Min-1 Mg-1 Research Articles

Enzymatic production of glucosamine and chitooligosaccharides using newly isolated exo-β-D-glucosaminidase having transglycosylation activity.

Exochitosanase secreting fungus (A. fumigatus IIT-004) was isolated from fish waste using 1 % (w/v) chitosan as sole carbon source after multistage screening. Chitosan-dependent exochitosanase enzyme production (6 IU ml−1) in log phase of growth (chitosan utilization rate 0.11 g g−1 cell h−1) was observed for Aspergillus fumigatus in chitosan minimal salt medium and there was no enzyme production in glucose medium. Enzyme production was found to be extracellular and subjected to purification by a number of steps like acetone fractionation as well as column chromatography. 40 % yield and 26-fold of enzyme purification was achieved after all the steps. Purified enzyme was characterized for optimum temperature, pH, ionic strength and substrate specificity. The K m and V max for purified exochitosanase enzyme was calculated to be 8 mg ml−1 and 5.2 × 10−6 mol mg−1 min−1. Enzyme was immobilized on polyacrylonitrile nanofibres membrane matrix by adsorption as well as amidination. Enzymatic production of glucosamine was achieved using various chitosan substrates by free/immobilized exochitosanase and compared. Isolated and purified exochitosanase also showed transglycosylation activity.

Purification and Molecular Characterization of an [FeFe]-Hydrogenase from <i>Thermotoga hypogea</i>

Thermotoga hypogea is a hyperthermophilic bacterium growing on carbohydrates and peptides, producing acetate, CO2 and H2 as major end products. Hydrogenase activity was detected in the cell-free extract of T. hypogea and a hydrogenase was purified to homogeneity. The purified enzyme was homotetrameric with a subunit of 65 kDa, and contained 16 atoms of Fe and 11.6 atoms of acid labile sulfur per mole subunit. The gene encoding the hydrogenase was sequenced, and four iron-sulfur clusters including the H-cluster were found to be present in the gene sequence, indicating it is an [FeFe]-hydrogenase. The enzyme was oxygen sensitive with a t1/2 of 3 min when exposed to air. It was thermostable with t1/2 values of approximately 40 and 15 min when incubated at 70°C and 85°C, respectively. The optimal temperature for the hydrogenase activity was about 85 °C. Both benzyl viologen (BV) and methyl viologen (MV) could serve as electron acceptors, and the apparent Vmax values were determined to be 1,142 and 607 μmol H2 oxidized min-1 mg-1, respectively. For H2 uptake activity, the apparent Km values for MV and BV were 0.17 and 0.24 mM, respectively. For H2 evolution activity, the apparent Km value for MV and apparent Vmax value were determined to be 1.1 mM and 192 μmol min-1 mg-1, respectively. Ferredoxin purified from T. hypogea served as electron carrier. The enzyme exhibited pH optima of 10.0 and 8.0 for the H2 uptake and evolution activities, respectively. The physiological function of the enzyme is proposed to catalyze the H2 evolution. Keywords: [FeFe]-hydrogenase, ferredoxin, iron-sulfur clusters, ferredoxin, hydrogen production, hyperthermophiles, Thermotoga hypogea.

Comparative Study on Adsorption of Pb(II) Ions by Alginate Beads and Mangrove-Alginate Composite Beads

The aim of the present study report on the adsorption performance of alginate bead (AB) and mangrove-alginate composite bead (MACB) bead adsorbents for the removal of Pb(II) ions from aqueous solution. The effects of pH and initial concentration with contact time on the adsorption properties of Pb(II) onto both adsorbent were investigated and were described by isotherm and kinetic studies. The isotherm adsorption data were fitted well to Freundlich isotherms for both beads and the maximum adsorption capacities of the AB and MACB beads were 29.02 mg g-1 and 10.84 mg g-1, respectively. The kinetics adsorption data were best described to a pseudo-second-order kinetic models showing that the MACB beads had a higher kinetic adsorption rate at 2.6084 g mg-1 min-1 compared to AB at 0.7043 g mg-1 min-1.

