Cloning and characterization of a putative β-glucosidase (NfBGL595) from Neosartorya fischeri

Whole genome sequence of Neosartorya fischeri NRRL181 revealed four putative GH1 β-glucosidases (BGLs). One BGL, NfBGL595 was successfully expressed and characterized. DNA sequence analysis revealed an open reading frame of 1590 bp, encoding a polypeptide of 529 amino acid residues. The gene was cloned in pET28a and overexpressed in Escherichia coli. The purified recombinant BGL showed high levels of catalytic activity, with V_max of 1693 U mg-protein⁻¹ and a K_m of 2.8 mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature and pH for enzyme activity were 40 °C and 6.0, respectively. The enzyme exhibited broad substrate specificity towards aryl glycosides including pNP-mannose, pNP-galactose, pNP-xylose, and pNP-cellobioside. A homology model of NfBGL595 was constructed based on the X-ray crystal structure of Trichoderma reesei BGL2. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose, shed light on the substrate specificity of N. fischeri BGL595 only towards aryl glycoside.     

Mutagenicity and metabolism of dimethylnitrosamine and benzo[α]pyrene in tissue homogenates from inbred syrian hamsters treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls

There are significant differences between mice and hamsters in polycyclic hydrocarbon and nitrosamine metabolism. Homogenates of liver, lung and intestinal mucosa from 6 strains of Syrian golden hamster were compared for their ability to metabolize benzo[α]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains MHA/SsLak, LSH/SsLak, CB/SsLak, PD4/Lak LHC/Lak and Lak:LVG (SYR) were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. Dimethyl-nitrosamine demethylase (DMND) was assayed using 1 mM DMN as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured using benzo[α]pyrene as substrate. MC does not induce AHH activity in hamster liver, but is an excellent inducer of enzymes converting BP to mutagens. This lack of correlation between increased AHH activity and increased metabolism of BP to mutagen in liver is in marked contrast to correlations seen in mice. MC induces AHH in hamster lung and intestinal mucosa. AR induces AHH in liver, lung and intestinal mucosa. Activity of DMND in liver is not affected by treatment of hamsters with BP or AR, but is repressed approx. 30% by treatment with MC.     

Binding of benzo(α)pyrene, ellipticine, and cis-parinaric acid to β-lactoglobulin: Influence of protein modifications

The binding of benzo()pyrene, ellipticine, and cis-parinaric acid to native, esterified, and alkylated -lactoglobulin was followed by enhancement of the ligand fluorescence. Three studied ligands bind to native or modified -lactoglobulin in apparent molar ratios varying between 1/8 and 2/1, with apparent dissociation constants in the range of 10^–8 M for ligand/-lactoglobulin complexes. The studied, chemically modified -lactoglobulin derivatives display higher binding affinities for all studied ligands, cis-parinaric acid excluded. The reductive alkylation of -NH₂ lysyl residues of -lactoglobulin increases the apparent molar ratios of benzo()pyrene and cis-parinaric acid, and decreases it for ellipticine. The esterified and native -lactoglobulin complexed to the investigated ligands display similar stoichiometries. Dynamic light scattering study of ligand--lactoglobulin complexes in solution shows the formation of aggregates: the apparent hydrodynamic radius value of -lactoglobulin dimer (3.4 nm) reaches 49, 46, and 74 nm upon addition and binding of benzo()pyrene, ellipticine, and cis-parinaric acid, respectively.     

Cloning,expression and characterization of Bombyx mori α1,6-fucosyltransferase

Although core α1,6-fucosylation is commonly observed in N-glycans of both vertebrates and invertebrates, the responsible enzyme, α1,6-fucosyltransferase, has been much less characterized in invertebrates compared to vertebrates. To investigate the functions of α1,6-fucosyltransferase in insects, we cloned the cDNA for the α1,6-fucosyltransferase from Bombyx mori (Bmα1,6FucT) and characterized the recombinant enzyme prepared using insect cell lines. The coding region of Bmα1,6FucT consists of 1737 bp that code for 578 amino acids of the deduced amino acid sequence, showing significant similarity to other α1,6-fucosyltransferases. Enzyme activity assays demonstrated that Bmα1,6FucT is enzymatically active in spite of being less active compared to the human enzyme. The findings also indicate that Bmα1,6FucT, unlike human enzyme, is N-glycosylated and forms a disulfide-bonded homodimer. These findings contribute to a better understanding of roles of α1,6-fucosylation in invertebrates and also to the development of the more efficient engineering of N-glycosylation of recombinant glycoproteins in insect cells.     

