Phenylmethylsulfonyl Fluoride Research Articles

Capsaicin reverses the inhibitory effect of licochalcone A/β-Arbutin on tyrosinase expression in b16 mouse melanoma cells.

Introduction:Melanin is synthesized by melanocytes, which are located in the basal layer of the skin. After synthesis, melanin is further deposited on the surface of the skin to form black spots or chloasma. Tyrosinase is a rate-limiting enzyme that plays an important role in melanogenesis. Currently, there are many drugs that inhibit tyrosinase expression to further reduce melanogenesis. Nevertheless, some of these could reverse the pharmacological effect of other drugs, when used simultaneously.Materials and Methods:B16 mouse melanoma cells were treated with the tyrosinase inhibitors licochalcone A and β-arbutin, alone or in combination with capsaicin, an alkaloid found in peppers. Cytotoxicity, melanin content, and tyrosinase activity and expression were determined.Results:Licochalcone A/β-arbutin inhibited tyrosinase expression and further hindered melanin synthesis when applied individually to B16 mouse melanoma cells. However, licochalcone A/β-arbutin combined with 50 μmol/L capsaicin enhanced the expression of tyrosinase in these cells and further increased melanin content.Conclusion:Our data implied that capsaicin could reverse the inhibitory effect of licochalcone A/β-arbutin on tyrosinase expression in B16 mouse melanoma cells.SUMMARY B16 mouse melanoma cells were treated with the tyrosinase inhibitors licochalcone A and β-arbutin, alone or in combination with capsaicin, an alkaloid found in peppers. Cytotoxicity, melanin content, and tyrosinase activity and expression were determined. Licochalcone A/β-arbutin inhibited tyrosinase expression and further hindered melanin synthesis when applied individually to B16 mouse melanoma cells. However, licochalcone A/β-arbutin combined with 50 μmol/L capsaicin enhanced the expression of tyrosinase in these cells and further increased melanin content. Our research implied that capsaicin could reverse the inhibitory effect of licochalcone A/β-arbutin on tyrosinase expression in B16 mouse melanoma cells. Abbreviations used: B16: B16 mouse melanoma cells; L-DOPA: 3, 4-L-dihydroxyphenylalanine; TYR: Tyrosinase; USP: United States Pharmacopeia; FBS: Fetal bovine serum; EDTA: Ethylenediaminetetraacetic acid; DMSO: Dimethyl sulfoxide; RPMI: Roswell Park Memorial Institute; MTT3: 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, NaOH: Sodium hydroxide; PBS: Phosphate-buffered saline; RIPA: Radio-immunoprecipitation assay; PMSF: Phenylmethanesulfonyl fluoride or phenylmethylsulfonyl fluoride; SDS: Sodium dodecyl sulfate, sodium salt; PVDF: Polyvinylidene fluoride; ECL: Enhanced chemiluminescence.

An aspartic-metalloprotease from an endemic plant tuber (Burnatia enneandra micheli): Purification and biochemical characterization

Background: The objective of this work was to isolate, optimize, and characterize protease from Burnatia enneandra which is an endemic plant found abundantly in the Far-Nord Region of Cameroon. The optimum condition to extract maximum quantity of protease from B. enneandra with respect to pH, the ratio (m/v), and agitation frequency was defined as 5.1%, 4%, and 100 rpm, respectively. Materials and Methods: The enzyme was purified using ammonium sulphate precipitation, double gel filtration chromatography sephadex G200 followed by sephadex G75 and the purified protease was further characterized. With an apparent molecular weight of 23 kDa on SDS-PAGE, the purified protease showed maximum activity at 5.1 and 40°C respectively for pH and temperature. Its activity was enhanced by metal ions such as Ca2+ and Ni2+, while Fe2+and Zn2+ showed significant inhibition. Results: B. enneandra protease activity was not affected by proteases inhibitors such as phenylmethylsulfonyl fluoride, aprotinin, and iodoacetamide but was strongly inhibited by Pepstatin A and ethylenediaminetetraacetic acid which allowed to classify this new protease as aspartic-metalloproteases. Using casein as substrate, protease from B. enneandra had a maximum rate of reaction (Vmax) and Michaelis-Menten constant (Km) of 64.935 (U/mL) and 373.941 (μg/mL), respectively. Abbreviations used: CCD: Central composite design; Km: Michaelis–Menten constant; Vmax: Maximum Velocity; PBD: Plackett–Burman design; PMSF: Phenylmethylsulfonyl floride; AAD: Absolute Average Deviation; AF: Accuracy Factor; BSA : Bovine serum albumin; BF: Bias factor; EDTA: Ethylene diamine tetraacetic acid; RSM: Response surface methodology; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis.