BIOCHEMICAL AND PHYLOGENETIC STUDIES OF CreD OF Corynebacterium glutamicum

CreD characterized as Mg2+-dependent phosphohydrolase with conserved HD domain was involved in 4-cresol metabolism in Corynebacterium glutamicum. Native molecular mass of 54 kDa suggested that the biological unit is a dimer. No deoxynucleotide triphosphate triphosphohydrolase (dNTPase) activity was detected for CreD. The apparent Km and Vmax values for 4-nitrophenyl phosphate were 0.35 mM and 16.23 M min-1 mg-1, respectively, while calculated values for kcat and kcat/Km were 0.4 s-1 and 1.14103 M-1 s-1, respectively. Among thiol group inhibitors, iodoacetic acid significantly inhibited phosphohydrolase activity. Sequence identity and phylogenetic analysis suggested universal existence of CreD homologues. Involvement of HD-domain hydrolase in aromatic degradation has not been reported before.

Biosorption of styrene from synthetic wastewater by sugar cane waste(Bagass)

In this work, styrene removal from wastewater by using sugarcane waste (bagasse) as an adsorbent was studied. Equilibrium isotherms and kinetics were determined; the effects of bagasse particle size and concentration, solutions pH, and temperature on the biosorption of styrene were studied in batch experiments. Adsorption equilibrium data was successfully fitted to Langmuir isotherms (R2=0.986) and Freundlich isotherms (R2=0.96). Also, the kinetics of biosorption was fitted to pseudo-second order equations (K2=0.00146 g mg-1 min-1, qe=24.5 mg g-1 for particle size range of 88-105 μm). According to the obtained results, an empirical equation was presented that could be used to calculate the percentage of styrene adsorption. The results showed that an increase in temperature caused a decrease in styrene removal. Moreover, maximum uptake was observed with NaOH-treated bagasse. It was found that an increase in average particle size decreased the biosorption rate. According to the calculated heat of adsorption, this sorption can be classified as a chemical biosorption. The optimum uptake was determined to be 88% at a pH equal to 12.1, a temperature of 35 oC, a particle size of 420-500 μm, and a bagasse concentration of 1 g L-1.

Purification and Characterization of Methionine Gamma Lyase-Deaminase (Mgld) from the Oral Pathogenic Organism Porphyromonas gingivalis

Porphyromonas gingivalis is an oral pathogenic bacterium that causes halitosis (bad breath) and periodontitis in humans. Halitosis manifests due to volatile sulfur compounds such as methylthiol, generated from methionine degradation by Methionine Gamma Lyase-Deaminase (Mgld). In this report, we cloned and expressed Porphyromonas gingivalis mgld using a bacterial expression system, and the purified homotetrameric enzyme was characterized by a novel isotope assay using L-[1-14C]-methionine as a substrate. Mgld is a PLP-dependent L-methionine C-S lyasedeaminase that cleaves the gamma-carbon-sulfur bond of L-methionine to methylthiol and forms the deaminated overall product α-ketobutyrate. We find that exogenous 3H-L-2-aminobutyrate is not deaminated into 3H-α-ketobutyrate under conditions of enzyme excess, supporting efficient transfer of bound intermediate(s) during L-methionine catabolism. The overall reaction to form α-ketobutyrate from L-methionine exhibits Km of 1.0 mM, Vmax of 5.27 μmol min-1 mg-1, and a monomeric kcat/Km of 3729.3 M-1 s-1. Mgld exhibits optimal activity above pH 8 and in a temperature range of 37°-50°C. Several compounds were tested for Mgld inhibition. The natural product DL-Propargylglycine stands out as the most effective Mgld inhibitor and thus may be useful for the control of halitosis. Mgld has almost no activity on N-formylmethionine, confirming the requirement of a free α-amino-nitrogen on the substrate to form a Schiff base with the enzyme.

