Chemoenzymatic synthesis of chiral 2,2'-bipyridine ligands and their N-oxide derivatives: applications in the asymmetric aminolysis of epoxides and asymmetric allylation of aldehydes.

A series of enantiopure 2,2'-bipyridines have been synthesised from the corresponding cis-dihydrodiol metabolites of 2-chloroquinolines. Several of the resulting hydroxylated 2,2'-bipyridines were found to be useful chiral ligands for the asymmetric aminolysis of meso-epoxides leading to the formation of enantioenriched amino alcohols (-->84% ee). N-oxide and N,N'-dioxide derivatives of these 2,2'-bipyridines, including separable atropisomers, have been synthesised and used as enantioselective organocatalysts in the asymmetric allylation of aldehydes to give allylic alcohols (-->86% ee).

Aerobic metabolism of 4-hydroxybenzoic acid in Archaea via an unusual pathway involving an intramolecular migration (NIH shift).

A novel haloarchaeal strain, Haloarcula sp. strain D1, grew aerobically on 4-hydroxybenzoic acid (4HBA) as a sole carbon and energy source and is the first member of the domain Archaea reported to do so. Unusually, D1 metabolized 4HBA via gentisic acid rather than via protocatechuic acid, hydroquinone, or catechol. Gentisate was detected in 4HBA-grown cultures, and gentisate 1,2-dioxygenase activity was induced in 4HBA-grown cells. Stoichiometric accumulation of gentisate from 4HBA was demonstrated in 4HBA-grown cell suspensions containing 2,2'-dipyridyl (which strongly inhibits gentisate 1,2-dioxygenase). To establish whether initial 1-hydroxylation of 4HBA with concomitant 1,2-carboxyl group migration to yield gentisate occurred, 2,6-dideutero-4HBA was synthesized and used as a substrate. Deuterated gentisate was recovered from cell suspensions and identified as 3-deutero-gentisate, using gas chromatography-mass spectrometry and proton nuclear magnetic resonance spectroscopy. This structural isomer would be expected only if a 1,2-carboxyl group migration had taken place, and it provides compelling evidence that the 4HBA pathway in Haloarcula sp. strain D1 involves a hydroxylation-induced intramolecular migration. To our knowledge, this is the first report of a pathway which involves such a transformation (called an NIH shift) in the domain Archaea.

The effect of intravenous haloperidol on QT interval dispersion in critically ill patients: comparison with QT interval prolongation for assessment of risk of Torsades de Pointes.

The objective of this study was to determine the effect of intravenous haloperidol on QT interval dispersion in critically ill patients and to compare increases in QT interval dispersion and QTc intervals in patients who developed haloperidol-induced Torsades de Pointes versus those in patients who did not. This was a case-controlled study of 30 critically ill patients who received intravenous haloperidol for delusional agitation. Cases were patients (n = 6) who developed Torsades de Pointes during haloperidol therapy. Controls were patients (n = 24) who did not experience haloperidol-induced Torsades dePointes. QTc intervals were measured and QT interval dispersion was calculated. Haloperidol prolonged QTc interval compared to pretreatment values in Torsades de Pointes patients (606 +/- 61 ms vs. 501 +/- 44 ms, p = 0.007) by a greater magnitude than in patients who did not experience Torsades de Pointes (507 +/- 60 ms vs. 466 +/- 44, p = 0.01). Twelve-lead analysis revealed that QT interval dispersion increased in patients who experienced Torsades de Pointes (from 63 +/- 11 to 95 +/- 22 ms, p = 0.03) but not in those who did not (62 +/- 18 vs. 60 +/- 26 ms, p = 0.66). Analysis of precordial leads only showed no significant haloperidol-associated increases in QTinterval dispersion in eithergroup. The odds of developing haloperidol-induced Torsades de Pointes were highest in patients with QTc interval > 521 ms during haloperidol therapy(odds ratio = 12.1). It was concluded that intravenous haloperidol prolongs QTc intervals in critically ill patients. The degree of prolongation is greater in patients who experience Torsades de Pointes. QT interval dispersion may be increased in patients who develop haloperidol-induced Torsades de Pointes compared with those who do not. However, these effects are dependent on the method of measurement (12 leads vs. precordial leads). In addition, the odds of haloperidol-induced Torsades de Pointes are higherin patients with QTc intervalprolongation compared with increased QT interval dispersion. Therefore, QTc interval determination remains preferable to QT interval dispersion as a means assessment of risk for haloperidol-induced Torsades de Pointes.

