Biomedical subjects

A Molinari

Publications and source records attributed to A Molinari.

At least 19 recordsLinked to original sources

PubMed2006

Cationic liposomes, loaded with m-THPC, in photodynamic therapy for malignant glioma.

In this study we investigated the feasibility of mixed liposomes formed by dimyristoyl-sn-glycero-phosphatidylcholine (DMPC) and cationic gemini surfactant (Gemini 1) loaded with the chlorin m-tetrahydroxyphenylchlorin (m-THPC), in photodynamic therapy (PDT) for glioma. To this aim, an in vitro study was carried out by employing various human glioblastoma cell lines (A172, DBTRG, LN229, U118). The following liposomal formulations were tested: (i) DMPC and Gemini 1; (ii) m-THPC in DMPC in the absence or (iii) in the presence of Gemini 1 in the molar ratio 8:2; 7:3, and 6:4. The presence of Gemini 1 significantly increased the intracellular uptake of chlorin in all cell tested although with a different extent: LN229>U118>A172>DBTRG. The cytotoxicity of chlorin-loaded liposomes was then tested by cloning efficiency performed on different cultures, before and after irradiation with laser light at 652nm, at a Fluence Rate of 200mW/s for 100s, with a total Fluence of 20J/cm(-2). In the absence of irradiation, the different liposomal formulations induced a cytotoxicity in less than 30% of glioblastoma cells. On the contrary, irradiation induced total destruction of all cultures treated with m-THPC/DMPC+Gemini 1 in the ratios 8:2, or 7:3, or 6:4.

Cell Line, Tumor↗

PubMed2006

Evaluation of sequential perfusion with Euro-Collins and Belzer solutions for pancreas preservation in rats.

OBJECTIVE: To investigate the effect of Euro-Collins and Belzer solutions in a sequential preservation of the pancreas. METHODS: Forty-five Wistar-EPM rats were divided into four groups, according to the solution used during preservation: (1) saline solution (SF): animals perfused and preserved with saline solution; (2) Euro-Collins group (C): animals perfused and preserved with Euro-Collins solution; (3) Belzer group (B): animals perfused and preserved with Belzer solution; (4) Euro-Collins/Belzer group (CB): animals perfused with equal parts of Euro-Collins and Belzer solutions sequentially and preserved with Belzer solution. After perfusion, the animals underwent pancreas resection and preservation with the respective substance at 4 degrees C. Amylase was measured in the preservation solution after 12, 24, 36, or 48 hours. Finally, the pancreas was analyzed histologically, and a statistical analysis was performed. RESULTS: Groups SF and C showed the highest amylase levels in the preservation solution during all periods. The levels were higher than in groups C and CB (P = .05). Amylase levels were similar in groups B and CB to 24 hours (P = .05). Histological analysis was significant for analysis of pancreas islet cells and edema. Groups B and CB were histologically similar (P = .001) and different from groups SF and C. CONCLUSION: Sequential perfusion using Euro-Collins and Belzer solutions was effective for pancreas preservation in rats up to 24 hours.

Adenosine↗

PubMed2006

A randomized study comparing filgrastim versus lenograstim versus molgramostim plus chemotherapy for peripheral blood progenitor cell mobilization.

We conducted a prospective randomized clinical trial to assess the mobilizing efficacy of filgrastim, lenograstim and molgramostim following a disease-specific chemotherapy regimen. Mobilization consisted of high-dose cyclophosphamide in 45 cases (44%), and cisplatin/ifosfamide/etoposide or vinblastine in 22 (21%), followed by randomization to either filgrastim or lenograstim or molgramostim at 5 microg/kg/day. One hundred and three patients were randomized, and 82 (79%) performed apheresis. Forty-four (43%) patients were chemonaive, whereas 59 (57%) were pretreated. A median number of one apheresis per patient (range, 1-3) was performed. The median number of CD34+ cells obtained after mobilization was 8.4 x 10(6)/kg in the filgrastim arm versus 5.8 x 10(6)/kg in the lenograstim arm versus 4.0 x 10(6)/kg in the molgramostim arm (P=0.1). A statistically significant difference was observed for the median number of days of growth factor administration in favor of lenograstim (12 days) versus filgrastim (13 days) and molgramostim (14 days) (P<0.0001) and for the subgroup of chemonaive patients (12 days) versus pretreated patients (14 days) (P<0.001). In conclusion, all three growth factors were efficacious in mobilizing peripheral blood progenitor cells with no statistically significant difference between CD34+ cell yield and the different regimens, and the time to apheresis is likely confounded by the different mobilization regimens.

