Degradation of Benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

Electronic structures of collagen model polymers: (Gly-Pro)n, (Gly-Hyp)n, (Ala-Pro)n, (Ala-Hyp)n, (Gly-Pro-Gly)n, (Gly-Hyp-Gly)n, (Gly-Pro-Pro)n, and (Gly-Pro-Hyp)n

In order to investigate the relationship existing between the electronic structures of collagen and its biochemical functions in vivo, the semiempirical CNDO/2 SCF MO calculations were carried out on several model polymers of collagen, (Gly-Pro)_n, (Gly-Hyp)_n, (Ala-Pro)_n, (Ala-Hyp)_n, (Gly-Pro-Gly)_n, (Gly-Hyp-Gly)_n, (Gly-Pro-Pro)_n and (Gly-Pro-Hyp)_n. Geometries of the skeleton of these polymers were assumed to be the same as those of poly(l-proline) I (cis) and II (trans) and the calculations were performed only on infinite polymers in a single chain. The results show that the cis form is always more stable than the trans form for all the polymers treated. This energy difference between the cis and trans forms depends, for example, on the kind of amino acid residue, Gly or Ala, but this could not be seen in the Pro or Hyp residue. The flexibility or mobility of the collagen structure was explained using the energy difference between the cis and trans forms of the polymers, i.e. the cis-trans conversion of the collagen was discussed in connection with the energy difference. The reason why the collagen has the constitution of (Gly-Pro-Hyp)_n is briefly discussed.     

Computational study on the mechanisms of action of the potential anticancer drug trans-isopropylaminedimethylaminedichloroplatinum (trans-IPADMADP) and its cis isomer with DNA purine bases

The monofunctional and bifunctional bindings of the potential anticancer drug trans-isopropylaminedimethylaminedichloroplatinum (trans-IPADMADP) and its cis isomer to purine base in DNA are explored by using density functional theory and IEF-PCM solvation models. The computed lowest free energy barrier in the aqueous solution is 14.0/11.6 kcal/mol (from trans-Pt-chloroaqua complex to trans-/cis-monoadduct) for guanine(G), and 11.7/13.3 kcal/mol (from trans-Pt-chloroaqua complex to trans-/cis-monoadduct) for adenine(A). Our calculations demonstrate that the trans reactant complexes (or isolated reactants) can generate trans- or cis-monoadducts via similar trigonal bipyramidal transition state structures, suggesting that the monoadducts can subsequently close to form the bifunctional intrastrand Pt-DNA adducts and simultaneously distort DNA in the similar way as cisplatin. Our calculations show that Pt(isopropylamine)(dimethylamine)G₂²⁺ head-to-head path has the lowest free energy of activation at 17.6 kcal/mol, closely followed by the Pt(isopropylamine)(dimethylamine)GA²⁺ head-to-head path at 19.6 kcal/mol when the monofunctional cis-Pt-G complex serves as the reactant; while the Pt(isopropylamine)(dimethylamine)G₂²⁺ head-to-tail adduct has the lowest barrier of 20.5 kcal/mol, closely followed by the Pt(isopropylamine)(dimethylamine)GA²⁺ head-to-tail adduct at 23.0 kcal/mol if the monofunctional trans-Pt-G complex is the reactant.The calculated relatively lower activation energy barrier than that of cisplatin theoretically confirm that trans-[PtCl₂(isopropylamine)(dimethylamine)] is a potential anticancer drug as described by experiment.     

Cis-trans isomerization of proline in the peptide (his 105-Val 124) of ribonuclease a containing the primary nucleation site

Conformational energy calculations have been carried out to determine the relative stabilities of the C-terminal sequence 105–124 of ribonuclease A, withcis andtrans forms, respectively, of Asn 113-Pro 114. Thecis form of Pro 114 is the one that occurs in the native protein. This peptide contains the sequence 106–118, which, on the basis of both theoretical and experimental studies, is thought to constitute the primary nucleation site for the folding of ribonuclease A. It is shown that both conformations of the isolated peptide (with Pro 114 in thecis andtrans forms, respectively) are of approximately equal stability. Both forms have similar conformations from residues 105–110 and 118–124, while they differ in the bend region involving residues 111–117. Calculations have also been carried out to deduce the possible low-energy paths for the interconversion between thecis andtrans forms of both Pro 114 and Pro 117. It is shown that there are two low-energy paths (with a minimum activation energy of 16.5 kcal/mole) for the interconversion of Pro 114. Attractive nonbonded interaction energies stabilize the transition state on these paths. Only one relatively low-energy path (with an activation energy of 18 kcal/mole) could be found for the isomerization of Pro 117, which occur in thetrans form in the native protein; in this case, allcis forms have significantly higher energy than thetrans form. These calculations thus show that native-like forms for the isolated peptide can exist with Pro 114 in either thecis or thetrans form and that these forms are readily interconvertible.     

