Generally, proteases in medicinal plants had different therapeutic effects such as anti-inflammatory effect; modulate the immune response and inhibitory effect toward tumor growth. In this study, protease was purified and characterized from miswak roots, as medicinal plant and natural toothbrush.
Results
Physical and chemical characterization of cysteine protease P1 were studied such as pH optimum (6.5), optimum temperature (50?°C), thermal stability (50?°C) and Km (3.3?mg azocasein/ml). The enzyme digested some proteins in the order of caseine > haemoglobin > egg albumin >gelatin > bovine serum albumin. Hg2+ had strong inhibitory effect on enzyme activity compared with other metal ions. Kinetic of inhibition for determination the type of protease was studied. Iodoactamide and p-Hydroximercuribenzaoic acid (p-HMB) caused strong inhibitory effect on enzyme activity indicating the enzyme is cysteine protease.
Conclusions
The biochemical characterization of this enzyme will be display the suitable conditions for using of this enzyme in toothpaste in the future and the enzyme may be used in other applications.
Thauera linaloolentis 47Lol uses the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. The conversion of linalool to geraniol had been observed in carbon-excess cultures, suggesting the presence of a 3,1-hydroxyl-Δ1-Δ2-mutase (linalool isomerase) as responsible enzyme. To date, only a single enzyme catalyzing such a reaction is described: the linalool dehydratase/isomerase (Ldi) from Castellaniella defragrans 65Phen acting only on (S)-linalool.
Results
The linalool isomerase activity was located in the inner membrane. It was enriched by subcellular fractionation and sucrose gradient centrifugation. MALDI-ToF MS analysis of the enriched protein identified the corresponding gene named lis that codes for the protein in the strain with the highest similarity to the Ldi. Linalool isomerase is predicted to have four transmembrane helices at the N-terminal domain and a cytosolic domain. Enzyme activity required a reductant for activation. A specific activity of 3.42?±?0.28 nkat mg * protein?1 and a kM value of 455?±?124 μM were determined for the thermodynamically favored isomerization of geraniol to both linalool isomers at optimal conditions of pH 8 and 35 °C.
Conclusion
The linalool isomerase from T. linaloolentis 47Lol represents a second member of the enzyme class 5.4.4.4, next to the linalool dehydratase/isomerase from C. defragrans 65Phen. Besides considerable amino acid sequence similarity both enzymes share common characteristics with respect to substrate affinity, pH and temperature optima, but differ in the dehydratase activity and the turnover of linalool isomers.
An increased incidence of fungal infections, both invasive and superficial, has been witnessed over the last two decades. Candida species seem to be the main etiology of nosocomial fungal infections worldwide with Candida albicans, which is commensal in healthy individuals, accounting for the majority of invasive Candida infections with about 30-40% of mortality.
Results
New aromatic and heterocyclic esters 5a-k of 1-aryl-3-(1H-imidazol-1-yl)propan-1-ols 4a-d were successfully synthesized and evaluated for their anti-Candida potential. Compound 5a emerged as the most active congener among the newly synthesized compounds 5a-k with MIC value of 0.0833 μmol/mL as compared with fluconazole (MIC value >1.6325 μmol/mL). Additionally, molecular modeling studies were conducted on a set of anti-Candida albicans compounds.
Conclusion
The newly synthesized esters 5a-k showed more potent anti-Candida activities than fluconazole. Compounds 7 and 8 revealed significant anti-Candida albicans activity and were able to effectively satisfy the proposed pharmacophore geometry, using the energy accessible conformers (Econf?<?20 kcal/mol).
β-Glucosidase is claimed as a key enzyme in cellulose hydrolysis. The cellulosic fibers are usually entrapped with hemicelluloses containing xylose. So there is ongoing interest in searching for glucose- and xylose-stimulated β-glucosidases to increase the efficiency of hydrolysis of cellulosic biomass.
