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1.
Pharmacol Res ; 203: 107173, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38580186

RESUMO

Our recent multi-omics studies have revealed rich sources of novel bioactive proteins and polypeptides from marine organisms including cnidarians. In the present study, we initially conducted a transcriptomic analysis to review the composition profile of polypeptides from Zoanthus sociatus. Then, a newly discovered NPY-like polypeptide-ZoaNPY was selected for further in silico structural, binding and virtually pharmacological studies. To evaluate the pro-angiogenic effects of ZoaNPY, we employed an in vitro HUVECs model and an in vivo zebrafish model. Our results indicate that ZoaNPY, at 1-100 pmol, enhances cell survival, migration and tube formation in the endothelial cells. Besides, treatment with ZoaNPY could restore a chemically-induced vascular insufficiency in zebrafish embryos. Western blot results demonstrated the application of ZoaNPY could increase the phosphorylation of proteins related to angiogenesis signaling including PKC, PLC, FAK, Src, Akt, mTOR, MEK, and ERK1/2. Furthermore, through molecular docking and surface plasmon resonance (SPR) verification, ZoaNPY was shown to directly and physically interact with NPY Y2 receptor. In view of this, all evidence showed that the pro-angiogenic effects of ZoaNPY involve the activation of NPY Y2 receptor, thereby activating the Akt/mTOR, PLC/PKC, ERK/MEK and Src- FAK-dependent signaling pathways. Furthermore, in an excision wound model, the treatment with ZoaNPY was shown to accelerate the wound healing process in mice. Our findings provide new insights into the discovery and development of novel pro-angiogenic drugs derived from NPY-like polypeptides in the future.


Assuntos
Cnidários , Peptídeos , Receptores de Neuropeptídeo Y , Animais , Humanos , Camundongos , Movimento Celular/efeitos dos fármacos , Quinase 1 de Adesão Focal/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Ligantes , Simulação de Acoplamento Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Neuropeptídeo Y/metabolismo , Neuropeptídeo Y/farmacologia , Peptídeos/farmacologia , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Receptores de Neuropeptídeo Y/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/efeitos dos fármacos , Quinases da Família src/metabolismo , Peixe-Zebra , Cnidários/química , Fosfoinositídeo Fosfolipase C/efeitos dos fármacos , Fosfoinositídeo Fosfolipase C/metabolismo
2.
Biol Chem ; 401(8): 945-954, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32229648

RESUMO

Ants (Hymenoptera, Apocrita, Aculeata, Formicoidea) comprise a well-succeeded group of animals. Like bees and wasps, ants are mostly venomous, having a sting system to deliver a mixture of bioactive organic compounds and peptides. The predatory giant ant Dinoponera quadriceps belongs to the subfamily Ponerinae that includes one of the largest known ant species in the world. In the present study, low molecular weight compounds and peptides were identified by online peptide mass fingerprint. These include neuroactive biogenic amines (histamine, tyramine, and dopamine), monoamine alkaloid (phenethylamine), free amino acids (e.g. glutamic acid and proline), free thymidine, and cytosine. To the best of our knowledge, most of these components are described for the first time in an ant venom. Multifunctional dinoponeratoxin peptide variants (pilosulin- and ponericin-like peptides) were characterized that possess antimicrobial, hemolytic, and histamine-releasing properties. These venom components, particularly peptides, might synergistically contribute to the overall venom activity and toxicity, for immobilizing live prey, and for defending D. quadriceps against aggressors, predators, and potential microbial infection.


Assuntos
Venenos de Formiga/química , Peptídeos/química , Animais , Formigas , Peso Molecular
3.
Zygote ; 28(1): 72-79, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31662126

RESUMO

This study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA-dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA-dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA-dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA-dLCr 1:25 and the controls. The DNA-dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA-dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA-dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.


Assuntos
Venenos de Crotalídeos/farmacologia , DNA/química , Dissulfetos/química , Embrião de Mamíferos/fisiologia , Fertilização in vitro/veterinária , Fragmentos de Peptídeos/química , Transfecção/métodos , Animais , Bovinos , Células Cultivadas , Venenos de Crotalídeos/química , DNA/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Fragmentos de Peptídeos/metabolismo
4.
J Biol Chem ; 293(5): 1536-1549, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29255091

RESUMO

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.


Assuntos
Antibacterianos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular , Venenos de Crotalídeos , Crotalus , Escherichia coli , Fragmentos de Peptídeos , Pseudomonas aeruginosa , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Membrana Celular/química , Membrana Celular/metabolismo , Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacologia , Escherichia coli/química , Escherichia coli/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Ressonância de Plasmônio de Superfície
5.
Biol Chem ; 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32087061

RESUMO

Ants (Hymenoptera, Apocrita, Aculeata, Formicoidea) comprise a well-succeeded group of animals. Like bees and wasps, ants are mostly venomous, having a sting system to deliver a mixture of bioactive organic compounds and peptides. The predatory giant ant Dinoponera quadriceps belongs to the subfamily Ponerinae that include one of the largest known ant species in the world. In the present study, low molecular weight compounds and peptides were identified by on-line peptide mass fingerprint. These include neuroactive biogenic amines (histamine, tyramine, and dopamine), monoamine alkaloid (phenethylamine), free amino acids (e.g., glutamic acid and proline), free thymidine and cytosine. To the best of our knowledge most of these components are described for the first time in an ant venom. Multifunctional dinoponeratoxin peptides variants (pilosulin- and ponericin-like peptides) were characterized that possess antimicrobial, hemolytic, and histamine-releasing properties. These venom components, particularly peptides, might synergistically contribute to the overall venom activity and toxicity, for immobilizing live prey, and defending D. quadriceps against aggressors, predators and potential microbial infection.

6.
Arch Toxicol ; 93(1): 189-206, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30334080

RESUMO

We previously reported a novel toxic peptide identified from the anthozoan Protopalythoa variabilis transcriptome which is homologous to a novel structural type of sodium channel toxin isolated from a parental species (Palythoa caribaeorum). The peptide was named, according to its homologous, as Pp V-shape α-helical peptide (PpVα) in the present study. Through molecular docking and dynamics simulation, linear and hairpin folded PpVα peptides were shown to be potential voltage-gated sodium channel blockers. Nowadays, sodium channel blockers have been the mainstream of the pharmacological management of epileptic seizures. Also, sodium channel blockers could promote neuronal survival by reducing sodium influx and reducing the likelihood of calcium importation resulting in suppressing microglial activation and protecting dopaminergic neurons from degeneration. The folded PpVα peptide could decrease pentylenetetrazol (PTZ)-induced c-fos and npas4a expression level leading to reverse PTZ-induced locomotor hyperactivity in zebrafish model. In vitro, the folded PpVα peptide protected PC12 cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via activating heme oxygenase-1 (HO-1) and attenuating inducible nitric oxide synthase (iNOS) expression. In vivo, PpVα peptide suppressed the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish and, importantly, prevented the 6-OHDA-induced excessive ROS generation and subsequent dopaminergic neurons loss. This study indicates that the single S-S bond folded PpVα peptide arises as a new structural template to develop sodium channel blockers and provides an insight on the peptide discovery from cnidarian transcriptome to potentially manage epilepsy and neurodegenerative disorders.


Assuntos
Antozoários/química , Anticonvulsivantes/farmacologia , Fármacos Neuroprotetores/farmacologia , Peptídeos/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo , Sequência de Aminoácidos , Animais , Heme Oxigenase (Desciclizante)/metabolismo , Locomoção , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Óxido Nítrico Sintase Tipo II/metabolismo , Oxidopamina/efeitos adversos , Células PC12 , Pentilenotetrazol/efeitos adversos , Peptídeos/síntese química , Estrutura Terciária de Proteína , Ratos , Espécies Reativas de Oxigênio/metabolismo , Peixe-Zebra
7.
Arch Toxicol ; 93(6): 1745-1767, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31203412

