Speed-up hyperspheres homotopic path tracking algorithm for PWL circuits simulations.

In the present work, we introduce an improved version of the hyperspheres path tracking method adapted for piecewise linear (PWL) circuits. This enhanced version takes advantage of the PWL characteristics from the homotopic curve, achieving faster path tracking and improving the performance of the homotopy continuation method (HCM). Faster computing time allows the study of complex circuits with higher complexity; the proposed method also decrease, significantly, the probability of having a diverging problem when using the Newton-Raphson method because it is applied just twice per linear region on the homotopic path. Equilibrium equations of the studied circuits are obtained applying the modified nodal analysis; this method allows to propose an algorithm for nonlinear circuit analysis. Besides, a starting point criteria is proposed to obtain better performance of the HCM and a technique for avoiding the reversion phenomenon is also proposed. To prove the efficiency of the path tracking method, several cases study with bipolar (BJT) and CMOS transistors are provided. Simulation results show that the proposed approach can be up to twelve times faster than the original path tracking method and also helps to avoid several reversion cases that appears when original hyperspheres path tracking scheme was employed.

Laplace transform homotopy perturbation method for the approximation of variational problems.

This article proposes the application of Laplace Transform-Homotopy Perturbation Method and some of its modifications in order to find analytical approximate solutions for the linear and nonlinear differential equations which arise from some variational problems. As case study we will solve four ordinary differential equations, and we will show that the proposed solutions have good accuracy, even we will obtain an exact solution. In the sequel, we will see that the square residual error for the approximate solutions, belongs to the interval [0.001918936920, 0.06334882582], which confirms the accuracy of the proposed methods, taking into account the complexity and difficulty of variational problems.

Modified hyperspheres algorithm to trace homotopy curves of nonlinear circuits composed by piecewise linear modelled devices.

We present a homotopy continuation method (HCM) for finding multiple operating points of nonlinear circuits composed of devices modelled by using piecewise linear (PWL) representations. We propose an adaptation of the modified spheres path tracking algorithm to trace the homotopy trajectories of PWL circuits. In order to assess the benefits of this proposal, four nonlinear circuits composed of piecewise linear modelled devices are analysed to determine their multiple operating points. The results show that HCM can find multiple solutions within a single homotopy trajectory. Furthermore, we take advantage of the fact that homotopy trajectories are PWL curves meant to replace the multidimensional interpolation and fine tuning stages of the path tracking algorithm with a simple and highly accurate procedure based on the parametric straight line equation.

Approximate Solutions for Flow with a Stretching Boundary due to Partial Slip.

The homotopy perturbation method (HPM) is coupled with versions of Laplace-Padé and Padé methods to provide an approximate solution to the nonlinear differential equation that describes the behaviour of a flow with a stretching flat boundary due to partial slip. Comparing results between approximate and numerical solutions, we concluded that our results are capable of providing an accurate solution and are extremely efficient.

Crystallographic and FTIR spectroscopic evidence of changes in Fe coordination upon reduction of the active site of the Fe-only hydrogenase from Desulfovibrio desulfuricans.

Fe-only hydrogenases, as well as their NiFe counterparts, display unusual intrinsic high-frequency IR bands that have been assigned to CO and CN(-) ligation to iron in their active sites. FTIR experiments performed on the Fe-only hydrogenase from Desulfovibrio desulfuricans indicate that upon reduction of the active oxidized form, there is a major shift of one of these bands that is provoked, most likely, by the change of a CO ligand from a bridging position to a terminal one. Indeed, the crystal structure of the reduced active site of this enzyme shows that the previously bridging CO is now terminally bound to the iron ion that most likely corresponds to the primary hydrogen binding site (Fe2). The CO binding change may result from changes in the coordination sphere of Fe2 or its reduction. Superposition of this reduced active site with the equivalent region of a NiFe hydrogenase shows a remarkable coincidence between the coordination of Fe2 and that of the Fe ion in the NiFe cluster. Both stereochemical and mechanistic considerations suggest that the small organic molecule found at the Fe-only hydrogenase active site and previously modeled as 1,3-propanedithiolate may, in fact, be di-(thiomethyl)-amine.

