RESUMO
Malaria is a parasitic infectious disease caused by Plasmodium sp, which was responsible for about 409 thousand deaths only in 2019. The clinical manifestations in patients with malaria, which may include fever and anemia and that can occasionally lead to the death of the host, are mainly associated to the asexual blood stage of parasite. The discovery of novel compounds active against stages of the intraerythrocytic cell cycle has been the focus of many researches seeking for alternatives to the control of malaria. The antimalarial effect of a native cationic polypeptide from the venom of a South American rattlesnake named crotamine, with ability of targeting and disrupting the acidic compartments of Plasmodium falciparum parasite, was previously described by us. Herein, we extended our previous studies by investigating the internalization and trafficking of crotamine in P. falciparum-infected erythrocytes at different blood-stages of parasites and periods of incubation. In addition, the effects of several pharmacological inhibitors in the uptake of this snake polypeptide with cell-penetrating properties were also assessed, showing that crotamine internalization was dependent on ATP generated via glycolytic pathway. We show here that crotamine uptake is blocked by the glycolysis inhibitor 2-deoxy-D-glucose, and the most efficient internalization is observed at trophozoite stage of parasite after at least 30 min of incubation. The present data provide important insights into biochemical pathway and cellular features determined by the parasite cycle, which may be underlying the internalization and effects of cationic antimalarials as crotamine.
Assuntos
Venenos de Crotalídeos/química , Eritrócitos , Peptídeos , Plasmodium falciparum , Animais , Crotalus , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Peptídeos/farmacologia , América do SulRESUMO
In recent years, egg production has had an intense growth in Brazil, and Brazilian egg consumption per capita has significantly increased in the last decade. To reduce sanitary and financial risks, decisions regarding the production and health status of the flock must be made based on objective criteria. Our aim was to determine the main "input" variables for the prediction of egg production performance in commercial laying breeder flocks using an ANN model. The software NeuroShellClassifier and NeuroShell Predictor were used to build the ANN. A total of 26 egg-production traits were selected as input variables and eight as output variables. A database of 44,120 Excel cells was generated. For the training and validation of the models, 74.9% and 25.1% of the data were used, respectively. The accuracy of the ANN models was calculated and compared using the analysis of coefficient of multiple determination (R2), mean squared error (MSE), and an assessment of uniform scatter in the residual plots. The models for the outputs "weekly egg production," "weekly incubated egg,", "accumulated commercial egg," and "viability" showed an R2 greater than 0.8. Other models yielded R2 values lower than 0.8. The ANN predicts adequately eight egg-production traits in the breeders of commercial laying hens. The method is an option for data management analysis in the egg industry, providing estimates of the relative contribution of each input variable to the outputs.(AU)
Assuntos
Animais , Galinhas , Redes Neurais de Computação , Ovos/análise , Produtos Avícolas/análise , Simulação por ComputadorRESUMO
Effects of adding different concentrations of melatonin (10-7 , 10-9 and 10-11 M) to maturation (Experiment 1; Control, IVM + 10-7 , IVM + 10-9 , IVM + 10-11 ) and culture media (Experiment 2; Control, IVC + 10-7 , IVC + 10-9 , IVC + 10-11 ) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10-9 M) from Experiments 1-2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM + 10-9 , IVC + 10-9 , IVM/IVC + 10-9 ). In Experiment 1, maturated oocytes from Control and IVM + 10-9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC + 10-7 (43.5%; 56.7%) and IVC + 10-9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC + 10-9 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC + 10-9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM/IVC + 10-9 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC + 10-9 treatment also had a higher mRNA expression of antioxidant gene (SOD2) compared to the Control, as well as the heat shock protein (HSPB1) compared to the IVM + 10-9 . Reactive oxygen species production was greater in the IVM/IVC + 10-9 treatment group. In conclusion, the 10-9 M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.
