BBA - General Subjects (v.1619, #1)

Oxidative stress, NO and smooth muscle cell extracellular superoxide dismutase expression by Pontus Strålin; Håkan Jacobsson; Stefan L Marklund (1-8).
Oxygen free radicals apparently play important roles in diseases of the blood vessel wall and increased secretion of superoxide radicals occurs in many situations. The vascular wall contains large amounts of extracellular superoxide dismutase (EC-SOD). The synthesis of the enzyme by the smooth muscle cells (SMC) is modulated by cytokines, growth factors, and vasoactive factors.Here we studied the effects of oxidants (pyrogallol, xanthine oxidase, Cu and Fe), antioxidants (SOD, catalase, and ascorbate), glutathione modulation (n-acetylcysteine and buthionine sulfoximine) and nitric oxide on EC-SOD expression by human vascular SMCs. Generally, the responses in EC-SOD synthesis were small, and no changes were noted in mRNA levels. High concentrations of some of the agents caused reductions in EC-SOD synthesis, mostly concomitantly with toxic effects on the cells. Cell cultures are normally ascorbate deficient, and addition of ascorbate to approach physiological levels doubled the EC-SOD content. Iron ions up-regulated EC-SOD synthesis but also blocked the secretion of the enzyme. Only down-regulation was found by NO-releasing compounds.In conclusion, there is limited response to oxidant stress of EC-SOD synthesis by SMCs on a cell-autonomous level. The synthesis appears mainly regulated by factors coordinating concerted tissue responses.
Keywords: Superoxide dismutase; Atherosclerosis; Vascular smooth muscle cell; Free radical; Ascorbate; Nitric oxide;

Substrate specificity and kinetics for VP14, a carotenoid cleavage dioxygenase in the ABA biosynthetic pathway by Steven H Schwartz; Bao C Tan; Donald R McCarty; William Welch; Jan A.D Zeevaart (9-14).
The plant growth regulator, abscisic acid (ABA), is synthesized via the oxidative cleavage of an epoxy-carotenoid. Specifically, a double bond is cleaved by molecular oxygen and an aldehyde is formed at the site of cleavage in both products. The Vp14 gene from maize encodes an oxidative cleavage enzyme for ABA biosynthesis and the recombinant VP14 protein catalyzes the cleavage reaction in vitro. The enzyme has a strict requirement for a 9-cis double bond adjacent to the site of cleavage (the 11–12 bond), but shows some plasticity in other features of carotenoids that are cleaved. A kinetic analysis with the 9-cis isomer of five carotenoids displays several substrate activity relationships. One of the carotenoids was not readily cleaved, but inhibited the cleavage of another substrate in mixed assays. Of the remaining four carotenoids used in this study, three of the substrates have similar V max values. The V max for the cleavage of one carotenoid substrate was significantly higher. Molecular modeling and several three-dimensional quantitative substrate-activity relationship programs were used to analyze these results. In addition to a 9-cis double bond, the presence and orientation of the ring hydroxyl affects substrate binding or the subsequent cleavage. Additional variations that affect substrate cleavage are proposed.
Keywords: Abscisic acid; Apocarotenoid; Carotenoid cleavage dioxygenase; Quantitative substrate activity relationship;

In this report the kinetics of CO recombination to ferrocytochrome c in which Met80 has been oxidized to a sulfoxide are examined. Transient optical difference spectra suggest that the species formed immediately after photolysis contains a five-coordinate high spin heme. Single wavelength transient absorption data show triphasic kinetics with rate constants of (2.1±0.08)×104, (2.0±0.01)×103, and 57±0.01 s−1. The data suggest a model for ligand recombination in which the methionine sulfoxide and CO compete for binding to the five-coordinate heme with rate constants of (2.1±0.08)×104 and (2.0±0.01)×103 s−1, respectively. Carbon monoxide then binds to the population of cytochrome c containing the methionine sulfoxide with a rate constant of 57 s−1. In addition, the slower than expected rate of methionine sulfoxide recombination (much smaller rate constant than expected for a ligand restricted to the distal heme pocket) is attributed to hydrogen bonding between the unbound methionine sulfoxide and Tyr68.
Keywords: Cytochrome c; Ligand binding; Heme protein; Flash photolysis;