A novel detergent additive: Organic solvent- and thermo-alkaline-stable recombinant subtilisin

In the present work, subtilisin gene from Bacillus subtilis PTTC 1023 was synthesized, cloned into the vector pD441-NH and expressed in E. coli BL21 (DE3). Recombinant subtilisin was purified in a single-step procedure by affinity chromatography. The molecular mass of the purified protein was determined to be about 40kDa by SDS-PAGE. The optimum pH and temperature values of its proteolytic activity were 10.5 and 50°C, respectively and retained more than 70% and 89% of its activity in pH range of 7–12 and 30–60°C, respectively. Enzyme purity was estimated to be about 200- fold greater than that of the crude extract and subtilisin had a specific activity of 56.16U/mg, with a yield of about 87.9%. It was completely inhibited by phenylmethanesulfonyl fluoride, which strongly suggests its belonging to serine protease family. Interestingly, subtilisin protease displayed a significant compatibility with commercial detergents, and tolerance organic solvents, metallic ions and surfactants. The findings obtained demonstrated that protease of B. subtilis could potentially be used in future applications as an additive in detergent formulations.

Induction of proteases secreted by fusarium oxysporum F. SP. Dianthi in the presence of carnation root cell walls. Biochemical characterization of a serine protease

We determined that protease activity is secreted by Fusarium oxysporum f. sp. dianthi race 2 (Fod) in vitro. The cell wall extracted from resistant and susceptible cultivars of carnation (Dianthus caryophyllus L.) was used as an inducer. The activity increased in the presence of the two cultivars, and higher levels were detected with the susceptible cultivar. One of the three proteases secreted by Fod was purified using ion exchange (DEAE-cellulose), gel filtration (Sephadex G-75), and affinity chromatographies (Benzamidine Sepharose 6B). Using protease inhibitors, the purified enzyme was classified as a trypsin-like serine protease that was inhibited by phenylmethanesulfonyl fluoride, diisopropylfluorophosphate and the soybean trypsin inhibitor. In addition, the enzyme was biochemically characterized and determined to have an optimal pH of 8.0, an optimal temperature of 50°C, stability from pH 6 to 8, temperature stability between 40 to 60°C, a molecular weight of 54 kDa, and an isoelectric point of 4.4. The values of the Km and Vmax kinetic constants were found to be 0.31 mg/ml and 24.7 µmol/min, respectively.

Characterization of a robust serine protease from Bacillus subtilis K-1.

Process apt microbial proteases due to their wide range industrial applications have become the focus of intense scientific research during recent years. Considering the hostile process milieu, the proteases intended for application must be robust enough to withstand the extremes of temperature and pH, and presence of organic solvents and other potential enzyme inhibitors. Current study presents the characterization of a robust protease from a previously isolated bacterium Bacillus subtilis K-1 (BSK-1). Purification of BSK-1 protease (5.21-fold) was achieved to homogeneity by salt (ammonium sulfate) precipitation, and ion-exchange (diethyl-aminoethyl-sephadex) and size exclusion chromatography (Sephadex G-100). Molecular weight of BSK-1 protease was determined by SDS-PAGE analysis (42 kDa). Though the optimum temperature and pH for BSK-1 protease activity was 50 °C and 10, respectively, but, the protease exhibited remarkable activity and stability over elevated temperatures (60-80 °C) and a broad pH range (pH 7-11). Protease showed resistance towards several organic solvents/other potential enzyme inhibitors. Drastic activity loss in presence of phenylmethylsulfonyl fluoride indicated that the enzyme is a serine protease. Kinetic parameters (Km and Vmax ) for BSK-1 protease were found to be 0.14 mg ml-1 and 1176 mg min-1 , respectively. Putative amino acid sequence of BSK-1 protease (derived from nucleotide sequence of protease gene) suggested that the enzyme belonged to peptidases S8/S53 super family with multidomain of S8. BSK-1 protease being stable under harsh conditions may serve a model system for understanding the molecular basis of stability, and may help designing novel proteases that are suitable for industrial applications.