Covalent binding of benzo[a]pyrene to cytochrome P-450βNF-B2 and other proteins in reconstituted mixed-function oxidase systems

A reconstituted mixed-function oxidase system, containing the major β-naphthoflavone-induced isozyme of rat liver cytochrome P-450 bound benzo[a]pyrene covalently in the presence of NADPH. NADPH-cytochrome P-450 reductase was required for binding and a maximum rate of adduct formation was obtained at 8 units of reductase per nmol cytochrome P-450. Phosphatidylcholine inhibited this reaction. Benzo[a]pyrene was bound to the cytochrome, but not to the reductase, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Approximately 6 molecules of benzo[a]pyrene bound to each molecule cytochrome P-450 during prolonged incubations. No binding occurred when the β-naphthoflavone-induced isozyme of cytochrome P-450 was replaced by the major isozyme induced by phenobarbital, but both cytochromes incorporated benzo[a]pyrene to approximately the same extent when they were incubated together in the presence of the reductase and NADPH. Metabolically activated benzo[a]pyrene also bound covalently to purified epoxide hydrodrolase, when this enzyme was added to the reconstituted mixed-function oxidase system.     

Nuclear metabolism of benzo(a)pyrene and of (±)-trans-7,8-dihydroxy-7,8,-dihydrobenzo(a)pyrene: Comparative chromatographic analysis of alkylated DNA

Rat liver nuclei were incubated with [¹⁴C]benzo(a)pyrene (BP) or [³H](±)-trans-7,8-dihydrodiol of BP (³H-BP-7,8-diol) in the presence of a NADPH-generating system. The nuclei were able to form from BP the 9,10-, 4,5- and 7,8-dihydrodiols, the 3,6- and 1,6-quinones as well as the 3- and 9-phenols. The total nuclear metabolism was stimulated 11-fold by prior administration to the rats of 3-methylcholanthrene (3MC). BP-7,8-dihydrodiol formation, under these circumstances, was enhanced 29-fold. The rat liver nuclei were also able to form from [³H]BP-7,8-diol, (±)-7β,8α-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 1), (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 2), as well as three unknown metabolites. Diol epoxides 1 and 2 represented 23 and 65% of the total metabolites produced during the control nuclear incubation. Pretreatment of the rats with 3MC resulted in 4-fold increase in nuclear metabolic activity. Under the latter circumstances, the diol epoxides 1 and 2 represented 43 and 38%, respectively, of the total nuclear metabolites. Incubation of liver nuclei with labeled BP or BP-7,8-diol in the presence of NADPH resulted in alkylation of DNA. The alkylated deoxyribonucleosides were separated by Sephadex LH-20 chromatography. Two peaks of radioactivity were noted after incubation with the parent polycyclic hydrocarbon while only one peak was seen after incubation with the diol derivative. These results emphasize the importance of nuclei in the metabolism of BP and in the subsequent alkylation of DNA, reactions which may be related to mutagenesis or carcinogenesis.     

Combinatorial screening of polymer precursors for preparation of benzo[α] pyrene imprinted polymer: an ab initio computational approach