Palladium and rhodium complexes of a chiral pincer ligand derived from 1,3-trans disubstituted cyclohexane

Reactions of cis- and trans-1,3-bis(di-t-butylphosphinomethyl)cyclohexane (cis- and trans-PCyP), with rhodium and palladium chlorides afforded C_s and C₁ symmetrical pincer complexes, RhHCl(cis-PCyP), RhHCl(trans-PCyP), [(COD)Rh(μ-Cl)₂RhH(trans-PCyP)], PdCl(cis-PCyP), and PdCl(trans-PCyP), where the PCyP ligands are coordinated in a meridianal fashion through the two phosphorus atoms and the cyclometalated C-1 carbon of the cyclohexane ring. The rhodium complexes were structurally characterized by X-ray diffraction. Isomers of the RhHCl(PCyP) and PdCl(PCyP) complexes were studied by DFT calculations.     

Effect of double bond geometry in sphingosine base on the antioxidant function of sphingomyelin

We previously showed that sphingomyelin (SM) inhibits peroxidation of phosphatidylcholine (PC) and cholesterol. Since SM uniquely has a trans unsaturation in its sphingosine base, we investigated whether this feature is important for its antioxidant function. Substitution of the natural trans Δ⁴-double bond with a cis double bond (cis-SM), however, increased SM’s ability to inhibit Cu²⁺-mediated 16:0-18:2 PC oxidation by up to eightfold. Dihydro-SM, which lacks the double bond, was equally effective as trans-SM. In contrast to its effect in the sphingosine base, the presence of a cis double bond in the N-acyl group of trans-SM was not protective. cis-SM also inhibited the oxidation of cholesterol by FeSO₄/ascorbate more efficiently than the trans isomer. The enhanced protective effect of cis-SM is selective for metal ion-promoted oxidation, and appears to arise from a decrease in the effective concentration of metal ions. These studies show that the trans double bond of SM is not essential for its antioxidant effects.     

Theoretical study of the spectroscopic and nonlinear optical properties of <Emphasis Type="Italic">trans</Emphasis>- and <Emphasis Type="Italic">cis</Emphasis>-4-hydroxyazobenzene

We investigate the molecular structure, vibrational and electronic absorption spectra, and electronic hyperpolarizabilities of trans and cis isomers of 4-hydroxyazobenzene (HOAB) via density functional theory. Results show that the azo dye exhibits a high third-order nonlinear optical response and good optical transparency. Both the basis set and the functional are important influences on the results obtained when calculating the absorption spectrum and NLO response. We also study the effect of the solvent on the electronic absorption spectrum to assess the ability of the functional to reproduce the experimental spectrum in combination with a suitable solvent model. Our calculations show that the SMD model of Truhlar et al. handles the electrostatic and the non-electrostatic effects of hydrogen-bonding solvents on the absorption spectrum better than the traditional polarizable continuum model does. In addition, our results indicate that the dye trans-HOAB exhibits a high second hyperpolarizability and excellent optical transparency. Also, although the second hyperpolarizability of cis-HOAB is much lower than that of trans-HOAB, it is non-negligible when calculating the optical nonlinearity of HOAB under an optical pump. We also examine the effect of frequency dispersion on second harmonic generation. This study provides the basis for further research on the spectroscopic and nonlinear optical properties of novel azo dyes and other π-conjugated compounds.     

Heritability and tissue specificity of expression quantitative trait loci

Variation in gene expression is heritable and has been mapped to the genome in humans and model organisms as expression quantitative trait loci (eQTLs). We applied integrated genome-wide expression profiling and linkage analysis to the regulation of gene expression in fat, kidney, adrenal, and heart tissues using the BXH/HXB panel of rat recombinant inbred strains. Here, we report the influence of heritability and allelic effect of the quantitative trait locus on detection of cis- and trans-acting eQTLs and discuss how these factors operate in a tissue-specific context. We identified several hundred major eQTLs in each tissue and found that cis-acting eQTLs are highly heritable and easier to detect than trans-eQTLs. The proportion of heritable expression traits was similar in all tissues; however, heritability alone was not a reliable predictor of whether an eQTL will be detected. We empirically show how the use of heritability as a filter reduces the ability to discover trans-eQTLs, particularly for eQTLs with small effects. Only 3% of cis- and trans-eQTLs exhibited large allelic effects, explaining more than 40% of the phenotypic variance, suggestive of a highly polygenic control of gene expression. Power calculations indicated that, across tissues, minor differences in genetic effects are expected to have a significant impact on detection of trans-eQTLs. Trans-eQTLs generally show smaller effects than cis-eQTLs and have a higher false discovery rate, particularly in more heterogeneous tissues, suggesting that small biological variability, likely relating to tissue composition, may influence detection of trans-eQTLs in this system. We delineate the effects of genetic architecture on variation in gene expression and show the sensitivity of this experimental design to tissue sampling variability in large-scale eQTL studies.     

