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Biomedical subjects

A De Luca

Publications and source records attributed to A De Luca.

At least 109 records · Page 6Linked to original sources
PubMed1999

Interaction between the pRb2/p130 C-terminal domain and the N-terminal portion of cyclin D3.

An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using the yeast two-hybrid system. Further analysis restricted the epitope responsible for the binding within the 74 N-terminal amino acids of cyclin D3, independent of the LXCXE amino acid motif present in the D-type cyclin N-terminal region. In a coprecipitation assay in T98G cells, a human glioblastoma cell line, the C-terminal domain of pRb2/p130 was able to interact solely with cyclin D3, while the corresponding portion of pRb interacted with either cyclin D3 or cyclin D1. In T98G cells, endogenous cyclin D3-associated kinase activity showed a clear predisposition to phosphorylate preferentially the C-terminal domain of pRb2/p130, rather than that of pRb. This propensity was also confirmed in LAN-5 human neuroblastoma cells, where phosphorylation of the pRb2/p130 C-terminal domain and expression of cyclin D3 also decreased remarkably in the late neural differentiation stages.

Adenovirus E1A Proteins↗
PubMed1999

Does HHV-8 have a protective role on the development of HIV encephalopathy? Italian HIV-Seroconversion Study.

OBJECTIVE: To evaluate risk factors for HIV encephalopathy and whether Kaposi's sarcoma (KS) and coinfection with human herpesvirus 8 (HHV-8) protect against this disease in a cohort of HIV seroconverters. METHODS: Individuals with known dates of HIV seroconversion belonging to different HIV exposure categories (intravenous drug users, homosexual men, heterosexual contacts) were recruited by 17 clinical centers throughout Italy. Antibodies to HHV-8 lytic antigens were detected in a subgroup of participants using an immunofluorescence assay. Risk factors for HIV encephalopathy were evaluated using Cox proportional models. The association between KS or HHV-8 infection and HIV encephalopathy was evaluated using standard statistical techniques. RESULTS: During the study period, 485 of the 1,520 participants developed acquired immunodeficiency syndrome, 38 of whom developed HIV encephalopathy. HHV-8 serologic status was determined for 390 participants. Male gender, injecting drug use, and low CD4 T-cell count were associated with HIV encephalopathy; none of the 63 participants with KS developed this disease. The risk of HIV encephalopathy did not differ significantly by HHV-8 serologic status. CONCLUSIONS: HIV encephalopathy was found to be associated with male gender and intravenous drug use. The risk increased at lower CD4 T-cell counts. Although HIV encephalopathy occurred less frequently in patients with KS, no association with HHV-8 infection was found.

AIDS Dementia Complex↗
PubMed1999

Detection of dendritic cells in the non-obese diabetic (NOD) mouse islet pancreas infiltrate is correlated with Th2-cytokine production.

We investigate the role played by dendritic cells (DCs) in the non-obese diabetic (NOD) mouse pancreas. The early peri-islet, nondestructive infiltration phase, and intra-islet, destructive infiltration phase, which immediately precedes overt diabetes, are studied. Results show that infiltrating cells are Ia-b, ICAM-1, and, mainly, MIDC-8 immunoreactive (ir). These data from silica-treated animals and ultrastructural observations strongly support the hypothesis that DCs are both Ia-b-ir and ICAM-1-ir and that they exert a pivotal role during the period of early infiltration. This is a novel finding for NOD mice and increases the interest for this protective cell type during the rather complex islet infiltration process. Moreover, the cytokine profile demonstrates that Th2 protective cytokines are specific for peri-islet infiltrate. Disappearance of DCs from the infiltrate is concomitant with both the formation of intra-islet infiltration and the increase in proinflammatory Th1 cytokine levels. This further supports the hypothesis that DCs may exert a protective role against diabetes development.

Age Factors↗
PubMed1999

Aging-associated down-regulation of ClC-1 expression in skeletal muscle: phenotypic-independent relation to the decrease of chloride conductance.

