PubMed Health⌕ Search

Biomedical subjects

S Marchetti

Publications and source records attributed to S Marchetti.

At least 19 recordsLinked to original sources
PubMed2006

Calorimetric and dynamic light-scattering investigation of cationic surfactant--DNA complexes.

By means of combined calorimetric and dynamic light-scattering measurements, we have investigated the conformational behavior of DNA chains after thermal melting in the presence of a cationic surfactant at different concentrations, up to a surfactant-to-phosphate group molar ratio close to unity. Both the specific heat capacity, C(ex)(p) and the hydrodynamic radius R of the DNA chains provide support for the existence of two structural arrangements with different thermal stabilities, coexisting in the bulk solution. Although a component remains an elongated unfolded DNA chain originated in the thermal denaturation, the second component, consisting of DNA-surfactant complexes, assumes a compact structure with an average size of about 80 nm, whose thermal denaturation occurs at temperatures higher than 100 degrees C.

Animals↗
PubMed2006

Apoptosis and erythroid differentiation triggered by Bcr-Abl inhibitors in CML cell lines are fully distinguishable processes that exhibit different sensitivity to caspase inhibition.

Imatinib targets the Bcr-Abl oncogene that causes chronic myelogenous leukemia (CML) in humans. Recently, we demonstrated that besides triggering apoptosis in K562 cells, imatinib also mediated their erythroid differentiation. Although both events appear to proceed concomitantly, it is not known at present whether or not imatinib-induced apoptosis and differentiation are interdependent processes. Hence, we investigated the requirements for Bcr-Abl inhibitor-mediated apoptosis and erythroid differentiation in several established and engineered CML cell lines. Imatinib triggered apoptosis and erythroid differentiation of different CML cell lines, but only apoptosis exhibited sensitivity to ZVAD-fmk inhibition. Conversely, the p38 mitogen-activated protein (MAP) kinase inhibitor, SB202190, significantly slowed down erythroid differentiation without affecting caspase activation. Furthermore, imatinib and PD166326, another Bcr-Abl inhibitory molecule, triggered erythroid differentiation of K562 cell clones, nevertheless resistant to Bcr-Abl inhibitor-induced apoptosis. Finally, short hairpin RNA inhibitor (shRNAi) silencing of caspase 3 efficiently inhibited caspase activity but had no effect on erythroid differentiation, whereas silencing of Bcr-Abl mimicked imatinib or PD166326 treatment, leading to increased apoptosis and erythroid differentiation of K562 cells. Taken together, our findings not only demonstrate that Bcr-Abl inhibitor-mediated apoptosis and differentiation are fully distinguishable events, but also that caspases are dispensable for erythroid differentiation of established CML cell lines.

Apoptosis↗
PubMed2006

A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis.

The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA+SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMA-induced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.

Base Sequence↗
PubMed2005

Dielectric behavior of DNA in water-organic co-solvent mixtures.

The radiowave dielectric dispersions of DNA in different water-organic co-solvent mixtures have been measured in the frequency range from 100 kHz to 100 MHz, where the polarization mechanism is generally attributed to the confinement of counterions within some specific lengths, either along tangential or perpendicular to the polyion chain. The dielectric dispersions have been analyzed on the basis of two partially different dielectric models, a continuum counterion fluctuation model proposed by Mandel and a discrete charged site model, proposed by Minakata. The influence of the quality of the solvent on the dielectric parameters has been investigated in water-methanol and water-glycerol mixtures at different composition, by varying the permittivity (m) and the viscosity eta of the solvent phase. The analysis of the dielectric spectra in solvents where electrostatic and hydrodynamic interactions vary with the solvent composition suggests that both the two models are able, in principle, to account for the observed high-frequency dielectric behavior. However, while some certain assumptions are necessary about the polyion structure within the Mandel model, no structural prerequisite is needed within the Minakata model, where the polarization mechanism invoked considers a radial counterion exchange with the outer medium, which is largely independent of the local polyion conformation.

DNA↗
PubMed2005

DNA condensation induced by cationic surfactant: a viscosimetry and dynamic light scattering study.