Deproteinization of Distillery Yeast Biomass Waste by Protease-producing Bacillus megaterium PB 4

The distillery yeast biomass (DYB), a waste by-product from distilleries is one of the under-utilized protein sources to be exploited for the production of protein hydrolysates. In this study, a neutral protease produced from Bacillus megaterium PB4 isolated from agro wastes enriched soil exhibited significant deproteinization of distillery yeast biomass to yield protein hydrolysates. Among three protease producing strains isolated, the maximum protease production of 120.3 ± 1.4 U mL-1 was exhibited by PB4 grown in Luria-Bertani (LB) broth supplemented with casein at pH 7.0 and 30°C. The minimal media supplemented with various carbon and nitrogen sources, glucose and peptone significantly improved the protease production in PB4. The effect of metal ions such as Zn2+, Mg2+, Mn2+, Cu2+ at 0.5 and 1.0 mM final concentration in the media on protease production indicated that Zn2+ (1.0 mM) was favorable in enhancing the protease production. The Km and Vmax of PB4 protease for casein substrate was 0.6 U ml-1 and 217.3 μM min-1 mg-1 protein, respectively. The deproteinization rate for distillery yeast biomass was 84 and 76.4% using culture and crude enzyme extracted from PB4, respectively. To the best of our knowledge, deproteinization of distillery yeast biomass using proteolytic bacterial isolate has never been demonstrated before. The experimental results in the present study suggest that Bacillus megaterium PB4, can be utilized for the eco-friendly, economical production of protein hydrolysates and may be considered as a potential candidate in various biotechnological applications.

Comparative Investigation of Glutathione S-transferase (GST) in Different Crops and Purification of High Active GSTs from Onion (Allium cepa L.)

Glutathione S-transferases (GSTs; EC 2.5.1.18) are abundant proteins encoded by a highly divergent ancient gene family with protective functions through detoxification and non-catalytic roles as carriers of cytotoxins. In this work, we investigated the GST activities in seedlings of 38 crops to obtain a plant with high active GST for further study. In screening of 38 crops, onion seedling showed the highest GST activity (483.54 nmol min-1mg-1 protein) followed by wheat(372.89 nmol min-1mg-1 protein), barley (253.44 nmol min-1mg-1 protein), rice (244.12 nmol min-1mg-1 protein) and proso millet (173.34 nmol min-1mg-1 protein). Carrot seedling showed the lowest activity (3.63 nmol min-1mg-1 protein).In onion plants, both root and leaf showed high GST activity. Onion bulb GSTs were separated DEAE cellulose column chromatography, and purified by affinity chromatography (S-hexyl glutathione-agarose). Three GST peaks were found to elute at 67, 107 and 140mM of KCl gradient solution, and were named as GSTa, GSTb and GSTc. Among the three GSTs, GSTa, GSTb and GSTc contained 9.58, 61.45 and 28.97% of total activity, respectively. In purification, GSTa, GSTb and GSTc had specific activities of 9075, 17259 and 19868 nmol min-1 mg-1 protein, respectively, along with yield of 2.48, 3.17 and 1.28,and purification fold of 15.8, 29.9 and 34.5, respectively. The purity and molecular mass of the fraction was examined by SDS-PAGE. The silver staining of the purified GSTa, GSTb and GSTc indicated that final product of GSTb and GSTc were highly purified and migrated as a single band on SDS-PAGE with an apparent molecular mass of 27 kDa. However, GSTa eluted with Glyoxalase-I (Gly-I) and to purify GSTa, more methodological application are suggested.

Continuous Removal of Lead from Aqueous Solutions by Ca(II) Imprinted Chitosan Microspheres Packed Column

The continuous removal of lead from aqueous solutions by Ca(II) imprinted chitosan (Ca(II)-CS) microspheres packed column is carefully investigated in this work. The modified dose-response model and the Thomas model are exploited to evaluate dynamic behaviors of breakthrough curves. The results show 0.257 min of space time is enough for a Ca(II) imprinted chitosan microspheres packed column to realize deep purification of water containing Pb(II). The Thomas rate constants are higher than 3 mL min-1 mg-1. The Modified dose-response model is feasible to predict the breakthrough curve. The reusability is confirmed during a multi-cycle adsorption-desorption process.