RESUMO

Venoms from marine animals have been recognized as a new emerging source of peptide-based therapeutics. Several peptide toxins from sea anemone have been investigated as therapeutic leads or pharmacological tools. Venom complexity should be further highlighted using combined strategies of large-scale sequencing and data analysis which integrated transcriptomics and proteomics to elucidate new proteins or peptides to be compared among species. In this work, transcriptomic and proteomic analyses were combined to identify six groups of expressed peptide toxins in Zoanthus natalensis. These include neurotoxin, hemostatic and hemorrhagic toxin, protease inhibitor, mixed function enzymes, venom auxiliary proteins, allergen peptides, and peptides related to the innate immunity. Molecular docking analysis indicated that one expressed Zoanthus Kunitz-like peptide, ZoaKuz1, could be a voltage-gated potassium channels blocker and, hence, it was selected for functional studies. Functional bioassays revealed that ZoaKuz1 has an intrinsic neuroprotective activity in zebrafish model of Parkinson's disease. Since pharmacological blockade of KV channels is known to induce neuroprotective effects, ZoaKuz1 holds the potential to be developed in a therapeutic tool to control neural dysfunction by slowing or even halting neurodegeneration mediated by ion-channel hyperactivity.


Assuntos
Venenos de Cnidários/genética , Venenos de Cnidários/toxicidade , Peptídeos/genética , Peptídeos/toxicidade , Proteômica , Anêmonas-do-Mar/genética , Transcriptoma , Alérgenos/genética , Alérgenos/toxicidade , Animais , Antiparkinsonianos/farmacologia , Hemostáticos , Humanos , Simulação de Acoplamento Molecular , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/genética , Neurotoxinas/toxicidade , Bloqueadores dos Canais de Potássio/farmacologia , Inibidores de Proteases/farmacologia , Dobramento de Proteína , Peixe-Zebra
8.
J Proteome Res ; 17(2): 891-902, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29285938

RESUMO

Palythoa caribaeorum (class Anthozoa) is a zoanthid that together jellyfishes, hydra, and sea anemones, which are venomous and predatory, belongs to the Phyllum Cnidaria. The distinguished feature in these marine animals is the cnidocytes in the body tissues, responsible for toxin production and injection that are used majorly for prey capture and defense. With exception for other anthozoans, the toxin cocktails of zoanthids have been scarcely studied and are poorly known. Here, on the basis of the analysis of P. caribaeorum transcriptome, numerous predicted venom-featured polypeptides were identified including allergens, neurotoxins, membrane-active, and Kunitz-like peptides (PcKuz). The three predicted PcKuz isotoxins (1-3) were selected for functional studies. Through computational processing comprising structural phylogenetic analysis, molecular docking, and dynamics simulation, PcKuz3 was shown to be a potential voltage gated potassium-channel inhibitor. PcKuz3 fitted well as new functional Kunitz-type toxins with strong antilocomotor activity as in vivo assessed in zebrafish larvae, with weak inhibitory effect toward proteases, as evaluated in vitro. Notably, PcKuz3 can suppress, at low concentration, the 6-OHDA-induced neurotoxicity on the locomotive behavior of zebrafish, which indicated PcKuz3 may have a neuroprotective effect. Taken together, PcKuz3 figures as a novel neurotoxin structure, which differs from known homologous peptides expressed in sea anemone. Moreover, the novel PcKuz3 provides an insightful hint for biodrug development for prospective neurodegenerative disease treatment.


