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Biomedical subjects

C Guarnieri

Publications and source records attributed to C Guarnieri.

At least 19 recordsLinked to original sources
PubMed2005

Green tea modulation of inducible nitric oxide synthase in hypoxic/reoxygenated cardiomyocytes.

Hypoxia/reoxygenation (H/R) is one of the causes of the increased expression of inducible nitric oxide synthase (iNOS) in cardiomyocytes. Since an aberrant NOS induction has detrimental consequences, we evaluated the effect of a green tea extract (GTE) on the NOS induction and activity in H/R-cardiomyocytes to define a nutritional strategy. Cultured rat cardiomyocytes were exposed to H/R in the presence of two concentrations of a green tea extract (GTE), which is reported to inhibit NOS expression and activity in different cells. In cultured cardiomyocytes two NOS isoforms were constitutively expressed, but only iNOS was induced by H/R. GTE supplementation at the lowest concentration, comparable to that in human plasma after dietary consumption, was ineffective, while the highest, comparable to that achievable by dietary supplements, counteracted the effect of H/R on iNOS induction and activity. It is necessary to verify in humans the relationship between the modulation of NO production and green tea dietary consumption.

Animals↗
PubMed2002

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).

Acetylcholine↗
PubMed2002

Evidence of oxidative imbalance in long-term liver transplant patients.

BACKGROUND: Oxidative stress in patients undergoing liver transplantation results both from the pre-existing cirrhosis and ischaemia-reperfusion injury related to surgery. Previous studies have provided information limited to the immediate post-operative period. It remains to be established whether this oxidative imbalance is reversed in a longer time. AIM, METHODS AND PATIENTS: This study aimed to compare plasma concentrations of thiobarbituric acid-reactant substances and alpha-tocopherol in 20 cirrhotic patients before liver transplantation and 22 patients in whom transplant had been carried out at least 6 months previously. Thirty healthy age and sex-matched volunteers served as controls (cross-sectional study). Five patients were evaluated before and after liver transplantation (longitudinal study). RESULTS AND CONCLUSIONS: Pre-transplant patients showed greater thiobarbituric acid-reactant substances and lower alpha-tocopherol levels than controls. Transplanted patients presented lower thiobarbituric acid-reactant substances and greater alpha-tocopherol levels than cirrhotic patients without reaching, however, the levels observed in controls. No correlations were found between oxidative parameters and liver tests. Hypertransaminasaemia, liver disease recurrence, and rejection episodes did not significantly influence the oxidative parameters. In the longitudinal study, transplantation induced a significant decrease in plasma thiobarbituric acid-reactant substances and a rise in alpha-tocopherol. Although a long-term improvement in the oxidative injury observed in cirrhotic patients occurs after liver transplantation, mild oxidative stress persists even in successfully transplanted patients.

Adult↗
PubMed2001

Effect of polyamine depletion on caspase activation: a study with spermine synthase-deficient cells.

Activation of the caspase proteases represents a central point in apoptosis. The requirement for spermine for the processes leading to caspase activation has been studied in transformed embryonic fibroblasts obtained from gyro (Gy) mutant male mice. These cells lack spermine synthase activity and thus provide a valuable model to study the role of spermine in cell processes. Gy fibroblasts do not contain spermine and have a higher spermidine content. However, when compared with fibroblasts obtained from normal male littermates (N cells), Gy fibroblasts were observed to grow normally. The lack of spermine did not affect the expression of Bcl-2, and caspases 3 and 9 were activated by etoposide in both N and Gy cells, indicating that spermine is dispensable for caspase activation. Spermine deficiency did not significantly influence caspase activity in cells treated with etoposide, cycloheximide or staurosporine, but sensitized the cells to UV irradiation, which triggered significantly higher caspase activity in Gy cells compared with N cells. alpha-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis that is able to deplete cells of putrescine and spermidine, but usually does not influence spermine content, was able to produce a more complete polyamine depletion in Gy cells. This depletion, which included spermine deficiency, dramatically increased caspase activation and cell death in Gy fibroblasts exposed to UV irradiation. On the other hand, in either N or Gy cells, DFMO treatment did not influence caspase activity triggered by staurosporine, but inhibited it when the inducers were cycloheximide or etoposide. In Gy cells depleted of polyamines by DFMO, polyamine replenishment with either spermidine or spermine was sufficient to restore caspase activity induced by etoposide, indicating that, in this model, polyamines have an interchangeable role in supporting caspase activation. Therefore, spermine is not required for such activation, and the effect and specificity of polyamine depletion on caspase activity may be very different, depending on the role of polyamines in the specific death pathways engaged by different stimuli. Some inducers of apoptosis, for example etoposide, absolutely require polyamines for caspase activation, yet the lack of polyamines, particularly spermine, strongly increases caspase activation when induced by UV irradiation.

