Probing nucleon strangeness with neutrinos: Nuclear model dependences.
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Journal Article↗
Biomedical subjects
A Molinari
Publications and source records attributed to A Molinari.
28833, a selective kynurenine 3-hydroxylase inhibitor. Effects on the kynurenine pathway metabolites in the gerbil.
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Animals↗
Liposomal targeting of leukaemia HL60 cells induced by transferrin-receptor endocytosis.
A liposomal carrier system able to interact specifically with HL60 leukaemia cells was produced using small unilamellar liposomes made of pure phospholipids chemically cross-linked to human transferrin. The conjugation of transferrin to liposomes was carried out using N-succinimidyl 3-(2-pyridyldithio)-propionate and 2-iminothiolane as activating agents for the liposomes and the protein. The reaction occurred under conditions set to covalently link on the surface of a single vesicle a limited number (one to ten) of transferrin molecules, as verified by means of electron microscopy and immunoenzymic measurements. Before conjugation, the ultrastructure of the liposomes, and the content and distribution of the amino groups within the bilayer, were determined. The reactivity of the liposomes towards amino-derivatizing or thiolating compounds was also measured. Kinetic spectroscopic measurements confirmed that the distribution of the phosphatidylethanolamine in the vesicle bilayer is asymmetrical: 22% of phosphatidylethanolamine was found exposed to the external surface of the liposomes and accessible to the cross-linker. The modified liposomes were able to interact specifically with the cells and to be internalized by active receptor-mediated endocytosis, as demonstrated by the full inhibition of internalization induced by free transferrin.
Endocytosis↗
A modified spectroscopic method for the determination of the transbilayer distribution of phosphatidylethanolamine in soya-bean asolectin small unilamellar vesicles.
A spectroscopic kinetic approach for determining the relative concentrations of phosphatidylethanolamine (PE) exposed on the external and internal layers of small unilamellar vesicles (SUVs) used as a model system and prepared by sonication of purified soya-bean asolectin is proposed, based on the use of 2,4,6-trinitrobenzenesulphonic acid (TNBS) and N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The known reactions between PE and TNBS and/or SDPD were used, separately or in combination, to derivatize PE in preformed vesicles. We have observed that mixing SUVs with excess TNBS results in a biphasic time course. Kinetic analysis of the data supports the conclusion that external PE is rapidly derivatized (fast phase) with a half-time of 2 min. In the next (slow) phase (half-time 70 min), TNBS permeates the vesicle membrane and also reacts with PE molecules facing the internal liposomal compartment. Under the experimental conditions chosen, SPDP reacted with only the external PE molecules. The reaction of SUVs first derivatized with SPDP and then with TNBS further demonstrates that the two phases, observed with TNBS, are due to modification of external and internal PE. Approx. 30% of PE was found to be facing the external bulk phase, thus confirming the asymmetric distribution of the molecules in SUVs. The maximum number of thiol arms covalently linked by means of SPDP modification of PE on the surface of a single liposome was estimated at about 10(2).
Cross-Linking Reagents↗
Prolonged half-life in the circulation of a chemical conjugate between a pro-urokinase derivative and human serum albumin.
Pro-urokinase is a natural plasminogen activator that displays a clot-lysis activity through a fibrin-dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. Like tissue-type plasminogen activator and two-chain urokinase-type plasminogen activator, pro-urokinase has a very short half-life in circulation. It has been described that conjugation of serum albumin with pro-urokinase in plasma may occur that could protect this protein from degradation. In this study we describe the insertion of an extra cysteine residue in the N-terminal end of des-(C11-K135)-pro-urokinase (delta 125-proUK), a pro-urokinase deletion mutant lacking amino acids 11-135. We have expressed and purified the new mutein [H5K, S9C, N10T] des-(C11-K135)-pro-urokinase (Cys-delta 125-pro-urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys-delta-pro-urokinase. The purified conjugate obtained has a lower specific amidolytic activity (72,000 U/mg) than unconjugated Cys-delta 125-pro-urikinase (240,000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of delta 125-pro-urokinase. We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection. The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half-life in the circulation, with respect to pro-urokinase and delta 125-pro-urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro-urokinase.
Amino Acid Sequence↗
Genetic amniocentesis in biamniotic twin pregnancies by a single transabdominal insertion of the needle.