The H2 sensor of Ralstonia eutropha. Biochemical characteristics, spectroscopic properties, and its interaction with a histidine protein kinase.

Previous genetic studies have revealed a multicomponent signal transduction chain, consisting of an H(2) sensor, a histidine protein kinase, and a response regulator, which controls hydrogenase gene transcription in the proteobacterium Ralstonia eutropha. In this study, we isolated the H(2) sensor and demonstrated that the purified protein forms a complex with the histidine protein kinase. Biochemical and spectroscopic analysis revealed that the H(2) sensor is a cytoplasmic [NiFe]-hydrogenase with unique features. The H(2)-oxidizing activity was 2 orders of magnitude lower than that of standard hydrogenases and insensitive to oxygen, carbon monoxide, and acetylene. Interestingly, only H(2) production but no HD formation was detected in the D(2)/H(+) exchange assay. Fourier transform infrared data showed an active site similar to that of standard [NiFe]-hydrogenases. It is suggested that the protein environment accounts for a restricted gas diffusion and for the typical kinetic parameters of the H(2) sensor. EPR analysis demonstrated that the [4Fe-4S] clusters within the small subunit were not reduced under hydrogen even in the presence of dithionite. Optical spectra revealed the presence of a novel, redox-active, n = 2 chromophore that is reduced by H(2). The possible involvement of this chromophore in signal transduction is discussed.

Kinetic characterization of Desulfovibrio gigas hydrogenase upon selective chemical modification of amino acid groups as a tool for structure-function relationships.

The effect of amino acid residues modification of Desulfovibrio gigas hydrogenase on different activity assays is reported. The first method consisted in the modification of glutamic and aspartic acid residues of the enzyme with ethylenediamine in order to change the polarity of certain regions of the protein surface. The second method consisted in the modification of histidine residues with a Ru complex in order to change the acid-base properties of the histidine residues. The implication of these modifications in the enzyme kinetics has been studied by measuring in parallel the activities of para/ortho hydrogen conversion, deuterium/hydrogen exchange and dyes reduction with hydrogen. Our experimental data support some hypothesis based on the three-dimensional structure of this enzyme: (a) electrostactic interactions between the hydrogenase and the redox partner play an essential role in the kinetics; (b) the histidine ligand and the surrounding acidic residues of the distal [4Fe4S] cluster form the recognition site of the redox partner of the hydrogenase; and (c) histidine residues are involved in the hydron transfer pathway of the hydrogenase.

C-terminal extension of the H2-activating subunit, HoxH, directs maturation of the NAD-reducing hydrogenase in Alcaligenes eutrophus.

Formation of enzymatically active [NiFe] hydrogenases is dependent on a number of posttranslational steps, including metal attachment to a precursor of the catalytic subunit, truncation of a small C-terminal peptide from the precursor, and oligomerisation of the subunits. Two amino acid replacements were introduced by site-directed mutagenesis at the C-terminal proteolytic cleavage site of HoxH, the Ni-containing subunit of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus H16. Replacement of Ala465, the first residue of the 24-amino-acid cleaved polypeptide, by Pro yielded a form of HoxH that was blocked in C-terminal proteolysis. This HoxH subunit, although capable of binding Ni, was blocked in formation of a stable tetrameric holoenzyme. In the second mutant, the C-terminal extension of HoxH was eliminated by substituting the Ala codon for a translational stop codon. Although this mutant subunit was able to form the oligomeric holoenzyme, it was devoid of Ni. Both mutant proteins contained only traces of H2-activating functions. H2-dependent reduction of NAD and benzylviologen, and D2/H+-exchange activity were almost completely abolished, while the NADH oxidoreductase activity, mediated by the diaphorase moiety of the hydrogenase, was retained. These results allow the following conclusions: the C-terminal extension of HoxH is neccessary to direct specific Ni insertion into the hydrogenase; subunit assembly to the holoenzyme is not dependent on Ni insertion; and a precursor with the C-terminal peptide is not competent for assembly.