A combinatorial screening procedure was used for the selection of polymer precursors in the preparation of molecularly imprinted polymer (MIP), which is useful in the detection of the air pollution marker molecule benzo[a]pyrene (BAP). Molecular imprinting is a technique for the preparation of polymer materials with specific molecular recognition receptors. The preparation of imprinted polymers requires polymer precursors such as functional monomer, cross-linking monomer, solvent, an initiator of polymerization and thermal or UV radiation. A virtual library of functional monomers was prepared based on interaction binding scores computed using HyperChem Release 8.0 software. Initially, the possible minimum energy conformation of the monomers and BAP were optimized using the semi-empirical (PM3) quantum method. The binding energy between the functional monomer and the template (BAP) was computed using the Hartree-Fock (HF) method with 6-31 G basis set, which is an ab initio approach based on Moller-Plesset second order perturbation theory (MP2). From the computations, methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) were selected for preparation of BAP imprinted polymer. The larger interaction energy (ΔE) represents possibility of more affinity binding sites formation in the polymer, which provides high binding capacity. The theoretical predictions were complimented through adsorption experiments. There is a good agreement between experimental binding results and theoretical computations, which provides further evidence of the validity of the usefulness of computational screening procedures in the selection of appropriate MIP precursors in an experiment-free way.     

Purification and characterisation of β-N-acetylhexosaminidase from the butter clam, saxidomus purpuratus

A β-N-acetylhexosaminidase [EC 3.2.1.30] has been purified ～98-fold from an extract of the digestive organs of Saxidomus purpuratus by using ammonium sulfate fractionation, and chromatography on Toyopearl HW-50, CM-cellulose, and Sepharose 4B. The purified enzyme, the molecular weight of which was estimated to be ～66,000 by gel filtration, was composed of two sub-units of molecular weight 30,000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified enzyme had a pH optimum of 3.8 and an optimum temperature of 55°, and its activity was enhanced ～2-fold in the presence of 0.1m sodium chloride. The Michaelis constants toward p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucoside and -galactoside were 1.2 × 10^?4 and 1.3 × 10^?4m, respectively.     

Purification and characterisation of glutathione S-transferases from the freshwater clam Corbicula fluminea (Müller)

Glutathione S-transferases (GSTs) are involved in the phase II detoxification metabolism. To provide a molecular basis for their use as biomarkers of pollution, cytosolic GSTs from the freshwater clam Corbicula fluminea have been purified by glutathione-Sepharose affinity chromatography, anion-exchange chromatography (AEC) and reversed-phase (RP) HPLC. SDS-PAGE of visceral mass (VM) affinity-purified extracts revealed four subunits with apparent molecular masses (MW) of 30.2, 29.2, 28.5 and 27.2 kDa. Analysis by non-denaturing PAGE revealed three acidic dimeric proteins with apparent MW of 64, 55 and 45 kDa, named GSTc1, GSTc2 and GSTc3, respectively, based on their elution order by AEC. Only GSTc2 and GSTc3 exhibited GST activity towards 1-chloro-2,4-dinitrobenzene. A tissue-specific subunit pattern was obtained by RP-HPLC of affinity-purified extracts from VM and gills (GI): three major peaks were resolved, one of which was common to both tissues. MW of each VM subunit was determined by electrospray ionisation-mass spectrometry: 23602+/-1 Da for the major subunit and 23289+/-1 Da for the minor ones. Immunoblot analysis revealed all subunits from both tissues were related to the Pi-class GSTs. In addition, minor VM subunits were slightly related to the Mu-class ones. The interest of such molecular studies in biomonitoring programs is discussed.     

Structure and mechanism of error-free replication past the major benzo[a]pyrene adduct by human DNA polymerase κ

Benzo[a]pyrene (BP) is a well-known and frequently encountered carcinogen which generates a bulky DNA adduct (+)-trans-10S-BP-N²-dG (BP-dG) in cells. DNA polymerase kappa (polκ) is the only known Y-family polymerase that bypasses BP-dG accurately and thus protects cells from genotoxic BP. Here, we report the structures of human polκ in complex with DNA containing either a normal guanine (G) base or a BP-dG adduct at the active site and a correct deoxycytidine. The structures and supporting biochemical data reveal a unique mechanism for accurate replication by translesion synthesis past the major bulky adduct. The active site of polκ opens at the minor groove side of the DNA substrate to accommodate the bulky BP-dG that is attached there. More importantly, polκ stabilizes the lesion DNA substrate in the same active conformation as for regular B-form DNA substrates and the bulky BPDE ring in a 5′ end pointing conformation. The BP-dG adducted DNA substrate maintains a Watson–Crick (BP-dG:dC) base pair within the active site, governing correct nucleotide insertion opposite the bulky adduct. In addition, polκ''s unique N-clasp domain supports the open conformation of the enzyme and the extended conformation of the single-stranded template to allow bypass of the bulky lesion. This work illustrates the first molecular mechanism for how a bulky major adduct is replicated accurately without strand misalignment and mis-insertion.     

