Search PubMed⌕ Search

Biomedical subjects

E Bucci

Publications and source records attributed to E Bucci.

At least 73 records · Page 4Linked to original sources
PubMed1995

Role of nitric oxide scavenging in peripheral vasoconstrictor response to beta beta cross-linked hemoglobin.

Transfusion with many crosslinked hemoglobin solutions causes an increase in arterial pressure that may be mediated by scavenging of nitric oxide (NO). If so, we postulated that inhibiting synthesis of NO after hemoglobin transfusion would fail to cause vasoconstriction ordinarily seen with such inhibition. In pentobarbital anesthetized cats, we tested whether administration of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), produced peripheral vasoconstriction after isovolemic exchange transfusion with hemoglobin to the same extent as occurs with L-NAME infusion in time controls and in controls matched for reduced hematocrit (17%) with albumin transfusion. Bovine hemoglobin was treated aerobically with bis-(3,5-dibromosalicyl)fumarate to produce beta beta-81 lysine crosslinks. Hemoglobin exchange transfusion increased mean arterial blood pressure and there was no further increase after L-NAME. In contrast, L-NAME increased pressure in the time controls and albumin controls. Hemoglobin transfusion decreased intestinal and renal blood flow, and there was no further decrease after L-NAME. In contrast, L-NAME decreased intestinal and renal blood flow in time controls and albumin controls. With L-NAME pretreatment in a separate group of cats, there was little further increase in arterial pressure or visceral vasoconstriction after hemoglobin transfusion. We conclude that the increase in arterial blood pressure after isovolemic crosslinked hemoglobin transfusion is best explained by scavenging of NO in intestinal and renal vascular beds.

Animals↗
PubMed1995

Renal and systemic-hemodynamic response to isovolemic exchange transfusion with hemoglobin cross-linked with bis (3,5-dibromosalicyl) fumarate or albumin.

Experiments were done in anesthetized rats to determine systemic hemodynamic and renal functional effects of isovolemic exchange transfusion with either 5% albumin or hemoglobin cross-linked with bis (3,5-dibromosalicyl) fumarate (XLHb) in volumes ranging from 1 to 6.3 ml.100 gm-1. Hematocrit decreased in proportion to increasing exchange volumes with either fluid. Exchange with increasing volumes of albumin led to progressive decreases in blood pressure. Exchange of 1 ml.100 gm-1 of XLHb was associated with an increase in blood pressure, whereas with larger exchanges, blood pressure returned to and was maintained at control values even for exchanges as large as 6.3 ml.100 gm-1. An increase of similar magnitude in glomerular filtration rate occurred with both fluids. Net and fractional sodium excretion (FENa) increased significantly with both transfusion fluids; the increase was significantly larger for XLHb than for albumin. Maximal FENa excretion with albumin was about 8% but exceeded 6% with XLHb. Pretreatment with indomethacin (5 mg.kg-1.day-1 for 3 days) did not blunt the diuresis that occurred with an exchange of 2 ml.100 gm-1 XLHb. It is concluded that 5% XLHb, as compared with 5% albumin, better supports systemic blood pressure, especially as exchange volume increases, possibly as a result of hemoglobin-induced increased vascular tone. Although a decrease in hematocrit may play a role in the diuresis observed with either fluid, the greater diuresis with XLHb must be due to some additional factor; the mechanism does not appear to involve prostaglandins.

Animals↗
PubMed1995

"In vitro" and in animal model studies on a double virus-inactivated factor VIII concentrate.

To improve the safety of plasma derived factor VIII (FVIII) concentrate, we introduced a final super heat treatment (100 degrees C for 30 min) as additional virus inactivation step applied to a lyophilized, highly purified FVIII concentrate (100 IU/mg of proteins) already virus inactivated using the solvent/detergent (S/D) method during the manufacturing process. The efficiency of the super heat treatment was demonstrated in inactivating two non-lipid enveloped viruses (Hepatitis A virus and Poliovirus 1). The loss of FVIII procoagulant activity during the super heat treatment was of about 15%, estimated both by clotting and chromogenic assays. No substantial changes were observed in physical, biochemical and immunological characteristics of the heat treated FVIII concentrate in comparison with those of the FVIII before heat treatment.

