ATP stimulates Ca2+ oscillations and contraction in airway smooth muscle cells of mouse lung slices

In airway smooth muscle cells (SMCs) from mouse lung slices, > or =10 microM ATP induced Ca2+ oscillations that were accompanied by airway contraction. After approximately 1 min, the Ca2+ oscillations subsided and the airway relaxed. By contrast, > or =0.5 microM adenosine 5'-O-(3-thiotriphosphate) (nonhydrolyzable) induced Ca2+ oscillations in the SMCs and an associated airway contraction that persisted for >2 min. Adenosine 5'-O-(3-thiotriphosphate)-induced Ca2+ oscillations occurred in the absence of external Ca2+ but were abolished by the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin. Adenosine, AMP, and alpha,beta-methylene ATP had no effect on airway caliber, and the magnitude of the contractile response induced by a variety of nucleotides could be ranked in the following order: ATP = UTP > ADP. These results suggest that the SMC response to ATP is impaired by ATP hydrolysis and mediated via P2Y(2) or P2Y(4) receptors, activating phospholipase C to release Ca2+ via the inositol 1,4,5-trisphosphate receptor. We conclude that ATP can serve as a spasmogen of airway SMCs and that Ca2+ oscillations in SMCs are required to sustain airway contraction.     

Spirobipyridopyrans, spirobinaphthopyrans, indolinospiropyridopyrans, indolinospironaphthopyrans and indolinospironaphtho-1,4-oxazines: synthesis, study of X-ray crystal structure, antitumoral and antiviral evaluation

The novel racemic indolinospirobenzopyrans (5-7), indolinospironaphthopyrans (11-14) and indolinospironaphtho-1,4-oxazine (17) were synthesized by an aldol type of condensation of 1',3',3'-trimethyl-2 '-methyleneindoline and its 5-substituted derivatives with an appropriately substituted hydroxybenzaldehyde, hydroxynaphthaldehyde or nitrosonaphthol. An unequivocal proof of the stereostructures of 9 and 17 was obtained by the single-crystal X-ray diffraction method. A substituted indoline ring and the benzopyran ring in 9 and the naphtho-1,4-oxazine moiety in 17 are interconnected via the common chiral atom and positioned almost perpendicularly to each other. The five-membered 2,3-dihydropyrrolo moiety of the indoline ring adopts an envelope conformation in both structures. Of all the compounds of this series, spirobipyridopyran (1) inhibited specifically the growth of human melanoma (HBL) (IC(50): 0.9 microM) cells but not the growth of normal fibroblasts (WI38). Indolinospirobenzopyrans (8-10) showed significant cytostatic activities against all tumor cell lines. However, these compounds also exhibited a cytotoxic effect on normal human fibroblasts. The indolinospirobenzopyrans 4, 6-8, 10 and the indolinospironaphtho-1,4-oxazine 16 showed, albeit modest, selectivity as antiviral agents against varicella-zoster virus (VZV) and/or cytomegalovirus (CMV) (EC(50) within the concentration range of 1.0-12.6 microM).     

Interfacial phospholipids inhibit ozone-reactive absorption-mediated cytotoxicity in vitro

The intrapulmonary distribution of inhaled ozone (O(3)) and induction of site-specific cell injury are related to complex interactions among airflow patterns, local gas-phase concentrations, and the rates of O(3) flux into, and reaction and diffusion within, the epithelial lining fluid (ELF). Recent studies demonstrated that interfacial phospholipid films appreciably inhibited NO(2) absorption. Because surface-active phospholipids are present on alveolar and airway interfaces, we investigated the effects of interfacial films on O(3)-reactive absorption and acute cell injury. Compressed films of dipalmitoyl-glycero-3-phosphocholine (DPPC) and rat lung lavage lipids significantly reduced O(3)-reactive absorption by ascorbic acid, reduced glutathione, and uric acid. Conversely, unsaturated phosphatidylcholine films did not inhibit O(3) absorption. We evaluated O(3)-mediated cell injury using a human lung fibroblast cell culture system, an intermittent tilting exposure regimen to produce a thin covering layer, and nuclear fluorochrome permeability. Exposure produced negligible injury in cells covered with MEM. However, addition of AH(2) produced appreciable (<50%) cell injury. Film spreading of DPPC monolayers necessitated the use of untilted regimens. Induction of acute cell injury in untilted cultures required both AH(2) plus very high O(3) concentrations. Addition of DPPC films significantly reduced cell injury. We conclude that acute cell injury likely results from O(3) reaction with ELF substrates. Furthermore, interfacial films of surface-active, saturated phospholipids reduce the local dose of O(3)-derived reaction products. Finally, because O(3) local dose and tissue damage likely correlate, we propose that interfacial phospholipids may modulate intrapulmonary distribution of inhaled O(3) and the extent of site-specific cell injury.     

