RESUMO
Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90-95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin.
Assuntos
Calotropis , Cisteína Proteases , Sefarose , Quimosina , Cisteína , Caseínas , Cisteína Proteases/metabolismo , Concentração de Íons de Hidrogênio , Enzimas Imobilizadas/metabolismoRESUMO
Antimicrobial peptides (AMPs) have been reported to be promising alternatives to chemical preservatives. Thus, this study aimed to characterise AMPs generated from the hydrolysis of wheat gluten proteins using latex peptidases of Calotropis procera, Cryptostegia grandiflora, and Carica papaya. The three hydrolysates (obtained after 16 h at 37 °C, using a 1:â25 enzyme: âsubstrate ratio) inhibited the growth of Aspergillus niger, A. chevalieri, Trichoderma reesei, Pythium oligandrum, Penicillium sp., and Lasiodiplodia sp. by 60-90%, and delayed fungal growth on bread by 3 days when used at 0.3 g/kg. Moreover, the specific volume and expansion factor of bread were not affected by the hydrolysates. Of 28 peptides identified, four were synthesised and exhibited activity against Penicillium sp. Fluorescence and scanning electron microscopy suggested that the peptides damaged the fungal plasma membrane. Bioinformatics analysis showed that no peptide was toxic and that the antigenic ones had cleavage sites for trypsin or pepsin.
Assuntos
Calotropis , Látex , Peptídeos Antimicrobianos , Aspergillus niger , Pão , Peptídeo Hidrolases , PeptídeosRESUMO
BACKGROUND: Previous studies have shown that latex proteins from Plumeria pudica (LPPp) have anti-inflammatory and antioxidant activity. Therefore, the aim of this study was to evaluate the effects in rats of LPPp on ligature-induced periodontitis, an inflammatory disease. METHODS: The animals were divided into groups: saline (animals without induction of periodontitis), periodontitis (induced periodontitis and untreated) and LPPp (induced periodontitis and treated with 40 mg/kg). The following parameters were evaluated after 20 consecutive days of treatment: gingival bleeding index (GBI), probing pocket depth (PPD), alveolar bone height (ABH) and gingival myeloperoxidase (MPO) activity. In the hepatic tissue, malondialdehyde (MDA), glutathione (GSH) and histopathological alterations were evaluated. Blood levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. RESULTS: Significant reduction in GBI, PPD and gingival MPO activity and ABH was seen in animals treated with LPPp compared with periodontitis. Values of GSH, MDA, ALT and histopathological evaluation were preserved in animals treated with LPPp. CONCLUSIONS: Treatment with LPPp improved clinical aspects of periodontitis, reduced the blood and hepatic alterations and prevented alveolar bone loss. Data suggest that LPPp have potential for treatment of periodontitis.
Assuntos
Perda do Osso Alveolar , Apocynaceae , Periodontite , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/etiologia , Perda do Osso Alveolar/prevenção & controle , Animais , Apocynaceae/metabolismo , Látex/metabolismo , Látex/farmacologia , Látex/uso terapêutico , Periodontite/tratamento farmacológico , Periodontite/patologia , Ratos , Ratos WistarRESUMO
Peruvianin-I is a cysteine peptidase (EC 3.4.22) purified from Thevetia peruviana. Previous studies have shown that it is the only germin-like protein (GLP) with proteolytic activity described so far. In this work, the X-ray crystal structure of peruvianin-I was determined to a resolution of 2.15 Å (PDB accession number: 6ORM) and its specific location was evaluated by different assays. Its overall structure shows an arrangement composed of a homohexamer (a trimer of dimers) where each monomer exhibits a typical ß-barrel fold and two glycosylation sites (Asn55 and Asn144). Analysis of its active site confirmed the absence of essential amino acids for typical oxalate oxidase activity of GLPs. Details of the active site and molecular docking results, using a specific cysteine peptidase inhibitor (iodoacetamide), were used to discuss a plausible mechanism for proteolytic activity of peruvianin-I. Histological analyses showed that T. peruviana has articulated anastomosing laticifers, i.e., rows of cells which merge to form continuous tubes throughout its green organs. Moreover, peruvianin-I was detected exclusively in the latex. Because latex peptidases have been described as defensive molecules against insects, we hypothesize that peruvianin-I contributes to protect T. peruviana plants against herbivory.
