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1.
PLoS One ; 15(1): e0227626, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945130

RESUMO

BACKGROUND: Autism spectrum disorder (ASD) is a complex group of heterogeneous neurodevelopmental disorders the prevalence of which has been in the rise in the past decade. In an attempt to better target the basic causes of ASD for diagnosis and treatment, efforts to identify reliable biomarkers related to the body's metabolism are increasing. Despite an increase in identifying biomarkers in ASD, there are none so far with enough evidence to be used in routine clinical examination, unless medical illness is suspected. Promising biomarkers include those of mitochondrial dysfunction, oxidative stress, energy metabolism, and apoptosis. METHODS AND PARTICIPANTS: Sodium (Na+), Potassium (K+), glutathione (GSH), glutathione-s-transferase (GST), Creatine kinase (CK), lactate dehydrogenase (LDH), Coenzyme Q10, and melatonin (MLTN) were evaluated in 13 participants with ASD and 24 age-matched healthy controls. Additionally, five ratios, which include Na+/K+, GSH:GST, CK:Cas7, CoQ10: Cas 7, and Cas7:MLTN, were tested to measure their predictive values in discriminating between autistic individuals and controls. These markers, either in absolute values, as five ratios, or combined (9 markers + 5 ratios) were subjected to a principal component analysis and multidimensional scaling (MDS), and hierarchical clustering, which are helpful statistical tools in the field of biomarkers. RESULTS: Our data demonstrated that both PCA and MDS analysis were effective in separating autistic from control subjects completely. This was also confirmed through the use of hierarchical clustering, which showed complete separation of the autistic and control groups based on nine biomarkers, five biomarker ratios, or a combined profile. Excellent predictive value of the measured profile was obtained using the receiver operating characteristics analysis, which showed an area under the curve of 1. CONCLUSION: The availability of an improved predictive profile, represented by nine biomarkers plus the five ratios, inter-related different etiological mechanisms in ASD and would be valuable in providing greater recognition of the altered biological pathways in ASD. Our predictive profile could be used for the diagnosis and intervention of ASD.

2.
Metab Brain Dis ; 35(1): 215-224, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31625070

RESUMO

The current study evaluated the protective and therapeutic potency of bee pollen in ameliorating the toxic effects of methylmercury (MeHg), by measuring certain biochemical parameters related to neurotransmission, neuroinflammation, apoptosis, and glutamate excitotoxicity in the male neonate brain. Healthy, pregnant female rats (N = 40) were randomly divided into 5 groups, each comprising10 male neonates, as follows: (i) neonates delivered by control mothers; (ii) neonates delivered by MeHg-treated mothers who received 0.5 mg/kg BW/day MeHg via drinking water from gestational day 7 till postnatal day 7; (iii) neonates delivered by bee pollen treated mothers who received 200-mg/kg BW bee pollen from postnatal day 0 for 4 weeks; (iv) protective group of neonates delivered by MeHg and bee pollen-treated mothers, who continued to receive bee pollen until day 21 at the same dose, and (v) therapeutic group of neonates delivered by MeHg- treated mothers followed by bee pollen treatment, wherein they received 200-mg/kg BW bee pollen from postnatal day 0 for 4 weeks. Selected biochemical parameters in brain homogenates from each group were measured. MeHg-treated groups exhibited various signs of brain toxicity, such as a marked reduction in neurotransmitters (serotonin (5-HT), nor-adrenalin (NA), dopamine (DA)) and gamma aminobutyric acid (GABA) and elevated levels of interferon gamma (IFN-γ), caspase-3, and glutamate (Glu). Bee pollen effectively reduced the neurotoxic effects of MeHg. Minimal changes in all measured parameters were observed in MeHg-treated animals compared to the control group. Therefore, bee pollen may safely improve neurotransmitter defects, inflammation, apoptosis, and glutamate excitotoxicity.