Cloning, heterologous expression, and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris by Michele A. McGuirl; Jennifer C. Lee; Julia G. Lyubovitsky; Chalita Thanyakoop; John H. Richards; Harry B. Gray; Jay R. Winkler (23-28).
The cytochrome (cyt) c′, cyt c 556, and cyt c 2 genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c′ and cyt c 556 have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b 562, in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the FeII and FeIII states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native FeII-cyt c 556 is more stable than the corresponding state of FeIII-cyt c 556 (ΔΔG f°=22 kJ/mol).
Keywords: Cytochrome c′; Cytochrome c 556; Protein folding; Rhodopseudomonas palustris; Spectroscopy;

Species of the fish genus Carassius survive prolonged anoxia. Nitric oxide (NO) regulates cerebral blood flow in these fish during normoxic conditions whereas adenosine is the main vasoregulating molecule during anoxia. We investigated the calcium ion dependence of Carassius auratus brain NO synthase (NOS) as a function of pH. The physiological pH decrease from 7.2 to 6.8, which takes place during anoxia, greatly decreases NOS activity. This strong pH dependence is mainly due to variation of the calcium sensitivity of the enzyme. The EC50 is 0.15 μM at pH 7.2 and 2.1 μM at pH 6.8 for the soluble enzyme. The particulate enzyme is also dependent on pH variations. The reduced sensitivity to calcium ions at acidic pH decreases both NO and H2O2 production, saving the cells by suppression of the formation of potentially toxic nitrogen and oxygen species. Modulation of NOS activity by variation of its calcium affinity within the range of physiological pH constitutes an important and rapid mechanism to control the formation of NO and H2O2 during normoxia–anoxia and anoxia–normoxia transitions.
Keywords: Nitric oxide synthase; Brain; Hypoxia; pH dependence; Ca2+ dependence; Carassius auratus;

Apoptosis-inducing neurotoxicity of dopamine and its metabolites via reactive quinone generation in neuroblastoma cells by M Emdadul Haque; Masato Asanuma; Youichirou Higashi; Ikuko Miyazaki; Ken-ichi Tanaka; Norio Ogawa (39-52).
Neurotoxic properties of l-dopa and dopamine (DA)-related compounds were assessed in human neuroblastoma SH-SY5Y cells with reference to their structural relationship. l-Dopa and its metabolites containing two free hydroxyl residues on their benzene ring showed toxicity in the cell, which was prevented by superoxide dismutase (SOD) and reduced glutathione (GSH), but not by catalase. Furthermore, a synthetic derivative of DA, 3-hydroxy-4-methoxyphenethylamine (HMPE) containing methoxy residue at position 4 in the benzene ring, exerted partial cytotoxicity, which was not prevented by SOD, GSH or catalase. However, the metabolites containing methoxy residue at position 3 failed to show a toxic effect in the SH-SY5Y cells. Moreover, DA induced apoptotic cell death, which was observed by nuclear and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and measurement of caspase-3 activity; this compound up-regulated apoptotic factor p53 while down-regulating anti-apoptotic factor Bcl-2. In the cell-free in vitro electron spin resonance (ESR) spectrometry, DA possessing two hydroxyl groups showed generation of DA-semiquinone radicals, which were markedly prevented by addition of SOD or GSH but not by catalase. On the other hand, methylation of one of the hydroxyl residues on the benzene ring of DA converted DA to an unoxidizable compound (3-MT or HMPE), and caused it to lose the property to produce semiquinone radicals. It has been previously reported that SOD acting as a superoxide:semiquinone oxidoreductase prevents quinone formation, and that reduced GSH through forming a complex with DA-quinone prevents quinone binding to the thiol group of the intact protein. Therefore, the present results suggest that DA and its metabolites containing two hydroxyl residues exert cytotoxicity mainly due to generation of highly reactive quinones.
Keywords: l-Dopa; Dopamine; Quinone; Apoptosis;