The compaction of DNA induced by two simple amphiphiles, cetyltrimethylammonium bromide [CTAB] and dodecyldimethylamine oxide [DDAO], has been investigated by means of combined viscosity and dynamic light scattering measurements, to demonstrate the formation of soluble DNA/surfactant complexes, undergoing a coil-globule transition, upon the increase of the amphiphile concentration. In both of the two systems investigated, the complexation process reaches a maximum for a value of the surfactant to DNA phosphate groups molar ratio of about X = 1. Below this critical concentration, the coil and the globule state coexist in the solution, as clearly shown by the bimodal size distribution obtained from the light scattering intensity correlation functions. Some suggestions are given to support a molecular mechanism responsible for the complex formation, both in the case of a cationic surfactant (CTAB) and of a pH-dependent neutral or cationic amphiphile (DDAO), where the hydrophobic interactions play an important role.

Animals↗
PubMed2004

Inducible expression of a MAP kinase phosphatase-3-GFP chimera specifically blunts fibroblast growth and ras-dependent tumor formation in nude mice.

The p42/p44 mitogen activated protein kinase (MAPK) pathway participates in a wide range of cellular programs including proliferation, migration, differentiation, and survival. Specific pharmacological inhibitors, like PD98059 and U0126, are often used to inhibit p42/p44 MAPK signaling. However, these inhibitors are not appropriate to study the function of these kinases in whole organisms. We thus developed an inducible system designed to inhibit p42/p44 MAPK activity through the expression of a phosphatase specific for these two kinases, the MAPK phosphatase 3 (MKP-3). A fibroblast cell line was established in which MKP-3 expression is controlled by tetracycline. Tetracycline-induced MKP-3 resulted in partial de-phosphorylation of p42/p44 MAPKs in serum-stimulated cells. However, we could improve MKP-3 stability and thereby the rate of MAPK de-phosphorylation, when the C-terminal end of MKP-3 was fused to the green fluorescent protein (GFP). Importantly, the fusion of GFP to MKP-3 did not alter the specificity of the phosphatase towards its MAPK substrates. We further show that conditional expression of MKP-3-GFP in this fibroblast cell line results in the inhibition of: (a) the phosphorylation of the p42/p44 MAPK substrates Elk1 and HIF-1alpha, (b) vascular endothelial growth factor (VEGF), cyclin D1, and c-fos gene transcription in response to MAPK pathway activation, and (c) cell proliferation. Finally, the MKP-3-GFP inducible cell line was transformed by Ha-ras and injected into nude mice. Treatment of mice with the tetracycline analog doxycycline resulted in a large delay in tumor emergence and growth as compared to the untreated control group, indicating that MKP-3-GFP activity is maintained in vivo. Altogether, these results show that inducible expression of MKP-3-GFP constitutes a valuable tool to study the role of p42/p44 MAPKs in various cellular responses in both cultured cell and animal models, a tool that may also be used to block unwanted cell growth in pathological conditions.

Animals↗
PubMed2004

Bacterial and viral DNA in periodontal disease: a study using multiplex PCR.

Recent studies have suggested an association between periodontal disease and the presence of Herpesviruses, in particular: Epstein-Barr virus (EBV) and Human Cytomegalovirus (CMV) (Contreras et al., 1999--Contreras et al., 2000--Slots et al., 2000--Ting et al., 2000). In the work reported in this paper, we use a multiplex Polymerase Chain Reaction (PCR) to compare the presence of Herpesviruses and putative bacterial pathogens in patients with periodontal disease and in healthy individuals. Direct detection of microorganisms with PCR is shown to offer significant advantages in terms of time, effort and cost. The study detected no statistically significant differences between the prevalence of EBV and CMV in patients and controls. The failure to replicate previous findings may be due to differences in the age composition and the geographical and social origins of the study groups. The study detected a significant excess of HSV-1 in periodontal patients. This suggests that the role of Herpesviruses in the pathogenesis of periodontal disease deserves further investigation. The bacterial assay confirmed the results of previous studies showing a strong association between periodontitis and the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia.

Actinobacillus Infections↗
PubMed2003

Age-specific seroprevalence of Human Herpesvirus 8 in Mediterranean regions.

