EPR investigations of the iron domain in neuromelanin.

The interactions between iron and neuromelanin (NM) have been studied by means of EPR spectroscopy. The variable temperature EPR spectral features of a specimen of NM extracted from normal human midbrains clearly indicate that iron is present as polynuclear oxy-hydroxy ferric aggregates as well as isolated Fe(III) centres. Ferric oxy-hydroxy phases are typical of the iron storage proteins ferritin and hemosiderin, but the comparison of the variable temperature EPR spectra of ferritin and NM highlights significant differences between the two iron(III)oxy-hydroxy domains. Moreover, further investigations on melanin models synthesised in the presence of either ferritin or a ferric salt as iron sources suggest that the same pathway of formation and inclusion of the polynuclear iron oxide is operating in NM and in the model systems, whatever is the source of iron.

Q-band EPR investigations of neuromelanin in control and Parkinson's disease patients.

New insights into the understanding of the changes induced in the iron domain of neuromelanin (NM) upon development of Parkinson's disease (PD) have been gained by electron paramagnetic spectroscopy (EPR). The results of this study are compared with a previously reported variable temperature analysis of X-band EPR spectra of a NM specimen obtained from control brain tissues. The availability of high sensitivity instruments operating in the Q-band (34.4 GHz) allows us to deal with the low amounts of NM available from PD brains. The organization of iron in NM is in the form of polynuclear superparamagnetic/antiferromagnetic aggregates, but the lack of one or more signals in the EPR spectra of NM from PD suggests that the development of the pathology causes NM to decrease its ability to bind iron. Furthermore, the detection of the Mn(II) signal in the Q-band spectra is exploited as an additional internal probe to assess minor structural differences in iron domains of PD and control NM specimens.

A novel 19F-NMR method for the investigation of the antioxidant capacity of biomolecules and biofluids.

A new assay for the measurement of the antioxidant capacity of biomolecules by high resolution 19F-NMR spectroscopy is presented here. This method is based on the use of trifluoroacetanilidic detectors, namely trifluoroacetanilide, N-(4-hydroxyphenyl)-trifluoroacetamide and 2-hydroxy-4-trifluoroacetamidobenzoic acid. Upon hydroxyl radical attack, such fluorinated detectors yield trifluoroacetamide and trifluoroacetic acid that can be quantitatively determined by 19F-NMR spectroscopy. Trifluoroacetamide was found to be a reliable reporter of hydroxyl radical attack on the fluorinated detectors, whereas N-(4-hydroxyphenyl)-trifluoroacetamide was found to be the most sensitive detector amongst the ones considered. Therefore, N-(4-hydroxyphenyl)-trifluoroacetamide has been used in competition experiments to assess the antioxidant capacity of a number of low and high molecular weight antioxidants. The antioxidant capacity of a given compound has been scaled in terms of an adimensional parameter, kF, that represents the ratio between the scavenger abilities of the fluorinated detector and the competitor. kF values obtained for low-molecular-mass compounds fall in the range 0.17 < kF < 1.5 and are in good agreement with second order rate constants (k2OH) for the reaction of the antioxidant with hydroxyl radicals. The kF value for serum albumin is much larger (46.9) than that predicted from the reported k2OH value. This finding supports the view that the protein can very effectively scavenge hydroxyl radicals as well as secondary radicals. Human blood serum showed that its antioxidant capacity is even higher than that shown by aqueous solutions of albumin at physiologic concentration suggesting a further contribution from other macromolecular serum components.

Nuclear magnetic relaxation dispersion profiles of substantia nigra pars compacta in Parkinson's disease patients are consistent with protein aggregation.

Nuclear Magnetic Relaxation field-cycling relaxometry is a technique, able to report on water mobility in tissues. By means of this technique, post-mortem specimens from both controls and idiopathic Parkinson's disease patients have been investigated. Results show different relaxometric behavior between the groups, which is consistent with protein aggregation in Parkinson's disease specimens.

Relationship between Solid State NMR Parameters and X-ray Structural Data in Tricadmium Phosphates.