Assuntos
Apoptose/efeitos dos fármacos , Blastocisto , Técnicas de Cultura Embrionária/veterinária , Proteínas de Choque Térmico HSP27/efeitos dos fármacos , Melatonina/farmacologia , Superóxido Dismutase/efeitos dos fármacos , Animais , Bovinos , Meios de Cultura/farmacologia , Feminino , Fertilização in vitro/veterinária , Glutationa/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/análiseRESUMO
Crotamine is a natural polypeptide from snake venom which delivers nucleic acid molecules into cells, besides having pronounced affinity for negatively charged membranes and antifungal activity. We previously demonstrated that crotamine derived short linear peptides were not very effective as antifungal, although the non-structured recombinant crotamine was overridingly more potent compared to the native structured crotamine. Aiming to identify the features necessary for the antifungal activity of crotamine, two linear short peptides, each comprising half of the total positively charged amino acid residues of the full-length crotamine were evaluated here to show that these linear peptides keep the ability to interact with lipid membrane model systems with different phospholipid compositions, even after forming complexes with DNA. Interestingly, the presence of cysteine residues in the structure of these linear peptides highly influenced the antifungal activity, which was not associated to the lipid membrane lytic activity. In addition to the importance of the positive charges, the crucial role of cysteine residues was noticed for these linear analogs of crotamine, although the tridimensional structure and lipid membrane lytic activity observed only for native crotamine was not essential for the antifungal activity. As these peptides still keep the ability to form complexes with DNA molecules with no prejudice to their ability to bind to lipid membranes, they may be potentially advantageous as membrane translocation vector, as they do not show lipid membrane lytic activity and may harbor or not antifungal activity, by keeping or not the semi-essential amino acid cysteine in their sequence.
Assuntos
Antifúngicos/química , Peptídeos Penetradores de Células/química , Venenos de Crotalídeos/química , Sequência de Aminoácidos , Animais , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candida/crescimento & desenvolvimento , Peptídeos Penetradores de Células/isolamento & purificação , Peptídeos Penetradores de Células/farmacologia , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Crotalus/metabolismo , Cisteína/química , DNA/química , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Cinética , Testes de Sensibilidade Microbiana , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Ligação Proteica , Eletricidade Estática , Relação Estrutura-Atividade , Trichosporon/efeitos dos fármacos , Trichosporon/crescimento & desenvolvimento , Lipossomas Unilamelares/químicaRESUMO
We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 µM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.
Assuntos
Antimaláricos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Venenos de Crotalídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Venenos de Serpentes/química , Laranja de Acridina/metabolismo , Sequência de Aminoácidos , Animais , Antimaláricos/isolamento & purificação , Transporte Biológico , Carbocianinas/química , Peptídeos Penetradores de Células/isolamento & purificação , Células Cultivadas , Cloroquina/farmacologia , Venenos de Crotalídeos/isolamento & purificação , Crotalus/metabolismo , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Concentração Inibidora 50 , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Coloração e Rotulagem , Vacúolos/efeitos dos fármacos , Vacúolos/parasitologiaAssuntos
Doença Granulomatosa Crônica/complicações , Doença Granulomatosa Crônica/genética , NADPH Oxidases/genética , Tuberculose Pulmonar/imunologia , Adulto , Antituberculosos/uso terapêutico , Biópsia , Brasil , Feminino , Humanos , Mutação , Fagócitos/enzimologia , Fagócitos/patologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/patologia , Adulto JovemRESUMO
Despite the unquestionable importance of the highly cationic feature of several small polypeptides with high content of positively charged amino acids for their biological activities, positively charged peptides do not necessarily have the capacity to cross the cell membranes. Interestingly, we found that crotamine, a positively charged amphiphilic peptide from the South American rattlesnake venom, has a unique cell-penetrating property with affinity for acidic vesicles, besides a well-characterized antimicrobial and antitumoral activities. In spite of a remarkable in vitro antifungal activity of crotamine against Candida spp., no significant effect of this peptide could be observed in the course of Candida albicans and Candida krusei infection on Caenorhabditis elegans asssed in vivo. These experiments, in which the nematode C. elegans was used as a living host, suggested, however, the potential anthelmintic activity of crotamine because of its uptake by the worms and accumulation in their acidic compartments. As described in the present work, this lysosomotropic property is consistent with a previously proposed mechanism of toxicity of crotamine on mammalian tumoral cell lines. This study also allowed us to propose the cationic peptides with lysosomotropic property, as crotamine, as a potential new class of anthelmentics with ability to overcome the challenging problems of drug resistance.