Toxoplasma gondii infections in cats from Paraná, Brazil: seroprevalence, tissue distribution, and biologic and genetic characterization of isolates

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete environmentally resistant oocysts. The prevalence of T. gondii was determined in 58 domestic cats from 51 homes from Santa Isabel do Ivai, Parana State, Brazil where a water-associated outbreak of acute toxoplasmosis had occurred in humans. Antibodies to T. gondii were found with the modified agglutination test in 49 of 58 (84.4%) cats at a serum dilution of 1:20. Tissues (brain, heart, and skeletal muscle) of 54 of these cats were bioassayed in T. gondii-free, laboratory-reared cats; T. gondii oocysts were excreted by 33 cats that were fed feline tissues. Brains from these 54 cats were bioassayed in mice; T. gondii was isolated from 7. Skeletal muscles and hearts of 15 cats were also bioassayed in mice; T. gondii was isolated from skeletal muscles of 9 and hearts of 13. The results indicate that T. gondii localizes in muscle tissue more than the brains of cats. In total there were 37 T. gondii isolates from 54 cats. Most isolates of T. gondii were virulent for mice. Genotyping of the 37 isolates of T. gondii, using the SAG2 locus, revealed that 15 isolates were type I and 22 were type III. The absence of type II genotype in cats in this study is consistent with the previous studies on T. gondii isolates from Brazil and is noteworthy because most T. gondii isolates from the United States are type II. These findings support the view that Brazilian and North American T. gondii isolates are genetically distinct. This is the first report of genotyping of T. gondii isolates from the domestic cat.     

Cloning,purification and characterization of a thermostable β-galactosidase from Thermotoga naphthophila RUK-10

A novel β-galactosidase gene (Tnap1577) from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 was cloned and expressed in Escherichia coli BL21 (DE3) cells to produce β-galactosidase. The recombinant β-galactosidase was purified in three steps: heat treatment to deactivate E. coli proteins, Ni-NTA affinity chromatography and Q-sepharose chromatography. The optimum temperatures for the hydrolysis of o-nitrophenyl-β-d-galactoside (o-NPG) and lactose with the recombinant β-galactosidase were found to be 90 °C and 70 °C, respectively. The corresponding optimum pH values were 6.8 and 5.8, respectively. The molecular mass of the enzyme was estimated to be 70 kDa by SDS-PAGE analysis. Thermostability studies showed that the half-lives of the recombinant enzyme at 75 °C, 80 °C, 85 °C and 90 °C were 10.5, 4, 1, and 0.3 h, respectively. Kinetic studies on the recombinant β-galactosidase revealed K_m values for the hydrolysis of o-NPG and lactose of 1.31 mM and 1.43 mM, respectively. These values are considerably lower than those reported for other hyperthermophilic β-galactosidases, indicating high intrinsic affinity for these substrates. The recombinant β-galactosidase from Thermotoga naphthophila RUK-10 also showed transglycosylation activity in the synthesis of alkyl galactopyranoside. This additional activity suggests the enzyme has potential for broader biotechnological applications beyond the degradation of lactose.     

Cloning,expression and biochemical characterization of a GH1 β-glucosidase from Cellulosimicrobium cellulans

β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase ccbgl1b gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of Cellulosimicrobium cellulans and expressed in Escherichia coli BL21 cells. The sequence contained an open reading frame of 1494?bp, encoded a polypeptide of 497?amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57?kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55?°C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides p-nitrophenyl-β-D-glucopyranoside (pNPβGlc), followed by p-nitrophenyl-β-D-galactopyranoside (pNPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from C. cellulans works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation.     