Aromatic dioxygenases: molecular biocatalysis and applications.

Aromatic dioxygenases have been found to catalyse single and tandem oxidation reactions of conjugated polyenes. Rational selection and design of dioxygenases, allied to substrate shape, size and substitution pattern, has been used to control regiochemistry and stereochemistry during the oxygenation process. The resulting enantiopure bioproducts have been increasingly utilised as precursors for new and alternative routes in chiral synthesis.

Regioselectivity and enantioselectivity of naphthalene dioxygenase during arene cis-dihydroxylation: control by phenylalanine 352 in the alpha subunit.

The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the alpha subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl cis-3,4-dihydrodiol [77 and 60% (-)-(3S,4R), respectively] to that formed by wild-type NDO [>98% (+)-(3R,4S)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene cis-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoikuyae B8/36. A recombinant Escherichia coli strain expressing the F352V variant of NDO and the enantioselective toluene cis-dihydrodiol dehydrogenase from Pseudomonas putida F1 was used to produce enantiomerically pure (-)-biphenyl cis-(3S,4R)-dihydrodiol and (-)-phenanthrene cis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively.

Left ventricular thrombus and subsequent thromboembolism in patients with severe systolic dysfunction.

STUDY OBJECTIVES: To determine the frequency of left ventricular (LV) thrombi by echocardiography and to define the predictors of LV thrombus and subsequent thromboembolism. DESIGN: Retrospective case-control design. SETTING: Single tertiary care center. PATIENTS: Twenty-eight patients with LV thrombus in a consecutive series of 144 patients with severe LV dysfunction and follow-up period for a mean of 27.6 months. MEASUREMENTS AND RESULTS: Thirty-five clinical and echocardiographic variables were evaluated. The mean age of patients with (n = 28) vs patients without (n = 116) LV thrombus was 50.3 +/- 11.0 years vs 54.2 +/- 11.1 years (p = 0.09), with 22 patients (78.6%) and 78 patients (67.2%) being male (p = 0.24), respectively. The mean ejection fraction (EF) for those with vs those without LV thrombus was 17.5 +/- 5.5 vs 20.0 +/- 6.9 (p = 0. 08), with 16 patients (57.1%) and 42 patients (36.2%) having an EF < 20% (p = 0.04), respectively. The groups were similar with respect to other baseline characteristics, comorbid illnesses, and drug therapies other than anticoagulants. All 28 patients with LV thrombus (100%) and 54 of those without LV thrombus (46.6%) were treated with warfarin. Ischemic etiology of the cardiomyopathy (odds ratio, 4.78; 95% confidence interval, 1.51 to 15.11; p = 0.008) and increased LV internal diastolic dimension (LVIDD; odds ratio, 1.10; 95% confidence interval, 1.03 to 1.18; p = 0.004) were found to be independent predictors of thrombus formation. Peripheral embolism occurred in 5 patients (17.9%) vs 13 patients (11.2%) of those with and without LV thrombi, respectively (p = 0.35). Ischemic etiology of the cardiomyopathy (odds ratio, 3.79; 95% confidence interval, 1. 13 to 12.64; p = 0.03) and EF (odds ratio, 0.91; 95% confidence interval, 0.82 to 1.00; p = 0.04) were found to be independent predictors of systemic embolization. The patients with an embolic event suffered a significantly higher mortality (7 of 18 patients; 38.9%) during the follow-up period when compared to those without an embolic event (13 of 126 patients; 10.3%; p < 0.0001). CONCLUSIONS: We conclude that ischemic cardiomyopathy and dilated LV chamber sizes (LVIDD > 60 mm) are independently associated with LV thrombi. A peripheral embolic event is related to poor long-term survival in this patient group.

Telomerase activity in Indian patients with carcinomas of the aerodigestive tract.

The activation of telomerase, a ribonucleoprotein maintaining telomeric length, might represent an additional required event in the multigenetic process of tumorigenesis in human cancer. To investigate whether telomerase activity is a prerequisite or a useful indicator of malignant potential, we assayed the enzyme in squamous cell carcinomas and tried to observe any correlation with clinical staging and histopathological grading. We have studied telomerase activity in 23 samples of squamous cell carcinomas of the aerodigestive tract and in 22 corresponding samples of adjoining tissues using the telomerase repeat amplification assay. Telomerase activity was detected in 100% of the tumor samples studied. The telomerase activity increased with tumor grading, but was not statistically significant. Low levels of enzyme activity were also detected in 60.86% of the adjoining normal tissue samples. Reactivation of telomerase may play an important role in the carcinogenesis of aerodigestive tract tumors. Detection of enzyme activity in the adjoining normal tissue is suggestive of microinvasion of tumor cells and/or early activation of telomerase in the progression towards cancer, before possible pathological identification.