Results
A thermostable β-glucosidase gene (Bglp) was cloned from Anoxybacillus flavithermus subsp. yunnanensis E13T and characterized. Optimal enzyme activity was observed at 60 °C and pH 7.0. Bglp was relatively stable at 60 °C with a 10-h half-life. The kinetic parameters Vmax and Km for p-nitrophenyl-β-D-glucopyranoside (pNPG) were 771?±?39 μmol/min/mg and 0.29?±?0.01 mM, respectively. The activity of Bglp is dramatically stimulated by glucose or xylose at concentrations up to 1.4 M. After Bglp was added to Celluclast® 1.5 L, the conversion of sugarcane bagasse was 48.4?±?0.8%, which was much higher than of Celluclast® 1.5 L alone. Furthermore, Bglp showed obvious advantages in the hydrolysis when initial concentrations of glucose and xylose are high.
Conclusions
The supplementation of BglP significantly enhanced the glucose yield from sugarcane bagasse, especially in the presence of high concentrations of glucose or xylose. Bglp should be a promising candidate for industrial applications.
Previous studies have demonstrated that members of Trichoderma are able to generate appreciable amount of extracellular amylase and glucoamylase on soluble potato starch. In this study the α-amylase was purified and characterized from Trichoderma pseudokoningii grown on orange peel under solid state fermentation (SSF).
Results
Five α-amylases A1-A5 from Trichodrma pseudokoningii were separated on DEAE-Sepharose column. The homogeneity of α-amylase A4 was detected after chromatography on Sephacryl S-200. α-Amylase A4 had molecular weight of 30 kDa by Sephacryl S-200 and SDS-PAGE. The enzyme had a broad pH optimum ranged from 4.5 to 8.5. The optimum temperature of A4 was 50 °C with high retention of its activity from 30 to 80 °C. The thermal stability of A4 was detected up to 50 °C and the enzyme was highly stable till 80 °C after 1 h incubation. All substrate analogues tested had amylase activity toward A4 ranged from 12 to 100% of its initial activity. The Km and Vmax values of A4 were 4 mg starch/ml and 0.74 μmol reducing sugar, respectively. The most of metals tested caused moderate inhibitory effect, except of Ca2+ and Mg2+ enhanced the activity. Hg2+ and Cd+?2 strongly inhibited the activity of A4. EDTA as metal chelator caused strong inhibitory effect.
Conclusions
The properties of the purified α-amylase A4 from T. pseudokoningii meet the prerequisites needed for several applications.
The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields.
Results
Here we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, β-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl.
Conclusions
The biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes.
The emergence of bacterial resistance is a major public health problem. It is essential to develop and synthesize new therapeutic agents with better activity. The mode of actions of certain newly developed antimicrobial agents, however, exhibited very limited effect in treating life threatening systemic infections. Therefore, the advancement of multi-potent and efficient antimicrobial agents is crucial to overcome the increased multi-drug resistance of bacteria and fungi. Cancer, which remains as one of the primary causes of deaths and is commonly treated by chemotherapeutic agents, is also in need of novel and efficacious agents to treat resistant cases. As such, a sequence of novel substituted benzamides was designed, synthesized and evaluated for their antimicrobial and anticancer activities.
Methodology
All synthesized compounds were characterized by IR, NMR, Mass and elemental analysis followed by in vitro antimicrobial studies against Gram-positive (Staphylococcus aureus), Gram-negative (Salmonella typhi and Klebsiella pneumoniae) bacterial and fungal (Candida albicans and Aspergillus niger) strains by the tube dilution method. The in vitro anticancer evaluation was carried out against the human colorectal carcinoma cell line (HCT116), using the Sulforhodamine B assay.
Results, discussion and conclusion
Compound W6 (MICsa, st, kp?=?5.19 µM) emerged as a significant antibacterial agent against all tested bacterial strains i.e. Gram-positive (S. aureus), Gram-negative (S. typhi, K. pneumoniae) while compound W1 (MICca, an?=?5.08 µM) was most potent against fungal strains (A. niger and C. albicans) and comparable to fluconazole (MIC?=?8.16 µM). The anticancer screening demonstrated that compound W17 (IC50?=?4.12 µM) was most potent amongst the synthesized compounds and also more potent than the standard drug 5-FU (IC50?=?7.69 µM).