3.
Saudi J Biol Sci ; 26(2): 301-307, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31485169

RESUMO

In the present study, Peroxidase from date palm (Phoenix dactylifera) leaves was purified to homogeneity by three-step procedure including aqueous two-phase system, hydrophobic and Ion-exchange chromatography. The enzyme migrated as single band on SDS-PAGE giving molecular weight of 68 ±â€¯3 kDa. The purification factor for purified date palm peroxidase was 68 with high 41% yield. Enzymatic assays together with far-UV circular dichroism (CD), intrinsic and extrinsic fluorescence studies were carried out to monitor the structural stability of date palm and horseradish peroxidase (HRP) against various pH and temperatures. Activity measurements illustrated different pH stability for date palm and HRP. Both peroxidases are more susceptible to extreme acidic conditions as suggested by 4 & 15 nm red shift in date palm and HRP, respectively. Secondary structure analysis using far UV-CD exhibited predominance of α-helical (43.8%) structure. Also, pH induces loss in the secondary structure of date palm peroxidase. Thermal stability analysis revealed date palm peroxidase is more stable in comparison to HRP. In summary, date palm peroxidases could be promising enzymes for various applications where extreme pH and temperature is required.

4.
J Enzyme Inhib Med Chem ; 34(1): 672-683, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30821525

RESUMO

Some new 3H-quinazolin-4-one derivatives were synthesised and screened for anticancer, antiphospholipases, antiproteases, and antimetabolic syndrome activities. Compound 15d was more potent in reducing the cell viabilities of HT-29 and SW620 cells lines to 38%, 36.7%, compared to 5-FU which demonstrated cell viabilities of 65.9 and 42.7% respectively. The IC50 values of 15d were ∼20 µg/ml. Assessment of apoptotic activity revealed that 15d decreased the cell viability by down regulating Bcl2 and BclxL. Moreover, compounds, 8j, 8d/15a/15e, 5b, and 8f displayed lowered IC50 values than oleanolic acid against proinflammatory isoforms of hGV, hG-X, NmPLA2, and AmPLA2. In addition, 8d, 8h, 8j, 15a, 15b, 15e, and 15f showed better anti-α-amylase than quercetin, whereas 8g, 8h, and 8i showed higher anti-α-glucosidase activity than allopurinol. Thus, these compounds can be considered as potential antidiabetic agents. Finally, none of the compounds showed higher antiproteases or xanthine oxidase activities than the used reference drugs.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Síndrome Metabólica/tratamento farmacológico , Peptídeo Hidrolases/metabolismo , Fosfolipases/antagonistas & inibidores , Quinazolinonas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HT29 , Humanos , Síndrome Metabólica/metabolismo , Estrutura Molecular , Fosfolipases/metabolismo , Quinazolinonas/síntese química , Quinazolinonas/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
5.
Cell Mol Biol (Noisy-le-grand) ; 64(13): 55-62, 2018 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-30403596

RESUMO

Many enzymes are involved in numerous pathologies which are related to metabolic reactions and inflammatory diseases such as pancreatic lipase, α-amylase, α-glucosidase and xanthine oxidase and secreted phospholipases A2 (Group IIA, V and X), respectively. Therefore, inhibiting these enzymes offer the potential to block production of more inflammatory substances and decrease the risk factors for cardiovascular diseases. The purpose of this study was to investigate some potent, bioavailable and selective inhibitors of some catalytic proteins implicated to metabolic syndrome and their antioxidant effects from various solvent extracts of R. frangula leaves. The anti-inflammatory, obesity, diabete and XO potentials were evaluated through analyses of inhibition activities of corresponding metabolites.The water extract exhibited an important inhibitory effect on human, dromedary and stingray sPLA2-G IIA achieved an IC50 of 0.16±0.06, 0.19±0.05 and 0.07±0.01 mg/mL, respectively. Likewise, the same fraction demonstrated the highest pancreatic lipase inhibitory activity using two different substrates. Indeed, 50% of dromedary pancreatic lipase inhibition was demonstrated for 5 min and 15 min using olive oil and TC4 substrates, respectively. Besides, it was established that methanolic extract had more effective inhibitory lipase activity than ORLISTAT used as a specific inhibitor of gastric, pancreatic and carboxyl ester lipase for treating obesity, with an IC50 of 5.51±0.27 and 91.46±2.3 µg/mL, respectively. In the case of α-amylase, α-glucosidase and xanthine oxidase, the crude methanolic extract showed a potential inhibitory effect with an IC50 of 45±3.45, 3±0.15 and 27±1.71 µg/mL, respectively. Conclusively, R. frangula leaves extracts showed a potential value of some sPLA2, some metabolic enzymes and XO inhibitors as anti-inflammatory and metabolic syndrome drugs.


