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Biomedical subjects

A Molinari

Publications and source records attributed to A Molinari.

At least 37 records · Page 2Linked to original sources
PubMed2000

Characterization of surface layer proteins from different Clostridium difficile clinical isolates.

In a previous study we suggested that two surface proteins of a Clostridium difficile strain were involved in the formation of a regularly assembled surface layer (S-layer) external to the cell wall. In the present paper six C. difficile strains isolated from cases and healthy carriers were studied. By using freeze-etching and negative staining techniques two superimposed structurally different lattices were detected on the cell surface of the different C. difficile strains. In each strain, the outer S-layer lattice was arranged in a square symmetry and the inner S-layer lattice in hexagonal symmetry. The S-layer proteins from the different strains were isolated and characterized. Each strain showed two distinct S-layer glycoproteins ranging in molecular mass 36-56 kDa. Antigenic cross-reactivity among the S-layer proteins of higher molecular masses extracted from each strain was demonstrated whereas no antigenic relationship was observed among the different S-layer proteins of lower molecular masses. N-terminal sequence analysis showed the presence of common structural motifs conserved among the high S-layer proteins as well as among the low S-layer proteins. These data indicate that the presence of S-layer on C. difficile strains is common and that its glycoprotein subunits show a certain degree of heterogeneity.

Adult↗
PubMed2000

New antineoplastic prenylhydroquinones. Synthesis and evaluation.

Several prenylhydroquinones have been prepared through Diels-Alder condensation, further functionalized or degraded chemically and then evaluated for their cytotoxic activity against some neoplastic cultured cell lines. A number of them have shown IC50 values under the microM level.

Antineoplastic Agents↗
PubMed2000

Detection of P-glycoprotein in the nuclear envelope of multidrug resistant cells.

P-glycoprotein is a plasma membrane efflux pump which is responsible for multidrug resistance of many cancer cell lines. A number of studies have demonstrated the presence of P-glycoprotein molecules, besides on the plasma membrane, also in intracellular sites, such as the Golgi apparatus and the nucleus. In this study, the presence and function of P-glycoprotein in the nuclear membranes of human breast cancer cells (MCF-7 WT) and their multidrug resistant variants (MCF-7 DX) were investigated. Electron and confocal microscopy immunolabelling experiments demonstrated the presence of P-glycoprotein molecules in the nuclear membranes of MCF-7 DX cells. Moreover, the labelling pattern was strongly dependent on pH values of the incubation buffer. At physiological pH (7.2), a strong labelling was detected in the cytoplasm and the nuclear matrix in both sensitive and resistant MCF-7 cells. By raising the pH to 8.0, the P-glycoprotein molecules were easily detected in the cytoplasm (transport vesicles and Golgi apparatus), plasma and nuclear membranes exclusively in MCF-7 DX cells. Furthermore, drug uptake and efflux studies, performed by flow cytometry on isolated nuclei in the presence of the P-glycoprotein inhibitor cyclosporin A, suggested the presence of a functional P-glycoprotein in the nuclear membrane, but not in the nuclear matrix, of drug resistant cells. Therefore, P-glycoprotein in the nuclear envelope seems to represent a further defense mechanism developed by resistant cells against antineoplastic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Molecular determinants of the physicochemical properties of a critical prion protein region comprising residues 106-126.

Prion diseases are marked by the cerebral accumulation of conformationally modified forms of the cellular prion protein (PrP(C)), known as PrP(res). The region comprising the residues 106-126 of human PrP seems to have a key role in this conformational conversion, because a synthetic peptide homologous with this sequence (PrP106-126) adopts different secondary structures in different environments. To investigate the molecular determinants of the physicochemical characteristics of PrP106-126, we synthesized a series of analogues including PrP106-126 H(D), PrP106-126 A and PrP106-126 K, with l-His-->d-His, His-->Ala and His-->Lys substitutions respectively at position 111, PrP106-126 NH(2) with amidation of the C-terminus, PrP106-126 V with an Ala-->Val substition at position 117, and PrP106-126 VNH(2) with an Ala-->Val substitution at position 117 and amidation of the C-terminus. The analysis of the secondary structure and aggregation properties of PrP106-126 and its analogues showed the following. (1) His(111) is central to the conformational changes of PrP peptides. (2) Amidation of the C-terminal Gly(126) yields a predominantly random coil structure, abolishes the molecular polymorphism and decreases the propensity of PrP106-126 to generate amyloid fibrils. (3) PrP106-126 V, carrying an Ala-->Val substitution at position 117, does not demonstrate a fibrillogenic ability superior to that of PrP106-126. However, the presence of Val at position 117 increases the aggregation properties of the amidated peptide. (4) Amyloid fibrils are not required for neurotoxicity because the effects of PrP106-126 NH(2) on primary neuronal cultures were similar to those of the wild-type sequence. Conversely, astroglial proliferation is related to the presence of amyloid fibrils, suggesting that astrogliosis in prion encephalopathies without amyloid deposits is a mediated effect rather than a direct effect of disease-specific PrP isoforms.

