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M Magnani

Publications and source records attributed to M Magnani.

At least 19 recordsLinked to original sources
PubMed1997

Up-regulation of the ubiquitin-conjugating and proteolytic systems in murine acquired immunodeficiency syndrome.

Lymphoproliferation, chronic B-cell activation resulting in hypergammaglobulinemia, and profound immunodeficiency are prominent features of retrovirus-induced murine acquired immunodeficiency syndrome (murine AIDS). Here we demonstrate that in murine AIDS the ATP-dependent and ubiquitin-dependent proteolytic system is strongly affected, at least in the lymph nodes of infected mice. Solid-phase immunochemical assays show that the ubiquitin-conjugate pools increase by about threefold 10 weeks after infection, then decline slightly 15 weeks after infection to a twofold increase. Accumulation of ubiquitin conjugates is accompanied by induction of the ubiquitin-conjugating pathway, involving several carrier-protein isozymes (E2), mainly 14-kDa E2 and 17-kDa E2. Furthermore, accumulation of ubiquitin conjugates and induction of the conjugating system are coincident with an increase in the proteolytic activity supported by the 26S proteolytic complex. However, 15 weeks after infection, when the conjugation rate and levels of ubiquitin conjugates decrease, proteasome activity returns to values similar to those of the control, suggesting that a higher proteosomal activity is no longer needed. The concerted induction of the ubiquitin-conjugating and proteolytic systems in murine AIDS apparently does not involve the breakdown of viral products nor is it supported by virus-coded events, but probably arises as a cellular response to viral infection.

Adenosine Triphosphate↗
PubMed1997

Molecular bases of hexokinase deficiency.

Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1; HK) deficiency is a rare disease where the predominant clinical effect is nonspherocytic hemolytic anemia. We have previously shown that the only patient for which hexokinase deficiency has been so far investigated at molecular level is a double heterozygote carrying a T1667 --> C substitution on one HK type I allele and a 96 bp deletion (concerning nucleotides 577 to 672 in the HK cDNA sequence) in the other allele. To investigate whether these mutations found in the patient with the hexokinase variant referred to as 'HK-Melzo' could be associated with hexokinase deficiency, we have expressed in E. coli the wild-type human hexokinase type I and two different mutants carrying the T --> C nucleotide substitution at position 1667 and the nt 577-672 deletion, respectively. Wild-type human recombinant hexokinase is expressed in bacterial cells as a soluble catalytically active enzyme that, upon purification to homogeneity, exhibited the same kinetic properties of human placenta hexokinase type I. Both mutant hexokinases were also expressed as soluble recombinant proteins under the same conditions, but they showed an impaired catalytic activity with respect to the wild-type enzyme. In particular, the T1667 --> C substitution, causing the amino acid change from Leu529 to Ser, is responsible for the complete loss of the hexokinase catalytic activity, while the 96 bp deletion causes a drastic reduction of the hexokinase activity. Taken together, both mutations explain the hexokinase deficiency found in the patient with the 'HK-Melzo' variant.

Amino Acid Sequence↗
PubMed1997

Identification of alpha-spectrin domains susceptible to ubiquitination.

Previously, we demonstrated that alpha-spectrin is a substrate for the ubiquitin system and that this conjugation is a dynamic process (Corsi, D., Galluzzi, L., Crinelli, R., and Magnani, M. (1995) J. Biol. Chem. 270, 8928-8935). In this study, we mapped the sites of ubiquitination on erythrocyte alpha-spectrin. A peptide map of digested alpha-spectrin, previously submitted to in vitro 125I-ubiquitin conjugation, revealed the presence of four distinct labeled bands with Mr 40,000, 36,000, 29,000, and 25,500. Western blotting experiments using antibodies against each alpha-spectrin domain revealed that only IgG anti-alphaIII domain recognized the 125I-labeled ubiquitin peptide of 29 kDa, whereas the IgG anti-alphaV domain recognized the Mr 40,000 125I-ubiquitin-labeled peptide. The other two labeled bands of Mr 36,000 and Mr 25,500 were identified as tetra and tri multiubiquitin chains. Ubiquitination of the alphaIII and alphaV domains was further confirmed by anti-alpha-spectrin domain immunoaffinity chromatography. Endoprotease Lys C-digested spectrin conjugated previously to 125I-ubiquitin was incubated with antibodies against each trypsin-resistant domain of alpha-spectrin. Gamma counting of the radiolabeled antigen-antibody complexes purified by protein A chromatography showed labeling in the IgG anti-alphaIII and anti-alphaV complexes alone. Domain alphaIII is not associated with any known function, whereas domain alphaV contains the nucleation site for the association of the alpha and beta chains. Ubiquitination of the latter domain suggests a role for ubiquitin in the modulation of the stability, deformability, and viscoelastic properties of the erythrocyte membrane.