Contrasting effects of a nonionic surfactant on the biotransformation of polycyclic aromatic hydrocarbons to cis-dihydrodiols by soil bacteria.

The biotransformation of the polycyclic aromatic hydrocarbons (PAHs) naphthalene and phenanthrene was investigated by using two dioxygenase-expressing bacteria, Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae B8/36, under conditions which facilitate mass-transfer limited substrate oxidation. Both of these strains are mutants that accumulate cis-dihydrodiol metabolites under the reaction conditions used. The effects of the nonpolar solvent 2,2,4, 4,6,8,8-heptamethylnonane (HMN) and the nonionic surfactant Triton X-100 on the rate of accumulation of these metabolites were determined. HMN increased the rate of accumulation of metabolites for both microorganisms, with both substrates. The enhancement effect was most noticeable with phenanthrene, which has a lower aqueous solubility than naphthalene. Triton X-100 increased the rate of oxidation of the PAHs with strain 9816/11 with the effect being most noticeable when phenanthrene was used as a substrate. However, the surfactant inhibited the biotransformation of both naphthalene and phenanthrene with strain B8/36 under the same conditions. The observation that a nonionic surfactant could have such contrasting effects on PAH oxidation by different bacteria, which are known to be important for the degradation of these compounds in the environment, may explain why previous research on the application of the surfactants to PAH bioremediation has yielded inconclusive results. The surfactant inhibited growth of the wild-type strain S. yanoikuyae B1 on aromatic compounds but did not inhibit B8/36 dioxygenase enzyme activity in vitro.

Torsades de Pointes associated with intravenous haloperidol in critically ill patients.

In this retrospective case-control study, 8 of 223 consecutive patients (3.6%) treated with intravenous haloperidol developed torsades de pointes, and were compared with 41 patients randomly selected as controls. The likelihood of torsades de pointes associated with intravenous haloperidol is significantly greater in patients receiving > or = 35 mg over 24 hours or in those with a QTc interval of >500 ms, or both.

Effect of combined nitrogen on the expression of nitrate reductase and nitrite reductase in Azorhizobium caulinodans.

In aerobically grown Azorhizobium caulinodans strain IRBG 46, in vivo expression of nitrate reductase (NR) and nitrite reductase (NiR) requires the presence of either nitrate or nitrite. On the contrary mere microaerobic conditions are sufficient for the expression of NR and NiR, however, addition of nitrate to the growth medium enhanced the activities of the enzymes. Optimum concentration of nitrate for maximum expression of NR and NiR activities was different in aerobic and microaerobic conditions. Nitrite was released into the medium both in aerobic and microaerobic conditions beyond a particular concentration of nitrate in the medium. Dissimilatory nitrate reduction was affected to a lesser extent by ammonium compared to assimilatory nitrate reduction.

Effect of nitrate on nitrogen fixation and nitrate assimilation in Azorhizobium-Sesbania rostrata symbiotic system.

Root nodule formation was inhibited by 30% and 50% respectively at low concentration of 1 mM and 2 mM nitrate, while stem nodule formation was enhanced by 50% only at 1 mM nitrate. The nodule specific nitrogenase activity decreased with the increasing concentration of nitrate. At 1 mM nitrate nitrogenase activity per plant stem nodule was not affected, but it was less than 50% in the root nodules as compared to control. Increasing concentration of nitrate increased in vivo activity of nitrate reductase (NR) significantly in stem, root nodules and leaves. Nodule cytosolic NR utilized both NADH and succinate as electron donor, but not reduced MV. However bacteroidal NR utilised reduced MV as reductant more efficiently than succinate.

Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038.

The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.

Evidence for the glycosylation of porcine serum transferrin at a single site located within the C-terminal lobe.

In this study we report the number and location of the glycans on PST. Urea PAGE and SDS-PAGE have been used to follow the enzymatic removal of sialic acids and of glycans from PST and the masses of native and deglycosylated PST have been determined by electrospray mass spectrometry. The results are consistent with the presence of a single biantennary glycan chain. As amino acid sequence analysis demonstrated the absence of a glycosylated asparagine at position 25, the glycosylation site is restricted to Asp-497.

Chemical and enzyme-catalysed synthesis of quinoline arene hydrates.

