenced. The effect of AaSG34 on DENV2 transmission was investigated in Stat1-deficient mice. The intradermal inoculation of infectious mosquito saliva induced hemorrhaging in the Stat1-deficient mice; however, saliva from the AaSG34-silenced mosquitoes did not induce hemorrhaging, suggesting that AaSG34 enhances DENV2 transmission. This is the first report to demonstrate that the protein AaSG34 promotes DENV2 replication in mosquito salivary glands and enhances the transmission of the virus to the mammalian host.


Assuntos
Aedes/virologia , Vírus da Dengue/fisiologia , Dengue/transmissão , Proteínas e Peptídeos Salivares/farmacologia , Animais , Dengue/patologia , Dengue/virologia , Vírus da Dengue/crescimento & desenvolvimento , Feminino , Proteínas de Insetos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mosquitos Vetores/virologia , Interferência de RNA , Replicação Viral
6.
DNA Cell Biol ; 38(8): 773-785, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31339741

RESUMO

Pierisin-5 protein (pie-5) belongs to a family of proteins possessing DNA-dependent ADP-ribosyltransferase activity, which can induce apoptotic cell death. The baculovirus-mediated expression vector system (BEVS) has been commonly used for in vitro expression of heterologous protein subunits for basic scientific research, in addition to the development and production of diagnostics and vaccines. In this study, a new method for the in vitro expression of the cytotoxic protein was established using the baculovirus expression system. The antiproliferative and apoptotic effect of the novel recombinant pierisin-5 protein (rpie-5) was investigated in different human cancer cell lines, such as HeLa, HepG2, and AGS. Cloning, in vitro overexpression, and purification of the rpie-5 protein were performed by using BEVS in Sf21 (Spodoptera frugiperda) insect cell line. The rpie-5 protein exhibits cytotoxicity in all the cell lines, but HeLa (IC50 0.6 µg/mL) was more sensitive when compared with HepG2 (IC50 1.9 µg/mL) and AGS (IC50 3.7 µg/mL) cell lines. The cytotoxic effects of rpie-5 lead to apoptotic cell death in cancer cells and resulted in nuclear fragmentation, enlargement of the nucleus, loss of mitochondrial membrane potential, and finally release of lactose dehydrogenase (LDH) enzyme from the cell membrane. This study reports the molecular mechanism of apoptotic cell death through the upregulation of Bax (Bcl-2 family activating protein-X), Bad, APAF-1 (apoptotic protease activating factor-1), Cyt-c, and caspase-3/9 and the downregulation of Bcl-2 (B-cell lymphoma 2) in rpie-5-treated cancer cells. The study concludes that rpie-5 has p53-independent apoptosis in HepG2 cells and p53-dependent apoptosis in HeLa and AGS cell lines. In the future, this study helps to understand the molecular mechanism of rpie-5 to induction of apoptosis and cell death.


Assuntos
ADP Ribose Transferases/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Proteínas Recombinantes/farmacologia , ADP Ribose Transferases/genética , Animais , Apoptose/fisiologia , Baculoviridae/genética , Linhagem Celular Tumoral , Clonagem Molecular , Humanos , Proteínas de Insetos/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/genética , Células Sf9 , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/genética
7.
Biochim Biophys Acta Biomembr ; 1861(10): 183023, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31325418

RESUMO

Pore-forming antimicrobial peptides (AMPs) are attracting interest as cytolytic antibiotics and drug delivery agents with potential use for targeting cancer cells or multidrug-resistant pathogens. Ceratotoxin A (CtxA) is an insect-derived cytolytic AMP with 36 amino acids that is thought to protect the eggs of the medfly Ceratitis capitata against pathogens. Single channel recordings using planar lipid bilayers have shown that CtxA forms pores with well-defined conductance states resembling those of alamethicin; it also forms one of the largest pores among the group of ceratotoxins. In this work, we modified CtxA at its N-terminus with an azide group and investigated its pore-forming characteristics in planar lipid bilayer experiments. We demonstrate the possibility to target specific lipids by carrying out click reactions in-situ on lipid membranes that display a dibenzocyclooctyne (DBCO) moiety on their head group. As a result of covalent linkage of the peptides to the bilayer, pore-formation occurs at 10-fold reduced peptide concentration and with a reduced dependence on the transmembrane voltage compared to unlinked CtxA-azide peptides or native CtxA peptides.


Assuntos
Azidas/farmacologia , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Sequência de Aminoácidos , Aminoácidos , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Azidas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HeLa , Humanos , Proteínas de Insetos/química , Células KB , Bicamadas Lipídicas/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/química
8.
Oxid Med Cell Longev ; 2019: 7897584, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31198493

RESUMO

Bombyx batryticatus is a known traditional Chinese medicine (TCM) utilized to treat convulsions, epilepsy, cough, asthma, headaches, and purpura in China for thousands of years. This study is aimed at investigating the antiepileptic effects of protein-rich extracts from Bombyx batryticatus (BBPs) on seizure in mice and exploring the protective effects of BBPs against H2O2-induced oxidative stress in PC12 cells and their underlying mechanisms. Maximal electroshock-induced seizure (MES) and pentylenetetrazole- (PTZ-) induced seizure in mice and the histological analysis were carried out to evaluate the antiepileptic effects of BBPs. The cell viability of PC12 cells stimulated by H2O2 was determined by MTT assay. The apoptosis and ROS levels of H2O2-stimulated PC12 cells were determined by flow cytometry analysis. Furthermore, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), lactate dehydrogenase (LDH), and glutathione (GSH) in PC12 cells were assayed by ELISA and expressions of caspase-3, caspase-9, Bax, Bcl-2, PI3K, Akt, and p-Akt were evaluated by Western blotting and quantitative real-time polymerase chain reaction (RT-qPCR) assays. The results revealed that BBPs exerted significant antiepileptic effects on mice. In addition, BBPs increased the cell viability of H2O2-stimulated PC12 cells and reduced apoptotic cells and ROS levels in H2O2-stimulated PC12 cells. By BBPs treatments, the levels of MDA and LDH were reduced and the levels of SOD and GSH-Px were increased in H2O2-stimulated PC12 cells. Moreover, BBPs upregulated the expressions of PI3K, Akt, p-Akt, and Bcl-2, whereas they downregulated the expressions of caspase-9, caspase-3, and Bax in H2O2-stimulated PC12 cells. These findings suggested that BBPs possessed potential antiepileptic effects on MES and PTZ-induced seizure in mice and protective effects on H2O2-induced oxidative stress in PC12 cells by exerting antioxidative and antiapoptotic effects via PI3K/Akt signaling pathways.


Assuntos
Anticonvulsivantes/farmacologia , Bombyx/química , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Insetos/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Convulsões/tratamento farmacológico , Animais , Anticonvulsivantes/administração & dosagem , Antioxidantes/farmacologia , Apoptose , Sobrevivência Celular , Convulsivantes/toxicidade , Eletrochoque/efeitos adversos , Peróxido de Hidrogênio/toxicidade , Proteínas de Insetos/farmacologia , Masculino , Malondialdeído/metabolismo , Camundongos , Fármacos Neuroprotetores/administração & dosagem , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Pentilenotetrazol/toxicidade , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Convulsões/etiologia , Convulsões/metabolismo , Convulsões/patologia , Transdução de Sinais
9.
Int J Biol Macromol ; 136: 1153-1160, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226372

RESUMO

Effects of a beetle antifreeze proteins (AFP) from Dendroides canadensis (DAFP-1) on a model freeze-labile enzyme, lactate dehydrogenase (LDH), were investigated under freezing and thawing conditions. The presence of DAFP-1 can effectively protect the enzymatic activity of LDH upon repeated freezing and thawing and the protective role of DAFP-1 is more significant than that of bovine serum albumin (BSA), a common protectant for freeze-labile proteins. The results of circular dichroism (CD) spectroscopy suggest that the presence of DAFP-1 provides protection to the denaturation of LDH under freezing and thawing. The molecular dynamics (MD) simulation of DAFP-1 and LDH suggests that DAFP-1 interacts with LDH using its ice-binding surface (IBS) and mainly through its arginine residues. A mutant of DAFP-1, where all the arginine residues were replaced by alanine residues, lost its effect in protecting LDH under freezing and thawing. The results demonstrated that DAFP-1 is an effective protectant for a freeze-labile protein under freezing and thawing and the arginine residues in DAFP-1 are important for its protective role. By correlating the protective effect of an AFP with its structure, new insights in the identification and development of effective protectants for freeze-labile proteins were provided.


