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Biomedical subjects

E D'Ambrosio

Publications and source records attributed to E D'Ambrosio.

At least 19 recordsLinked to original sources
PubMed2005

Upper limits on a stochastic background of gravitational waves.

The Laser Interferometer Gravitational-Wave Observatory has performed a third science run with much improved sensitivities of all three interferometers. We present an analysis of approximately 200 hours of data acquired during this run, used to search for a stochastic background of gravitational radiation. We place upper bounds on the energy density stored as gravitational radiation for three different spectral power laws. For the flat spectrum, our limit of omega0 < 8.4 x 10(-4) in the 69-156 Hz band is approximately 10(5) times lower than the previous result in this frequency range.

Journal Article↗
PubMed2005

Limits on gravitational-wave emission from selected pulsars using LIGO data.

We place direct upper limits on the amplitude of gravitational waves from 28 isolated radio pulsars by a coherent multidetector analysis of the data collected during the second science run of the LIGO interferometric detectors. These are the first direct upper limits for 26 of the 28 pulsars. We use coordinated radio observations for the first time to build radio-guided phase templates for the expected gravitational-wave signals. The unprecedented sensitivity of the detectors allows us to set strain upper limits as low as a few times 10(-24). These strain limits translate into limits on the equatorial ellipticities of the pulsars, which are smaller than 10(-5) for the four closest pulsars.

Journal Article↗
PubMed2003

Inhibitors of DNA methylation and histone deacetylation activate cytomegalovirus promoter-controlled reporter gene expression in human glioblastoma cell line U87.

The expression of many cellular genes is modulated by DNA methylation and histone acetylation. These processes can influence malignant cell transformation and are also responsible for the silencing of DNA constructs introduced into mammalian cells for therapeutic or research purposes. As a better understanding of these biological processes may contribute to the development of novel cancer treatments and to study the complex mechanisms regulating gene silencing, we established a cellular system suitable to dissect the mechanisms regulating DNA methylation and histone acetylation. For this purpose, we stably transfected the neuroblastoma cell line U87 with a cytomegalovirus promoter-driven reporter gene construct whose expression was analyzed following treatment with the DNA methylation inhibitor 5'-aza-2'-deoxycytidine or histone deacetylation inhibitor trichostatin A. Both substances reactivated the silenced cytomegalovirus promoter, but with different reaction kinetics. Furthermore, whereas the kinetics of reactivation by trichostatin A did not substantially change over the time range considered (5 days), reactivation induced by 5'-aza-2'-deoxycytidine showed profound differences between day 1 and longer time points. We showed that this effect is related to the down-regulation of DNA replication by 5'-aza-2'-deoxycytidine. Finally, we have shown that the simultaneous administration of trichostatin A and 5'-aza-2'-deoxycytidine results in reactivation of the CMV promoter according to a cooperative, not synergistic or additive, mechanism. In conclusion, our cellular system should represent a powerful tool to investigate the complex mechanisms regulating gene silencing and to identify new anticancer drugs.

Animals↗
PubMed2001

The length of telomeric G-rich strand 3'-overhang measured by oligonucleotide ligation assay.

A typical G-rich telomeric DNA strand, which runs 5'-->3' toward the chromosome ends, protrudes by several nucleotides in lower eukaryotes. In human chromosomes long G-rich 3'-overhangs have been found. Apart from the standard G-rich tail, several non-canonical terminal structures have been proposed. However, the mechanism of long-tail formation, the presence and the role of these structures in telomere maintenance or shortening are not completely understood. In a search for a simple method to accurately measure the 3'-overhang we have established a protocol based on the ligation of telomeric oligonucleotide hybridized to non-denatured DNA under stringent conditions (oligonucleotide ligation assay with telomeric repeat oligonucleotide). This method enabled us to detect a large proportion of G-rich single-stranded telomeric DNA that was as short as 24 nt. Nevertheless, we showed G-tails longer than 400 nt. In all tested cells the lengths ranging from 108 to 270 nt represented only 37% of the whole molecule population, while 56-62% were <90 nt. Our protocol provides a simple and sensitive method for measuring the length of naturally occurring unpaired repeated DNA.

Blotting, Southern↗
PubMed2001

Absence of association between a variant of the mast cell chymase gene and atopic dermatitis in an Italian population.