Enzyme changes during seed storage in groundnut (Arachis hypogaea L.)

A change in enzyme activity in seeds due to ageing is a topic of scientific importance. Vigour is essentially a physiological phenomenon influenced by the reserved metabolites, enzyme activities and growth regulators. The exact cause of loss of seed vigour and viability is still unknown as deterioration of seed is a complex process. In the presence of oxygen, ageing of seed can lead to peroxidative changes in polyunsaturated fatty acids. The free radical -induced non-enzymatic peroxidation, which has the potential to damage membrane, is likely to be a primary cause of deterioration of stored seeds. Certain anabolic enzymes help in maintaining viability while some catabolic enzymes decrease viability. The seed catalase and peroxidase activity seem to be decreased during storage. The results revealed that the peroxidase enzyme activity decreased from 0.236 to 0.444 OD 10 min-1 when storage period increased. A decrease in catalase activity from 0.454 to 0.444 ?g H2O2 mg-1 min-1 followed by a small increase from 0.434 to 0.452 ?g H2O2 mg-1 min-1 was observed during storage. But the activity of lipase enzyme increased from 0.236 to 0.231 meq min-1g-1 of sample when the storage period was increased. The study would help to know the deterioration pattern of stored groundnut seeds.

Induction of D-xylose uptake and expression of NAD(P)H-linked xylose reductase and NADP + -linked xylitol dehydrogenase in the oleaginous microalga Chlorella sorokiniana.

BackgroundThe heterotrophic and mixotrophic culture of oleaginous microalgae is a promising process to produce biofuel feedstock due to the advantage of fast growth. Various organic carbons have been explored for this application. However, despite being one of the most abundant and economical sugar resources in nature, D-xylose has never been demonstrated as a carbon source for wild-type microalgae. The purpose of the present work was to identify the feasibility of D-xylose utilization by the oleaginous microalga Chlorella sorokiniana.ResultsThe sugar uptake kinetic analysis was performed with 14C-labeled sugars and the data showed that the D-glucose induced algal cells (the alga was heterotrophically grown on D-glucose and then harvested as D-glucose induced cells) exhibited a remarkably increased D-xylose uptake rate. The maximum D-xylose transport rate was 3.8 nmol min−1 mg−1 dry cell weight (DCW) with Km value of 6.8 mM. D-xylose uptake was suppressed in the presence of D-glucose, D-galactose and D-fructose but not L-arabinose and D-ribose. The uptake of D-xylose activated the related metabolic pathway, and the activities of a NAD(P)H-linked xylose reductase (XR) and a unique NADP+-linked xylitol dehydrogenase (XDH) were detected in C. sorokiniana. Compared with the culture in the dark, the consumption of D-xylose increased 2 fold under light but decreased to the same level with addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), indicating that extra chemical energy from the light-dependent reaction contributed the catabolism of D-xylose for C. sorokiniana.ConclusionsAn inducible D-xylose transportation system and a related metabolic pathway were discovered for microalga for the first time. The transportation of D-xylose across the cell membrane of C. sorokiniana could be realized by an inducible hexose symporter. The uptake of D-xylose subsequently activated the expression of key catalytic enzymes that enabled D-xylose entering central metabolism. Results of this research are useful to better understand the D-xylose metabolic pathway in the microalga C. sorokiniana and provide a target for genetic engineering to improve D-xylose utilization for microalgal lipid production.