We present a technique to aspirate amniotic fluid from both sacs in biamniotic twin pregnancies using a single abdominal insertion with a spinal needle. It was successful in 48 out of 55 cases of biamniotic twin pregnancies referred to our perinatal unit between 1985 and 1994. The single insertion technique was used when the inter-amniotic membrane was clearly evident and two separate free amniotic fluid pools could be reached by the operator with a single puncture. An adequate amount of amniotic fluid was sampled from both sacs to make a cytogenetic diagnosis in all cases. There were four fetuses with trisomy 21 in three twin pregnancies. In two cases, only one twin was affected whilst the co-twin was normal, so that a selective feticide was performed. No miscarriages due to genetic amniocentesis were reported. After 1990, all genetic amniocenteses in biamniotic twin pregnancies (except for one case due to late booking) were performed between 14 and 15 weeks of gestation and with all cases except one, it was possible to sample both twins by a single puncture. We suggest that early amniocentesis (14-15 weeks) by a single abdominal puncture could be a reliable and safe alternative to first-trimester chorionic villus sampling in twin pregnancies.
Amniocentesis↗
Ultrastructural and spectroscopic methods in the study of anthracycline-membrane interaction.
Observations on the interaction of anthracycline antibiotics with the plasma membrane, performed by morphological, ultrastructural, microanalytical and spectroscopic methods, are reported and discussed in this review. Scanning and transmission electron microscopy analysis indicates that doxorubicin (DOX) induces dose-dependent modifications of the cell morphology and membrane ultrastructure of human erythrocytes. The formation of intramembrane particle-free domains on both the fracture faces of the plasma membrane suggests that the DOX molecules become incorporated within the lipid bilayer. Electron energy-loss spectroscopy measurements reveal a reduction in the P/C ratio in treated membranes, probably due to a phospholipid "dilution" following the incorporation of DOX molecules. The radiowave dielectric spectroscopy indicates modifications induced by DOX in the passive electrical properties of the membrane. In particular, the decrease in membrane conductivity suggests that the interaction of the drug with the membrane lipids can affect the function of specific ion channels. The results obtained allow us to propose a structural model of the DOX-membrane interaction, in which DOX molecules self-associate in the phospholipid bilayer. The DOX incorporation induces remarkable changes in the structural and functional properties of the plasma membrane, strengthening the hypothesis that this drug can also exert its cytotoxic action at the membrane level.
Animals↗
Noninhibitory binding of human interleukin-2-activated natural killer cells to the germ tube forms of Candida albicans.
During incubation in vitro with yeast or germ tube forms of Candida albicans, only 2 to 6% of freshly isolated human natural killer (NK) cells (> 85% CD16+, CD56+, CD3-; < 15% CD3+; cytolytic for the NK-susceptible target K562 but not for the NK-resistant target DAUDI), were seen to interact with the fungal cells. As seen under the electron microscope, the contact area had a limited extent and was narrow, and neither the surface nor the intracytoplasmic organization of the NK cell was altered. In contrast, more than 30% of interleukin-2-activated NK (LAK) cells (> 96% CD16+, CD56+, CD3-; 1.5% CD3+; cytolytic for both K562 and DAUDI targets) interacted closely with the fungus. This interaction was particularly extensive with the surface of the fungal germ tube that was intimately enveloped by villous protrusions from the lymphocyte surface. The fungus-interacting LAK cell also showed a remarkable redistribution of surface microvilli and polarization of cytoplasmic organelles, such as the Golgi apparatus, centrioles, and granules, toward the area of fungal contact. Together with the elevated cytolytic potential against the K562 and DAUDI targets, all the morphological data suggested the presence of a potentially active lytic machinery in the fungus-interacting LAK cell. Nonetheless, two independent assays for anticandidal activity did not show consistent killing or fungal growth inhibition by either fresh NK or LAK cells. While offering direct evidence of the strong interaction between human LAK cells and the germ tubes, precursors of tissue-invasive hyphal forms of C. albicans, our observations also suggest that this interaction may not be sufficient to kill the fungus or arrest its growth.
Antigens, CD↗
P-glycoprotein expression in the Golgi apparatus of multidrug-resistant cells.
The surface and intracellular expression of mdrI-P-glycoprotein in parental drug-sensitive human breast cancer cells (MCF-7) and their multidrug-resistant (MDR) variants has been studied by using the monoclonal antibodies (MAbs) MM4.17 and MRK-16, which recognize 2 different epitopes of the drug efflux pump molecule. Fluorescence microscopic observations showed that P-glycoprotein, in addition to being located at the cell surface, can also be found in the Golgi apparatus of resistant cells. To confirm this finding, Golgi apparatus and P-glycoprotein were double-labelled with wheat-germ agglutinin (WGA) and MAb MM4.17. Laser scanning confocal microscopy indicated that, in MDR cells, Adriamycin mainly accumulated cytoplasmically in a perinuclear region. This accumulation proved to be modulated by pre-treatment with verapamil or ATP depletion. Moreover, the vital staining of Adriamycin-treated MDR cells, performed with the fluorescent lipid N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]- 6-aminocaproyl sphingosine (C6-NBD-ceramide), revealed that the anthracyclinic antibiotic was located in the Golgi apparatus. All these results indicate that the drug transporter is located in the Golgi apparatus, in which Adriamycin molecules also accumulate.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Lonidamine-mediated respiratory changes in rat heart myocytes: a re-examination of the functional response of mitochondrial cytochrome c oxidase.
