Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

Immobilization and phytoavailability of cadmium in variable charge soils. I. Effect of phosphate addition

The effect of phosphate on the surface charge and cadmium (Cd) adsorption was examined in seven soils that varied in their variable-charge components. The effect of phosphate on immobilization and phytoavailability of Cd from one of the soils, treated with various levels of Cd (0–10 mg Cd kg^–1 soil), was further evaluated using mustard (Brassica juncea L.) plants. Cadmium immobilization in soil was evaluated by a chemical fractionation scheme. Addition of phosphate, as KH₂PO₄, increased the pH, negative charge and Cd adsorption by the soils. Of the seven soils examined, the three allophanic soils (i.e., Egmont, Patua and Ramiha) exhibited greater increases in phosphate-induced pH, negative charge and Cd²⁺ adsorption over the other four non-allophanic soils (i.e., Ballantrae, Foxton, Manawatu ad Tokomaru). Increasing addition of Cd enhanced Cd concentration in plants, resulting in decreased plant growth (i.e., phytotoxicity). Addition of phosphate effectively reduced the phytotoxicity of Cd. There was a significant inverse relationship between dry matter yield and Cd concentration in soil solution. Addition of phosphate decreased the concentration of the soluble + exchangeable Cd fraction but increased the concentration of inorganic-bound Cd fraction in soil. The phosphate-induced alleviation of Cd phytotoxicity can be attributed primarily to Cd immobilization due to increases in pH and surface charge.     

Reversible and time-dependent inhibition of the hepatic cytochrome P450 steroidal hydroxylases by the proestrogenic pesticide methoxychlor in rat and human

Methoxychlor, a currently used pesticide, is demethylated and hydroxylated by several hepatic microsomal cytochrome P450 enzymes. Also, methoxychlor undergoes metabolic activation, yielding a reactive intermediate (M*) that binds irreversibly and apparently covalently to microsomal proteins. The study investigated whether methoxychlor could inhibit or inactivate certain liver microsomal P450 enzymes. The regioselective and stereoselective hydrox-ylation of testosterone and the 2-hydroxylation of estradiol (E₂) were utilized as markers of the P450 enzymes inhibited by methoxychlor. Both reversible and time-dependent inhibition were examined. Coincubation of methoxychlor and testosterone with liver microsomes from phenobarbital treated (PB-microsomes) male rats, yielded marked diminution of 2α- and 16α-testosterone hydroxylation, indicating strong inhibition of P4502C11 (P450h). Methoxychlor moderately inhibited 2β-, 7α-, 15α-, 15β-, and 16β-hydroxylation and androstenedi-one formation. There was only a weak inhibition of 6β-ydroxylation of testosterone. The methox-ychlor-mediated inhibition of 6β-hydroxylation was competitive. By contrast, when methoxychlor was permitted to be metabolized by PB-microsomes or by liver microsomes from pregnenolone-16α-car-bonitrile treated rats (PCN-microsomes) prior to addition of testosterone, a pronounced time-dependent inhibition of 6β-hydroxylation was observed, suggesting that methoxychlor inactivates the P450 3A isozyme(s). The di-demethylated methoxychlor (bis-OH-M) and the tris-hydroxy (ca-techol) methoxychlor metabolite (tris-OH-M) inhibited 6β-hydroxylation in PB-microsomes competitively and noncompetitively, respectively; however, these methoxychlor metabolites did not exhibit a time-dependent inhibition. Methoxychlor inhibited competitively the formation of 7α-hydroxytestosterone (7α-OH-T) and 16α-hydroxy-testosterone (16α-OH-T) but exhibited little or no time-dependent inhibition of generation of these metabolites, indicating that P450s 2A1, 2B1/B2, and 2C11 were inhibited but not inactivated. Methoxychlor inhibited in a time-dependent fashion the 2-hydroxylation of E2 in PB-microsomes. However, bis-OH-M exhibited solely reversible inhibition of the 2-hydroxylation, supporting our conclusion that the inactivation of P450s does not involve participation of the demethylated metabolites. Both competitive inhibition and time-dependent inactivation of human liver P450 3A (6β-hydroxylase) by methoxychlor, was observed. As with rat liver microsomes, the human 6β-hydroxylase was inhibited by bis-OH-M and tris-OH-M competitively and noncompetitively, respectively. Testosterone and estradiol strongly inhibited the irreversible binding of methoxychlor to microsomal proteins. This might explain the “clean” competitive inhibition by methoxychlor of the 6β-OH-T formation when the compounds were coin-cubated. Glutathione (GSH) has been shown to interfere with the irreversible binding of methoxychlor to PB-microsomal proteins. The finding that the coincubation of GSH with methoxychlor partially diminishes the time-dependent inhibition of 6β-hydroxylation provides supportive evidence that the inactivation of P450 3A isozymes by methoxychlor is related to the formation of M*.     