Adjuvants, Immunologic↗

PubMed2006

Pharmacokinetics of nelfinavir in HIV-1-infected pregnant and nonpregnant women.

AIMS: To compare steady-state nelfinavir (NFV) pharmacokinetics in pregnant and nonpregnant HIV-infected women. METHODS: Twenty-five pregnant HIV-infected women were selected from an ongoing observational study evaluating the pharmacokinetics of antiretroviral agents during pregnancy. Twenty of them were in the third and five in the second trimester. Data for the control group of 21 HIV-infected nonpregnant women were taken from a previous multicentre pharmacokinetic trial. All the participating women achieved steady-state plasma concentrations while on a highly active antiretroviral therapy (HAART) regimen including NFV (1250 mg bid) and two nucleoside reverse transcriptase inhibitors (NRTIs). Blood samples for NFV measurement were collected predose (C(trough)) and at 0.5, 1, 2, 3, 4, 5, 6, 8 and 12 h post dose. RESULTS: During the third trimester of pregnancy NFV AUC(0-12 h) median (range) values were 25.76 (12.61-42.74) microg h(-1) ml(-1), and were 32.49 (19.16-63.81) microg h(-1) ml(-1) in the control group [mean difference - 9.30 microg h(-1) ml(-1); 95% confidence interval (CI) -15.76, -2.83; P < 0.05). Median oral clearance (CL/F) was significantly higher in pregnant women than in the control group (48.5 l h(-1), range 29.3-99.1 l h(-1) vs. 38.5 l h(-1), range 19.6-65.2 l h(-1); mean difference 12.6 l h(-1); 95% CI 3.3, 21.9) but the difference disappeared when CL/F was adjusted for body weight. C(trough) was significantly (P < 0.01) lower in pregnant compared with nonpregnant women (median 0.8 microg ml(-1), range 0-2.6 microg ml(-1) vs. 1.5 microg ml(-1), range 0.5-4.9 microg ml(-1); mean difference -1.0 microg ml(-1); 95% CI -1.7, -0.31). The median elimination half-life of NFV observed during pregnancy was 3.7 h (range 1.4-6.6 h), compared with 5.2 (range 3.1-10.1 h) in the control group (mean difference -1.7; 95% CI -2.8, -0.51). CONCLUSIONS: Our results indicate that women in the later stages of pregnancy may be exposed to subtherapeutic concentrations of NFV. Thus, adjustments in drug dosage or frequency of administration may be required.

Female↗

PubMed2006

Influence of complexation with cyclodextrins on photo-induced free radical production by the common sunscreen agents octyl-dimethylaminobenzoate and octyl-methoxycinnamate.

The influence of complexation with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) or beta-cyclodextrin (beta-CD) on the photo-induced production of free radicals by the sunscreen agents octyl-dimethylaminobenzoate (ODAB), oxybenzone (OB) and octyl-methoxycinnamate (OMC) was investigated. The formation of radical species during irradiation was detected by spin-trapping electron paramagnetic resonance (EPR) spectroscopy. 2,2,6,6-tetramethylpiperidine-1-oxyl, nitroxide radical (TEMPO) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used a spin-traps. Following the 4-h illumination with simulated sunlight, OB did not generate radicals. On the other hand, photoexcitation of solutions containing ODAB or OMC produced a marked decrease (>40%) of the TEMPO signal intensity, demonstrating the formation of carbon-centred radicals. In addition, the results obtained on irradiation of ODAB solutions containing DMPO as spin-trap indicated the generation of oxygen-centred radicals. Complexation of ODAB with HP-beta-CD and OMC with beta-CD markedly inhibited (>64%) the formation of free radicals generated by the sunscreens on exposure to simulated sunlight. Therefore, inclusion of ODAB and OMC into the cyclodextrin cavities minimizes their photosensitising potential.

Chromatography, High Pressure Liquid↗

PubMed2005

Invasive properties of multidrug resistant human melanoma cells.