Assuntos
Anti-Helmínticos/toxicidade , Caenorhabditis elegans/efeitos dos fármacos , Venenos de Crotalídeos/química , Animais , Anti-Helmínticos/química , Anti-Helmínticos/isolamento & purificação , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Candida albicans/fisiologia , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/toxicidadeRESUMO
Previous studies have demonstrated that cells from both multi-drug-resistant tuberculosis (MDR-TB) and non-tuberculous mycobacteria (NTM) patients respond poorly to mycobacterial antigens in vitro. In the present study, we compared the in vitro response of cells isolated from sensitive TB (NR-TB)-, MDR-TB- and NTM-infected patients. Analysis of T cell phenotype ex vivo revealed that both MDR-TB and NTM patients present an increased percentage of CD4(+) CD25(+-) forkhead box protein 3 (FoxP3)(+) and CD4(+) CD25(+) CD127(-) regulatory T (T(reg) ) cells when compared to NR-TB. Increased numbers of T(reg) cells and interleukin (IL)-10 serum levels were detected in MDR-TB, whereas elevated serum transforming growth factor (TGF)-ß was found in the NTM group. Cells of MDR-TB patients stimulated with early secretory antigenic target (ESAT)-6, but not purified protein derivative (PPD), showed a lower frequency of CD4(+) /interferon (IFN)-γ(+) T cells and enhanced CD4(+) CD25(+) FoxP3(+) , CD4(+) CD25(+) CD127(-) and CD4(+) CD25(+) IL-10(+) T cell population. In addition, increased IL-10 secretion was observed in cultured MDR-TB cells following ESAT-6 stimulation, but not in NR-TB or NTM patients. In vitro blockade of IL-10 or IL-10Rα decreased the CD4(+) CD25(+) FoxP3(+) frequencies induced by ESAT-6 in MDR-TB, suggesting a role of IL-10 on impaired IFN-γ responses seen in MDR-TB. Depletion of CD4(+) CD25(+) T lymphocytes restored the capacity of MDR-TB T cells to respond to ESAT-6 in vitro, which suggests a potential role for T(reg) /T regulatory 1 cells in the pathogenesis of MDR-TB. Together, our results indicate that although the similarities in chronicity, NTM- and MDR-TB-impaired antigenic responses involve different mechanisms.
Assuntos
Tolerância Imunológica , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Tuberculose Resistente a Múltiplos Medicamentos/imunologia , Tuberculose Pulmonar/imunologia , Adulto , Idoso , Antígenos de Bactérias/imunologia , Antígenos CD/metabolismo , Proteínas de Bactérias/imunologia , Células Cultivadas , Citocinas/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunofenotipagem , Isoniazida/uso terapêutico , Masculino , Pessoa de Meia-Idade , Rifampina/uso terapêutico , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Adulto JovemRESUMO
The classical renin-angiotensin system (RAS) consists of enzymes and peptides that regulate blood pressure and electrolyte and fluid homeostasis. Angiotensin II (Ang II) is one of the most important and extensively studied components of the RAS. The beneficial effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of hypertension and heart failure, among other diseases, are well known. However, it has been reported that patients chronically treated with effective doses of these inhibitors do not show suppression of Ang II formation, suggesting the involvement of pathways alternative to ACE in the generation of Ang II. Moreover, the finding that the concentration of Ang II is preserved in the kidney, heart and lungs of mice with an ACE deletion indicates the important role of alternative pathways under basal conditions to maintain the levels of Ang II. Our group has characterized the serine protease elastase-2 as an alternative pathway for Ang II generation from Ang I in rats. A role for elastase-2 in the cardiovascular system was suggested by studies performed in heart and conductance and resistance vessels of normotensive and spontaneously hypertensive rats. This mini-review will highlight the pharmacological aspects of the RAS, emphasizing the role of elastase-2, an alternative pathway for Ang II generation.
Assuntos
Angiotensina II/biossíntese , Sistema Cardiovascular/metabolismo , Sistema Renina-Angiotensina/fisiologia , Serina Endopeptidases/fisiologia , Angiotensina I/biossíntese , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Quimases/metabolismo , Humanos , Camundongos , Ratos , Serina Endopeptidases/farmacologiaRESUMO
The classical renin-angiotensin system (RAS) consists of enzymes and peptides that regulate blood pressure and electrolyte and fluid homeostasis. Angiotensin II (Ang II) is one of the most important and extensively studied components of the RAS. The beneficial effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of hypertension and heart failure, among other diseases, are well known. However, it has been reported that patients chronically treated with effective doses of these inhibitors do not show suppression of Ang II formation, suggesting the involvement of pathways alternative to ACE in the generation of Ang II. Moreover, the finding that the concentration of Ang II is preserved in the kidney, heart and lungs of mice with an ACE deletion indicates the important role of alternative pathways under basal conditions to maintain the levels of Ang II. Our group has characterized the serine protease elastase-2 as an alternative pathway for Ang II generation from Ang I in rats. A role for elastase-2 in the cardiovascular system was suggested by studies performed in heart and conductance and resistance vessels of normotensive and spontaneously hypertensive rats. This mini-review will highlight the pharmacological aspects of the RAS, emphasizing the role of elastase-2, an alternative pathway for Ang II generation.