Assuntos
Antozoários/química , Venenos de Cnidários/isolamento & purificação , Neurotoxinas/isolamento & purificação , Peptídeos/isolamento & purificação , Bloqueadores dos Canais de Potássio/isolamento & purificação , Transcriptoma , Alérgenos/química , Alérgenos/isolamento & purificação , Animais , Antozoários/patogenicidade , Antozoários/fisiologia , Sítios de Ligação , Venenos de Cnidários/química , Venenos de Cnidários/toxicidade , Sequenciamento de Nucleotídeos em Larga Escala , Larva/efeitos dos fármacos , Larva/fisiologia , Locomoção/efeitos dos fármacos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neurotoxinas/química , Neurotoxinas/toxicidade , Oxidopamina/antagonistas & inibidores , Oxidopamina/farmacologia , Peptídeos/química , Peptídeos/toxicidade , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/toxicidade , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Peixe-Zebra
9.
Biol Chem ; 399(2): 187-196, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28976889

RESUMO

The crude venom of the giant ant Dinoponera quadriceps is a cocktail of polypeptides and organic compounds that shows antiparasitic effects against Trypanosoma cruzi, the causative agent of Chagas disease. In order to investigate the venom-derived components responsible for such antitrypanosomal activity, four dinoponeratoxins (DnTxs) were identified, namely M-PONTX-Dq3a, -Dq3b, -Dq3c and -Dq4e, that are diverse in size, net charge, hydrophobicity and propensity to interact with eukaryote cell membranes. These peptides were tested against epimastigote, trypomastigote and amastigote forms of benznidazole (Bz)-resistant Y strain of T. cruzi and in mammalian host cells. The M-PONTX-Dq3a and -Dq4e inhibited all developmental forms of T. cruzi, including amastigotes, the responsible form for the maintenance of infection on chronic phase of the disease. The M-PONTX-Dq3a showed the highest selectivity index (SI) (80) and caused morphological alterations in T. cruzi, as observed by scanning electron microscopy (SEM), and induced cell death through necrosis, as seen by multiparametric flow cytometry analysis with specific biochemical markers. Altogether, the D. quadriceps venom appears as a source for the prospection of trypanocidal peptides and the M-PONTX-Dq3a arises as a candidate among the dinoponeratoxin-related peptides in the development of compounds against Chagas disease.


Assuntos
Peptídeos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Formigas , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Testes de Sensibilidade Parasitária , Peptídeos/química , Peptídeos/isolamento & purificação , Relação Estrutura-Atividade , Tripanossomicidas/química , Tripanossomicidas/isolamento & purificação , Trypanosoma cruzi/citologia
10.
Parasitology ; 145(8): 1059-1064, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29208061

RESUMO

Cathelicidins are antimicrobial peptides produced by humans and animals in response to various pathogenic microbes. Crotalicidin (Ctn), a cathelicidin-related vipericidin from the South American Crotalus durissus terrificus rattlesnake's venom gland, and its fragments have demonstrated antimicrobial and antifungal activity, similarly to human cathelicidin LL-37. In order to provide templates for the development of modern trypanocidal agents, the present study evaluated the antichagasic effect of these four peptides (Ctn, Ctn[1-14], Ctn[15-34] and LL-37). Herein, Ctn and short derived peptides were tested against the epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi Y strain (benznidazole-resistant strain) and cytotoxicity in mammalian cells was evaluated against LLC-MK2 lineage cells. Ctn inhibited all T. cruzi developmental forms, including amastigotes, which is implicated in the burden of infection in the chronic phase of Chagas disease. Moreover, Ctn showed a high selective index against trypomastigote forms (>200). Ctn induced cell death in T. cruzi through necrosis, as determined by flow cytometry analyses with specific molecular probes and morphological alterations, such as loss of membrane integrity and cell shrinkage, as observed through scanning electron microscopy. Overall, Ctn seems to be a promising template for the development of antichagasic agents.


Assuntos
Fragmentos de Peptídeos/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Venenos de Crotalídeos/farmacologia , Citometria de Fluxo , Haplorrinos , Concentração Inibidora 50 , Microscopia Eletrônica de Varredura , Trypanosoma cruzi/ultraestrutura , Catelicidinas
11.
Mar Drugs ; 16(6)2018 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899267

