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Biomedical subjects

E Marchesi

Publications and source records attributed to E Marchesi.

At least 19 recordsLinked to original sources
PubMed2006

Floral scent diversity is differently expressed in emitted and endogenous components in Petunia axillaris lines.

BACKGROUND AND AIMS: Among the subspecies of Petunia axillaris are various lines emitting sensorially different scents. Analysis of variations in floral scent among genetically close individuals is a powerful approach to understanding the mechanisms for generating scent diversity. METHODS: Emitted and endogenous components were analysed independently to gain information about evaporation and endogenous production in 13 wild lines of P. axillaris. A dynamic headspace method was used to collect emitted components. Endogenous components were extracted with solvent. Both of these sample types were subjected to quantitative and qualitative analysis by gas chromatography (GC)-flame ionization detector (FID) and GC-mass spectrometry (MS). KEY RESULTS AND CONCLUSIONS: Whereas the profiles of emitted compounds showed qualitative homogeneity, being mainly composed of methyl benzoate with quantitative variation, the profiles of endogenous compounds showed both qualitative and quantitative variation. A negative correlation was found between the evaporation ratio and boiling point of each compound examined. Lower boiling point compounds were strongly represented in the emitted component, resulting in the reduction of qualitative variation in floral scent. In conclusion, floral scent diversity results from variation in both the endogenous production and the evaporation rate of the individual volatile compounds.

Flowers↗
PubMed2001

Inhibition of promyelocytic leukemia (PML)/retinoic acid receptor-alpha and PML expression in acute promyelocytic leukemia cells by anti-PML peptide nucleic acid.

The fusion protein promyelocytic leukemia (PML)/retinoic acid receptor (RAR)alpha is tightly linked to the pathogenesis of acute promyelocytic leukemia (APL); hence, it represents a tumor-associated, transformation-related molecule. In this study, three anti-PML adamantyl-conjugated peptide nucleic acid (PNA) oligomers previously described as in vitro inhibitors of PML/RARalpha translation were combined and used to block PML/RARalpha synthesis in NB4 cells. Cationic liposomes were used to achieve sufficient delivery of PNAs into the cells. Upon treatment of cells with the liposome/PNA mixture, enhanced cellular uptake of PNA (approximately 5-fold compared with control) was obtained. Concomitantly, a substantial reduction (>90%) of the expression of PML/RARalpha was observed when all of the three PNAs were used together. This resulted in a dramatic effect on the number and viability of NB4 cells in culture after 48 h of treatment. This phenomenon was preceded by induction of apoptosis that could be observed 24 h after treatment. No sign of granulocytic differentiation was observed after treatment. These effects were also noted on other leukemic cell lines that express PML but not the fusion transcript. These results show that it is possible to deliver PNA into hematopoietic cells and obtain specific gene inhibition, and they suggest that a growth inhibitory effect on acute promyelocytic leukemia cells can be obtained through the block of PML/RARalpha and PML expression.

Antineoplastic Agents↗
PubMed2001

Sensitivity to the abl inhibitor STI571 in fresh leukaemic cells obtained from chronic myelogenous leukaemia patients in different stages of disease.

STI571 (CGP57148B) is an inhibitor of BCR/ABL, the cause of chronic myeloid leukaemia (CML). A difference exists between CML patients in chronic phase, in which responses to STI571are durable, and patients in blast crisis, who generally experience only transient responses. Leukaemic cells from six CML patients from whom samples could be obtained during chronic phase and at the time of blast crisis (BC) were compared for sensitivity to STI571, using an in vitro assay. BC samples showed a sensitivity similar to that obtained during chronic phase, suggesting that no substantial intrinsic resistance to STI571 was present in BC.

Adult↗
PubMed2000

Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification.

The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenic bcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/abl inhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 microM) 10-fold higher than the IC50 (0.1 microM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3-like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro. (Blood. 2000;95:1758-1766)

Adenosine Triphosphate↗
PubMed2000

Differential recognition of a BCR/ABL peptide by lymphocytes from normal donors and chronic myeloid leukemia patients.