Melanoma cells exhibit a high level of intrinsic or acquired resistance to the cytotoxic agents often associated with the over-expression of drug transporters such as P-glycoprotein (P-gp). In this in vitro study, we investigated the possible relationship between P-gp and CD44, the cell adhesion molecule involved in metastasis and tumor progression of melanoma cells. CD44 expression appeared to be similar in the parental sensitive M14 WT cells and in their resistant counterparts M14 ADR cells. Double-labeling of cryosectioned cells showed that P-gp and CD44 were transported from the synthesis loci to the cell periphery by different vesicles and began to coalesce in proximity of the plasma membrane; thus, P-gp and CD44 seemed to reach together the cell surface. Moreover, P-gp and CD44 appeared to be associated with ERM proteins. The invasive activities of both M14 WT and M14 ADR cells were analyzed by the "transwell chamber invasion" assay. M14 WT cells revealed low capacity to traverse the filters, both in the absence (motility) and in the presence (invasion) of a Matrigel coating. In comparison, M14 ADR cells displayed significantly higher motility and invasion. SEM observations showed that sensitive cells employed lamellar cytoplasmic extrusions to pass through the filter pores whereas resistant cells elongated along the hole through globular processes. In conclusion, the results herein reported suggest that drug resistance in melanoma cells appears associated with a more aggressive behaviour. P-gp and CD44 might cooperate to confer this more invasive phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Self-organized formation of rhomboidal nanopyramids on fcc(110) metal surfaces.

We report on the far from equilibrium self-organized morphologies obtained after Xe ion irradiation of the Rh(110) and Cu(110) surfaces. Here we experimentally identify by means of high resolution LEED a novel interfacial state characterized by a rhomboidal pyramid islanding with majority steps oriented along nonequilibrium low-symmetry directions. The formation of the novel rhomboidal pyramid state and the transition to the well-known rippled phases results from a delicate interplay of kinetic processes which are controlled by acting on temperature, ion flux, and impact energy.

Journal Article↗

PubMed2004

High serum osteoprotegerin levels in patients with hyperthyroidism: effect of medical treatment.

This study was aimed at evaluating serum osteoprotegerin (OPG) concentrations in a cohort of patients with hyperthyroidism before and after methimazole (MMI) treatment. One hundred fourteen hyperthyroid patients [93 with Graves disease (GD) and 21 with toxic nodular goitre (TNG)] and 68 matched for sex and age healthy subjects were evaluated for serum free-thyroxine (FT4), free-triiodiothyronine (FT3), thyrotropin (TSH), TSH receptor antibodies (TRAb), bone alkaline phosphatase (BALP), C-telopeptides of type-1 collagen (CrossLaps), OPG levels, and bone mineral density (BMD). In hyperthyroid patients, the biochemical evaluations were performed before and after 6 and 12 months of MMI treatment, whereas BMD was measured at baseline and after 12 months of treatment. Hyperthyroidism was more severe in GD than TNG patients. Serum OPG levels were found to be significantly higher in hyperthyroid patients than in the healthy subjects (4.3 pmol/l, range: 1.6-12.0, vs. 2.2 pmol/l, range: 1.4-6.0; P < 0.001), the values being higher in GD patients than TNG. A significant correlation between serum OPG levels and age was found in the healthy subjects (r: 0.48; P < 0.001) but not in hyperthyroid patients (r: -0.03; P = 0.8). In the healthy subjects, serum OPG levels were also positively correlated with both serum FT4 (r: 0.23; P = 0.03) and FT3 (r: 0.24; P = 0.04) levels. In hyperthyroid patients, however, serum OPG was still correlated with FT3 levels (r: 0.38; P < 0.001), whereas the correlation with serum FT4 was lost (r: 0.19; P = 0.06). In hyperthyroid patients, but not in the healthy subjects, serum OPG levels were correlated positively with CrossLaps (r: 0.20; P = 0.03) and negatively with BALP (r: -0.24; P = 0.01) and BMD (r: -0.33; P = 0.01). After 6 months of MMI treatment, serum OPG concentrations decreased significantly in TNG patients (from 3.5 pmol/l, range: 1.6-8.0, to 2.3 pmol/l, range: 1.0-4.3; P < 0.001), whereas a not significant change in OPG levels occurred in GD patients (from 4.8 pmol/l, range: 1.8-12.0, to 4.2 pmol/l, range: 1.0-14.0; P = 0.7). At Month 12 of treatment, serum OPG concentrations were significantly lower than those measured at baseline in both TNG (2.5 pmol/l, range: 1.0-3.1, vs. 3.5 pmol/l, range: 1.6-8.0; P < 0.001) and GD (2.1 pmol/l, range: 1.0-8.6, vs. 4.8 pmol/l, range: 1.8-12.0; P < 0.001). At this time, no significant differences in serum OPG, CrossLaps, and BALP values were found between patients and control subjects. At the end of follow-up, BMD was higher than those measured at baseline but still significantly lower than those measured in the control subjects. This study shows that hyperthyroid patients have serum OPG concentrations significantly higher in comparison with euthyroid subjects, in relation to thyroid hormone excess and high bone turnover. Medical treatment of hyperthyroidism normalizes serum OPG levels in temporal relationship with the normalization of bone metabolism markers, even in presence of persistent abnormal bone structure as determined by ultrasonography.