Animals↗
PubMed2001

Genotype-phenotype correlation for thiopurine S-methyltransferase in healthy Italian subjects.

OBJECTIVE: The aim of the present study was to estimate the concordance rate between erythrocyte thiopurine methyltransferase (TPMT) activity and genotype at the TPMT locus in an Italian population sample. METHODS: The TPMT phenotype and genotype were determined in an unrelated population of 103 Italian healthy blood donors. Erythrocyte TPMT activity was measured with a radiochemical assay using 12.5 microM S-adenosyl-L-(methyl-14C)-methionine and 4 mM 6-mercaptopurine. The genotyping assay was based on restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) methods. RESULTS: All subjects had detectable TPMT activity. The activity of TPMT varied 2.8-fold between the 5th and 95th percentile. This variation was neither age (P = 0.63) nor gender (P = 0.44) regulated and the frequency distribution of TPMT activity is compatible with a polymorphic distribution. The presence of the four most common defective alleles, i.e. TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C, was examined through the entire phenotyped population. Ninety-two subjects did not carry any of the tested mutations. Eleven individuals were heterozygous for one of the mutant alleles and had a TPMT activity lower than 30 pmol/min/mg. Eight subjects were TPMT*1/TPMT*3A, two TPMT*1/TPMT*3C and one was TPMT*1/TPMT*2. The TPMT*3B allele was not detected in the samples analysed. CONCLUSION: There was a concordance of 97% between genotype and phenotype. All the heterozygotes had an intermediate phenotype. However, the wide variation range in TPMT activity detected in the wild-type homozygotes indicates that other genetic or epigenetic factors influence the TPMT phenotype.

Adult↗
PubMed2000

Polyamines directly induce release of cytochrome c from heart mitochondria.

Cytochrome c release from mitochondria to the cytosol represents a critical step in apoptosis, correlated to the activation of the caspase cascade. In this report, we show that addition of micromolar concentrations of polyamines to isolated rat heart mitochondria induces the release of cytochrome c. Spermine, which is effective at concentrations of 10-100 microM, is more potent than spermidine, whereas putrescine has no effect up to 1 mM. The release of cytochrome c caused by spermine is a rapid, saturable and selective process that is independent of mitochondria damage. Spermine, unlike polylysine, is able to release a discrete amount of cytochrome c from intact, functional mitochondria. The cytochrome c-releasing power of spermine is not affected by cyclosporin A, differently from the effect of permeability transition inducers. In a cardiac cell-free model of apoptosis, the latent caspase activity of cytosolic extracts from cardiomyocytes could be activated by cytochrome c released from spermine-treated heart mitochondria. These data indicate a novel mechanism of cytochrome c release from the mitochondrion, and suggest that prolonged and sustained elevation of polyamines, characteristic of some pathologies such as heart hypertrophy, could be involved in the development of apoptosis.

Animals↗
PubMed2000

Emerin presence in platelets.

Emerin is an almost ubiquitous protein which is abnormal in X-linked Emery-Dreifuss muscular dystrophy (EMD), a syndrome characterized by muscle weakness, joint contractures and cardiac arrhythmia. Emerin is localized in the cells at the nuclear rim and its function is still unknown. In some models, emerin has also been described in the cytoplasm; however, its presence outside the nucleus is still matter of debate. We report the presence of emerin in circulating normal human platelets and its absence in platelets from X-linked EMD patients. Since platelets are cytoplasmic fragments derived from megakaryocytes, the presence of emerin in platelets confirms cytoplasmic localization of this protein, probably related to specific functions. We found also that emerin is present in the cytoplasm of megakaryocytes, while it is absent in circulating granulocytes.