RESUMO

Marine invertebrates, such as sponges, tunicates and cnidarians (zoantharians and scleractinian corals), form functional assemblages, known as holobionts, with numerous microbes. This type of species-specific symbiotic association can be a repository of myriad valuable low molecular weight organic compounds, bioactive peptides and enzymes. The zoantharian Protopalythoa variabilis (Cnidaria: Anthozoa) is one such example of a marine holobiont that inhabits the coastal reefs of the tropical Atlantic coast and is an interesting source of secondary metabolites and biologically active polypeptides. In the present study, we analyzed the entire holo-transcriptome of P. variabilis, looking for enzyme precursors expressed in the zoantharian-microbiota assemblage that are potentially useful as industrial biocatalysts and biopharmaceuticals. In addition to hundreds of predicted enzymes that fit into the classes of hydrolases, oxidoreductases and transferases that were found, novel enzyme precursors with multiple activities in single structures and enzymes with incomplete Enzyme Commission numbers were revealed. Our results indicated the predictive expression of thirteen multifunctional enzymes and 694 enzyme sequences with partially characterized activities, distributed in 23 sub-subclasses. These predicted enzyme structures and activities can prospectively be harnessed for applications in diverse areas of industrial and pharmaceutical biotechnology.


Assuntos
Antozoários/enzimologia , Organismos Aquáticos/enzimologia , Produtos Biológicos , Enzimas/genética , Animais , Antozoários/genética , Organismos Aquáticos/genética , Biocatálise , Biotecnologia/métodos , Enzimas/metabolismo , Química Verde/métodos , Indústrias/métodos , Transcriptoma
12.
J Biochem Mol Toxicol ; 31(11)2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28815806

RESUMO

Crotamine is defensin-like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom-derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B-conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB-crotamine was as low as 4 µM, which effectively kill zebrafish larvae in less than 10 min. With non-lethal concentrations (<1 µM), crotamine caused malformation in zebrafish embryos, delayed or completely halted hatching, adversely affected embryonic developmental programming, decreased the cardiac functions, and attenuated the swimming distance of zebrafish. The RhoB-crotamine translocated across vitelline membrane and accumulated in zebrafish yolk sac. These results demonstrate the sensitive responsivity of zebrafish to trial crotamine analogues for the development of novel therapeutic peptides with improved safety, bioavailability, and efficacy profiles.


Assuntos
Venenos de Crotalídeos/toxicidade , Rodaminas/química , Testes de Toxicidade/métodos , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacocinética , Embrião não Mamífero/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Coração/efeitos dos fármacos , Coração/embriologia , Larva/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Rodaminas/farmacocinética , Distribuição Tecidual
13.
Biochim Biophys Acta ; 1850(6): 1253-60, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25731980

RESUMO

BACKGROUND: Animal venoms contain a diverse array of proteins and enzymes that are toxic toward various physiological systems. However, there are also some practical medicinal uses for these toxins including use as anti-bacterial and anti-tumor agents. METHODS: In this study, we identified a nine-residue cryptic oligopeptide, KRFKKFFKK (EVP50) that is repeatedly encoded in tandem within vipericidin sequences. RESULTS: EVP50 displayed in vivo potent lethal toxicity to zebrafish larvae (LD50=6 µM) when the peptide's N-terminus was chemically conjugated to rhodamine B (RhoB). In vitro, RhoB-conjugated EVP50 (RhoB-EVP50) exhibited a concentration-dependent cytotoxic effect toward MCF-7 and MDA-MB-231 breast cancer cells. In MCF-7 cells, the RhoB-EVP50 nonapeptide accumulated inside the cells within minutes. In the cytoplasm, the RhoB-EVP50 induced extracellular calcium influx and intracellular calcium release. Membrane budding was also observed after incubation with micromolar concentrations of the fluorescent EVP50 conjugate. CONCLUSIONS: The conjugate's interference with calcium homeostasis, its intracellular accumulation and its induced membrane dysfunction (budding and vacuolization) seem to act in concert to disrupt the cell circuitry. Contrastively, unconjugated EVP50 peptide did not display neither toxic nor cytotoxic activities in our in vivo and in vitro models. GENERAL SIGNIFICANCE: The synergic mechanism of toxicity was restricted to the structurally modified encrypted vipericidin nonapeptide.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Catelicidinas/farmacologia , Oligopeptídeos/farmacologia , Rodaminas/farmacologia , Venenos de Víboras/química , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Neoplasias da Mama/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Catelicidinas/isolamento & purificação , Catelicidinas/metabolismo , Catelicidinas/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Larva/efeitos dos fármacos , Dose Letal Mediana , Células MCF-7 , Dados de Sequência Molecular , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/metabolismo , Oligopeptídeos/toxicidade , Rodaminas/metabolismo , Rodaminas/toxicidade , Fatores de Tempo
14.
Zygote ; 24(1): 48-57, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25532535