31P and (113)Cd MAS NMR spectra of solid beta'-tricadmium phosphate (beta'-TCdP) show a number of highly resolved resonances that agree well with the number of independent crystallographic sites indicated by the results of X-ray diffraction studies. A correlation of the (31)P chemical shifts with the crystallographic sites for the six different PO(4)(3)(-) groups in the unit cell of beta'-TCdP has been obtained by a method based on the computation of bond strength at oxygen atoms in phosphate moieties. The assignment of the (113)Cd resonances has been carried out on the basis of the relationship between the asymmetry of the chemical shift tensor (evaluated by analysis of the spinning side bands intensities in the MAS spectrum) and a geometric parameter related to the distortion from the bipyramidal trigonal coordination at each cadmium center. Samples of tricadmium phosphate with different degrees of magnesium substitution for cadmium were investigated by (31)P MAS NMR, (113)Cd MAS NMR, and X-ray diffraction. The results of these investigations showed that the magnesiums distribute randomly in the cadmium sites, inducing a marked decrease in the order of the structure.

Bone marrow transplantation and thymopoietin pentapeptide treatment in two infants with immunodeficiency with predominant T-cell defects.

Two infants with immunodeficiency with predominant T-cell defects received transplants of HLA-identical bone marrow cells along with thymopoietin pentapeptide (TP-5) treatment and no prior immunosuppressive therapy. Both patients achieved durable engraftment with early reconstitution of cell-mediated immunity. The study of cell surface antigens with monoclonal antibodies (MoAb) revealed that the early appearance of T-cell subsets defined by OKT4 and OKT8 MoAb occurred. Neither of the patients showed any signs or symptoms of graft versus host disease over a 1-year period. This experience suggests that patients with T-cell deficiency who do not benefit from thymic hormones alone can be successfully treated by bone marrow transplantation. The association of TP-5 with bone marrow transplantation seems to induce an early and stable reconstitution and to protect against fatal post-transplant infection.

Viral infections in transfusion-dependent patients with beta-thalassemia major: the predominant role of cytomegalovirus.

For 9 months, 38 transfusion-dependent patients with beta-thalassemia, ranging in age from 3.4 to 19.1 years, were observed for serologic evidence of viral infections, by the collection of serial serum samples. Seventy-six age-matched healthy subjects, two for each patient, were followed as controls. Samples taken at the beginning, middle, and end of the study were tested against 18 viral antigens by complement fixation (CF). In addition, tests for antibodies to HIV, Epstein-Barr virus, hepatitis A virus, and markers for hepatitis B virus were performed. When changes in the antibody titer on CF tests (greater than or equal to 2-fold increase or decrease) or persistently high titers (greater than or equal to 64) were revealed, specific enzyme immunoassays (EIAs) for IgM and IgA antibodies were performed concomitant with CF tests in all sera. When symptomatic infections occurred, viral cultures and/or direct detection of antigens were carried out by immunofluorescence methods, EIA, or latex agglutination tests. Thalassemic patients and controls had similar (p greater than 0.05) overall rates of serologically confirmed viral infections (53 versus 132), but the former group had a higher (p less than 0.01) incidence of cytomegalovirus (CMV) infections (9 versus 4). CMV infections were associated in the thalassemic patients with hepatitis (2 cases), lymphadenitis (2 cases), and upper respiratory tract infection (1 case), while the remaining cases of CMV had a subclinical course. Moreover, the thalassemic patients had a lower (p less than 0.01) incidence of symptomatic infections (27 versus 110) than controls. Therefore, this study showed that both symptomatic and subclinical CMV infections may occur often in thalassemic patients, who otherwise have subclinical viral infections at an overall rate similar to that in healthy subjects.

Metal complexes as allosteric effectors of human hemoglobin: an NMR study of the interaction of the gadolinium(III) bis(m-boroxyphenylamide)diethylenetriaminepentaacetic acid complex with human oxygenated and deoxygenated hemoglobin.

The boronic functionalities on the outer surface of the Gd(III) bis(m-boroxyphenylamide)DTPA complex (Gd(III)L) enable it to bind to fructosamine residues of oxygenated glycated human adult hemoglobin. The formation of the macromolecular adduct can be assessed by NMR spectroscopy via observation of the enhancement of the solvent water proton relaxation rate. Unexpectedly, a strong binding interaction was also observed for the oxygenated unglycated human adult hemoglobin, eventually displaying a much higher relaxation enhancement. From relaxation rate measurements it was found that two Gd(III)L complexes interact with one hemoglobin tetramer (KD = 1.0 x 10(-5) M and 4.6 x 10(-4) M, respectively), whereas no interaction has been observed with monomeric hemoproteins. A markedly higher affinity of the Gd(III)L complex has been observed for oxygenated and aquo-met human adult hemoglobin derivatives with respect to the corresponding deoxy derivative. Upon binding, a net change in the quaternary structure of hemoglobin has been assessed by monitoring the changes in the high-resolution 1H-NMR spectrum of the protein as well as in the Soret absorption band. On the basis of these observations and the 11B NMR results obtained with the diamagnetic La(III)L complex, we suggest that the interaction between the lanthanide complex and deoxygenated, oxygenated, and aquo-met derivatives of human adult hemoglobin takes place at the 2, 3-diphosphoglycerate (DPG) binding site, through the formation of N-->B coordinative bonds at His143beta and His2beta residues of different beta-chains. The stronger binding to the oxygenated form is then responsible for a shift of the allosteric equilibrium toward the high-affinity R-state. Accordingly, Gd(III)L affinity for oxygenated human fetal hemoglobin (lacking His143beta) is significantly lower than that observed for the unglycated human adult tetramer.