The covalent linkage of a viologen to a flavoprotein reductase transforms it into an oxidase.

Chemical cross-linkage of the positively charged viologen N-methyl-N'-(aminopropyl)-4-4'-bipyridinium dibromide (APMV) to the enzyme ferredoxin-NADP+ reductase from the cyanobacterium Anabaena PCC 7119 has been performed using the carbodiimide 1-ethyl[3-(3-dimethylaminopropyl)]carbodiimide. 0.5-1 mol, depending on the preparation, is introduced for each mol enzyme. The residue involved in the covalent linkage with the viologen, Glu139, has been identified using HPLC separation of the modified proteolytic peptides and subsequent sequencing. Modification of the enzyme changes its catalytic specificity since it is able to react directly with oxygen; this is observed by a high NADPH oxidase activity, which is completely absent in the native enzyme. More important, this new enzymic activity is indicative of the intramolecular electron transfer between the natural redox cofactor FAD and the artificially introduced viologen. Electrons can also flow in the reverse direction, from the viologen to the FAD group, then to NADP+, when the reaction is performed using glassy-carbon electrodes to reduce the viologen. Cyclic voltammetry experiments have shown that there is a small catalytic current between the electrode and the enzyme which is not observed in the native enzyme.

H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase: mechanism of H2 formation analyzed using hydrogen isotopes.

H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase catalyzes the reversible dehydrogenation of N5,N10-methylenetetrahydromethanopterin (CH2 = H4MPT) to N5,N10-methenyltetrahydromethanopterin (CH = H4MPT+) and H2. In D2O both HD and D2 are formed from CH2 = H4MPT and in H2O both HD and H2 from CD2 = H4MPT. Evidence is presented that HD is not an intermediate in the formation of D2 and H2, respectively.

Protein conformational changes in tetraheme cytochromes detected by FTIR spectroelectrochemistry: Desulfovibrio desulfuricans Norway 4 and Desulfovibrio gigas cytochromes c3.

The conformational change coupled to the redox processes of two tetraheme cytochromes c3 from bacteria of the genus Desulfovibrio have been studied by UV-vis and FTIR difference spectroscopy combined with protein electrochemistry. Two pairs of equivalent hemes were found in Desulfovibrio desulfuricans Norway 4 cytochrome c3 by UV-vis spectroelectrochemical redox titration in an optically transparent thin-layer electrochemical cell. In contrast to this, Desulfovibrio gigas cytochrome c3 showed a UV-vis difference spectrum for the highest potential heme different from that of the others. The redox titrations were monitored by FTIR difference spectroscopy using the same spectroelectrochemical cell. They show that in both cytochromes the overall redox process from the fully oxidized (III4) to the fully reduced oxidation state (II4), III4<==>II4, proceeds via an intermediate oxidation stage (III2II2) which is formed after the second electron uptake. The small amplitude of the difference signals in the reduced-minus-oxidized FTIR difference spectra obtained for the overall redox process in both Desulfovibrio cytochromes indicates a very small conformational change induced by the redox transition. Nevertheless, by application of potential steps from the fully oxidized or reduced form to the midwave potential (as obtained from the UV-vis redox titrations), the reduced-minus-oxidized IR difference spectra corresponding to the intermediate redox transitions (III4<==>III2II2 and III2II2<==>II4) were obtained, reflecting separately the contributions of the high- and low-potential heme pairs to the overall redox-induced conformational change. The overall redox process and both intermediate redox transitions were fully reversible. In the spectral region between 1500 and 1200 cm-1 the IR difference spectra of both cytochromes show several signals previously observed in the reduced-minus-oxidized IR difference spectra of spinach cytochrome b559 and iron-protoporphyrin IX-bis(imidazole) model compounds [Berthomieu, C., Boussac, A., Mäntele, W., Breton, J., & Nabedryk, E. (1992) Biochemistry 31, 11460-11471]. Moreover, Raman spectra of Desulfovibrio vulgaris cytochrome c3 and cytochrome b5 show signals attributed to Raman active heme skeletal modes at nearly the same positions [Kitagawa, T., Kyogoyu, Y., Izuka, T., Ikeda-Saito, M., & Yamanaka, T. (1975) J. Biochem. 78, 719-728], thus allowing their assignment to signals arising from heme vibrational modes. Comparatively strong IR difference signals at 1618 cm-1, which are tentatively assigned to phenylalanine residues, were found in D. desulfuricans cytochrome c3. In the spectra of D. gigas cytochrome c3, IR signals at 1614 cm-1 were detected only for the first redox transition (III4<==>III2II2).(ABSTRACT TRUNCATED AT 400 WORDS)