Amides↗
PubMed1999

Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts.

Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.

3T3 Cells↗
PubMed1999

Photochemically driven models of oxygenases based on the use of iron porphyrins.

Photoexcited iron porphyrins can be used to mimic the catalytic activity of cytochrome P-450 oxygenases both in the reduction of halogenated alkanes and in the oxidation of hydrocarbons by O2 itself at room temperature and atmospheric pressure. The results reported indicate that there is a similarity and a complementarity of photonic activation with other more conventional methods of activation of model systems of oxygenases. In fact, by irradiating at suitable wavelengths, it is possible to induce redox reactions which mimic those of natural oxygenases, avoiding the difficulties caused by parallel processes which could be expected when chemical reagents are used. These processes occur with good turnover values of the photocatalyst and in mild temperature and pressure conditions. By controlling the reaction environment, it is possible to address the selectivity of the process. In this regard, the very recent results obtained after heterogenization of the photocatalysts inside membranes of Nafion are particularly promising for the development of new biomimetic photocatalysts in heterogeneous or organized systems.

Animals↗
PubMed1999

Brain concentrations of kynurenic acid after a systemic neuroprotective dose in the gerbil model of global ischemia.

1. Kynurenic acid (KYNA) is a kynurenine metabolite and a broad spectrum excitatory amino acid antagonist that has been shown to be neuroprotective in models of cerebral ischemia, when administered exogenously. However, the actual concentration required in the CNS to evoke significant neuroprotection has never been assessed. 2. The purpose of this study was to address this question in the gerbil model of forebrain ischemia. KYNA (400-1600 mg/kg) or vehicle were administered i.p. 15 min before 5 min bilateral carotid occlusion. 3. Seven days after reperfusion, ischemia-induced hippocampal nerve cell loss (95% in vehicle-treated) was significantly lower in KYNA-treated gerbils (65% and 52% at 1000 and 1200 mg/Kg, respectively, P < 0.01). Treatment with 1000 mg/kg produced brain KYNA concentrations that were dramatically elevated (135.9 and 42.3 microM in CSF and whole brain, vs 0.032 and 0.16 microM in controls, at 15 min after ischemia), as measured in a separate group of transcardially-perfused gerbils. Cerebral KYNA concentrations tended to return to basal values 2 hours after reperfusion. 4. These results indicate that KYNA has a marked neuroprotective effect in a model of forebrain ischemia. This activity is associated with KYNA concentrations in the brain and CSF that are compatible with the in vitro affinity of the compound for ionotropic glutamate receptors.

Animals↗
PubMed1999

Interactions of anthracyclines with zwitterionic phospholipid monolayers at the air-water interface.

The present note describes the use of surface pressure measurements (Langmuir monolayer technique) for the analysis of interactions of two different anthracyclines (adriamycin and daunorubicin) with a non-ionic, zwitterionic phospholipid monolayer, at the air-water interface. Because the surface membrane of the cell is the first barrier encountered by the anthracyclines in the treatment of cancer, drug-membrane interactions studied in model (monolayers or bilayers) and natural systems play an important role in the understanding of the bioactivity properties of these molecules. We report here the rate constants of the adsorption process of adriamycin and daunorubicin in the presence of a zwitterionic phospholipid monolayer at the air-water interface. Because interactions with the lipid monolayer strongly depend on the molecular packing of the lipid, we investigated this process at a relatively low surface pressure (7 mN/m), the interactions being favoured by the gaseous and liquid expanded structure of the lipid monolayer. The apparent molecular area of these molecules during the insertion into the lipid film and their interactions with the phospholipid polar head groups was evaluated and the estimated percentage of anthracyclines at the interface after adsorption into the lipid monolayer is briefly discussed. The rate constants for the adsorption and desorption process at the water-monolayer interface have been calculated on the basis of a single-exponential model. The observed difference of these parameters for daunorubicin and adriamycin suggests a different interaction of these anthracyclines during the adsorption to and/or penetration across the phospholipid monolayer.

