Presence of interleukin-2 receptor gamma chain transcript in AML blasts.
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Blotting, Northern↗
Biomedical subjects
A Rigo
Publications and source records attributed to A Rigo.
Purification and characterization of amine oxidase from soybean seedlings.
A simple and rapid procedure for purification of soybean seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation was purified by ion-exchange chromatography on a cellulose phosphate column and batch affinity chromatography on 6-aminohexyl-Sepharose. Cyclohexylamine, a competitive inhibitor, was utilized to elute the enzyme. A homogeneous enzyme was obtained with a yield higher than 25%, the content of minor components being < or = 2%. The M(r) estimated by gel filtration is 113,000 and 77,000 by sodium lauryl sulfate-polyacrylamide gel electrophoresis. The enzyme is a dimer and contains two Cu2+ ion per molecule. Its EPR spectrum is typical of Cu2+ in a tetragonal symmetry. The enzyme oxidizes cadaverine at high rate, the specific activity being 4.3 mukat/mg. Molecular, spectroscopic, and kinetic properties of this enzyme are reported.
Amine Oxidase (Copper-Containing)↗
Uptake and life time of fluoride ion in rats by 19F-NMR.
19F nuclear magnetic resonance (NMR) was utilized to obtain information on the uptake and half-life time of fluoride ion in rats. Changes in tissue fluoride level after acute loading were monitored over time in blood and tissue homogenates obtained from liver and brain. The rate of fluoride elimination from various tissues was roughly similar, following in all cases a first-order kinetic rate law. The F- concentration in brain was about 20% of that found in liver, indicating a reduced fluoride diffusion across the blood-brain barrier. In vivo F- spectra were obtained in rat brain in few minutes with a good signal-to-noise ratio; this confirms the possibility of extending the use of F- as a probe of biomolecules to in vivo applications.
Animals↗
IL-8 mRNA expression and IL-8 production by acute myeloid leukemia cells.
Purified leukemic cells from 30 acute myeloid leukemia (AML) cases at diagnosis were investigated for the presence of interleukin 8 (IL-8) mRNA by Northern blot analysis. IL-8 specific transcripts were detected in uncultured blasts in 14/30 cases, 10/14 from patients with M4-M5 and 4/14 from cases with M0-M3 morphology. The transcript expression was associated with the detection of IL-8 molecule in blast cells by immunostaining performed on cytospin preparations. After 24-hour culture, a strong up-regulation or the appearance in cases negative before culture of IL-8 mRNA was observed in all cases tested, and culture supernatants contained high amounts of IL-8. Our data demonstrate that leukemic cells in AML are equipped with the functional apparatus for IL-8 production. Since IL-8 displays a wide range of biological activities, including the regulation of some membrane molecules relevant to adhesion and migration processes, its production by AML blasts might be of relevance for the pattern of leukemic growth.
Acute Disease↗
Alpha (p55) and beta (p75) chains of the interleukin-2 receptor are expressed by AML blasts.
In the present study, we have investigated the leukemic cells obtained from 16 patients with acute myeloid leukemia (AML) at diagnosis for the membrane expression of p55 (alpha) and p75 (beta) interleukin-2 receptor (IL-2R) chains using specific monoclonal antibodies (mAbs), as well as for the presence of their transcripts using Northern blot analysis. In addition, immunoprecipitation of the p75 membrane molecule with TU27 and Mik-beta 1 mAbs was carried out in selected cases. The p75 IL-2R beta transcripts were detected in all cases, whereas the membrane p75 molecule was demonstrable by flow cytometry in three cases. However, data from the immunoprecipitation analysis suggest that the lack of the p75 IL-2R detection by flow cytometry might be caused by the low density of molecules per cell rather than the fact that the specific mRNA is not translated into the p75 surface molecule. In addition, a consistent membrane positivity with an anti-p55/CD25 mAb, present on fresh uncultured blasts in 37.5% of the cases, became detectable after short-term culture in 75% of cases. In each individual case, a strict correlation was found between membrane CD25 reactivity and the expression of p55 mRNA. Taken together, our data suggest that the expression of both alpha (p55) and beta (p75) IL-2R molecules is a common feature of leukemic cells in AML, and provide new arguments for reassessing the possible role of IL-2 in leukemic growth.