OBJECTIVE: Human herpesvirus 8 (HHV8) is believed to be transmitted mainly by sexual contact; epidemiological data from Africa show, however, that non-sexual transmission routes may also play an important role. To evaluate better the distribution of HHV8 infection in the Mediterranean area, we performed an age-specific seroprevalence study. METHODS: Sera were collected from subjects from different geographical areas. The sera were analyzed by immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA). A total of 1083 patients were studied, 667 patients from various regions of Italy and 416 from Albania. The patients were stratified into six age groups. Multivariate logistic regression was used to evaluate associations between HHV8 and demographic data. RESULTS: An overall seropositivity rate of 17.6% was observed. The highest rate was observed in Sardinia (25.0%) and the lowest was found in Albania (13.9%). The prevalence rate increased linearly with age, from 9.7% in patients belonging to the 0-14 years age group to 26.3% for patients more than 59 years old. Seropositivity for HHV8 was significantly associated with membership of the 59 years-plus age group. Rates of seropositivity were significantly higher in patients from central southern Italy (OR = 1.7) and Sardinia (OR = 1.8) than in patients from Albania. CONCLUSIONS: The data suggest that HHV8 is widespread in the Mediterranean area, including regions like Albania that have not been previously investigated. The statistically significant association between HHV8 seropositivitity and increasing age suggests that non-sexual transmission routes may be involved in the spread of the virus.

Adolescent↗
PubMed2003

HHV 8 seroprevalence and transmission within Albanian family groups.

The recently discovered Human Herpesvirus 8 (HHV 8) is associated with all clinical forms of Kaposi's sarcoma. While early research suggested that the virus was transmitted sexually and that it was present only in KS patients, more recent studies seem to show that infection with the virus is more common than once thought, presenting differing distribution patterns in different geographical areas. In this study we analyze seroprevalence and transmission of HHV 8 in a sample of 86 family groups from Albania. Participants were selected among families requesting routine pre-expatriation medical examinations at the Poliambulatorio Padre "L. Monti" in Tirana. Specimens were collected from 180 healthy individuals and tested for the presence of a specific antibody. Antibody anti-HHV-8 detection was performed by immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA). The study found an overall rate of HHV 8 seroprevalence of 20.0%. In 4.5% of couples the male and female were both positive, in 30.2% at least one partner was positive; (in 17.4% only the male was positive; in 12.8% only the female). These results support the hypothesis that HHV 8 spreads via multiple transmission routes.

Adolescent↗
PubMed2002

Effect of physical activity on the pharmacokinetics of ceftazidime in mice.

BACKGROUND: The aim of the present work was to assess comparatively the pharmacokinetic profile of ceftazidime (CAZ) in trained and non-trained mice. METHODS: The study was performed on 256 mice divided at random into four groups: long-term physically trained mice with (E1a) and without (E1b) physical activity prior to the administration of CAZ, and untrained mice with (E2a) and without (E2b) physical activity prior to the administration of the antibiotic. CAZ was administered intramuscularly (25 mg/kg) to all mice, and blood samples were obtained at different time points. The plasma concentrations of CAZ were determined by HPLC and analyzed by non-compartmental models. RESULTS: The area under the curves in groups E1a and E2a (27.3 and 22.9 microg x ml(-1) x h, respectively) were different compared to the other groups [(E1b) = 11.1 and (E2b) = 15.6 microg x ml(-1) x h; p < 0.05]. Differences were observed between the concentration-time of CAZ in E1a compared to E1b, E1a versus E2a, E1a versus E2b, E1b versus E2a and E1b versus E2b (p < 0.05). CONCLUSION: Physical activity performed prior to CAZ administration modified the pharmacokinetic profile of the drug administered to mice.

Animals↗
PubMed2001

Impact of thymidine phosphorylase surexpression on fluoropyrimidine activity and on tumour angiogenesis.

Tumoral thymidine phosphorylase (TP) appears to play a dual role by being involved in neoangiogenesis and by activating 5FU prodrugs at the tumoral target site. The aim of the study was to investigate more thoroughly these potential physiological and pharmacological roles of TP. A rat carcinoma cell line (PROb) was transfected with TP/PD-ECGF in order to study the effect of the overexpression of this enzyme (1) on the sensitivity of cells to 5'DFUR and 5FU in vitro and (2) on tumour growth in vivo by using a syngenic tumour model in the BDIX rat (hepatic tumours, sub-cutaneous tumours). Cytotoxic effects of 5'DFUR, and to a lesser extent those of 5FU, were enhanced in TP clones as compared to control cells: there was a highly significant correlation between TP activity and in vitro sensitivity to 5'DFUR (r2= 0.91, P = 0.0002, n = 8) and, to a lesser extent, to 5FU (r2= 0.49, P = 0.053, n = 8). The impact of TP transfection on tumour growth was relatively modest and concerned only the initial stages of tumour expansion. Staining of TP tumours for endothelial (factor VIII) cells was always higher than controls. The staining ratio (TP/controls) tended to be reduced as tumours increased in size. The stability of TP expression was checked both in vitro (TP activity measurement) and in vivo (RT-PCR determinations) and there was no loss of TP expression over time which could be advanced to explain the progressive weakening of the impact of TP overexpression on both tumour growth and neoangiogenesis.