Cloning and characterization of a novel intracellular serine protease (IspK) from Bacillus megaterium with a potential additive for detergents

A new intracellular serine protease gene of Bacillus megaterium, ispK, encoding a protein composed of 332 amino acid residues with a predicted pI of 4.7 was cloned into Escherichia coli. The deduced amino acid sequence of IspK showed 49–56% similarity with the other microbial intracellular serine proteases described in the literature. The enzyme was effectively purified by one-step chromatography after heat-treatment, and showed a homogeneous band corresponding to 35kDa by SDS-PAGE analysis. Amino acid analysis showed that 16 amino acids of the N-terminus of IspK were removed by post-translational protease activity. The optimum pH and temperature of IspK were 6.0–7.0 and 50°C, respectively. In the presence of 2mM of Ca2+ ion, the optimum temperature was increased to 65°C and thermostability (t1/2) increased 32.9-fold from 3.3min to 108.5min at 60°C. The enzyme was activated by Ca2+ and Mg2+, almost completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and EDTA, but tolerant to nonionic surfactants, such as, Triton X-100 or Tween 80. IspK efficiently hydrolyzed natural proteins, such as, casein and hemoglobin, and improved blood stain removal. These results suggest IspK can be used as a useful additive for detergent formulations and for deproteinizations.

Extraction, purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

ABSTRACTA protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ∼ 28 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co2+, Ni2+, Hg2+, and Zn2+ while its activity has increased in the presence of Ca2+ and Mg2+. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its Vmax and Km determined using casein as a substrate were 94.34 U/mL and 349.07 µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.

Expression, purification and enzymatic properties of a β-N-acetylhexosaminidase from brown planthopper, Nilaparvata lugens

A previous study demonstrated that a β-N-acetyl-d-hexosaminidase (NlHex4) from the brown planthopper could serve as a new potential target for biopesticides due to its requirement for molting, and lethal phenotypes were observed via RNA interference. However, the enzymatic properties of NlHex4 remain unclear. In the present study, NlHex4 was expressed, purified and characterized. Recombinant NlHex4 was functionally expressed in baculovirus-infected insect cells and purified with a molecular weight of approximately 70kDa. Recombinant NlHex4 had an optimal pH and temperature of 6.0 and 60°C, respectively. Moreover, recombinant NlHex4 exhibited thermal stability at 50°C for at least 1h. Furthermore, the activity of recombinant NlHex4 was inhibited by the metal ions Fe2+ and Fe3+ and the serine protease inhibitor phenylmethanesulfonyl fluoride. These results reveal the enzymatic properties of NlHex4 and provide a basis for future investigation of the interaction between HEX and chitin oligosaccharide.

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 it is extensively metabolized into glucuronides (GCs). The aim of this study was to test HT and GC vasculoprotective effects and the underlying mechanisms using aorta rings from 8-week-old male Wistar rats. In the absence of oxidative stress, incubation with 100 μM HT or GC for 5 min did not exert any vasorelaxing effect and did not influence the vascular function. Conversely, in condition of oxidative stress [upon incubation with 500 μM tert-butylhydroperoxide (t-BHP) for 30 min], preincubation with HT or GC improved acetylcholine-induced vasorelaxation compared with untreated samples (no t-BHP). This protective effect was lost for GC, but not for HT, when a washing step (15 min) was introduced between preincubation with HT or GC and t-BHP addition, suggesting that only HT enters the cells. In agreement, bilitranslocase inhibition with 100 μM phenylmethanesulfonyl fluoride for 20 min reduced significantly HT, but not GC, effect on the vascular function upon stress induction. Moreover, GC protective effect (improvement of endothelium-dependent relaxation in response to acetylcholine) in oxidative stress conditions was reduced by preincubation of aorta rings with 300 μM D-saccharolactone to inhibit β-glucuronidase, which can deconjugate polyphenols. 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 can be deconjugated into HT that is transported through the cell membrane by bilitranslocase to protect vascular function.