Circadian genes in a blind subterranean mammal III: molecular cloning and circadian regulation of cryptochrome genes in the blind subterranean mole rat, Spalax ehrenbergi superspecies

The blind subterranean mole rat superspecies Spalax ehrenbergi is an extreme example of mammalian adaptation to life underground. Though this rodent is totally visually blind, harboring a drastically degenerated subcutaneous rudimentary eye, its daily activity rhythm is entrainable to LD cycles. This indicates that it confers light information to the clock, as has been previously shown by the authors in behavioral studies as well as by molecular analyses of its Clock/MOP3 and its three Per genes. The Cryptochrome (Cry) genes found in animals and plants act both as photoreceptors and as essential components of the negative feedback mechanism of the biological clock. To further understand the circadian system of this unique mammal, the authors cloned and characterized the open reading frame of Spalax Cry1 and Cry2. The Spalax CRY1 protein is significantly closer to the human homolog than to the mice one, in contrast to the evolutionary expectations. They have found two isoforms of Cry2 in Spalax, which differ in their 5' end of the open reading frame and defined their expression in Spalax populations. They found a large and significant excess of heterozygotes of sCry2 (sCry2L/S genotype). Both sCry1 and sCry2 mRNAs were found in the SCN, the eye, the harderian gland, as well as in a wide range of peripheral tissues. Their expression pattern under different LD conditions has also been analyzed. As was already shown for other circadian genes, despite being blind and living in darkness, the Cry genes of Spalax behave in a similar, though not identical, pattern as in sighted animals. Once again, the results indicate that the uniquely hypertrophied harderian gland of Spalax plays a key role in its circadian system.     

Iron oxide particles for molecular magnetic resonance imaging cause transient oxidative stress in rat macrophages

Iron oxide particles are a promising marker in molecular magnetic resonance imaging. They are used to label distinct cell populations either in vitro or in vivo. We investigated for the first time whether small citrate-coated very small superparamagnetic iron oxide particles (VSOPs) can lead to an increase in cellular oxidative stress. We incubated rat macrophages (RAW) in vitro with iron oxide particles. We observed a massive uptake of VSOPs measured both with atomic absorption spectroscopy and with NMR, which could be visualized by confocal laser scanning microscopy. After incubation, cells were lysed and the levels of malonyldialdehyde (MDA) and protein carbonyls were determined. We found a significant increase in both MDA and protein carbonyl levels after incubation with the particles. Surprisingly, 24 h after incubation, a significant indication of oxidative stress could no longer be observed. The increase in oxidative stress seems to be transient and closely linked to the incubation procedure. The iron chelator desferal and the intracellular spin trap PBN caused a significant reduction in oxidative stress to almost control levels. This indicates that the augmentation of oxidative stress is closely linked to the free iron during incubation. Proliferation assays showed that incorporation of VSOPs did not lead to long-term cytotoxic effects even though the iron oxide particles remained in the cell. Magnetic labeling of cells with VSOPs seems to cause transient oxidative conditions not affecting cellular viability and seems to be a usable approach for molecular magnetic resonance imaging.     

Advanced anaerobic bioconversion of lignocellulosic waste for bioregenerative life support following thermal water treatment and biodegradation by Fibrobacter succinogenes