Polyaniline coating on carbon fiber fabrics for improved hexavalent chromium removal

Carbon fabrics (CFs) loaded with 5.0, 10.0, 15.0 and 20.0 wt% polyaniline (PANI) (PANI/CF) prepared by soaking carbon fiber fabrics in 1.0 wt% PANI m-cresol solution have demonstrated superior hexavalent chromium (Cr(VI)) removal performance compared to the as-received CFs. The PANI/CF nanocomposites were noticed to remove Cr(VI) from solutions with an initial Cr(VI) concentration of 1.0 mg L-1 within 15 min, which is faster than the conventional active carbon (6 h) and the as-received CFs (>1 h). A better Cr(VI) removal efficiency was observed in the acidic solutions than in the basic solutions. A pseudo-second-order behavior was justified for the PANI/CF with a much higher removal rate (0.06 g mg-1 min-1) than the reported ~0.03 g mg-1 min-1 for the active carbon. The adsorption isotherm study demonstrated that the adsorbents follow the Langmuir model with a calculated maximum adsorption capacity of 18.1 mg g-1 for the 10.0 wt% PANI/CF. The Cr(VI) removal mechanisms explored by FT-IR and XPS involve the reduction of Cr(VI) to Cr(III) by PANI. The PANI/CF adsorbents have demonstrated easy recycling capability for up to five cycles with a Cr(VI) removal rate at above 91%.

Identification and characterization of a ginsenoside-transforming β-glucosidase from Pseudonocardia sp. Gsoil 1536 and its application for enhanced production of minor ginsenoside Rg2(S).

The ginsenoside Rg2(S), which is one of the pharmaceutical components of ginseng, is known to have neuroprotective, anti-inflammation, and anti-diabetic effects. However, the usage of ginsenoside Rg2(S) is restricted owing to the small amounts found in white and red ginseng. To enhance the production of ginsenoside Rg2(S) as a 100 gram unit with high specificity, yield, and purity, an enzymatic bioconversion method was developed to adopt the recombinant glycoside hydrolase (BglPC28), which is a ginsenoside-transforming recombinant β-glucosidase from Pseudonocardia sp. strain Gsoil 1536. The gene, termed bglPC28, encoding β-glucosidase (BglPC28) belonging to the glycoside hydrolase family 3 was cloned. bglPC28 consists of 2,232 bp (743 amino acid residues) with a predicted molecular mass of 78,975 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The optimum conditions of the recombinant BglPC28 were pH 7.0 and 37°C. BglPC28 can effectively transform the ginsenoside Re to Rg2(S); the K m values of PNPG and Re were 6.36±1.10 and 1.42±0.13 mM, respectively, and the V max values were 40.0±2.55 and 5.62±0.21 µmol min−1 mg−1 of protein, respectively. A scaled-up biotransformation reaction was performed in a 10 L jar fermenter at pH 7.0 and 30°C for 12 hours with a concentration of 20 mg/ml of ginsenoside Re from American ginseng roots. Finally, 113 g of Rg2(S) was produced from 150 g of Re with 84.0±1.1% chromatographic purity. These results suggest that this enzymatic method could be usefully exploited in the preparation of ginsenoside Rg2(S) in the cosmetics, functional food, and pharmaceutical industries.