Lipase Activity in the Larval Midgut of Rhynchophorus palmarum: Biochemical Characterization and the Effects of Reducing Agents.

Lipases have key roles in insect lipid acquisition, storage, and mobilization and are also fundamental to many physiological processes in insects. Lipids are an important component of insect diets, where they are hydrolyzed in the midgut lumen, absorbed, and used for the synthesis of complex lipids. The South American palm weevil Rhynchophorus palmarum is one of the most important pests on commercial palm plantations. However, there are few studies about lipid digestion for this insect. In this work, we have described the biochemical characterization of the lipase activity in the posterior midgut of the R. palmarum palm weevil. Lipase activity was highest between the temperatures of 37 °C and 45 °C and at pH 6.5. Lipase activity was also sensitive to variations in salt and calcium concentrations. Lipases have been described structurally as enzymes with the Ser-His-Asp Catalytic Triad, containing an active serine. The serine protease inhibitor PMSF (phenylmethane sulfonyl fluoride) inhibited the lipases from R. palmarum, demonstrating the importance of a serine residue for this activity. The ability of the lipases to hydrolyze p-Nitrophenyl esters with different chain lengths has revealed the activities of a broad range of substrates. The lipase activities of R. palmarum increased in the presence of reduced glutathione (GSH) and dithiothreitol (DTT), while in the presence of oxidized glutathione (GSSG), activities were drastically reduced. To our knowledge, this study has provided the first information about lipase activity in the R. palmarum palm weevil.

Effects of arachidonyl-2'-chloroethylamide (ACEA) on the protective action of various antiepileptic drugs in the 6-Hz corneal stimulation model in mice.

Accumulating evidence indicates that cannabinoid CB1 receptor ligands play a pivotal role in seizures, not only in preclinical studies on animals, but also in clinical settings. This study was aimed at characterizing the influence of arachidonyl-2′-chloroethylamide (ACEA–a selective cannabinoid CB1 receptor agonist) co-administered with phenylmethylsulfonyl fluoride (PMSF) on the anticonvulsant potency of various antiepileptic drugs (clobazam, lacosamide, levetiracetam, phenobarbital, tiagabine and valproate) in the 6-Hz corneal stimulation model. Psychomotor seizures in male albino Swiss mice were evoked by a current (32 mA, 6 Hz, 3 s stimulus duration) delivered via corneal electrodes. Potential adverse effects produced by the antiepileptic drugs in combination with ACEA+PMSF were assessed using the chimney test (motor performance), passive avoidance task (remembering and acquisition of learning), and grip-strength test (muscular strength). Brain concentrations of antiepileptic drugs were measured by HPLC to exclude any pharmacokinetic contribution to the observed effect. ACEA (5 mg/kg, i.p.) + PMSF (30 mg/kg, i.p.) significantly potentiated the anticonvulsant potency of levetiracetam (P<0.05), but not that of clobazam, lacosamide, phenobarbital, tiagabine or valproate in the 6-Hz corneal stimulation model. Moreover, ACEA+PMSF did not significantly affect total brain concentrations of levetiracetam in mice. No behavioral side effects were observed in animals receiving combinations of the studied antiepileptic drugs with ACEA+PMSF. In conclusion, the combined administration of ACEA+PMSF with levetiracetam is associated with beneficial anticonvulsant pharmacodynamic interaction in the 6-Hz corneal stimulation model. The selective activation of cannabinoid CB1 receptor-mediated neurotransmission in the brain may enhance levetiracetam-related suppression of seizures in epilepsy patients, contributing to the efficacious treatment of epilepsy in future.

