An erythroid-specific exon is present in the human hexokinase gene.
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DNA, Complementary↗
Biomedical subjects
M Magnani
Publications and source records attributed to M Magnani.
Expression, purification, and characterization of a recombinant erythroid-specific hexokinase isozyme.
Hexokinase type I (HK I; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the predominant glucose-phosphorylating enzyme in red blood cells, exists in human erythrocytes in multiple molecular forms that differ in isoelectric point and are separable by ion-exchange chromatography. The major forms, designated HK Ia, Ib and Ic, have similar kinetic properties but are characterized by different age-dependent decay and different intracellular distribution in reticulocytes. HK Ib, which elutes between HK I and HK II in the DEAE ion-exchange chromatography, appears to be unique to RBCs and different from any other hexokinase isozyme previously described. Indeed, Murakami and Piomelli recently reported the presence of a specific HK isozyme (named HKr) expressed in K562 cells and in human reticulocytes and, moreover, the resolution of the human HK I gene structure provided the direct evidence of an erythroid-specific exon 1. To further investigate the microheterogeneity of HK I in human RBCs we established a prokaryotic expression system for the HKr isozyme, using the pET plasmid, inducible with IPTG. The recombinant HKr, expressed in bacterial cells as a catalytically active enzyme, was purified to homogeneity by a combination of DEAE ionexchange chromatography followed by hydrophobic interaction chromatography and dye-ligand affinity chromatography. The kinetic and chromatographic properties of the homogeneous recombinant HKr suggest that this erythroid-specific HK isozyme in fact corresponds to the HK isoform previously described in human RBCs and referred to as HK Ib.
Erythrocytes↗
Use of the Parental Bonding Instrument to compare interpretations of the parental bond by adolescent girls with restricting and binge/purging anorexia nervosa.
The Parental Bonding Instrument (PBI) was administered to 62 adolescent female patients (mean age 14.3): 35 with restricting-type anorexia nervosa (RAN), and 27 with binge/purging-type anorexia nervosa (B/PAN) according to the DSM-IV criteria, all at onset and initial diagnosis. The PBI was also administered to a control group (55 subjects) and 22 patients with Crohn's disease or celiac disease. The three groups were matched for age and socioeconomic status. The RAN and B/PAN patients gave significantly different interpretations of the parental bond (PB): for the former, it was adequate with both parents, for the latter it was inadequate, especially with the father. The fact that these differences exist at the onset of anorexia prior to any possible effect of therapy suggests that its structure is determined by different family dynamics.
Adolescent↗
Expression of hexokinase mRNA in human hippocampus.
The expression of hexokinase messenger RNA was evaluated in human hippocampus using in situ hybridization technique. The message showed an uneven distribution with high levels present in the granular cell layer of the dentate gyrus and CA3 region. The detection of specific transcripts was also observed in the lateral geniculate nucleus, the dentate polymorphic cell layer and the parahippocampal gyrus. The data suggest that, in the hippocampus, the expression of hexokinase is higher in neurons than in glial cells and that the rate of glucose metabolism may display considerable variations in the different subregions of this area.
Adult↗
Enzymatic properties of overexpressed human hexokinase fragments.
Full-length hexokinase (HK; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), a truncate form of the enzyme lacking the first 11 amino acids (HK-11aa) and the 50 kDa C-terminal half ('mini'-HK) containing the catalytic domain, were overexpressed and purified to homogeneity to investigate the influence of the N-terminal region of human hexokinase type I (HK) on its regulatory properties. All forms of the enzyme are catalytically active with the HK-11aa being the most active. All the forms of HK showed the same affinity for glucose and MgATP and were also inhibited by glucose 6-phosphate (Glc 6-P) competitively vs. MgATP with similar Kis (28.5-37 microM). Glucose 1,6-bisphosphate (Glc 1,6-P2) was also a strong inhibitor of all HKs without significant differences among the different truncate forms of the enzyme (Kis 49.5-59 microM). At low concentrations (0-3 mM), Pi was able to reverse the sugar phosphate inhibition of the full-length HK and HK-11aa but not of the 'mini'-HK. In contrast, at high concentrations Pi was an inhibitor of all the hexokinases investigated. These findings confirm that Pi has a low affinity binding site on the C-terminal of HK while counteracts glucose 6-phosphate inhibition by binding to or requiring the N-terminal half of the enzyme. The first 11 N-terminal amino acids influence the specific activity of HK but are unable to affect the kinetic properties investigated.