Blood Platelets↗
PubMed1999

Nitric oxide can function as either a killer molecule or an antiapoptotic effector in cardiomyocytes.

Caspase enzymes are a family of cysteine proteases that play a central role in apoptosis. Recently, it has been demonstrated that caspases can be S-nitrosylated and inhibited by nitric oxide (NO). The present report shows that in chick embryo heart cells (CEHC), NO donor molecules such as S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione, spermine-NO or sodium nitroprusside inhibit caspase activity in both basal and staurosporine-treated cells. However, the inhibitory effect of NO donors on caspase activity is accompanied by a parallel cytotoxic effect, that precludes NO to exert its antiapoptotic capability. N-Acetylcysteine (NAC) at a concentration of 10 mM blocks depletion of cellular glutathione and cell death in SNAP-treated CEHC, but it poorly affects the ability of SNAP to inhibit caspase activity. Consequently, in the presence of NAC, SNAP attenuates not only caspase activity but also cell death of staurosporine-treated CEHC. These data show that changes in the redox environment may inhibit NO-mediated toxicity, without affecting the antiapoptotic capability of NO, mediated by inhibition of caspase enzymes. NO may thus be transformed from a killer molecule into an antiapoptotic agent.

Acetylcysteine↗
PubMed1999

Spermine triggers the activation of caspase-3 in a cell-free model of apoptosis.

Polyamines are ubiquitous organic cations required for cell proliferation. However, some evidence suggested that their excessive accumulation can induce apoptosis. We show here that, in a post-nuclear extract from U937 cells, the addition of spermine triggers the death program, represented by cytochrome c exit from mitochondria, the dATP-dependent processing of pro-caspase-3 and the onset of caspase activity. Spermine is more effective than spermidine, whereas putrescine has no effect. Polyamine acetylation abolishes their pro-apoptotic power. These data demonstrate a direct mechanism responsible for polyamine toxicity and also suggest that an excessive elevation of free polyamines could be involved in the transduction of a death signal.

Animals↗
PubMed1999

Molecular mechanisms of response to low oxygen tension in the vascular wall.

A number of human diseases are linked to local reduced oxygen availability. Hypoxemia, the condition in which oxygen partial pressure in blood falls below 40 mmHg, generates a distress which leads the cells in the vascular wall to activate a genetic program inducing a homeostatic response. The effectiveness of this response is conditioned by the degree and duration of the hypoxic stress and depends on the equilibrium among several factors which are worked out mainly in the vascular endothelial cell layer. Among them are vasoconstrictors such as angiotensin II, endothelins, prostaglandins and thromboxans, and vasodilators such as nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor. A present challenge of the research is understanding the physiological and pathophysiological relevance of the growing body of data collected, disclosing the potential therapeutical application of the basic knowledge in this field.

Animals↗
PubMed1999

Genetic polymorphisms influence variability in benzene metabolism in humans.

