Rhodopsin photoproducts in 2D crystals

The published electron microscope and X-ray structures of rhodopsin have made available a detailed picture of the inactive dark state of rhodopsin. Yet, the photointermediates of rhodopsin that ultimately lead to the activated receptor species still await a similar analysis. Such an analysis first requires the generation and characterization of the photoproducts that can be obtained in crystals of rhodopsin. We therefore studied with Fourier-transform infrared (FTIR) difference spectroscopy the photoproducts in 2D crystals of bovine rhodopsin in a p22(1)2(1) crystal form. The spectra obtained by cryotrapping revealed that in this crystal form the still inactive early intermediates batho, lumi, and meta I are similar to those obtained from rhodopsin in native disk membranes, although the transition from lumi to meta I is shifted to a higher temperature. However, at room temperature, the formation of the active state, meta II, is blocked in the crystalline environment. Instead, an intermediate state is formed that bears some features of meta II but lacks the specific conformational changes required for activity. Despite being unable to activate its cognate G protein, transducin, to a significant extent, this intermediate state is capable of interacting with functional transducin-derived peptides to a limited extent. Therefore, while unable to support formation of rhodopsin's active state meta II, 2D p22(1)2(1) crystals proved to be very suitable for determining 3D structures of its still inactive precursors, batho, lumi, and meta I. In future studies, FTIR spectroscopy may serve as a sensitive assay to screen crystals grown under altered conditions for potential formation of the active state, meta II.     

Synthesis of aromatic compounds containing a 1,1-dialkyl-2-trifluoromethyl group, a bioisostere of the tert-alkyl moiety

1,1-Dialkyl-2-perfluoroalkyl compounds, which are potential metabolically stable bioisosteres of the tert-alkyl moiety, have been synthesized from the corresponding tertiary alcohols using titanium (IV) chloride-dimethylzinc or trimethylaluminium as the source of the methyl group. The synthetic methods proved to be versatile for synthesizing 1,1-dimethyl-2,2,2-trifluoroethyl compounds and analogs, including compounds containing aromatic and heterocyclic rings.     

Stability‐indicating spectrofluorimetric method for the assay of riboflavin and photoproducts: Kinetic applications

A stability‐indicating spectrofluorimetric method has been developed for the simultaneous assay of riboflavin (RF) and photoproducts, formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF) in aqueous solution. The method is based on the extraction of LC formed in acid solution and LC and LF formed in alkaline solution with chloroform at pH 2.0 and their assay by fluorescence measurements at 478 and 530 nm, respectively. The aqueous phase, on readjustment of the pH to 6.5, is used to extract FMF with chloroform and its assay is carried out at 530 nm. The aqueous phase is then used for the assay of RF at 530 nm. The proposed method gives more accurate results for the assay of RF compared to those of the United States Pharmacopeia (USP) spectrofluorimetric method which does not take into account the presence of RF photoproducts having similar fluorescence characteristics. The proposed method along with the USP method has been applied to the study of the kinetics of photolysis of RF, assay of stored commercial vitamin preparations and their radiated samples. The results show that the USP method does not distinguish between the fluorescence of RF and its photoproducts, and, therefore, gives erroneous results with about 11% excess in the quantity of the vitamin compared to that of the proposed method. This is due to the interference of the fluorescence of photoproducts in the assay of RF. The method has been validated for various analytical parameters according to the guideline of the International Council for Harmonization (ICH).     

Oxidation of aromatic compounds in soil

The oxidation ofp-hydroxybenzoic acid, quinic acid, vanillin and coumarin in soil was studied. With vanillin, and particularly with coumarin, the lag phase for oxygen consumption was longer and the rate of oxygen consumption attained more than one peak. In soil preincubated with the relevant substrate, the second dose of the same substrate was oxidized more rapidly. If the soil was preincubated with glucose, the lag phase was also shortened and oxygen consumption was raised with all aromatic substrates.     

Simple aromatic compounds containing propenone moiety show considerable dual COX/5-LOX inhibitory activities

For the development of safer anti-inflammatory agents, simple aromatic compounds containing propenone moiety were prepared and evaluated for their dual COX/5-LOX inhibitory activities. Among the 17 prepared compounds, most of the compounds exhibited considerable COX/5-LOX inhibitory activities. Especially compound C(15) showed the most significant dual COX/5-LOX inhibitory activity.     

Augmentation of H2 photoproduction in Rhodopseudomonas palustris by N-heterocyclic aromatic compounds

Increases of 23- (5.6 mmol acetylene reduced mg dry wt^–1) and 16- (4 mmol acetylene reduced mg dry wt^–1) fold in nitrogenase activity and 12- (671 l H₂ mg dry wt^–1 h^–1) and 6- (349 l mg dry wt^–1 h^–1) fold in H₂ photoproduction in Rhodopseudomonas palustris JA1 over 24 h were achieved with pyrazine 2-carboxylate (3 mM) and 3-picoline (3 mM), respectively, and were higher than earlier reports of enhancement (1.5 to 5- fold) in biological H₂ production using various alternative methods.     