Excretory/secretory proteases and mechanical movement of Anisakis pegreffii infective larvae in the penetration of BALB/c mice gastrointestine

Anisakiasis is a human parasitic disease caused by infection with the infective larvae of Anisakis. Accidental infection in humans causes the gastrointestinal pathophysiological effects of mechanical tissue damage by migrating larvae. The mechanism of the infective larval invasion and migration is suspected to involve larval excretory/secretory proteases and motility. This study demonstrates the penetration rate of the infective larvae of Anisakis pegreffii in mouse gastrointestine depends on the time after infection, and that only 15% of larvae remain in the gastrointestinal tract 3 h after infection. Strong activities of matrix metalloproteinases (MMPs) and serine proteases, especially plasmin, were found in the excretory/secretory products of A. pegreffii; these can be inhibited by ONO-4817 and phenylmethylsulfonyl fluoride, respectively. The protease activity was also significantly decreased in another 1 h of cultivation of larvae in fresh 0.9% normal saline (NS) after previous cultivation for 48 h in NS. The motility scores of larvae were significantly lower after 48 h of cultivation in NS. The penetration rate of A. pegreffii larvae in the gastrointestine of infected mice sequentially were 90% in the freshly prepared, 68% in serine protease inhibited, 55% in MMPs inhibited larvae, and 16% in larvae cultivated in NS for 48 h. Therefore, this study demonstrates that MMPs and serine proteases excreted and secreted by A. pegreffii and the mechanical movement of infective larvae participate in the penetration of the gastrointestine of mice after infection.

Biochemical characterization of purified lipoxygenase from sesame (Sesamum indicum)

ABSTRACTA 66.3 kDa lipoxygenase (LOX) with optimum pH of 8.0 and temperature of 35 °C was purified from Sesamum indicum. Fe2+and Ca2+ could stimulate the enzyme activity by 160 and 141%, respectively, while Co2+ highly reduced it to 20%. The LOX activity was decreased at 1.5 mM of cetyltrimethylammonium bromide (CTAB) and β-mercapto ethanol, and 2.5 mM of phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA). The enzyme showed Km = 1.01 μM and Vmax = 0.054 μmol.min−1 toward linoleic acid as the substrate. The IC50 values for LOX inhibitors including potassium cyanide (KCN) (270 µM), β-carotene (48 µM), and ascorbic acid (132 µM) were determined.

In vitro characterization of jellyfish venom fibrin(ogen)olytic enzymes from Nemopilema nomurai

BackgroundBecause jellyfish are capable of provoking envenomation in humans, they are considered hazardous organisms. Although the effects of their toxins are a matter of concern, information on the venom components, biological activity and pathological mechanisms are still scarce. Therefore, the aim of the present study was to investigate a serine protease component of Nemopilema nomurai jellyfish venom (NnV) and unveil its characteristics.MethodsTo determine the relationship between fibrinolytic activity of NnV and the serine protease, fibrin zymography was performed using metalloprotease and serine protease inhibitors. The biochemical characterization of serine proteases of NnV were determined by the amidolytic assay. Fractions with fibrinolytic activity were obtained by DEAE cation exchange column.ResultsNnV displayed fibrinolytic activities with molecular masses of approximately 70, 35, 30, and 28 kDa. The fibrinolytic activity of NnV was completely obliterated by phenylmethylsulfonyl fluoride, a prototype serine protease inhibitor. Based on amidolytic assays using chromogenic substrates specific for various kinds of serine proteases, NnV predominantly manifested a chymotrypsin-like feature. Its activity was completely eliminated at low pH (< 6) and high temperatures (> 37 °C). Some metal ions (Co2+, Cu2+, Zn2+ and Ni2+) strongly suppressed its fibrinolytic activity, while others (Ca2+ and Mg2+) failed to do so. Isolation of a serine protease with fibrionolytic activity from NnV revealed that only p3 showed the fibrinolytic activity, which was completely inhibited by PMSF.ConclusionThe present study showed that N. nomurai jellyfish venom has a chymotrypsin-like serine protease with fibrinolytic activity. Such information might be useful for developing clinical management of jellyfish envenomation and pharmacological agents with therapeutic potential for thrombotic diseases in the future.

Proteolytic activities in cortex of apical parts of Vicia faba ssp. minor seedling roots during kinetin-induced programmed cell death.