Animals↗
PubMed1995

Myxoma of the jaws with an unusual intracellular banded structure.

Myxoma of the jaw is a rare odontogenic tumor, probably derived from the dental apparatus. The case described here was studied by both light and electron microscopic methods. Stellate cells with branching processes were reported in an abundant acid mucopolysaccharide stroma, and ultrastructurally these cells were seen to be fibroblast. Occasionally myxoblasts and macrophages were reported in the tumor. No odontogenic epithelium was identified, while an intracellular synthesis of cross-striated fibrils was observed. The ultrastructural features suggest that the connective tissue cell is the proliferating component of the tumor and is very similar to a fibroblast.

Adult↗
PubMed1994

Reversible anergy in circulating lymphocytes of cancer patients during interleukin-2 therapy.

Interleukin-2 plays a crucial role in enhancing the antitumor immune response. Clinical trials, mainly in renal cell carcinoma and melanoma patients, have been carried out with encouraging results. Recent reports demonstrated that interleukin-2 therapy may depress the immune response either in vitro or in vivo. We decided to monitor, in nine renal cancer patients, the proliferative responses and the parallel variations in Ca2+ homeostasis of peripheral blood lymphocytes collected before, during and after the first cycle of a 3-day interleukin-2 systemic administration. The proliferative response to phytohemagglutinin or concanavalin A significantly dropped early during interleukin-2 infusion. Consistently, an impairment in mobilizing Ca2+, either from internal stores or via influx from outside, was observed. Results obtained with a mAb-alpha CD3 molecular complex strongly suggested that the TCR/CD3 signal transduction pathway was defective. In contrast, no major variations were observed in the general machinery controlling Ca2+ homeostasis nor in the total Ca(2+)-releasable pool. Patients' lymphocytes, cultured in vitro for 3 days in medium alone, showed an almost complete recovery in their ability to respond to mitogens. In conclusion, we show that interleukin-2 administration in cancer patients induces a reversible state of anergy in circulating lymphocytes, assessed both by the reduction in the proliferative response and the block of the mitogen-activated intracellular Ca2+ signalling.

Calcium↗
PubMed1994

Treatment of advanced renal cell cancer with sequential intravenous recombinant interleukin-2 and subcutaneous alpha-interferon.

Starting from in vitro studies suggesting synergistic antitumour activity against renal cell cancer (RCC) of recombinant interleukin-2 (rIL-2) and alpha-interferon (IFN), a phase II trial was initiated to test the clinical activity of this combination. The two cytokines were administered sequentially, with the aim of reducing the risk of additive toxicity and enhancing the immunological reaction against the tumour. The original treatment schedule consisted of rIL-2 18 x 10(6) U/m2/day by continuous intravenous infusion for 120 h days 1-5, and alpha-IFN 2b, at a flat dose of 9 x 10(6) U by subcutaneous or intramuscular injection thrice in a week, from day 8 to 28. Treatment was planned to be continued for six or more 28-day cycles, depending on clinical response. 12 patients were treated according to this schedule; as some cardiovascular toxicity was experienced in this set of patients, 11 further patients were treated with half-dose rIL-2 (i.e. 9 x 10(6) U/m2/day). 17 out of 23 enrolled patients completed at least one cycle of treatment and were evaluated for response. We observed six major responses [one complete response (CR) + five partial responses (PR)] for an objective response rate of 35% [95% confidence interval (CI) 17-59%]. 5 additional patients achieved stabilisation of disease; one of them reached CR after surgical extirpation of a lung mass. Sites of response included lung, nodes and bone. Duration of response is 12+ months for CR; 17, 16, 12+, 9 and 9 months for PRs. Median survival is 16 months. Response was not significantly different between full-dose and half-dose rIL-2. Considering stable disease (SD) as responses, there seemed to be a higher chance of response for patients with smaller tumour burden (P = 0.032). The toxicity of rIL-2 treatment, mainly cardiovascular, was substantial; 9 patients experienced severe cardiotoxicity, consisting of major arrhythmias, myocardial ischaemia, reduction of ejection fraction measured with heart radionuclide scan, and were excluded from continuing treatment. Other rIL-2-related toxicities forcing exclusion from the study were severe thrombocytopenia (1 case), and generalised exfoliative dermatitis requiring steroids (1 case). Otherwise, treatment was well tolerated; rIL-2-related toxicities promptly recovered after rIL-2 discontinuation in the majority of cases, and no treatment-related deaths were reported. The half-dose rIL-2 regimen was significantly less toxic in terms of hypotension (P = 0.014), fever (P = 0.014), oliguria (P = 0.042), serum creatinine elevation (P = 0.009) and prothrombin time elongation (P = 0.038).(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1993