The cytochrome P450s are monooxygenases that insert oxygen functionalities into a wide variety of organic substrates with high selectivity. There is interest in developing efficient catalysts based on the “peroxide shunt” pathway in the cytochrome P450s, which uses H2O2 in place of O2/NADPH as the oxygenation agent. We report on our initial studies using cytochrome c peroxidase (CcP) as a platform to develop specific “peroxygenation” catalysts.
Results
The peroxygenase activity of CcP was investigated using 1-methoxynaphthalene as substrate. 1-Methoxynaphthalene hydroxylation was monitored using Russig’s blue formation at standard reaction conditions of 0.50 mM 1-methoxynaphthalene, 1.00 mM H2O2, pH 7.0, 25°C. Wild-type CcP catalyzes the hydroxylation of 1-methoxynaphthalene with a turnover number of 0.0044?±?0.0001 min-1. Three apolar distal heme pocket mutants of CcP were designed to enhance binding of 1-methoxynaphthalene near the heme, constructed, and tested for hydroxylation activity. The highest activity was observed for CcP(triAla), a triple mutant with Arg48, Trp51, and His52 simultaneously mutated to alanine residues. The turnover number of CcP(triAla) is 0.150?±?0.008 min-1, 34-fold greater than wild-type CcP and comparable to the naphthalene hydroxylation activity of rat liver microsomal cytochrome P450. While wild-type CcP is very stable to oxidative degradation by excess hydrogen peroxide, CcP(triAla) is inactivated within four cycles of the peroxygenase reaction.
Conclusions
Protein engineering of CcP can increase the rate of peroxygenation of apolar substrates but the initial constructs are more susceptible to oxidative degradation than wild-type enzyme. Further developments will require constructs with increased rates and selectivity while maintaining the stability of wild-type CcP toward oxidative degradation by hydrogen peroxide.
Colony stimulating factors (CSFs) are endogenous cytokines that have key roles in proliferation and differentiation of hematopoietic progenitor cells and in regulation of mature blood cells performance. The CSFs families members are widely used for therapeutic purposes in many field include microbial infections, in cancer chemotherapy, alzheimer disease, hematopoiesis process, and for some neutropenia- related pathologies. Crown ethers are chemical compounds with therapeutic application that can affect the colony formation in vitro. The primary objective of the present study is to evaluate the effect of TDN (novel crown ether) on colony formation of red bone marrow cells in incubation with lung tissues cells.
Method
In this study, bone marrow cells and lung tissue cells of Balb/C were used as a source of hematopoietic stem cells and a source to production colony-stimulating factors, respectively. These cells were incubated with TDN separately and together.
Results
Briefly, the results of this study show that the effects of TDN has excitatory in concentrations lower than 50 µg/ml on colony formation and greater than 50 µg/ml is toxic to cells and it was inhibited the colony formation. Maximum stimulatory and inhibitory effects are shown in 50 and 400 µg/ml of crown ether and no colony was observed in the latter concentration.
Conclusion
The results from this study indicate that TDN significantly able to stimulate the colon formation while increased concentrations of TDN is inhibited colony formation by induction toxic effects due to excessive production of free radicals.
A series of 2-(1H-benzo[d]imidazol-2-ylthio)-N-(substituted 4-oxothiazolidin-3-yl) acetamides was synthesized and characterized by physicochemical and spectral means. The synthesized compounds were evaluated for their in vitro antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Candida albicans and Aspergillus niger by tube dilution method. The in vitro cytotoxicity study of the compounds was carried out against human colorectal (HCT116) cell line. The most promising anticancer derivatives (5l, 5k, 5i and 5p) were further docked to study their binding efficacy to the active site of the cyclin-dependent kinase-8.
Results
All the compounds possessed significant antimicrobial activity with MIC in the range of 0.007 and 0.061 µM/ml. The cytotoxicity study revealed that almost all the derivatives were potent in inhibiting the growth of HCT116 cell line in comparison to the standard drug 5-fluorouracil. Compounds 5l and 5k (IC50 = 0.00005 and 0.00012 µM/ml, respectively) were highly cytotoxic towards HCT116 cell line in comparison to 5-fluorouracil (IC50 = 0.00615 µM/ml) taken as standard drug.