Adult↗

PubMed2004

Early complications in endovascular treatment of abdominal aortic aneurysm.

OBJECTIVE: The incidence of perioperative complications during endovascular repair of abdominal aortic aneurysm (EVAR) is reported in limited series. The aim of this study is to evaluate a multi-center survey of unexpected intraprocedural critical events of EVAR. METHODS: A questionnaire relative to intraprocedural complications during EVAR was sent to major vascular surgery divisions in Italy. Eleven answered to the survey. The data obtained are relative to 1696 procedures. RESULTS: A wide range of incidence of critical events was observed, from a lower value of 2.7% to a higher value of 68.8% (mean 21.16%). The problems relative to the insertion phase of the delivery system were 7.7%. Endoleaks were reported in 5.5% of cases. Stent graft release was problematic in 0.4% of cases and in another 0.4% there was a problem in shaft retrieval. Unintentional coverage of renal or polar arteries occurred in 0.8% of the procedures ; hypogastric arteries were unwillingly excluded in 2.7% of cases. Aortic or iliac artery rupture had an incidence of 0.7% ; arterial dissection occurred in 0.9%, atheroembolism in 0.5%, lower limb ischemia due to graft limb kinks in 0.7% and to occlusion in 0.9%. CONCLUSIONS: Perioperative critical events represent a serious problem only in few cases of EVAR ; they are common but in many cases not predictable ; in most circumstances they can be easily corrected with adjunctive manoeuvres during the same procedure. There is a highly significant correlation between the total workload and the incidence of critical events ; these do not appear to be related to the learning curve.

Angioscopy↗

PubMed2003

Saquinavir induces stable and functional expression of the multidrug transporter P-glycoprotein in human CD4 T-lymphoblastoid CEMrev cells.

BACKGROUND: The multidrug transporter P-glycoprotein (P-gp) is expressed in HIV-1 target cells, in a range of pharmacological barriers and in AIDS-associated tumours. P-gp substrates include HIV-1 protease inhibitors (PIs) and anticancer drugs, which are efficiently effluxed from multidrug-resistant (MDR) cells. OBJECTIVES: The aim of this study was to investigate the effect on human CD4 T-lymphoblastoid CEMrev cells of saquinavir and other PIs in terms of P-gp expression and to characterize the functional and biochemical patterns of PI-induced P-gp molecules. METHODS: CEMrev cells no longer expressing detectable amounts of P-gp were cultured for a prolonged period in the presence of 10 microg/mL saquinavir (CEMsaq10) and tested for P-gp expression and function. Subsequently, CEMsaq10 cells were transferred into medium containing 15 microg/mL saquinavir (CEMsaq15) and cultured for several months. These cell lines were continuously monitored for P-gp expression, function and immunochemical patterns. A similar strategy was adopted to determine whether other PIs, such as ritonavir and indinavir, were able to induce P-gp expression in CEMrev cells. RESULTS: Compared with the drug-diluent control, the exposure of CEMrev cells to 10 microg/mL saquinavir induced, in a consistent fraction of cells (45-50%), de novo expression of functioning P-gp molecules. The transfer of CEMsaq10 cells to 15 microg/mL saquinavir was associated with a dramatic increase in P-gp expression and function (85-90% of CEMsaq15 cells expressed P-gp and effluxed P-gp dye substrates). These saquinavir-induced P-gp molecules included 75-kDa molecules as well as the classical 170-kDa form of P-gp, suggesting induction of a particular isoform of P-gp termed mini-P-glycoprotein. Conversely, ritonavir and indinavir induced transient P-gp expression in a small percentage of the CEMrev cells. CONCLUSIONS: Treatment of human CD4 T-lymphoblastoid CEMrev cells with saquinavir caused over-expression of functioning P-gp molecules. This de novo acquired MDR phenotype, which differed from that induced by other PIs, was stable, as expression and activity of P-gp were observed in CEMsaq10 and CEMsaq15 cells during prolonged in vitro culturing, even in drug-free conditions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

What is the relationship between P-glycoprotein and adhesion molecule expression in melanoma cells?

