Volume 43, Issue 6

Aim This study was evaluated the effects of N-acetylcysteine (NAC) and calcium hydroxide (Ca(OH) 2 ) on the expression levels of matrix metalloproteinase -2, -9 (MMP-2, -9) and tissue inhibitor metalloproteinase -1, -2 (TIMP-1, -2) in lipopolysaccharide (LPS)-stimulated human macrophages. Methods Human monocyte precursor cells (THP-1) were differentiated into macrophage-adherent cells and were stimulated with LPS for 24 h. Then individually incubated with NAC or Ca(OH) 2 for 24, 48 and 72 h. Following incubation, protein expression and mRNA levels of MMP-2, -9 and TIMP-1, -2 were evaluated using enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Data were statistically analysed using two-way ANOVA, to followed by Bonferroni test at α=0.05. Results NAC significantly decreased mRNA expression and protein levels of MMP-9, while Ca(OH) 2 decreased mRNA expression alone at 24 h. NAC and Ca(OH) 2 decreased mRNA expression of MMP-2 at 24 h, while NAC increased this expression at 48 h. Although NAC and Ca(OH) 2 decreased the mRNA expression of TIMP-1, -2 at 24 h, only NAC increased mRNA expression of TIMP-1 at 48 h. Conclusion At the early stages of inflammation, NAC and Ca(OH) 2 have anti-inflammatory effects on macrophages.

Objective The aim of this study was to synthesize ten 1,4-dihydropyridine (DHP) derivatives in which substituted cyclohexane rings were fused to the DHP ring and to determine how different ester groups and the benzoyl substituent introduced in 4-phenyl ring affected their calcium channel blocking activity. Methods A microwave-assisted one-pot method was applied for the synthesis of compound 1–5 according to a modified Hantzsch reaction. The benzoyl moiety was introduced in the 4-phenyl ring of these dihydropyridines by refluxing with benzoyl chloride in acetone in the presence of anhydrous potassium carbonate. Synthesized products were characterized by elemental analysis, IR, 1 H-NMR and 13 C-NMR spectroscopy. The inhibitory actions of compounds 1–10 on calcium channel blocking activity were tested on isolated rat aorta preparations. Results The obtained pharmacological results showed that although all compounds are potent relaxing agents on isolated rat aorta smooth muscle, introduction of a benzoyloxy substitiuent on the phenyl ring (compound 6–10 ) decreased the relaxant effect of these compunds. Conclusion The reported 1,4-DHP derivatives have calcium channel blocking activity on rat aorta smooth muscle.

Background Mucor hiemalis is a dimorphic fungus that efficiently produces ethanol from different sugars; however, the yield of ethanol production highly depends on the fermentation conditions. Objective The conditions for obtaining a high ethanol production yield were optimized in this study. Materials and methods A response surface methodology was used to optimize pH, temperature, and time of ethanolic fermentation by M. hiemalis . Additionally, wheat flour was enzymatically hydrolyzed and the hydrolysate solution with high glucose concentration was fermented by the fungus. Results The optimum pH, temperature, and time were 5.5, 30°C, and 36 h, respectively. Maximum ethanol and glycerol yields were 0.48 and 0.06 g/g, respectively. The biomass yield was between 0.01 and 0.16 g/g of consumed glucose. The results showed that the fungus was able to produce ethanol in a medium containing 5.5% (v/v) ethanol, while higher ethanol concentration prevented further production of ethanol. Conclusion At the optimized conditions, the fungus was able to consume glucose with the concentration of 140 g/L and produce ethanol with a yield of 0.45 g/g, which was comparable to that by Saccharomyces cerevisiae.

Objective Since the soluble enzymes can not be used in repeated reactions and are not stable in operational conditions and not suitable for continuous processes, this study aimed the covalent immobilization of Bacillus licheniformis protease ( BL P) onto Eupergit CM. Methods Optimum conditions for immobilization were determined by changing the conditions individually. The proteins and L-tyrosine were determined by UV/VIS spectrophotometer. Results The immobilization resulted in 100% immobilization and 107.7% activity yields. The optimum pH (7–8) and the optimum temperature (70°C) have not changed after immobilization. The K m values for free and immobilized enzyme were 26.53 and 37.59 g/L, while the V max values were 2.84 and 3.31 g L-Tyrosine/L·min, respectively. The immobilized enzyme has not lost its initial activity during the repeated 20 uses and 20 days of storage. The milk proteins were hydrolyzed in 2 h by using immobilized enzyme. The pH of the milk dropped from 6.89 to 6.53, the color was clearer but there was no change in the smell or the taste. Conclusion Consequently, it can be said that the immobilized BL P obtained can be used for industrial purposes.