RESUMO

The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 µM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 µM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 µM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 µM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 µM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1-10 µM for 6-24 h.


Assuntos
Blastocisto/efeitos dos fármacos , Blastocisto/fisiologia , Venenos de Crotalídeos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Animais , Aquaporina 3/genética , Blastocisto/citologia , Bovinos , Venenos de Crotalídeos/administração & dosagem , Venenos de Crotalídeos/farmacocinética , Feminino , Fertilização in vitro , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 3/genética , Masculino , ATPase Trocadora de Sódio-Potássio/genética
15.
J Assist Reprod Genet ; 33(10): 1405-1413, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27515309

RESUMO

PURPOSE: Crotamine is capable of penetrating cells and embryos and transfecting cells with exogenous DNA. However, no studies are available regarding its uptake by parthenogenetic (PA) embryos or its use for transfection in in vitro fertilized (IVF) embryos. This study aimed to determine the translocation kinetics of crotamine into PA and IVF bovine embryos and assess its effect over in vitro development of PA embryos. Moreover, crotamine-DNA complexes were used to test the transfection ability of crotamine in bovine IVF zygotes. METHODS: PA and IVF embryos were exposed to labeled crotamine for four interval times. Embryo toxicity was assayed over PA embryos after 24 h of exposure to crotamine. Additionally, IVF embryos were exposed to or injected with a complex formed by crotamine and pCX-EGFP plasmid. RESULTS: Confocal images revealed that crotamine was uptaken by PA and IVF embryos as soon as 1 h after exposure. Crotamine exposure did not affect two to eight cells and blastocyst rates or blastocyst cell number (p > 0.05) of PA embryos. Regarding transfection, exposure or injection into the perivitelline space with crotamine-DNA complex did not result in transgene-expressing embryos. Nevertheless, intracytoplasmic injection of plasmid alone showed higher expression rates than did injection with crotamine-DNA complex at days 4 and 7 (p < 0.05). CONCLUSIONS: Crotamine is able to translocate through zona pellucida (ZP) of PA and IVF embryos within 1 h of exposure without impairing in vitro development. However, the use of crotamine does not improve exogenous DNA expression in cattle embryos, probably due to the tight complexation of DNA with crotamine.


Assuntos
Blastocisto/citologia , Peptídeos Penetradores de Células/administração & dosagem , Venenos de Crotalídeos/administração & dosagem , Técnicas de Cultura Embrionária , Animais , Blastocisto/efeitos dos fármacos , Bovinos , Embrião de Mamíferos , Feminino , Fertilização in vitro , Partenogênese/efeitos dos fármacos , Partenogênese/genética , Zigoto
16.
Exp Parasitol ; 148: 73-80, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25450774

