Phenylmethylsulfonyl Fluoride Research Articles

Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

Partial characterization of digestive proteases in adults of bigclaw river shrimp Macrobrachium carcinus

The present research was focused to characterize the digestive proteases in Macrobrachium carcinus adults using biochemical and electrophoretic techniques. Our results showed that the alkaline proteolytic activity from males and females did not show significant differences ( P > 0.05) between them, the optimum pH for digestive proteases is 8, and is very stable to changes in alkaline pH (8 and 10). The optimum temperature for alkaline proteases is 45oC and is stable from 25 to 45oC. The activity was totally inhibited with phenylmethylsulfonyl fluoride (PMSF), additionally the inhibition with trypsin soybean inhibitor 1 (SBT1) and tosyl-lysine-methyl ketone (TLCK) indicate high effect over serine proteases. Eight active bands were found using sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) zymogram (range 17.8-94.0 kDa), which were partially inhibited with ovalbumin (Ovo), SBT1, phenanthroline (Phen), tosyl-phenylalanine-methyl ketone (TPCK), TLCK and ethylenediaminetetraacetic acid (EDTA), indicating an omnivorous digestive capacity, which remarks that the mainly alkaline protease in M. carcinus hepatopancreas are trypsin like enzymes.

Purification and characterization of a novel extracellular serine-protease with collagenolytic activity from Aspergillus tamarii URM4634

An extracellular serine-protease from Aspergillus tamarii URM4634 was purified and characterized. The possibility of using Aspergillus tamarii URM4634 protease in detergent formulations and collagenolytic activity was investigated. The protease demonstrated excellent stability at pH range 7.0–11.0, the optimum being at pH 9.0. The enzyme was stable at 40 °C for 180 min, enhanced by Mg++ and Ca++, but inhibited by Zn++, and strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggested as serine-protease. The azocasein substrate result showed Km = 0.434 mg/mL and Vmax = 7.739 mg/mL/min. SDS-PAGE and azocasein zymography showed that the purified alkaline protease (2983.8 U/mg) had a molecular mass of 49.3 kDa. The enzyme was purified by column chromatography using Sephadex A50 resin. The proteolytic activity was activated by SDS (sodium dodecyl sulfate), Tween-80, Tween 20 and Triton-100. This study demonstrated that A. tamarii URM4634 protease has potent, stable and compatible collagenolytic activity to the desired level in local laundry detergent brands compared with similar enzymes produced by solid-state fermentation. This protease can thus be chosen as an option in both the food industry to tenderization meat and the detergent industry to washing process.

Characterization of a metagenomic serine metalloprotease and molecular docking studies

Abstract Functional screening of marine metagenomic library resulted in a single protease positive clone (GUSK-1) containing a 4.0 kbps insert. The DNA insert was sub-cloned using pET-22b expression vector system. Phenylmethylsulfonyl fluoride (PMSF) caused 28% inhibition of protease activity, while 60% inhibition was observed with Disodium ethylenediaminetetraacetic acid (EDTA-Na2) suggesting it to be a serine metalloprotease. The pH and temperature optima for protease activity were found to be 10 and 70 °C. Bivalent metal cations such as Mg2+, Fe2+, Mn2+, and Ca2+ enhanced the protease activity indicating their possible role either at the catalytic site or in the stabilization of the enzyme. Additionally, common organic solvents viz. isopropanol, ethanol, methanol, butanol, chloroform, and benzene also improved the protease activity. Sequence analysis of the DNA insert demonstrated an open reading frame (ORF) of 861 bps encoding 286 amino acids corresponding to a protease belonging to transpeptidase superfamily. In silico docking revealed interesting interactions of this serine metalloprotease with a gp41 protein of HIV-1 and cell adhesion protein of Listeria monocytogenes. Therefore, the novel characteristics of this protease make it a potential candidate for various biotechnological and pharmaceutical industries.