5'-Nucleotidase activity of lymphocyte plasma membranes. Effect of concanavalin A]

The 5'-nucleotidase properties of isolated lymphocyte plasma membranes from young pig mesenteric nodes are described; nucleosides-5'-monophosphates are the substrates of this specific enzyme. Concanavalin A inhibits this enzyme; on the same membranes this mitogen does not affect alkaline phosphatase and activates the membrane bound (Ca2+) ATPase. The 5'-nucleotidase inhibition is due to a specific interaction of Con A with carbohydrate groups of the membrane; its high positive cooperativity suggests that the lectin promotes reorganization of the membrane bound 5'-nucleotidase. Solubilization of the 5'-nucleotidase does not prevent the effect of Con A and the solubilized enzyme is firmly bound by Con A-Sepharose 4B; these results suggest that Con A inhibits the enzyme by a direct interaction and that 5'-nucleotidase can be considered as an eventual receptor for the lectin.     

Detection of plasmid-mediated quinolone resistance in clinical isolates of Enterobacteriaceae strains in Hamadan,West of Iran

Plasmid mediated quinolone resistance (PMQR) determinants have arisen as a significant concern in recent years. The aim of this study was screening of resistant-clinical isolates to fluoroquinolone antibiotics and detection of qnr and aac(6′)-Ib-cr genes.For this purpose we collected 100 fluoroquinolone-resistant Enterobacteriaceae which were from 3 hospitals in Hamadan, west provinces of Iran, between October 2012 and June 2013. The all samples were identified by biochemical tests and confirmed by PCR method. Antimicrobial susceptibility to 14 antimicrobial agents including levofloxacin and ciprofloxacin were determined by disk diffusion methods and ciprofloxacin MIC was obtained by broth microdilution method as Clinical Laboratory Standards Institute (CLSI) recommendations. The isolates were screened for the presence of qnrA, qnrB, qnrS and aac(6′)-Ib-cr genes using PCR assay. Among the screened isolates, 64 strains (64%) of Escherichia coli, 23 strains (23%) of Klebsiella pneumoniae, 13 strains (13%) of Proteus mirabilis were collected as quinolone-resistant isolates. out of 100 isolates, two (2%) were positive for qnrS, seventeen (17%) isolates were positive for qnrB and we did not find qnrA gene in any of the isolates. There were also 32 positive isolates for aac(6′)-Ib-cr determinant. We described the prevalence of qnr and aac(6′)-Ib-cr genes in fluoroquinolone-resistant Enterobacteriaceae in Hamadan city. The carriage rate of multidrug-resistant Enterobacteriaceae in healthy people in Hamadan City is extremely high. Moreover, genes encoding transferable quinolones, in particular aac(6′)-Ib-cr, are highly prevalent in these strains.     

Verification of a virtual fields method to extract the mechanical properties of human optic nerve head tissues in vivo

We aimed to verify a custom virtual fields method (VFM) to estimate the patient-specific biomechanical properties of human optic nerve head (ONH) tissues, given their full-field deformations induced by intraocular pressure (IOP). To verify the accuracy of VFM, we first generated ‘artificial’ ONH displacements from predetermined (known) ONH tissue biomechanical properties using finite element analysis. Using such deformations, if we are able to match back the known biomechanical properties, it would indicate that our VFM technique is accurate. The peripapillary sclera was assumed anisotropic hyperelastic, while all other ONH tissues were considered isotropic. The simulated ONH displacements were fed into the VFM algorithm to extract back the biomechanical properties. The robustness of VFM was also tested against rigid body motions and noise added to the simulated displacements. Then, the computational speed of VFM was compared to that of a gold-standard stiffness measurement method (inverse finite element method or IFEM). Finally, as proof of principle, VFM was applied to IOP-induced ONH deformation data (obtained from one subject’s eye imaged with OCT), and the biomechanical properties of the prelamina and lamina cribrosa (LC) were extracted. From given ONH displacements, VFM successfully matched back the biomechanical properties of ONH tissues with high accuracy and efficiency. For all parameters, the percentage errors were less than 0.05%. Our method was insensitive to rigid body motions and was also able to recover the material parameters in the presence of noise. VFM was also found 125 times faster than the gold-standard IFEM. Finally, the estimated shear modulus for the prelamina and the LC of the studied subject’s eye were 33.7 and 63.5 kPa, respectively. VFM may be capable of measuring the biomechanical properties of ONH tissues with high speed and accuracy. It has potential in identifying patient-specific ONH biomechanical properties in the clinic if combined with optical coherence tomography.