Objective The aim of this study was to determine the total protein amounts, protein profiles, in vitro cytotoxicities, antimicrobial activities and hemolytic effects of skin secretions of the Lissotriton vulgaris and Triturus ivanbureschi . Methods Skin secretions were obtained, clarified, supernatants snap-frozen then lyophilized. Total protein amounts were determined by BCA assay kit. Protein profiles were revealed by the SDS-PAGE. The cytotoxicity and antimicrobial activity were determined by using MTT assay and minimum inhibitory concentration (MIC) method. Hemolytic effects were measured on rabbit red blood cells. Results Lissotriton vulgaris and T. ivanbureschi skin secretions have totally 18 and 20 protein fractions. IC 50 values were detected between 1.40 and 40.28 μg/mL. The MIC results were found between 7.8 and 250 μg/mL. Lissotriton vulgaris skin secretion showed low hemolytic effect while T. ivanbureschi skin secretion showed high hemolytic effect. Conclusion This study is the first report showing the potential of L. vulgaris and T. ivanbureschi skin secretions for cytotoxicity, antimicrobial and hemolytic activity as an alternative therapeutic approach for traditional uses. Further studies need to focus on purification of the active components from these skin secretions and mode of action on cancer cell lines and microorganisms as anti-agents.

Objective Industrial effluents and agriculture biomass are main environmental hazards which are facing by developing country like Pakistan. Along with various other industrial applications, laccases are also involved in the oxidation of various industrial hazardous compounds to detoxify them. This study was designed to produce and purify laccase from ascomyceteous fungi, i.e. Alternaria alternata through solid stat fermentation. Materials and methods Abundantly available Sarkanda grass “ Saccharum spontaneum ” was used as agro-waste substrate for laccase production from fungus A. alternata . Previously only white rot fungi are familiar for laccase production and almost no work has been done on laccase production by A. alternata. In this research work, different physical and chemical parameters were optimized for maximum laccase production through solid state fermentation (SSF). Results Enzyme was purified and its molecular weight was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Maximum laccase activity (21.87±0.0115 unit/mL) was detected on 7th day of incubation having pH 5 of the medium at 35°C. None of the added metal ions increased laccase production. Galactose and “yeast extract” used as optimum carbon and nitrogen source for highest laccase production. Conclusion A monomeric protein (laccase) having approximately 51 kDa molecular weight obtained after SDS-PAGE.

Objective The presence of dyes in the water is toxic and harmful to human body so, it must be removed from the water. In the present study, the removal of methyl violet (MV) from aqueous solutions using brown algae “Padina sanctae-crucis” was investigated. Materials and methods The rate of adsorption was investigated under various parameters such as contact time (5–200), pH (2–11), dye concentration (10–60 mg/L), amount of adsorbent (0.25–5 g/L) and temperature (25–45°C). Results The maximum adsorption was achieved in 10 mg/L, pH=8 and adsorbent dose 2 g/L and 80 min contact time for removal of MV from aqueous solutions. Kinetic studies showed that the pseudo second-order model describes adsorbent kinetic behavior better. Besides, experimental data have been modeled using Langmuir and Freundlich isotherms and the results showed that both models are proper to describe adsorption isotherm behavior. In addition, the equilibrium study shows that the adsorption was physical and favorable. Moreover, a thermodynamic study revealed that the adsorption process is exothermic and spontaneously in nature. Furthermore, Maximum adsorption capacity using adsorbent was 10.02 mg/g. Conclusions It could be concluded that the P. sanctae-crucis biomass is a good adsorbent for removing MV dyes from aqueous solutions.

Objectives This study investigated the protective effect of dexpanthenol (DEX) in the septic shock model of rats with biochemical parameters. Methods 12–15 weeks old male 32 Wistar rats has been used for this study. Sepsis was induced by a single intraperitoneal injection of lipopolysaccharide (LPS) (5 mg/kg) and treatment groups received single intraperitoneal injection of DEX (500 mg/kg) just 30-min before. The blood and tissue samples were obtained 16 h later of LPS intervention under the ketamine and xylasine (50 and 5 mg/kg, respectively) anesthesia. Results Giving alone DEX did not alter any physiologic levels of biochemical markers. Induction of sepsis resulted in a marked increase in ALT, AST, urea, creatinine, lactate, procalcitonin, TNF-α, IL-1β levels to show the tissue damage. In all serum parameters, liver’s GSH, CAT levels and kidney’s CAT, GSH, MDA and NO levels have ameliorated by DEX treatment in sepsis group. Conclusion Along with the standard therapy of sepsis, DEX can be used as a safe way of restoring (anti)oxidant status of kidney and liver tissues. It can be effective to control cytokine pathway, to decrease procalcitonin and regulate the metabolic process of sepsis, such as lactate.