A number of studies have reported that increased P-glycoprotein expression in drug-resistant tumour cells may be associated with decreased expression of a family of surface glycoproteins. However, despite its potential biological and clinical relevance, this phenomenon has not been extensively studied. In this study the phenotypic alterations that are associated with the acquisition of the multidrug-resistant phenotype in tumour cells, together with drug transporter overexpression, were investigated in human melanoma cells. The expression of cell adhesion molecules was analysed in a panel of multidrug-resistant melanoma cell lines (M14Dx) showing different degrees of resistance to doxorubicin and different levels of the expression of the drug transporter P-glycoprotein. In particular, expression of intercellular adhesion molecule-1 (ICAM-1), CD44, very late activation antigen (VLA)-5 and VLA-2 was determined by flow cytometry in the different resistant cell lines. A progressive downregulation of all the adhesion molecules examined was revealed in M14Dx cells, in parallel with an increasing level of expression of the drug transporter P-glycoprotein. The results obtained raise the question of the role of P-glycoprotein in the invasive and metastatic behaviour of tumour cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells.

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Photoinitiated catalysis in Nafion membranes containing palladium(II) meso-tetrakis(N-methyl-4-pyridyl)porphyrin and iron(III) meso-tetrakis-(2,6-dichlorophenyl)porphyrin for O2-mediated oxidations of alkenes.

Immobilisation of both palladium(II) meso-tetrakis(N-methyl-4-pyridyl)porphyrin (PdTMPyP4+) and iron(III) meso-tetrakis(2,6-dichlorophenyl)porphyrin (FeTDCPP+) in the same membrane of Nafion creates a new composite system, in which the photoexcited palladium complex induces the O2-mediated oxidation of cyclohexene to the corresponding allylic hydroperoxide and the iron porphyrin works as a catalyst for specific oxygenations of cyclohexene and cyclooctene. The role of PdTMPyP4+ is to induce the photoactivation of O2 with visible light (lambda > 500 nm) to generate singlet oxygen (1O2) by means of energy transfer from the excited triplet state. Consequently, the 1O2-mediated oxidation of cyclohexene to cyclohexenyl hydroperoxide can be realised with a selectivity greater than 90%. Spectroscopic and photophysical investigations show that the tetracationic palladium porphyrin is mainly fixed to the external part of the Nafion membrane, it is characterised by a triplet-state lifetime significantly higher than that in the solution phase. The monocationic FeTDCPP+ is able to diffuse into the anionic cavities of Nafion, where it works as a catalyst for O2-mediated autooxidation processes that are initiated by the photogenerated hydroperoxides. These processes continue in the dark for many hours giving cyclohex-2-en-1-ol and trans-cyclohexane-1,2-diol monoethyl ether as main oxidation products. The presence of this ether, indirectly, reveals the formation of cyclohexene epoxide which undergoes a nucleophilic attack by ethanol and epoxide opening because of the strong acidic environment inside Nafion. The good photocatalytic efficiency of the oxidation process is demonstrated by an overall quantum yield of 1.1, as well as by a turnover value of 4.7 x 10(3) with respect to the iron porphyrin. When cyclooctene is present as co-substrate, it also undergoes oxygenation. In contrast to what was observed for cyclohexene, cyclooctene epoxide can be accumulated in a significant amount. As far as the stability of the system is concerned, FeTDCPP+ undergoes about 1% degradation during the process, while the Nafion matrix can be utilised several times without observable modification.

Alkenes↗

PubMed2001

New selective cytotoxic diterpenylquinones and diterpenylhydroquinones.