Binding Sites↗
PubMed1997

Targeting antiviral nucleotide analogues to macrophages.

Macrophages are important target cells for human immunodeficiency virus type 1 (HIV-1) infection. We have developed a drug targeting system for the selective delivery of phosphorylated nucleoside analogues to these phagocytosing cells. This system is based on the possibility of encapsulating the phosphorylated drugs into autologous erythrocytes and on the subsequent selective modification of their membranes to promote macrophage recognition and phagocytosis. Targeted delivery of phosphorylated nucleoside analogues to human, feline, and murine macrophages inhibits the infectivity of HIV-1, feline immunodeficiency virus, and LP-BM5 viruses more efficiently than the administration of the corresponding nucleoside analogues. In vivo administration of 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) encapsulated into autologous erythrocytes to LP-BM5-infected mice was found to reduce infectivity and disease progression. Furthermore, the simultaneous administration of AZT or ddC produced additive antiviral effects. The possibility of using red cells as drug targeting systems was useful for the design, synthesis, and delivery of new antiviral nucleoside analogues. As a prototype of these new drugs, di-(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophospha te (AZTp2AZT) was prepared. Although this drug in solution has the same antiviral activity as AZT, when administered encapsulated into erythrocytes it was several times more efficient in inhibiting the infectivity of human, feline, and murine immunodeficiency viruses. Thus, the availability of a drug targeting system for the selective delivery of antivirals to macrophages offers an additional possibility for the development of new drugs and of new combination antiviral therapies.

Animals↗
PubMed1997

Increased microbicidal activity of human monoblastoid cells upon long-term exposure to dideoxycytidine.

2',3'-Dideoxycytidine (ddC) is a nucleoside analogue currently used in AIDS therapy. We had previously found that long term exposure of U937 human monoblastoid cells to ddC induces the selection of drug-resistant cells (U937-R). In the present work we investigated some important biochemical properties and functional activities of these resistant cells. The results obtained show that U937-R maintained the properties of cell aggregation, adhesion and differentiation. Basal respiration, protein kinase C activity, superoxide anion release and intracellular free calcium were all increased in the drug-resistant line. Phagocytosis of fungi (Candida albicans) and bacteria (Staphylococcus aureus and Salmonella anatum) were similar in U937 and U937-R cells. Killing of C. albicans was significantly higher in drug-resistant cells (29.07 +/- 2.23% of killing vs 19.07 +/- 2.01 in the control; p < 0.001). Similarly, the bacterial killing was enhanced in U937-R cells (34.07 +/- 8.06% vs 22.60 +/- 4.41% in the control; p < 0.05). Thus, the results presented in this paper provide evidence of an increased microbicidal activity of human monocytic cells upon long term exposure to ddC, most likely due to an increased oxidative metabolism.

Blood Bactericidal Activity↗
PubMed1997

Red blood cells as delivery system for recombinant HSV-1 glycoprotein B: immunogenicity and protection in mice.

The immunotherapeutic potential of autologous red blood cells (RBC) coupled to the secretory form of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB1s) was examined with a mouse model of HSV-1 infection. C57BL/6 mice were immunized intraperitoneally with gB1s (0.05 microgram per dose) linked to RBC, or mixed with Freund's complete or bound to AlPO4 adjuvants (0.5 microgram per dose). Mice immunized with RBC coupled gB1s were protected against lethal and latent HSV-1 infection, and developed an anti-HSV antibody response, as measured by ELISA and HSV-1 neutralization assays, similar or higher than that elicited by the same antigen in Freund's complete adjuvant, which suggested that autologous RBC coupled to gB1s may provide an effective and safe method of immunization against HSV infection.