The role of genetic polymorphism in modulating urinary excretion of two benzene metabolites, i.e. trans,trans-muconic acid (t,t-MA) and S-phenylmercapturic acid (PMA), has been investigated in 59 non-smoking city bus drivers, professionally exposed to benzene via vehicle exhausts. Exposure to benzene was determined by personal passive samplers (mean +/- SD = 82.2 +/- 25.6 micrograms/m3), while internal dose and metabolic rate were evaluated by measuring urinary excretion of unmodified benzene (mean +/- SD = 361 +/- 246 ng/l), t,t-MA (mean +/- SD = 602 +/- 625 micrograms/g creatinine), and PMA (mean +/- SD = 5.88 +/- 4.76 micrograms/g creatinine). Genetic polymorphism at six loci encoding cytochrome-P450-dependent monooxygenases (CYP2E1 and CYP2D6), glutathione-S-transferases (GSTT1, GSTP1 and GSTM1) and NAD(P)H:quinone oxidoreductase (NQOR) was determined by polymerase chain reaction-based methods. No evidence emerged for a possible role of CYP2E1, GSTM1 and GSTP1 polymorphisms in determining the wide differences observed in the rate of benzene biotransformation. Conversely, a significantly higher t,t-MA urinary excretion was found to be correlated to, GSTT1 null genotype, and a significantly lower PMA excretion was detected in the subjects lacking NQOR activity and in the CYP2D6 extensive-metabolizers. Many biological (i.e. age and body burden) or lifestyle factors (i.e. rural or urban residence, use of paints and solvents, medication, alcohol and coffee intake), also taken into account as potential confounders, did not influence the correlations found. These findings suggest that CYP2D6, GSTT1 and NQOR polymorphisms contribute in explaining the metabolic variability observed in our sample. Therefore, these polymorphisms should be regarded as potential risk factors for benzene-induced adverse health effects.

Acetylcysteine↗
PubMed1998

Inhibition of etoposide-induced apoptosis with peptide aldehyde inhibitors of proteasome.

Recent investigations have indicated the involvement of proteasome in programmed cell death. The present studies show that although peptide aldehyde inhibitors of proteasome are by themselves weak inducers of apoptosis, they inhibit the apoptotic effect of the anticancer drug etoposide in rat thymocytes. Acetyl-Leu-Leu-norvalinal (LLnV-al) and other related peptide aldehydes inhibited the increase in caspase activity and DNA fragmentation that followed treatment with etoposide and their effect was related to their potency as proteasome inhibitors. To inhibit etoposide-induced apoptosis, LLnV-al must be present within 3 h of treatment with etoposide, in the same way as the inhibitor of protein synthesis cycloheximide must be. Etoposide caused a rapid accumulation of p53 protein that was not inhibited by LLnV-al, which was also a strong inducer of p53. Peptide aldehydes were also weak activators of caspase activity, suggesting that the same mechanism, i.e. the blocking of proteasome function, both triggers apoptosis and inhibits the effect of etoposide. These results are consistent with a model in which proteasome is selectively involved in the pathway used by etoposide to induce cell suicide.

Aldehydes↗
PubMed1998

Simultaneous detection of reduced and oxidized glutathione in tissues and mitochondria by capillary electrophoresis.

We have developed a rapid and precise method for glutathione quantitation by capillary electrophoresis, that allows a low amount of both redox forms to be measured. Small fragments of rat heart or liver tissues (20 mg wet weight) and the corresponding mitochondria (1 mg protein) were homogenized in 1% perchloric acid and the acid-soluble phase ultrafiltered by centrifugation with a microconcentrator (Mr cut-off 3000 Da). The analysis was performed at a constant temperature (28 degrees C) using a Beckman P/ACE System 2100, equipped with a UV absorbance detector set to 200 nm. The limit of quantitation in heart tissue was 1.8 microM for GSH and 1.2 microM for GSSG. Myocardial concentrations of GSH and GSSG were 8.1 +/- 2.6 and 0.45 +/- 0.15 (nmol/mg protein +/- S.D.), respectively. The ratio of GSH to GSSG was 17.8 +/- 1.3 for heart tissue, whereas it was much higher (>100) in the mitochondria. An oxidative stress decreased the myocardial tissue GSH/GSSG ratio, indicating that the CE analysis of both glutathione forms is also a useful method to study biological redox modification.

Animals↗
PubMed1998

Increase of neuronal nitric oxide synthase in rat skeletal muscle during ageing.

Nitric oxide synthases (NOS) are different widely expressed enzymes which produce the molecular messenger nitric oxide. The neuronal isoform of NOS (nNOS) is involved in several processes of the cell metabolism, most of which are, at present, not fully understood (neurotransmission, smooth muscle motility, myoblast and myocyte biology and others). In skeletal muscle nNOS is present mainly at the plasmalemma, where it is attached to the dystrophin-related proteins; in fact, in pathologies involving dystrophin, nNOS is altered as well. We report that in aged rats the nNOS amount in skeletal muscle increases both in the soluble and microsomal fractions and that an additional intracytoplasmic localisation appears.