Animals↗
PubMed2001

Phase I metabolism of ganstigmine. Rat, dog, monkey and human liver microsomal extracts investigated by liquid chromatography electrospray tandem mass spectrometry.

Ganstigmine, a new acetylcholinesterase inhibitor, was incubated with rat liver microsomes and the resulting metabolites were identified by high-performance liquid chromatographic/mass spectrometric (HPLC/MS) and HPLC/MS/MS analyses. The results showed the formation of eight main metabolites, among which geneseroline and molecules corresponding to mono-hydroxylated, demethylated and reduced ganstigmine. The metabolic profile drawn for humans, dog and monkey showed a pattern very similar to that of rat: only in the case of liver dog microsomes higher amounts of geneseroline and of a metabolite identified as demethylated and reduced drug were detected.

Alkaloids↗
PubMed2001

Isolation and characterization of an endonuclease synthesized by barley (Hordeum vulgare L.) uninucleate microspores.

Few biochemical and molecular details are available on microspore growth and development. In this work, a nuclease was partially purified from diffusates of barley (Hordeum vulgare L.) microspores by using concanavalin-A as ligand. The chromatographic preparation contained a 34-kDa protein with nucleolytic activity; the enzyme (called BMN: barley microspore nuclease) was very stable at pH > 8.0 and temperatures below 50 degrees C. Activity was highest at pH 5.6 and increased almost exponentially with temperature until a breakpoint between activity and stability was reached at 70 degrees C. Although BMN was able to cleave RNA, the enzyme showed a remarkable preference for DNA, especially in the single-stranded form. The best homopolymeric substrates were poly(dA) and poly(A), whereas poly(dC), poly(G) and poly(I) were almost completely uncleaved. When incubated with intact nuclei, BMN caused a nucleosomal DNA ladder of approximately 200 bp. On the basis of DNA laddering, substrate specificity, Mg2+ -dependence and best performance at apoplastic pH, BMN can be referred to as a putative apoptotic nuclease involved in pollen development.

Apoptosis↗
PubMed2001

Improvement of the bone-pin interface strength in osteoporotic bone with use of hydroxyapatite-coated tapered external-fixation pins. A prospective, randomized clinical study of wrist fractures.

BACKGROUND: Achieving adequate fixation strength in osteoporotic bone is a challenge. In this study, we examined the use of hydroxyapatite-coated tapered external-fixation pins for the fixation of wrist fractures in patients with osteoporosis. METHODS: Twenty female patients with osteoporosis and a fracture of the wrist were divided into two paired groups and randomized to receive either standard tapered pins or hydroxyapatite-coated tapered pins. Two pins were inserted in the distal part of the radius, two pins were inserted in the second metacarpal, and an external fixation device was mounted. All fixation devices were removed six weeks after surgery. RESULTS: The mean pin-insertion torque (and standard deviation) was 461 +/- 254 Nmm in the group managed with standard pins and 332 +/- 176 Nmm in the group managed with hydroxyapatite-coated pins (p = 0.01). The mean pin-extraction torque was 191 +/- 155 Nmm in the group managed with standard pins and 600 +/- 214 Nmm in the group managed with hydroxyapatite-coated pins (p < 0.0001, power 95%). The mean extraction torque was lower than the corresponding insertion torque at each pin position in the group managed with standard pins (p < 0.05), whereas the mean extraction torque was higher than the corresponding insertion torque at each pin position in the group managed with hydroxyapatite-coated pins (p = 0.001). Two patients managed with standard pins and no patient managed with hydroxyapatite-coated pins had a pin-track infection. Pain during pin removal did not differ between the two groups. CONCLUSIONS: The present study showed that hydroxyapatite-coated tapered external-fixation pins provided improved fixation in the treatment of wrist fractures in patients with osteoporosis.

Aged↗
PubMed2000

Intercodon dinucleotides affect codon choice in plant genes.