Arene hydrates of quinoline have been synthesized by enzyme-catalysed benzylic and allylic hydroxylation of dihydroquinolines in growing cultures of the fungus Mortierella isabellina and in liver microsomal preparations. A preference for allylic hydroxylation was generally observed in eukaryotic systems. Evidence of epoxidation of dihydroarenes by both the fungal and animal enzyme systems was also obtained. The chemical synthesis of these arene hydrates (5-hydroxy-5,6-dihydroquinoline, 6-hydroxy-5,6-dihydroquinoline, 7-hydroxy-7,8-dihydroxyquinoline, and 8-hydroxy-7,8-dihydroquinoline) from the corresponding hydroxy-5,6,7,8-tetrahydroquinolines, has also been accomplished.

Biotransformation of 1,2-dihydronaphthalene and 1,2-dihydroanthracene by rat liver microsomes and purified cytochromes P-450. Formation of arene hydrates of naphthalene and anthracene.

Both 1,2-dihydronaphthalene and 1,2-dihydroanthracene were hydroxylated at the benzylic (1-) or the allylic (2-) position by rat liver microsomes and purified cytochrome P-450 enzymes to yield "arene hydrates". Two other classes of metabolites were formed, the dehydrogenation products naphthalene and anthracene, and trans-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and its anthracene analog as products of the classical expoxide pathway. Regioselectivity (hydroxylation at benzylic or allylic positions) and stereoselectivity (hydroxylation at pro-R or pro-S hydrogen atoms) during metabolism of dihydroarenes to yield arene hydrates were found to be dependent upon the nature of the inducing agents used during pretreatment of the rats and thus the level of particular P-450 enzymes. This selectivity was more pronounced for anthracene than for naphthalene. Naphthalene and anthracene were formed enzymatically by direct dehydrogenation of the dihydro compounds rather than by dehydration of the arene hydrate metabolites. A general mechanism involving the intermediacy of benzylic and resonance-stabilized allylic carbon radicals can account for the formation of both enzyme-catalyzed hydroxylation (arene hydrate) and dehydrogenation (arene) metabolites of dihydroarene substrates.

Adenine photodimerization in deoxyadenylate sequences: elucidation of the mechanism through structural studies of a major d(ApA) photoproduct.

The mechanism of the photodimerization of adjacent adenine bases on the same strand of DNA has been elucidated by determining the structure of one of the two major photoproducts that are formed by UV irradiation of the deoxydinucleoside monophosphate d(ApA). The photoproduct, denoted d(ApA)*, corresponds to a species of adenine photodimer first described by Pörschke (Pörschke, D. (1973) J.Am.Chem.Soc. 95, 8440-8446). From a detailed examination of its chemical and spectroscopic properties, including comparisons with the model compound N-cyano-N1-(1-methylimidazol-5-yl)formamidine, it is deduced that d(ApA)* contains a deoxyadenosine unit covalently linked through its C(8) position to C(4) of an imidazole N(1) deoxyribonucleoside moiety bearing an N-cyanoformamidino substituent at C(5). On treatment with acid, d(ApA)* is degraded with high specificity to 8-(5-amino-imidazol-4-yl)adenine whose identity has been confirmed by independent chemical synthesis. It is concluded that the primary event in adenine photodimerization entails photoaddition of the N(7)-C(8) double bond of the 5'-adenine across the C(6) and C(5) positions of the 3'-adenine. The azetidine species thus generated acts as a common precursor to both types of d(ApA) photoproduct which are formed from it by competing modes of azetidine ring fission.

Biotransformation of the Pentahydroxy Flavone Quercetin by Rhizobium loti and Bradyrhizobium Strains (Lotus).

Lotus rhizobia catabolized quercetin in an arabinose-based medium via a novel form of C-ring cleavage, yielding phloroglucinol and protocatechuic acid. Conservation of the A and B rings of the flavone suggests that a chalcone could be formed as a transient intermediate.

Preferential modification of guanine bases in DNA by dimethyldioxirane and its application to DNA sequencing.

From gel sequencing experiments with 32P-end-labelled oligodeoxyribonucleotides, it is shown that treatment of DNA with the powerful oxidant dimethyldioxirane, followed by heating in piperidine, causes selective strand scission at the sites of guanine bases. The same specificity for cleavage at guanine was observed with a 45-mer labelled at either the 3'- or 5'-end and with a single and double stranded 34-mer. On account of its speed and operational simplicity, modification with dimethyldioxirane is proposed as a practicable alternative to conventional chemical sequencing procedures for locating guanine bases in DNA.