Showing papers in "Biofactors in 1997"

Lipoic acid increases de novo synthesis of cellular glutathione by improving cystine utilization

Abstract: Lipoic acid (thiotic acid) is being used as a dietary supplement, and as a therapeutic agent, and is reported to have beneficial effects in disorders associated with oxidative stress, but its mechanism of action remains unclear. We present evidence that lipoic acid induces a substantial increase in cellular reduced glutathione in cultured human Jurkat T cells human erythrocytes, C6 glial cells, NB41A3 neuroblastoma cells, and peripheral blood lymphocytes. The effect depends on metabolic reduction of lipoic acid to dihydrolipoic acid. Dihydrolipoic acid is released into the culture medium where it reduces cystine. Cysteine thus formed is readily taken up by the neutral amino acid transport system and utilized for glutathione synthesis. By this mechanism lipoic acid enables cystine to bypass the xc- transport system, which is weakly expressed in lymphocytes and inhibited by glutamate. Thereby lipoic acid enables the key enzyme of glutathione synthesis, gamma-glutamylcysteine synthetase, which is regulated by uptake-limited cysteine supply, to work at optimum conditions. Flow cytometric analysis of freshly prepared human peripheral blood lymphocytes, using monobromobimane labeling of cellular thiols, reveals that lipoic acid acts mainly to normalize a subpopulation of cells severely compromised in thiol status rather than to increase thiol content beyond physiological levels. Hence lipoic acid may have clinical relevance in restoration of severely glutathione deficient cells.

Basic mechanisms of antioxidant activity

Abstract: A book originating from an annual meeting of the Oxygen Society (Charleston, South Carolina, 1995) [7] and dedicated to Paul Hochstein in occasion of his retirement is entitled The Oxygen Paradox: the preface and some interesting articles define The Oxygen Paradox as the dual effects of oxygen, in a manner reminiscent of Fridovich’s article 20 years before in The American Scientist, Oxygen: Boon and Bane [14]. It is clear that in both instances an equilibrium is referred to and that this equilibrium is encompassed by, on the one hand, the production of oxygen-centered radicals (concept today extended to sulfur-, nitrogen-, and carbon-centered radicals) and, on the other hand, the effective removal of these species, as accomplished by disproportionation to less reactive (non radical) molecules, interception by small antioxidant molecules, or specific antioxidant enzymes. Our current view of cellular antioxidant defenses can be categorized into primary and secondary defense systems. The primary defenses consist of a variety of antioxidant enzymes (superoxide dismutases, catalase, glutathione peroxidases, etc.) and a broad range of small antioxidant molecules. Secondary defenses include proteolytic and lipolytic enzymes as well as the DNA repair systems. Of growing interest are the small antioxidant molecules (some of them vitamins), such as α-tocopherol, ascorbic acid, carotenoids, coenzyme Q, uric acid, vitamin A, melatonin, and aminoindoles. Also of physiological and/or pharmacological interest are small molecule antioxidants, such as flavonoids and polyphenols, those contained in herbal antioxidants, and α-lipoic acid. A comprehensive and authoritative treatise on the chemical, biological, and clinical aspects of natural antioxidants, providing an in-depth account of current knowledge on these naturally occurring compounds, was recently published [4]. Likewise, synthetic antioxidants are currently being developed as therapeutic agents against oxidative stress. Such compounds include, among others, derivatives of natural antioxidants (e.g., α-tocopherol analogs), phenolic antioxidants (such as Probucol and Nitecapone), 21 aminosteroids or lazaroids, sulfhydryl-containing compounds (thiazolidine, ebselen, dithiolethiones), and low-molecular weight mimics of superoxide dismutase [19]. Examples of tissuedirected α-tocopherol analogs are the water-soluble, quaternary ammonium tocopherol derivatives, which accumulate in heart tissue, and the 2,3-dihydrobenzofuran-5-ol derivatives, which penetrate brain tissue. These compounds have potential use as cardioprotectors and against cerebrovascular diseases, respectively [20].

Breakdown of oxidized proteins as a part of secondary antioxidant defenses in mammalian cells

Abstract: The degradation of oxidized proteins is an essential part of antioxidant defenses against free radical attack. Selective degradation of oxidatively damaged proteins allows proteolytic systems to function directly in the removal of useless cellular debris and therefore prevent the accumulation of potentially toxic fragments or large aggregates of cross-linked proteins. The degradation of oxidized proteins in dividing mammalian cells after hydrogen peroxide treatment has been demonstrated. Cells are able to increase proteolysis rates after treatment with moderate levels of oxidants. The role of proteasome in the removal of oxidized proteins has been demonstrated by treatment of cells with an anti-sense oligodesoxynucleotide to the proteasome C2 subunit gene. This treatment decreases the proteasome content of the cells and prevents increased proteolysis rates after hydrogen peroxide treatment. Thus proteasome clearly plays a role in the removal of oxidized proteins. As part of antioxidant defenses the proteasome provides a second line of defense against the numerous radicals and oxidants which contact cells during their lifetime. The degradation of oxidatively damaged proteins enables cells to recover from a moderate oxidant attack.