The feasibility of nearly-complete conversion of lignocellulosic waste (70% food crops, 20% faecal matter and 10% green algae) into biogas was investigated in the context of a life support project. The treatment comprised a series of processes, i.e., a mesophilic laboratory scale CSTR (continuously stirred tank reactor), an upflow biofilm reactor, a fiber liquefaction reactor employing the rumen bacterium Fibrobacter succinogenes and a hydrothermolysis system in near-critical water. By the one-stage CSTR, a biogas yield of 75% with a specific biogas production of 0.37 l biogas g(-1) VSS (volatile suspended solids) added at a RT (hydraulic retention time) of 20-25 d was obtained. Biogas yields could not be increased considerably at higher RT, indicating the depletion of readily available substrate after 25 d. The solids present in the CSTR-effluent were subsequently treated in two ways. Hydrothermal treatment (T approximately 310-350 degrees C, p approximately 240 bar) resulted in effective carbon liquefaction (50-60% without and 83% with carbon dioxide saturation) and complete sanitation of the residue. Application of the cellulolytic Fibrobacter succinogenes converted remaining cellulose contained in the CSTR-effluent into acetate and propionate mainly. Subsequent anaerobic digestion of the hydrothermolysis and the Fibrobacter hydrolysates allowed conversion of 48-60% and 30%, respectively. Thus, the total process yielded biogas corresponding with conversions up to 90% of the original organic matter. It appears that particularly mesophilic digestion in conjunction with hydrothermolysis at near-critical conditions offers interesting features for (nearly) complete and hygienic carbon and energy recovery from human waste in a bioregenerative life support context.     

Characterization of a T-DNA insertion mutant for the protein import receptor atToc33 from chloroplasts

In Arabidopsis thaliana, the Toc34 receptor component of the chloroplast import machinery is encoded by two independent but highly homologous genes, atToc33 and atToc34. We have isolated a T-DNA insertion mutant of atToc33 which is characterized by a pale phenotype, due to reductions in the levels of photosynthetic pigments, and alterations in protein composition. The latter involve not only chloroplast proteins but also some cytosolic polypeptides, including 14-3-3 proteins which, among other functions, have been proposed to be cytosolic targeting factors for nucleus-encoded chloroplast proteins. Within the chloroplast, many, though not all, proteins of the photosynthetic apparatus, as well as proteins not directly involved in photosynthesis, are found in significantly reduced amounts in the mutant. However, the accumulation of other chloroplast proteins is unaffected. This suggests that the atToc33 receptor is responsible for the import of a specific subset of nucleus-encoded chloroplast proteins. Supporting evidence for this conclusion was obtained by antisense repression of the atToc34 gene in the atToc33 mutant, which results in an exacerbation of the phenotype.Communicated by R. Hagemann     

Pyranose oxidase identified as a member of the GMC oxidoreductase family

Fungal pyranose oxidase is a flavoenzyme whose preferred substrate among several monosaccharides is D-glucose. After a comprehensive analysis of conserved features in a structure-based multiple sequence alignment of homologous proteins, we could classify this enzyme into the GMC oxidoreductase family. The identified homology also suggests a three-dimensional protein structure similar to the functionally related glucose oxidase.     

Determinants of enzymatic specificity in the Cys-Met-metabolism PLP-dependent enzymes family: crystal structure of cystathionine gamma-lyase from yeast and intrafamiliar structure comparison

The crystal structure of cystathionine gamma-lyase (CGL) from yeast has been solved by molecular replacement at a resolution of 2.6 A. The molecule consists of 393 amino acid residues and one PLP moiety and is arranged in the crystal as a tetramer with D2 symmetry as in other related enzymes of the Cys-Met-metabolism PLP-dependent family like cystathionine beta-lyase (CBL). A structure comparison with other family members revealed surprising insights into the tuning of enzymatic specificity between the different family members. CGLs from yeast or human are virtually identical at their active sites to cystathionine gamma-synthase (CGS) from E. coli. Both CGLs and bacterial CGSs exhibit gamma-synthase and gamma-lyase activities depending on their position in the metabolic pathway and the available substrates. This group of enzymes has a glutamate (E333 in yeast CGL) which binds to the distal group of cystathionine (CTT) or the amino group of cysteine. Plant CGSs use homoserine phosphate instead of O-succinyl-homoserine as one substrate. This is reflected by a partially different active site structure in plant CGSs. In CGL and CBL the pseudosymmetric substrate must dock at the active site in different orientations, with S in gamma-position (CBL) or in delta-position (CGL). The conserved glutamate steers the substrate as seen in other CGLs. In CBLs this position is occupied by either tyrosine or hydrophobic residues directing binding of CTT such that S is in the in gamma-position. In methionine gamma-lyase a hydrophic patch operates as recognition site for the methyl group of the methionine substrate.