Redox, haem and CO in enzymatic catalysis and regulation

The present paper describes general principles of redox catalysis and redox regulation in two diverse systems. The first is microbial metabolism of CO by the Wood-Ljungdahl pathway, which involves the conversion of CO or H2/CO2 into acetyl-CoA, which then serves as a source of ATP and cell carbon. The focus is on two enzymes that make and utilize CO, CODH (carbon monoxide dehydrogenase) and ACS (acetyl-CoA synthase). In this pathway, CODH converts CO2 into CO and ACS generates acetyl-CoA in a reaction involving Ni·CO, methyl-Ni and acetyl-Ni as catalytic intermediates. A 70 ? (1 ?=0.1?nm) channel guides CO, generated at the active site of CODH, to a CO 'cage' near the ACS active site to sequester this reactive species and assure its rapid availability to participate in a kinetically coupled reaction with an unstable Ni(I) state that was recently trapped by photolytic, rapid kinetic and spectroscopic studies. The present paper also describes studies of two haem-regulated systems that involve a principle of metabolic regulation interlinking redox, haem and CO. Recent studies with HO2 (haem oxygenase-2), a K+ ion channel (the BK channel) and a nuclear receptor (Rev-Erb) demonstrate that this mode of regulation involves a thiol-disulfide redox switch that regulates haem binding and that gas signalling molecules (CO and NO) modulate the effect of haem.     

Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight

Planta - Overexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin in Malus . Decreased flavonoid concentrations but unchanged flavonoid class composition were...     

Repair synthesis and sedimentation analysis of DNA of human cells exposed to dimethylnitrosamine and activated dimethylnitrosamine

The capacity of dimethylnitrosamine(DMN), and of DMN activated by a NADPH-fortified mouse liver microsomal preparation, to elicit DNA alterations in cultured human fibroblasts was examined. A maximum induction of DNA repair synthesis, estimated by unscheduled incorporation of tritiated thymidine, occurred following 60-minute incubation of the human cells with DMN activated by a NADPH-fortified mouse liver microsomal preparation. A low level of DNA repair activity followed exposure to DMN alone, or to DMN mixed with the microsomal preparation without NADPH or without O₂. The extent of DNA damage, estimated by velocity sedimentation of DNA through alkaline sucrose gradients, was maximum following treatment with DMN mixed with the NADPH-fortified microsomal preparation. The combined application of $\text{in vitro}$ activation systems and estimation of DNA repair synthesis in cultured cells may be exploited in the detection of precarcinogens.     

Aziridine-2,3-dicarboxylate inhibitors targeting the major cysteine protease of Trypanosoma brucei as lead trypanocidal agents

The protozoan parasite Trypanosoma brucei causes Human African trypanosomiasis, which is fatal if left untreated. Due to the toxicity of currently used drugs and emerging drug resistance, there is an urgent need for novel therapies. The major trypanosome papain-like cysteine protease expressed by the parasite (e.g., rhodesain in T. b. rhodesiense) is considered an important target for the development of new trypanocidal drugs. Series of aziridine-2,3-dicarboxylate-based cysteine protease inhibitors have been tested, most of them inhibiting rhodesain in the low micromolar range. Among these, only dibenzyl aziridine-2,3-dicarboxylates display trypanocidal activity being equipotent to the drug eflornithine. The Leu-Pro-containing aziridinyl tripeptides 13a-f are the most promising as they are not cytotoxic to macrophages up to concentrations of 125microM.     

Profiling of adipokines secreted from human subcutaneous adipose tissue in response to PPAR agonists

The role of PPARs in the regulation of human adipose tissue secretome has received little attention despite its potential importance in the therapeutic actions of PPAR agonists. Here, we have investigated the effect of selective PPARgamma, PPARalpha, and PPARbeta/delta agonists on the production of adipokines by human subcutaneous adipose tissue. Antibody arrays were used to measure secreted factors in media from cultured adipose tissue explants. Sixteen proteins were produced in significant amounts. Activation of PPARs regulated the production of five proteins. Treatments with the three PPAR agonists decreased the secretion of leptin and interleukin-6. PPARalpha and beta/delta agonists markedly enhanced hepatocyte growth factor secretion whereas PPARbeta/delta down-regulated angiogenin and up-regulated TIMP-1 release. Hepatocyte growth factor, interleukin-6, and TIMP-1 are chiefly expressed in cells from the stromal vascular fraction whereas angiogenin is expressed in both adipocytes and cells from the stromal vascular fraction. Our data show that PPAR agonists modulate secretion of bioactive molecules from the different cell types composing human adipose tissue.     