Impaired Forebrain Cellular Bioenergetics Following Acute Exposure to Ammonia

Introduction: The main purpose of this study was to report on the acute neurotoxicity of ammonia, using cellular respiration and ATP as surrogate metabolic biomarkers. Cellular respiration (mitochondrial O2 consumption), ATP and glutathione (GSH) were measured in forebrain specimens after intraperitoneal administration of 3.8 to 28.8 μmol/g ammonia to Taylor Outbred mice; the lethal dose was ≥ 30 μmol/g. Methods: Cortical fragments were collected in phosphate-buffered saline plus 5 mM glucose and immediately processed for measuring O2 consumption, using a phosphorescence O2 analyzer. Cellular ATP was determined with the luciferase/luciferin system. Cellular GSH was labeled with monobromobimane and the bimane derivatives were separated on HPLC and detected by fluorescence. In the presence of the tissue specimen in a vial sealed from air, O2 concentration declined linearly with time, confirming zero-order kinetics of O2 consumption. This process was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. Results: For untreated mice, cellular respiration was 0.32 ± 0.12 μM O2 min-1 mg-1 (n=36 mice), ATP was 238 ± 45 pmol.mg-1 (n=9 mice) and GSH was 300 ± 50 pmol.mg-1 (n=6 mice). For treated mice, cellular respiration was 0.26 ± 0.09 μM O2 min-1 mg-1 (n=22 mice; 19% lower, p=0.034), ATP was 183 ± 34 pmol mg-1 (n=9 mice; 23% lower, p=0.008) and GSH was 320 ± 160 pmol mg-1 (n=6 mice, p=0.700). Conclusion: Acute ammonia intoxication significantly lowered forebrain cellular bioenergetics. These changes would be are difficult to monitor at lethal doses, but the results are expected to be more pronounced.

High-level expression of a novel thermostable and mannose-tolerant β-mannosidase from Thermotoga thermarum DSM 5069 in Escherichia coli

BackgroundMannan is one of the primary polysaccharides in hemicellulose and is widely distributed in plants. β-Mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. Therefore, the mannose-tolerant β-mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fields as above.ResultsA β-mannosidase gene (Tth man5) of 1,827 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 608 amino acid residues, and was over-expressed in Escherichia coli BL21 (DE3). The results of phylogenetic analysis, amino acid alignment and biochemical properties indicate that the Tth Man5 is a novel β-mannosidase of glycoside hydrolase family 5. The optimal activity of the Tth Man5 β-mannosidase was obtained at pH 5.5 and 85°C and was stable over a pH range of 5.0 to 8.5 and exhibited 2 h half-life at 90°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 4.36±0.5 mM and 227.27±1.59 μmol min-1 mg-1, 58.34±1.75 mg mL-1 and 285.71±10.86 μmol min-1 mg-1, respectively. The kcat/Km values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 441.35±0.04 mM-1 s-1 and 41.47±1.58 s-1 mg-1 mL, respectively. It displayed high tolerance to mannose, with a Ki value of approximately 900 mM.ConclusionsThis work provides a novel and useful β-mannosidase with high mannose tolerance, thermostability and catalytic efficiency, and these characteristics constitute a powerful tool for improving the enzymatic conversion of mannan through synergetic action with other mannan-degrading enzymes.

Biological endpoints, enzyme activities, and blood cell parameters in two anuran tadpole species in rice agroecosystems of mid-eastern Argentina

Different biological variables of tadpoles, including survival, development and growth rates, and biomarkers [cholinesterases, glutathione-S-transferases (GST), and blood cell morphology] were evaluated in two anuran species, Scinax squalirostris (Hylidae) and Leptodactylus mystacinus (Leptodactylidae), using in situ experimental chambers in a rice field (RF) sprayed with insecticide Lambda-cyhalothrin (LTC) by aircraft in Santa Fe Province, Argentina. We found a significant decrease in body weight (0.62 ± 0.04 g) of L. mystacinus and an increased development rate of S. squalirostris in individuals from RF (41 ± 1; Gosner) with respect to individuals from the reference site (RS: 0.93 ± 0.04 g and 37 ± 0; respectively). In S. squalirostris, individuals from RF mean values of butyrylcholinesterase activities decreased at 48 (4.09 ± 0.32 nmol min-1 mg-1 of TP) and 96 h (3.74 ± 0.20 nmol min-1 mg-1 of TP), whereas inhibition of acetylcholinesterase was observed at 96 h (47.44 ± 2.78 nmol min-1 mg-1 of TP). In L. mystacinus from RF, an induction of acetylcholinesterase activity was observed at 96 h (36.01 ± 1.09 nmol min-1 mg-1 of TP). Glutathione-S-transferase levels varied between species, being higher in L. mystacinus individuals but lower in S. squalirostris from RF at 48 (272.29 ±11.78 and 71.87 ± 1.70 nmol min-1 mg-1 of TP; respectively) and 96 h (279.25 ± 13.06 and 57.62 ± 4.58 nmol min-1 mg-1 of TP, respectively). Blood cell parameters revealed a lower number of mitotic cells (MC: 0.36 ± 0.31%o for S. squalirostris and 0.08 ± 0.05 %o for L. mystacinus) and higher number of eosinophils (E: 3.45 ± 1.75 %o for S. squalirostris and 7.64 ± 0.98 %o for L. mystacinus) in individuals from the RF than in individuals from the RS (MC: 2.55 ± 0.74 %o for S. squalirostris and 1.87 ± 0.72%o for L. mystacinus; and E: 0.13 ± 0.09 for S. squalirostris and 3.20 ± 0.80 for L. mystacinus). Overall, our results demonstrate the existence of apparent differences in sensitivity between species in a series of sublethal responses to short-term exposure in RF after the application of Lambda-cyhalothrin. We suggest that the integral use of biological endpoints (development and growth) together with biomarkers (cholinesterase, GST, and blood cell parameters) may be a promising integral procedure for investigating pesticide exposure in wild frog populations.

