Role of the midgut gland in purine excretion in the robber crab,Birgus latro (Anomura: Coenobitidae)

White fecal strands of Birgus latro are composed of small spherules of uric acid with a mean diameter of 1.6 ± 0.6 μm. Large numbers of membrane‐bound spherules with concentric lamellae are present in the R cells of the midgut gland, so we suggest that lengths of white feces are produced by coordinated secretion of these spherules into the lumen of the midgut gland tubules. There are four cell types in the tubules with embryonic (E) cells at the distal tip, B cells in a narrow band at the distal end and R cells making up the bulk of the tubules and gland. F cells are sparsely scattered among the R cells. Midgut gland tissue was assayed for activities of xanthine dehydrogenase and xanthine oxidase, the two forms of xanthine oxidoreductase. Contrary to previous reports, we found that the midgut gland of B. latro contains only high activities of xanthine dehydrogenase. If proteinase inhibitors were omitted from the assays, however, significant activity of xanthine oxidase was measured, a result we regard as an artifact attributable to the partial conversion of xanthine dehydrogenase to xanthine oxidase by endogenous proteinases. R cells were demonstrated to contain peroxisomes, which may be involved in lipid metabolism rather than synthesis of uric acid. J. Morphol. 241:227–235, 1999 © 1999 Wiley‐Liss, Inc.     

Pyrogallol inhibits the growth of lung cancer Calu-6 cells via caspase-dependent apoptosis

Pyrogallol (PG) is a polyphenol compound and a known O₂⁻ generator. We evaluated the effects of PG on the growth and apoptosis of human pulmonary adenocarcinoma Calu-6 cells. PG decreased the viability of Calu-6 cells in a dose- and time-dependent manner. The induction of apoptosis by PG was accompanied by the loss of mitochondrial membrane potential (ΔΨ_m), cytochrome c release from mitochondria and activation of caspase-3 and caspase-8. All tested caspase inhibitors, especially the pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from PG-induced cell death. Rescue was accompanied by inhibition of caspase-3 activation and PARP cleavage. Treatment with Z-VAD also prevented the loss of mitochondrial membrane potential (ΔΨ_m). In conclusion, PG inhibits the growth of Calu-6 cells via caspase-dependent apoptosis.     

EPR studies of the Mo-enzyme aldehyde oxidoreductase from Desulfovibrio gigas: An application of the Bloch-Wangsness-Redfield theory to a system containing weakly-coupled paramagnetic redox centers with different relaxation rates

Electron transfer proteins and redox enzymes containing paramagnetic redox centers with different relaxation rates are widespread in nature. Despite both the long distances and chemical paths connecting these centers, they can present weak magnetic couplings produced by spin-spin interactions such as dipolar and isotropic exchange. We present here a theoretical model based on the Bloch-Wangsness-Redfield theory to analyze the dependence with temperature of EPR spectra of interacting pairs of spin 1/2 centers having different relaxation rates, as is the case of the molybdenum-containing enzyme aldehyde oxidoreductase from Desulfovibrio gigas. We analyze the changes of the EPR spectra of the slow relaxing center (Mo(V)) induced by the faster relaxing center (FeS center). At high temperatures, when the relaxation time T₁ of the fast relaxing center is very short, the magnetic coupling between centers is averaged to zero. Conversely, at low temperatures when T₁ is longer, no modulation of the coupling between metal centers can be detected.     

Assembly of non-natural electron transfer conduits in the cytochrome P450 system: a critical assessment and update of artificial redox constructs amenable to exploitation in biotechnological areas

The high plasticity of the active-site cavity of cytochromes P450, permitting reactivity toward a vast array of compounds, makes these enzymes attractive targets for biotechnological application. Escalating attention in this area is driven by remarkable progress in the rational design by DNA shuffling of self-sufficient, multi-domain P450/electron donor constructs simplifying the composition of biocatalytic systems. Moreover, versatile approaches were undertaken to supersede the well-established, NAD(P)H-steered proteinaceous redox chains by cost-effective alternative electron transfer conduits constituted of organometallic mediators or photoactivatable redox triggers. Electrochemical techniques have proven particularly useful: employing different types of carbon- and metal-based electrodes for the fabrication of biosensors, the continuing challenge was to optimize the conductive properties of these devices by creating biocompatible interfaces for transferring electrons between sensor surfaces and redox proteins. The present review provides a critical update of the most significant breakthroughs in innovative manipulation of the redox machinery, giving an impulse to exploitation of P450s in fields such as the production of fine chemicals, drug processing, medicinal diagnostics and remediation of biotopes contaminated with harmful environmental pollutants.     