Adjuvants, Immunologic↗
PubMed1996

Encapsulation, metabolism and release of 2-fluoro-ara-AMP from human erythrocytes.

2-Fluoro-ara-AMP (fludarabine phosphate) is a purine analogue with anti-neoplastic activity in lymphoproliferative malignancies. Fludarabine phosphate activity and toxicity is schedule-dependent; multiple daily administrations (for five days) are more effective than single dose. We have encapsulated fludarabine phosphate in human erythrocytes and found that it is slowly released as fludarabine for more than four days. Encapsulated fludarabine phosphate does not affect erythrocyte metabolism and is rapidly converted by erythrocyte enzymes both to fludarabine with a Km of 0.4 mM and a Vmax of 20 nmol/min per g hemoglobin and to fludarabine diphosphate and triphosphate. The apparent Km for fludarabine monophosphate in the phosphorylation reaction was 0.4 mM and the Vmax 40 nmol/min per g hemoglobin. In the phosphorylation of 2-fluoro-ara-AMP to the di- and triphosphate derivatives, ATP was the phosphate donor with apparent Km of 0.12 and 1.0 mM, respectively. During incubations of 2-fluoro-ara-AMP-loaded erythrocytes at 37 degrees C fludarabine was found in equilibrium between the erythrocyte and the culture medium suggesting that permeation of the erythrocyte membrane is not rate-limiting. Thus, fludarabine phosphate-loaded erythrocytes might be used as a slow-delivery system for fludarabine administration in the treatment of lymphoid malignancies.

Adenosine Diphosphate↗
PubMed1996

Inhibition of murine AIDS by reduced glutathione.

The imbalance of the redox state in cells and body fluids in HIV-1-infected patients may result in progression of the disease as well as in immunologic disfuctions. In this report, we have evaluated whether the direct administration of high doses of reduced glutathione (GSH) exerts any antiviral activity and/or improves immune functions in a murine immunodeficiency animal model. Intramuscular administration of 50 or 100 mg GSH/mouse for five consecutive days weekly to LP-BM5-infected mice did not show local or systemic signs of acute toxicity. During the first 3 weeks from infection, a period in which clinical signs of disease were not yet detectable, GSH significantly reduced the viral load in lymph nodes and spleen as evaluated by a PCR semiquantitative assay of the proviral DNA content. At 10 weeks a GSH concentration-dependent reduction of splenomegaly, lymphadenopathy and hypergammaglobulinemia was evident in all treated mice. Evaluation of proviral DNA content showed that GSH was effective in inhibiting LP-BM5 infectivity in lymph nodes, spleen, and bone marrow at 100 mg/day, while it was less effective when administered at 50 mg/day. At 10 weeks some animals receiving the highest GSH dose died, thus only the mice receiving 50 mg GSH were followed up to 15 weeks without signs of toxicity. In this case, almost not significant differences among infected untreated or treated animals were observed. Thus, GSH is effective in reducing the proviral DNA load in the first period of infection. These data and the failure of sulfhydril supplementation to further counteract the progression of disease after 10 weeks of infection suggest that combinations of GSH and other antiviral agents may be useful for improving current antiviral therapies.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Inhibition of murine AIDS by combination of AZT and dideoxycytidine 5'-triphosphate.