English

The adsorption capacities of iron oxide, aluminum oxide, and activated carbon sorbents for the removal of copper ions were compared by down flow method. For this purpose, the 20 mg L-1aqueous solution of Cu (II) with initial pH of 6.5 was fed through three different packed columns at bed depth of 10 cm and flow rate of 1.85 ml min-1. The experimental breakthrough curves were drawn from the adsorption data of packed bed columns. The breakthrough time comparison of the three columns revealed that the uptake capacity of Cu (II) was highest for activated carbon (16.24 mg g-1) followed by aluminum oxide (5.60 mg g-1) and iron oxide (5.41 mg g-1). The maximum amounts of Cu (II) removal were attained as follows: 94.0% for aluminum oxide, 95.2% for iron oxide, and 99.7% for activated carbon. The computed adsorption capacity per unit bed volume (N0) and the kinetic constant (KAB) were, respectively 1202, 1045 (mg L-1) and 3 × 10-4, 4 × 10-4 (L mg-1 min-1) for iron oxide and aluminum oxide. Regression coefficient value (0.98) indicated that Thomas model fitted well with the experimental data for the iron oxide and aluminum oxide sorbents. The theoretical adsorption capacities obtained by Thomas model were in good agreement with experimental capacities determined by adsorption data. In a short time after running the experiment, the equilibrium concentration of Cu (II) reached to 1.2 mg L-1 using these sorbents. Hence, the packed bed column with continuous down flow method can be successfully applied for the removal of heavy metals from contaminated waters in practical use.   Key words: Adsorption, breakthrough curve, continuous down flow, copper (II), packed bed column, Thomas model, Bohart-Adams, Yoon-Nelson.

Bioenergetic study of murine hepatic tissue treated in vitro with atorvastatin

Atorvastatin (a 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitor) is a widely used cholesterol-lowering drug, which is recognized for its potential hepatotoxicity. This study investigated in vitro effects of this agent on hepatic tissue respiration, ATP content, caspase activity, urea synthesis and histology. Liver fragments from Taylor Outbred and C57Bl/6 mice were incubated at 37°C in Krebs-Henseleit buffer continuously gassed with 95% O2: 5% CO2 in the presence and absence of atorvastatin. Phosphorescence O2 analyzer that measured dissolved [O2] as a function of time was used to monitor cellular mitochondrial O2 consumption. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin was used to monitor caspase activity. The rates of hepatocyte respiration (μM O2 min-1 mg-1) in untreated samples were 0.15 ± 0.07 (n = 31). The corresponding rates for samples treated with 50 nM (therapeutic concentration), 150 nM or 1.0 μM atorvastatin for ≤13 h were 0.13 ± 0.05 (n = 19), p = 0.521. The contents of hepatocyte ATP (pmol-1 mg-1) in untreated samples were 40.3 ± 14.0 and in samples treated with 1.0 μM atorvastatin for ≤4.5 h were 48.7 ± 23.9 (p = 0.7754). The concentrations of urea (mg/dL mg-1, produced over 50 min) for untreated samples were 0.061 ± 0.020 (n = 6) and for samples treated with 1.0 μM atorvastatin for ≤6 h were 0.072 ± 0.022 (n = 6), p = 0.3866. Steadily, hepatocyte caspase activity and histology were unaffected by treatments with up to 1.0 μM atorvastatin for ≤6 h. Thus, the studied murine model showed preserved hepatocyte function and structure in the presence of high concentrations of atorvastatin.