A theoretical estimate of the energy barriers between stable conformations of the proline dimer.

Semi-empirical energy calculations for an internal Pro-Pro dimer are presented that take into account the nature of the flexibility of the proline ring due to its puckering. Calculations show that three stable conformations are available for the dimer: the cis (ω = 0°, ψ = 160°); the trans (ω = 180°, ψ = 160°, also referred to as trans′); and the cis′ (ω = 180°, ψ = ?40°) conformations. The best conformational pathways between these stable conformations are determined. Calculations also show that the barrier for cis′–trans′ conversion is of the same order of magnitude as that for cis–trans conversion.     

Importance of the sphingosine base double-bond geometry for the structural and thermodynamic properties of sphingomyelin bilayers

The precise role of the sphingosine base trans double bond for the unique properties of sphingomyelins (SMs), one of the main lipid components in raftlike structures of biological membranes, has not been fully explored. Several reports comparing the hydration, lipid packing, and hydrogen-bonding behaviors of SM and glycerophospholipid bilayers found remarkable differences overall. However, the atomic interactions linking the double-bond geometry with these thermodynamic and structural changes remained elusive. A recent report on ceramides, which differ from SMs only by their hydroxyl headgroup, has shown that replacing the trans double bond of the sphingosine base by cis weakens the hydrogen-bonding potential of these lipids and thereby alters their biological activity. Based on data from extensive (a total 0.75 μs) atomistic molecular dynamics simulations of bilayers composed of all-trans, all-cis, and a trans/cis (4:1 ratio) racemic mixture of sphingomyelin lipids, here we show that the trans configuration allows for the formation of significantly more hydrogen bonds than the cis. The extra hydrogen bonds enabled tighter packing of lipids in the all-trans and trans/cis bilayers, thus reducing the average area per lipid while increasing the chain order and the bilayer thickness. Moreover, fewer water molecules access the lipid-water interface of the all-trans bilayer than of the all-cis bilayer. These results provide the atomic basis for the importance of the natural sphingomyelin trans double-bond conformation for the formation of ordered membrane domains.     

Studies on fluorescence polarization of 1-acyl-2-cis- or trans-parinaroyl sn-3-glycerophosphorylcholines in model systems and microsomal membranes

Fluorescent lecithin probes containing cis- or trans-parinaric acid (PnA) at the 2-position cis-parinaroylphosphatidylcholine (cis-PnPC) and trans-parinaroyl phosphatidylcholine (trans-PnPC)) showed similar behavior to that of the free cis- or trans-parinaric acids (cis-PnA or trans-PnA) in bilayer vesicles of synthetic saturated lecithins. Transition temperatures detected by cis-PnPc were about 1°C lower than those observed with trans-PnPc. In mixed lecithin vesicles, the trans-PnPc probe monitored a higher temperature melting component than did the cis-probe. Both probes were readily incorporated into microsomal membranes and into sonicated vesicles prepared from the microsomal phospholipids. With either cis- or trans-PnPc no change in polarization ratio was observed for microsomal membranes between 40°C and 0°C but this ratio increased with decreasing temperature between 0°C and ?5°C. However, vesicles of extracted phospholipids showed a continuous increase in polarization ratio with decreasing temperature between 20°C and ?15°C with trans-PnPc and bewteen 5°C and ?15°C with cis-PnPc. These results suggest that the two lecithin probes monitor different environments in the membranes and phospholipid vesicles prepared from them.     

The trans-to-cis proline isomerization in E. coli Trx folding is accelerated by trans prolines

The conserved fold of thioredoxin (Trx)-like thiol/disulfide oxidoreductases contains an invariant cis-proline residue (P76 in Escherichia coli Trx) that is essential for Trx function and that is responsible for the folding rate-limiting step. E. coli Trx contains four additional prolines, which are all in the trans conformation in the native state. Notably, a recent study revealed that replacement of all four trans prolines in Trx by alanines (Trx variant Trx1P) further slowed the rate-limiting step 25-fold, indicating that one or several of the four trans prolines accelerate the trans-to-cis transition of P76 in Trx wild-type (wt). Here, we characterized the folding kinetics of Trx variants containing cisP76 and one or several of the natural trans prolines of Trx wt with NMR spectroscopy. First, we demonstrate that the isomerization reaction in Trx1P is a pure two-state transition between two distinct tertiary structures, in which all observed NMR resonances changes follow the same first-order kinetics. Moreover, we show that trans-P68 is the critical residue responsible for the faster folding of wt Trx relative to the single-proline (P76) variant Trx1P, as the two-proline variant Trx2P(P76P68) already folds seven times faster than Trx1P. trans-P34 also accelerates trans-to-cis isomerization of P76, albeit to a smaller extent. Overall, the results demonstrate that trans prolines can significantly modulate the kinetics of rate-limiting trans-to-cis proline isomerization in protein folding. Finally, we discuss possible mechanisms of acceleration and the potential significance of a protein-internal folding acceleration mechanism for Trx in a living cell.     