Isolation and 13C-NMR characterization of an insoluble proteinaceous fraction from substantia nigra of patients with Parkinson's disease.

Neuromelanin is a dark brown pigment suspected of being involved in the pathogenesis of Parkinson's disease. This pigment can be isolated from normal human substantia nigra by a procedure that includes an extensive proteolytic treatment. In this study we used such a procedure to extract the neuromelanin pigment from a pool of substantia nigra from patients affected by Parkinson's disease. 13C Cross polarization magic angle spinning nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy were used to characterize the solid residue obtained from the extraction procedure. We found that the pigment extracted from the substantia nigra of parkinsonian patients was mainly composed of highly cross-linked, protease-resistant, lipo-proteic material, whereas the neuromelanin macromolecule appears to be only a minor component of this extract. A synthetic model of melanoprotein has been prepared by enzymatic oxidation of dopamine in the presence of albumin. Once it has undergone the same proteolytic treatment, this model system yields a 13C-NMR spectrum which is similar to that observed for the parkinsonian midbrain extract. These results are consistent with the view that oxidative stress has a relevant role in the pathogenesis of Parkinson's disease.

Contrast agents for magnetic resonance angiographic applications: 1H and 17O NMR relaxometric investigations on two gadolinium(III) DTPA-like chelates endowed with high binding affinity to human serum albumin.

The relaxometric properties of two Gd(III) DTPA-like complexes (DTPA = diethylenetriamine-N,N',N,N"'-pentaacetic acid) bearing different substituents for binding to human serum albumin (HSA) are compared. In spite of the structural differences of the recognition synthon and of the residual electric charge, the two chelates display an analogous binding affinity for the serum protein. Upon formation of the adducts with HSA, the exchange rates of the coordinated water appear slowed down by an amount corresponding to ca. 50% of the rates found for the free complexes. The relaxivity of [Gd(BOM)3DTPA (H2O)]2- is significantly higher than that of MS-325 either in the free complex or in the macromolecular adduct. Finally, the effect of pH on the stability of the HSA adducts and on the values of their relaxivities has been investigated.

Intestinal obstruction at the onset of acute lymphoblastic leukemia in a child.

Surgical complications need not be fatal in acute leukemia. If these are promptly diagnosed and properly treated, the prognosis will improve. This report deals with a case of acute lymphoblastic leukemia presenting with an acute abdomen following surgery for choledochal cyst. A peripheral blood smear and examination of the bone marrow revealed acute lymphoblastic leukemia. The child received transfusions of blood and platelets. Pretreatment with prednisolone was started as therapy for leukemia, and 2 days later, the patient underwent surgery. Therapy was continued until the general condition allowed a more aggressive form of treatment. Complete remission was achieved, and the patient is still in good health 48 months after diagnosis and 15 months after discontinuation of treatment. The favorable outcome in this child shows that prompt surgery is sometimes an essential step in the treatment of childhood leukemia.

Italian registry of patients off therapy after childhood acute lymphoblastic leukemia. Results after first phase of data collection.

The Italian Registry of Off-Therapy patients after childhood tumors now includes 760 subjects with acute lymphoblastic leukemia. These patients were all removed from treatment by December 31, 1981, and were followed in 35 different institutions. All the children have received multiple-drug treatment, combined, in 79.7% of the cases, with cranial irradiation. Thirty-nine (5%) experienced a relapse before treatment suspension. Total duration of antileukemic therapy ranges between 18 and 131 months (median, 38). At the last updating (December 31, 1981), 699 subjects were alive, 6 were lost to follow-up, and 55 had died. Life-table analysis shows that 90.8% were alive and 77% were alive in continuous complete remission at 36 months, whereas at 66 months, the cumulative proportions were 88% and 75.5%, respectively. One hundred thirty-six of 760 relapses after therapy suspension were reported: 83 in male patients and 53 in female patients (P less than 0.01). The longest interval between relapse and treatment suspension was 64 months. Six of 55 died in continuous complete remission 3 to 44 months after treatment suspension. Five births of apparently normal babies to female patients have been reported. A general outline of the project and the future program are given.