Respiratory activity of intact cardiac myocytes isolated from rats treated with lonidamine (LND) has been examined under conditions where cytochrome oxidase turns over at its maximal rate. Compared to myocytes isolated from control rat hearts, those treated with LND displayed a 60% increase in the cytochrome oxidase-dependent rate of respiration; electron microscopy revealed, in agreement with the literature, that the membrane structure of the mitochondrion had become disorganized. The increase in the rate of oxygen consumption was correlated with the (partial) impairment of the membrane ability to maintain the proton electrochemical potential gradient which normally inhibits oxidase activity. Results are discussed with reference to previous reports showing no effect of LND on cytochrome c oxidase activity. The evidence reported better clarifies the contribution of cytochrome oxidase to the demonstrated energetic failure displayed by cells treated with LND.
Animals↗
Effects of sequential combinations of N-methylformamide with adriamycin on cultured melanoma cells (M14).
The effects of sequential combinations of N-methylformamide (NMF) with adriamycin (ADM) on a human melanoma cell line (M14) were investigated. The results obtained can be summarized as follows: (i) When NMF was administered before ADM (NMF-->ADM), a decrease of ADM-induced cytotoxicity and intracellular ADM content was revealed. Conversely, the reverse combination (ADM-->NMF) produced a remarkable reduction of cell survival accompanied by a significant intracellular drug retention. (ii) The fluorescence microscopy revealed that in NMF- and in NMF-->ADM-treated cells the actin microfilament distribution appeared to be very similar to that in control cells. On the contrary, a disorganization of the microfilament architecture was observed in ADM-->NMF-treated cells. (iii) The microtubule organization was significantly altered by NMF. This effect appeared to be even more evident in M14 cells treated with the combination ADM-->NMF. (iv) Confocal microscopy observations showed an intracytoplasmic retention of ADM in ADM-->NMF-treated cells. These findings suggest that the cytoskeletal changes induced by NMF might interfere with the ADM efflux mechanisms, accounting for the high drug level and low survival detected in ADM-->NMF-treated cells.
Antineoplastic Agents↗
Chromosome 14 maternal uniparental disomy in the euploid cell line of a fetus with mosaic 46,XX/47,XX,+14 karyotype.
We investigated the parental origin of the extra chromosome 14 and of the two chromosomes 14 of the euploid cell line, in a case of fetal mosaicism 46,XX/47,XX+14 diagnosed at amniocentesis. Molecular analysis of five polymorphic loci of the short tandem repeat type was performed. Markers D14S43 and D14S49 showed the presence of maternal uniparental disomy of chromosome 14 in the apparently normal cell line. The distribution of the markers analysed along the chromosome suggests maternal heterodisomy with a large isodisomic segment in the telomeric region, possibly caused by meiotic crossing-over.
Adult↗
Intracellular localization of the antitumour drug adriamycin in living cultured cells: a confocal microscopy study.
The intracellular distribution of the anthracyclinic antibiotic adriamycin in living cultured cells has been investigated by confocal microscopy. In human melanoma cells (M14), adriamycin was localized inside the nuclei. When adriamycin-treated M14 cells were allowed to recover in drug-free medium, a complete efflux of the drug from the nucleus was revealed. In recovered cells, a weakly fluorescent signal was observed in the perinuclear region. When M14 cells were recovered in a medium containing colcemid, a microtubule depolymerizing agent, the drug transport from the nucleus to the cell periphery appeared to be inhibited, suggesting that the microtubule network is strongly involved in drug transport mechanisms. In multidrug-resistant (MDR) cells the intracellular location of adriamycin was shown to be noticeably different from that of the parental wild-type cells. In particular, in resistant human breast carcinoma cells (MCF-7), adriamycin appeared to be exclusively located within the cytoplasm whereas the nuclei were shown to be completely negative. When adriamycin treatment was performed in association with MDR revertants, such as Lonidamine (inhibitor of the energy metabolism) or verapamil (inhibitor of the P-glycoprotein efflux pump), a marked enhancement of the cytoplasmic signal was observed in resistant cells. Under these conditions, adriamycin appeared concentrated in the perinuclear region, but the nuclei were still negative. Confocal microscopy proved to be a very useful method for the study of the intracellular transport of fluorescent substances, such as anthracyclinic antibiotics, and for the investigation of the multidrug resistance phenomenon in tumour cells.
Breast Neoplasms↗
Modulation of cell surface-associated mannoprotein antigen expression in experimental candidal vaginitis.