In order to clarify the mechanism underlying the reduction of resting membrane chloride conductance (gCl) during aging, the levels of mRNA encoding the principal skeletal muscle chloride channel, ClC-1, were measured. Total RNA samples isolated from tibialis anterior muscles of aged (24-29 months old) and adult (3-4 months old) rats were examined for ClC-1 expression using Northern blot analysis, and macroscopic gCl was recorded from extensor digitorum longus muscle fibers from each adult and aged rat in vitro using a two intracellular microelectrode technique. Although interindividual variability was observed, aged rats exhibited a parallel reduction of both gCl and ClC-1 mRNA expression as compared to adult rats. A linear correlation exists between individual values of ClC-1 mRNA and gCl. These results provide evidence that ClC-1 is the main determinant of sarcolemmal gCl and demonstrate that the decrease of gCl observed during aging is associated with a down-regulation of ClC-1 expression in muscle.

Aging↗
PubMed1999

EGF-related peptides are involved in the proliferation and survival of MDA-MB-468 human breast carcinoma cells.

A majority of human breast carcinomas co-express the epidermal growth factor (EGF)-like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor alpha (TGF-alpha). MDA-MB-468 breast carcinoma cells express CR, AR and TGFalpha, while SK-BR-3 cells express CR and TGF-alpha. Anti-sense phosphorothioate oligodeoxynucleotides (AS S-oligos) directed against either CR or TGF-alpha inhibit the proliferation of both cell lines. A 40-50% growth inhibition was observed at a 2-microM concentration of each AS S-oligo. Treatment with the AR AS S-oligo also resulted in a significant inhibition of MDA-MB-468 anchorage dependent growth (ADG). No significant growth inhibition was observed when MDA-MB-468 or SK-BR-3 cells were treated with a mis-sense S-oligo. The AS S-oligos inhibited the expression of AR, CR or TGF-alpha proteins and mRNAs, as assessed by immuno-cytochemistry and semi-quantitative RT-PCR. An additive growth-inhibitory effect was observed when MDA-MB-468 cells were treated with a combination of EGF-related AS S-oligos. Indeed, treatment of MDA-MB-468 cells with a combination of AR, CR and TGF-alpha AS S-oligos resulted in about 70% growth inhibition at a concentration of 0.7 microM each. Finally, treatment of MDA-MB-468 cells with a combination either of the 3 AS S-oligos or of an EGF receptor-blocking antibody (MAb 225) and either CR, AR or TGFalpha AS S-oligos resulted in a significant increase in DNA fragmentation. Our data suggest that the EGF-related peptides are involved in the proliferation and survival of breast carcinoma cells.

Amphiregulin↗
PubMed1999

Effects of HMG-CoA reductase inhibitors on excitation-contraction coupling of rat skeletal muscle.

3-Hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors currently used as cholesterol-lowering drugs produce side effects in patients, one of which is myopathy. In the present study we compared the effect of a 3-month chronic treatment with two different compounds, simvastatin and pravastatin, on the excitation-contraction coupling of rat skeletal muscle fibers, the mechanism which links membrane depolarization to the movements of cytosolic Ca2+ from intracellular stores. The voltage threshold for mechanical activation of extensor digitorum longus muscle fibers in response to depolarizing pulses of various durations was studied in vitro by the two intracellular microelectrode method in 'point' voltage clamp mode. Simvastatin (5-50 mg/kg) modified the mechanical threshold of striated fibers in a dose-dependent manner. The muscle fibers of rats treated with 10 mg/kg and 50 mg/kg of simvastatin needed significantly less depolarization to contract than did untreated fibers at each pulse duration, suggesting that levels of cytosolic Ca2+ were higher. Consequently, the rheobase voltage for fiber contraction was significantly shifted toward more negative potentials with respect to controls by 2.4 mV and 7.1 mV in the 10 mg/kg and 50 mg/kg simvastatin-treated animals, respectively. Pravastatin treatment at 100 mg/kg did not produce any alteration of excitation-contraction coupling since the rheobase voltage was similar to that of controls. The different physicochemical properties of the two drugs may underlie the different effect observed because lipophilic agents, such as simvastatin, have been shown to affect sterol biosynthesis in many tissues, whereas the hydrophilic pravastatin is hepato-selective.

Animals↗
PubMed1999

Independent prognostic role of p16 expression in lung cancer.