Assuntos
Proteínas Anticongelantes/farmacologia , Besouros , Congelamento , Proteínas de Insetos/farmacologia , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Animais , Proteínas Anticongelantes/química , Proteínas Anticongelantes/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Desnaturação Proteica/efeitos dos fármacos
10.
Biomed Pharmacother ; 115: 108813, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31054505

RESUMO

Our objective was to investigate whether a thermostable protein fraction (TPF) obtained from the larvae of Musca domestica, which contains cecropin family AMPs, is effective in treating senna leaf (Folium Sennae)-induced diarrhea in mice and its possible underlying mechanism. We did the experiments both in vitro and in vivo. Firstly, lipopolysaccharide (LPS) was used to induce inflammation in RAW 264.7 macrophages. The expression level of nitric oxide (NO) and tumor necrosis factor (TNF)-α was assessed using kits and immunofluorescence assay. A mice model of total diarrhea was established using a decoction of Folium Sennae. Levels of interleukin (IL)-6 and IL-1ß in mice serum and of TNF-α in the supernatant of jejunal tissue homogenate were measured using commercially available ELISA kits. Hematoxylin-eosin staining was employed to evaluate pathological lesions, and immunohistochemistry was used for determining IL-1ß, IL-6, and TNF-α expression levels. Results display that TPF markedly inhibited NO and TNF-α production in LPS-stimulated RAW 264.7 macrophages in vitro. Moreover, TPF significantly lowered the diarrhea index (DI) in diarrheic mice; when TPF was administered at a high dose (120 mg/kg) to mice, in comparison with mice in the model group, DI was markedly reduced. TPF could also decrease the expression levels of some pro-inflammatory factors, high dose TPF treated mice were with the reduction of (202.29 ± 18.58) pg/ml (tumor necrosis factor alpha, TNF-α), (53.69 ± 7.83) pg/ml (interleukin (IL)-1ß, IL-1ß), (48.44 ± 3.77) pg/ml (IL-6I, L-6) to the model separately. In comparison with berberine hydrochloride, which was used as the positive control in this study, TPF could confer better intestinal protection. To conclude, our results demonstrate that TPF has potent anti-inflammatory activities in vitro and antidiarrheal effects on mice with Folium Sennae-induced diarrhea.


Assuntos
Antidiarreicos/farmacologia , Diarreia/induzido quimicamente , Moscas Domésticas/química , Proteínas de Insetos/farmacologia , Animais , Antidiarreicos/química , Diarreia/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Temperatura Alta , Proteínas de Insetos/química , Intestino Delgado/efeitos dos fármacos , Larva/química , Lipopolissacarídeos/toxicidade , Camundongos , Óxido Nítrico/metabolismo , Estabilidade Proteica , Células RAW 264.7 , Fator de Necrose Tumoral alfa/antagonistas & inibidores
11.
Insect Biochem Mol Biol ; 110: 60-68, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31051236