Dose-Response Relationship, Drug↗
AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: homologous erythrocytes as a delivery system for preferential immunization with putative protective antigens.
Feline immunodeficiency virus (FIV) is a useful model for testing of criteria for AIDS vaccine development. In the protocol we adopted, we used a primary isolate of FIV as a source of antigen and, for challenge, plasma from cats infected with the homologous virus never passaged in vitro. Cat erythrocytes (RBC) were coated with the surface components of freshly harvested and purified FIV by means of biotin-avidin-biotin bridges and used to immunize specific-pathogen-free cats (four doses at monthly intervals; total amount of FIV antigen administered per cat, approximately 14 microg). Immunized cats developed moderate levels of antibodies directed mainly to surface components of the virion and clearly evident lymphoproliferative responses. Four months after the last dose of immunogen, FIV-immunized cats and control cats immunized with bovine serum albumin-coated RBC were challenged. Judged from the results of the subsequent 12-month follow-up, FIV-immunized cats exhibited at least some degree of protection. However, following rechallenge, most of the FIV-immunized animals became virus positive in spite of a booster immunogen dose given 2 months before the second challenge.
AIDS Vaccines↗
Competitive PCR for quantification of BM5d proviral DNA in mice with AIDS.
Murine AIDS in C57BL/6 mice is caused by a unique mixture of murine leukemia viruses. We report the use of a competitive PCR to detect and quantitate BM5d proviral DNA. This assay allowed discrimination among endogenous wild-type murine retroviruses and BM5d sequences. Furthermore, the method was subsequently used to evaluate the amount of BM5d in infected mice and in infected AZT (zidovudine)-treated mice, providing an effective way to quantitatively evaluate drug efficacy in the murine AIDS model.
Animals↗
An ex vivo erythrocyte model for investigating oxidative metabolism.
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Animals↗
[The heart of anorexic adolescents].
BACKGROUND: Anorexia nervosa (AN) is often associated with cardiac changes, such as thinning of the left ventricle (LV), reduction of LV mass, abnormalities of mitral valve function and systolic dysfunction. Some authors have reported QT interval prolongation and sudden death in these patients. METHODS: We studied 23 adolescent females, aged 14.7 +/- 2 years (mean +/- SD), with AN. Serum electrolytes, proteins and albumin were measured in all patients. Electrocardiogram, Doppler-echocardiogram and chest X-rays were also performed on the same day. Eighteen patients were also examined via indirect calorimetry (difference from basal metabolic rate) and 21 underwent dosage of thyroid hormones. RESULTS: The patients, who were of normal height (159 +/- 7.4 cm), were underweight (36 +/- 4.8 kg) and had a body mass index (BMI) of less than 19 (14.2 +/- 1.3). Serum electrolytes, proteins, albumin and chest X-rays were substantially normal in all patients; 74% of them showed reduction of FT3. The calorimetry was reduced (-27.1 +/- 10.6%) with the exception of one patient. Resting heart rate was 58 +/- 12 bpm. We found normal values for PR, QRS, QT (0.41 +/- 0.03 s1/2) and QTc intervals (0.40 +/- 0.03 s1/2) and QT dispersion (40.9 +/- 14.1 ms). Echocardiography showed a reduction in the dimensions of the interventricular septum (52% of patients), LV free wall (61%), left atrium (31%) and LV mass (61%). Fractional shortening was normal in all but one patient. In 61% of cases, there was mild or moderate pericardial effusion that was clinically silent and inversely related to BMI (r = -0.38, p 0.08, ns), to calorimetry (r = -0.56, p < 0.0055), to FT3 (r = -0.53, p < 0.05) and to sodium concentration (r = -0.43, p 0.04). CONCLUSIONS: Teen-agers with AN often show a reduction in LV thickness and mass, as well as clinically silent pericardial effusion that is inversely related to BMI, calorimetry, FT3 and sodium serum concentrations. We did not find any prolongation of QTc interval or of QT dispersion.
Adolescent↗
Erythrocyte engineering for drug delivery and targeting.