Effect of intramolecular cross-links on the enthalpy and quaternary structure of the intermediates of oxygenation of human hemoglobin.

We have reported [Bucci, E., Fronticelli, C., & Grycznski, Z. (1991) Biochemistry 30, 3195-3199] that in human and bovine hemoglobins the release of heat at the subsequent steps of oxygenation is not constant. This is especially evident in the binding of the third O2 molecule, which is an endothermic event. This phenomenon was attributed to peculiar conformations of the intermediates of oxygenation, not included in the fundamental R/T transition of the system. To test this hypothesis, we have explored the effect of conformational constraints on the thermodynamics of the intermediates of oxygenation. The assumption was that intramolecular constraints would stabilize the intermediates into conformations similar to the R and T forms reducing the variability of their enthalpies. We have analyzed the temperature dependence of the oxygen binding isotherms of human hemoglobin cross-linked either between the beta 82 or between the alpha 99 lysines by bis(3,5-dibromosalicyl)fumarate. The measurements were perfomred at pH 9.0 in 0.1 M borate buffer in order to avoid thermal effects due to oxygen-linked binding of anions and protons. The data were analyzed singularly by local procedures and simultaneously using global procedures. The two cross-links had opposite effects. The cross-link between the beta-subunits decreased while that between the alpha-subunits increased the endothermic behavior of the third step of oxygenation. Also, the cross-link between the beta-subunits increased the fractional amount of the triligated species at intermediate stages of oxygenation, while that between the alpha-subunits decreased this quantity to hardly detectable values.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Recombinant human hemoglobin: modification of the polarity of the beta-heme pocket by a valine67(E11)-->threonine mutation.

Using the mutagenesis and a gene expression system previously described [Fronticelli et al. (1991) J. Protein Chem. 10, 495-501], we have replaced Val67E11 in the distal heme pocket of the beta-chains of hemoglobin with Thr. The valine to threonine substitution is isosteric and only modifies the polarity of the beta-heme environment. The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom. Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin. Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step. The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to C1- were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

A new front-face optical cell for measuring weak fluorescent emissions with time resolution in the picosecond time scale.

Recent developments of ultrafast fluorimeters allow measuring time-resolved fluorescence on the picosecond time scale. This implies one is able to monitor lifetimes and anisotropy decays of highly quenched systems and of systems that contain fluorophores having lifetimes in the subnanosecond range; both systems that emit weak signals. The combination of weak signals and very short lifetimes makes the measurements prone to distortions which are negligible in standard fluorescence experiments. To cope with these difficulties, we have designed a new optical cell for front-face optics which offers to the excitation beam a horizontal free liquid surface in the absence of interactions with optical windows. The new cell has been tested with probes of known lifetimes and anisotropies. It proved very useful in detecting tryptophan fluorescence in hemoglobin. If only diluted samples are available, which cannot be used in front-face optics, regular square geometry can still be utilized by inserting light absorbers into a cuvette of 1 cm path length.