Acetogenic bacteria : what are the in situ consequences of their diverse metabolic versatilities?

Abstract: The four decades of the now classic studies by Harland G. Wood and Lars G. Ljungdahl lead to the resolution of the autotrophic acetyl-CoA 'Wood/Ljungdahl' pathway of acetogenesis. This pathway is the hallmark of acetogens, but is also used by other bacteria, including methanogens and sulfate-reducing bacteria, for both catabolic and anabolic purposes. Thus, the pathway is wide spread in nature and plays an important role in the global turnover of carbon. Because most historical studies with acetogens focused on the biochemistry of the acetyl-CoA pathway, the metabolic diversity and ecology of acetogens remained largely unexplored for many years. Although acetogens were initially conceived to be a somewhat obscure bacteriological group with limited metabolic capabilities, it is now clear that acetogens are arguably the most metabolically diverse group of obligate anaerobes characterized to date. Their anaerobic metabolic arsenal includes the capacity to oxidize diverse substrates, including aromatic, C1, C2, and halogenated compounds, and engage a large number of alternative energy-conserving, terminal electron-accepting processes, including classic fermentations and the dissimilation of inorganic nitrogen. In this regard, one might consider acetogens on a collective basis as the pseudomonads of obligate anaerobes. By virtue of their diverse metabolic talents, acetogens can be found in essentially all habitats. This review evaluates the metabolic versatilities of acetogens relative to both the engagement (regulation) of the acetyl-CoA pathway and the ecological roles likely played by this bacteriological group.

Pathways for oxidation of low density lipoprotein by myeloperoxidase: tyrosyl radical, reactive aldehydes, hypochlorous acid and molecular chlorine

Abstract: Many lines of evidence implicate oxidation of low density lipoprotein (LDL) in the pathogenesis of atherosclerosis, a chronic inflammatory disease. The physiologically relevant mechanisms have not been identified, but phagocytic white cells may play an important role because macrophage-rich lesions characterize the disorder. Recent studies have shown that myeloperoxidase, a heme enzyme secreted only by phagocytes, is present in human atherosclerotic tissue. The enzyme is a potent catalyst of LDL oxidation in vitro, it co-localizes with macrophages in lesions, and it generates products that are detectable in atherosclerotic plaque. These findings suggest that myeloperoxidase may promote LDL oxidation in the artery wall. This article reviews the enzyme's ability to generate a range of oxidants, including tyrosyl radical, reactive aldehydes, hypochlorous acid and molecular chlorine. These products have the potential to damage host molecules as well as microbes, suggesting a mechanism that may contribute to atherosclerotic vascular disease.

Enzymology of the fermentation of acetate to methane by Methanosarcina thermophila.

Abstract: Biologically-produced CH4 derives from either the reduction of CO2 or the methyl group of acetate by two separate pathways present in anaerobic mierobes from the Archaea domain. Elucidation of the pathway for CO2 reduction to CH4, the first to be investigated, has yielded several novel enzymes and cofactors. Most of the CH4 produced in nature derives from the methyl group of acetate. Methanosarcina thermophila is a moderate thermophile which ferments acetate by reducing the methyl group to CH4 with electrons derived from oxidation of the carbonyl group to CO2. The pathway in M. thermophila is now understood on a biochemical and genetic level comparable to understanding of the CO2-reducing pathway. Enzymes have been purified and characterized. The genes encoding these enzymes have been cloned, sequenced, transcriptionally mapped, and their regulation defined on a molecular level. This review emphasizes recent developments concerning the enzymes which are unique to the acetate fermentation pathway in M. thermophila.