Conclusion
The molecular docking studies of potent anticancer compounds 5l, 5k, 5i and 5p showed their putative binding mode and significant interactions with cyclin-dependent kinase-8 as prospective agents for treating colon cancer.
Etravirine (ETV) was approved as the second generation drug for use in individuals infected with HIV-1 in 2008 by the U.S. FDA with its unique antiviral activity, high specificity, and low toxicity. However, there are some shortcomings of the existing synthetic routes, such as the long reaction time and poor yield.
Results
This article describes our efforts to develop an efficient, practical, microwave-promoted synthetic method for one key intermediate of ETV, which is capable of being operated on a scale-up synthesis level. Through this optimized synthetic procedure, the amination reaction time decreased from 12 h to 15 min and the overall yield improved from 30.4 to 38.5%.
Conclusion
Overall, we developed a practical synthesis of ETV via a microwave-promoted method, and the synthetic procedure could be amenable to scale-up, and production costs could be significantly lowered.
The defensive capacities of plant protease Inhibitors (PI) rely on inhibition of proteases in insect guts or those secreted by microorganisms; and also prevent uncontrolled proteolysis and offer protection against proteolytic enzymes of pathogens.
Methods
An array of chromatographic techniques were employed for purification, homogeneity was assessed by electrophoresis. Specificity, Ki value, nature of inhibition, complex formation was carried out by standard protocols. Action of SNTI on insect gut proteases was computationally evaluated by modeling the proteins by threading and docking studies by piper using Schrodinger tools.
Results
We have isolated and purified Soap Nut Trypsin Inhibitor (SNTI) by acetone fractionation, ammonium sulphate precipitation, ion exchange and gel permeation chromatography. The purified inhibitor was homogeneous by both gel filtration and polyacrylamide gel electrophoresis (PAGE). SNTI exhibited a molecular weight of 29 kDa on SDS-PAGE, gel filtration and was negative to Periodic Acid Schiff’s stain. SNTI inhibited trypsin and pronase of serine class. SNTI demonstrated non-competitive inhibition with a Ki value of 0.75?±?0.05×10-10 M. The monoheaded inhibitor formed a stable complex in 1:1 molar ratio. Action of SNTI was computationally evaluated on larval gut proteases from Helicoverpa armigera and Spodoptera frugiperda. SNTI and larval gut proteases were modeled and docked using Schrodinger software. Docking studies revealed strong hydrogen bond interactions between Lys10 and Pro71, Lys299 and Met80 and Van Der Waals interactions between Leu11 and Cys76amino acid residues of SNTI and protease from H. Armigera. Strong hydrogen bonds were observed between SNTI and protease of S. frugiperda at positions Thr79 and Arg80, Asp90 and Gly73, Asp2 and Gly160 respectively.
Conclusion
We conclude that SNTI potentially inhibits larval gut proteases of insects and the kinetics exhibited by the protease inhibitor further substantiates its efficacy against serine proteases.
Green approach in synthesizing metal nanoparticles has gain new interest from the researchers as metal nanoparticles were widely applied in medical equipment and household products. The use of plants in the synthesis of nanoparticles emerges as a cost effective and eco-friendly approach. A green synthetic route for the production of stable silver nanoparticles (Ag-NPs) by using aqueous silver nitrate as metal precursor and Artocarpus elasticus stem bark extract act both as reductant and stabilizer is being reported for the first time.
Results
The resultant Ag-NPs were characterized by UV–vis spectroscopy, powder X-Ray diffraction, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and Fourier-transform infra-red (FT-IR). The morphological study by TEM and SEM shows resultant Ag-NPs in spherical form with an average size of 5.81 ± 3.80, 6.95 ± 5.50, 12.39 ± 9.51, and 19.74 ± 9.70 nm at 3, 6, 24, and 48 h. Powder X-ray diffraction showed that the particles are crystalline in nature, with a face-centered cubic structure. The FT-IR spectrum shows prominent peaks appeared corresponds to different functional groups involved in synthesizing Ag-NPs.
Conclusions
Ag-NPs were synthesized using a simple and biosynthetic method by using methanolic extract of A. elasticus under room temperature, at different reaction time. The diameters of the biosynthesis Ag-NPs depended on the time of reaction. Thus, with the increase of reaction time in the room temperature the size of Ag-NPs increases. From the results obtained in this effort, one can affirm that A. elasticus can play an important role in the bioreduction and stabilization of silver ions to Ag-NPs.