Assuntos
Glicoproteínas/química , Proteínas de Plantas/química , Thevetia/química , Thevetia/metabolismo , Domínio Catalítico , Simulação de Acoplamento Molecular , Estrutura Quaternária de Proteína , ProteóliseRESUMO
The Chikungunya virus (CHIKV) is an arbovirus transmitted to humans through mosquito bites and can cause a series of symptoms ranging from a benign febrile illness to severe neurological conditions. We report the identification of CHIKV in a serum sample from an elderly woman with febrile illness and severe arthralgia in Brazil. The occurrence was found of peripheral polyneuropathy affecting the upper and lower limbs evidenced by electroneuromyographic findings. The patient was treated with a corticoid associated with methotrexate, suggesting that the pathophysiological basis of the case in question may be related to an immune-mediated response by T cells and inflammatory cytokines. This finding reinforces the need to be aware of the emergence of neuroinfections related to CHIKV and effective diagnoses for the early detection of neurological alterations, favoring the clinical management of these patients.
Assuntos
Febre de Chikungunya/complicações , Polineuropatias/virologia , Idoso , Brasil , Feminino , Humanos , Imunossupressores/uso terapêutico , Metotrexato/uso terapêutico , Polineuropatias/tratamento farmacológicoRESUMO
The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow's milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30â¯min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow's milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow's milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy.
Assuntos
Caseínas/metabolismo , Hipersensibilidade a Leite/patologia , Peptídeo Hidrolases/metabolismo , Animais , Apocynaceae/enzimologia , Calotropis/enzimologia , Carica/enzimologia , Caseínas/imunologia , Bovinos , Humanos , Hidrólise , Látex/metabolismo , Masculino , Camundongos , Leite/metabolismo , Hipersensibilidade a Leite/imunologia , Hipersensibilidade a Leite/veterinária , Proteínas do Soro do Leite/imunologia , Proteínas do Soro do Leite/metabolismoRESUMO
The germin-like protein (GLP) purified from Thevetia peruviana, Peruvianin-I, is the only one described as having proteolytic activity. Therefore, the goal of this study was to investigate the structural features responsible for its enzymatic activity. Although the amino acid sequence of Peruvianin-I showed high identity with other GLPs, it exhibited punctual mutations, which were responsible for the absence of oxalate oxidase activity. The phylogenetic analysis showed that Peruvianin-I does not belong to any classification of GLP subfamilies. Moreover, Peruvianin-I contains a catalytic triad found in all plant cysteine peptidases. Molecular docking simulations confirmed the role of the catalytic triad in its proteolytic activity. Synchrotron radiation circular dichroism assays confirmed that Peruvianin-I was stable at pH ranging from 5.0 to 8.0 and that it presented significant structural changes only above 60⯰C. The addition of iodoacetamide caused changes in its native conformation, but only a slight effect was observed after adding a reducing agent. This study reports an unusual protein with germin-like structure, lacking typical oxalate oxidase activity. Instead, the proteolytic activity observed suggests that the protein is a cysteine peptidase. These structural peculiarities make PeruvianinI an interesting model for further understanding of the action of laticifer fluids in plant defense.
Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteólise , Thevetia/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Oxirredutases/metabolismo , Filogenia , Inibidores de Proteases/farmacologia , Substâncias Redutoras/química , Análise de Sequência de Proteína , Especificidade por Substrato/efeitos dos fármacos , TemperaturaRESUMO
Transitory allergies to cow milk proteins in infants or adults have become a public health problem. Although extensively or partially hydrolyzed cow milk protein formulas are available, these products are costly. Therefore, studies into innovative enzymes to digest cow milk proteins are needed. Danaus plexippus gut peptidases were purified and examined with regard to cow milk protein hydrolysis. The peptidases hydrolyzed caseins and whey proteins. However, after heat treatment, there was a significant improvement in ß-lactoglobulin hydrolysis. The hydrolyzed cow milk proteins were not recognized by anti-casein antibodies and only reacted slightly with antibodies against whey proteins. This performance was better than that of partially hydrolyzed formulas and similar to that of an extensively hydrolyzed formula. These results suggest that D. plexippus gut peptidases are suitable and innovative enzymes to produce hypoallergenic cow milk protein formulas.