OBJECTIVES: The cyclin-dependent kinase p16 (also known as Ink4A, Mts1, Cdkn2, and Cdkn4i) has been proposed as a tumor suppressor gene mapped on chromosome segment 9p21. This study evaluated p16 protein expression in 135 lung cancer specimens and investigated potential genetic alterations occurring in this gene. RESULTS: We found altered p16 immunohistochemical expression to be a frequent event in lung cancer and to be independent of either the histologic type or any other clinical-pathologic feature. Western blot analyses performed on about one third of the specimens correlated highly with these results. In addition, we found p16 immunohistochemical expression to be a favorable prognostic factor in lung cancer in that its reduction or loss correlated with a worse outcome for the patients. Polymerase chain reaction amplification and direct sequencing of p16 exons 1 and 2 revealed no mutations, indicating that p16-altered expression in lung cancer is not necessarily linked to mutational events of these genes. CONCLUSIONS: We conclude that p16-altered expression is both an independent and frequent event in lung cancer and may have an important role in tumorigenesis and in malignant progression of a significant proportion of these cancers. However, the actual incidence and relevance of p16 mutations in this neoplasm continues to be debated, and its analysis seems inconclusive. Our results suggest a prognostic role for the immunodetection of this protein on formalin-fixed and paraffin-embedded specimens. They further suggest its routine use in the evaluation of the frequently unpredictable behavior of lung cancer.

Adenocarcinoma↗
PubMed1999

The role of pRb2/p130 protein in diagnosing lung carcinoma on fine needle aspiration biopsies.

The retinoblastoma gene family is composed of three members: the retinoblastoma gene, one of the most studied tumor suppressor genes, and two related genes: p107 and pRb2/p130. These proteins are also known as the pocket proteins due to a unique structural and functional domain composed of subdomains A and B separated by a spacer region that is highly conserved among each of the proteins. These proteins exhibit unique growth suppressive properties that are cell type specific, suggesting that although the pocket proteins may complement each other, they are not fully functionally redundant. With the development of antibodies recognizing these three proteins it is now possible to detect expression in formalin-embedded specimens. Recent studies on 235 lung cancers, using immunohistochemical techniques, suggested an independent role for Rb2/p130 in the development and/or progression of human lung carcinoma. We found a statistically significant inverse relationship between the histological grading (degree of malignant potential) and the expression of pRb/p105, p107 and pRb2/p130 in squamous cell carcinomas, meaning that an increase in grading resulted in a significant decrease in protein expression. This phenomenon was particularly evident for pRb2/p130 (p < .0001) which had the highest percentage of undetectable levels in all the specimens examined and the tightest inverse correlation (p value) with both the histological grading and PCNA expression in the most aggressive tumor types, suggesting an important role for pRb2/p130 in the pathogenesis and progression of certain lung cancers. We further explored the expression of pRb2/p130 protein in routine archival FNAB cytological material from 30 Patients with lung cancer using immunocytochemical techniques, comparing protein expression with tumor type. Two pathologists evaluated the staining pattern and scored the percentage of positive cells. Of the 30 neoplasms, 27 displayed a positive staining for pRb2/p130. In particular, we detected pRb2/p130 in 9 (100%) squamous carcinomas, 11 (84%) adenocarcinomas, 5 (100%) BAC, and 2 (66%) SCC. The percentage of positive nuclei varied in different tumors with the highest expression level in adenocarcinomas. Immunocytochemistry represents a sensitive method for detection of pRb2/p130 expression in cytological or archival specimens, and the level of detection seems to be comparable to paraffin sections. Therefore, this methodology could be used in the preoperative evaluation of routine cytological specimens in order to improve the diagnostic and prognostic evaluation of lung cancer patients.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed1999

Higher content of insulin-like growth factor-I in dystrophic mdx mouse: potential role in the spontaneous regeneration through an electrophysiological investigation of muscle function.