RESUMO

Antimicrobial peptides (AMPs) are important immune effectors in insects. Bacteria have a limited number of ways to resist AMPs, and AMP-resistance is often costly. Recently, it has become clear that AMP activities in vitro and in vivo differ. Although some studies have followed the in vivo survival of AMP resistant pathogens, studying a pathogen resistant to the AMPs of that particular host has never been reported. Here, we infected the mealworm beetle Tenebrio molitor with Staphylococcus aureus strains that were evolved in vitro in the presence of one or two antimicrobial peptides from T. molitor. We found that the Tenebrio immune system could clear mutant Tenecin resistant strains at least as efficiently as sensitive controls. The bacterial load of Tenecin resistant S. aureus segregated by mutation. Strains with mutations in both the pmt and rpo operons showed the highest in vivo survival and therefore showed the lowest fitness cost amongst the evolved resistance mutations. In contrast, Tenecin resistant strains with mutations in the nsa and rpo operons showed much lower survival within the hosts. Our study shows that Tenecin resistant strains are phagocytosed at a lower rate. The nsa/rpo mutants were phagocytosed at a higher rate than other Tenecin resistant S. aureus strains. The differences in resistance against AMPs and phagocytosis did not translate into changes in virulence. AMP resistance, while a prerequisite for an infection in vertebrates, does not provide a survival advantage to S. aureus in a host environment that is dominated by AMPs.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Imunidade Inata , Proteínas de Insetos/farmacologia , Staphylococcus aureus/fisiologia , Tenebrio/imunologia , Animais , Feminino , Masculino , Fagocitose/imunologia
12.
Microb Pathog ; 132: 335-342, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31100407

RESUMO

The aim of this study was to evaluate the effects of Bacillus subtilis and Bacillus atrophaeus on Galleria mellonella immunity challenged by Candida albicans. Firstly, we analyzed the susceptibility of G. mellonella to bacilli (vegetative and sporulating forms). It was found that both vegetative and sporulating forms were not pathogenic to G. mellonella at a concentration of 1 × 104 cells/larva. Next, larvae were pretreated with two species of Bacillus, in the vegetative and sporulating forms, and then challenged with C. albicans. In addition, the gene expression of antimicrobial peptides (AMPs) such as Gallerimycin, Gloverin, Cecropin-D and Galiomicin was investigated. Survival rates increased in the Bacillus treated larvae compared with control larvae inoculated with C. albicans only. Cells and spores of Bacillus spp. upregulated Gloverin, Galiomicin and Gallerimycin genes in relation to the control group (PBS + PBS). When these larvae were infected with C. albicans, the group pretreated with spores of B. atrophaeus and B. subtilis showed a greater increase in expression of Galiomycin (49.08-fold and 13.50-fold) and Gallerimycin (27.88-fold and 68.15-fold), respectively, compared to the group infected with C. albicans only (p = 0.0001). After that, we investigated the effects of B. subtilis and B. atrophaeus on immune system of G. mellonella evaluating the number of hemocytes, quantification of melanization, cocoon formation and colony forming units (CFU) count. Hemocyte count increased in response to stimulation by Bacillus, and a higher increase was achieved when larvae were inoculated with B. subtilis spores (p = 0.0011). In the melanization assay, all groups tested demonstrated lower production of melanin compared to that in the phosphate-buffered saline (PBS) group. In addition, full cocoon formation was observed in all groups analyzed, which corresponded to a healthier wax worm. Hemolymph culture revealed higher growth of B. atrophaeus and B. subtilis in the groups inoculated with spores. We concluded that spores and cells of B. atrophaeus and B. subtilis stimulated the immune system of G. mellonella larvae and protected them of C. albicans infection.


Assuntos
Bacillus/fisiologia , Candida albicans/patogenicidade , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade , Lepidópteros/imunologia , Alcaloides/genética , Alcaloides/metabolismo , Alcaloides/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/fisiologia , Contagem de Colônia Microbiana , Defensinas/genética , Defensinas/metabolismo , Defensinas/farmacologia , Modelos Animais de Doenças , Expressão Gênica/genética , Hemócitos/imunologia , Hemócitos/metabolismo , Hemolinfa , Interações entre Hospedeiro e Microrganismos/genética , Sistema Imunitário , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Larva/imunologia , Larva/microbiologia , Lepidópteros/genética , Lepidópteros/microbiologia , Proteínas/genética , Proteínas/metabolismo , Proteínas/farmacologia , Quinolinas/metabolismo , Quinolinas/farmacologia , Esporos Bacterianos , Taxa de Sobrevida
13.
Dev Comp Immunol ; 98: 6-12, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30898519

