A Phanerochaete chrysosporium mutant defective in lignin degradation as well as several other secondary metabolic functions

A pleiotropic mutant of Phanerochaete chrysosporium 104-2 lacking phenol oxidase and unable to form fruit bodies and a revertant strain 424-2 were isolated after UV mutagenesis. Strains 104-2 and 424-2 had no apparent dysfunction in primary metabolism with glucose as a carbon source. Unlike the wild type strain and strain 424-2, strain 104-2 was unable to evolve ¹⁴CO₂ from ¹⁴C ring, side chain and 3-O-¹⁴C-methoxy labeled lignin. In addition, strain 104-2 was unable to evolve ¹⁴CO₂ from a variety of lignin model compounds including ¹⁴C-4-methoxy labeled veratrylglycerol--guaiacyl (V) ether, -¹⁴C-guaiacylglycerol--guaiacyl ether (VI), as well as 1-(¹⁴C-4-methoxy, 3-methoxyphenyl)1,2 propene (III) and 1-(¹⁴C-4-methoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IV). The addition of peroxidase/H₂O₂ to cultures of strain 104-2 did not alter its capacity to degrade the labeled lignins. A variety of unlabeled lignin model compounds previously shown to be degraded by the wild type organism including -aryl ether dimers and diaryl propane dimers were also not degraded by the mutant 104-2. The revertant strain 424-2 regained the capacity to degrade these compounds. The substrates described are degraded by oxygen requiring system(s) expressed during the secondary phase of growth, suggesting this pleiotropic mutant is possibly defective in the onset of postprimary metabolism. The inability of the mutant to produce the secondary metabolite veratryl alcohol and to elaborate enzymes in the veratryl alcohol biosynthetic pathway supports this hypothesis.Abbreviations GLC gas liquid chromatography - TMSi trimethylsilyl - MS mass spectrometry - LDS lignin degrading system     

Quantitative structure–activity relationship study on some benzodiazepine derivatives as anti-Alzheimer agents

A QSAR study was performed in an attempt to explore the pharmacophore of some benzodiazepine derivatives as anti-Alzheimer agents for the inhibition of -secretase. The study, which used the electrotopological state atom (ETSA) index, which encodes electronic and topological information, reveals the importance of two phenyl rings—one substituted and another unsubstituted, for the inhibition of the enzyme. Fluorine substitution on the substituted phenyl ring has an important contribution to the activity. R-configurations of the aliphatic chain substituents provide the exact conformation of the molecules to enter into the binding pockets of the receptor(s).Figure General structure of benzodiazepine containing -secretase inhibitors     

AIPAR: ab initio parametrization of intermolecular potentials for computer simulations

An unambiguous, fully ab initio and automated technique denoted AIPAR (ab initio parametrization) implemented in the SJBR program has been proposed to yield intermolecular interaction potentials between polar molecules and water. The AIPAR procedure has been applied to several organic molecules covering a wide range of structure and functional groups, namely methanol, acetone (propanone), methanethiol (methyl mercaptan), imidazole (1,3-diazole), oxazole and furan. The AIPAR-derived sets of parameters compare well with the empirical OPLS ones, mainly when the all-atoms model is employed in the OPLS procedure. Monte Carlo simulations were performed for an aqueous solution of methanol and for an equimolar binary mixture methanol–water using the AIPAR and OPLS parameters. The thermodynamic and geometric results obtained with the parameters obtained with the AIPAR procedure compare favorably with the OPLS simulations, even for the binary mixture, demonstrating the precision, robustness and transferability of the parameters obtained with the AIPAR procedure.Figure Superimposed configurations of water (without the hydrogen atoms) around the methanol molecule obtained with the AGOA procedure.     

Determination of fuzzy logic membership functions using genetic algorithms: application to structure–odor modeling

Fuzzy logic has been used as a tool in structure–camphoraceous odor relationships. The data base studied included 99 molecules. The rules used to discriminate between camphor and non camphor molecules lead to 77% correct discrimination. Such rules account for the shape and the size of the molecule. Their adjustment by means of genetic algorithms led to 84% correct discrimination between camphor and non-camphor molecules.Figure Membership function for the chosen variables     

Modulation of Adenosine 5′-Monophosphate Adsorption Onto Aqueous Resident Pyrite: Potential Mechanisms for Prebiotic Reactions