RESUMO

Cyclophilin (CyP), a peptidyl-prolyl cis/trans isomerase, is a key molecule with diverse biological functions that include roles in molecular chaperoning, stress response, immune modulation, and signal transduction. In this respect, CyP could serve as a potential drug target in disease-causing parasites. Previous studies employing proteomics techniques have shown that the TcCyP19 isoform was more abundant in a benznidazole (BZ)-resistant Trypanosoma cruzi population than in its susceptible counterpart. In this study, TcCyP19 has been characterized in BZ-susceptible and BZ-resistant T. cruzi populations. Phylogenetic analysis revealed a clear dichotomy between Cyphophilin A (CyPA) sequences from trypanosomatids and mammals. Sequencing analysis revealed that the amino acid sequences of TcCyP19 were identical among the T. cruzi samples analyzed. Southern blot analysis showed that TcCyP19 is a single-copy gene, located in chromosomal bands varying in size from 0.68 to 2.2 Mb, depending on the strain of T. cruzi. Northern blot and qPCR indicated that the levels of TcCyP19 mRNA were twofold higher in drug-resistant T. cruzi populations than in their drug-susceptible counterparts. Similarly, as determined by two-dimensional gel electrophoresis immunoblot, the expression of TcCyP19 protein was increased to the same degree in BZ-resistant T. cruzi populations. No differences in TcCyP19 mRNA and protein expression levels were observed between the susceptible and the naturally resistant T. cruzi strains analyzed. Taken together, these data indicate that cyclophilin TcCyP19 expression is up-regulated at both transcriptional and translational levels in T. cruzi populations that were in vitro-induced and in vivo-selected for resistance to BZ.


Assuntos
Ciclofilinas/genética , Nitroimidazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Mapeamento Cromossômico , Ciclofilinas/química , Ciclofilinas/classificação , Ciclofilinas/metabolismo , DNA de Protozoário/análise , DNA de Protozoário/química , Resistência a Medicamentos , Dosagem de Genes , Regulação da Expressão Gênica , Genoma de Protozoário , Filogenia , RNA Mensageiro/metabolismo , RNA de Protozoário/análise , RNA de Protozoário/química , Alinhamento de Sequência , Análise de Sequência de DNA , Trypanosoma cruzi/química , Trypanosoma cruzi/efeitos dos fármacos
17.
Toxins (Basel) ; 16(6)2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38922129

RESUMO

Polyamines (PAs) are polycationic biogenic amines ubiquitously present in all life forms and are involved in molecular signaling and interaction, determining cell fate (e.g., cell proliferation, dif-ferentiation, and apoptosis). The intricate balance in the PAs' levels in the tissues will determine whether beneficial or detrimental effects will affect homeostasis. It's crucial to note that endoge-nous polyamines, like spermine and spermidine, play a pivotal role in our understanding of neu-rological disorders as they interact with membrane receptors and ion channels, modulating neuro-transmission. In spiders and wasps, monoamines (histamine, dopamine, serotonin, tryptamine) and polyamines (spermine, spermidine, acyl polyamines) comprise, with peptides and other sub-stances, the low molecular weight fraction of the venom. Acylpolyamines are venom components exclusively from spiders and a species of solitary wasp, which cause inhibition chiefly of iono-tropic glutamate receptors (AMPA, NMDA, and KA iGluRs) and nicotinic acetylcholine receptors (nAChRs). The first venom acylpolyamines ever discovered (argiopines, Joro and Nephila toxins, and philanthotoxins) have provided templates for the design and synthesis of numerous analogs. Thus far, analogs with high potency exert their effect at nanomolar concentrations, with high se-lectivity toward their ionotropic and ligand receptors. These potent and selective acylpolyamine analogs can serve biomedical purposes and pest control management. The structural modification of acylpolyamine with photolabile and fluorescent groups converted these venom toxins into use-ful molecular probes to discriminate iGluRs and nAchRs in cell populations. In various cases, the linear polyamines, like spermine and spermidine, constituting venom acyl polyamine backbones, have served as cargoes to deliver active molecules via a polyamine uptake system on diseased cells for targeted therapy. In this review, we examined examples of biogenic amines that play an essential role in neural homeostasis and cell signaling, contributing to human health and disease outcomes, which can be present in the venom of arachnids and hymenopterans. With an empha-sis on the spider and wasp venom acylpolyamines, we focused on the origin, structure, derivatiza-tion, and biomedical and biotechnological application of these pharmacologically attractive, chemically modular venom components.