Chemistry Techniques, Analytical↗
PubMed1993

Effect of disordered hemes on energy transfer rates between tryptophans and heme in myoglobin.

Our recent linear dichroism study of heme transitions (Gryczynski, Z., E. Bucci, and J. Kusba. 1993. Photochem. Photobiology. in press) indicate that heme cannot be considered a planar oscillator when it acts as an acceptor of radiationless excitation energy transfer from tryptophan. The linear nature of the heme absorption transition moment in the near-UV region implies a strong dependence of the transfer rate factors on the relative angular position of the heme and tryptophan, i.e., on the kappa 2 orientation parameter of the Förster equation. Using the atomic coordinates of SW myoglobin we have estimated the variation of kappa 2 parameter as a function of the heme absorption transition moment direction. The simulations proved that transfer is very efficient and anticipates lifetimes in the picosecond range. Also, they showed that transfer is very sensitive to rotations of the heme around its alpha-gamma-meso-axis, which may reduce the efficiency of transfer to almost zero values, producing lifetimes very similar to those of free tryptophan, in the nanosecond range. Comparisons between the lifetime values reported in the literature and those here estimated suggest that natural heme disorder, in which heme is rotated 180 degrees around its meso axis, is at the origin of the nanosecond lifetimes found in myoglobin systems.

Animals↗
PubMed1993

Linear dichroism study of metalloporphyrin transition moments in view of radiationless interactions with tryptophan in hemoproteins.

We measured the linear dichroism of several metalloporphyrins embedded in stretched polyvinyl alcohol (PVA) films to estimate the orientation of the absorption transition moments, which in hemoproteins are relevant to the radiationless energy transfer between tryptophan and heme. The metalloporphyrins were derivatives of protoporphyrin IX (PPIX), namely Fe(3+)-PPIX (ferric-heme) and Fe2+CO-PPIX (CO-heme), Mg-PPIX (Mg-heme) and Zn-PPIX (Zn-heme). Measurements were conducted between 300 and 700 nm. In all cases the linear dichroism was wavelength dependent, indicating the presence of several transition moments with different orientations. We focused our attention on the near-UV (300-380 nm) and Soret (380-450 nm) absorption bands. Deconvolution in terms of Gaussian components gave three components between 380 and 450 nm and only one in the 300-380 nm region. Deconvolution of the near-UV and Soret spectra of oxy-, deoxy- and carbonmonoxyhemoglobin gave very similar results, suggesting a very similar orientation of the various transition moments in the free and protein-embedded hemes. It should be stressed that the single 300-380 nm band is the only one responsible for the overlap integral that regulates the energy transfer from tryptophan to heme in hemoproteins (Gryczynski et al., Biophys. J. 63, 648-653, 1992). The dichroism of this single band indicated that its transition moment is oriented at about 60 degrees from the alpha-gamma meso-axis of the heme moiety. We conclude that the heme should be considered a linear oscillator when it acts as acceptor of energy transfer from tryptophans.

Binding Sites↗
PubMed1992

Renal excretion of pseudo-cross-linked human, porcine and bovine hemoglobins.

UNLABELLED: The present experiments were done to determine plasma retention, urinary excretion, and acute renal effects of bolus administration (20 mg/100 g), of hemoglobins (human, bovine and porcine) whose tetrameric structure was stabilized by a pseudo-cross-link between the beta-chains. Standard renal clearance studies showed that these preparations had no immediate adverse effects on GFR or ERPF, however, urine flow and electrolyte excretion commonly increased. Plasma disappearance curves best fit a double-exponential and showed components with half-times of 28 +/- 3 and 257 +/- 43 min. A variable fraction, 57 +/- 6%, appeared in the urine, in phase with the early-fast component of plasma disappearance. This in vivo finding of extensive urinary excretion conflicted with sedimentation velocity analysis of these preparations which indicated a homogenous preparation with mean molecular weight of a stable and therefore, unfilterable tetramer. It appears that the glomerular filter selects for permeant species not easily detected by sedimentation velocity analysis. The different species may reflect the presence, within the PXLs of conformational-slow-equilibria among isomeric forms with different degrees of filterability. IN CONCLUSION: It is important, useful and simple to determine urinary excretion as a means to ascertain molecular stability in vivo.