RESUMO

The Bombyx mori cocoon/silk possesses many immune-related components, including protease inhibitors, seroins, and antimicrobial peptides, which likely help to protect the pupating larva from infection. However, the natural antimicrobial activity of the B. mori cocoon/silk is still too weak for biomedical applications. With the goal of enhancing this natural activity, we constructed a transgenic vector to overexpress the B. mori antimicrobial peptide Gloverin2 (BmGlv2) under control of the silk gland-specific Serion1 promoter. Transgenic silkworms were generated via embryo microinjection. A low level of BmGlv2 was expressed in the non-transgenic silk gland, but BmGlv2 was efficiently overexpressed and proteolytically activated in the transgenic line. Overexpressed BmGlv2 was secreted and incorporated into the silk during spanning without affecting cocoon/silk formation. Moreover, the transgenic cocoon/silk had significantly greater inhibitory activity against bacteria and fungi than the non-transgenic cocoon/silk. This strategy could help enhance the antimicrobial performance and biomedical application of silk.


Assuntos
Anti-Infecciosos/metabolismo , Bombyx/genética , Perfilação da Expressão Gênica , Proteínas de Insetos/genética , Seda/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Anti-Infecciosos/farmacologia , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Sequência de Bases , Bombyx/metabolismo , Fungos/efeitos dos fármacos , Fungos/crescimento & desenvolvimento , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Larva/genética , Larva/metabolismo , Seda/metabolismo , Seda/farmacologia
14.
Microbes Environ ; 34(2): 155-160, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-30905896

RESUMO

Aphids have a mutualistic relationship with the bacterial endosymbiont Buchnera aphidicola. We previously reported seven cysteine-rich peptides in the pea aphid Acyrthosiphon pisum and named them Bacteriocyte-specific Cysteine-Rich (BCR) peptides; these peptides are exclusively expressed in bacteriocytes, special aphid cells that harbor symbionts. Similar symbiotic organ-specific cysteine-rich peptides identified in the root nodules of leguminous plants are named Nodule-specific Cysteine-Rich (NCR) peptides. NCR peptides target rhizobia in the nodules and are essential for symbiotic nitrogen fixation. A BacA (membrane protein) mutant of Sinorhizobium is sensitive to NCR peptides and is unable to establish symbiosis. Based on the structural and expressional similarities between BCR peptides and NCR peptides, we hypothesized that aphid BCR peptides exhibit antimicrobial activity, similar to some NCR peptides. We herein synthesized BCR peptides and investigated their antimicrobial activities and effects on the bacterial membrane of Escherichia coli. The peptides BCR1, BCR3, BCR5, and BCR8 exhibited antimicrobial activities with increased membrane permeability. An sbmA mutant of E. coli, a homolog of bacA of S. meliloti, was more sensitive to BCR peptides than the wild type. Our results suggest that BCR peptides have properties that may be required to control the endosymbiont, similar to NCR peptides in legumes.


Assuntos
Anti-Infecciosos/farmacologia , Afídeos/metabolismo , Cisteína/química , Proteínas de Insetos/farmacologia , Peptídeos/farmacologia , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Afídeos/microbiologia , Buchnera/fisiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/genética , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Insetos/síntese química , Proteínas de Insetos/química , Mutação , Peptídeos/síntese química , Peptídeos/química , Sinorhizobium meliloti/efeitos dos fármacos , Sinorhizobium meliloti/genética , Simbiose
15.
Insect Biochem Mol Biol ; 108: 24-31, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30885802

RESUMO

Cuticle tanning occurs in insects immediately after hatching or molting. During this process, the cuticle becomes dark and rigid due to melanin deposition and protein crosslinking. In insects, different from mammals, melanin is synthesized mainly from dopamine, which is produced from DOPA by the enzyme DOPA decarboxylase. In this work, we report that the silencing of the RpAadc-2 gene, which encodes the putative Rhodnius prolixus DOPA decarboxylase enzyme, resulted in a reduction in nymph survival, with a high percentage of treated insects dying during the ecdysis process or in the expected ecdysis period. Those treated insects that could complete ecdysis presented a decrease in cuticle pigmentation and hardness after molting. In adult females, the knockdown of AADC-2 resulted in a reduction in the hatching of eggs; the nymphs that managed to hatch failed to tan the cuticle and were unable to feed. Despite the failure in cuticle tanning, knockdown of the AADC-2 did not increase the susceptibility to topically applied deltamethrin, a pyrethroid insecticide. Additionally, our results showed that the melanin synthesis pathway did not play a major role in the detoxification of the excess (potentially toxic) tyrosine from the diet, an essential trait for hematophagous arthropod survival after a blood meal.