Polyphenols produced during red wine ageing

Abstract: Over the past few years, it has been accepted that a moderate red wine consumption is a factor beneficial to human health. Indeed, people of France and Italy, the two major wine-producing European countries, eat a lot of fatty foods but suffer less from fatal heart strokes than people in North-America or in the northern regions of Europe, where wine is not consumed on a regular basis. For a time, ethanol was thought to be the "good" chemical species hiding behind what is known as the "French paradox". Researchers now have turned their investigations towards a family of natural substances called "polyphenols", which are only found in plants and are abundant in grapes. It is well known that these molecules behave as radical scavengers and antioxidants, and it has been demonstrated that they can protect cholesterol in the LDL species from oxidation, a process thought to be at the origin of many fatal heart attacks. However, taken one by one, it remains difficult to demonstrate which are the best polyphenols as far as their antioxidant activities are concerned. The main obstacle in that kind of research is not the design of the chemical and biological tests themselves, but surprisingly enough, the limited access to chemically pure and structurally elucidated polyphenolic compounds. In this article, particular attention will be paid to polyphenols of red wine made from Vitis vinifera cultivars. With respect to the "French paradox", we address the following question: are wine polyphenolic compounds identical to those found in grapes (skin, pulp and seed), or are there biochemical modifications specifically taking place on the native flavonoids when a wine ages? Indeed, structural changes occur during wine conservation, and one of the most studied of those changes concerns red wine colour evolution, called "wine ageing". As a wine ages, it has been demonstrated that the initially present grape pigments slowly turn into new more stable red pigments. That phenomenon goes on for weeks, months and years. Since grape and wine polyphenols are chemically distinct, their antioxidant activities cannot be the same. So, eating grapes might well lead to beneficial effects on human health, due to the variety and sometimes large amounts of their polyphenolic content. However, epidemiological surveys have focused on wines, not on grapes....

The Eastern and Western branches of the Wood/Ljungdahl pathway: How the East and West were won

Abstract: Figure 1 is a transmission electron micrograph of Clostridium ljungdahlii [1]. It, like Acetobacterium woodii [2], was named after someone who made great contributions to the understanding of how anaerobes fix carbon dioxide. C. ljungdahlii was named after Lars Ljungdahl; A. woodii after Harland Wood. I had the good fortune to train under both Lars and Harland and have continued research in this area for approximately fifteen years, learning and gaining greater and greater appreciation for the art of anaerobes. This review introduces the concept of the Eastern and Western branches of the Wood/Ljungdahl pathway. The Eastern branch is important in one-carbon metabolism of all organisms; the Western branch is unique to anaerobic microorganisms that use the Wood/Ljungdahl pathway for fixing carbon dioxide or carbon monoxide to generate cell carbon or for synthesizing acetate (acetogens) or methane (aceticlastic methanogens) to generate energy. In focusing on the biochemistry and enzymology of this pathway, I, unfortunately, must risk leaving the important ecology and physiology of these organisms in the lurch. They are responsible for the turnover of a trillion tons of acetic acid per year. They convert a variety of compounds including sugars, aromatic compounds, and inorganic compounds like CO and H2 and CO2 into acetic acid which is then used by methane producing bacteria to make natural gas. They are important in soil microbiology and, in the biology of organisms that house them in their digestive system, like humans, termites and ruminants like cows and sheep [3–5]. The story of acetogens can be traced back to the ancient times when someone discovered that apple or grape juice could be fermented, first to alcohol and then to acetic acid, producing cider vinegar, which contains 3–6% acetic acid. The word acetum is the Latin word for vinegar. Acetic acid was isolated in pure form by Stahl in 1700. Over the years, it has been found to be an excellent solvent for many organic compounds and some inorganic compounds. It also is essential in the production of cellulose acetate and is used widely in the textile and rubber industries.

Biogenic 4‐hydroxy‐2‐nonenal activates transcription factor AP‐1 but not NF‐κB in cells of the macrophage lineage

Abstract: A large spectrum of pro-oxidant agents, including molecules with lipoperoxidative effect, can modulate gene expression through modification of the DNA binding activity of the transcription factors activator protein 1 (AP-1) and nuclear factor kappa B (NF-kappa B). In this study the effect on these redox-sensitive factors by 4-hydroxy-2-nonenal (HNE), a major aldehydic product of lipid peroxidation, was examined in two cell lines of the macrophage type. Incubation in the presence of microM concentrations of the aldehyde led to a rapid increase of AP-1 binding with a transient maximum 30 min from HNE addition to the culture medium in both cell lines. On the contrary, HNE did not stimulate nuclear translocation of NF-kappa B. The diverging effect of HNE on the two transcription factors is likely related to the demonstrated differential activation pathway of AP-1 and NF-kappa B in macrophages. The HNE-induced activation of AP-1 suggests the aldehyde's involvement in the regulatory mechanisms of cell proliferation and differentiation.

Evidence for a functional relevance of the selenocysteine residue in mammalian thioredoxin reductase

Abstract: Human thioredoxin reductase was recently shown to contain a TGA encoded selenocysteine residue at the penultimate position of its amino acid chain. Depending on the availability of selenium during biosynthesis, an authentic selenocysteine-containing or a selenium-free enzyme truncated at the penultimate position is expected to be formed. Correspondingly, the enzymatic activity should be altered by selenium restriction, if the selenocysteine residue is functionally important. In order to check the catalytic role of the selenocysteine residue, four different human cell lines were grown in selenium deficient media or with adequate selenium supplementation (40 nM sodium selenite) and thioredoxin reductase activity was measured as NADPH-dependent DTNB reduction or thioredoxin-mediated insulin reduction. Thioredoxin reductase activities, like glutathione peroxidase activities, were consistently higher in selenium supplemented cells, whereas glutathione reductase activity was not affected by the selenium. The dose-response was similar for thioredoxin reductase and glutathione peroxidase, but the recovery of glutathione peroxidase activity upon selenium supplementation was faster than with thioredoxin reductase. Also the increase of glutathione peroxidase activities was substantially higher than that of thioredoxin reductase (400-1200% versus a maximum of 250%). These observations clearly indicate a catalytic role of the selenocysteine residue in the thioredoxin reductase, but suggest either the existence of a selenium-unresponsive isoenzyme or a residual disulfide reductase activity in the selenium-free truncated protein made under conditions of selenium deficiency.

The mechanism of the hypochlorite‐induced lipid peroxidation

Abstract: The article reviews data related to the role of exogenic hypochlorite (HOCl/OCl-) and hypochlorite produced by myeloperoxidase catalysis in initiation of lipid peroxidation (LPO) in phospholipid membranes and human blood lipoproteins (LP). It has been shown that HOCl/OCl- promotes free radical lipid oxidation in liposomes and LP that is followed by the formation of LPO products; hydroperoxides, conjugated dienes, TBARS, and fluorescent products. Water soluble reactive substances (.O2-, H2O2, Fe2+) which can be present in the reaction mixture as a dopant are not the source of free radicals and do not participate in HOCl/OCl(-)-induced LPO at the initiation step. The main reaction of HOCl/OCl- with unsaturated lipid is probably the generation of chlorohydrins. However, this reaction is not accompanied by generation of free radicals and LPO. HOCl/OCl- reacts efficiently with TBARS of aldehydic nature. It is likely that the reaction proceeds without the participation of free radicals. Among the compounds of a peroxide nature (hydro-, dialkyl-, diacyl-, alkyl-acyl-peroxide groups and epoxides) only hydroperoxides react with HOCl/OCl-. This reaction is accompanied by the production of free radicals (but not singlet oxygen), probably alkoxyl radicals, which may play a role in the initiation of HOCl/OCl(-)-induced LPO.

Whole plasma oxidation assay as a measure of lipoprotein oxidizability

Abstract: Lipoprotein oxidation induced in vitro in whole plasma is expected to be a more relevant model of the lipoprotein oxidation in the arterial wall than the in vitro oxidation of single isolated lipoproteins, e.g., low density lipoprotein (LDL). However, it is unclear, whether the oxidizability of whole plasma may serve as an adequate measure of the oxidizability of plasma lipoproteins. We measured the oxidizability of whole plasma diluted 150-fold as an absorbance increase at 234 nm known to reflect the level of conjugated dienes in the samples. Plasma oxidation was induced by Cu(II), 2,2 0 -azobis- (2-amidinopropane) hydrochloride (AAPH), lipoxygenase or myeloperoxidase + H2O2. Oxidizability of human plasma measured in the presence of Cu(II) was found to correlate with the oxidizability of LDL measured in the common Cu(II)- based LDL oxidation assay. The plasma oxidizability also correlated positively with plasma oxidizable fatty acid and negatively with plasma antioxidant content. Supplementation of human plasma with different antioxidants (albumin, urate, ascorbate, bilirubin, -tocopherol and ubiquinol-10) in vitro decreased its oxidizability. Supplementation of Watanabe heritable hyperlipidaemic rabbits with different antioxidants (vitamin E, ubiquinone-10, probucol, carvedilol) in vivo lowered the oxidizability of rabbit plasma in comparison with rabbits fed standard diet. When plasma from hyperlipidaemic patients with or without coronary heart disease and from age-matched healthy controls was studied, the plasma oxidizability was found to be highest in the patients with coronary heart disease and lowest in the controls. Taken together, these data indicate that the plasma oxidation assay (i) provides information similar to that obtained using the common LDL oxidation assay, (ii) upgrades the latter, taking into account the effect of hydrophilic antioxidants on lipoprotein oxidation and characterizing the oxidizability of all plasma lipoproteins, and (iii) offers important practical advantages, such as fast and simple sample processing, low amount of plasma required and avoidance of artefactual oxidation during lipoprotein isolation. We propose the measurement of plasma oxidizability at 234 nm as an adequate practical index of the oxidizability of plasma lipoproteins.

The role of oxidative stress in the long‐term glycation of LDL

Abstract: Advanced glycation is a major pathway for the posttranslational modification of plasma and tissue proteins. The initiating reaction is the nonenzymatic addition of sugars such as glucose to the primary amino groups of proteins, i.e., mainly to lysine residues. These "early" Schiff base and Amadori products then undergo a series of inter- and intramolecular rearrangements to produce the "late" products termed advanced glycation end products (AGEs). Incubation of LDL with glucose or glucose-6-phosphate produces AGE moieties on both the lipid and apolipoprotein B components. In addition, we tried to generate AGE-LDL by reaction with AGE-peptides (< 10 kD) obtained by enzymatic digestion of long-term glycated fibronectin as a model for connective tissue AGE-peptides. AGE-formation can be assessed by monitoring of fluorescence (370/440 nm) which is easily differentiated from the much lower autofluorescence of oxidized low density lipoproteins (oxLDL). Alternatively, AGE formation was detected by an AGE-specific ELISA using antibodies elicited in rabbits against bovine AGE-RNAse. In the present study we investigated the influence of oxidative stress on the long-term glycation of LDL and the modulation of LDL-oxidation by AGE-modification. We observed (a) that the rate of AGE formation is reduced by BHT/EDTA both on LDL and serum albumin (glycation vs. glycoxidation), (b) long-term glycated LDL is more readily oxidized than unglycated LDL, (c) oxLDL is more prone to AGE-modification, (d) AGE-modification of LDL strongly alters its epitope spectrum and (e) that aminoguanidine at higher concentrations (1-10 mM) inhibits copper-catalyzed LDL oxidation in the way of a classical antioxidant.

Cellular regulation by protein phosphorylation: a historical overview.

Abstract: The regulation of cellular processes requires a myriad of commands, positive and negative, that must be tightly coordinated to keep all the reactions that take place under control. In particular, to make sure that no crucial event will occur at inappropriate times or out of phase. We know today that most of the signals that are used to orchestrate these reactions, the switches that must be turned on or off, rely on reversible protein tyrosine phosphorylation. This article will provide a rapid historical overview of the regulation of cellular processes by reversible protein phosphorylation, then over some more recent aspects of this field, particularly as they relate to tyrosine phosphatases. Fifty years ago, very little was known about cellular regulation and nothing at all, of course, about a possible involvement of protein phosphorylation/dephosphorylation in these processes. But in the mid-50s, it was shown that protein phosphorylation could serve as a means to regulate the activity of glycogen phosphorylase, discovered by Parnas in Poland and Carl and Gerty Cori in the US in the mid-30s (for review, see [1]). The muscle enzyme was thought to have an absolute requirement for adenylic acid for activity until, in 1943, Arda Green in Cori’s lab crystallized it in a form that was active without added AMP. They called this form phosphorylase a, and very logically, assumed that it contained covalently-bound AMP. They further thought that it had to be the native form of the enzyme because, when left standing in crude extracts, it was rapidly converted to the earlier species which they called phosphorylase b. However, if that hypothesis were correct, AMP would have to be released in the reaction, but they found none. Furthermore, no AMP or adenine or ribose could be detected in the “native” enzyme, using the most sensitive microbiological assays available at that time. They knew that the enzyme existed in two forms but did not know how these two forms differed and, strangely actually dropped the problem.

Analysis of polyphenols and resveratrol in Italian wines

Abstract: A realistic estimate of total phenols in white wines with the Folin–Ciocalteau essay can be obtained only after isolation of the phenolic fraction from other compounds having a synergistic interference with the essay [15,19]. The polyphenolic content in white wine is much lower than in red wine, and in most cases it lies between 30 and 120 mg/l [10,13]. In this article we chose to point our attention on the composition of red wines, referring mainly on the mean data contained in our database [4] of Italian mono-variety red wines (Table 1). The total phenols reactive to Folin–Ciocalteau, do vary according to our data from 264 to 3451 mg/l in different red wines. This index is relatively stable for some years during the ageing of bottled wines,

cDNA and deduced polypeptide sequence of a mouse selenoprotein P

Abstract: An 11-day embryonic Swiss Webster/NIH mouse cDNA library was screened with a partial murine selenoprotein P cDNA probe and a murine selenoprotein-P-type cDNA 1 clone of 2075 bp lenght was obtained. The clone contained a 5 0 -leader sequence of 132 bp length, the selenoprotein P coding frame, and 803 base pairs in the 3 0 untranslated region. Alignment and RNA folding studies revealed the presence of two well conserved selenocysteine inserting motifs in the 3 0 flanking region. The deduced polypeptide sequence comprises 380 residues including ten selenocysteines. Identical amino acid residues in homologous positions are 86%, 71%, and 64% when compared to the previously reported selenoprotein P sequences of rat, man, and cattle, respectively. The comparatively low similarity between the selenoprotein P sequences reported so far leaves open the question whether they belong to the same molecular clade.

Development of genetic approaches for the methane‐producing archaebacterium Methanococcus maripaludis

Abstract: Methanococcus maripaludis is a strict anaerobe that utilizes H2 or formate as an electron donor for CO2 reduction to methane. Recent progress in development of genetic systems in this archaebacterium makes it an excellent model system for molecular and biochemical studies. This progress includes development of methods for growth on solid medium, enriching auxotrophic mutants, efficient transformation, and random insertional inactivation of genes. Genetic markers for both puromycin and neomycin resistance are available. Lastly, a shuttle vector has been constructed from a cryptic methanococcal plasmid. These technical advances made it possible to utilize genetic approaches for the study of autotrophic CO2 assimilation in methanococci.

Farnesyl: proteintransferase inhibitors as agents to inhibit tumor growth.

Abstract: Ras, a signal-transducing protein involved in mediating growth factor-stimulated proliferation, is mutationally activated in over 30% of human tumors. To be functional Ras must bind to the inner surface of the plasma membrane, with post-translational lipid modifications being necessary for this localization. The essential, first modification of Ras is farnesylation catalyzed by the enzyme farnesyl: proteintransferase (FPTase). Inhibitors of FPTase (FTIs) are currently being tested to determine if they are capable of tumor growth inhibition. Here we describe our efforts, along with those of other groups, in testing the biological and biochemical effects of FTIs.

Antioxidants and atherosclerosis: an overview.

Abstract: There is substantial evidence that the early stages of atherosclerosis are comprised of a series of oxidative processes that accompany the known risk factors attributed to abnormal plasma lipid profiles. The current opinion in this field holds that hyperlipidemia along with enhanced oxidation of lipoprotein lipids are separate but interacting factors giving rise to the formation of atherogenic lesions [23]. Evidence in support of the lipid hypothesis is substantial, shown in recent years by the dramatic reduction in coronary artery disease arising from lipid lowering therapies ‐ particularly cholesterollowering drugs. However, progression of atherosclerosis even with aggressive lipid-lowering therapies indicates that other factors are involved in a substantial segment of the population. There are certainly genetic factors that determine predisposition to disease based on abnormalities in lipid metabolism and deposition, however, other genetically determined as well as genetically independent processes appear to be of importance. These include the formation and elimination of oxidants and their effects on vascular tissues, as these oxidants can be derived either via metabolic processes or by external sources. The oxidant-mediated processes thought to be of importance in early atherogenesis are described in Table 1. These steps apply to the oxidation of serum lipoproteins as well as to vascular tissues, and interaction of lipoproteins with vascular cells through oxidative events appears to be central to the formation of atherosclerotic lesions. Each of these events are characteristic of the generally accepted processes involved in atherosclerosis. Oxidants have been shown to produce atherogenic responses independent of lipid levels and the mechanisms of injury include many of the biological events described for atherogenesis. Injury can be manifested by acute damage to cell components and vascular cell death, however, this is less likely than the non-lethal injury that is produced by oxidized LDL or by the inflammatory response encountered in vivo which causes perturbations in cell function. Support for the role of oxidants in the development of atherosclerosis also comes from recent clinical trials demonstrating antioxidant protection beyond that afforded by lipid lowering therapies [13,19]. The therapeutic potential of antioxidants can be demonstrated in many of the key steps described for early atherosclerosis. The steps of interest include: 1) Injury to the vessel wall, lipoprotein-derived oxidants have been shown to damage the endothelium through direct reactions with membranes [25], and cytoskeletal components [16] leading to overt injury or to apoptosis [11]. Lipid peroxides, derived aldehydes and cholesterol oxides are postulated as likely causative agents [7,9].

Wine and mortality

Abstract: In the present chapter the question of possible different effects of beer wine and spirits in data from the Copenhagen City Heart Study will be addressed.... The results...strongly suggest that in addition to the common effect of ethanol there are--within the studied range of drinking--different factors influencing health in the three types of beverages.... [We find] that only wine drinking clearly reduces both risk of dying from cardio- and cerebrovascular disease and risk of dying from other causes.... (EXCERPT)

Microbial and plant metabolism of NO.

Abstract: During microbial denitrification, NO is produced by reduction of nitrite by either the reduced high spind1 hemes in a unique reductase (NIR) or at the expense of a blue copper protein that transfers electrons that move first to a type I copper and then to a type II copper in a unique trimeric NIR. This latter type of NIR is also produced by several denitrifying filamentous fungi. Reduction of NO is then carried out by either a specific cytochromebc complex NOR in denitrifying bacteria or a unique cytochrome P-450 in denitrifying filamentous fungi. NO is also produced by an anomalous reaction of a molybdoprotein, nitrate reductase (NAR), acting on an odd substrate, NO 2 . NO is also reduced by a multiheme NIR that serves physiologically for reduction of NO2 to NH3. This type NIR reduces NO to either N2O, if only partially reduced, or NH3, if fully reduced, when it encounters NO. This multiheme NIR is very sensitive to cyanide. Transcription of the genes for NIR and NOR production in a denitrifier is activated by NO, a process that also requires the presence of the gene product, a transcriptional activator, NnrR. Capacity for producing, releasing and degrading NO is widespread in the biological world. A number of bacteria and fungi continually form and dispose of the gas. Plants are also known to liberate NO, but the degree of their involvement is not yet established. In contrast to the oxidative, L-arginine-dependent mechanism for producing NO (converted in turn to NO 2 and NO 3 )i n animals, microbes and the few plants studied generate NO by reductive reactions catalyzed by metalloenzymes. The set of natural phenomena that appear to yield NO more often than any other is called microbial denitrification - a series of anaerobic or alternative respiratory reactions for which widely distributed, usually aerobic bacterial species form branched, post quinone, electron transfer chains. Even when switched from aerobic to denitrifying respiration, these bacteria are likely to continue using the standard components of electron transfer chains to carry out the pre-quinone transfers. Unique enzymes are then produced and combined to bring about orderly and sequential reduction of NO 3 ,N O 2 ,N O and N2O to yield N2. A number of denitrifiers lack the final reductase and thus accumulate N 2 O( 1). These steps drive proton translocation, and several are linked to phosphorylation (2). Some of the NO so formed evolves continually and unceasingly into the atmosphere and other environments. Study of the denitrification processes has led to better understanding of the interactions of NO with metalloenzymes and of the influence of NO on sulfate reduction, nitrogen fixation and methanogenesis (3-6). Together, reduction of NO 2 to NO, and of NO to N2O, constitute the pivotal events in the global nitrogen cycle. Otherwise, standing stocks of fixed nitrogen would suffice for

The effect of chronic treatment with NO donors during intimal thickening and fatty streak formation

Abstract: Intimal thickening in arteries is considered as a site of predilection for atherosclerosis. We investigated whether oral application of the nitric oxide (NO) donors SPM-5185 (N-nitratopivaloyl-S-(N'-acetylalanyl)-cysteine ethylester, 10 mg/kg body weight/b.i.d.) and molsidomine (pro-drug of 3-morpholino-sydnonimine (SIN-1), 10 mg/kg body weight/day) can retard intimal thickening and changes in vascular reactivity induced by a silicone collar positioned around the carotid artery of rabbits. Intimal thickening was significantly inhibited by SPM-5185 (cross-sectional area 18 +/- 6 vs. 44 +/- 10 x 10(-3) mm2; P < 0.05), but not by molsidomine (28 +/- 6 vs. 35 +/- 9 x 10(-3) mm2), which is a donor of both NO and superoxide anions. In organ chamber studies collaring was associated with a decreased sensitivity to acetylcholine (ACh). SPM-5185 evoked a tendency towards normalization of the pD2 of ACh in collared arteries. We also investigated whether chronic nitric oxide (NO) treatment affected vascular reactivity and fatty streak development in the rabbit aorta. During 16 weeks rabbits received 150 g/day of a standard diet, or diets with 0.3% cholesterol, with 0.02% molsidomine (10 mg/kg body weight/day) or with the combination. The NO donor enhanced the area of fatty streaks, without affecting hypercholesterolemia. Moreover, it desensitized the smooth muscle cells of the rabbit aorta to vasodilators acting via the cytoplasmic guanylate cyclase and suppressed the capacity of the endothelial cells to release NO in response to muscarinic receptor stimulation. This suggested that chronic exposure to large quantities of NO caused a negative feedback, with selective decreases of both the endothelial capacity to generate NO and the responsiveness to vasodilators operating via cyclic GMP. In conclusion, we demonstrated that exogenous NO can decrease intimal hyperplasia in vivo. However, prolonged in vivo treatment with a donor of NO enhanced atherosclerosis in hypercholesterolemic rabbits.

Inhibition of platelet activity with red wine and grape products

Abstract: Platelets are known to contribute to the development of coronary artery atherosclerotic disease by several mechanisms [1]. Platelets are also primarily involved in forming the occlusive thrombus in the narrowed coronary artery that leads to fatal or non-fatal myocardial infarction (heart attack) [1]. It is known that patients with diabetes, hypertension, elevated LDL cholesterol, or patients who smoke have hyperactive platelets [2]. This is likely to exacerbate the atherothrombotic process. A number of clinical trials have shown that aspirin, a platelet inhibitor, given daily significantly reduces the incidence of symptoms of coronary artery disease and heart attacks [3]. It seems reasonable to assume that by taking “something” on a daily basis that turns down platelet activity, one could reduce the contribution of platelets to the development and sequelae of coronary artery disease.

Vasorelaxation, endothelium, and wine

Abstract: The mechanism of the cardiovascular benefits of wine is not fully understood, and indeed, may be manifold [1–3]. We have evidence, at least in vitro, that there are compounds present in grapes and wines, particularly red wines, as well as many other food plants, which cause endotheliumdependent relaxation (EDR) of blood vessels via the arginine/nitric oxide/cyclic GMP pathway [4,5]. Nitric oxide (NO) was discovered in 1980 by Furchgott and Zawadzki [6], who coined the term endothelium-derived relaxing factor (EDRF). In 1987, Furchgott [7] and Ignarro et al. [8], working in separate laboratories, simultaneously reported that EDRF was indeed nitric oxide. Since that time, NO has been shown to play important roles in many physiological systems. NO is generated in endothelial cells by the Ca2+-calmodulin-dependent enzyme NO synthase (NOS), acting on its substrate, the amino acid L-arginine. NO is released from the endothelial cell (both luminal and abluminal sides) and easily traverses the distance to the vascular smooth muscle cell, where it diffuses in and acts on its target enzyme, guanylate cyclase. Guanylate cyclase, in turn, causes generation of cyclic GMP, which ultimately leads to vasorelaxation. NO donor drugs such as nitroglycerin deliver NO directly to the smooth muscle cell to cause vasodilation, thus bypassing the endothelial cell. NO exhibits several potentially significant cardiovascular protective effects:

Quantitative determination of oLAb titers in various animal species

Abstract: It is generally accepted, that lipid peroxidation plays a pathogenic role in atherosclerosis. Furthermore, recent studies indicate that antibodies directed against oxidative modifications of Low Density Lipoprotein (oLAb) contribute to atherosclerotic processes and may have some function in other disorders. These antibodies have been determined predominantly in humans, because assays for oLAb measurement use species specific anti IgG conjugates. From such assay designs it is not possible to get directly comparable data from various animal species. Main advantages of comparable data between animal species are that results of animal experiments can be interpreted using human calibrators and that results of immunisations and production of monoclonal antibodies are directly comparable not only within, but also between animal species. The aim of this study was to find a modification for ELISAs for oLAb determination, which allows to measure sera of various animal species simultaneously. Microtitration plates were coated with oxidised LDL and blocked with bovine serum albumine. Human and animal sera were then pipetted into the plate in logarithmic serial dilutions and incubated for 2 h at 37 degrees C. After washing, a protein A horse-radish peroxidase conjugate (Biomakor, Israel) was added to each well in a dilution of 1:20,000. The incubation conditions had to be optimized to achieve reliable results. After another washing step, the assay was developed with TMB. Absorptions were read at 450 nm in a microplate photometer. Following the manufacturers incubation instructions, which recommended a duration of 1 h at room temperature, the system did not work optimally. No binding of protein A to IgG molecules bound to oxidised LDL could be observed, if the system was incubated at 37 degrees C. In our hands, best results were achieved for several animal species, if the conjugate was incubated for two hours at 2-4 degrees C in a refrigerator. Under these conditions, assay sensitivity was the same as in the standard method, which uses anti-species IgG conjugates. The protein A modification of oLAb allows direct reading of animal oLAb titres from human calibrators. With this method, results of animal experiments can be interpreted on the basis of the situation in humans. Preliminary results obtained show that immunisation experiments with oxidised LDL give serum titres in animals, which are in the same order of magnitude as human sera with high oLAb concentrations. The results of this study, in accordance with findings of other authors, give further indications that atherosclerotic processes are influenced by the specific immune system.

The effects of insulin, transferrin and androgens on rat prostate explants in serum‐free organ culture

Abstract: A model previously developed in our laboratory to culture rat prostate explants in serum-free chemically-defined medium was used to evaluate the direct influence of potential regulators. The aim of the present work was to verify the effects of insulin (I) and transferrin (Tr), two hormones considered as essential in other serum-free culture systems, and three androgenic hormones, since the prostate is known to be androgen-dependent. Explants of rat prostate were cultured for five days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air-5% CO2). The addition of Tr (50 micrograms/ml) had no effect, but I (5 micrograms/ml) significantly increased DNA synthesis. This influence was amplified by combination of the two hormones. However, protein synthesis was only slightly stimulated. Testosterone (T) or androstanediol significantly increased DNA synthesis when compared to corresponding control values at five days. In combination with I plus Tr, each hormone showed potentiated effects, particularly T with a twofold increase over day 0 values. When dihydrotestosterone was added singly, the incorporation of 3H-thymidine was stimulated by 300% over control values at five days, and by 100% over values in uncultured explants. This influence was maximal since it was not improved by I plus Tr. Protein synthesis was increased significantly by the triple combination. In addition, each androgen as well as the combination of I plus Tr had a positive influence on explant morphology. The above conditions optimize the present culture system and establish its usefulness as a valuable tool to study the direct influence of different effectors in prostate metabolism and to eventually identify putative cancer markers.