Programmed cell death (PCD) is a crucial process in plant development. In this paper, proteolytically related aspects of kinetin-induced PCD in cortex cells of Vicia faba ssp. minor seedlings were examined using morphological, fluorometric, spectrophotometric, and fluorescence microscopic analyses. Cell viability estimation after 46μM kinetin treatment of seedling roots showed that the number of dying cortex cells increased with treatment duration, reaching maximum after 72h. Weight of the apical root segments increased with time and was about 2.5-fold greater after 96h, while the protein content remained unchanged, compared to the control. The total and cysteine-dependent proteolytic activities fluctuated during 1-96-h treatment, which was not accompanied by the changes in the protein amount, indicating that the absolute protein amounts decreased during kinetin-induced PCD. N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), and Z-Leu-Leu-Nva-H (MG115), the respective cysteine, serine, and proteasome inhibitors, suppressed kinetin-induced PCD. PMSF significantly decreased serine-dependent proteolytic activities without changing the amount of proteins, unlike NEM and MG115. More pronounced effect of PMSF over NEM indicated that in the root apical segments, the most important proteolytic activity during kinetin-induced PCD was that of serine proteases, while that of cysteine proteases may be important for protein degradation in the last phase of the process. Both NEM and PMSF inhibited apoptotic-like structure formation during kinetin-induced PCD. The level of caspase-3-like activity of β1 proteasome subunit increased after kinetin treatment. Addition of proteasome inhibitor MG-115 reduced the number of dying cells, suggesting that proteasomes might play an important role during kinetin-induced PCD.

Biochemical properties of a serine protease from Aspergillus flavus and application in dehairing

The proteases are enzymes produced by several filamentous fungi with important biotechnological applications. In this work, a protease from Aspergillus flavus was characterized. The culture filtrate of A. flavus was purified to homogeneity by Sephacryl S-200 column chromatography followed by CM–cellulose. The molecular weight of the purified enzyme was estimated to be approximately 32 kDa by SDS–PAGE. The enzyme hydrolysed BTpNA (N-α-benzoyl-dl-tyrosyl-p-nitroanilide), azo-casein and casein as substrates. Optimal temperature and pH were 55 °C and 6.5, respectively. The enzyme was stimulated by Mg2+, Ca2+, Zn2+ and inhibited by Hg2+ and Ag2+ and Cu2+. The protease showed increased activity with detergents, such as Tween 80 and Triton X, and was stable to the reducing agents, such as β-mercaptoethanol. The protease activity was strongly inhibited in the presence of phenylmethylsulfonyl fluoride, indicating it is a serine protease. The enzyme entrapped in calcium alginate beads retained its activity for longer time and could be reused up to 10 times. The thermostability was increased after the immobilization and the enzyme retained 100% of activity at 45 °C after 60 min of incubation, and 90% of residual activity at 50 °C after 30 min. In contrast, the free enzyme only retained 10% of its residual activity after 60 min at 50 °C. The enzymatic preparation was demonstrated to be efficient in the capability of dehairing without destruction of the hide. The remarkable properties such as temperature, pH and immobilization stability found with this enzyme assure that it could be a potential candidate for industrial applications.

A Long-Term Treatment with Arachidonyl-2'-Chloroethylamide Combined with Valproate Increases Neurogenesis in a Mouse Pilocarpine Model of Epilepsy.

Rational polytherapy in the treatment of refractory epilepsy has been the main therapeutic modality for several years. In treatment with two or more antiepileptic drugs (AEDs), it is of particular importance that AEDs be selected based on their high anticonvulsant properties, minimal side effects, and impact on the formation of new neurons. The aim of the study was to conduct an in vivo evaluation of the relationship between treatments with synthetic cannabinoid arachidonyl-2′-chloroethylamide (ACEA) alone or in combination with valproic acid (VPA) and hippocampal neurogenesis in a mouse pilocarpine model of epilepsy. All studies were performed on adolescent male CB57/BL mice with using the following drugs: VPA (10 mg/kg), ACEA (10 mg/kg), phenylmethylsulfonyl fluoride (PMSF—a substance protecting ACEA against degradation by fatty acid hydrolase, 30 mg/kg), pilocarpine (PILO, a single dose of 290 mg/kg) and methylscopolamine (30 min before PILO to stop peripheral cholinergic effects of pilocarpine, 1 mg/kg). We evaluated the process of neurogenesis after a 10-day treatment with ACEA and VPA, alone and in combination. We observed a decrease of neurogenesis in the PILO control group as compared to the healthy control mice. Furthermore, ACEA + PMSF alone and in combination with VPA significantly increased neurogenesis compared to the PILO control group. In contrast, VPA 10-day treatment had no impact on the level of neurons in comparison to the PILO control group. The combination of ACEA, PMSF and VPA considerably stimulated the process of creating new cells, particularly neurons, while chronic administration of VPA itself had no influence on neurogenesis in the mouse pilocarpine model of epilepsy. The obtained results enabled an in vivo evaluation of neurogenesis after treatment with antiepileptic drugs in an experimental model of epilepsy.