SUMMARY: A combination of antiretroviral drugs acting on different cell types (lymphocytes and macrophages) was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS). In a first experiment, C57BL/6 mice were infected with a single i.p. administration of LP-BM5 and treated with 0.125 or 0.25 mg/ml AZT in drinking water for 3 months. AZT treatment was found to reduce lymphadenopathy (60 and 65 percent, respectively), splenomegaly (37 and 50 percent, respectively), and hypergammaglobulinemia (6 and 50 percent, respectively). Furthermore, at the highest AZT concentration, BM5d proviral DNA content in lymph nodes and in the spleen showed a reduction of 78 and 70 percent, respectively, compared to untreated animals. In a second experiment, infected mice were treated with AZT (0.25 mg/ml in drinking water) and with 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) encapsulated into autologous erythrocytes for macrophage protection. Combined treatments resulted in a further reduction of lymphadenopathy (a further 33 percent with respect to the single treatment of AZT) and splenomegaly (a further 28 percent respect to the single treatment of AZT) but not of gammaglobulinemia. Proviral DNA in lymph nodes and spleen showed a reduction of 82 and 77 percent, respectively, compared to infected mice. Stimulation index of T cells was also significantly increased in animals receiving both treatments versus AZT only. In conclusion, the selective administration of antiviral drugs that preferentially protect different cell types seems to provide additional advantages compared to single-agent therapy.

Animals↗
PubMed1996

Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection.

The infectivity and replication of human (HIV-1), feline (FIV), and murine (LP-BM5) immunodeficiency viruses are all inhibited by several nucleoside analogues after intracellular conversion to their triphosphorylated derivatives. At the cellular level, the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations a new nucleoside analogue homodinucleotide, di(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphat e (AZTp2AZT), was designed and synthesized. AZTp2AZT was a poor in vitro inhibitor of HIV reverse transcriptase, although it showed antiviral and cytotoxic activities comparable to those of the parent AZT when added to cultures of a HTLV-1 transformed cell line. AZTp2AZT encapsulated into erythrocytes was remarkably stable. Induction of erythrocyte-membrane protein clusterization and subsequent phagocytosis of AZTp2AZT-loaded cells allowed the targeted delivery of this impermeant drug to macrophages where its metabolic activation occurs. The addition of AZTp2AZT-loaded erythrocytes to human, feline, and murine macrophages afforded almost complete in vitro protection of these cells from infection by HIVBa-L, FIV, and LP-BM5, respectively. Therefore, AZTp2AZT, unlike the membrane-diffusing azidothymidine, acts as a very efficient antiretroviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.

Animals↗
PubMed1996

Azidothymidine homodinucleotide-loaded erythrocytes and bioreactors for slow delivery of the antiretroviral drug azidothymidine.

A new Azidothymidine derivative, di-(thymidine-3'- azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphate (AZTp2AZT), was encapsulated in human erythrocytes according to a conservative procedure of hypotonic shock-isotonic resealing and reannealing. Like in erythrocyte lysates supplemented with 1 mM ATP, intact red cells too were found to convert AZTp2AZT to 3'-Azido-3'-deoxythymidine which was then released linearly in plasma. The major metabolic pathway involved in this conversion was the symmetrical hydrolysis of AZTp2AZT to yield two 3'-Azido-3'- deoxythymidine-5'-phosphate molecules which were then dephosphorylated to 3'-Azido-3'-deoxythymidine. At late times of incubation, also a limited asymmetrical hydrolysis of AZTp2AZT became apparent in the intact erythrocytes, yielding 3'-Azido-3'-deoxythymidine-5'-diphosphate that was then converted to the triphosphorylated derivative. Therefore, erythrocytes loaded with AZTp2AZT act "in vitro" as bioreactors ensuring sustained and potentially useful release of 3'-Azido-3'-deoxythymidine.

Dideoxynucleotides↗
PubMed1996

High-level expression and purification of a human "mini"-hexokinase.

Human hexokinase type I is a 100-kDa enzyme with the catalytic site located in the C-terminal domain. We had previously expressed this domain in Escherichia coli, however only a small amount of the recombinant enzyme was catalytically active. To overcome this problem we have now expressed the "mini"-hexokinase using the pET expression system. An average of 1000 U of enzyme per liter of culture was obtained. The recombinant enzyme was purified to homogeneity by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The enzyme was unstable under ultrafiltration; thus, a multicolumn purification procedure was developed in order to avoid the ultrafiltration steps. The recombinant "mini"-hexokinase was found to have the same kinetic properties as the entire enzyme. Using the method described, the enzyme can be obtained in sufficient quantities for biophysical and biochemical investigations.

Blotting, Western↗
PubMed1995

2',3'-Dideoxycytidine metabolism in a new drug-resistant cell line.