The BCR/ABL oncogenic fusion protein transforms normal bone marrow stem cells into neoplastic cells. It has been shown that peptides derived from the junctional region of this oncogenic fusion protein can be recognized by human T-lymphocytes obtained from normal donors. In this study, we investigated the immunogenicity in patients with chronic myeloid leukemia (CML) of a 17 mer b3/a2 Bcr/abl peptide (B/A1), which was shown to induce proliferative responses in lymphocytes from normal donors. A total of 56 CML patients in chronic phase were studied. Twenty-two patients were studied at diagnosis without any treatment (group I). Fourteen patients were receiving IFN (group II), 14 patients were being treated with hydroxyurea (group III), and 6 patients were on different regimens (group IV). Patients were initially assessed for general immunological competence using both in vivo and in vitro assays. Patients were also selected for the expression of HLA-DR0401, the HLA specificity known to present peptide B/A1 to CD4 lymphocytes. With the exception of the six patients in group IV, the results of all these assays (in vitro phytohemagglutinin/tetanus toxoid responses, in vivo skin reaction to ubiquitous antigens) in CML patients did not significantly differ from those obtained in normal donors, thus excluding the presence of generalized immunosuppression. Eight patients with HLA-DR0401 and a b3/a2 type of fusion were identified and further studied. In these eight patients dendritic cells were obtained from adherent peripheral blood mononuclear cells and used to stimulate CD4 lymphocytes. No patient developed a specific response to the bcr/abl peptide, although patients' lymphocytes proliferated in response to a promiscuous tetanus toxoid peptide in all but one case. In contrast, response to the bcr/abl peptide was observed in seven of eight HLA-DR0401 healthy donors tested. These data suggest that immunocompetent, HLA-DR0401+ CML patients are unable to respond to peptide B/A1, at difference from healthy donors. The implication of these results for the immunotherapy of CML is discussed.

Cell Division↗
PubMed1999

In vivo eradication of human BCR/ABL-positive leukemia cells with an ABL kinase inhibitor.

BACKGROUND: The leukemia cells of approximately 95% of patients with chronic myeloid leukemia and 30%-50% of adult patients with acute lymphoblastic leukemia express the Bcr/Abl oncoprotein, which is the product of a fusion gene created by a chromosomal translocation [(9:22) (q34;q11)]. This oncoprotein expresses a constitutive tyrosine kinase activity that is crucial for its cellular transforming activity. In this study, we evaluated the antineoplastic activity of CGP57148B, which is a competitive inhibitor of the Bcr/Abl tyrosine kinase. METHODS: Nude mice were given an injection of the Bcr/Abl-positive human leukemia cell lines KU812 or MC3. Tumor-bearing mice were treated intraperitoneally or orally with CGP57148B according to three different schedules. In vitro drug wash-out experiments and in vivo molecular pharmacokinetic experiments were performed to optimize the in vivo treatment schedule. RESULTS: Treatment schedules administering CGP57148B once or twice per day produced some inhibition of tumor growth, but no tumor-bearing mouse was cured. A single administration of CGP57148B caused substantial (>50%) but short-lived (2-5 hours) inhibition of Bcr/Abl kinase activity. On the basis of the results from in vitro wash-out experiments, 20-21 hours was defined as the duration of continuous exposure needed to block cell proliferation and to induce apoptosis in these two leukemia cell lines. A treatment regimen assuring the continuous block of the Bcr/Abl phosphorylating activity that was administered over an 11-day period cured 87%-100% of treated mice. CONCLUSION: These data indicate that the continuous block of the oncogenic tyrosine kinase of Bcr/Abl protein is needed to produce important biologic effects in vivo.

Animals↗
PubMed1999

The nature of prooxidant activity of vitamin C.