Assuntos
Inibidores Enzimáticos/farmacologia , Enzimas/metabolismo , Inflamação/enzimologia , Extratos Vegetais/farmacologia , Folhas de Planta/química , Rhamnus/química , Animais , Humanos , Concentração Inibidora 50 , Metanol/química , Solventes , Ácido Taurodesoxicólico/farmacologia
6.
J Mol Neurosci ; 65(3): 327-335, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29946915

RESUMO

MeHg is a widely distributed environmental toxicant with harmful effects on the developing and adult nervous system. This study aimed to evaluate the therapeutic and protective efficacy of pollen grain in improving the toxic effects of MeHg, through the measurement of selected biochemical parameters linked to oxidative stress, energy metabolism, and neurotransmission in brain homogenates of male pups' neonates. Forty healthy pregnant female rats were randomly divided into five groups, and after delivery, each group was consisting of 10 male neonates: (1) neonates delivered by control mothers, (2) neonates delivered by bee pollen treated mothers who received bee pollen at the dose of 200-mg/kg body weight from postnatal day 0 for 4 weeks, (3) neonates delivered by MeHg-treated mothers who received MeHg at the dose of 0.5 mg/kg/day via drinking water from gestational day 7 till postnatal day 7 of delivery, (4) therapeutic group: neonates delivered by MeHg-treated mothers followed by bee pollen treatment who received bee pollen at the dose of 200-mg/kg body weight from postnatal day 0 for 4 weeks, and (5) protective group: neonates delivered by MeHg and bee pollen-treated mothers. Mothers continued receiving the bee pollen at the same dose until day 21. Biochemical parameters linked to oxidative stress and energy metabolism and neurotransmission were investigated in brain homogenates of neonates from all the five groups. MeHg treatment showed an increase in oxidative stress markers like lipid peroxidation and catalase activity coupled with a non-significant decrease in glutathione level. Impaired energy metabolism was ascertained via the inhibition of creatine kinase and lactate dehydrogenase activities. Dramatic decrease of Mg2+ and K+ concentrations confirmed the neurotransmission defect. Interestingly, the bee pollen treatment was highly effective in restoring the catalase, lactate dehydrogenase, and creatine kinase activities in addition to normalizing the levels of Mg2+, K+, lipid peroxidation, and glutathione. Overall, the exposure to MeHg during the developing brain stages was highly effective to show signs and symptoms of neuronal toxicity. Furthermore, it has been concluded that bee pollen can be used safely to ameliorate oxidative stress, poor detoxification as well as metal ion defects, and neuronal death as a critical mechanisms involved in the etiology of numerous neurological disorders.


Assuntos
Antioxidantes/uso terapêutico , Apiterapia/métodos , Poluentes Ambientais/toxicidade , Compostos de Metilmercúrio/toxicidade , Fármacos Neuroprotetores/uso terapêutico , Pólen/química , Efeitos Tardios da Exposição Pré-Natal/tratamento farmacológico , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Feminino , Masculino , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/etiologia , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Ratos , Ratos Wistar
7.
J Mol Neurosci ; 65(3): 265-276, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29931502

RESUMO

Autism spectrum disorder (ASD) is a neuro-behavioral syndrome with a broad spectrum of different mechanisms and etiologies that are caused by abnormal brain development. To date, no highly reliable and effective diagnostic biomarker to assess ASD is available so far. The present study investigated the predictivity potential of some suggested markers in ASD diagnosis focusing onto the relative ratios of several plasma biomarkers of electron transport chain function, and mitochondrial metabolism in 41 patients with ASD evaluated for behavior deficits measured using Childhood Autism Rating Scales (CARS). The control matched for further 41 healthy subjects. The relation of these relative ratios to ASD severity was also examined, as well as their ability to distinguish ASD children from neurotypical children. All predictive ratios were found to be markedly altered and correlated in ASD patients. However, no ratio was connected with autism severity. Interestingly, MRCC-I/caspase-7, GSH/GST, and MRCC-I/COQ10 were the most distinctive relative ratios between neurotypical controls and ASD patients and may thereby be useful biomarkers for early diagnosis of ASD. Overall, this investigation proves that relative ratios of numerous mitochondrial biomarkers might be predictive and efficient to differentiate between neurotypical children and ASD.