Acute Disease↗
Effect of phosphate ion on the activity of bovine plasma amine oxidase.
The system bovine plasma amine oxidase-polyamine-phosphate ion was investigated by activity measurements and 31P NMR spectroscopy. Lineweaver-Burk plots showed that phosphate ion, under physiological conditions, is an apparent competitive inhibitor of bovine plasma amine oxidase. While NMR measurements of the T1 of 31P do not suggest the binding of phosphate to/or near the paramagnetic Cu(II) sites of bovine plasma amine oxidase, the chemical shift dependence of 31P on spermidine concentration indicates the formation of a spermidine-phosphate complex. The value of the dissociation constant of this complex was found 18.5 +/- 1.4 mM, at pH 7.2, by NMR, in good agreement with the value 17.0 +/- 0.8 mM calculated from activity measurements, assuming the enzyme activity is proportional to the free amine concentration, under second order conditions. Our data suggest that the decrease of the free spermidine, due to the binding of phosphate ion, is responsible of the observed inhibition of bovine plasma amine oxidase.
Amine Oxidase (Copper-Containing)↗
Highly concentrated urine-purified Tac peptide fails to inhibit IL-2-dependent cell proliferation in vitro.
Tac peptide, i.e., the p55 chain of the human interleukin-2 receptor (IL-2R) complex, is detectable as a soluble from (sIL-2R) in normal sera and, at increased levels, in patients with different diseases. Since several immunological abnormalities are observed in most conditions associated with an increase in sIL-2R levels, a down-regulatory effect on IL-2-dependent functions has been postulated as a consequence of binding and functional block of IL-2 by the excess of sIL-2R. To test this hypothesis, we purified sIL-2R from the urine of a patient with hairy cell leukemia and investigated the possible inhibitory effect of this peptide on the in vitro IL-2-induced cell proliferation. The urine-purified molecule was detectable by the specific immunoassay utilized to measure the serum Tac peptide and was constructed by a single polypeptide of about 50 kDa which was able to bind IL-2. Experiments performed with the IL-2-dependent murine CTLL-2 cell line and with PHA-stimulated human peripheral blood mononuclear cells showed that the purified sIL-2R at concentrations up to about 300 nM was unable to block IL-2-dependent cell proliferation. According to these data, which can be explained by the low affinity for IL-2 of the p55 IL-2R chain, it seems unlikely that in vivo the soluble Tac peptide can exert a down regulatory effect on IL-2-induced phenomena through a functional block of IL-2.
Animals↗
ATP-stimulated glutamate-dependent calcium uptake by rat synaptosomes.
The entry of Ca2+ in rat synaptosomes was followed with a Ca(2+)-selective electrode. Extracellular ATP is necessary for the entry which is a function of synaptosomal protein, free Ca2+ and glutamate concentrations. Ketamine, glycine and kainate have negligible effect while quisqualate slightly inhibits the uptake of Ca2+ in the presence of glutamate. The added ATP is hydrolyzed by the synaptosomes through an ouabain-insensitive ecto-ATPase affected by the presence of Ca2+, glutamate and, to a slight extent, NMDA.
Adenosine Triphosphate↗
Added ATP influences some responses of rat synaptosomes to glutamate.
ATP added externally to rat synaptosomes activated uptake of both Ca2+ and glutamate which was partially accounted for by the uptake phenomena of synaptic vesicles and mitochondria, as shown by using specific inhibitors of the latter. Increasing concentrations of glutamate stimulated Ca2+ entry linearly, as shown by using 45Ca or a Ca-specific electrode. The processes of glutamate and Ca2+ uptake shared some common features and their ATP-dependence may be correlated with an ouabain-insensitive synaptosomal ectonucleotidase activity measured by a 31P-NMR or a luminometric technique. The ATP hydrolysis catalysed by the synaptosomes was activated by both Ca2+ and glutamate. The present synaptosomal activities may represent a model for studying the modulatory effects of ATP on the glutamatergic neurotransmission.