Electron spin resonance and electron nuclear double resonance studies of cation radicals derived from tocopherol model compounds

Electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) measurements were performed for the cation radicals obtained from the model compounds of α-, β-, γ- and δ-tocopherol (vitamin E) by oxidizing the tocopherol precursors in an AlCl₃-CH₂Cl₂ solution. The proton hyperfine coupling constants g-values were precisely determined. The ENDOR spectra of the cation radicals of α-, β-, γ- and δ-tocopherol models in CH₂Cl₂ at ?100°C clearly show 10, 6, 6 and 12 different proton hyperfine couplings, respectively. By varying the temperature, the ESR spectra of the α- and δ-tocopherol model cations exhibit line-width alternation phenomena characteristic of the hindered rotation of the OH group. However, neither the β- nor the γ-tocopherol model cation radical ESR spectra show any sign of an alternative line-width effect. These results are interpreted by assuming that the β- and γ-tocopherol model cations are stabilized in the trans and cis conformations, respectively. On tocopherol model cations are stabilized in the trans and cis conformations, respectively. On the other hand, both the α- and δ-tocopherol model cations exist as cis and trans isomers.     

Quantitative determination of diol metabolites of CS-670, a new antiinflammatory agent,by capillary column gas chromatography-mass spectrometry

CS-670(I), being developed as a non-steroidal anti-inflammatory agent, is a racemic prodrug. It has been found to be readily metabolized to active metabolites: trans and unsaturated mono-ols (trans-OH, unsaturated-OH). We report here a method for the quantitative determination of the eight diol stereoisomers excreted in urine after administration I. The diols were well separated and quantitated using capillary column GC-MS after a rather simple derivatization with diazomethane-trifluoroacetic anhydride. Sex differences in rats and species differences between rats and mice were observed in the metabolism of I: the trans-diols originating from trans-OH were predominantly excreted in male and female rat urine but the excretion rate was greater in the male rats; the cis-diols originating from cis mono-ol (cis-OH) were the major urinary metabolites in mice. The hydroxy groups were mainly introduced at the respective equatorial hydrogen atoms at the 4′-carbon of trans-OH and the 5′-carbon of cis-OH. The 4′- and 5′-hydroxy groups in the diols were in the cis conformation with respect to the original 2′-hydroxy group. As approximately 9% of the trans-diols were excreted in urine after administration of cis-OH to rats, the chiral inversion from cis-OH to trans-OH was suggested to occur through the saturated ketone intermediate.     

Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE

Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the γ-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration.     

In Vivo and in Vitro binding of platinum to metallothionein

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammine-platinum(II)). Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl₂ significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl₂ pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.     

Geometric specificity of porcine liver carboxylesterase and its application for the production of cis-arylacrylic esters

Porcine liver carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) hydrolyses trans isomers of three different methyl 3-arylacrylates approximately one order of magnitude faster than the corresponding cis isomers. This phenomenon can be used for preparative production of cis esters from their trans counterparts as exemplified by methyl cinnamate. A solution of commercial, predominantly trans methyl cinnamate was irradiated by ultraviolet light and the resultant mixture of trans and cis esters was passed through a column packed with immobilized esterase. The effluent contained mainly trans cinnamic acid and cis methyl cinnamate. The latter was then extracted with methylene chloride, and the cis ester was isolated by evaporating the solvent. By esterifying the co-produced trans acid, the process can be made continuous.     

Evidence for a 13,14-Cis Cycle in Bacteriorhodopsin

We discuss to what extent the vibrational spectra of bacteriorhodopsin that have been observed and assigned by Smith et al. (1, 2) by means of resonance Raman and by Gerwert and Siebert (EMBO (Eur. Mol. Biol. Organ.) J. In press) by means of infrared absorption experiments are in agreement with a photo-cycle of bacteriorhodopsin that involves the sequence BR, IO(all-trans) → K(13,14-cis) → L(13,14-cis) → M(13-cis) → N(13-cis) → O(all-trans). Our discussion is based on a quantumchemical modified neglect of diatomic overlap [MNDO] calculation of the vibrational spectra of the relevant isomers of the protonated retinal Schiff base. In particular, we investigated in these calculations the effects of different charge environments on the frequencies of the relevant C-C single bond stretching vibrations of these isomers.