It was recently reported that vitamin C (500 mg/day for 6 weeks) administered as a dietary supplement to healthy humans exhibits a prooxidant, as well as an antioxidant effect in vivo. Here we show that high intakes of vitamin C (500 mg/kg b.w. for 4 days) in the rat are able to markedly induce hepatic cytochrome P4502E1-linked monooxygenases, measured as p-nitrophenol hydroxylase activity and corroborated by means of Western blot analyses. Furthermore, using Electron Paramagnetic Resonance Spectroscopy (EPR) coupled to a spin-trapping technique, we have also found that this induction generates large amounts of the anion radical superoxide (O2-). Therefore we can conclude that the adverse prooxidant outcomes (i.e. oxidative DNA damage) associated to vitamin C supplementation, being associated to a typical reversible boosting effect (i.e. enzymatic induction), may be easily controlled by a discontinuous supply. However, since the induced P4502E1 isoforms by vitamin C are responsible for ethanol metabolism to highly reactive radicals, care should be taken even in moderate drinkers.

Animals↗
PubMed1999

Photoreduction of the fluorescent dye 2'-7'-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements.

The photoreduction of 2'-7'-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (lambda > 300 nm) 2'-7'-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/*OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.

Aerobiosis↗
PubMed1999

Medium effects on the antioxidant activity of dipyridamole.

Very strong medium effects have been observed when testing the antioxidant activity of dipyridamole (DP) in different media such as benzene, tert-butanol, methanol solutions and egg yolk lecithin unilamellar and multilamellar vesicles. Actually, dipyridamole behaves as a very poor antioxidant in benzene while its ability to inhibit the lipid peroxidation reaction increases with increasing solvent polarity, being the highest in lipid vesicles. This behavior can not be rationalized on the basis of the classical chain breaking mechanism which operates in the case of phenolic and amine antioxidants and involving the transfer of a hydrogen atom to peroxyl radicals. An explanation is instead given in terms of an electron transfer reaction which leads to the oxidation of DP by the chain carrying peroxyl radical to give the dipyridamole cation radical, DP+*, and the peroxyl anion LOO-, and whose rate constant is expected to increase in strongly polar media. EPR and electrochemical data supporting this interpretation have been collected.

Antioxidants↗
PubMed1999

Plasminogen activator inhibitor-1 and carotid intima-media thickening in patients with newly detected primary hypertension.

OBJECTIVE: To investigate the correlation between ultrasonographically evaluated intima-media thickness (IMT) of common carotid artery (CCA) and cardiovascular risk factors for subjects with newly detected, uncomplicated and untreated primary hypertension. METHODS: The study population consisted of 200 subjects (123 men and 77 women, aged 46+/-7.5 years). Blood pressure was measured in the clinical setting and by 24 h noninvasive ambulatory monitoring. Fasting levels of blood glucose, plasma lipids and lipoproteins, fibrinogen and plasminogen activator inhibitor (PAI)-1 were measured. Ultrasound examination included measurement of far-wall intima-media complex of CCA and morphologic evaluation of occurrence of plaques in carotid and femoral bifurcations. RESULTS: The prevalence of greater than normal IMT (mean IMT > or =0.80 mm) was 22%. Significant univariate correlations to the dichotomy between normal and greater than normal mean IMT were detected for age, smoking, level of LDL cholesterol, level of PAI-1 and total ultrasonographic score. Multivariate logistic regression analysis confirmed the associations between greater than normal mean IMT and plasma concentrations of LDL cholesterol and PAI-1 as well as total ultrasonographic score. CONCLUSION: Greater than normal IMT of CCA was more strictly related to other cardiovascular risk factors than it was to blood pressure and was strongly associated with the occurrence of atherosclerotic plaques in carotid and femoral arteries. The role of PAI-1 in intima-media thickening that is emerging suggests that fibrinolytic balance is an important determinant of vessel-wall homeostasis in hypertensive patients.

Adult↗
PubMed1997

Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis.