Atopic dermatitis (AD) is a chronic dermatitis which belongs to the group of atopy-related diseases together with asthma and rhinitis. IgE and mast cell chymase (MCC) play a key role in atopic or allergic inflammation of the skin. An association between AD and a genetic variant of the MCC has been reported in a Japanese population, but failure of confirmation has rendered this association questionable. We have tested for genetic association to an MCC variant in relation to AD in an Italian population. No significant association was found between AD and MCC genotypes. These data suggest that BstXI MCC polymorphism may not be involved in AD.

Adolescent↗
PubMed2001

Evidence for telomerase involvement in the angiogenesis of astrocytic tumors: expression of human telomerase reverse transcriptase messenger RNA by vascular endothelial cells.

OBJECT: Evidence from recent in vitro studies indicates that reactivation of telomerase, the enzyme that synthesizes the telomere ends of chromosomes, is a crucial event in the unlimited clonal expansion of endothelial cells that precedes the neoplastic conversion of these cells. It is known that high-grade gliomas express telomerase and that, in these neoplasms, proliferating endothelial cells may undergo transformational changes with development of sarcomatous components within the primitive tumor. To assess whether telomerase is involved in the endothelial cell proliferation that characterizes brain tumor angiogenesis, the authors investigated at the single-cell level the expression of messenger (m)RNA for the human telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) by vascular cells of astrocytic tumors. METHODS: The in situ hybridization (ISH) method was performed by processing histological sections with specific riboprobes for hTERT and for c-myc, an oncogene that is known to upregulate hTERT. Results of the ISH studies were compared with proliferative activity, as estimated by Ki-67 immunostaining. The expression of hTERT mRNA by vascular endothelial cells was related to the histological grade of the tumor because it was detected in five (29%) of 17 low-grade astrocytomas, nine (56%) of 16 anaplastic astrocytomas, and 19 (100%) of 19 glioblastomas multiforme (GBMs). Expression of c-myc mRNA was strictly correlated with that of hTERT mRNA. In low-grade astrocytomas and anaplastic astrocytomas, a dissociation was noted between hTERT mRNA expression and the proliferation rate of endothelial cells. Conversely, GBMs displayed a significant correlation between the level of hTERT mRNA expression and endothelial cell proliferation. Data from an in vitro assay in which human umbilical vein endothelial cells were stimulated to proliferate by adding vascular endothelial growth factor and an ISH study of newly formed vessels surrounding brain infarcts confirmed that expression of hTERT mRNA does not merely reflect the proliferative status of endothelial cells but represents a specific feature of brain tumor neovascularization. CONCLUSIONS: The results of this study are consistent with a role of telomerase in the angiogenesis of astrocytic tumors. Expression of hTERT mRNA by tumor vascular cells is an early event during the progression of astrocytic tumors, which precedes endothelial cell proliferation and may represent a first sign of dedifferentiation. Other than elucidating the mechanisms of tumor angiogenesis, these results encourage research on antitelomerase drugs for the treatment of malignant gliomas.

Adult↗
PubMed2000

In situ detection of telomerase catalytic subunit mRNA in glioblastoma multiforme.

Activation of telomerase may allow unlimited cell proliferation and immortalization. One of the telomerase protein subunits has a reverse transcriptase (hTERT) activity that is essential for telomerase function and regulation. In human gliomas, telomerase is frequently associated with malignant tumor progression. In our study, we investigated the expression of hTERT at the cellular level in 34 primary de novo glioblastoma multiforme (GBM) by in situ hybridization (ISH). The expression of hTERT in tumor tissue was also assessed by RT-PCR. In addition, telomerase activity measured by telomeric repeat amplification protocol (TRAP) and telomere length polymorphism assayed by telomere restriction fragment (TRF) Southern blot were investigated. We found that all GBM, including those with negative TRAP reaction, contained abundant amounts of cytoplasmic hTERT mRNA. Interestingly, the ISH analysis revealed that the hTERT mRNA was homogeneously expressed by the whole tumor cell population in about 60% of the GBM. In the remaining cases, hTERT was absent in subsets of tumor cells. TRF analysis, which shows that both TRAP-positive and TRAP-negative de novo GBM have elongated telomeres, further supports that telomerase activity is present in all de novo GBM. Correlations with tumor size and extent of necrosis suggest that hTERT reactivation is an early event in GBM development and that telomerase activity may be lost in subpopulations of neoplastic cells during tumor progression. Finally, ISH analysis of hTERT mRNA seems to provide a prognostic parameter for primary de novo GBM.

Adult↗
PubMed2000

Haemochromatosis gene mutations and risk of coronary artery disease.