The adsorption of adenosine 5-monophosphate (5-AMP) ontopyrite (FeS₂) and its modulation by acetate, an organic precursor of complex metabolic pathways, was studied in aqueousmedia that simulate primitive environments. 5-AMP adsorptionrequires divalent cations, indicating that a cationic bridge mediates its attachment to negatively charged sites of the mineral surface. The isotherm of 5-AMP adsorption exhibits a strong cooperative effect at low nucleotide concentrations inacetate-rich medium, whereas high levels of adsorption were only found at high nucleotide concentrations in a model of primitive seawater (acetate free). The modulating role of acetate is also evidenced in the presence of high dipolar moment molecules: dimethyl sulfoxide (Me₂SO) and dimethylformamide (DMF) strongly inhibit 5-AMP adsorption in acetate-rich media, whereas no effect of DMF was found in artificial seawater. The observation that exogenous divalentcations are not needed for acetate attachment onto FeS₂ reveals that organic acids can interact with the Fe²⁺ atoms in the mineral surface. All considered, the results showthat complex and flexible iron-sulfide/biomonomers interactionscan be modulated by molecules that accumulate in the interfacelayer.     

A combined steepest descent and genetic algorithm (SD/GA) approach for the optimization of solvation parameters

An accurate solvation model is essential for computer modeling of protein folding and other biomolecular self-assembly processes. Compared to explicit solvent models, implicit solvent models, such as the Poisson-Boltzmann (PB) with solvent accessible surface area model (PB/SA), offer a much faster speed—the most compelling reason for the popularity of these implicit solvent models. Since these implicit solvent models typically use empirical parameters, such as atomic radii and the surface tensions, an optimal fit of these parameters is crucial for the final accuracy of properties such as solvation free energy and folding free energy. In this paper, we proposed a combined approach, namely SD/GA, which takes the advantage of both local optimization with the steepest descent (SD), and global optimization with the genetic algorithm (GA), for parameters optimization in multi-dimensional space. The SD/GA method is then applied to the optimization of solvation parameters in the non-polar cavity term of the PB/SA model. The results show that the newly optimized parameters from SD/GA not only increase the accuracy in the solvation free energies for ～200 organic molecules, but also significantly improve the free energy landscape of a β-hairpin folding. The current SD/GA method can be readily applied to other multi-dimensional parameter space optimization as well.     

Recombinant human interferon γ exerts an anti-proliferative effect and modulates the expression of human leukocyte antigens A,B,C and DR in human urothelial cell lines

Summary In this study we have treated three malignant (TGrIII) and two pre-malignant (TGrII) urothelial cell lines with recombinant human interferon (rHu-INF). The malignant cells (HCV29-T112C1, Hu1703He and T24) were inhibited in growth by more than 50% after treatment with 100–1000 units of rHu-INF/ml for 4 days as compared to untreated controls. The growth of the pre-malignant cell lines (HCV29 and Hu609) was not influenced to the same extent in the presence of rHu-INF in the culture medium. Treatment with rHu-INF increased the expression of monomorphic human leukocyte antigens (HLA) A,B,C as well as ₂-microglobulin in all the cell lines tested, as demonstrated using a quantitative immunofluorescence assay. The tumourigenic cell lines increased their expression of HLA in a dose-dependent way, whereas treatment of the non-tumourigenic cells with higher concentrations of rHu-INF than 10 units/ml, did not increase the HLA-A,B,C expression further. None of the cell lines expressed HLA-DR unless treated with rHu-INF. No correlation between tumourigenicity and the dose of rHu-INF required for de novo induction of HLA-DR could be demonstrated. After removal of rHu-INF from the medium, the expression of HLA-DR gradually decreased in less than 14 days, indicating that the expression of HLA-DR is not constitutive but dependent upon the presence of rHu-INF. We conclude that human urothelial cells grown in vitro are sensitive to the anti-proliferative and major-histocompatibility-complex-modulating effects of rHu-INF, and that malignant urothelial cells are more sensitive than pre-malignant cells. Finally, our data indicate a possible role for rHu-INF in the management of human bladder cancer.     