Assuntos
Transtorno Autístico/sangue , Complexo I de Transporte de Elétrons/sangue , Metabolismo Energético , Adolescente , Adulto , Transtorno Autístico/metabolismo , Transtorno Autístico/patologia , Biomarcadores/sangue , Estudos de Casos e Controles , Caspase 7/sangue , Criança , Pré-Escolar , Glutationa/sangue , Glutationa Transferase/sangue , Humanos , Masculino , Mitocôndrias/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/sangue
8.
Int J Biol Macromol ; 117: 1140-1146, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29885399

RESUMO

A novel non-toxic phospholipase A2 was purified to homogeneity in a single chromatography step from the venom of Walterinnesia aegyptia, a monotypic elapid snake caught in Saudi Arabia, and its antimicrobial and hemolytic properties were evaluated as well. This enzyme, namely WaPLA2, is a homodimer with an estimated molecular mass of 30 kDa, and its NH2-terminal sequence exhibits a significant degree of similarity with PLA2 group-I. At optimal pH (8.5) and temperature (45 °C), the purified PLA2 exhibited a specific activity of 2100 U/mg, and it requires bile salts and Ca2+ for its activity. However, other cations such as Cd2+ and Hg2+ diminished the enzyme activity remarkably, thereby suggesting that the catalytic site arrangement has an exclusive structure for Ca2+ binding. Furthermore, WaPLA2 maintained almost 100% and 60% of its full activity in a pH range of 6.0-10 after 24 h incubation or after 60 min treatment at 70 °C, respectively. In the biological activity assays, WaPLA2 displayed potent indirectly hemolytic and antimicrobial activities that were strongly correlated. These promising findings encourage further in-depth research to understand the molecular mechanism of WaPLA2's antimicrobial properties for its possible use as a potential therapeutic lead molecule for treating infections.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Venenos Elapídicos/química , Venenos Elapídicos/farmacologia , Elapidae , Fosfolipases A2/química , Fosfolipases A2/farmacologia , Animais , Bactérias/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Multimerização Proteica
9.
Saudi J Biol Sci ; 25(3): 409-417, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29686504

RESUMO

An extracellular lipase of a newly isolated S. aureus strain ALA1 (SAL4) was purified from the optimized culture medium. The SAL4 specific activity determined at 60 °C and pH 12 by using olive oil emulsion or TC4, reached 7215 U/mg and 2484 U/mg, respectively. The 38 NH2-terminal amino acid sequence of the purified enzyme starting with two extra amino acid residues (LK) was similar to known staphylococcal lipase sequences. This novel lipase maintained almost 100% and 75% of its full activity in a pH range of 4.0-12 after a 24 h incubation or after 0.5 h treatment at 70 °C, respectively. Interestingly, SAL4 displayed appreciable stability toward oxidizing agents, anionic and non-ionic surfactants in addition to its compatibility with several commercial detergents. Overall, these interesting characteristics make this new lipase promising for its application in detergent industry.

10.
Cell Mol Biol (Noisy-le-grand) ; 64(1): 103-106, 2018 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-29412802

RESUMO

The gut and the liver are closely linked to each other, as changes in the gut microbiota can play a significant role in the development of many liver diseases. Gut bacteria respond rapidly to changes in diet and thus can affect the liver through their metabolites. The impact of a high lipid diet on the liver in the presence of an altered gut flora modulated by ampicillin was investigated. The study was performed on 30 male Western albino rats randomly divided into 3 groups: control (phosphate buffered saline treated), group II (ampicillin 50 mg/kg for three weeks to induce microbiota alterations and fed on standard diet) and group III (same dose of ampicillin and fed on a lipid rich diet). Stool samples were collected for qualitative determination of bacteria. Serum hepato-specific markers, in addition to Glutathione (GSH), Lipid peroxidase (MDA), Glutathione-S- transferase(GST), and vitamin C in liver tissues, were measured. Altered gut microbiota significantly increased the level of the hepato-specific marker MDA and reduced the GST, GSH and vitamin C levels. However, animals fed a lipid rich diet displayed a more significant shift in hepatic markers and antioxidants. Moreover, a new switch in composition of the gut bacteria was observed by feeding the lipid rich diet. Our study showed that bacterial overgrowth in the gut can be associated with liver dysfunction and that a high lipid diet can promote the overgrowth of some liver damaging microflora during antibiotic treatment.