A preparation of murine liver fragments for in vitro studies: liver preparation for toxicological studies

BackgroundThe aim of this study was to develop liver tissue preparation suitable for investigating toxins. Hepatocyte respiration, ATP content, urea synthesis, caspase activity and morphology were measured as a function of in vitro incubation time. Mice were anesthetized by sevoflurane inhalation. Small liver fragments were then rapidly excised and incubated at 37°C in Krebs-Henseleit buffer (continuously gassed with 95% O2: 5% CO2) for up to 6 h. Phosphorescence O2 analyzer was used to determine the rate of cellular mitochondrial O2 consumption (kc, μM O2 min-1 mg-1). Cellular ATP was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence.FindingsRespiration was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. The values of kc (mean ± SD) for 0≤ t ≤6 h were 0.15 ± 0.02 μM O2 min-1 mg-1 (n = 18, coefficient of variation, CV = 13%), ATP content 131 ± 69 pmol mg-1 (1≤ t ≤6 h, n = 16, CV = 53%), synthesized urea 0.134 ± 0.017 mg/dL mg-1 in 50 min (0≤ t ≤6 h, n = 14, CV = 13%), and AMC peak area 62,540 ± 26,227 arbitrary units mg-1 (1≤ t ≤6 h, n = 3, CV = 42%). Hepatocyte morphology and organelles were reasonably persevered.ConclusionsThe described liver tissue preparation demonstrates stable hepatocyte structure, ultrastructure and biomarkers for up to 6 h, permitting in vitro studies.

Lung tissue bioenergetics and caspase activity in rodents

BackgroundThis study aimed to establish a suitable in vitro system for investigating effects of respiratory pathogens and toxins on lung tissue bioenergetics (cellular respiration and ATP content) and caspase activity. Wistar rats and C57Bl/6 mice were anesthetized by sevoflurane inhalation. Lung fragments were then collected and incubated at 37°C in a continuously gassed (with 95% O2:5% CO2) Minimal Essential Medium (MEM) or Krebs-Henseleit buffer. Phosphorescence O2 analyzer that measured dissolved O2 concentration as a function of time was used to monitor the rate of cellular mitochondrial O2 consumption. Cellular ATP content was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence. Lung histology and immunostaining with anti-cleaved caspase-3 antibody were also performed.ResultsFor Wistar rats, the values of kc and ATP for 0 < t ≤ 7 h (mean ± SD) were 0.15 ± 0.02 μM O2 min-1 mg-1 (n = 18, coefficient of variation, Cv = 13%) and 131 ± 69 pmol mg-1 (n = 16, Cv = 53%), respectively. The AMC peak areas remained relatively small despite a ~5-fold rise over 6 h. Good tissue preservation was evident despite time-dependent increases in apoptotic cells. Lung tissue bioenergetics, caspase activity and structure were deleterious in unoxygenated or intermittently oxygenated solutions. Incubating lung tissue in O2 depleted MEM for 30 min or anesthesia by urethane had no effect on lung bioenergetics, but produced higher caspase activity.ConclusionsLung tissue bioenergetics and structure could be maintained in vitro in oxygenated buffer for several hours and, thus, used as biomarkers for investigating respiratory pathogens or toxins.