Bioanalysis of sulprostone, a prostaglandin E2 analogue and selective EP3 agonist, in monkey plasma by liquid chromatography-tandem mass spectrometry

Sulprostone is a potent prostaglandin E2 (PGE2) analogue and one of the first identified selective G-protein-coupled receptor 3 (EP3) agonists. It has been investigated as a potential antiulcer agent and frequently used in the research of EP3 antagonist. To assist pharmacokinetic and pharmacodynamic studies, a rapid and sensitive LC-MS/MS method was developed and qualified for the quantitation of sulprostone in monkey plasma. Using electrospray ionization mass spectrometry, an ammonium adduct in positive mode was chosen for analysis which had seven times of the sensitivity of the depronated ion in negative mode. Latanoprost, a prostaglandin F2α analogue, was used as the internal standard while good sensitivity and chromatography were obtained on a 2.6 μm core-shell column with pentafluorophenyl stationary phase. An assay dynamic range of 2 to 4000 ng/mL was achieved with a sample volume of 25 μL plasma on a Sciex API4000 instrument with simple protein precipitation. Several esterase inhibitors including sodium fluoride (NaF), phenylmethanesulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), paraoxon and dichlorvos as well as wet ice conditions were explored for the stabilization of sulprostone in monkey plasma. The developed method was successfully applied for the evaluation of pharmacokinetics of sulprostone after intravenous administration of 0.5 mg/kg to cynomolgus monkey.

Purification and characterization of an organic solvent-tolerant and detergent-stable lipase from Haloferax mediterranei CNCMM 50101

The present study investigates the purification and biochemical characterization of an extracellular lipase (HML) from Haloarchaea Haloferax mediterranei strain ATS1, isolated from the Sebkha (Medea, Algeria). The pure protein was obtained with ammonium sulfate precipitation (40–70%)-dialysis and UNO Q-6 FPLC, and characterized biochemically. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 45,011.09 Da. It showed optimum lipase activity at pH 7 and 60 °C. HML showed a higher specific activity on triacylglycerols with long-chains fatty acids, indicating that HML is a true lipase. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggested its belonging to the serine lipase family. The Km and Vmax for HML toward olive oil were 1.01 mM and 1195 U/mg, respectively. Compared to Lipolase, HML displayed an elevated organic solvent tolerance, an outstanding stability to surfactants, oxidizing, and auxiliary agents, a considerable compatibility with various commercialized laundry detergents, and wash performance analysis revealed that it could remove oil-stains effectively. Overall, HML has a number of attractive properties that make it a potential promising candidate for the synthesis of non-aqueous peptides and detergent formulations.

Hemolytic reactions in the hemolymph of bivalve Sinonovacula constricta show complement-like activity

The complement-like hemolysis method was used to determine the total complement-like activity of the plasma of Sinonovacula constricta. In this study, the effects of both physical and chemical conditions on complement hemolysis of S. constricta were measured. Physical conditions included proportion (S. constricta plasma: 2% rabbit red blood cells), temperature, time, and incubation, while the chemical factors consisted of Lipopolysaccharide (LPS), Flagellin (FLA), Zymosan, Peptidoglycan (PGN), Phenylmethanesulfonyl fluoride (PMSF), Methylamine, and Poly (I: C). The results showed that LPS, flagellin, Zymosan and PGN could activate complement-like activity of S. constricta plasma and cause hemolysis. PMSF and methylamine inhibited complement-like activity, resulting in the disappearance of hemolysis. Poly (I: C) had no effect on plasma complement-like activity. When the reaction temperature was less than 50 °C, hemolytic activity would increase following an increase in temperature. The ratio of plasma to rabbit blood cells had a great impact on the rate of hemolysis. Additionally, incubation with low speed oscillation could improve the hemolysis rate. It is indicated that the hemolytic reactions in the hemolymph of bivalve S. constricta show complement-like activity. The results contribute to further research on immune function of complement in bivalve.

In vitro thrombolytic activity of a purified fibrinolytic enzyme from Chlorella vulgaris

A fibrinolytic enzyme was produced by microalga Chlorella vulgaris cultivated in autotrophic and mixotrophic conditions added corn steep liquor, purified by a single chromatographic step, then biochemical characterization and in vitro thrombolytic activity was performed. Maximum cell concentration (1637.45 ± 15 mg L−1) and productivity (181.93 mg L−1 day−1) was obtained in mixotrophic culture using 1% corn steep liquor. Enzyme-extracted microalgal biomass was purified by acetone precipitation and DEAE Sephadex anion exchange chromatography up to 2 fold with recovery of 4.0%. After purification, fibrinolytic activity was 1834.6 U mg−1 and 226.86 mm2 by spectrophotometry and fibrin plate assays, respectively. SDS-PAGE results exhibited a protein band of about 45 kDa and fibrinolytic band was detected by fibrin zymography. Enzyme activity was enhanced in the presence of Fe2+ and inhibited by phenylmethane sulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA), which suggest it to be a metal-dependent serine protease. The extract also showed a red blood cell lysis <4% and in vitro thrombolytic activity of 25.6% in 90 min of reaction. These results indicate that the fibrinolytic enzyme from C. vulgaris may have potential applications in the prevention and treatment of thrombosis.