Isotachophoresis as a useful tool for monitoring neurological complications of acute leukaemia in children.

Cerebrospinal fluid proteins from 42 children with acute lymphoblastic leukaemia were analysed by isotachophoresis. The isotachopherograms of cerebrospinal fluid taken from patients undergoing central nervous system prophylaxis with neurological complications showed an increase of several peaks (albumin, prealbumin, and an unidentified peak), and changes in the globulin zone, compared with those from patients who had completed central nervous system prophylaxis for at least six months. The most striking finding was that these alterations were not associated with any other biochemical changes in the cerebrospinal fluid, as assayed by routine analysis. Isotachophoresis may be useful in the monitoring of therapy in children affected with acute lymphoblastic leukaemia.

Post-transfusional human retrovirus infection in 41 Italian beta-thalassemic patients.

BACKGROUND: Previous studies have demonstrated that HTLV-I is present in Italy both in endemic form in Southern Apulia and in epidemic form among the population of intravenous drug addicts. In the present paper we intend to evaluate the risk for transfusional HTLV-I transmission in our country, as well as the already known risk for HIV1. METHODS: A population of 41 polytransfused Italian beta-thalassemic patients was examined by serological methods and PCR (polymerase chain reaction) for human retrovirus infection. Genomic DNA from PBMCs was analyzed by PCR with primer pairs specific for the HTLV-I gag, pol and env regions, and the HTLV-II env region. RESULTS: Two patients were found to be weakly seroreactive to p19 and p24 HTLV-I/HTLV-II proteins by Western blot. The analysis of genomic DNA from PBMCs by PCR revealed sequence homology to HTLV-I only in these two patients. On the contrary, PCR with primer pairs specific for HTLV-II showed no beta-thalassemic patient was infected by this retrovirus. Surprisingly, Western blot analysis for detecting anti-HIV1 antibodies in these polytransfused subjects showed a seropositivity in two patients (not the same found to be infected with HTLV-I) in spite of a screening for HIV1 antibodies in the blood bank. CONCLUSIONS: These findings suggest that in Italy polytransfused people should still be considered at risk for HIV1 as well as HTLV-I infection, even if the incidence cannot be evaluated from such a small sample. The authors stress the importance of a through medical history of potential blood donors to eliminate possibly infected subjects.

Superoxide production by neutrophils in children with malignant tumors treated with recombinant human granulocyte colony-stimulating factor.

BACKGROUND: Human recombinant granulocyte colony-stimulating factor (rhG-CSF), widely used to combat chemotherapy-induced neutropenia, stimulates both in vivo and in vitro intra- and extra-cellular O2- production in human polymorphonuclear cells (PMNs). PATIENTS AND METHODS: Twelve patients with solid tumors or acute lymphoblastic leukemia were treated during induced aplasia with rhG-CSF (5 micrograms/kg/day). Intra- and extracellular O2- production by PMNs isolated from these patients after 5 days of rhG-CSF therapy was assessed following both fMLP and PMA stimulation. RESULTS: All patients showed a rise in PMN count; administration of rhG-CSF enhanced intra- and extracellular O2- release after fMLP but not after PMA stimulation. CONCLUSIONS: rhG-CSF potentiates in vivo O2- production by PMNs stimulated with receptor-mediated agonists via G-protein (e.g. fMLP), but not by those stimulated with agonists that bypass receptors via protein kinase C (e.g. PMA).

Radioisotope assessment of heart damage in hypertransfused thalassaemic patients.

Twenty-five thalassaemic patients treated with repeated blood transfusion (BT) and intensive iron removal therapy were studied by echocardiography and rest/stress equilibrium gated radionuclide angiocardiography (EGNA). Stress left ventricular ejection fraction (LVEF) showed an important negative correlation with number of BTs (r = -0.75). Abnormal values of stress LVEF were measured after 200 BTs: these data demonstrate the effectiveness of stress LVEF in the follow-up of patients who have undergone repeated BTs and the clinical importance of intensive chelation therapy. Peak filling rate did not show diagnostic value in the early detection of iron cardiotoxicity. However, its inverse correlation with BT (r = -60) indicates that iron overload depresses the diastolic parameters.