Assuntos
Dieta Hiperlipídica , Microbioma Gastrointestinal , Fígado/metabolismo , Ampicilina/farmacologia , Animais , Antibacterianos/farmacologia , Ácido Ascórbico/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Fígado/enzimologia , Masculino , Peroxidases/metabolismo , Ratos
11.
J Enzyme Inhib Med Chem ; 32(1): 1143-1151, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28856929

RESUMO

Elevated blood glucose and increased activities of secreted phospholipase A2 (sPLA2) are strongly linked to coronary heart disease. In this report, our goal was to develop small heterocyclic compound that inhibit sPLA2. The title compounds were also tested against α-glucosidase and α-amylase. This array of enzymes was selected due to their implication in blood glucose regulation and diabetic cardiovascular complications. Therefore, two distinct series of quinoxalinone derivatives were synthesised; 3-[N'-(substituted-benzylidene)-hydrazino]-1H-quinoxalin-2-ones 3a-f and 1-(substituted-phenyl)-5H-[1,2,4]triazolo[4,3-a]quinoxalin-4-ones 4a-f. Four compounds showed promising enzyme inhibitory effect, compounds 3f and 4b-d potently inhibited the catalytic activities of all of the studied proinflammatory sPLA2. Compound 3e inhibited α-glucosidase (IC50 = 9.99 ± 0.18 µM); which is comparable to quercetin (IC50 = 9.93 ± 0.66 µM), a known inhibitor of this enzyme. Unfortunately, all compounds showed weak activity against α-amylase (IC50 > 200 µM). Structure-based molecular modelling tools were utilised to rationalise the SAR compared to co-crystal structures with sPLA2-GX as well as α-glucosidase. This report introduces novel compounds with dual activities on biochemically unrelated enzymes mutually involved in diabetes and its complications.


Assuntos
Inibidores Enzimáticos/farmacologia , Fosfolipases A2 Secretórias/antagonistas & inibidores , Quinoxalinas/farmacologia , alfa-Glucosidases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Fosfolipases A2 Secretórias/metabolismo , Quinoxalinas/síntese química , Quinoxalinas/química , Relação Estrutura-Atividade
12.
Molecules ; 21(12)2016 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-27918459

RESUMO

Some novel hydrazone derivatives 6a-o were synthesized from the key intermediate 4-Chloro-N-(2-hydrazinocarbonyl-phenyl)-benzamide 5 and characterized using IR, ¹H-NMR, 13C-NMR, mass spectroscopy and elemental analysis. The inhibitory potential against two secretory phospholipase A2 (sPLA2), three protease enzymes and eleven bacterial strains were evaluated. The results revealed that all compounds showed preferential inhibition towards hGIIA isoform of sPLA2 rather than DrG-IB with compounds 6l and 6e being the most active. The tested compounds exhibited excellent antiprotease activity against proteinase K and protease from Bacillus sp. with compound 6l being the most active against both enzymes. Furthermore, the maximum zones of inhibition against bacterial growth were exhibited by compounds; 6a, 6m, and 6o against P. aeruginosa; 6a, 6b, 6d, 6f, 6l, 6m, 6n, and 6o against Serratia; 6k against S. mutans; and compounds 6a, 6d, 6e, 6m, and 6n against E. feacalis. The docking simulations of hydrazones 6a-o with GIIA sPLA2, proteinase K and hydrazones 6a-e with glutamine-fructose-6-phosphate transaminase were performed to obtain information regarding the mechanism of action.


Assuntos
Antibacterianos/farmacologia , Endopeptidase K/antagonistas & inibidores , Hidrazonas/síntese química , Hidrazonas/farmacologia , Inibidores de Fosfolipase A2/farmacologia , Inibidores de Proteases/farmacologia , Antibacterianos/síntese química , Bacillus/crescimento & desenvolvimento , Benzamidas/química , Enterococcus faecalis/crescimento & desenvolvimento , Simulação de Acoplamento Molecular , Inibidores de Fosfolipase A2/síntese química , Inibidores de Proteases/síntese química , Espectroscopia de Prótons por Ressonância Magnética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Serratia/crescimento & desenvolvimento , Streptococcus mutans/crescimento & desenvolvimento , Relação Estrutura-Atividade
13.
Biotechnol Appl Biochem ; 63(3): 378-90, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-25828848