A new series of diterpenylquinone/hydroquinones has been prepared by Diels-Alder cycloaddition between three labdanic diterpenoids (myrceocommunic acid, methyl myrceocommunate, and myrceocommunyl acetate) and p-benzoquinone or 1,4-naphthoquinone. Influences of the quinone/hydroquinone fragment and other structural features, such as the different functionalities in the terpenic core, are considered in relation to the cytotoxicity toward neoplastic cells and the selectivity of these diterpenylnaphthoquinones/hydroquinones and anthraquinones. Several compounds showed IC50 values under the micromolar level, and four of these derivatives were evaluated at the NCI screening panel. The results showed an important selectivity toward renal cancer lines, identifying these compounds as a very promising group of antineoplastics.

Animals↗

PubMed2001

1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells.

The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r = 0.993, P < 0.001). Similar ML levels were measured in drug-induced apoptotic cells (A approximately 30-40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter < or = 1.0 microm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, 'biochemically active' phase of programmed cell death.

Apoptosis↗

PubMed2001

Immunohistochemical visualization of newly formed quinolinate in the normal and excitotoxically lesioned rat striatum.

Intracerebral infusion of 3-hydroxyanthranilate (3HANA) rapidly increases the brain content of the endogenous excitotoxin quinolinate (QUIN). QUIN formation from 3HANA is readily prevented by coadministration of the specific 3-hydroxyanthranilate oxygenase inhibitor 4-chloro-3HANA (4-Cl-3HANA). This experimental paradigm was used to identify the cell populations which are responsible for the rapid de novo production of QUIN in the rat striatum in vivo. Rats received an intrastriatal infusion of 3HANA, alone or together with equimolar 4-Cl-3HANA, for 1 h. Striatal QUIN immunoreactivity (ir) was assessed immunohistochemically, using an antibody against protein-conjugated QUIN. This antibody displayed no significant crossreactivity with compounds structurally or functionally related to QUIN. QUIN-ir cells were detected after infusion with > or =300 microM 3HANA, but not in naïve striata or after co-infusion of 4-Cl-3HANA. Cellular staining was also abolished by preabsorption of the antibody with protein-conjugated QUIN. In the normal striatum, QUIN-ir was detected exclusively in cells of an apparent microglial morphology. When examined in the excitotoxically lesioned striatum, 3HANA-induced QUIN-ir localized exclusively to OX42-ir cells of an activated microglial/macrophage morphology. These data indicate that microglia and macrophages are the major source of QUIN in the rat striatum when hyperphysiological concentrations of 3HANA are used to drive QUIN synthesis. Comparison with earlier biochemical and immunohistochemical studies suggests that the enzyme responsible for microglial QUIN production is a distinct 3-hydroxyanthranilate oxygenase with high capacity and low affinity for 3HANA.

3-Hydroxyanthranilic Acid↗

PubMed2001

Immunogold localisation of P-glycoprotein in supported lipid bilayers by transmission electron microscopy and atomic force microscopy.

In this study, purified P-glycoprotein molecules, a membrane drug pump responsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997). The insertion of proteins into planar supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir-Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic force microscopy (AFM). The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and their stability under different environmental conditions (vacuum, air and water). P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Intracellular P-glycoprotein in multidrug resistant tumor cells.

The overexpression of the drug-efflux molecular pump P-glycoprotein (P-gp) may confer to tumor cells the multidrug resistant (MDR) phenotype, which is one of the causes of cancer chemotherapy failure. By investigating several in vitro models of human tumor cells, we observed that P-gp, in addition to its localization on the plasma membrane, can also be found intracellularly. In particular, by using immunocytochemical and cytofluorimetric methods, we revealed that in MDR breast cancer cells (MCF-7) a significant level of P-gp was expressed in the Golgi apparatus, which is the major site of accumulation of the antitumoral compound doxorubicin. Moreover, we demonstrated the intracellular location of P-gp in three stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. Double immunofluorescence labelling and immunoelectron microscopy revealed, also in this tumor cell type, the location of P-gp in the Golgi apparatus where it seems to play a pivotal role in intracellular drug transport. Finally, we analyzed the expression, localization and function of drug transport proteins in human colon carcinoma lines (LoVo) exhibiting different degrees of intrinsic or drug-induced resistance. We found that only MDR LoVo cells expressed P-gp on the plasma membrane while both low-level drug resistant clonal LoVo cells and MDR LoVo cells appeared to be positive for intracellular P-gp. Our findings suggest a functional role of the intracytoplasmic P-gp in the transport and sequestration of drugs. This represents a complementary protective mechanism of tumor cells against cytotoxic agents.

ATP Binding Cassette Transporter, Subfamily B↗