The identification of mutations in the haemochromatosis gene (HFE) (C282Y and H63D) provides the unique opportunity to test whether genetic variants that are associated with tissue iron accumulation may influence the risk of coronary atherosclerosis. To this aim the prevalence of C282Y and H63D mutations was determined in 174 patients with angiographically documented CAD (>50% stenosis) and history of MI, 187 healthy free-living individuals and 142 blood donors. C282Y and H63D mutations were not found to be more frequent in coronary patients as compared to controls. Moreover, these HFE variants were unrelated to the severity of coronary atherosclerosis. These findings did not provide evidence of an association between HFE mutations and the presence of coronary atherosclerosis or its major ischaemic complications, thus indicating that HFE mutations are poor genetic markers of coronary risk.

Adult↗
PubMed1999

Reduction of IFN-gamma and IL-2 production by peripheral lymphocytes of HIV-exposed seronegative subjects.

OBJECTIVES: In order to evaluate the role played by cytokine profile as a co-factor involved in the resistance to HIV infection in couples serodiscordant for HIV, we studied HIV-seronegative subjects with multiple unprotected sexual exposures. DESIGN AND METHODS: Twenty-one HIV-exposed seronegative subjects (HEPS), their 21 HIV-seropositive partners and 10 HIV-seronegative unexposed individuals were studied for T helper (Th) types 1-2 cell pattern and CCR5 receptor. RESULTS: Twelve out of 21 HIV-seropositive partners of HEPS showed a CD4 cell count below 200 lymphocytes/microl. HIV strains were isolated from peripheral blood mononuclear cells (PBMC) in 17 patients (81%): seven subjects with syncytium-inducing strains and 10 with non-syncytium-inducing isolates. Low Th1 cytokine production and high levels of IL-4 were detected in HIV-seropositive subjects. A significant reduction of IL-2 and IFN-gamma expression in the CD4 and CD8 cells of HEPS was found compared with HIV-seronegative unexposed individuals. Similar levels of low IL-4 were present in both HEPS and controls. The partial deletion of a single allele (wild type/delta32) of CCR5 was found in only one HEPS. CONCLUSION: The downregulated Th1 profile we observed in HEPS could be related to a cellular anergy state with a protective role in the transmission rate of HIV. Low levels of IL-2 and IFN-gamma could be involved in a low-grade activation state of CD4 lymphocytes. A decrease of IFN-gamma levels could render macrophage cells incapable of antigen presentation, thus resulting in a reduction of the cell-to-cell spread of infection.

CD4-Positive T-Lymphocytes↗
PubMed1999

Induction of telomerase activity in v-myc-transformed avian cells.

Telomerase activity is detectable in the majority of tumors or immortalized cell lines, but is repressed in most normal human somatic cells. It is generally assumed that reactivation of telomerase prevents the erosion of chromosome ends which occurs in cycling cells and, hence, hinders cellular replicative senescence. Here, we show that the expression of v-Myc oncoprotein by retroviral infection of telomerase-negative embryonal quail myoblasts and chicken neuroretina cells is sufficient for reactivating telomerase activity, earlier than telomere shortening could occur. Furthermore, the use of a conditional v-Myc-estrogen receptor protein (v-MycER) causes estrogen-dependent expression of detectable levels of telomerase activity in recently infected chick embryo fibroblasts and neuroretina cells. We conclude that the high levels of telomerase activity in v-Myc-expressing avian cells are not the mere consequence of transformation or of a differentiative block, since v-Src tyrosine kinase, which prevents terminal differentiation and promotes cell transformation, fails to induce telomerase activity.

Animals↗
PubMed1999

Telomerase expression in intracranial tumours: prognostic potential for malignant gliomas and meningiomas.

AIM: To evaluate the diagnostic value of telomerase expression in intracranial tumours. METHODS: 98 surgical specimens from different neoplasms were analysed by the telomeric repeat amplification protocol (TRAP) and the presence of telomerase compared with the histological diagnosis and the proliferation index. RESULTS: A high degree of positivity for telomerase activity was found in glioblastomas and atypical/anaplastic meningiomas. Telomerase activity was poorly detected in anaplastic astrocytomas. CONCLUSIONS: The TRAP assay seems to be a valuable index for identifying meningeal tumours with aggressive behaviour.

Adolescent↗
PubMed1998

Increased sensitivity and reproducibility of TRAP assay by avoiding direct primers interaction.