Assuntos
Dopa Descarboxilase/fisiologia , Proteínas de Insetos/fisiologia , Tegumento Comum/fisiologia , Rhodnius/enzimologia , Rhodnius/crescimento & desenvolvimento , Animais , Dopa Descarboxilase/farmacologia , Feminino , Inativação Metabólica , Proteínas de Insetos/farmacologia , Inseticidas , Melaninas/metabolismo , Muda/fisiologia , Nitrilos , Pigmentação/fisiologia , Piretrinas , Reprodução , Rhodnius/genética , Tirosina/metabolismo
16.
Int J Biol Macromol ; 128: 782-795, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711561

RESUMO

Liver diseases are serious life-threating conditions that should be controlled. Here, we identify a protein fraction from royal-jelly (RJ) that represents the most effective composite against CCl4-induced hepatotoxicity and HepG2 cell growth. Two closely related proteins were purified from this fraction by a new simple method and identified by MALDI-TOF MS as major RJ protein 2 (MRJP2) and its predicted isoform X1. The in silico assessment (3D structures and functions) of these proteins were performed using Iterative Threading ASSEmbly Refinement (I-TASSER) analysis and RAMPAGE program. These two purified proteins were able to relieve the necrotic hepatocytes (by 60.4%) via reducing tumor necrosis factor (TNF)-α, mixed lineage kinase domain-like protein (MLKL) and intracellular reactive species. The latter effects associated with improving hepatocyte functions. Furthermore, they revealed the potent anticancer effect via induction of caspase-dependent apoptosis and controlling the expression of both Bcl-2 and p53 in HepG2 cells. Thus, MRJP2 and its isoform X1 can be a promising dual strategy for fighting hepatic injury and cancer in future animal and human studies.


Assuntos
Citoproteção/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Proteínas de Insetos/farmacologia , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Antígeno Ki-67/metabolismo , Masculino , Modelos Moleculares , Necrose/patologia , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/farmacologia , Ratos , Relação Estrutura-Atividade
17.
Prep Biochem Biotechnol ; 49(3): 279-285, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30767702

RESUMO

In this study, we report a novel member of the attacin family from Hermetia illucens. The cDNA clone encoding the attacin-like protein was isolated by screening a cDNA library prepared from immunized fat body. The complete 510 bp cDNA of HI-attacin was predicted to encode a protein of 169 amino acids with a molecular weight of 17.7 kDa. The putative mature protein of H. illucens attacin (HI-attacin) had 50% identity with that of Bactrocera dorsalis attacin B. Phylogenetic analysis revealed that the HI-attacin was separated from the other dipteran attacins with a high bootstrap percentage. Compared to that in the other dipteran attacins, the G1 domain of HI-attacin was shorter and the sequence of the G2 domain of HI-attacin was more conserved than that of the G1 domain. We produced the recombinant attacin (rHI-attacin) protein using a prokaryotic expression system to confirm its antibacterial character. The rHI-attacin was produced as inclusion bodies and refolded by on-column refolding. rHI-attacin exhibited antibacterial activity against both Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Using real-time PCR, the expression of HI-attacin was was barely detected before the immunization, but was mostly evident in the fat body after immunization.


Assuntos
Antibacterianos/farmacologia , Dípteros/química , Proteínas de Insetos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Corpo Adiposo/química , Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Filogenia , RNA/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência
18.
Lab Chip ; 19(5): 837-844, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30698187