Assuntos
Inseticidas , Poliaminas , Venenos de Aranha , Vespas , Animais , Poliaminas/química , Venenos de Aranha/química , Venenos de Aranha/toxicidade , Inseticidas/farmacologia , Inseticidas/química , Inseticidas/toxicidade , Humanos , Aranhas
18.
Biochim Biophys Acta ; 1818(11): 2707-17, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22749950

RESUMO

A novel class of cell-penetrating, nucleolar-targeting peptides (NrTPs), was recently developed from the rattlesnake venom toxin crotamine. Based on the intrinsic fluorescence of tyrosine or tryptophan residues, the partition of NrTPs and crotamine to membranes with variable lipid compositions was studied. Partition coefficient values (in the 10(2)-10(5) range) followed essentially the compositional trend POPC:POPG≤POPG

Assuntos
Nucléolo Celular/metabolismo , Venenos de Crotalídeos/metabolismo , Lipídeos de Membrana/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Fluorescência , Microscopia Confocal , Dados de Sequência Molecular , Ligação Proteica
19.
J Xenobiot ; 13(1): 121-131, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36976159

RESUMO

Fragrance compounds (synthetic fragrances or natural essential oils) comprise formulations of specific combinations of individual materials or mixtures. Natural or synthetic scents are core constituents of personal care and household products (PCHPs) that impart attractiveness to the olfactory perception and disguise the unpleasant odor of the formula components of PCHPs. Fragrance chemicals have beneficial properties that allow their use in aromatherapy. However, because fragrances and formula constituents of PCHPs are volatile organic compounds (VOCs), vulnerable populations are exposed daily to variable indoor concentrations of these chemicals. Fragrance molecules may trigger various acute and chronic pathological conditions because of repetitive human exposure to indoor environments at home and workplaces. The negative impact of fragrance chemicals on human health includes cutaneous, respiratory, and systemic effects (e.g., headaches, asthma attacks, breathing difficulties, cardiovascular and neurological problems) and distress in workplaces. Pathologies related to synthetic perfumes are associated with allergic reactions (e.g., cutaneous and pulmonary hypersensitivity) and potentially with the perturbation of the endocrine-immune-neural axis. The present review aims to critically call attention to odorant VOCs, particularly synthetic fragrances and associated formula components of PCHPs, potentially impacting indoor air quality and negatively affecting human health.

20.
Toxicon ; 234: 107274, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37657514

RESUMO

Crotalicidin is a cathelicidin-related anti-infective (antimicrobial) peptide expressed in the venom glands of the South American rattlesnake Crotalus durissus terrificus. Congener peptides of crotalicidin, named vipericidins, are found in other pit vipers inhabiting South America. Crotalicidin is active against bacteria and pathogenic yeasts and has anti-proliferative activity for some cancer cells. The structural dissection of crotalicidin produced fragments (e.g., Ctn [15-34]) with multiple biological functionalities that mimic the native peptide. Another structural characteristic of crotalidicin and congeners is a unique repetitive stretch of amino acid sequences in tandem embedded in their primary structures. One of the encrypted vipericidn peptides (Ctn [1-9]) was synthesized, and the analog covalently conjugated with rhodamine B (RhoB-Ctn [1-9]) displayed considerable antimicrobial activity and selective cytotoxicity. Methods to evaluate antimicrobial peptides' toxicity include lysis of red blood cells (hemolysis) in vitro and cytotoxicity of healthy cultured cells (e.g., fibroblasts). Here, as a non-conventional model of toxicity, the bovine oocytes were exposed to two standardized concentrations of RhoB-Ctn [1-9], and embryo viability and development at its first stage of cleavage (division of cells) and blastocyst formation were evaluated. Oocytes treated with peptide at 10 and 40 µM induced cleavage rates of 44.94% and 51.53%, resulting in the formation of blastocysts of 7.07% and 11.73%, respectively. Light sheet microscopy and in silico prediction analysis indicated that RhoB-Ctn [1-9] peptide interacts with zona pellucida and internalizes into bovine oocytes and developing embryos. The ADMET prediction estimated good bioavailability of RhoB-Ctn [1-9]. In conclusion, the peptide appeared harmless to bovine oocytes and, remarkably, activated the parthenogenesis in vitro.

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