A new procedure for the encapsulation of non-diffusible drugs into human erythrocytes was developed. With as little as 50 ml of blood and by using a new apparatus, it was possible to encapsulate a variety of biologically active compounds into erythrocytes in 2 h at room temperature and under blood-banking conditions. The process, which is based on two sequential hypotonic dilutions of washed red cells followed by concentration with a haemofilter and resealing of red cells, allows a 35-50% cell recovery and approx. 30% encapsulation of added drugs. The resulting processed erythrocytes have a normal survival in vivo and can be modified further, with the same apparatus, to increase their recognition by tissue macrophages to perform as a drug-targeting system. The new equipment designed and built for this procedure was named 'Red Cell Loader'.
Cell Membrane Permeability↗
Use of nonvolatile buffers in liquid chromatography/mass spectrometry: advantages of capillary-scale particle beam interfacing.
The use of nonvolatile buffers like phosphate and citrate is usually incompatible with mass spectrometric detection or may heavily interfere with ion generation. In the case of conventional HPLC flow rates, the continuous buffer deposition produces fouling of the mass spectrometer ion source and may worsen the performance of any LC/MS interface as well. Our research group demonstrated that reducing the mobile phase flow rate in a particle beam interface improves the nebulization and enhances the overall performance. We have also assumed that such a modification may better handle potentially instrument harmful solvents or nonvolatile HPLC buffers. Since the presence of salts does not interfere with the electron ionization process, the particle beam interface can be considered, in principle, particularly suitable for those chromatographic separations that benefit from nonvolatile buffers. The microscale flow rate interface, developed in our laboratory, generates an aerosol from 1 microL/min of mobile phase flow rate. Under these conditions, the absolute amount of a nonvolatile buffer actually introduced into the system is approximately 1/1000 of that carried by a conventional HPLC column, slowing its deposition. This assumption was verified with a real-world application requiring the use of a phosphate buffer at a maximum concentration of 10 mM. It involved the determination of dexamethasone, an anti-inflammatory drug, in human blood after its administration to a patient. Several crucial mass spectral and chromatographic parameters were monitored during a 35-day period of intense use. Neither appreciable signal modification nor evident buffer deposition was observed during the test period, with only a normal and limited run-by-run and day-by-day variation of the instrument response.
Anti-Inflammatory Agents↗
Antiretroviral effect of combined zidovudine and reduced glutathione therapy in murine AIDS.
A combination of antiretroviral drugs acting at different points in the virus replication cycle was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS). Intramuscular administration of high doses of reduced glutathione (GSH, 100 mg/mouse/day) and AZT (0.25 mg/ml in drinking water) was found to reduce lymphoadenopathy (92%), splenomegaly (80%), and hypergammaglobulinemia (90%) significantly more than AZT alone. Combined treatment resulted in a reduction in proviral DNA content of 69, 66, and 60%, respectively, in lymph nodes, spleen, and bone marrow. Furthermore, the stimulation index of B cells was also significantly higher in animals receiving GSH and AZT whereas additional responses were not observed in the T cell stimulation index and blood lymphocyte phenotype analyses. In conclusion, the administration of high doses of GSH and AZT, a new combination of antiviral drugs, seems to provide additional advantages compared to single-agent therapy.
Animals↗
2',3'-Dideoxycytidine cytotoxicity in human macrophages.
Human macrophages when cultured for several weeks in the presence of therapeutically relevant 2',3'-dideoxycytidine (ddC) concentrations show a time-dependent decay in mitochondrial DNA content. This decay is associated with a reduction of Rhodamine 123 fluorescence, a marker for mitochondrial membrane potential suggesting that impairment of mitochondrial functions occurs. Mitochondrial metabolic impairment was confirmed by direct evaluation of lactate production, which is markedly increased in cells treated with ddC. The activity of protein kinase C and intracellular free Ca2+ upon addition of phorbol 12-myristate 13-acetate (PMA) were lower in the drug-treated cells compared to controls. A 50% reduction in O2-release was also found upon PMA stimulation. Fluorescent latex beads, yeast and bacteria phagocytosis were normal, but intracellular bacteria killing was markedly impaired in ddC-exposed macrophages. Thus, ddC exerts a delayed mitochondrial toxicity also on differentiated macrophages with impairment of several metabolic properties and O2 production causing a reduced ability of these phagocytic cells to kill phagocytosed bacteria.
Anti-HIV Agents↗
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↗
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↗
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↗
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↗
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↗