The monoclonal antibody (MAb) AF1 recognizes an oligosaccharide epitope present on highly immunogenic and immunomodulatory mannoproteins (MP) of Candida albicans. The expression of this epitope (AF1-MP) during experimental candidal vaginitis was studied in two strains of C. albicans (3153 and CA-2) which were equally vaginopathic but differed in the mode of hypha formation in the vagina. In both strains, immunofluorescence of vaginal samples, taken 1 h after challenge, revealed an intense, MAb AF1-specific labelling of the yeast cells. This labelling was very scarce in fungal cells taken at 24 h and on subsequent days during the development of filamentous forms. Electron-microscopic gold immunolabelling observations showed that molecules carrying AF1-MP spanned the entire cell wall in the initial yeast cells but were absent on the cell surface and in the outermost, capsular layer of the cell wall of the germ tubes and filamentous forms. In both strains, at any time and for any form of intravaginal growth, AF1-MP was clearly expressed in the cytoplasm and cytoplasmic vesicles, and was fully incorporated into the inner layers of the cell wall. As seen by immunofluorescence, the vaginal fluid from C. albicans-infected rats did not hinder the expression of AF1-MP on the yeast cells surface in vitro. In electron-microscopic gold immunolabelling, a hypha-specific MAb (3D9) labelled the surface of the hyphal but not of the yeast cells of C. albicans harvested from rat vagina. Overall, these data strongly suggest that cell surface expression of MP antigen is modulated during intravaginal growth and morphogenesis of C. albicans.
Animals↗
Inactivation of high and low molecular weight single-chain urokinase-type plasminogen activator (pro-urokinase) by thrombin in the presence of thrombomodulin.
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Enzyme Precursors↗
Differential cell surface expression of mannoprotein epitopes in yeast and mycelial forms of Candida albicans.
The ultrastructural localization of mannoprotein constituents (MP) of the cell wall of yeast and hyphal forms of Candida albicans was studied by immunoelectron microscopy. To this aim, two monoclonal antibodies (mAbs AF1 and 1D10), recognizing distinct oligomannoside epitopes of MP molecules, and a second antibody coupled to colloidal gold, were employed. Preembedding methods revealed the presence of both AF1- and, albeit to a lesser intensity, 1D10-epitopes within the fibrillar, capsular layer of yeast cells of the fungus, provided this capsule was preserved and stabilized by treatment of whole cells with Concanavalin A. These cell surface-associated MP were absent in hyphal cells, despite the presence in these cells of a capsular layer not different in form and thickness from that present in yeast cells. Postembedding methods showed that both yeast and hyphal forms of growth of C. albicans synthesized the relevant mannoproteins and similarly incorporated them into inner layers of the cell wall. Apparently, however, the "export" of these MP to the outermost, capsular layer occurred in yeast but not in hyphal cells. These ultrastructural data, coupled with previous biochemical ones, emphasize form-associated patterns of MP expression on Candida cell surface. Given the value of MP as main immunogenic components of Candida, this differential expression could be a means by which the fungus evades from, or attenuates host's response.
Candida albicans↗
Thrombomodulin is a cofactor for thrombin degradation of recombinant single-chain urokinase plasminogen activator "in vitro" and in a perfused rabbit heart model.
Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) to a two-chain derivative (tcu-PA) fibrinolytically inactive. This reaction was accelerated in vitro by purified rabbit lung thrombomodulin in equimolar complex with thrombin. Polyclonal antibodies to rabbit thrombomodulin prevented this effect. We also observed that heparin and other sulfated polysaccharides had an accelerating effect on thrombin cleavage of recombinant scu-PA. Their effect was concentration-dependent and then reversed at high levels. The effect of heparin and heparan sulfate was independent and synergic with respect to thrombomodulin. All observations except the effect of heparin, could be confirmed in a Langendorff isolated rabbit heart model. From competition experiments carried out with scu-PA derivatives and mutants, we postulate that the amino-terminal sequence of rscu-PA, containing the epidermal growth factor (EGF)-like and the kringle domains is involved in the cofactor effect of thrombomodulin on scu-PA inactivation by thrombin. We conclude that a regulatory mechanism of scu-PA inactivation is present at the cell surface.
Animals↗
Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies.
Prourokinase is a plasminogen activator of 411 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction. The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain. In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (delta 125-prourokinase) in which 125 amino acids of the N-terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-1 for delta 125-prourokinase and 0.25 h-1 for prourokinase). Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure, non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and delta 125-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria. After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160,000 and 250,000 IU/mg protein, respectively, for prourokinase and delta 125-prourokinase. As with prourokinase, the deletion mutant delta 125-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.
Amino Acid Sequence↗