Background β-Cyanoalanine synthase plays essential roles in germinating seeds, such as in cyanide homeostasis. Methods β-Cyanoalanine synthase was isolated from sorghum seeds, purified using chromatographic techniques and its biochemical and catalytic properties were determined. Results The purified enzyme had a yield of 61.74% and specific activity of 577.50 nmol H 2 S/min/mg of protein. The apparent and subunit molecular weight for purified β-cyanoalanine synthase were 58.26±2.41 kDa and 63.4 kDa, respectively. The kinetic parameters with sodium cyanide as substrate were 0.67±0.08 mM, 17.60±0.50 nmol H 2 S/mL/min, 2.97×10 −1 s −1 and 4.43×10 2 M −1 s −1 for K M , V max , k cat and k cat /K M , respectively. With L-cysteine as substrate, the kinetic parameters were 2.64±0.37 mM, 63.41±4.04 nmol H 2 S/mL/min, 10.71×10 −1 s −1 and 4.06×10 2 M −1 s −1 for K M , V max , k cat and k cat /K M , respectively. The optimum temperature and pH for activity were 35°C and 8.5, respectively. The enzyme retained more than half of its activity at 40°C. Inhibitors such as HgCl 2 , EDTA, glycine and iodoacetamide reduced enzyme activity. Conclusion The biochemical properties of β-cyanoalanine synthase in germinating sorghum seeds highlights its roles in maintaining cyanide homeostasis.

Aims In order to shed light of characterizations of centipede Scolopendra subspinipes mutilans venom, a two novel full-lengths of alpha-like-neurotoxin and one metalloproteinase cDNAs derived from the maxilllipeds RNA of centipede S. subspinipes mutilans were isolated, and, respectively, named as SsuTA1, SsuTA2 and SsuMPs. Materials and methods The SsuTA1, SsuTA2 and SsuMPs were cloned from the S. subspinipes mutilans using the rapid amplification of cDNA ends methods. Results In the current study, SsuTA1 and SsuTA2 were, respectively, composed of 82 amino acid residues and 106 amino acid residues. Deduced protein sequence of SsuTA1 shared high homology with that of SsuTA2, one major difference was the C-terminal 24-residue extension in SsuTA2. An abundance of cysteine residues and several adjacent beta-sheets were observed in SsuTA1 and SsuTA2. SsuMPs had 594 amino acid residues containing with a molecular mass of 68.29 kDa. The primary sequence analysis indicated that the SsuMPs contains a zinc-binding motif (HEIGHSLGLAHS) and methionine-turn motif (YIM). Phylogenetic analysis revealed early divergence and independent evolution of SsuTA1 and SsuTA2 from other α-neurotoxins. Conclusion The results suggested that centipede S. subspinipes mutilans is an ancient member of venomous arthropods, but its venom exhibits novel scenario.

Objective The insecticidal activity of Bacillus thuringiensis directly depends on the yield of delta-endotoxins. In this study, various nutritional and cultural parameters influencing delta-endotoxin synthesis by a local isolate of B. thuringiensis Se13 were investigated using Taguchi methods. Methods In the first experiment, four factors, incubation period, incubation temperature, initial pH and medium, each at four levels, were selected and an orthogonal array layout of L 16 was carried out. In the second experiment, Taguchi’s orthogonal array method of L 27 was used to evaluate the effects of the different concentration of medium components. Taguchi’s signal–noise ratio and variance analysis were applied to determine the effect of the factors. After each experiment, verification studies were carried out using determined optimum conditions. Results The optimum conditions for incubation period, incubation temperature, initial pH, and medium determined as 72 h, 30°C, pH 9, and M4 medium, respectively. In the second experiment, soybean flour (5%), glucose (5%), KH 2 PO 4 (0.3%), K 2 HPO 4 (0.1%), MgSO 4 (0.4%) were determined as the optimum conditions. The delta-endotoxin yield was elevated to 1559.25 μg mL −1 when the factors were adjusted to optimum level. Conclusion Optimization using the Taguchi method appeared to be a good choice for the overproduction of delta-endotoxin.

Soybean products are popular because of its taste, digestibility, and health benefits. However, soybean lacks vitamin, mainly the low water-soluble vitamin B 12 . This study investigated the effects of fermentation conditions on the synthesis of vitamin B 12 , production of metabolites, and growth of Lactobacillus reuteri and Propionibacterium shermainii in fermented soy-milk. A Lotka Volterra model was successfully employed to describe the competition relationship between the two microorganisms under various fermentation conditions. A quadratic function between the ratio of interaction coefficients and vitamin B 12 content was found. Higher vitamin B 12 in soy-milk can be produced when the ratio of interaction coefficients approach to one. Compared with other fermented soybean products, fermented soy-milk contains more acetate, ethanol, and propionic acid. This study successfully demonstrated a mathematical model to enhance soy-milk vitamin B 12 production.