Adenosine Triphosphate↗
Isolation of amine oxidase from bovine plasma by a two-step procedure.
A novel method for isolation of amine oxidase from bovine plasma is reported; it involves a two-step procedure, namely ammonium sulfate fractionation and affinity chromatography with elution by aniline, which is a competitive inhibitor of the enzyme. A homogeneous enzyme, characterized by a specific activity of 0.44 U/mg, was obtained with a yield higher than 50%.
Amine Oxidase (Copper-Containing)↗
Modification of some markers of inflammation during treatment for acute respiratory exacerbation in cystic fibrosis.
An objective approach for monitoring the treatment of acute pulmonary exacerbation in cystic fibrosis was evaluated. Eleven biochemical markers of inflammation (erythrocyte sedimentation rate, neutrophil count, C-reactive protein, alpha-1 antitrypsin, haptoglobin, ceruloplasmin, fibronectin, alpha-1 glycoprotein, alpha-2 macroglobulin, C3, granulocyte elastase and anti-Pseudomonas IgG) were measured in blood serum and plasma from 46 cystic fibrosis patients with chronic Pseudomonas aeruginosa colonization before and after treatment. The overall outcome in each patient was evaluated by means of a pondered sum of clinical, chest X-ray and lung function scores. Biochemical markers were related to the overall clinical improvement: haptoglobin, ceruloplasmin, fibronectin and alpha-1 glycoprotein showed a good sensitivity (64-70%), specificity (60-70%) and positive predictive value (86-89%). Granulocyte elastase showed a similar sensitivity (67%) and positive predictive value (85%) but a lower specificity (33%). The negative predictive value was generally poor (32-39%). Our data suggest that the combined measurement of some markers of inflammation and of conventional clinical parameters, may help in evaluating the efficacy of anti-infective treatment in cystic fibrosis.
Acute Disease↗
The CD4 molecule belongs to the phenotypic repertoire of most cases of acute myeloid leukemia.
In the present study fresh leukemic cells obtained from 23 patients with acute myeloid leukemia (AML; FAB subtypes: three M1, five M2, two M3, five M4, eight M5) were investigated for the membrane expression of the CD4 molecule by cytofluorimetric analysis with an anti-CD4 monoclonal antibody (mAb). In 15 cases the presence of the CD4 mRNA was also investigated using Northern blot analysis. Membrane expression of the CD4 molecule was demonstrated in 19 out of 23 cases, and it was found to be weaker than in CD4+ lymphocytes and monocytes obtained from normal controls. Full-length CD4 mRNA was detected in 12 out of 15 (80%) cases, and AML cells positive for CD4 mRNA expression also expressed the CD4 antigen. Since the CD4 molecule expressed by T cells is associated with p56lck, a member of the src family of intracellular tyrosine kinases, we investigated whether the CD4 molecule expressed by myeloid blasts is also associated with a tyrosine kinase activity. In vitro kinase assays performed on anti-CD4 immunoprecipitates from lysates of myeloid leukemia cells from four CD4+ cases were negative for the presence of a tyrosine kinase activity. This finding was not due to the lack of expression of members of the src family since we were able to detect at least p60src and p59fyn in myeloid leukemia cells. According to our results, the CD4 molecule seems to belong to the phenotypic repertoire of most AML, irrespective of their FAB subtypes. However, in myeloid blasts this molecule is not associated with a tyrosine kinase activity as it occurs in T lymphocytes.
Blotting, Northern↗
19F NMR study of the interactions of fluoride with superoxide dismutase and hemoglobin in erythrocytes.
F- added to an erythrocyte suspension shows two separated resonances arisen from intra and extracellular compartments. Cu, Zn superoxide dismutase dominates the longitudinal relaxation rate of the intracellular F- resonance, while diamagnetic interactions with hemoglobin contribute mainly to the transversal relaxation rate.