Purification, biochemical, and molecular characterization of novel protease from Bacillus licheniformis strain K7A

A novel extracellular alkaline protease, called SAPHM, from Bacillus licheniformis strain K7A was purified by four steps procedure involving heat treatment (30 min at 70 °C) followed by ammonium sulfate precipitation (40–70%)-dialysis, UNO Q-12 FPLC, and ZORBAX PSM 300 HPLC, and submitted to biochemical characterization assays. The purified enzyme is a monomer of molecular mass of 30,325.12 Da. It was completely inhibited by phenylmethanesulfonyl fluoride (PMSF)and diiodopropyl fluorophosphates (DFP), which strongly suggested its belonging to the serine protease family. Its sequence of the 26 NH2-terminal residues showed high homology with those of Bacillus proteases. The purified enzyme was optimally active at pH 10 and temperature 70 °C. Its catalytic efficiency was higher than those of Alcalase and Thermolysin. SAPHM exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively. Data suggest also that SAPHM may be considered as potential candidate for future applications in non-aqueous peptide biocatalysis because it possesses an elevated organic solvent resistance. The sapHM gene encoding SAPHM was cloned, sequenced, and expressed in Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rSAPHM) were similar to those of native one. The deduced amino acid sequence showed strong homology with other Bacillus proteases. The highest sequence identity value (97%) was obtained with APRMP1 protease from Bacillus licheniformis strain MP1, with only 9 aa of difference.

Involvement of bilitranslocase and beta-glucuronidase in the vascular protection by hydroxytyrosol and its glucuronide metabolites in oxidative stress conditions

Olive oil vascular benefits have been attributed to hydroxytyrosol (HT). However, HT biological actions are still debated because of its high metabolisation into glucuronides (GC). The aim of this study was to test HT and GC vasculoprotective effects on aorta rings from male Wistar rats. Vasorelaxant dose-responses to acetylcholine (ACh) or sodium nitroprusside (SNP) were realized in presence/absence of tert-butylhydroperoxide (t-BHP) (30 min, 500 μM). To prove this antioxidative effect, DHE staining was realized on phenol-treated aortas in oxidative stress conditions. Implication of bilitranslocase, a phenol transporter, and β-glucuronidase, implicated in deconjugation, were assessed using phenylmethanesulfonyl fluoride (PMSF, 100 μM, 20 min) and saccharolactone (SAL, 300 μM, 60 min), respectively. In addition, β-glucuronidase protein expression was assessed by western blot after using the same experimental procedures. In presence of oxidative stress, HT and GC enhanced endothelium-dependent and -independent relaxation to ACh and SNP compared to t-BHP-treated aortas. This effect was associated with a significant reduction in DHE staining for phenol-treated aortas compared to t-BHP-treated aortas. However, this protective effect was lost for GC when a washing step was introduced between phenols incubation and t-BHP stress. Bilitranslocase inhibition with PMSF reduced significantly HT but not GC effect on the vascular function upon stress induction. Moreover, GC protective effect in oxidative stress conditions was reduced by pre-incubation of aorta rings with SAL and was not associated with a reduction in β-glucuronidase protein expression. Finally, only HT was detected by high-pressure liquid chromatography in aorta rings incubated with GC and t-BHP. These results suggest that in conditions of oxidative stress, GC has to be deconjugated into HT that is transported through the cell membrane by bilitranslocase to produce its protective effect on vascular function.

Purification and characterization of Stenotrophomonas maltophilia-derived l-amino acid ester hydrolase for synthesizing dipeptide, isoleucyl-tryptophan

In the present study, we purified α-amino acid ester hydrolase (AEH) from cell-free extracts of the Stenotrophomonas maltophilia strain HS1. The approximately 70-kDa AEH from S. maltophilia HS1 (SmAEH) was homogeneous in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and was present as a tetramer in gel-filtration experiments. The activity of the SmAEH enzyme was then determined by monitoring the synthesis of the antihypertensive agent dipeptide isoleucyl-tryptophan (Ile-Trp) from isoleucyl methyl ester (Ile-OMe) and tryptophan (Trp). In these experiments, SmAEH had wide substrate specificity for acyl donors, such as Gly-OMe, β-Ala-OMe, Pro-OMe and Trp-OMe and Ile-OMe, and maximal activity were observed under conditions of pH 9.0 and 30°C. SmAEH also showed the greatest stability at pH 9.0, whereas its activity was reduced by 40% after 10-min incubation at approximately 50°C. In subsequent activity assays in the presence of various metal ions, Ag+ strongly inhibited enzyme activity. Finally, SmAEH activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), suggesting that the protein is a serine protease.