Bis(2-methyltetrazole)dichlorocopper(II) is a layered coordination polymer built from dinuclear chloro-bridged Cu units linked by bridging tetrazoles. The first structurally and magnetically characterized complex of 2-monosubstituted tetrazoles

The data on synthesis, crystal structure, magnetic susceptibility and thermal properties of coordination compound of copper(II) chloride with 2-methyltetrazole (2mtet) of Cu(2mtet)₂Cl₂ composition are reported. The Cu atom environment forms an elongated octahedron, with two 2-methyltetrazole ligands (N4 bound) and two Cl atoms in the equatorial positions. Symmetry related 2-methyltetrazole ligand and Cl atom are in the axial positions. One of the two 2-methyltetrazole molecules of the asymmetric unit exhibits bridging properties being linked to two Cu atoms through two N atoms (i.e., N4 and N1) of the tetrazole ring, while the other ligand molecule is coordinated in monodentate fashion via one tetrazole N4 atom. The Cu-octahedra form dinuclear building bricks by sharing edges with equatorial and axial Cl atoms. These dinuclear units are linked together via bridging 2-methyltetrazole ligands to form infinite layers parallel to the plane. Magnetic properties of Cu(2mtet)₂Cl₂ and the data of quantum-chemical calculations of molecular electrostatic potential and energies of hydronation of nitrogen atoms for 2mtet using B3LYP/6-31G^* level of theory are in agreement with the structural data obtained.     

Data consistency,yield and maintenance coefficient for the growth of Candida lypoytica and Pseudomonas aeruginosa on n-hexadecane

Summary When more than the minimum number of variables are measured, and measurement error is taken into account, the results of parameter estimation depend on which of the measured variables are selected for this purpose. The reparameterization of Pirt's models for growth produces multiresponse models with common parameters. By using the covariate adjustment technique, a unit variate linear model with covariates is obtained. This allows a combined point and interval estimates of biomass energetic yield and maintenance coefficient to be obtained using standard multiple regression programmes. When this method was applied using form I and form II of the Pirt's models, good combined estimates were obtained and compared. Using data from the literature for Candida lipolytica produced reliable results. However, for Pseudomonas aeruginosa, which has been known to produce intermediate products, a modified Pirt's model is required for a good estimate of the biomass energetic yield.Nomenclature a Mole of ammonia per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - b Moles of oxygen per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - c Moles of water per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon; no of covariates included in model - d Moles of carbon dioxide per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - e _i Error terms in Eqs. (6–8) - l Atomic ratio of oxygen to carbon in organic substrate, dimensionless - m Atomic ratio of hydrogen to carbon in organic substrate, dimensionless - m _e Rate of organic substrate consumption for maintenance, g equiv. of available electrons in biomass (h) or kcal/Kcal of biomass(h) - n Atomic ratio of oxygen to carbon in biomass, dimensionless - p Atomic ratio of hydrogen to carbon in biomass, dimensionless - Q _{CO 2} Rate of evolution of carbon dioxide, g moles/g dry wt (h) - Q _{O 2} Rate of oxygen consumption, g moles/g dry wt (h) - Q _s Rate of organic substrate consumption g/g dry wt (h) - q Atomic ratio of nitrogen to carbon in biomass, dimensionless - r Atom ratio of hydrogen to carbon in products, dimensionless; the number of parameters of interest - s Atomic ratio of oxygen to carbon in products, dimensionless - t Atomic ratio of nitrogen to carbon in products, dimensionless - r Mean of k responses in Eq. (10) - x _ki Kth response in the ith observation - y _c Biomass carbon yield (fraction of organic substrate carbon in biomass), dimensionless - z _i Covariate matrix - z Fraction of organic substrate carbon in products, dimensionless - a _i Parameters associated with covariates - _s Reductance degree of biomass, equivalents of available electrons per gram atom carbon - Reductance degree of organic substrate, equivalents of available electrons per gram atom carbon - Fraction of energy in organic substrate which is evolved as heat, dimensionless - Fraction of available electrons transferred to biomass; biomass energetic yield - True growth yield - Specific growth rate, h^-1 - _p Fraction of available electrons incorporated into products; product energetic yield - Correlation coefficient - Mass fraction carbon - ² Mean square error of model (10)     

Immunization of cross-bred cattle against Hyalomma anatolicum anatolicum by purified antigens

Extracts prepared from unfed larvae of Hyalomma anatolicum anatolicum were purified by immunoaffinity chromatography using anti-gut IgG as ligand. Affinity purified antigen (Aff-GHLAg) was used to immunize cross-bred (Bos taurus×B. indicus) calves of 6–7 months of age. Immunized calves rejected 70.6% larvae, 54.5% nymphs and 61.9% adults. No significant changes in the engorged weight of females was observed; however, significant decrease in the engorgement weight of larvae and nymphs was recorded. There was a significant decrease in the emerging nymphs (p<0.05) and adults (p<0.01) of the tick stages fed on immunized animals. SDS-PAGE analysis revealed three antigenic proteins of 100, 59.4 and 37kDa responsible for induction of resistance in the host.     