In this work, 710 CDSs corresponding to over 290 000 codons equally distributed between Brassica napus, Arabidopsis thaliana, Lycopersicon esculentum, Nicotiana tabacum, Pisum sativum, Glycine max, Oryza sativa, Triticum aestivum, Hordeum vulgare and Zea mays were considered. For each amino acid, synonymous codon choice was determined in the presence of A, G, C or T as the initial nucleotide of the subsequent triplet; data were statistically analysed under the hypothesis of an independent assortment of codons. In 33.4% of cases, a frequency significantly (P: = 0.01) different from that expected was recorded. This was mainly due to a pervasive intercodon TpA and CpG deficiency. As a general rule, intercodon TpAs and CpGs were preferably replaced by CpAs and TpGs, respectively. In several instances, codon frequencies were also modified to avoid homotetramer and homotrimer formation, to reduce intercodon ApCs downstream (1,2) GG or AG dinucleotides, as well as to increase GpA or ApG intercodons under certain contexts. Since TpA, CpG and homotetra(tri)mer deficiency directly or indirectly accounted for 77% of significant variation in the codon frequency, it can be concluded that codon usage mirrors precise needs at the DNA structure level. Plant species exhibited a phylogenetically-related adaptation to structural constraints. Codon usage flexibility was reflected in strikingly different arrays of optimum codons for probe design.

Base Composition↗
PubMed2000

Synthesis, conformational analysis, and biological activity of C-thioribonucleosides related to tiazofurin.

The syntheses of furanthiofurin [5beta-D-(4'-thioribofuranosyl)furan-3-carboxamide, 1] and thiophenthiofurin [5beta-D-(4'-thioribofuranosyl)thiophene-3-carboxamide, 2], two C-thioribonucleoside analogues of tiazofurin, are described. Direct trifluoroacetic acid-catalyzed C-glycosylation of ethyl furan-3-carboxylate with 1-O-acetyl-2,3,5-tri-O-benzyl-4-thio-D-ribofuranose gave 2- and 5-glycosylated regioisomers, as a mixture of alpha and beta anomers. Ethyl 5-(2,3,5-tri-O-benzyl)-beta-D-(4'-thioribofuranosyl)furan-3-carboxylate (6beta) was debenzylated and then converted into the corresponding amide (furanthiofurin) by reaction with ammonium hydroxide. A similar C-glycosylation of ethyl thiophene-3-carboxylate with 1,2,3,5-tetra-O-acetyl-4-thio-D-ribofuranose catalyzed by stannic chloride afforded an anomeric mixture of 2- and 5-glycosylated regioisomers. Deacetylation of ethyl 5-(2,3,5-tri-O-acetyl)-beta-D-(4'-thioribofuranosyl)thiophene-3-carboxylate (13beta) with methanolic ammonia and treatment of the ethyl ester with ammonium hydroxide gave thiophenthiofurin. The glycosylation site and anomeric configuration were established by (1)H NMR spectroscopy. Thiophenthiofurin was found to be cytotoxic in vitro toward human myelogenous leukemia K562, albeit 39-fold less than thiophenfurin, while furanthiofurin proved to be inactive. K562 cells incubated with thiophenthiofurin resulted in inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) and an increase in IMP pools with a concurrent decrease in GTP levels. From computational studies it was deduced that, among the C-nucleoside analogues of tiazofurin, activity requires an electrophilic sulfur adjacent to the C-glycosidic bond and an energetically favorable conformer around chi = 0 degrees. Among these, the more constrained (least flexible) compounds (tiazofurin and thiophenfurin) are more active than the less constrained thiophenthiofurin. Those compounds which contain a nucleophilic oxygen in place of the thiazole or thiophene (oxazofurin, furanfurin, and furanthiofurin) show the least activity.

Antineoplastic Agents↗
PubMed2000

A new acyclic heterodinucleotide active against human immunodeficiency virus and herpes simplex virus.

The most common therapies against human herpes virus (HSV-1) and human immunodeficiency virus (HIV-1) infectivity are based on the administration of nucleoside analogues. Acyclovir (ACV) is the drug of choice against HSV-1 infection, while the acyclic nucleoside phosphonate analogue PMPA has shown marked anti-HIV activity in a phase I and II clinical studies. As monocyte-derived macrophages are assumed to be important as reservoirs of both HSV-1 and HIV-1 infection, new approaches able to inhibit replication of both viruses in macrophages should be welcome. ACVpPMPA, a new heterodinucleotide consisting of both an antiherpetic and an antiretroviral drug bound by a phosphate bridge, was synthesized and encapsulated into autologous erythrocytes modified to increase their phagocytosis by human macrophages. ACVpPMPA-loaded erythrocytes provided an effective in vitro protection against both HSV-1 and HIV-1 replication in human macrophages.

Animals↗