Purification and characterization of a fibrinolytic enzyme from tempeh bongkrek as an alternative of thrombolytic agents

Tempeh is one of Indonesia’s traditional foods that contain fibrinolytic enzymes. Tempeh bongkrek shows very strong activity among various tempeh. The fibrinolytic enzymes of bongkrek tempeh are obtained by steps of purification i.e, ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The fibrinolytic enzymes has been successfully purified with a yield of 4.37%, specific activity of 3,361 U / mg and purification fold of 44.02. SDS PAGE analysis showed that the enzyme was purified in to single band with estimated molecular mass of 75.82 kDa. The purified enzyme has optimum pH of 7 and optimum temperature of 50°C and pH stability between pH 4 - 7 with temperature stability from 30°-50°C. The fibrinolytic activity is increased with addition of CaCl2 but inhibited with CuSO4, phenylmethylsulfonyl fluoride (PMSF), sodium dodecyl sulfate (SDS), and ethylenediaminetetraacetic acid (EDTA).

Simultaneous determination of triamcinolone hexacetonide and triamcinolone acetonide in rabbit plasma using a highly sensitive and selective UPLC–MS/MS method

An ultra-high pressure liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was successfully developed and qualified for the simultaneous determination of triamcinolone hexacetonide (TAH) and triamcinolone acetonide (TAA, the active metabolite of TAH) in rabbit plasma. To prevent the hydrolysis of TAH to TAA ex vivo during sample collection and processing, we evaluated the effectiveness of several esterase inhibitors to stabilize TAH in plasma. Phenylmethanesulfonyl fluoride (PMSF) at 2.0 mM was chosen to stabilize TAH in rabbit plasma. The developed method is highly sensitive with a lower limit of quantitation of 10.0 pg/mL for both TAA and TAH using a 300 μL plasma aliquot. The method demonstrated good linearity, accuracy, precision, sensitivity, selectivity, recovery, matrix effects, dilution integrity, carryover, and stability. Linearity was obtained over the range of 10–2500 pg/mL. Both intra- and inter-run coefficients of variation were less than 9.1% and accuracies across the assay range were all within 100 ± 8.4%. The run time is under 5 minutes. The method was successfully implemented to support a rabbit pharmacokinetic study of TAH and TAA following a single intra-articular administration of TAH (Aristospan®).

A serine carboxypeptidase-like acyltransferase catalyzes synthesis of indole-3-acetic (IAA) ester conjugate in rice (Oryza sativa)

Indole-3-acetic acid (IAA) conjugation is one of mechanisms responsible for regulation of free auxin levels in plants. A new member of the serine carboxypeptidase-like (SCPL) acyltransferases family from Oryza sativa has been cloned and characterized. 1-O-indole-3-acetyl-β-D-glucose (1-O-IAGlc): myo-inositol acyltransferase (IAInos synthase) is an enzyme of IAA ester conjugates biosynthesis pathway that catalyzes transfer of IAA moiety from 1-O-IAGlc to myo-inositol forming IA-myo-inositol (IAInos). The OsIAA-At cDNA has been cloned and expressed using yeast and bacterial expression systems. Proteins produced in Saccharomyces cerevisiae and Escherichia coli contained 483 and 517 amino acids, respectively. The enzyme functionally expressed in both expression systems exhibits 1-O-IAGlc-dependent acyltransferase activity. Analysis of amino acid sequence confirmed that rice IAInos synthase belongs to the SCPL protein family. Recombinant IAInos synthases produced in yeast and bacterial expression systems have been partially characterized and their properties have been compared to those of the native enzyme obtained from 6-days-old rice seedlings by biochemical approach. The oligosaccharide component of the protein enzyme is not necessary for its catalytic activity. The native enzyme showed the lowest specific activity of 5.01 nmol min−1 mg−1 protein, whereas the recombinant enzymes produced in yeast and bacteria showed specific activity of 18.75 nmol min−1 mg−1 protein and 18.09 nmol min−1 mg−1 protein, respectively. The KM values for myo-inositol were similar for all three forms of the enzyme: 1.38, 0.83, 1.0 mM for native, bacterial and yeast protein, respectively. Both recombinant forms of IAInos synthase and the native enzyme also have the same optimal pH of 7.4 and all of them are inhibited by phenylmethylsulfonyl fluoride (PMSF), specific inhibitor of serine carboxypeptidases.