Beta-galactosidase (EC 3.2.1.23), a commercially important enzyme, catalyses the hydrolysis of β-1,3- and β-1,4-galactosyl bonds of polymer or oligosaccharidesas well as transglycosylation of β-galactopyranosides. Due to catalytic properties; β-galactosidase might be useful in the milk industry to hydrolyze lactose and produce prebiotic GOS. The purpose of this study is to characterize β-galactosidase mutants from B. subtilis.
Results
Using error prone rolling circle amplification (epRCA) to characterize some random mutants of the β-galactosidase (LacA) from B. subtilisVTCC-DVN-12-01, amino acid A301 and F361 has been demonstrated significantly effect on hydrolysis activity of LacA. Mutants A301V and F361Y had markedly reduced hydrolysis activity to 23.69 and 43.22 %, respectively. Mutants the site-saturation of A301 reduced catalysis efficiency of LacA to 20–50 %, while the substitution of F361 by difference amino acids (except tyrosine) lost all of enzymatic activity, indicating that A301 and F361 are important for the catalytic function. Interestingly, the mutant F361Y exhibited enhanced significantly thermostability of enzyme at 45–50 °C. At 45 °C, LacA-361Y retained over 93 % of its original activity for 48 h of incubation, whereas LacA-WT and LacA-301Vwere lost completely after 12 and 24 h of incubation, respectively. The half-life times of LacA-361Y and LacA-301 V were about 26.8 and 2.4 times higher, respectively, in comparison to the half-life time of LacA-WT. At temperature optimum 50 °C, LacA-361Y shows more stable than LacA-WT and LacA-301 V, retaining 79.88 % of its original activities after 2 h of incubation, while the LacA-WT and LacA-301 V lost all essential activities. The half-life time of LacA-361Y was higher 12.7 and 9.39 times than that of LacA-WT and LacA-301 V, respectively. LacA-WT and mutant enzymes were stability at pH 5–9, retained over 90 % activity for 72 h of incubation at 30 °C. However, LacA-WT showed a little bit more stability than LacA-301 V and LacA-361Y at pH 4.
Conclusions
Our findings demonstrated that the amino acids A301V and F361 play important role in hydrolysis activity of β -galactosidase from B. subtilis. Specially, amino acid F361 had noteworthy effect on both catalytic and thermostability of LacA enzyme, suggesting that F361 is responsible for functional requirement of the GH42 family.
The extracts from the aerial parts of Portulaca quadrifida have been reported to show the total flavonoid content, antioxidant and antibacterial activities.
Results
Our results revealed that the total flavonoid content of methanol and chloroform extracts is 2.335?±?0.0097 and 1.7312?±?0.0082 mgQE/100 g respectively. The two extracts also showed good antioxidant activity and total phenolic content as well as weak to moderate antibacterial activity against some bacteria.
Conclusions
The extracts the aerial parts of the P. quadrifida showed good total flavonoid content, DPPH radical scavenging activity and antibacterial activity. In addition to this, the extracts also showed the presence of some important compounds by phytochemical analysis.
Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP.
Methods
Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP.
Results
In this work we used, after several optimization reactions, creatine kinase isoforms as well as ?NEK7 protein kinase in the absence or presence of ATP analogous inhibitors to validate this new luminescence method.
Conclusion
With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.
The carbapenem subclass of β-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of β-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 → 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb).
Results
Here, we report the X-ray crystal structures of MtbL,D-transpeptidase-2 (LdtMt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of LdtMt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by β-lyases.
Conclusion
The results presented here demonstrate biapenem and tebipenem bind to the outer cavity of LdtMt2, covalently inactivate the enzyme, and subsequently degrade via an S-conjugate elimination mechanism. We discuss structure based drug design based on the findings and propose that the S-conjugate elimination can be leveraged to design novel agents to deliver and locally release antimicrobial factors to act synergistically with the carbapenem carrier.