Objectives The role of oral bacteria in the etiopathogenesis of odontogenic cysts (OC) is controversial. Immune response is regulated by the cytokines secreted during infection. This study aims to describe the association in between bacteria and levels of cytokines in OC. Methods Infected OC fluid samples were obtained from 25 odontogenic keratocysts and 14 radicular cysts (RC). Bacteria detection was performed by polymerase chain reaction on bacterial 16S rRNA genes. Cytokine levels in OC fluids were determined using “luminex” method. Results Porphyromonas gingivalis was the most common bacteria in all samples (41.03%). Bacteria species number was higher in RCs. The significant difference was detected in terms of interleukine (IL)-1β levels to the number of bacteria contained in cyst fluids (p<0.05). IL1-β level of cyst fluid group containing three or more species of bacteria increased compared with cyst fluid group containing two types of bacteria (p<0.05). IL-1β level was high in cyst fluids with Campylobacter rectus and Treponema denticola or with three or more bacteria species. IL-1β level was higher in the cyst fluids with Enterococcus faecalis negative than E. faecalis positives. Conclusions Our results suggest that species and the number of bacterium may differ IL-1β levels in the OC fluid.

Background Modern agriculture recognizes soil biota as major contributors for availabilities of nitrogen and phosphorus to plants. Centralizing focus on exopolymer production of these living entities is need of time to emphasize their impact on soil structural restoration and heavy metal intoxication. Material and methods Mung bean rhizosphere collected from 25 locations was serially diluted and poured onto MY agar plates that were incubated for 120 h at 25°C to isolate bacteria having watery mucoidal appearance. Liquid broths of secluded cultures were then tested for optical scattering and were treated with ethanol to precipitate Exopolysaccharides (EPS) for their physicochemical characterization. Results Anion-exchange and high-performance size exclusion chromatographic analysis indicated two main monosaccharides, Mannose (52%) and Glucose (29%) fractions of EPS. EPS have substantial (0.2%) protein contents, capacity related to emulsify several hydrophobic substances. 0.5% EPS solution had low viscosity with pseudoplastic behaviour, least suspended particles producing less turbid solutions. Conclusion Six strains (M 2 , M 3 , M 11 , M 16 , M 19 , and M 22 ) secreted noticeably greater amounts of exopolymers than other strains. Organic nature and pseudoplasticity of these exopolymers helps in soil structural restoration, sulfates and phosphates helps in heavy metals detoxication.

Background Benzo(a)pyrene (BaP), a high molecular weight pentacyclic aromatic hydrocarbon, is a priority pollutant of extreme concern. Bacillus subtilis BMT4i (MTCC 9447) degrades BaP through chromosomally encoded pathway. Nevertheless, inadequate information is available on BaP degradation pathway in genus Bacillus despite of its species being shown as potent BaP degrader. The objective of this study was to elucidate BaP degradation pathway in B. subtilis strain BMT4i by identifying metabolites through UHPLC-MS. Materials and methods Batch experiments were conducted to characterize metabolic pathway of BaP in the bacterium B. subtilis BMT4i. The metabolites were separated and characterized by UHPLC-MS. Results The major intermediates of BaP metabolism that had accumulated in the culture media after 15 days of incubation were benzo(a)pyrene-11,12-epoxide, 7,8,9,10-tetrahydrobenzo[pqr]tetraphene-7,8,9,10-tetraol, benzo(a)pyrene-cis-7,8-dihydrodiol, 8-carboxy-7-hydroxy pyrene, chrysene-4 or 5-carboxylic acid, cis-4-(8-hydroxypyrene-7yl)-2-oxobut-3-enoic acid, hydroxymethoxybenzo(a)pyrene and dimethoxybenzo(a)pyrene. Among above, 8-carboxy-7-hydroxy pyrene, chrysene-4 or 5-carboxylic acid, and cis-4-(8-hydroxypyrene-7yl)-2-oxobut-3-enoic acid are ring cleavage products of BaP. Conclusion The identified metabolites indicated that BMT4i initially oxidized BaP with monooxygenases and dioxygenases at C-11,12 or and C-7,8 and C-9,10 positions, suggesting operation of multiple pathways for BaP degradation in B. subtilis . Further studies are essential to find out whether the entire biodegradation process in B. subtilis results into metabolic detoxification of BaP or not.