Presence and inter-individual variability of carboxylesterases (CES1 and CES2) in human lung

Lungs are pharmacologically active organs and the pulmonary drug metabolism is of interest for inhaled drugs design. Carboxylesterases (CESs) are enzymes catalyzing the hydrolysis of many structurally different ester, amide and carbamate chemicals, including prodrugs. For the first time, the presence, kinetics, inhibition and inter-individual variations of the major liver CES isozymes (CES1 and CES2) were investigated in cytosol and microsomes of human lungs from 20 individuals using 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD) as substrates the rates of hydrolysis (Vmax) for pNPA and 4-MUA, unlike FD, were double in microsomes than in cytosol. In these cellular fractions, the Vmax of pNPA, as CES1 marker, were much greater (30–50-fold) than those of FD, as a specific CES2 marker. Conversely, the Km values were comparable suggesting the involvement of the same enzymes. Inhibition studies revealed that the FD hydrolysis was inhibited by bis-p-nitrophenylphosphate, phenylmethanesulfonyl fluoride, and loperamide (specific for CES2), whereas the pNPA and 4-MUA hydrolysis inhibition was limited. Inhibitors selective for other esterases missed having any effect on above-mentioned activities. In cytosol and microsomes of 20 lung samples, inter-individual variations were found for the hydrolysis of pNPA (2.5–5-fold), FD or 4-MUA (8–15-fold). Similar variations were also observed in CES1 and CES2 gene expression, although determined in a small number (n = 9) of lung samples. The identification of CES1 and CES2 and their variability in human lungs are important for drug metabolism and design of prodrugs which need to be activated in this organ.

Nagarse treatment of cardiac subsarcolemmal and interfibrillar mitochondria leads to artefacts in mitochondrial protein quantification

IntroductionIn the heart, subsarcolemmal (SSM), interfibrillar (IFM) and perinuclear mitochondria represent three subtypes of mitochondria. The most commonly used protease during IFM isolation is the nagarse, however, its effect on the detection of mitochondrial proteins is still unclear. Therefore, we investigated whether nagarse treatment influences the quantification of mitochondrial proteins. MethodsSSM and IFM were isolated from hearts of mice and rats. During IFM isolation, nagarse activity was either stopped by centrifugation (common protocol, IFM+N) or inhibited by phenylmethylsulfonyl fluoride (PMSF, IFM+N+I). The amounts of proteins located in different mitochondrial compartments (outer membrane: mitofusin 1 (MFN1) and 2 (MFN2); intermembrane space: p66shc; inner membrane (connexin 43 (Cx43)), and of protein deglycase DJ-1 were determined by Western blot. ResultsMFN2 and Cx43 were found predominantly in SSM isolated from mouse and rat hearts. MFN1 and p66shc were present in similar amounts in SSM and IFM+N, whereas the level of DJ-1 was higher in IFM+N compared to SSM. In IFM+N+I samples from mice, the amount of MFN2, but not that of Cx43 increased. Nagarse or nagarse inhibition by PMSF had no effect on oxygen consumption of SSM or IFM. DiscussionWhereas the use of the common protocol indicates the localization of MFN2 predominantly in SSM, the inhibition of nagarse by PMSF increases the signal of MFN2 in IFM to that of in SSM, indicating an underestimation of MFN2 in IFM. Therefore, protease sensitivity should be considered when assessing distribution of mitochondrial proteins using nagarse-based isolation.

A calcium-stimulated serine peptidase from a true-branching cyanobacterium, Westiellopsis ramosa sp. nov.