Modeling of adsorption in nanopores

Adsorption in nonporous materials has been studied using Grand Canonical Monte Carlo simulations. We discuss three types of materials: (a) a model of cylindrical pores with smooth walls, representing MCM-41 like materials, (b) a model of cylindrical pores with regular structured walls (model of carbon nanotubes) and (c) a material with crystalline wall structure (zeolites). Typical problems related to the stability of adsorbed layers have been analyzed. We have shown that the mechanism of adsorption is strongly dependent on the structure of the pore walls. In the case of amorphous walls it may lead to metastable configurations. In nanotubes, the ordered corrugation structure of walls determines the low temperature structure of the adsorbed system. In 3D ordered porous system, such as zeolites, the mechanism of adsorption is mostly determined by characteristic sites of adsorption.Figure Adsorbed atoms and energy fluctuations at the pressure of the first layer formation of krypton atoms: (a) instantaneous numbers of adsorbed atoms (per nm² of the pore wall) as a function of the time of simulation (Monte Carlo steps) observed in a relatively long run, (b) the bimodal distribution of the energy fluctuations is a consequence of the behavior of the systems as shown in (a).     

β-Globin gene polymorphism in Saudis — triple Hpa I fragments

Summary In an attempt to investigate -globin gene polymorphism in the Saudi population, DNA samples were analysed using restriction endonucleases, Mst II and Hpa I. Both ^A and ^S genes showed extensive polymorphism and were found to be linked to 13.0 kb, 7.6 kb, 7.0 kb, and 5.6 kb Hpa I fragments. Three DNA samples out of a total of 436 analysed, had -globin gene linked to three Hpa I fragments of the sizes 13.0 kb, 7.6 kb, and 5.6 kb. In this paper we present our findings and discuss the possibility of multiple -globin loci.     

Hydrophobic regions on protein surfaces. Derivation of the solvation energy from their area distribution in crystallographic protein structures.

For the first time, a direct approach for the derivation of an atomic solvation parameter from macromolecular structural data alone is presented. The specific free energy of solvation for hydrophobic surface regions of proteins is delineated from the area distribution of hydrophobic surface patches. The resulting value is 18 cal/(mol.A2), with a statistical uncertainty of +/-2 cal/mol.A2) at the 5% significance level. It compares favorably with the parameters for carbon obtained by other authors who use the the crystal geometry of succinic acid or energies of transfer from hydrophobic solvent to water for small organic compounds. Thus, the transferability of atomic solvation parameters for hydrophobic atoms to macromolecules has been directly demonstrated. A careful statistical analysis demonstrates that surface energy parameters derived from thermodynamic data of protein mutation experiments are clearly less confident.     

The influence of auxins,light and cell differentiation on solasodine production bySolanum eleagnifolium Cav. calli

The effect of auxins, light and cellular production ofSolanum eleagnifolium Cav. calli were studied. 2,4-dichlorophenoxyacetic acid (4.5 M) was the plant growth regulator used for calli initiation and this produced the highest solasodine concentration. The solasodine concentration in darkness was significantly lower than that achieved under a photoperiod of 16 h. Differentiated tissue obtained by adequate hormonal balance (several ratios of 3-indolebutyric acid to 6-benzylaminopurine) produced higher yields of solasodine than non-differentiated tissue. 3-indolebutyric acid (2.5 M) and 6-benzylaminopurine (8.8 M) increased the productivity of solasodine by 100%.Abbreviations BAP 6-benzylaminopurine - KIN Kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - NAA 1-naphtaleneacetic acid - IBA 3-indolebutyric acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - DW dry weight - GI      

β-Alanine Release from the Adult and Developing Hippocampus Is Enhanced by Ionotropic Glutamate Receptor Agonists and Cell-Damaging Conditions

The release of the inhibitory amino acid -alanine was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice, using a superfusion system. The release was enhanced by -alanine itself and the structural analogs taurine and -aminobutyrate. It was dependent on Na⁺, but independent of Ca²⁺ in both mature and immature hippocampus, being thus mostly mediated by uptake carriers operating in an outward direction. The release was potentiated in the developing mice, but not affected in the adults, by the ionotropic glutamate receptor agonists N-methyl-D-aspartate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and tetrazolylglycine in a receptor-mediated manner. Cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals, greatly enhanced -alanine release at both ages, but more markedly in the adults. The great amounts of -alanine, together with the inhibitory amino acids taurine and -aminobutyrate, released simultaneously with the excitatory amino acids in the hippocampus may constitute an important protective mechanism against excitotoxicity, which leads to neuronal death.     