2',3'-Dideoxycytidine (ddC) is a nucleoside analogue that inhibits human immunodeficiency virus type 1 (HIV-1) replication in vitro and is currently used in the therapy of acquired immune deficiency syndrome (AIDS). This compound exerts a delayed cytotoxicity due to inhibition of mitochondrial DNA (mDNA) synthesis. Long-term exposure of U937 human monoblastoid cells to ddC resulted in a time- and concentration-dependent decrease in mDNA content and Rhodamine 123 fluorescence. However, after 2 months on 0.1 microM ddC, a drug-resistant cell line (U937-R) with 66% of the normal amount of mDNA was isolated. ddC transport in U937 and U937-R cell lines was similar. In contrast, U937-R accumulated ddC phosphorylated derivatives at a much lower rate and to a reduced concentration into acid-soluble material. The rate of 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) formation in U937-R cells was almost one-third of that measured in normal cells, although the rate of ddCTP catabolism was similar in both cell lines. Dideoxyliponucleotide (ddCDP-choline and ddCDP-ethanolamine) formation was also much slower (between one-half and one-third as fast) in U937-R than in control cells, although catabolism occurred at similar rates. ddC was phosphorylated by a cytoplasmic deoxycytidine kinase in both cell lines. This enzyme showed Km values for ddC of 80 +/- 7 and 140 +/- 9 microM in U937 and U937-R cells respectively. Furthermore, Vmax was 12 +/- 1.1 and 7.8 +/- 0.5 pmol/min per mg of protein in U937 and U937-R. Thus resistance to ddC toxicity may be due to cells' decreased ability to accumulate intracellular ddC anabolites, which may depend on cytoplasmic deoxycytidine kinase.

Biological Transport↗
PubMed1995

Ubiquitin is conjugated to the cytoskeletal protein alpha-spectrin in mature erythrocytes.

Ubiquitination of red blood cell (RBC) proteins was investigated by encapsulation of 125I-ubiquitin into human erythrocytes using a procedure of hypotonic dialysis, isotonic resealing, and reannealing. Incubation (37 degrees C, up to 2 h) of 125I-ubiquitin-loaded cells resulted in the recovery of 125I-ubiquitin with the cytosolic proteins (9.22 +/- 0.4 micrograms/ml RBC) and conjugated to membrane proteins (2.18 +/- 0.05 micrograms/ml RBC). This conjugation was time-dependent, and the predominant membrane protein band that became labeled showed an apparent molecular mass of 240 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Western blotting experiments with three different anti-ubiquitin antibodies revealed that this protein is also ubiquitinated in vivo. Cell-free experiments have shown that fraction II (a DEAE-bound protein fraction eluted by 0.5 M KCl) prepared from both mature erythrocytes and reticulocytes is able to conjugate ubiquitin to this protein. Ubiquitin conjugation was ATP-dependent (Km 0.09 mM), time-dependent, and fraction II-dependent (8 +/- 0.5 pmol of 125I-ubiquitin/h/mg of fraction II). Isolation of the major RBC membrane protein that is ubiquitinated was obtained by using biotinylated ubiquitin. Membrane proteins, once ubiquitinated with this derivative, were extracted and purified by affinity chromatography on immobilized avidin. The major components retained by the column were two peptides of molecular masses 220 and 240 kDa. Both proteins are recognized by a monoclonal anti-spectrin antibody, but only the 240-kDa component is detected by streptavidin peroxidase conjugate. That indeed the ubiquitinated membrane protein of 240-kDa is alpha-spectrin was confirmed by immunoaffinity chromatography using 125I-ubiquitin and a monoclonal anti-spectrin antibody. Antigen-antibody complexes were purified by protein A chromatography and analyzed by SDS-PAGE and autoradiography. Again two bands of 240 and 220 kDa were eluted (alpha- and beta-spectrin), but only one band corresponding to the electrophoretic mobility of alpha-spectrin was detected by autoradiography. Thus, alpha-spectrin is a substrate for the ATP-dependent ubiquitination system, suggesting that the cytoskeleton is covalently modified by ubiquitination both in reticulocytes and mature RBC.

Biotin↗