RESUMO

In this study, a new strain, ALA1, was identified as Staphylococcus aureus by biochemical tests, and its 16S ribosomal DNA sequence was isolated from dromedary milk. ALA1 lipase production was optimized in shake flask experiments and measured with varying pH (3-11), temperature (20-55 °C) and substrate concentrations. The maximum lipase production was recorded at pH 8 and 30 °C for up to 30 H of culture period for the S. aureus ALA1 strain. Among the substrates tested, selected carbon sources, xylose, nitrogen source, yeast extract, and olive oil (1%) were suitable for maximizing lipase production. The effects of surfactants were investigated and showed that Tween 20, Tween 80, and Triton X-100 prevented lipase production. Interestingly, isolate ALA1 was able to grow in high concentrations of benzene or toluene (up to 50% (v/v)). Moreover, the lipolytic activity of the S. aureus ALA1 lipase was stimulated by diethyl ether, whereas almost 100% of S. aureus ALA1 lipase activity was retained in 25% acetone, acetonitrile, benzene, 2-propanol, ethanol, methanol, or toluene. Because of its stability in organic solvent, the S. aureus ALA1 lipase was used as a biocatalyst to synthesize high levels of added value molecules. S. aureus ALA1 lipase could be considered as an ideal choice for applications in detergent formulations because of its high stability and compatibility with various surfactants, oxidizing agents, and commercial detergents.


Assuntos
Biocatálise , Indústrias , Lipase/química , Lipase/metabolismo , Compostos Orgânicos/farmacologia , Solventes/farmacologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/isolamento & purificação , Detergentes/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Esterificação , Lipólise , Oxidantes/farmacologia , Tensoativos/farmacologia
14.
Indian J Biochem Biophys ; 52(2): 179-88, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26118130

RESUMO

Lipases are the enzymes of choice for laundry detergent industries, owing to their triglyceride removing ability from the soiled fabric, which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In this study, a novel thermo-alkaline lipase-producing strain identified as Bacillus stearothermophilus was isolated from the soil samples of olive oil mill. Enhanced lipase production was observed at 55 degrees C, pH 11 and after 48 h of incubation. Among the substrates tested, xylose (a carbon source), peptone (a nitrogen source) and olive oil at a concentration of 1% were suitable substrates for enhancing lipase production. MgSO4 and Tween-80 were suitable substrates for maximizing lipase production. The enzyme was purified to homogeneity by a single CM-Sephadex column chromatography and revealed molecular mass of 67 kDa. The enzyme (BL1) was active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 11.0, exhibited maximal activity at 55 degreesC and retained more than 70% of its activity after incubation at 70 degrees C or pH 13 for 0.5 h or 24 h, respectively. The enzyme hydrolyzed both short and long-chain triacylglycerols at comparable rates. BL1 was studied in a preliminary evaluation for use in detergent formulation solutions. This novel lipase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40 degrees C, and good stability towards oxidizing agents. Additionally, the enzyme showed excellent stability and compatibility with various commercial detergents, suggesting its potential as an additive in detergent formulations.


Assuntos
Álcalis/metabolismo , Detergentes/metabolismo , Geobacillus stearothermophilus/enzimologia , Lipase/metabolismo , Estabilidade Enzimática
15.
Int J Biol Macromol ; 62: 537-42, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24120965

RESUMO

In the present study, we have purified the group V phospholipase from the heart of cartilaginous fish stingray Dasyatis pastinaca and compared its biochemical properties with group IIA (sPLA2-IIA) and IB (sPLA2-IB) phospholipases previously purified from pancreas and intestine, respectively. Group V phospholipase (sPLA2-V) was purified to homogeneity by heat treatment, ammonium sulphate precipitation and RP-HPLC. The N-terminal sequence of the purified sPLA2-V exhibits a high degree of homology with those of mammal. The enzyme was found to be monomeric with a molecular mass estimation of 14 kDa. The specific activity of the purified enzyme, measured at pH 8 and 37 °C was 52 U/mg. Like sPLA2-IB and sPLA2-IIA, the sPLA2-V is found to be stable between pH 3 and 11 after 30 min of incubation. The purified sPLA2-V retained 65% of its activity after 10 min of incubation at 70 °C and it absolutely requires Ca(2+) for enzymatic activity. In addition it displayed high tolerance to organic solvents. Kinetic parameters Kmapp, kcat and the deduced catalytic efficiency (kcat/Kmapp) of the purified group-V, -IB and -IIA PLA2s were determined using phosphatidylethanolamine (PE), phosphatidylcholine (PC) or phosphatidylserine (PS) as substrate. The three enzymes hydrolyze the zwiterionic PE and PC substrates more efficiently than anionic PS substrate.