The details of a precise, accurate, and sensitive spectrophotometric method for measuring catalase activity are presented here. The assay was established for biological samples and depends on the rapid formation of a stable and colored carbonato-cobaltate (III) complex. Samples exhibiting catalase activity are incubated with hydrogen peroxide solution for 2 min prior to rapid mixing of the incubation enzymatic reaction mixture with cobalt-bicarbonate reagent, which assesses non-reacting hydrogen peroxide. Catalase activity is always directly proportional to the rate of dissociation of hydrogen peroxide. Hydrogen peroxide acts to oxidize cobalt (II) to cobalt (III) in the presence of bicarbonate ions; this process ends with the production of a carbonato-cobaltate (III) complex ([Co (CO3)3]Co). The formed end product has two maximum absorbance peaks: 440 nm and 640 nm. The 440-nm peak has been utilized for assessing catalase activity.
Results
The catalase activity results of the current method for erythrocyte lysate homogenates were computationally identical to those of the dichromate method (r?=?0.9950). The coefficient of variation was calculated to determine the imprecision of the current assay. The within-run and between-run results were 2.96 and 3.83%, respectively.
Conclusion
This method is appropriate for analyzing bacteria, red blood cells and liver and kidney tissue homogenates.
There is considerable interest from a regulatory and public health perspective in harmful and potentially harmful constituents in tobacco products, including smokeless tobacco products (STPs). A wide range of commercial STPs from the US and Sweden, representing 80–90 % of the 2010 market share for all the major STP categories in these two countries, were analysed for the IARC Group 2A carcinogen acrylamide. These STPs comprised the following styles: Swedish loose and portion snus, US snus, chewing tobacco, moist snuff, dry snuff, soft pellet, hard pellet and plug.
Results
Acrylamide was detected in all the products tested and quantified in all but one product. Concentrations ranged from 62 to 666 ng/g wet weight basis (WWB). The average levels of acrylamide (WWB) by type of STP were not significantly different (p > 0.05) except for US snus which had, on average, greater levels but with a very wide range of individual levels according to the manufacturer. Acrylamide levels in STPs were significantly and positively correlated with pH, but not with levels of either reducing sugars or ammonia nitrogen. Levels of acrylamide increased by sixfold or more (on a dry weight basis) during manufacture of a snus sample and then decreased during subsequent storage for up to 22 weeks. Acrylamide generation in tobacco generally appears to occur at lower temperatures, but longer time scales than found with food production.
Conclusions
Acrylamide is a common contaminant of STPs, formed through heat treatment of tobacco. Our data show that exposure to acrylamide from consumption of STPs is small compared with exposure from food consumption or cigarette smoking.
1,2-Dichlorobenzene (1,2-DCB) is a benzene-derived molecule with two Cl atoms that is commonly utilized in the synthesis of pesticides. 1,2-DCB can be absorbed by living creatures and its effects on naturally-occurring enzymatic systems, including the effects on Ca2+-ATPases, have been poorly studied. Therefore, we aimed to study the effect of 1,2-DCB on the Ca2+-ATPase from sarcoplasmic reticulum (SERCA), a critical regulator of intracellular Ca2+ concentration.
Results
Concentrations of 0.05–0.2 mM of 1,2-DCB were able to stimulate the hydrolytic activity of SERCA in a medium-containing Ca2+-ionophore. At higher concentrations (0.25–0.75 mM), 1,2-DCB inhibited the ATP hydrolysis to ~80 %. Moreover, ATP hydrolysis and Ca2+ uptake in a medium supported by K-oxalate showed that starting at 0.05 mM,1,2-DCB was able to uncouple the ratio of hydrolysis/Ca2+ transported. The effect of this compound on the integrity of the SR membrane loaded with Ca2+ remained unaffected. Finally, the analysis of phosphorylation of SERCA by [γ-32P]ATP, starting under different conditions at 0° or 25 °C showed a reduction in the phosphoenzyme levels by 1,2-DCB, mostly at 0 °C.
Conclusions
The temperature-dependent decreased levels of phosphoenzyme by 1,2-DCB could be due to the acceleration of the dephosphorylation mechanism – E2P?·?Ca2 state to E2 and Pi, which explains the uncoupling of the ATP hydrolysis from the Ca2+ transport.