Air↗
PubMed1999

Disinsertion of the medial rectus following pterygium surgery: signs and management.

The clinical characteristics, intraoperative findings and management of two cases of "lost" medial recti during surgery for recurrent pterygium are described. The lost muscles are classified in different groups according to their etiopathogenesis. In the cases reported, the muscles were found retroinserted with extensive proliferation of fibrous tissue. This complication was resolved after finding the muscle, liberating the surrounding fibrous tissue and reattachment in its original insertion. Two aspects stand out: The CAT scan to determine the location of the muscle and the usefulness of topical anesthesia to facilitate recognition of the muscle during the surgical procedure.

Adult↗
PubMed1998

Temporal and spatial changes of quinolinic acid immunoreactivity in the gerbil hippocampus following transient cerebral ischemia.

Quinolinic acid (QUIN) is an endogenous neurotoxin which originates from the kynurenine pathway of tryptophan metabolism. An increase of brain QUIN level occurs in several degenerative and inflammatory disorders, but the cellular source of QUIN is still a matter of controversy. In the present study, the gerbil model of transient global ischemia was used to investigate the time course and the cellular localization of QUIN immunoreactivity. Neurodegeneration was evident in the subiculum and in the CA1 area of the hippocampus 4, 7 and 14 days after ischemia. QUIN positive cells, with microglia-like morphology, appeared in the subiculum and in the CA1, 4 days after ischemia. At 7 days post-ischemia they extended to the whole CA1, disappearing at 14 days. Neither neurodegeneration nor QUIN positive cells could be detected in ischemic gerbils sacrificed at 1 and 2 days after ischemia and in sham-operated animals. These findings suggest that microglia-like cells infiltrating the degenerating areas of the hippocampus represent the major source of QUIN following transient ischemia in the gerbil. Thus, in situ production of QUIN in vulnerable brain regions may contribute to the pathophysiological mechanisms of delayed brain injury.

Animals↗
PubMed1998

Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells.

The intracellular location of the MDR1 gene product, known as P-glycoprotein (P-gp), has been detected by flow cytometry in 3 stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express P-gp on the plasma membrane. In addition, MDR1 mRNA expression was revealed by RT-PCR in the same cell lines. Immunofluorescence microscopy, performed by using the same 2 monoclonal antibodies (MM4.17 and MRK-16) as employed in the flow-cytometric analysis, revealed the presence of P-gp intracytoplasmically, in a well-defined perinuclear region. Double immunofluorescence labelling and immunoelectron microscopy strongly suggested the location of the transporter molecule in the Golgi apparatus. The same observations have been obtained on a primary culture from a metastasis of human melanoma. Analysis of the expression of another membrane transport protein, the multidrug-resistance-related protein (MRP1), showed that it was present in the cytoplasm of all the melanoma cell lines examined. MRP1 also showed Golgi-like localization. The study by laser scanning confocal microscopy on the intracellular localization of the anti-tumoral agent doxorubicin (DOX) during the drug-uptake and -efflux phases, indicated the Golgi apparatus as a preferential accumulation site for the anthracyclinic antibiotic. P-gp function modulators (verapamil and cyclosporin A) were able to modify DOX intracytoplasmic distribution and to increase drug intracellular concentration and cytotoxic effect in melanoma cells. On the contrary, MRP1 modulators (probenecid and genistein) did not significantly influence either DOX efflux and distribution or the sensitivity of melanoma cells to the cytotoxic drug.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Characterization of a clonal human colon adenocarcinoma line intrinsically resistant to doxorubicin.