Schistosoma japonicum: Cloning, expression and characterization of a gene encoding the α5-subunit of the proteasome

The development of an effective vaccine against the schistosome is thought to be the most desirable means to control schistosomiasis, even though there is an effective means of chemotherapy with praziquantel. A full-length cDNA encoding the Schistosoma japonicum proteasome subunit alpha type 5 protein (SjPSMA5) was first isolated from 18-day-schistosomulum cDNAs. The cDNA had an open reading frame (ORF) of 747 bp and encoded 248 amino acids. Real-time quantitative RT-PCR analysis revealed that SjPSMA5 is up-regulated in 18-day and 32-day schistosomes, and the level of expression in male is around fourfold higher than that in female worms at 42 days. The SjPSMA5 was subcloned into pET28a(+) and expressed as inclusion bodies in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjPSMA5 (rSjPSMA5) was immunogenic. After immunization of BALB/c mice with rSjPSMA5, reductions of 23.29% and 35.24% were obtained in the numbers of worms and eggs in the liver, respectively. The levels of specific IgG antibodies and cells were significantly higher (P < 0.01) in the group vaccinated with rSjPSMA5 combined with Seppic 206 adjuvant than in the other groups, as detected by enzyme linked immunosorbent assay (ELISA) and flow cytometry. The study suggested that rSjPSMA5 induced partial immunoprotection against S. japonicum in BALB/c mice, and it could be a potential vaccine candidate against schistosomiasis.     

Induction of cytochrome P-450 and P-448 in the outer membrane of mouse liver nuclei by phenobarbital and benzo [α-]pyrene

This investigation confirms the presence of the inducible mixed function hydroxylase enzyme system in nuclear membranes. The cytochrome P-450 spectrum and demethylase activity, markers of the enzyme system, were used to define its localization to the outer membrane envelope. Intact BALB/c mouse liver nuclei isolated and purified in Mg⁺⁺ sucrose media of low ionic strength gave CO-dithionite reduced difference spectra of cytochrome P-450 and P-448. Phenobarbital induced P-450 by 40% while the carcinogenic hydrocarbon, benzo [α] pyrene, induced P-448 twofold. A corresponding increase was also observed in the microsomes of the same tissue preparations. No microsomal contamination of nuclear preparations was found. Intact nuclei stripped of their outer membrane by 0.5% Triton X-100 treatment resulted in a striking absence of the P-450 which, however, was found to be present in isolated outer nuclear membranes.     

Interaction of benzo[a]pyrene, 2,3,3′,4,4′,5 hexachlorobiphenyl (PCB 156) and cadmium on biomarker responses in flounder (Platichthys flesus L.)

Interactive effects of a mixed pollutant exposure on biomarker responses were studied in European flounder (Platichthys flesus L.). The model chemicals, benzo[a]pyrene (BaP, 2.5 mg kg^-1), 2,3,3′,4,4′5 hexachlorobiphenyl (PCB-156, 2.5 mg kg^-1), and cadmium (cadmium, 1 mg kg^-1), were administered to fish by subcutaneous injections. Biomarker responses were quantified both following administration of single chemicals and sequential combinations of the chemicals in pairs. Significant induction of CYP1A protein levels and corresponding ethoxyresorufin-O-deethylase (EROD) activities was observed in BaP and PCB treated flounder after 2 and 8 days, respectively. The strongest induction (44 fold) was caused by BaP. No further induction was observed after additional treatment with PCB 156. CYP1A induction caused by BaP was inhibited (40% compared with BaP treatment alone) in flounder pre treated with cadmium, whereas induction by PCB 156 appeared to be unaffected by pre treatment with cadmium. Flounder treated with cadmium only had significantly elevated hepatic levels of metallothionein (MT) after 15 days. Pre treatment with BaP and PCB prior to cadmium inhibited the MT induction (30-50%) compared with cadmium alone. Furthermore, significantly higher glutathione S transferase activities were observed in flounder administered cadmium alone, and in flounder treated with BaP or PCB 156 prior to cadmium. GST selenium independent peroxidase activities appeared to be unaffected by any of the treatments in the present study. The results indicate that chemical mixtures may affect biomarker responses differently from compounds administered alone, and that the sensitivity of both CYP1A and MT are influenced by pollutants other than their primary inducers.