Bactericidal and inhibitory effects of azole antifungal compounds on Mycobacterium smegmatis

Azole antifungals are central to therapy and act by inhibiting a cytochrome P450, sterol 14-demethylase and blocking normal sterol synthesis. Our recent identification of a mycobacterial sterol biosynthetic pathway led us to probe the efficacy of a range of these compounds against Mycobacterium smegmatis. Several showed equivalent or greater inhibitory effects to those against Candida albicans, and bactericidal activity was demonstrated for four compounds, clotrimazole, econazole, miconazole and tebuconazole. The major drug used clinically, fluconazole, was ineffective. The results are discussed in the light of the world-wide spread of tuberculosis, including drug-resistant forms and the requirement for new drugs.     

Biotransformation of N-substituted aromatic compounds in mammalian spermatozoa. Nonoxidative formation of N-hydroxy-N-arylacetamides from nitroso aromatic compounds

The metabolism of N-substituted aromatic compounds, i.e. aniline, acetanilide, N-hydroxyacetanilide, nitrosobenzene, and nitrobenzene in mammalian spermatozoa was investigated. In boar spermatozoa fortified with glucose, no acetylation, deacetylation, and monooxygenation of these compounds were found. Nitrobenzene was reduced slowly, but nitrosobenzene was a good substrate for this reductive activity. In the latter reaction, the products were N-hydroxyacetanilide, azoxybenzene, and an organic phase-nonextractable metabolite(s). Pyruvate was found to be involved in the formation of N-hydroxyacetanilide from nitrosobenzene, and the reaction occurred through a ping-pong mechanism. This enzymatic activity, located in the mid-piece fraction of spermatozoa, was enhanced by thiamine pyrophosphate and Mg2+ and inhibited by thiamine thiazolone pyrophosphate. N-Hydroxyacetanilide formed from [3(-13)C]pyruvate showed complete retention of the isotope at the methyl carbon of the molecule. 2-Nitrosofluorene and 4-nitrosobiphenyl were also transformed into the corresponding N-hydroxy-N-arylacetamides. N-Hydroxyacetanilide was also formed in rat and human spermatozoa. These facts suggest that the formation of N-hydroxy-N-arylacetamides from the nitroso aromatic compounds and pyruvate is mediated by a pyruvate dehydrogenase complex located in the mitochondria of spermatozoa. The formation of both azoxybenzene and the organic phase-nonextractable metabolite(s) was found to be a pyruvate-independent nonenzymatic reaction.     

Anaerobic oxidation of aromatic compounds and hydrocarbons

Aromatic compounds and hydrocarbons have in common a great stability due to resonance energy and inertness of CbondH and CbondC bonds. It has been taken for granted that the metabolism of these compounds obligatorily depends on molecular oxygen. Oxygen is required first to introduce hydroxyl groups into the substrate and then to cleave the aromatic ring. However, newly discovered bacterial enzymes and reactions involved in oxidation of aromatic and hydrocarbon compounds to CO(2) in the complete absence of molecular oxygen have been discovered. Of special interest are two reactions: the reduction of the aromatic ring of benzoyl-coenzyme A and the addition of fumarate to hydrocarbons. These reactions transform aromatic rings and hydrocarbons into products that can be oxidized via more conventional beta-oxidation pathways.     

Cucurbitacins and aromatic compounds from Crinodendron hookerianum

Cucurbitacins-D, -F, -H, and the new member 23,24-dihydrocucurbitacin F have been isolated from Crinodendron hookeranium. This is the first report of cucurbitacins in the Elaeocarpaceae. Other components isolated included gallic and ellagic acids, methyl 3-O-methylgallate and an ellagitannin.     

Application of FT-IR spectroscopy for control of the medium composition during the biodegradation of nitro aromatic compounds

Previous studies showed that cabbage leaf extract (CLE) added to the growth medium can noticeably promote the degradation of nitro aromatic compounds by specific consortium of bacteria upon their growth. For further development of the approach for contaminated soil remediation it was necessary to evaluate the qualitative and/or quantitative composition of different origin CLE and their relevance on the growth of explosives-degrading bacteria. Six CLE (different by species, cultivars and harvesting time) were tested and used as additives to the growth medium. It was shown that nitro aromatic compounds can be identified in the FT-IR absorption spectra by the characteristic band at 1,527 cm⁻¹, and in CLE by the characteristic band at 1,602 cm⁻¹. The intensity of the CLE band at 1,602 cm⁻¹ correlated with the concentration of total nitrogen (R ² = 0.87) and decreased upon the growth of bacteria. The content of nitrogen in CLE differed (0.22–1.00 vol.%) and significantly influenced the content of total carbohydrates (9.50–16.00% DW) and lipids [3.90–9.90% dry weight (DW)] accumulated in bacterial cells while the content of proteins was similar in all samples. Though this study showed quantitative differences in the composition of the studied CLE and the response of bacterial cells to the composition of the growth media, and proved the potential of this additive for remediation of contaminated soil. It was shown that analysis of CLE and monitoring of the conversion of nitro aromatic compounds can be investigated by FT-IR spectroscopy as well as by conventional chemical methods.