H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum. Studies of the catalytic mechanism of H2 formation using hydrogen isotopes.

H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase is a novel hydrogenase found in most methanogenic archaea. It catalyzes the reversible conversion of N5,N10-methylenetetrahydromethanopterin (CH2 = H4MPT) to N5,N10-methenyltetrahydromethanopterin (CH identical to H4MPT+) and dihydrogen; CH2 = H4MPT + H+<-->CH identical to H4MPT(+) + H2; delta G degrees ' = + 5 kJ/mol. In the following investigation, the formation of H2, HD and D2 was studied in experiments in which either the methylene group of CH2 = H4MPT or water were deuterium labelled. In the case of CD2 = H4MPT and H2O, the dihydrogen formed immediately after the start of the reaction was composed of approximately 50% HD and 50% of H2 at all pH tested. In the case of CH2 = H4MPT and D2O, the dihydrogen generated was composed of approximately 50% HD and 50% D2 at pD 5.7 and of approximately 85% HD and 15% D2 at pD 7.0. Evidence is presented that the enzyme catalyzes a CH identical to H4MPT(+)-dependent isotopic exchange between HD and H2O and between HD and D2O, yielding H2 and D2, respectively. A catalytic mechanism aimed to explain these findings is discussed.

Purification and characterization of two distinct lipases from Candida cylindracea.

We have purified and characterized two isoenzymes from a commercial lipase preparation of Candida cylindracea. The purification procedure includes ethanol precipitation and DEAE-Sephacel and Sephacryl HR 100 chromatographies. Lipase A and lipase B were purified 11-fold with a 5% and 21% recovery in activity, respectively. The enzymes have similar amino acid content, N-terminal sequence and molecular weight, but differ on neutral sugar content, hydrophobicity, presence of isoforms and stability to pH and temperature. They also show some differences in the substrate specificity.

Further characterization of the [Fe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757.

The properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans ATCC 7757, previously reported to be a single-subunit protein [Glick, B. R., Martin, W. G., and Martin, S. M. (1980) Can. J. Microbiol. 26, 1214-1223] were reinvestigated. The pure enzyme exhibited a molecular mass of 53.5 kDa as measured by analytical ultracentrifugation and was found to comprise two different subunits of 42.5 kDa and 11 kDa, with serine and alanine as N-terminal residues, respectively. The N-terminal amino acid sequences of its large and small subunits, determined up to 25 residues, were identical to those of the Desulfovibrio vulgaris Hildenborough [Fe]-hydrogenase. D. desulfuricans ATCC 7757 hydrogenase was free of nickel and contained 14.0 atoms of iron and 14.4 atoms of acid-labile sulfur/molecule and had E400, 52.5 mM-1.cm-1. The purified hydrogenase showed a specific activity of 62 kU/mg of protein in the H2-uptake assay, and the H2-uptake activity was higher than H2-evolution activity. The enzyme isolated under aerobic conditions required incubation under reducing conditions to express its maximum activity both in the H2-uptake and 2H2/1H2 exchange reaction. The ratio of the activity of activated to as-isolated hydrogenase was approximately 3. EPR studies allowed the identification of two ferredoxin-type [4Fe-4S]1+ clusters in hydrogenase samples reduced by hydrogen. In addition, an atypical cluster exhibiting a rhombic signal (g values 2.10, 2.038, 1.994) assigned to the H2-activating site in other [Fe]-hydrogenases was detected in partially reduced samples. Molecular properties, EPR spectroscopy, catalytic activities with different substrates and sensitivity to hydrogenase inhibitors indicated that D. desulfuricans ATCC 7757 periplasmic hydrogenase is a [Fe]-hydrogenase, similar in most respects to the well characterized [Fe]-hydrogenase from D. vulgaris Hildenborough.