Unbranched heterocytous cyanobacteria produce a number of serine peptidases. We have characterized several peptidases in the cell-free extracts of a true-branched N2-fixing cyanobacterium, Westiellopsis ramosa sp. nov. Upon substrate-gel zymography of intact filaments and heterocytes, five peptidase bands were resolved, whereas in vegetative cells, a single band was discernible. No band was detected in [Formula: see text]-grown cultures suggesting that the peptidases were present under diazotrophic conditions with much of them confined to heterocytes. Using salt precipitation and chromatography, a caseinolytic peptidase, called Wrp49, was purified which also demonstrated fibrinolytic activity. In SDS-PAGE, the purified peptidase was resolved into 17 and 27kDa fragments. The enzyme in its native state exhibited Mr≈49kDa, and digested gelatin in a substrate gel at a corresponding position. The enzyme showed amidolytic activity on a plasmin specific substrate, D-Val-Leu-Lys p-nitroanilide. Moreover, a trypsin specific substrate, N-benzoyl-DL-Arg p-nitroanilide was hydrolyzed at an apparent Km=0.195mM and Vmax=5×10-7 Ms-1. The enzyme was stable in a wide pH and temperature range. While Ca2+ stimulated the activity; phenylmethane sulfonyl fluoride, leupeptin, EDTA and chelants were inhibitory. The activity of the EDTA-inactivated enzyme was completely restored upon adding Ca2+, suggesting that both compounds competed with each other in modulating the enzyme activity. The enzyme showed similarities with a Ca2+ stimulated subtilisin-like serine peptidase of Anabaena variabilis ATCC 29413, but also presented several unique features of metallopeptidases, such as the chelant's response. Moreover, the N-terminal sequence (MTVENLARTGVGPGWR) did not match with any of the known peptidases.

In vitro antioxidant and antidiabetic activity of oligopeptides derived from different mulberry (Morus alba L.) cultivars

Background: Several plant peptides have been known to exhibit various pharmacological activities. However, the bioactivity of mulberry peptides is not yet reported. Objective: In the present study, the peptides from different mulberry cultivars, namely S1, V1, S1635, and Dudhiya, were isolated and assessed for their in vitro free radical scavenging capacity and inhibitory activity of two key enzymes (α-amylase [AA] and α-glucosidase [AG]) associated with type-II diabetes. Materials and Methods: The 0.5–3 kDa ranged oligopeptides were isolated using cation exchange chromatography and ultrafiltration system and further characterized by high-performance liquid chromatography and protein sequencer. The peptides were investigated for their in vitro antioxidant potential by 1,1-diphenyl-2 picrylhydrazyl, 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and nitric oxide scavenging capacity and reducing power, metal chelating, and anti-lipid peroxidation activity along with in vitro antidiabetic activity by AA and AG inhibition. Results: The results revealed that the peptides were found to possess significant free radical scavenging as well as AA and AG inhibitory activity in a dose-dependent manner. Overall, the peptide isolated from S1 cultivar exhibited the most promising therapeutic potential. Further, the variation in amino acid composition of the oligopeptides could be associated with the observed variation in their bioactivity. Conclusion: These natural peptides may constitute an important part of the antioxidant defense system as well as antidiabetic agent and further could be used for the formulation of functional food and nutraceuticals. Abbreviation Used: HPLC: High-performance liquid chromatography, PMSF: Phenyl methyl sulfonyl fluoride; IAA: Indoleacetic acid, ABA: Abscisic acid, GA3: Gibberellic acid, DPPH: 1,1-diphenyl-2 picrylhydrazyl, ABTS: 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, AG: α-glucosidase, AA: α-amylase, DNSA: 3,5-Dinitrosalicylic acid, AAE: Ascorbic acid equivalent, TBARS: Thiobarbituric acid-reactive species.

Anticoagulant, antiplatelet and fibrin clot hydrolyzing activities of flax seed buffer extract