Das Vorkommen von Kohlenhydraten im Ruhekern und während der Mitose

Zusammenfassung Kerne verschiedener tierischer Zellen und der Alge Acetabularia wurden mit Hilfe der HotchkissReaktion auf ihren Kohlenhydratgehalt geprüft. Hierbei ergab sich, daß sich nicht teilende Kerne in Zellen mit einer starken Eiweißsynthese (Ganglienkerne von Mäusen und Tauben, Leberkerne von Mäusen, Oocytenkerne von Cyclops, Diaptomus, Tipula und Ascaris) frei von cytochemisch erfaßbaren Kohlenhydraten sind (ausgenommen Acetabularia), während in sich teilenden Kernen (Oogonien- und Furchungskernen von Cyclops) Kohlenhydrate nachgewiesen werden können. Die Kohlenhydrate erscheinen im Kernsaft und in der aus dem Kernsaft intranukleär sich ausbildenden Spindel, sowie in den Centrosomen und Astrosphären. Der Kohlenhydratgehalt der Kerne wechselt je nach Funktion dieser. So ist die Hotchkiss-Reaktion bei Cyclops positiv in Oogonienkernen, negativ in Oocytenkernen, positiv in Oocytenkernen kurz vor der Reifeteilung, positiv in Furchungskernen, negativ in den Urgeschlechtszellen, die sich während der Embryonalentwicklung nicht mehr teilen. Diese Befunde weisen auf eine wesentliche Bedeutung der Kohlenhydrate für den extrachromosomalen Teilungsapparat, wie Spindel, Centrosom und Astrosphäre hin.Mit Unterstützung durch die Notgemeinschaft der Deutschen Wissenschaft.     

Growth-promoting nitrogen nutrition affects flavonoid biosynthesis in young apple (Malus domestica Borkh.) leaves

Enhanced shoot growth and a decrease in flavonoid concentration in apple trees grown under high nitrogen (N) supply was observed in previous studies, along with increasing scab susceptibility of cultivar "Golden Delicious" after high N nutrition. Several hypotheses have suggested that there is a trade-off between primary and secondary metabolism because of competition for common substrates, but nothing is known about regulation at the enzyme level. In this study, a set of experiments was performed to elucidate the effect of N nutrition on the activities of key enzymes involved in flavonoid biosynthesis (phenylalanine ammonia-lyase [PAL], chalcone synthase/chalcone isomerase [CHS/CHI}, flavanone 3-hydroxylase [FHT], flavonol synthase [FLS], dihydroflavonol 4-reductase [DFR]) and the accumulation of different groups of phenylpropanoids. The inhibition of flavonoid accumulation by high N nutrition could be confirmed, but the influence of N supply on the flavonoid enzymes CHS/CHI, FHT, DFR, and FLS was not evident. However, PAL activity seems to be downregulated, thus forming a bottleneck resulting in a generally decreased flavonoid accumulation. Furthermore, the response of the scab-resistant cultivar "Rewena" to high N nutrition was not as strong as that of the susceptible cultivar "Golden Delicious".     

Power to detect higher-order epistatic interactions in a metabolic pathway using a new mapping strategy

Epistatic interactions among quantitative trait loci (QTL) contribute substantially to the variation in complex traits. The main objectives of this study were to (i) compare three- vs. four-step genome scans to identify three-way epistatic interactions among QTL belonging to a metabolic pathway, (ii) investigate by computer simulations the power and proportion of false positives (PFP) for detecting three-way interactions among QTL in recombinant inbred line (RIL) populations derived from a nested mating design, and (iii) compare these estimates to those obtained for detecting three-way interactions among QTL in RIL populations derived from diallel and different partial diallel mating designs. The single-nucleotide polymorphism haplotype data of B73 and 25 diverse maize inbreds were used to simulate the production of various RIL populations. Compared to the three-step genome scan, the power to detect three-way interactions was higher with the four-step genome scan. Higher power to detect three-way interactions was observed for RILs derived from optimally allocated distance-based designs than from nested designs or diallel designs. The power and PFP to detect three-way interactions using a nested design with 5000 RILs were for both the 4-QTL and the 12-QTL scenario of a magnitude that seems promising for their identification.