Insulin-like growth factor-I (IGF-I) is known to promote proliferation and differentiation of muscle cells during growth and regeneration. Both these conditions are characterized by acquisition of specialized muscle functions, such as a large macroscopic chloride conductance (GCl), a parameter that is a target of growth hormone (GH)/IGF-I axis action on skeletal muscle. The present study has been aimed at evaluating the role of IGF-I in the spontaneous regeneration occurring in hind limb muscle of dystrophic mdx mouse. IGF-I levels have been measured in hind limb muscles, plasma and liver of mdx and control mice of 8-10 weeks and 5 months of age by radioimmunoassay. In parallel the biophysical and pharmacological properties of muscle chloride channels of extensor digitorum longus (EDL) muscle fibers of mice belonging to the same age-group have been measured electrophysiologically in vitro. At 8-10 weeks of age, significantly greater amounts of IGF-I were found in plasma and hind limb muscles of mdx mice with respect to controls. Such a difference was only just detectable and no longer statistically significant at 5 months of age. No differences were found in hepatic IGF-I levels at either age. The EDL muscle fibers of mdx mice at 8-10 weeks of age were characterized by higher GCl values and by a different pharmacological sensitivity to the enantiomers of 2-(p-chlorophenoxy)-propionic acid (CPP), specific chloride channel ligands, with respect to age-matched controls. However, these differences were no longer detected at 5 months of age. Our results suggest a role of IGF-I in the high regenerative potential of muscles from mdx mice and support the hypothesis that the biophysical and pharmacological properties of chloride channels of EDL muscle fibers are sensitive indices of the action of regeneration-promoting factors on muscle function.

2-Methyl-4-chlorophenoxyacetic Acid↗
PubMed1999

Effect of mexiletine on sea anemone toxin-induced non-inactivating sodium channels of rat skeletal muscle: a model of sodium channel myotonia.

The sea anemone toxin ATX II impairs skeletal muscle sodium channel inactivation, mimicking the persistent inward current observed in patients suffering from sodium channel myotonia. Mexiletine has beneficial effects on myotonia. To verify the efficiency of the drug on persistent inward current, we investigated the effect of 50 microM R(-)-mexiletine on sodium channels in cell-attached patches of rat skeletal muscle fibres, in the absence or presence of 2 microM ATX II. With the toxin, a proportion of channels displayed remarkable abnormal activity lasting the entire depolarisation, which resulted in a persistent inward current that represented up to 2.0% of the peak current. Mexiletine reduced by 75% the peak current elicited by depolarisation from -100 to -20 mV. This was due to the reduction by 60% of the maximal available peak current Imax and to the negative shift by -7 mV of steady-state inactivation. Mexiletine also greatly decreased the late current, but the effect was limited to 60% of reduction, comparable to that on Imax. Therefore mexiletine was able to block the ATX II-modified sodium channels, inhibiting the myotonia-producing persistent inward current.

Animals↗
PubMed1999

Increased hindrance on the chiral carbon atom of mexiletine enhances the block of rat skeletal muscle Na+ channels in a model of myotonia induced by ATX.

1 The antiarrhythmic drug mexiletine (Mex) is also used against myotonia. Searching for a more efficient drug, a new compound (Me5) was synthesized substituting the methyl group on the chiral carbon atom of Mex by an isopropyl group. Effects of Me5 on Na+ channels were compared to those of Mex in rat skeletal muscle fibres using the cell-attached patch clamp method. 2 Me5 (10 microM) reduced the maximal sodium current (INa) by 29.7+/-4.4 % (n=6) at a frequency of stimulation of 0.3 Hz and 65.7+/-4.4 % (n=6) at 1 Hz. At same concentration (10 microM), Mex was incapable of producing any effect (n=3). Me5 also shifted the steady-state inactivation curves by -7. 9+/-0.9 mV (n=6) at 0.3 Hz and -12.2+/-1.0 mV (n=6) at 1 Hz. 3 In the presence of sea anemone toxin II (ATX; 5 microM), INa decayed more slowly and no longer to zero, providing a model of sodium channel myotonia. The effects of Me5 on peak INa were similar whatever ATX was present or not. Interestingly, Me5 did not modify the INa decay time constant nor the steady-state INa to peak INa ratio. 4 Analysis of ATX-induced late Na+ channel activity shows that Me5 did not affect mean open times and single-channel conductance, thus excluding open channel block property. 5 These results indicate that increasing hindrance on the chiral atom of Mex increases drug potency on wild-type and ATX-induced noninactivating INa and that Me5 might improve the prophylaxis of myotonia.