Evidence for various calcium sites in human hair shaft revealed by sub-micrometer X-ray fluorescence by C Mérigoux; F Briki; F Sarrot-Reynauld; M Salomé; B Fayard; J Susini; J Doucet (53-58).
New information about calcium status in human scalp hair shaft, deduced from X-ray micro-fluorescence imaging, including its distribution over the hair section, the existence of one or several binding-types and its variation between people, is presented. The existence of two different calcium types is inferred. The first one corresponds to atoms (or ions) easily removable by hydrochloric acid, located in the cortex (granules), in the cuticle zone and also in the core of the medulla, which can reasonably be identified as calcium soaps. The second type consists of non-easily removable calcium atoms (or ions) that are located in the medulla wall, probably also in the cuticle, and rather uniformly in the cortex; these calcium atoms may be involved in Ca2+-binding proteins, and their concentration is fairly constant from one subject to another. In addition to its nonuniform distribution across the hair section, the second striking feature of the first type calcium content is its high variability from one subject to another, by up to a factor 10. We expect this information to be useful for analyzing in more detail the relationship between hair calcium and environmental and medical factors.
Keywords: Hair; Lipid; Calcium; Calcium soap; Calcium binding protein; X-ray fluorescence;

Lipid/DNA complexes or Lipoplexes have been characterized by various biochemical and biophysical methods to understand the physical basis of transfection. Here we have addressed the effect of cationic liposomes, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), on transcription of DNA templates in vitro. Transcriptional activity of DNA-dependent RNA polymerase at DNA templates complexed with the cationic lipid varied as a function of charge ratio of lipid/DNA. At low charge ratios of 0.3:1 lipid/DNA and up to 1:1, we observed stimulation in transcription, while at higher charge ratios of lipid/DNA 3:1, complete inhibition in the activity occurred. Cetyl tri-methyl ammonium bromide (CTAB), a cationic detergent, and polyethylenimine (PEI), a cationic polymer, also bring about similar changes although to a lesser extent. The stimulation in transcription motivated us to probe into the molecular nature of the lipid/DNA interactions by absorbance spectroscopy and circular dichroism (CD). Upon interaction with lipids, hyperchromicity and susceptibility to micrococcal nuclease has increased, which suggests that the DNA was partially denatured. On complexation with the cationic lipid (DOTAP), the magnitude of the positive band in CD spectra decreased, accompanied with a red shift, as a function of charge ratio. Results from spectroscopic and enzyme assays suggest that at low charge ratios DNA may be partially unwound.
Keywords: Transcription; Cationic liposome; RNA polymerase; Circular dichroism; Hyperchromicity;

13C NMR studies with aluminum (Al)-stressed Pseudomonas fluorescens revealed that the trivalent metal was secreted in association with oxalate and phosphatidylethanolamine (PE). These moieties were observed in the insoluble pellet obtained upon incubation of these resting cells in the presence of either Al-citrate or citrate. This extrusion process was concomitant with the utilization of either of these tricarboxylic acids as a substrate. While only minimal amounts of Al were secreted in the presence of such carbon source as glucose, succinate or oxaloacetate, oxalate did permit the efflux of Al. Neither α-ketoglutarate nor ethylenediaminetetraacetic acid (EDTA) was effective in dislocating Al from the cells. The elimination of Al from the cells did not appear to be affected by p-dinitrophenol (DNP) or dicyclohexylcarbodiimide (DCCD) or azide, but was sensitive to temperature, pH and cerulenin, an inhibitor of lipid synthesis. Thus, it appears that P. fluorescens detoxifies Al via its extrusion in association with oxalate and PE in a process that apparently does not necessitate the direct utilization of energy.
Keywords: Oxalate; Phosphatidylethanolamine (PE); Aluminum; Isocitrate lyase;