The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL driven neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL both in vitro and in vivo and to inhibit the growth of v-abl and bcr/abl transfectants, as well as the in vitro formation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34(+) cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies. All six BCR/ABL+ lines showed a dose dependent inhibition of their spontaneous proliferative rate, which was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations </=3 microM, with the exception of fibroblasts and CD34 cells. Proliferation inhibition was observed also when using fresh samples obtained from two Ph+ ALL and 12 consecutive CML patients. Induction of apoptosis was observed in these samples too. The activity of CGP57148B can be monitored in ex vivo isolated or cultured cells using a simple and reproducible assay, without the need for exogenously added growth factors. This molecule possibly exerts its effects through the inhibition of the kinase activity of BCR/ABL and the subsequent initiation of apoptosis, without inducing cell differentiation. Some normal cells are also affected. These data support the use of CGP57148B in initial clinical studies; possible toxic effects on BM and fibroblast-derived cells will have to be closely monitored. The in vivo monitoring of patients will have to be focused on the induction of apoptosis in leukemic cells.

Apoptosis↗
PubMed1997

Structural changes in the heart and carotid arteries in hypertensive patients associated with cardiovascular risk factors.

OBJECTIVE: To estimate the relationship between structural changes in the heart and in the carotid arteries in hypertensives and to analyze the correlations between these structural changes and cardiovascular risk factors. METHODS: We studied 76 subjects (39 men and 27 women, mean age 45+/-7 years) with mild-to-moderate untreated and uncomplicated hypertension. All of the subjects underwent ambulatory blood pressure monitoring, M-mode echocardiography for evaluation of their left ventricular mass and B-mode high-resolution ultrasonography to determine their carotid arterial wall thickness. RESULTS: The mean intimal plus medial thickness of the common carotid artery was found to be related significantly and independently to the left ventricular mass indexed by the body surface area. In multivariate analysis, age and the high-density lipoprotein cholesterol level were related strongly to the intimal plus medial thickness, whereas the clinic systolic blood pressure, average night-time systolic blood pressure and glycemia were the most important determinants of the left ventricular mass index. Logistic regression analysis suggested that the thickness of the posterior left ventricular wall was a stronger predictor of the carotid intima-medial thickness than were age and the high-density lipoprotein cholesterol level. CONCLUSION: The carotid wall thickness and left ventricular mass of hypertensives are related independently; nevertheless the main determinants of structural cardiac and vascular changes are probably different.

Adult↗
PubMed1996

In vitro transcription and translation inhibition by anti-promyelocytic leukemia (PML)/retinoic acid receptor alpha and anti-PML peptide nucleic acid.

Peptide nucleic acids (PNAs) complementary to the 15 bases around the fusion point of both genomic DNA and cDNA of the promyelocytic leukemia/retinoic acid receptor alpha (PML/ RAR alpha; P/R) hybrid gene present in acute promyelocytic leukemia cells were synthesized and shown by gel retardation experiments to specifically bind oligonucleotides corresponding to the fusion region of the P/R molecule. PNA was also able to successfully compete with anti-P/R DNA for duplex formation with P/R DNA and to displace the anti-P/R DNA from dsDNA. In vitro transcribed P/R RNA from two inserts of approximately 350 to approximately 700 bp were tested in gel acceleration experiments with fluorescein-conjugated PNA and showed stable binding (resistant to denaturing conditions) of PNA to the newly transcribed RNA. Control RNA or transcripts from the noncoding strand did not bind PNA. However, this PNA, although able to specifically clamp polymerase chain reaction, was incapable of inhibiting in vitro translation of the PML/RAR alpha mRNA, even when a bis-PNA was used. Therefore, a PNA was targeted against the start region of the P/R cDNA and against poly-purine regions of the gene. Specific inhibition of in vitro translation and transcription was shown, starting at concentrations as low as 100 nmol/L. When oligonucleotides presenting the same sequence were compared, PNA proved to be approximately 40 times more active. In conclusion, in vitro inhibition of translation and transcription of the P/R gene can be obtained with PNA; however, it is still necessary to target the ATG start region or poly-purine regions of the gene.