Estimating protein-ligand binding free energy: atomic solvation parameters for partition coefficient and solvation free energy calculation

Solvation energy calculation is one of the main difficulties for the estimation of protein-ligand binding free energy and the correct scoring in docking studies. We have developed a new solvation energy estimation method for protein-ligand binding based on atomic solvation parameter (ASP), which has been shown to improve the power of protein-ligand binding free energy predictions. The ASP set, designed to handle both proteins and organic compounds and derived from experimental n-octanol/water partition coefficient (log P) data, contains 100 atom types (united model that treats hydrogen atoms implicitly) or 119 atom types (all-atom model that treats hydrogen atoms explicitly). By using this unified ASP set, an algorithm was developed for solvation energy calculation and was further integrated into a score function for predicting protein-ligand binding affinity. The score function reproduced the absolute binding free energies of a test set of 50 protein-ligand complexes with a standard error of 8.31 kJ/mol. As a byproduct, a conformation-dependent log P calculation algorithm named ASPLOGP was also implemented. The predictive results of ASPLOGP for a test set of 138 compounds were r = 0.968, s = 0.344 for the all-atom model and r = 0.962, s = 0.367 for the united model, which were better than previous conformation-dependent approaches and comparable to fragmental and atom-based methods. ASPLOGP also gave good predictive results for small peptides. The score function based on the ASP model can be applied widely in protein-ligand interaction studies and structure-based drug design.     

Polar and nonpolar atomic environments in the protein core: Implications for folding and binding

Hydrophobic interactions are believed to play an important role in protein folding and stability. Semi-empirical attempts to estimate these interactions are usually based on a model of solvation, whose contribution to the stability of proteins is assumed to be proportional to the surface area buried upon folding. Here we propose an extension of this idea by defining an environment free energy that characterizes the environment of each atom of the protein, including solvent, polar or nonpolar atoms of the same protein or of another molecule that interacts with the protein. In our model, the difference of this environment free energy between the folded state and the unfolded (extended) state of a protein is shown to be proportional to the area buried by nonpolar atoms upon folding. General properties of this environment free energy are derived from statistical studies on a database of 82 well-refined protein structures. This free energy is shown to be able to discriminate misfolded from correct structural models, to provide an estimate of the stabilization due to oligomerization, and to predict the stability of mutants in which hydrophobic residues have been substituted by site-directed mutagenesis, provided that no large structural modifications occur. © 1994 Wiley-Liss, Inc.     

Refinement of the protein backbone angle ψ in NMR structure calculations

Cross-correlated relaxation rates involving the C-H dipolar interaction and the carbonyl (C) chemical shift anisotropy (CSA) have been measured using two complementary 3D experiments. We show that the protein backbone angle can be directly refined against such cross-correlated relaxation rates (^{H C,C}) and the three-bond H/D isotope effect on the C chemical shifts (³C _(ND)). By simultaneously using both experimental parameters as restraints during NMR structure calculations, a unique value for the backbone angle is defined. We have applied the new refinement method to the -Spectrin SH3 domain (a -sheet protein) and to the Sgs1p HRDC domain (an -helical protein) and show that the quality of the NMR structures is substantially improved, judging from the atomic coordinate precision and the Ramachandran map. In addition, the -refined NMR structures of the SH3 domain deviate less from the 1.8 Å crystal structure, suggesting an improved accuracy. The proposed refinement method can be used to significantly improve the quality of NMR structures and will be applicable to larger proteins.     

Lipase immobilized by adsorption

Summary Candida cylindraceae lipase was immobilized by adsorption to various hydrophilic and hydrophobic supports and studied with respect to the esterification rates of a primary and a secondary alcohol, respectively, in organic media. The reaction rates were compared with the rate of esterification with free lipase. When the secondary alcohol, (R, S)-1-phenylethanol, was used the highest reaction rates were measured for lipase adsorbed to the hydrophobic supports. When the primary alcohol, heptanol, was used free lipase exhibited the highest reaction rate. A kinetic explantation of these results is proposed.