Influence of the support on the reaction course of tributyrin hydrolysis catalyzed by soluble and immobilized lipases.

Lipases from different origins have been immobilized in supports chosen by its different aquaphilicity and used as biocatalysts for the hydrolysis of tributyrin. The changes of the concentration of tri-, di-, monobutyrin, glycerol, and butyric acid during the reactions catalyzed by soluble, as well as immobilized, lipases were evaluated by gas chromatography. The experimental data were fitted to a simple kinetic model for the sequential reaction of tributyrin hydrolysis. The calculated apparent rate constants were different for the biocatalysts used and were apparently related to diffusional effects and aquaphilicity of the supports. Maximal yields of dibutyrin were found with the soluble Candida lipase, whereas the highest yield of monobutyrin (90%) was obtained with the least aquaphylic derivative (Candida-Celite).

Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans.

The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM-1 cm-1, respectively. D. fructosovorans hydrogenase contains 11 +/- 1 iron atoms, 0.9 +/- 0.15 nickel atom and 12 +/- 1 acid-labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N-terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel(III), [3Fe-4S] and [4Fe-4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3-h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D. gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni-B species (g = 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.

Inhibition of Desulfovibrio gigas hydrogenase with copper salts and other metal ions.

The effect of several transition metals on the activity of Desulfovibrio gigas hydrogenase has been studied. Co(II) and Ni(II) at a concentration of 1 mM did not modify the activity of the enzyme; nor did they affect the pattern of activation/deactivation. Cu(II) inhibited the active hydrogenase, prepared by treatment with hydrogen, but had little effect on the 'unready' enzyme unless a reductant such as ascorbate was present, in which case inactivation took place either in air or under argon. Hg(II) also inactivated the enzyme irreversible in the 'unready' state without the requirement for reductants. The reaction of H2 uptake with methyl viologen was much more sensitive to inhibition than the H2/tritium exchange activity. EPR spectra of this preparation showed that the rates of decline were [3Fe-4S] signal greater than H2-uptake activity greater than Ni-A signal. Similar results were obtained when the protein was treated with Hg(II). The results demonstrate that the [3Fe-4S] cluster is not essential for H2-uptake activity with methyl viologen, but the integrity of [4Fe-4S] clusters is probably necessary to catalyze the reduction of methyl viologen with hydrogen. D. gigas hydrogenase was found to be highly resistant to digestion by proteases.

[Plants used in Mexico for treatment of oral diseases]. / Plantas empleadas en México para el tratamiento de afecciones estomatológicas.

This paper presents an inventory of 167 plants traditionally employed in Mexico for the treatment of ailments of stomatologic origin.

NADH Induces the Generation of Superoxide Radicals in Leaf Peroxisomes.

In peroxisomes isolated from pea leaves (Pisum sativum L.) the production of superoxide free radicals (O(2) (-)) by xanthine and NADH was investigated. In peroxisomal membranes, 100 micromolar NADH induced the production of O(2) (-) radicals. In the soluble fractions of peroxisomes, no generation of O(2) (-) radicals was observed by incubation with either NADH or xanthine, although xanthine oxidase was found located predominantly in the matrix of peroxisomes. The failure of xanthine to induce superoxide generation was probably due to the inability to fully suppress the endogenous Mn-superoxide dismutase activity by inhibitors which were inactive against xanthine oxidase. The generation of superoxide radicals in leaf peroxisomes together with the recently described production of these oxygen radicals in glyoxysomes (LM Sandalio, VM Fernández, FL Rupérez, LA del Río [1988] Plant Physiol 87: 1-4) suggests that O(2) (-) generation could be a common metabolic property of peroxisomes and further supports the existence of active oxygen-related rôles for peroxisomes in cellular metabolism.