Animals↗
PubMed1999

Nitrergic and purinergic mechanisms and their interactions for relaxation of the rat internal anal sphincter.

1. The NANC neuronal mechanisms for relaxations of the rat internal anal sphincter in response to electrical field stimulation (EFS) were studied in isolated preparations in the presence of atropine (1 microM), propranolol (3 microM) and phentolamine (3 microM). 2. EFS-induced relaxations were abolished by tetrodotoxin (1 microM) and reduced to 64% of control by the guanylate cyclase inhibitor ODQ (1 microM), but were not significantly reduced by the nitric oxide synthase inhibitor L-NAME (100 microM) or oxyhaemoglobin (10 microM). However, in the presence of tubocurarine (10 microM) or apamin (0.1 microM), L-NAME or oxyhaemoglobin greatly reduced or abolished EFS-induced relaxations. 3. The EFS-induced relaxations were mimicked by NO (10-100 microM) and by ATP (3-10 mM). The relaxations elicited by these agents were not affected by tetrodotoxin, L-NAME, tubocurarine or apamin. However, ATP-induced relaxations were reduced by the combination of L-NAME with tubocurarine or apamin. 4. Nicotine (10-100 microM) produced concentration-dependent relaxations that were abolished by tubocurarine (10 microM) or hexamethonium (200 microM). After desensitisation to nicotine (100 microM) and in its continued presence, the addition of L-NAME (100 microM) resulted in almost complete abolition of EFS-induced relaxations. 5. It is suggested that tubocurarine, hexamethonium and desensitisation to nicotine have an apamin-like action in the rat internal anal sphincter, the main effect being blockade of a purinergic component of the relaxant transmission process. 6. The findings suggest that both nitrergic and purinergic transmissions are involved in EFS-induced NANC relaxations of the rat internal anal sphincter, and there appears to be a complex interaction between these two pathways of transmission.

Adrenergic Antagonists↗
PubMed1999

Localization of a gene for familial patella aplasia-hypoplasia (PTLAH) to chromosome 17q21-22.

Patella aplasia-hypoplasia (PTLAH) is a rare genetic defect characterized by congenital absence or marked reduction of the patella. PTLAH can occur either as an isolated defect or in association with other malformations, and it characteristically occurs in the nail-patella syndrome and in some chromosome imbalances. We report the first evidence of linkage for isolated PTLAH in an extended Venezuelan family. After exclusion of the candidate chromosome regions where disorders associated with PTLAH have been mapped, a genomewide scan was performed that supported mapping of the disease locus within a region of 12 cM on chromosome 17q22. Two marker loci (D17S787 and D17S1604) typed from this region gave maximum LOD scores >3. Accordingly, multipoint analysis gave a maximum LOD score of 3.39, with a most likely location for the disease gene between D17S787 and D17S1604. Sequencing of the noggin gene, a candidate mapping between these markers, failed to reveal any mutation in affected subjects.

Adolescent↗
PubMed1999

Application of molecular methods for detection and transmission analysis of mycobacterium tuberculosis drug resistance in patients attending a reference hospital in Italy.

A molecular analysis of drug-resistant isolates of Mycobacterium tuberculosis was done in a population with a high prevalence of human immunodeficiency virus infection. Seventy-one consecutive isolates were tested for genotypic resistance to isoniazid, rifampicin, streptomycin, and ethambutol by polymerase chain reaction-single strand conformation polymorphism analysis and automated sequencing of target regions. Phenotypic and genotypic resistance to isoniazid, rifampicin, streptomycin, and ethambutol were detected in 23.4%, 11.2%, 7%, and 5.6% of isolates and in 87%, 88%, 40%, and 100% of resistant isolates, respectively. Specificity was 100% for all target regions. When rpoB, katG, and ahpC mutation analysis were combined, 86% of resistant isolates to any drug were identified. No mutations in inhA were found in isoniazid-resistant isolates. Molecular detection of drug resistance, particularly for isoniazid and rifampicin, may represent a sensitive and very specific technique. The strategy of selecting rpoB, katG, and ahpC to quickly identify most resistant isolates, with a relevant saving of resources, is warranted.

Drug Resistance, Microbial↗