Erythrocytes↗
Determination of superoxide dismutase activity by the polarographic method of catalytic currents in the cerebrospinal fluid of aging brain and neurologic degenerative diseases.
The activity of the superoxide dismutase was measured by the polarographic method of catalytic currents in the cerebrospinal fluid of patients with age-related neurologic degenerative diseases, namely, amyotrophic lateral sclerosis and Alzheimer's disease, and of a reference group of normal subjects. The superoxide dismutase activity was found to increase with age in reference subjects (r = 0.81) while no significant correlation was found in amyotrophic lateral sclerosis and Alzheimer's disease patients. The activity mean values were significantly lower (P less than 0.01) in patients with neurologic degenerative diseases than in the reference subjects. The changes of superoxide dismutase activity in the aging brain and in age-related neurologic degenerative diseases are discussed.
Adult↗
Effects of vitamin D metabolites and bisphosphonates on fibronectin release from monocyte-derived macrophages.
Fibronectin release from cultured macrophages, derived from human blood monocytes, was measured during incubation of the cells with increasing concentrations of vitamin D3 metabolites or of aminobutane bisphosphonate (AHButBP) or dichloromethylene bisphosphonate (Cl2MBP), two powerful inhibitors of bone resorption. The bisphosphonates significantly inhibited fibronectin release at 10(-8) M concentration and this inhibition was almost complete at 10(-5) M concentration. Opposite results were observed when the cells were incubated with vitamin D3 metabolites: the stimulation of fibronectin release was specific for 1,25-dihydroxyvitamin D3 relative to other vitamin D3 metabolites (1,25-dihydroxyvitamin D3 greater than 25-hydroxyvitamin D3 greater than 24,25-dihydroxyvitamin D3).
24,25-Dihydroxyvitamin D 3↗
A more objective approach to the evaluation of antimicrobial therapy in cystic fibrosis.
Thirteen biochemical markers of infection and inflammation were measured during anti-Pseudomonas therapy in cystic fibrosis (CF) patients with respiratory exacerbation. The assessment of some of these markers is thought to be helpful in the evaluation of efficacy of antibiotic therapy in CF.
Adolescent↗
Characterization of free and immobilized amine oxidases.
Bovine plasma amine oxidase was covalently bound to CH-Sepharose 4B by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The immobilized enzyme showed no significant change in specific activity when spermidine was the substrate, while the enzyme affinity toward benzylamine and propylamine increased significantly. Similarly, the pig kidney diamine oxidase physically adsorbed to Con A-Sepharose showed large changes in affinity toward substrates such as p-dimethylaminoethylbenzylamine with respect to the native enzyme. These changes are discussed in terms of active site modification as a consequence of the enzyme immobilization.
Amine Oxidase (Copper-Containing)↗
A kinetic study of the reactions between H2O2 and Cu,Zn superoxide dismutase; evidence for an electrostatic control of the reaction rate.
H2O2 was shown to reduce the copper ion of native bovine Cu,Zn superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) (ECu2+) and to oxidize the reduced enzyme (ECu+). The time-course of these processes was monitored by NMR measurement of the longitudinal relaxation rate of the water protons. A steady-state characterized by the same ratio [ECu2+]/[( EC2+] + [ECu+]) was obtained either by starting from the oxidized or the reduced enzyme. The kinetics of these processes appear to be quite complex, since different reactions between H2O2, or its reaction products, and the enzyme-bound copper control the reaction rate. The solution of the differential equations describing the kinetic processes showed that the oxidation and the reduction of the copper ion by H2O2 are first-order with respect to the copper ion itself only when these processes approach the steady-state. The rate constants of the reduction and oxidation reactions were calculated according to these equations and were found to have comparable values which are in the range 5-80 and 5-45 M-1.min-1, respectively, changing the pH from 5.6 to 7 at 0.21 M ionic strength. This result, together with the dependence of the reaction rates on pH and ionic strength, points to HO2- as the reactive species in both processes, and indicates that the electrostatic control of the access of the peroxide to the active site is the rate-determining step of the two redox reactions.
Animals↗