Assuntos
Elasmobrânquios/metabolismo , Fosfolipases A2 do Grupo IB/metabolismo , Fosfolipases A2 do Grupo II/metabolismo , Fosfolipases A2 do Grupo V/metabolismo , Sequência de Aminoácidos , Animais , Ácidos e Sais Biliares/farmacologia , Cálcio/química , Cálcio/farmacologia , Ativação Enzimática/efeitos dos fármacos , Fosfolipases A2 do Grupo IB/química , Fosfolipases A2 do Grupo II/química , Fosfolipases A2 do Grupo V/química , Fosfolipases A2 do Grupo V/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Alinhamento de Sequência , Solventes , Especificidade por Substrato , Temperatura Ambiente , Tripsina/metabolismo
16.
Indian J Biochem Biophys ; 50(3): 186-95, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23898481

RESUMO

A phospholipase A2 belonging to IIA group secretory PLA2 was isolated and purified to homogeneity from the intestine of common stingray (Dasyatis pastinaca) using acidic treatment (pH 1.5) and ammonium sulphate precipitation methods combined with single-column ion-exchange chromatography. The purified enzyme was found to be a glycosylated monomeric protein with a molecular mass of about 14 kDa. The stingray sPLA2-IIA had optimum activity at 45 degrees C, unlike known mammalian PLA2-IIAs, which show optimum activity at 37 degrees C. The purified enzyme exhibited a specific activity of 290 U/mg at optimal conditions (pH 9.5 and 45 degrees C) in the presence of 6 mM NaDC and 8 mM CaCl2 with egg yolk as substrate. The NH2-terminal sequence of the enzyme and some protein fragments obtained from its tryptic digestion were also determined. All sequences obtained were similar to those of sPLA2-IIA. The enzyme also showed good stability in the presence of organic solvents, acidic and alkaline pH media and high temperature conditions. Thus, the purified enzyme exhibited a number of unique and promising properties, making it a potential possible candidate for future applications in the treatment of phospholipid-rich industrial effluents and synthesis of useful preparations for the food production and processing industry.


Assuntos
Elasmobrânquios/metabolismo , Fosfolipases A2 do Grupo II/química , Fosfolipases A2 do Grupo II/isolamento & purificação , Intestinos/enzimologia , Animais , Ativação Enzimática , Estabilidade Enzimática , Especificidade por Substrato
17.
Int J Biol Macromol ; 57: 156-64, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23500664

RESUMO

We purified a protein from the dromedary small intestine that displayed potent bactericidal activity against Gram-positive bacteria, and identified it as group-IIA phospholipase A2 (DrPLA2-IIA) by NH2-terminal sequencing and enzymatic measurements. In fact, our findings revealed that the purified PLA2-IIA was a monomeric protein with a molecular mass of about 14 kDa. Pure enzyme has a specific activity of 329 ± 25 U/mg at optimal conditions (pH 9.5 and 45 °C) in the presence of 6 mM NaDC and 7 mM CaCl2 with egg yolk emulsion as substrate and binds with a higher affinity to PE than PS and PC. Furthermore, the DrPLA2-IIA activity was dependent on Ca(2+); other cations (Cd(2+), Co(2+), Fe(2+), Mg(2+), Mn(2+), and Zn(2+)) reduced the enzymatic activity notably, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca(2+). On the other hand, DrPLA2-IIA was highly bactericidal against Gram-positive bacteria with inhibition zones and IC50 values in the range of 21-27 mm and 3.7-8 µg/ml, respectively, whereas Gram-negative bacteria exhibited a much higher resistance. These observations suggest that the main physiological role of DrPLA2-IIA could be the defense of the intestine against bacterial infections.


Assuntos
Antibacterianos , Camelus , Bactérias Gram-Positivas/crescimento & desenvolvimento , Intestinos/enzimologia , Fosfolipases A2 , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Infecções Bacterianas/enzimologia , Metais/química , Metais/metabolismo , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Fosfolipases A2/metabolismo , Fosfolipases A2/farmacologia
18.
Appl Biochem Biotechnol ; 169(6): 1858-69, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23344945

RESUMO

The best known physiologic function of secreted phospholipase A2 (sPLA2) group IIA (sPLA2-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA2-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye's innate immune defense system. The antibacterial properties of sPLA2 have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA2 on several Gram-positive bacteria. Our results showed that the sPLA2 displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca(2+) is crucial for the activity of dromedary tear sPLA2 and to a less extent Mg(2+) ions. Given the presence of sPLA2 in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.