Base Sequence↗
PubMed1996

Protective effects of carvedilol, a vasodilating beta-adrenoceptor blocker, against in vivo low density lipoprotein oxidation in essential hypertension.

Low density lipoprotein (LDL) oxidation plays a crucial role in the development and progression of atherosclerosis and is enhanced in patients with essential hypertension. This finding has stimulated a search for antihypertensive drugs with high intrinsic antioxidant properties. We investigated the antihypertensive and antioxidant effects of carvedilol, a new vasodilating beta-adrenoceptor blocking agent in a group of patients with mild to moderate essential hypertension after 4-month treatment. Carvedilol administration markedly increased the resistance to oxidation of LDL isolated from treated patients to values comparable to those of control, nonhypertensive subjects. This effect was achieved despite a significant loss in LDL-associated vitamin E. The increased resistance of LDL to oxidation promoted by carvedilol was not related to the normalization of previously increased blood pressure (BP). Indeed, the administration of other conventional antihypertensive drugs, capable of decreasing arterial BP but without high intrinsic antioxidant properties, to a control group of matched hypertensive patients failed to ameliorate LDL oxidation parameters. Carvedilol treatment also reduced the extent of in vivo LDL oxidation, as reflected by the decrease in antioxidized LDL autoantibody titer. This effect as well was detected only in the group of carvedilol-treated hypertensive patients and not after the simple reduction in BP obtained with antihypertensive drugs different from carvedilol.

Adrenergic beta-Antagonists↗
PubMed1996

Carotid intima-media thickening and in vivo LDL oxidation in patients with essential hypertension.

Low density lipoproteins (LDL) from hypertensive patients are more prone to in vitro oxidation and undergo a more pronounced oxidation in vivo. Due to the pro-atherogenic activity of oxidatively modified LDL, the correlation between the carotid intima-media thickening (IMT) and the markers of in vivo LDL oxidation was investigated in hypertensive patients. A cross-sectional study on 101 normocholesterolemic patients with newly diagnosed and untreated essential hypertension was performed. The occurrence of in vivo LDL oxidation was evaluated by measuring the titers of autoantibodies against Cu(2+)-oxidised LDL (oxLDL) and malondialdehyde-derivatised LDL (MDA-LDL). The extent and degree of atherosclerosis and the IMT were measured by means of carotid and femoral ultrasonography with a duplex scanner equipped with a high resolution probe. We did not find significant correlations between in vivo LDL oxidation parameters and the extent of atherosclerotic lesion in the entire group of hypertensive patients. However, a significant direct correlation was detected between the carotid IMT and the titer of autoantibodies against both oxLDL and MDA-LDL in hypertensive patients without advanced atherosclerotic plaques. The results obtained support the hypothesis that enhanced LDL oxidation may be one of the pathophysiological events related to the formation and progression of early atherosclerotic lesions (IMT) in carotid arteries of hypertensive patients.

Adult↗
PubMed1996

Effect of sublingual nifedipine on left ventricular diastolic dysfunction in hypertensives: echo-Doppler study at rest and during handgrip.

Using digitized M-mode echocardiography, we evaluated the acute effect of nifedipine on left ventricular (LV) diastolic dysfunction in 30 untreated hypertensives, evaluated at rest and during handgrip, both before and 30' after nifedipine (20 mg sublingually). At rest, after nifedipine blood pressure and end-systolic wall stress significantly decreased and peak lengthening rate of LV diameter, peak thinning rate of LV posterior wall and Doppler E/A ratio increased. Before and after nifedipine handgrip induced significant increases of blood pressure, heart rate and end-systolic wall stress; diastolic parameters significantly decreased during basal handgrip and did not change during handgrip after nifedipine. Nifedipine induced an acute improvement of LV diastolic dysfunction, that persisted when afterload increased during handgrip. Therefore nifedipine seems to improve LV diastolic function not only by reducing afterload, but also through a direct action on the myocardium. Besides, these results demonstrate that LV diastolic abnormalities in hypertension are partly dynamic and reversible.

Administration, Sublingual↗