Structure of Cu2(indomethacin)4 and the reaction with superoxide in aprotic systems

The copper complex of indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole acetate), a common anti-inflammatory drug, was prepared and characterized. Crystal structure determination revealed the dimeric form of the 1:2 complex, namely Cu₂(indomethacin)₄ · L₂, in the unit cell. Suprisingly, the copper-copper distance (263 pm) was very close to metallic copper (256 pm). The two coordination sites in the copper-copper axis can be readily replaced by superoxide. An intriguing similarity to Cu₂(acetate)₄ was seen.Due to the lipophilic nature of the indomethacin ligand, this copper complex reacted with superoxide in aprotic solvents. The superoxide dismutating activity was successfully demonstrated in Me₂SO/water and acetonitrile/water mixtures using the nitro-blue tetrazolium assay and pulse radiolysis. The second-order rate constant of 6 · 10⁹ M^?1 · s^?1 in strictly aqueous systems dropped only slightly to 1.1 · 10⁹ M^?1 · s^?1 when aprotic solvents were used. This is the fastest rate constant ever observed for a copper-dependent dismutation of superoxide. The KO₂-induced lipid peroxidation in both erythrocytes and liver microsomes was suppressed by 70% in the presence of 1 · 10^?10 mol · ml^?1 of Cu₂(indomethacin)₄. The inhibitory action dropped to 25% when Cu₂Zn₂superoxide dismutase was employed. The formation of copper · indomethacin in rat serum after administration of indomethacin was shown in vitro and in vivo.     

The effect of substrate modification on binding of porcine pancreatic alpha amylase: hydrolysis of modified amylose containing D-allose residues

A modified amylose containing 10% of tritiated D-allose residues has been hydrolyzed by porcine pancreatic alpha amylase (PPA). This reaction produced a number of radioactive oligosaccharides of low molecular weight, including modified mono-, di-, and tri-saccharides, as well as larger products. Analysis of these products by chemical and enzymic methods identified D-allose, two isomers of modified maltose, and isomers of modified maltotriose. These results may be interpreted in terms of current PPA models to indicate that D-allose residues may be productively bound at all five subsites of the active site of the enzyme. The distribution of modified residues in these products, however, further suggests that productive binding of D-allose at the subsite where catalytic attack occurs (subsite 3) is less favorable than binding of D-glucose. These results are compared with results of a series of PPA substrates having modifications at C-3 and at other positions. Trends observed in enzyme hydrolysis of these modified substrates reflect factors that contribute to PPA catalysis, with respect to steric, electronic, and hydrogen-bonding interactions between enzyme and substrate.     

Protoporphyrin Treatment Modulates Susceptibility to Experimental Autoimmune Encephalomyelitis in miR-155-Deficient Mice

We previously identified heme oxygenase 1 (HO-1) as a specific target of miR-155, and inhibition of HO-1 activity restored the capacity of miR-155 ^-/- CD4⁺ T cells to promote antigen-driven inflammation after adoptive transfer in antigen-expressing recipients. Protoporphyrins are molecules recognized for their modulatory effect on HO-1 expression and function. In the present study, we investigated the effect of protoporphyrin treatment on the development of autoimmunity in miR-155-deficient mice. MiR-155-mediated control of HO-1 expression in promoting T cell-driven chronic autoimmunity was confirmed since HO-1 inhibition restored susceptibility to experimental autoimmune encephalomyelitis (EAE) in miR-155-deficient mice. The increased severity of the disease was accompanied by an enhanced T cell infiltration into the brain. Taken together, these results underline the importance of miR-155-mediated control of HO-1 expression in regulating the function of chronically-stimulated T cells in EAE.     

Biological rhythms in birds — development, insights and perspectives

The aim of this review is to show that probably the internal clock of precocial birds is imprinted in the prenatal period by exogenous factors (zeitgeber). The activity of organ functions occurs early during embryonic development, before this function is ultimately necessary to ensure the survival of the embryo. Prenatal activation of some functional systems may have a training effect on the postnatal efficiency.The development of physiological control systems is influenced by endogenous and exogenous factors during the late prenatal and early postnatal period: epigenetic adaptation processes play an important role in the development of animals; they have acquired characteristics which are innated but not genetically fixed. As a rule, the actual value during the determination period has a very strong influence on the set-point of the system. This will be explained using the example of thermoregulation.It is shown in detail that it seems to be possible to imprint the prenatal development of circadian rhythms by periodic changes of the light-dark cycle but not by rhythmic influence of acoustic signals.Altogether, there are more questions open than solved concerning the perinatal genesis of circadian rhythms in birds. Topics are given for the future research.     

Carbonic anhydrase subunits of the mitochondrial NADH dehydrogenase complex (complex I) in plants

The mitochondrial nicotinamide adenine dinucleotide, reduced (NADH) dehydrogenase complex (complex I) of plants has a molecular mass of about 1000 kDa and is composed of more than 40 distinct protein subunits. About three quarter of these subunits are homologous to complex I subunits of heterotrophic eukaryotes, whereas the remaining subunits are unique to plants. Among them are three to five structurally related proteins that resemble an archaebacterial γ-type carbonic anhydrase (γCA). The γCA subunits are attached to the membrane arm of complex I on the matrix-exposed side and form an extra spherical domain. At the same time, they span the inner mitochondrial membrane and are essential for assembly of the protein complex. Expression of the genes encoding γCA subunits is reduced if plants are cultivated in the presence of elevated CO₂ concentration. The functional role of these subunits within plant mitochondria is currently unknown but might be related to photorespiration. We propose that the complex I–integrated γCAs are involved in mitochondrial HCO₃⁻ formation to allow efficient recycling of inorganic carbon for CO₂ fixation in chloroplasts under high light conditions.     

Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies

Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance.