Oncogenic K-ras expression is associated with derangement of the cAMP/PKA pathway and forskolin-reversible alterations of mitochondrial dynamics and respiration.

The Warburg effect in cancer cells has been proposed to involve several mechanisms, including adaptation to hypoxia, oncogenes activation or loss of oncosuppressors and impaired mitochondrial function. In previous papers, it has been shown that K-ras transformed mouse cells are much more sensitive as compared with normal cells to glucose withdrawal (undergoing apoptosis) and present a high glycolytic rate and a strong reduction of mitochondrial complex I. Recent observations suggest that transformed cells have a derangement in the cyclic adenosine monophosphate/cAMP-dependent protein kinase (cAMP/PKA) pathway, which is known to regulate several mitochondrial functions. Herein, the derangement of the cAMP/PKA pathway and its impact on transformation-linked changes of mitochondrial functions is investigated. Exogenous stimulation of PKA activity, achieved by forskolin treatment, protected K-ras-transformed cells from apoptosis induced by glucose deprivation, enhanced complex I activity, intracellular adenosine triphosphate (ATP) levels, mitochondrial fusion and decreased intracellular reactive oxygen species (ROS) levels. Several of these effects were almost completely prevented by inhibiting the PKA activity. Short-time treatment with compounds favoring mitochondrial fusion strongly decreased the cellular ROS levels especially in transformed cells. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial complex I decrease, reduction of ATP formation, prevalence of mitochondrial fission over fusion, and thereby opening new approaches for development of anticancer drugs.

Are retinoids a promise for Alzheimer's disease management?

Retinoids regulate several physiological and pathological processes through the interaction with nuclear receptors. Retinoid-associated signaling which plays an essential role in neurodevelopment appears to remain active in the adult central nervous system (CNS), thus assuming a high significance in the context of neurodegeneration, and indeed retinoid analogs are thought to be promising therapeutic agents for the treatment of neurodegenerative disorders. The ability of retinoids to exert antioxidant effects, inhibit amyloid-ß (Aß) deposits and likely Aß-induced mitochondrial dysfunction, tau hyperphosphorylation, Aß-induced IL6 production and neuroinflammation, increase survival in hippocampal neurons, and reverse cognitive deficits in animal models of Alzheimer's disease (AD) is discussed. Although retinoids with their multi-target activity are revealing to be promising for management of AD which is a multifaceted biochemical phenomenon, timing as well as appropriate dosage and safety remain, however, a challenge. The end-stage lesions, namely senile plaques and neurofibrillary tangles, are, at present, considered an adaptive response to oxidative stress underlying AD, thus paradoxically late administration of retinoids could even suppress a protective mechanism by inhibiting Aß deposits.

Genetic and physical mapping of new EST-derived SSRs on the A and B genome chromosomes of wheat.

The availability of genetic maps and phenotypic data of segregating populations allows to localize and map agronomically important genes, and to identify closely associated molecular markers to be used in marker-assisted selection and positional cloning. The objective of the present work was to develop a durum wheat intervarietal genetic and physical map based on genomic microsatellite or genomic simple sequence repeats (gSSR) markers and expressed sequence tag (EST)-derived microsatellite (EST-SSR) markers. A set of 122 new EST-SSR loci amplified by 100 primer pairs was genetically mapped on the wheat A and B genome chromosomes. The whole map also comprises 149 gSSR markers amplified by 120 primer pairs used as anchor chromosome loci, two morphological markers (Black colour, Bla1, and spike glaucousness, Ws) and two seed storage protein loci (Gli-A2 and Gli-B2). The majority of SSR markers tested (182) was chromosome-specific. Out of 275 loci 241 loci assembled in 25 linkage groups assigned to the chromosomes of the A and B genome and 34 remained unlinked. A higher percentage of markers (54.4%), localized on the B genome chromosomes, in comparison to 45.6% distributed on the A genome. The whole map covered 1,605 cM. The B genome accounted for 852.2 cM of genetic distance; the A genome basic map spanned 753.1 cM with a minimum length of 46.6 cM for chromosome 5A and a maximum of 156.2 cM for chromosome 3A and an average value of 114.5 cM. The primer sets that amplified two or more loci mapped to homoeologous as well as to non-homoeologous sites. Out of 241 genetically mapped loci 213 (88.4%) were physically mapped by using the nulli-tetrasomic, ditelosomic and a stock of 58 deletion lines dividing the A and B genome chromosomes in 94 bins. No discrepancies concerning marker order were observed but the cytogenetic maps revealed in some cases small genetic distance covered large physical regions. Putative function for mapped SSRs were assigned by searching against GenBank nonredundant database using TBLASTX algorithms.

Regulation by the cAMP cascade of oxygen free radical balance in mammalian cells.

A study is presented of the effect of the cAMP cascade on oxygen metabolism in mammalian cell cultures. Serum-starvation of the cell cultures resulted in depression of the forward NADH-ubiquinone oxidoreductase activity of complex I, decreased content of glutathione, and enhancement of the cellular level of H2O2. Depressed transcription of cytosolic Cu/Zn-SOD 1, mitochondrial glutathione peroxidase and catalase was also observed. Activation of the cAMP cascade reversed the depression of the activity of complex I and the accumulation of H2O2. The effect of cAMP involved the cAMP-dependent protein kinase.

Comparison of two enzyme immunometric assays to measure tumor necrosis factor-alpha in human serum.

BACKGROUND: Tumor necrosis factor-alpha is a 17.5 kDa, 157 amino acid protein that is a potent lymphoid factor, which exerts cytotoxic effects on a wide range of tumor cells and other target cells. TNF-alpha has been suggested to play a pro-inflammatory role by influencing transendothelial migration of monocytes and elicits the expression of proteolytic enzymes by macrophages and smooth muscle cells within the atherosclerotic plaque. METHODS: We compared two methods for the quantitative determination of human tumor necrosis factor-alpha in serum samples. Either kit follows the same assay procedure. Serum samples do not need to be diluted before sampling. Standard is provided lyophilized and serial dilutions after reconstitution generate the standard point curves. The tests are enzyme immunometric assays based on a standard 96-well microtiter plate. The wells are coated with anti-human TNF-alpha antibody. RESULTS: The range of the standard curve is similar in both kits. It spans from 1000 through 15.6 pg/mL. The median TNF-alpha concentration in samples measured by Pierce assay (n=368) was 4.23 pg/mL, (range, 1.34-77.2 pg/mL). A very different median was obtained for the same specimen measured with the Titerzyme EIA (median, 176.96 pg/mL; range, 54.7-283.9 pg/mL; n=364). Substantial significant differences were observed between the two methods. CONCLUSION: Our results show that the two kits are unmatchable for results they can give when TNF-alpha concentrations are measured in serum samples. One reason of this disagreement could be the matrix effect or a cross-reactivity of one of the two methods. This study shows that the determination of human serum TNF-alpha needs to be standardized, especially when a comparison of results is required.

C-reactive protein and tumor necrosis factor-alpha in gestational hyperglycemia.

OBJECTIVES AND STUDY DESIGN: Increasing evidences support an inflammatory origin for gestational hyperglycemia. This paper aims at investigating, cross-sectionally and prospectively, the relationships between tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein (CRP) levels in normoglycemic and hyperglycemic pregnancies of women with and without conventional risk factors for gestational diabetes (GDM). RESULTS: Both at simple and multiple correlations TNF-alpha levels are associated to fasting insulin, homeostasis model assessment-insulin resistance (HOMA-IR) values and gestational hyperglycemia, while high sensitivity CRP (hsCRP) levels to body mass index (BMI). Furthermore, the TNF-alpha levels of the second trimester and their increments in the third trimester are significant predictors of insulin levels measured at 32-36 weeks in the subgroup of hyperglycemic women with < or = 35 yr, BMI <25 kg/m2 and the absence of a first-degree relative with Type 2 diabetes (respectively, beta=1.1; 95%CI 0.66-1.48; p=0.002 and beta=1.0; 95%CI 0.36-1.66; p=0.02), in a multiple regression model, after multiple adjustments. In a second cohort of women at low risk for GDM (<25 yr, BMI <25 kg/m2 and absence of a first-degree relative with Type 2 diabetes), 24-28 weeks TNF-alpha levels are highly associated with corresponding insulin and HOMA values in the same model (respectively, beta=0.27; 95%CI 0.11-0.43; p=0.001 and beta=0.30; 95%CI 0.14-0.46; p<0.001). CONCLUSIONS: The data support the developing hypothesis that low-grade systemic inflammation is associated to GDM, in particular for pregnant women without conventional risk factors for gestational hyperglycemia, whose insulin resistance seems less explainable.

Obesity or diabetes: what is worse for the mother and for the baby?

OBJECTIVES: The aim of the present study is to evaluate pregnancy outcomes in a cohort of Caucasian pregnant women in relation to their body mass index and glucose tolerance status; the role of central fat distribution, as indicated by waist-to-hip circumference ratio, was also considered. METHODS: Seven hundred women were studied; they had gestational diabetes or impaired glucose tolerance (250) or normoglycaemia (450). Among them 117 had pre-pregnancy overweight/obesity (44 were obese), 133 hyperglycaemia, but normal weight, and 117 hyperglycaemia and overweight/obesity (42 were obese). RESULTS: Hypertension, cesarean delivery and prevalence of large-for-gestational age babies were higher in obese (both with normoglycaemia and hyperglycaemia), mainly in those with greater gestational weight gain and central fat distribution (waist-to-hip ratio > 0.90). Normal weight hyperglycaemic women showed better outcomes than obese normoglycaemic women did. In a multiple logistic regression model, obesity (OR=10.6; 95% CI 5.00-22.54) was directly related to hypertension, and independent predictors of cesarean section were: gestational hyperglycaemia (OR=1.78; 95% CI 1.21-2.62), gestational weight gain (OR=1.06; 95% CI 1.02-1.10), and central obesity (OR=1.51; 95% CI 1.02-2.24), while obesity (OR=4.48; 95% CI 2.30-8.71) gestational weight gain (OR=1.08; 95% CI 1.03-1.12) and central fat distribution (OR=1.81: 95% CI 1.12-2.93) were directly related to delivering larger babies, after multiple adjustments. CONCLUSION: These results suggest that pre-pregnancy obesity and gestational hyperglycaemia were independent risk factors for different adverse pregnancy and neonatal outcomes, while central distribution of fat, and gestational weight gain play an additive adverse role on these outcomes.

Clinical characteristics and outcome of pregnancy in women with gestational hyperglycaemia with and without antibodies to beta-cell antigens.

AIMS: To evaluate the prevalence of beta-cell autoantibodies in women with gestational diabetes and impaired glucose tolerance, and identify clinical characteristics differentiating hyperglycaemic patients with and without autoantibodies. METHODS: One hundred and twenty-three pregnant patients with gestational diabetes, 84 with impaired glucose tolerance and 290 with normoglycaemia were evaluated for anti-islet cell antibodies, glutamic acid decarboxylase (GAD) autoantibodies, and the components of the metabolic syndrome. RESULTS: Autoantibody positivity was 8.9%, 17.9% and 0.3% in patients with diabetes, impaired tolerance and normoglycaemia, respectively. Hyperglycaemic patients with autoantibodies had lower body mass index, waist, weight gain at the time of the screening test and a lower percentage of previous pregnancies than those without autoantibodies. In addition, their fasting insulin values were significantly lower and inversely related to the presence of autoantibodies (odds ratio (OR) = 0.64; 95% confidence interval (CI) 0.42-0.96), the lowest values being found in anti-GAD+ patients. Autoantibody-positive women with diabetes were more frequently treated with insulin than negative patients (OR = 7.21; 95% CI 1.85-28.08). CONCLUSIONS: Autoantibody-positive women with gestational hyperglycaemia displayed fewer features of insulin resistance and required more frequent insulin therapy than negative women and presumably had presymptomatic Type 1 diabetes. If this conclusion is corroborated by the follow-up of larger series, clinical and immunological distinction of types of gestational hyperglycaemia would be useful.

Complex I and the cAMP cascade in human physiopathology.

A cAMP-dependent protein kinase (PKA) is localized in mammalian mitochondria with the catalytic site at the matrix side of the membrane where it phosphorylates a number of proteins. One of these is the 18 kDa(IP) subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene. Mitochondria have a Ca(2+)-inhibited phosphatase, which dephosphorylates the 18 kDa phosphoprotein of complex I. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18 kDa protein is associated with stimulation of complex I and overall respiratory activity with NAD-linked substrates. Mutations in the human NDUFS4 gene have been found, which in the homozygous state are associated with deficiency of complex I and fatal neurological syndrome.

The NADH: ubiquinone oxidoreductase (complex I) of the mammalian respiratory chain and the cAMP cascade.

Recent work has revealed cAMP-dependent phosphorylation of the 18-kDa IP subunit of the mammalian complex I of the respiratory chain, encoded by the nuclear NDUFS4 gene (chromosome 5). Phosphorylation of this protein has been shown to take place in fibroblast cultures in vivo, as well as in isolated mitochondria, which in addition to the cytosol also contain, in the inner-membrane matrix fraction, a cAMP-dependent protein kinase. Mitochondria appear to have a Ca2+-inhibited phosphatase, which dephosphorylates the 18-kDa phosphoprotein. In fibroblast and myoblast cultures cAMP-dependent phosphorylation of the 18-kDa protein is associated with potent stimulation of complex I and overall respiratory activity with NAD-linked substrates. Mutations in the human NDUFS4 gene have been found, which in the homozygous state are associated with deficiency of complex I and fatal neurological syndrome. In one case consisting of a 5 bp duplication, which destroyed the phosphorylation site, cAMP-dependent activation of complex I was abolished in the patient's fibroblast cultures. In another case consisting of a nonsense mutation, leading to termination of the protein after only 14 residues of the putative mitochondria targeting peptide, a defect in the assembly of complex I was found in fibroblast cultures.

Cyclic adenosine monophosphate-dependent phosphorylation of mammalian mitochondrial proteins: enzyme and substrate characterization and functional role.

A study is presented on cyclic adenosine monophosphate- (cAMP-) dependent phosphorylation of mammalian mitochondrial proteins. Immunodetection with specific antibodies reveals the presence of the catalytic and the regulatory subunits of cAMP-dependent protein kinase (PKA) in the inner membrane and matrix of bovine heart mitochondria. The mitochondrial cAMP-dependent protein kinase phosphorylates mitochondrial proteins of 29, 18, and 6.5 kDa. With added histone as substrate, PKA exhibits affinities for ATP and cAMP and pH optimum comparable to those of the cytosolic PKA. Among the mitochondrial proteins phosphorylated by PKA, one is the nuclear-encoded (NDUFS4 gene) 18 kDa subunit of complex I, which has phosphorylation consensus sites in the C terminus and in the presequence. cAMP promotes phosphorylation of the 18 kDa subunit of complex I in myoblasts in culture and in their isolated mitoplast fraction. In both cases cAMP-dependent phosphorylation of the 18 kDa subunit of complex I is accompanied by enhancement of the activity of the complex. These results, and the finding of mutations in the NDUFS4 gene in patients with complex I deficiency, provide evidence showing that cAMP-dependent phosphorylation of the 18 kDa subunit of complex I plays a major role in the control of the mitochondrial respiratory activity.

Nitric oxide production in living neurons is modulated by sphingosine: a fluorescence microscopy study.

An investigation was carried out into the possible effect of sphingosine (Sph) on nitric oxide (NO) production in living neurons. Differentiated granule cells were used in a dynamic videoimaging analysis of single cells labeled, simultaneously, with FURA-2 and the NO indicator 4,5-diaminofluorescein. The results demonstrate that Sph exerts a potent inhibitory effect on the Ca2+-dependent production of NO, without modifying the [Ca2+]i. The effect appears to be specific as neither ceramide nor Sph-1-phosphate had any effect on the NO and [Ca2+]i levels. The data demonstrate that Ca2+-dependent NO production is a specific Sph target in living granule cells, suggesting that this bioactive sphingoid plays a relevant role in neuronal NO signaling.

Dietary fat and gestational hyperglycaemia.

AIMS/HYPOTHESIS: The purpose of this study was to investigate the relation between life-style habits and glucose abnormalities in Caucasian women with and without conventional risk factors for gestational diabetes. METHODS: A total of 126 pregnant women with gestational diabetes, 84 with impaired glucose tolerance and 294 with normal glucose tolerance, identified by sequential screening, were interviewed to determine their usual weekly food pattern, amount of exercise, smoking habits and alcohol intake. RESULTS: Patients with glucose abnormalities were older and shorter in height and had significantly higher BMI before pregnancy, percentage of diabetic first-degree relatives and higher intake of saturated fat. Patients without known risk factors for gestational diabetes (i. e. younger than 35 years of age, BMI < 25 kg/m2, no first-degree diabetic relatives) included 40 with impaired glucose tolerance or gestational diabetes. In a multiple logistic regression model age, short stature, familial diabetes, BMI and percentages of saturated fat were associated with impaired glucose tolerance or gestational diabetes in all patients, after adjustment for gestational age. In patients without conventional risk factors only percentages of saturated fat (OR = 2.0; 95 %-CI = 1.2-3.2) and polyunsaturated fat (OR = 0.85; 95 %-CI = 0.77-0.92) were associated with gestational hyperglycaemia, after adjustment for age, gestational age and BMI. CONCLUSION/INTERPRETATION: Saturated fat has an independent role in the development of gestational glucose abnormalities. This role is more important in the absence of conventional risk factors suggesting that glucose abnormalities could be prevented during pregnancy, at least in some groups of women.

Casein phosphopeptides influence calcium uptake by cultured human intestinal HT-29 tumor cells.

We investigated the direct effects of casein phosphopeptides (CPP), which are formed by the proteolytic degradation of alpha- and beta-caseins, on calcium uptake by human HT-29 intestinal tumor cells, which undergo an enterocytically oriented differentiation in culture. A commercial preparation containing a mixture of purified CPP and an individual CPP of 25 amino acids, both containing the characteristic Ca(2+) binding motif, ser(P)-ser(P)-ser(P)-glu-glu, were employed. The study was performed at the single-cell level and on a cell population and measured the changes in cytosolic calcium concentration before and after CPP addition. In the presence of 2 mmol/L extracellular calcium, both CPP preparations induced a transient rise of free intracellular calcium ions, which did not influence ATP-induced release of calcium from intracellular stores, and which disappeared completely in the absence of extracellular calcium. Pretreatment of these cells with thapsigargin, which completely empties the intracellular calcium stores, did not abolish the cell responses to CPP. Repetitive stimulation of HT-29 cells with CPP always elicited a transient calcium rise, suggesting a lack of desensitization. The CPP-stimulated cytosolic calcium rise was dependent on CPP dose, in a seemingly nonsaturating mode, and on cell numbers. All of this is consistent with the hypothesis that CPP do not influence membrane-bound receptors or ion channels, but may act as calcium ionophores or calcium carriers across the membrane. The reported findings provide a new basis on which to assess the possibility that CPP enhance calcium absorption and bioavailability in animals.

Chronic low dose ethanol intake: biochemical characterization of liver mitochondria in rats.

Liver mitochondria were isolated from male rats exposed for 2 months to low doses of ethanol (3% v/v in drinking water), a condition not associated with tolerance or dependence. The results show no significant changes in the content of reduced or oxidized glutathione in the liver mitochondria of ethanol treated rats with respect to controls. However, a slight but significant increase in lipid peroxidation, accompanied by an increased content of oxidized proteins, was found in ethanol exposed animals. Mitochondrial content of cytochrome complexes was not significantly affected by ethanol intake. The specific enzymatic activity of cytochrome oxidase showed, however, a significant decrease in ethanol-treated rats. The slight mitochondrial alterations found in the liver of rats exposed chronically to low doses of ethanol might represent the beginning of a more extensive damage previously observed in rats exposed to high doses of this substance.

Ethanol-induced changes of intracellular thiol compartmentation and protein redox status in the rat liver: effect of tauroursodeoxycholate.

BACKGROUND/AIMS: Ethanol impairs cellular antioxidant defense and protein metabolism. Hydrophilic bile acids are protective against ethanol-induced cytotoxicity. This study investigated the compartmentation of intracellular thiol and protein redox status after acute ethanol intoxication in the liver and the effect of tauroursodeoxycholate pretreatment. METHODS: The concentrations of total glutathione, glutathione bound to proteins, sulfhydryl proteins, carbonyl proteins and malondialdehyde were measured in hepatic cytosol, mitochondria and nuclei after oral administration of 25% ethanol (4 g/kg) or isocaloric carbohydrate solution to rats. The metabolisms of ethanol and acetaldehyde were investigated by giving 4-methylpyrazole (1 mmol/kg i.p.) or cyanamide (15 mg/kg i.p.) 1 h prior to ethanol ingestion. One group of rats received tauroursodeoxycholate (12 mg/kg p.os) 1 h before ethanol ingestion. RESULTS: Ethanol significantly decreased the glutathione concentrations. Significant increases in glutathione bound to proteins, carbonyl protein and malondialdehyde concentrations were also noted, especially at the mitochondrial level. Enhanced carbonyl protein formation was also observed (p < 0.01). The inhibition of acetaldehyde metabolism, but not ethanol metabolism, exaggerated the alterations produced by ethanol. Pretreatment with tauroursodeoxycholate significantly reduced lipid and protein oxidation, particularly in mitochondria. By contrast, no changes were observed in glutathione content and compartmentation. CONCLUSIONS: Ethanol intoxication differentially impairs thiol and protein redox status in the subcellular fractions of rat liver. These alterations seem dependent on acetaldehyde rather than ethanol. Tauroursodeoxycholate administration protects proteins and lipids from ethanol-induced oxidative damage without influencing the glutathione content and compartmentation.

Oxidative protein damage in human diabetic eye: evidence of a retinal participation.

Considerable evidence indicates that the maintenance of protein redox status is of fundamental importance for cell function, whereas structural changes in proteins are considered to be among the molecular mechanisms leading to diabetic complications. In this study, protein redox status and antioxidant activity were investigated in the lens and vitreous of diabetic and nondiabetic subjects. A significantly lower content of sulphydryl proteins was found in lens and vitreous of diabetic patients than in those of non-diabetic and control subjects. Moreover, an increased formation of protein-bound free sulphydryls and carbonyl proteins, indices of oxidative damage to proteins, was noted in diabetic patients. All these parameters were shown to be altered particularly when diabetes was complicated with retinal alterations. In addition, glutathione peroxidase activity and ascorbic acid levels, known to exert important antioxidant functions in the eye compartment, were found to be significantly decreased in the lens of diabetic patients, especially in the presence of retinal damage. This study indicates an alteration of protein redox status in subjects affected by diabetes mellitus; lens and vitreous proteins were found to be oxidized to a greater extent in the presence of retinal disease, together with a marked decrease of eye antioxidant systems. These results suggest that oxidative events are involved in the onset of diabetic eye complications, in which the decrease in free radical scavengers was shown to be associated with the oxidation of vitreous and lens proteins. Protein oxidation may, therefore, represent an important mechanism in the onset of eye complications in diabetic patients.

Oxidative modification of proteins in chronic alcoholics.

Oxidative modification of proteins is of great importance because of their biological role in transport, enzyme activity, immune response and membrane fluidity. This study investigated the redox status of proteins in plasma, erythrocyte and erythrocyte ghosts of chronic alcoholics; a comparison with subjects affected by chronic viral hepatitis and healthy controls was also performed. Compared to the other groups, chronic active alcoholics showed significant increase of plasma, erythrocyte and erythrocyte ghost concentrations of carbonyl proteins, marker of protein oxidative damage. Also, a significant correlation was noted between daily alcohol intake and plasma levels of carbonyl proteins. The incubation of fresh human plasma with acetaldehyde, but not with ethanol, led to a significant increase of the carbonyl protein production. In conclusion, plasma, erythrocyte and membrane proteins are oxidatively modified in active chronic alcoholics; these changes seem to be related to acetaldehyde rather than ethanol toxicity.