Hemorrhagic metalloproteinase, Cc HSM‐III, isolated from Cerastes cerastes venom: Purification and biochemical characterization

Snake venom metalloproteinases are the most abundant toxins in Viperidae venoms. In this study, a new hemorrhagin, Cc HSM-III (66 kDa), was purified from Cerastes cerastes venom by gel filtration, ion exchange, and reversed-phase high-performance liquid chromatographies. The analysis of Cc HSM-III by liquid chromatography with a tandem mass spectrometry revealed 32 peptides sharing a homology with P-III metalloproteinases from Echis ocellatus snake venom. Cc HSM-III displays hemorrhagic activity with a minimal hemorrhagic dose of 5 μg, which is abolished by ethylene diamine tetracetic acid but not by phenylmethylsulfonyl fluoride. The mechanism underlying Cc HSM-III hemorrhagic activity is probably due to its extensive proteolytic activity against type IV collagen. Cc HSM-III induces local tissue damage and an inflammatory response by upregulating both matrix metalloproteinase 2 and 9 in skin of mice. Thus, Cc HSM-III may play a key role in the pathogenesis of C. cerastes envenomation.

Partial Purification of Alkaline Protease as Thrombolytic Agent from Mutant Strain Bacillus licheniformis EMS250-O-1

Thrombosis leads to myocardial infarction, stroke and other cardiovascular complications. Microbial thrombolytic agents such as urokinase, streptokinase etc. are used to treat complications related to thrombosis. To search for new microbial enzymes as thrombolytics having better efficacy and specificity, Bacillus licheniformis EMS-O-1 mutant strain was cultured in modified urea-molasses media followed by purification using ammonium sulphate precipitation and ultrafiltration through centricon tube of 100 MWCO value. The yield of crude enzyme was 11129.14 U/mg and after purification 40180.46 U/mg. Purification process increased the specific activity of purified enzyme to 12.28 fold with a recovery of 17.79%. The purified enzyme was a serine protease with molecular weight of 25.5 kDa as confirmed by irreversible inhibition of activity with phenylmethylsulfonyl fluoride (PMSF) followed by SDS-PAGE gel image and by LC-MS analyses. In vitro clot lysis assay of the purified enzyme exhibited 38.30% thrombolytic activity. The crude enzymes from the mutant strain EMS-O-1 were found to be stable up to 50oC and showed maximum stability between pH range 7.5 to 8.5. These findings signify that proteases produced by B. licheniformis mutant have the potential to be developed as a viable thrombolytic agent.Dhaka Univ. J. Pharm. Sci. 15(2): 135-141, 2016 (December)

Characterization of the Crude Alkaline Extracellular Protease of Yarrowia lipolytica YlTun15

Yarrowia lipolytica YlTun15 (GeneBank Acc. N° MF327143), isolated from farmed Dicentrarchus labrax’s gills, secretes an alkaline extracellular protease, which exhibited the highest activity at pH 9 and temperature 45°C. The enzyme activity extracted was tested in the presence of different ion metal and protein inhibitors. The enzyme activity increased in the presence of both Cu2+ (1 mM) and Mn2+ (5mM) in the medium. K+, Na2+, Mg2+ and Ca2+ had no effect on the enzyme activity while Ni+, Hg+, Zn2+ and Fe2+ decreased significantly its relative activity to 43.63%, 66.25%, 30.75% and 19.48% respectively at the 5 mM level. The enzyme was almost (activity=1.47%) inhibited by phenylmethylsulfonyl fluoride at the concentration of 5 mM. However, the protease activity was relatively constant in the presence of EDTA and SDS that may conclude that this enzyme was not a metalloprotease and belong to the serine protease category. After 18 months-storage at -20°C, the enzyme activity has decreased to 23.17%. This protease may have a potential application in food and detergent activity.