Animals↗
PubMed1992

Immobilized hemoglobin in the purification of hemoglobin-based oxygen carriers.

Chemically modified hemoglobins can be used as oxygen carriers in cell-free fluids provided that they have a low oxygen affinity and are stable towards dissociation into subunits. The latter species are undesirable because they are filtered rapidly through the kidneys, have renal toxicity and are characterized by a high oxygen affinity. A most important step in the preparation of hemoglobin-based oxygen carriers is therefore their purification from any dissociable material. Hemoglobin immobilized as alpha beta dimers on Sepharose lends itself naturally to this purpose as it is able to interact in a specific and reversible way with soluble alpha beta dimers. Hemoglobin affinity columns are very effective in the purification of cross-linked and pseudo-cross-linked human and bovine hemoglobin. The applicability of the technique is enhanced by the ease with which alpha beta dimers from different species cross-interact to yield hybrid alpha 2 beta 2 tetramers. It is shown that hemoglobin affinity columns may provide analytical information on the cross-linking reaction itself.

Animals↗
PubMed1992

Consequences of chemical modifications on the free radical reactions of human hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as blood substitutes and resuscitation fluids. We determined that HBOCs of specific types differ in their ability to generate or interact with free radicals. The differences do not correlate with oxygen affinity. Detailed comparisons with unmodified human hemoglobin, HbA0, were carried out with two cross-linked derivatives: HbA-FMDA, produced by the reaction of human oxyhemoglobin with fumaryl monodibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyhemoglobin with bis(3,5-dibromosalicyl) fumarate. Both derivatives had lower oxygen affinity than unmodified HbA0. As previously reported, exposure of oxyhemoglobin to H2O2 causes generation of free radicals capable of generating formaldehyde from dimethyl sulfoxide. Relative to the reaction catalyzed by 50 microM HbA (18.0 +/- 3.5 nmol/30 min/ml), the formaldehyde formation was roughly 70% for HbA-DBBF and 50% for HbA-FMDA under comparable conditions. More profound differences are exhibited at lower hemoglobin concentrations. Spectral changes of the HBOCs during the reaction differ qualitatively and occur at different rates. The HBOCs also differ in rates of hemoglobin-catalyzed NADPH oxidation and aniline hydroxylation, reactions mediated by reactive oxygen species. These results show that stereochemical differences brought about by chemical cross-linking alter the ability of HBOCs to generate radicals and to react with activated oxygen species. These studies also show that the ability of hemoglobin to produce activated species of oxygen can be enhanced or suppressed independently of oxygen affinity.

Aniline Compounds↗
PubMed1992

Rates of energy transfer between tryptophans and hemes in hemoglobin, assuming that the heme is a planar oscillator.

Using the Förster equations we have estimated the rate of energy transfer from tryptophans to hemes in hemoglobin. Assuming an isotropic distribution of the transition moments of the heme in the plane of the porphyrin, we computed the orientation factors and the consequent transfer rates from the crystallographic coordinates of human oxy- and deoxy-hemoglobin. It appears that the orientation factors do not play a limiting role in regulating the energy transfer and that the rates are controlled almost exclusively by the intrasubunit separations between tryptophans and hemes. In intact hemoglobin tetramers the intrasubunit separations are such as to reduce lifetimes to 5 and 15 ps/ns of tryptophan lifetime. Lifetimes of several hundred picoseconds would be allowed by the intersubunit separations, but intersubunits transfer becomes important only when one heme per tetramer is absent or does not accept transfer. If more than one heme per tetramer is absent lifetimes of more than 1 ns would appear.

Energy Transfer↗