Aging↗
PubMed1998

Inhibition of glucocorticoid-induced apoptosis with 5-aminoimidazole-4-carboxamide ribonucleoside, a cell-permeable activator of AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases that are believed to protect cells against environmental and nutritional stress. In the present study the hypothesis of a protective role for AMPK against thymocyte apoptosis has been tested. It is shown that AMPK is expressed in rat thymocytes that contain the transcript for the a1 isoform of the AMPK catalytic subunit and can be activated by treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a well-established activator of AMPK. AICAR is not toxic and prevents glucocorticoid-induced apoptosis in the same concentration range used to activate AMPK. At concentrations higher than 1 mM, AICAR fully restores cell viability and inhibits DNA laddering in dexamethasone-treated thymocytes. Furthermore, AICAR blocks the dexamethasone-induced activation of caspase 3-like enzymes, which are believed to play a pivotal role in apoptotic cell death. Activation of AMPK by oligomycin, which depletes thymocytes of ATP, is also correlated to inhibition of caspase 3-like activity in dexamethasone-treated cells. However, AICAR and oligomycin do not exert any protective action when apoptosis is induced by staurosporine. These results indicate that AICAR is a powerful inhibitor of glucocorticoid-induced apoptosis and suggest that AMPK activation may interfere with a step in the apoptotic cascade triggered by dexamethasone.

AMP-Activated Protein Kinases↗
PubMed1998

Systemically circulating oxidative species in human deep venous thrombosis.

To investigate the hypothesis that plasmatic changes of lipoperoxidative markers are associated with deep venous thrombosis (DVT), peripheral venous blood samples were obtained from 10 patients with venographically proven DVT before starting anticoagulant therapy, and 36+/-3 and 60+/-3 hours later. Values of myeloperoxidase (MPO), 4-hydroxynonenal (HNE) and malondialdehyde (MDA) were compared with those of 10 age-matched control subjects. Despite individual variations, mean plasma MPO level was higher in the DVT group (p < 0.01), as were average plasma MDA (p < 0.001) and HNE (p < 0.01) levels. Separate analysis of the DVT cases showed that higher values of MDA, HNE and MPO were found in patients with either co-morbid diseases or clinically silent pulmonary embolism (PE). Good evidence exists for considering DVT a condition associated with an apparently excessive free radical production not buffered by efficient defence systems. A role of DVT itself cannot be excluded, but PE or other co-morbid diseases may participate in the oxidative stress. If confirmed in a larger series of patients, these findings could shed new light on the plasmatic changes associated with the propagation and complications of DVT, which in turn could have therapeutic implications.

Adult↗
PubMed1997

Phosphatidylinositol 3-kinase is required for the induction of ornithine decarboxylase in leukemia cells stimulated to growth.

The involvement of phosphatidylinositol 3-kinase (PI3K) in the induction of ornithine decarboxylase (ODC) was investigated by using specific PI3K inhibitors. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, treatment with LY294002 inhibited cell growth and provoked a complete block of the induction of ODC activity (IC50 approximately 2 microM) and ODC protein. Some reduction in the accumulation of ODC mRNA was also observed, whereas ODC turnover was not affected significantly. Wortmannin, another specific inhibitor of PI3K, structurally unrelated to LY294002, also inhibited ODC induction with an IC50 of about 10 nM. These results indicate that PI3K activity is required for the induction of ODC, possibly affecting both ODC mRNA level and translation. Since p70 S6 kinase (p70S6K) is considered an important mediator of PI3K action in several experimental systems, the effect of rapamycin, which can lead to selective inhibition of p70S6K, was also investigated. Rapamycin inhibited p70S6K activity and produced ODC inhibiting effects similar to those elicited by LY294002. However, LY294002 and wortmannin at concentrations which inhibited almost completely PI3K activity did not decrease p70S6K activity, suggesting that p70S6K does not mediate the PI3K effects on ODC, but may lie on a separate pathway in this experimental model.

Androstadienes↗