Assuntos
Anticorpos/imunologia , Borboletas/enzimologia , Proteínas do Leite/química , Peptídeo Hidrolases/metabolismo , Adulto , Animais , Caseínas/química , Caseínas/imunologia , Bovinos , Feminino , Alimentos Formulados , Trato Gastrointestinal/enzimologia , Herbivoria , Temperatura Alta , Humanos , Hidrólise , Lactente , Lactoglobulinas/química , Proteínas do Leite/imunologia , Peptídeo Hidrolases/isolamento & purificação , Proteínas do Soro do Leite/química , Proteínas do Soro do Leite/imunologiaRESUMO
Hydrogen sulphide (H2S) is a gasotransmitter that participates in various physiological and pathophysiological processes within the gastrointestinal tract. We studied the effects and possible mechanism of action of H2S in secretory diarrhoea caused by cholera toxin (CT). The possible mechanisms of action of H2S were investigated using an intestinal fluid secretion model in isolated intestinal loops on anaesthetized mice treated with CT. NaHS and Lawesson's reagent and l-cysteine showed antisecretory activity through reduction of intestinal fluid secretion and loss of Cl- induced by CT. Pretreatment with an inhibitor of cystathionine-γ-lyase (CSE), dl-propargylglycine (PAG), reversed the effect of l-cysteine and caused severe intestinal secretion. Co-treatment with PAG and a submaximal dose of CT increased intestinal fluid secretion, thus supporting the role of H2S in the pathophysiology of cholera. CT increased the expression of CSE and the production of H2S. Pretreatment with PAG did not reverse the effect of SQ 22536 (an AC inhibitor), bupivacaine (inhibitor of cAMP production), KT-5720 (a PKA inhibitor), and AICAR (an AMPK activator). The treatment with Forskolin does not reverse the effects of the H2S donors. Co-treatment with either NaHS or Lawesson's reagent and dorsomorphin (an AMPK inhibitor) did not reverse the effect of the H2S donors. H2S has antisecretory activity and is an essential molecule for protection against the intestinal secretion induced by CT. Thus, H2S donor drugs are promising candidates for cholera therapy. However, more studies are needed to elucidate the possible mechanism of action.
Assuntos
Toxina da Cólera/antagonistas & inibidores , Diarreia/induzido quimicamente , Diarreia/tratamento farmacológico , Sulfeto de Hidrogênio/farmacologia , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Adenilil Ciclases/metabolismo , Animais , Toxina da Cólera/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Masculino , CamundongosRESUMO
OBJECTIVE: The aim of this study was to investigate whether experimental periodontitis cause changes to the renal tissues and imbalance in oxidative stress in kidneys. METHODS: Twenty-two female Wistar rats were separated into two groups: control and periodontitis. We assessed the following parameters: gingival bleeding index (GBI), tooth mobility, gum malondialdehyde (MDA), myeloperoxidase (MPO) activity, probing pocket depth (PPD), alveolar bone loss (ABL) for periodontal tissues; histomorphometric measures associated with renal corpuscle and histopathological aspects (evaluation of brush border) for kidneys; as also blood and urine biomarkers. Finally, we evaluated renal oxidative stress through glutathione (GSH) and MDA respectively. RESULTS: With regard to renal histomorphometry, significant differences were observed in all parameters assessed. In relation periodic acid Schiff (PAS) staining, disruption was observed of brush border in the periodontitis group in the renal tubules in comparison with the control group. The periodontitis group presented significantly higher MDA and lower GSH concentrations in the kidneys compared with animals without periodontitis. CONCLUSION: The induced periodontitis caused histomorphometric changes in renal tissues as well as disruption of the brush border in renal tubules, alterations associated with increase in oxidative stress in kidneys. However, these alterations were not sufficient to cause differences in the renal function markers.