Combinations of insulin secretagogue-derived metabolites were added to microgram amounts of mitochondria obtained from rat and mouse pancreatic islets and the INS-1 cell line, and the export of citric acid cycle intermediates was surveyed to study anaplerosis in insulin secretion. Cellular levels of metabolites were also measured. In mitochondria from all three tissues, malate production was the most responsive to various substrates. The export of citrate and isocitrate in the presence of pyruvate and most other substrates was small and their levels in intact cells did not change with any secretagogue, except in INS-1 cells where citrate increased slightly. Changes in α-ketoglutarate and glutamate export from mitochondria and levels in intact cells indicate that glutamate can be consumed as a fuel secretagogue, but it is not likely produced as a messenger in insulin secretion. The citrate level may not need to increase in order to provide increased malonyl-CoA for signaling insulin secretion. Unlike some cells, insulin cells probably obtain cytosolic NADPH equivalents by exporting them from mitochondria to the cytosol via a pyruvate malate shuttle or an isocitrate shuttle. Only fuels that can enhance anaplerosis via pyruvate or α-ketoglutarate can be insulin secretagogues.
Keywords: Rat pancreatic islet; Mouse pancreatic islet; INS-1 cell; Mitochondrion; Metabolite export; Cellular level of metabolite; Citric acid cycle; Anaplerosis; Pyruvate malate shuttle; NADP(H); Isocitrate shuttle; Glutamate; Malate aspartate shuttle; Insulin secretion;

The production of asparagine (N)-linked oligosaccharides is of vital importance in the formation of glycosylated proteins in eukaryotes and is mediated by the dolichol pathway. As part of studies to allow manipulation of this pathway, the gene coding for the production of the enzyme UDP N-acetylglucosamine:dolichol phosphate N-acetylglucosaminylphosphoryl transferase (GPT), catalysing the first step in the assembly of dolichol-linked oligosaccharides, was cloned from the filamentous fungus Aspergillus niger. Degenerate-PCR was used to amplify a 470-bp fragment of the gene, which was labelled as a probe to obtain a full-length clone from a genomic library of A. niger. This contained a 1557-bp open reading frame encoding a highly hydrophobic protein of 468 amino acids with a predicted molecular weight of 51.4 kDa. The gene contained two intron sequences and putative dolichol recognition sites (PDRSs) were present in the deduced amino acid sequence. Comparison with other eukaryotic GPTs revealed the A. niger GPT to share 45–47% identity with yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and 41–42% identity with mammals (mouse, hamster, human). Nested-PCR of a cDNA library was used to confirm the position of an intron. A complete cDNA clone of A. niger gpt was obtained by employing a recombinant PCR approach. This was used to rescue a conditional lethal mutant of S. cerevisiae carrying a dysfunctional gpt gene by heterologous expression, confirming that the gpt genes from A. niger and S. cerevisiae are functionally equivalent.
Keywords: Fungi; N-glycosylation; Recombinant PCR; Heterologous protein expression;

A comparative analysis of structure and spatial distribution of decorin in human leiomyoma and normal myometrium by Alessandra G.A Berto; Lucia O Sampaio; Célia R.C Franco; Roberto M Cesar; Yara M Michelacci (98-112).
Leiomyoma is a benign smooth muscle tumor of the uterus that affects many women in active reproductive life. It is composed by bundles of smooth muscle cells surrounded by extracellular matrix. We have recently shown that the glycosylation of extracellular matrix proteoglycans is modified in leiomyoma: increased amounts of galactosaminoglycans with structural modifications are present. The data here presented show that decorin is present in both normal myometrium and leiomyoma but tumoral decorin is glycosylated with longer galactosaminoglycan side chains. Furthermore, these chains contain a higher ratio d-glucuronate/l-iduronate, as compared to normal tissue. To determine if these changes in proteoglycan glycosylation correlates with modifications in the extracellular matrix organization, we compared the general structural architecture of leiomyoma to normal myometrium. By histochemical and immunofluorescence methods, we found a reorganization of muscle fibers and extracellular matrix, with changes in the distribution of glycoproteins, proteoglycans, and collagen. Thin reticular fibers, possibly composed by types I and III collagen, were replaced by thick fibers, possibly richer in type I collagen. Type I collagen colocalized with decorin both in leiomyoma and normal myometrium, in contrast to type IV collagen that did not. The relative amount of decorin was increased and the distribution of decorin and collagen was totally modified in the tumor, as compared to the normal myometrium. These findings reveal that not only decorin structure is modified in leiomyoma but also the tissue architecture changed, especially concerning extracellular matrix.
Keywords: Decorin; Dermatan sulfate; Chondroitin sulfate; Proteoglycan; Collagen; Leiomyoma;