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.     

Comparative analysis of leaf-type ferredoxin-NADP+ oxidoreductase isoforms in Arabidopsis thaliana

Physiological roles of the two distinct chloroplast-targeted ferredoxin-NADP⁺ oxidoreductase (FNR) isoforms in Arabidopsis thaliana were studied using T-DNA insertion line fnr1 and RNAi line fnr2 . In fnr2 FNR1 was present both as a thylakoid membrane-bound form and as a soluble protein, whereas in fnr1 the FNR2 protein existed solely in soluble form in the stroma. The fnr2 plants resembled fnr1 in having downregulated photosynthetic properties, expressed as low chlorophyll content, low accumulation of photosynthetic thylakoid proteins and reduced carbon fixation rate when compared with wild type (WT). Under standard growth conditions the level of F₀'rise' and the amplitude of the thermoluminescence afterglow (AG) band, shown to correlate with cyclic electron transfer (CET), were reduced in both fnr mutants. In contrast, when plants were grown under low temperatures, both fnr mutants showed an enhanced rate of CET when compared with the WT. These data exclude the possibility that distinct FNR isoforms feed electrons to specific CET pathways. Nevertheless, the fnr2 mutants had a distinct phenotype upon growth at low temperature. The fnr2 plants grown at low temperature were more tolerant against methyl viologen (MV)-induced cell death than fnr1 and WT. The unique tolerance of fnr2 plants grown at low temperature to oxidative stress correlated with an increased level of reduced ascorbate and reactive oxygen species (ROS) scavenging enzymes, as well as with a scarcity in the accumulation of thylakoid membrane protein complexes, as compared with fnr1 and WT. These results emphasize a critical role for FNR2 in the redistribution of electrons to various reducing pathways, upon conditions that modify the photosynthetic capacity of the plant.     

A novel insight into the regulation of light-independent chlorophyll biosynthesis in <Emphasis Type="Italic">Larix decidua</Emphasis> and <Emphasis Type="Italic">Picea abies</Emphasis> seedlings

Light-independent chlorophyll (Chl) biosynthesis is a prerequisite for the assembly of photosynthetic pigment–protein complexes in the dark. Dark-grown Larix decidua Mill. seedlings synthesize Chl only in the early developmental stages and their Chl level rapidly declines during the subsequent development. Our analysis of the key regulatory steps in Chl biosynthesis revealed that etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase and reduced 5-aminolevulinic acid synthesizing capacity. The level of the Chl precursor protochlorophyllide also declined in the developing larch cotyledons. Although the genes chlL, chlN and chlB encoding subunits of the light-independent protochlorophyllide oxidoreductase were constitutively expressed in the larch seedlings, the accumulation of the ChlB subunit was developmentally regulated and ChlB content decreased in the fully developed cotyledons. The efficiency of chlB RNA-editing was also reduced in the mature dark-grown larch seedlings. In contrast to larch, dark-grown seedlings of Picea abies (L.) Karst. accumulate Chl throughout their whole development and show a different control of ChlB expression. Analysis of the plastid ultrastructure, photosynthetic proteins by Western blotting and photosynthetic parameters by gas exchange and Chl fluorescence measurements provide additional experimental proofs for differences between dark and light Chl biosynthesis in spruce and larch seedlings.     

New pulsed EPR methods and their application to characterize mitochondrial complex I

Electron Paramagnetic Resonance (EPR) spectroscopy is the method of choice to study paramagnetic cofactors that often play an important role as active centers in electron transfer processes in biological systems. However, in many cases more than one paramagnetic species is contributing to the observed EPR spectrum, making the analysis of individual contributions difficult and in some cases impossible. With time-domain techniques it is possible to exploit differences in the relaxation behavior of different paramagnetic species to distinguish between them and separate their individual spectral contribution. Here we give an overview of the use of pulsed EPR spectroscopy to study the iron-sulfur clusters of NADH:ubiquinone oxidoreductase (complex I). While FeS cluster N1 can be studied individually at a temperature of 30 K, this is not possible for FeS cluster N2 due to its severe spectral overlap with cluster N1. In this case Relaxation Filtered Hyperfine (REFINE) spectroscopy can be used to separate the overlapping spectra based on differences in their relaxation behavior.