Intrinsic low-level resistance to anti-cancer drugs is a major problem in the treatment of gastrointestinal malignancies. To address the problem presented by intrinsically resistant tumours, we have isolated two monoclonal lines from LoVo human colon adenocarcinoma cells: LoVo/C7, which is intrinsically resistant to doxorubicin (DOX); and LoVo/C5, which shows the same resistance index for DOX as the mixed parental cell population. For comparison, we have included in the study a LoVo-resistant line selected by continuous exposure to DOX and expressing a typical multidrug resistant (MDR) phenotype. In these cell lines we have studied the expression and/or activity of a number of proteins, including P-glycoprotein 170 (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione (GSH)-dependent enzymes and protein kinase C (PKC) isoforms, which have been implicated in anti-cancer drug resistance. Intracellular DOX distribution has been assessed by confocal microscopy. The results of the present study indicate that resistance in LoVo/C7 cells cannot be attributed to alterations in P-gp, LRP or GSH/GSH-dependent enzyme levels. Increased expression of MRP, accompanied by alterations in the subcellular distribution of DOX, has been observed in LoVo/C7 cells; changes in PKC isoform pattern have been detected in both intrinsically and pharmacologically resistant cells.

ATP-Binding Cassette Transporters↗
PubMed1997

Influence of N-methylformamide on the intracellular transport of doxorubicin.

The polar solvent N-methylformamide proved to be capable of enhancing the cytotoxic potential of various antitumoral compounds, both in vitro and in vivo. In many cases, this ability depended on the sequence of treatment, and the enhancement of the cytotoxic effect occurred only when N-methylformamide administration succeeded anticancer drug treatment. The results obtained in the present study indicate that N-methylformamide interferes with the mechanisms of intracellular transport and efflux of the antitumoral drug doxorubicin. In particular, laser scanning confocal microscopy observations performed on melanoma cells (M14) after N-methylformamide administration revealed evident alterations of the microtubular network, including numerous interruptions of the microtubules. Moreover, when doxorubicin-treated cells were recovered in the presence of the polar solvent, the normal efflux of the anthracyclinic antibiotic appeared to be hampered, and the drug was localized mainly in well delimited perinuclear regions. Double staining experiments demonstrated the colocalization of the doxorubicin molecules and the WGA-stained regions as well as a close structural relationship between them and the microtubule system. These results indicate that N-methylformamide interferes with the doxorubicin transport inducing a damage in the microtubular network and the consequent persistence and entrapment of the drug in the regions likely occupied by the Golgi apparatus of tumor cells. This finding could account for the chemosensitizing properties exerted by N-methylformamide.

Antineoplastic Agents↗
PubMed1997

Lower levels of 1H MRS-visible mobile lipids in H-ras transformed tumorigenic fibroblasts with respect to their untransformed parental cells.

High resolution 1H MRS studies report increased mobile neutral lipid (MNL) signals in transformed and malignant as well as in some in vitro cultured embryonic cells. Nature, subcellular localization and biological function of MNL are still under debate. This work was aimed at assessing alterations induced in MNL signals of NIH-3T3 mouse embryo fibroblasts by transformation with human HJ-ras oncogene. Lower MRS-visible MNL levels were unexpectedly detected in ras-transformed, in vivo tumorigenic fibroblasts, with respect to their untransformed and non-tumorigenic parental cells. MRS, gas chromatography and chemical analysis on cells and their lipid extracts indicated that these spectral differences could hardly be attributed to different triacylglycerol, free fatty acids and total cholesterol levels or to changes in the fatty acyl degree of unsaturation and average chain length. Additional, possibly more relevant mechanisms of regulation of MNL mobility may implicate the extensive morphogenetic changes and reorganization of cytoskeleton components (notably actin) associated with ras-transformation.

Animals↗
PubMed1996

[Crocodile tears and retraction syndrome].

BACKGROUND: An association between crocodile tears and Duane's syndrome has been described in more than 50 cases. PATIENT: A 29-year-old man had tearing of his right eye whenever he ate something sour. At psychic weeping his right eye remained dry. In addition, he had Duane's retraction syndrome, also in the right eye. CONCLUSION: In this case of congenital crocodile tears with an ipsilateral Duane's syndrome the normal function of the facial muscles suggests that the abnormal coupling between the gustatory nucleus and the lacrimal gland is not located in the facial nerve proximal to the Ganglion geniculi, as in most acquired cases. Rather, the abnormal coupling may be located in the brainstem. The association with Duane's syndrome indicates that abnormal innervational coupling is present also in the ocular motor domaine.

Abducens Nerve↗