RESUMO

The spread of bacterial resistance against conventional antibiotics generates a great need for the discovery of novel antimicrobials. Polypeptide antibiotics constitute a promising class of antimicrobial agents that favour attack on bacterial membranes. However, efficient measurement platforms for evaluating their mechanisms of action in a systematic manner are lacking. Here we report an integrated lab-on-a-chip multilayer microfluidic platform to quantify the membranolytic efficacy of such antibiotics. The platform is a biomimetic vesicle-based screening assay, which generates giant unilamellar vesicles (GUVs) in physiologically relevant buffers on demand. Hundreds of these GUVs are individually immobilised downstream in physical traps connected to separate perfusion inlets that facilitate controlled antibiotic delivery. Antibiotic efficacy is expressed as a function of the time needed for an encapsulated dye to leak out of the GUVs as a result of antibiotic treatment. This proof-of-principle study probes the dose response of an archetypal polypeptide antibiotic cecropin B on GUVs mimicking bacterial membranes. The results of the study provide a foundation for engineering quantitative, high-throughput microfluidics devices for screening antibiotics.


Assuntos
Antibacterianos/análise , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Proteínas de Insetos/análise , Proteínas de Insetos/farmacologia , Técnicas Analíticas Microfluídicas/instrumentação , Lipossomas Unilamelares/química
19.
PLoS One ; 14(1): e0211433, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682157

RESUMO

BACKGROUND: PACAP and VIP are closely related neuropeptides with wide distribution and potent effect in the vasculature. We previously reported vasomotor activity in peripheral vasculature of male wild type (WT) and PACAP-deficient (KO) mice. However, female vascular responses are still unexplored. We hypothesized that PACAP-like activity is maintained in female PACAP KO mice and the mechanism through which it is regulated differs from that of male PACAP KO animals. METHODS: We investigated the vasomotor effects of VIP and PACAP isoforms and their selective blockers in WT and PACAP KO female mice in carotid and femoral arteries. The expression and level of different PACAP receptors in the vessels were measured by RT-PCR and Western blot. RESULTS: In both carotid and femoral arteries of WT mice, PACAP1-38, PACAP1-27 or VIP induced relaxation, without pronounced differences between them. Reduced relaxation was recorded only in the carotid arteries of KO mice as compared to their WT controls. The specific VPAC1R antagonist completely blocked the PACAP/VIP-induced relaxation in both arteries of all mice, while PAC1R antagonist affected relaxation only in their femoral arteries. CONCLUSION: In female WT mice, VPAC1 receptors appear to play a dominant role in PACAP-induced vasorelaxation both in carotid and in femoral arteries. In the PACAP KO group PAC1R activation exerts vasorelaxation in the femoral arteries but in carotid arteries there is no significant effect of the activation of this receptor. In the background of this regional difference, decreased PAC1R and increased VPAC1R availability in the carotid arteries was found.


Assuntos
Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Vasodilatação , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/fisiologia , Feminino , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/fisiologia , Proteínas de Insetos/farmacologia , Camundongos , Camundongos Knockout , Nitroprussiato/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/antagonistas & inibidores , Peptídeo Intestinal Vasoativo/farmacologia , Vasodilatação/efeitos dos fármacos
20.
Amino Acids ; 51(2): 311-318, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30377840

RESUMO

Anticancer peptides (ACPs) are biologically anticancer active molecules that are produced by mammals, plants, insects and microorganisms. Here, a new peptide (TC22) with the amino acid sequence MTVVLLLIVLPLLGGVHSSGIL was identified and characterized from the beetle Tribolium castaneum. We found it inhibited the growth and viability of HeLa and MCF-7 cells. Flow cytometry analysis demonstrated the TC22 induced HeLa cell apoptosis, and activated caspase-9 and caspase-3. Furthermore, TC22 led to ROS generation, and triggered p53 transcription and expression. Taken together, our results indicated that TC22 exhibited high anticancer capacity via activating p53, inducing ROS generation and through a mitochondrial pathway. This research provided a novel natural source peptide with strong anticancer capacity. These findings provide some novel insights on the potential candidate reagent in cancer treatment.


Assuntos
Anticarcinógenos/isolamento & purificação , Anticarcinógenos/farmacologia , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/farmacologia , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Tribolium/química , Animais , Anticarcinógenos/uso terapêutico , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Expressão Gênica , Genes p53/genética , Células HeLa , Humanos , Proteínas de Insetos/uso terapêutico , Células MCF-7 , Peptídeos/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Transcrição Genética
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