Telomeric Repeat Amplification Protocol (TRAP) is a sensitive procedure to measure telomerase activity in small samples of cell or tissue extracts. Due to the strict correlation between high levels of telomerase activity and neoplastic transformation, TRAP assay could provide an important diagnostic marker of malignancy. Although the original TRAP assay is very sensitive and some improvements have been described, occasional artifacts still persist in the modified procedures. Here we describe how changes in the sequence of the primer used for the amplification step enhance the reproducibility and sensitivity in the TRAP assay.

Animals↗
PubMed1997

A novel double nucleotide substitution in the HMG box of the SRY gene associated with Swyer syndrome.

We describe a novel double nucleotide substitution in the SRY gene of a 46,XY female with gonadal dysgenesis or Swyer syndrome. The SRY sequence was analysed by both the single-strand conformational polymorphism assay and direct DNA sequencing of products from the polymerase chain reaction. A double nucleotide substitution was identified at codon 18 of the conserved HMG box motif, causing an arginine to asparagine amino acid substitution. The altered residue is situated in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. Since the mutation abolishes one HhaI recognition site, the results were confirmed by HhaI restriction mapping. No other mutations were found in the remaining regions of the gene. The corresponding DNA region from the patient's brother was analysed and found to be normal. We conclude that the SRY mutation in the reported XY female occurred de novo and is associated with sex reversal.

Adult↗
PubMed1996

Simultaneous and different binding mechanisms of 4',6-diamidino-2-phenylindole to DNA hexamer (d(CGATCG))2. A 1H NMR study.

The solution structure of the complex between 4', 6-diamidino-2-phenylindole (DAPI) and DNA oligomer (d(CGATCG))2 at a 2:1 drug/duplex ratio has been characterized by combined use of proton one- and two-dimensional NMR spectroscopy, molecular mechanics, and molecular dynamics computations. Intermolecular nuclear Overhauser effects (NOEs), DNA structure perturbations, and resonance shifts induced by binding provide evidence that DAPI interacts with DNA hexamer by two different binding mechanisms, in fast exchange on the NMR time scale, without any significant distortion of the B-type conformation of DNA hexamer. The results indicate that the ligand binds into the minor groove of the central 5'-ATC-3' region of the hexamer and on the outside of the oligomer by a pi,pi-stacking interaction with the terminal C1:G6 base pairs. A model for both binding mechanisms that accounts for all experimental data was generated by molecular mechanics and dynamics calculations based on experimental NOEs. In the minor groove binding, N2 amino group of G2 precludes a deep insertion of phenyl ring of DAPI into the groove. Position and orientation of the drug in the external stacking interaction resemble those suggested for intercalation of DAPI between C:G base pairs.

Base Composition↗
PubMed1996

Differential expression of heat shock protein (HSP70) mRNAs in rat cells.

Heat shock treatment in PC12 cells induces the synthesis of two HSP70 transcripts of 2.55 and 3.05 kb in size, while only a 70-kDa protein is shown by SDS-PAGE. Using reverse transcription-polymerase chain reaction strategy, two mRNA species were amplified from heat-shocked PC12 cells and sequenced. Both cDNAs revealed the same open reading frame encoding a single predicted 641-amino-acid polypeptide of about 70 kDa. The two 3' untranslated regions of the two mRNAs were completely different in both length and composition. Expression of the mRNA of the two corresponding genes and that of another rat HSP70 family member was investigated in PC12, Rat1 cells, and lung fibroblasts. Northern blot analysis revealed that the 2.55- and the 3.05-kb related gene transcripts are differentially expressed in the rat cell lines tested, while the third member of the subfamily is not induced. The single-copy nature of the three genes is also confirmed by Southern blot analysis.

Animals↗
PubMed1996

Exclusion of Treacher Collins Franceschetti syndrome in a subject with tetralogy of Fallot and cryptorchidism.

Genotyping with flanking DNA markers was used to ascertain Treacher Collins Franceschetti syndrome (TCOF1) in a subject affected by tetralogy of Fallot and cryptorchidism. The proband's family consisted of a father and sister who were affected by the disease, and a healthy mother. Since cardiac malformation and cryptorchidism have been associated with the TCOF1 syndrome, the proband was suspected to be a carrier of the mutated gene. Microsatellite markers D5S527, SPARC and D5S519, which previously mapped the TCOF1 gene within a 2.1-cM interval on chromosome 5 (5q32-33.1), were used to follow the transmission of the TCOF1 mutated locus. Flanking markers D5S519 and D5S527 were informative and enabled us to exclude inheritance of a TCOF1 mutation to the proband, while showing that cardiac malformation and crytorchidism were unrelated in this patient.

Abnormalities, Multiple↗