Background: Flax seeds possess long array of medicinal qualities as it found to show beneficial effects on cardiovascular, hypertension, diabetes, cancer, inflammatory and autoimmune disorders. While, the therapeutic role of flax seed proteins on thrombotic disorder is least explored. Objective: The current study aims to identify the fibrin clot-dissolving, anticoagulant and antiplatelet properties of flaxseed buffer extract (FSBE) using Platelet Rich Plasma (PRP) and Platelet Poor Plasma (PPP). Materials and Methods: Flax Seed Buffer Extract (FSBE) proteins were characterized upon subjected to SDS-PAGE. The presence of cysteine protease in the extract was identified using colorimeter and zymography experiments. Anticoagulant activity was established using invitro recalcification time. While, the fibrinogenolytic, fibrinolytic and human plasma proteins degradation activities were proved using SDS-PAGE. Furthermore, antiplatelet activity was confirmed using Chronology dual channel whole blood/optical lumi aggregation system (Model-700). Toxicity studies were assayed using RBC cells and albino mice. Results: FSBE showed divergent protein banding pattern from the molecular mass ranging 200 kDa-14 kDa under both reduced and non-reduced conditions. The FSBE showed proteolytic activity as it hydrolyzed casein and gelatin with the specific activity 0.180 and 0.201 units/mg/min respectively. The proteolytic activity of FSBE was completely abolished only by IAA but PMSF, 1, 10 Phenanthroline and EDTA did not, revealing the presence of cysteine protease in the extract. FSBE showed anticoagulant activity as it enhanced the clotting time from control 220s to 320s. Furthermore, FSBE showed antiplatelet activity by inhibiting ADP and Epinephrine induced platelet aggregation and the observed aggregation inhibition was found to be 63.6% and 16.3% respectively. In addition, it hydrolyzed specifically Aα and Bβ chains of human fibrinogen and all the chains of human fibrin clot. The fibrinogenolytic activity was inhibited by IAA but PMSF, EDTA, 1, 10 Phenanthroline did not, suggesting the role of cysteine proteases. Interestingly, FSBE did not cause edema and hemorrhage in the experimental mice and did not hydrolyze RBC cells suggesting its nontoxic properties. Abbreviations used: FSBE: Flaxseed buffer extract; PMSF: Phenylmethylsulfonyl fluoride; IAA: Iodoacetic acid; EDTA: Ethylenediaminetetraacetic acid; PRP: Platelet-rich plasma; PPP: Platelet-poor plasma; INR: International normalized ratio; MED: Minimum edema dose; MHD: Minimum hemorrhagic dose; APTT: Activated partial thromboplastin time; PT: Prothrombin time; HMWK: High-molecular-weight kininogen.

Evidence for the Involvement of COX-2/VEGF and PTEN/Pl3K/AKT Pathway the Mechanism of Oroxin B Treated Liver Cancer.

Background:Oroxin B (OB) is one of flavonoids isolated from traditional Chinese herbal medicine Oroxylum indicum (L.) Vent. Recent studies suggest that flavonoids have obvious anti-liver tumors effect, but the precise molecular mechanism is still unclear.Objective:The current study was performed to investigate the antitumor effects of OB on human hepatoma cell line SMMC-772 and explore the part of molecular mechanisms in this process.Materials and Methods:MTT method, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and flow cytometry were utilized to detect the inhibition of proliferation and the apoptosis after treating OB in of SMMC-7721 cells. The mRNA and proteins expressions of COX-2, vascular endothelial growth factor (VEGF), phosphatidylinositol-3-kinase (PI3K), p-AKT, and PTEN were measured by a real-time polymerase chain reaction and Western Blot method.Results:The results showed that OB inhibited proliferation of SMMC-7721 cell in a dose-dependent manner, and induced its apoptosis. Moreover, OB unregulated PTEN and downregulated COX-2, VEGF, p-AKT, and PI3K.Conclusion:Our results demonstrated that OB significantly inhibits proliferation and induce apoptosis, which may be strongly associated with the inhibiting COX-2/VEGF and PTEN/PI3K/AKT pathway signaling pathway in SMMC-7721 cells, OB potentially be used as a novel therapeutic agent for liver cancer.SUMMARY OB (Oroxin B) is one of the effective flavonoid components of traditional Chinese medicine O. indicum (L.)OB can inhibite the proliferation and promoted apoptosis of the human hepatoma cell line SMMC 7721OB plays a role of antitumor effect may to regulate COX 2/VEGF and PTEN/PI3K/AKT pathways directly or indirectly. Abbreviations used: OB: Oroxin B; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; COX-2: cyclooxygenase-2; PI3K: phosphatidylinositol 3 kinase; PTEN: Phosphatase and tensin homolog deleted on chromosome ten; VEGF: Vascular endothelial growth factor; RT-PCR: Reverse transcription polymerase chain reaction; DAPI: Diamidino 2 phenylindole; PBS: Phosphate buffer saline; FITC: Fluorescein isothiocyanate; PI: Propidium Iodide; RIPA: Radio immunoprecipitation assay lysis buffer; PMSF: Phenylmethanesulfonyl fluoride; PAGE: Polyacrylamide gel electrophoresis.