Assuntos
Antibacterianos/farmacologia , Camelus/imunologia , Camelus/microbiologia , Fosfolipases A2 do Grupo II/farmacologia , Staphylococcus/efeitos dos fármacos , Staphylococcus/fisiologia , Lágrimas/enzimologia , Animais , Antibacterianos/isolamento & purificação , Cátions Bivalentes/farmacologia , Sinergismo Farmacológico , Fosfolipases A2 do Grupo II/isolamento & purificação , Humanos , Camundongos , Muramidase/farmacologia , Coelhos , Lágrimas/imunologia , Lágrimas/microbiologia
19.
Appl Biochem Biotechnol ; 168(5): 1277-87, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22956299

RESUMO

Stingray phospholipase A(2) group IIA (SPLA(2)-IIA) was recently isolated and purified to homogeneity from the intestine of the common stingray Dasyatis pastinaca, suggesting that this enzyme plays an important role in systemic bactericidal defense. The present study showed that SPLA(2)-IIA was highly bactericidal against Gram-positive bacteria with inhibition zones and minimal inhibitory concentration values in the range of 13-25 mm and 2-8 µg/ml, respectively, whereas Gram-negative bacteria exhibited a much higher resistance. The bactericidal efficiency of SPLA(2)-IIA was shown to be unaffected by high protein and salt concentrations, but dependent upon the presence of calcium ions, and then correlated to the hydrolytic activity of membrane phospholipids. Importantly, we showed that stingray phospholipase A(2) group IIA presents no cytotoxicity after its incubation with MDA-MB-231 cells. SPLA(2)-IIA may be considered as a future therapeutic agent against bacterial infections.


Assuntos
Antibacterianos/farmacologia , Elasmobrânquios , Intestinos/enzimologia , Fosfolipases A2 , Animais , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hidrólise , Testes de Sensibilidade Microbiana , Fosfolipases A2/química , Fosfolipases A2/metabolismo , Fosfolipases A2/farmacologia , Fosfolipídeos/química , Proteínas/química , Sais/química
20.
J Neuroinflammation ; 9: 74, 2012 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-22531301

RESUMO

BACKGROUND: Recent clinical observations suggest that certain gut and dietary factors may transiently worsen symptoms in autism. Propionic acid (PA) is a short chain fatty acid and an important intermediate of cellular metabolism. Although PA has several beneficial biological effects, its accumulation is neurotoxic. METHODS: Two groups of young Western albino male rats weighing about 45 to 60 grams (approximately 21 days old) were used in the present study. The first group consisted of oral buffered PA-treated rats that were given a neurotoxic dose of 250 mg/kg body weight/day for three days, n = eight; the second group of rats were given only phosphate buffered saline and used as a control. Biochemical parameters representing oxidative stress, energy metabolism, neuroinflammation, neurotransmission, and apoptosis were investigated in brain homogenates of both groups. RESULTS: Biochemical analyses of brain homogenates from PA-treated rats showed an increase in oxidative stress markers (for example, lipid peroxidation), coupled with a decrease in glutathione (GSH) and glutathione peroxidase (GPX) and catalase activities. Impaired energy metabolism was ascertained through the decrease of lactate dehydrogenase and activation of creatine kinase (CK). Elevated IL-6, TNFα, IFNγ and heat shock protein 70 (HSP70) confirmed the neuroinflammatory effect of PA. Moreover, elevation of caspase3 and DNA fragmentation proved the pro-apoptotic and neurotoxic effect of PA to rat pups CONCLUSION: By comparing the results obtained with those from animal models of autism or with clinical data on the biochemical profile of autistic patients, this study showed that the neurotoxicity of PA as an environmental factor could play a central role in the etiology of autistic biochemical features.


Assuntos
Transtorno Autístico/induzido quimicamente , Encéfalo/metabolismo , Modelos Animais de Doenças , Propionatos/toxicidade , Animais , Transtorno Autístico/fisiopatologia , Encéfalo/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Propionatos/metabolismo , Distribuição Aleatória , Ratos
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