Proton Magnetic Relaxation in Ethane Diol-Water Solutions of Haemoglobin

Ethane diol was added to aqueous solutions of bovine and human met(FeUI)- and CO-haemoglobin in order to extend the temperature range for nuclear magnetict relaxation measurements below freezing point. No significant difference in the relaxation rates was found on adding ethane diol except in the case of human met- and CO-haemog1obin, which may be . ascribed to chang·es in the hydration sheath. The energies of activation derived :firom Arrhenius plots of the relaxation rates for bovine Hb are also independent (within ± 50/o) of ethane diol. It is concluded that the gross conformation of the haem pocket is not altered by addition of ethane diol, so that measurements can be done down to - 30 °c

Proton Magnetic Relaxation in Ethane Diol-Water Solutions of Haemoglobin

Ethane diol was added to aqueous solutions of bovine and human met(FeUI)- and CO-haemoglobin in order to extend the temperature range for nuclear magnetict relaxation measurements below freezing point. No significant difference in the relaxation rates was found on adding ethane diol except in the case of human met- and CO-haemog1obin, which may be . ascribed to chang·es in the hydration sheath. The energies of activation derived :firom Arrhenius plots of the relaxation rates for bovine Hb are also independent (within ± 50/o) of ethane diol. It is concluded that the gross conformation of the haem pocket is not altered by addition of ethane diol, so that measurements can be done down to - 30 °c

Hepatic Cytochrome P-450. A Proton Magnetic Relaxation Study of Microsomal, Solubilized and Partially Reconstituted Enzyme System

The longitudiJ:ial proton magnetic relaxation times, Ti, were measured from -5 to 40 °c for microsomal, solubilized and reconstituted cytochrome P-450 obtained from phenobarbital-induced rat livers. The paramagnetic contribution to the rates was derived by subtraction of the rates measured on dithionite-CO-reduced samples. The same values were obtained for microsomal P-450 on reduction with NADPH. PMR titratio.n by KCN yielded a dissociation constant of about 30 mM. This is three orders of magnitude larger than for metmyoglobin. It is concluded that the measured PMR rates are most likely due to the P-450 (and P-420) haem-iron while the 300/o non-haem iron found in both the microsomal and s olubilized P-450 is .ineffective for the PMR rates. These rates increase several times on isotopic dilution (D20 for H20) with the microsomes and diminish for the solubilized samples. Microsomes show 170/o residual, encaged, H20. Most of their paramagnetic PMR rate is due to the parama.gnetic iron located on the outside of microsomes. This is demonstrated by measurements with deuterated samples to which 190/o H20 had been added. Hence, the solubilized P-450 is homogeneous regarding PMR, but the microsomes are not

Hepatic Cytochrome P-450. A Proton Magnetic Relaxation Study of Microsomal, Solubilized and Partially Reconstituted Enzyme System

The longitudiJ:ial proton magnetic relaxation times, Ti, were measured from -5 to 40 °c for microsomal, solubilized and reconstituted cytochrome P-450 obtained from phenobarbital-induced rat livers. The paramagnetic contribution to the rates was derived by subtraction of the rates measured on dithionite-CO-reduced samples. The same values were obtained for microsomal P-450 on reduction with NADPH. PMR titratio.n by KCN yielded a dissociation constant of about 30 mM. This is three orders of magnitude larger than for metmyoglobin. It is concluded that the measured PMR rates are most likely due to the P-450 (and P-420) haem-iron while the 300/o non-haem iron found in both the microsomal and s olubilized P-450 is .ineffective for the PMR rates. These rates increase several times on isotopic dilution (D20 for H20) with the microsomes and diminish for the solubilized samples. Microsomes show 170/o residual, encaged, H20. Most of their paramagnetic PMR rate is due to the parama.gnetic iron located on the outside of microsomes. This is demonstrated by measurements with deuterated samples to which 190/o H20 had been added. Hence, the solubilized P-450 is homogeneous regarding PMR, but the microsomes are not

A Proton Magnetic Relaxation Study of tpe Interaction between Methaemoglobin and Inositol Hexaphosphate

Inositol hexaphosphate is the strongest allosteric effector even for the metform of haemoglobin. Its effects upon the quaternary structure of the tetramer have been studied in relation to the overall conformational state(s) of the haem-pockets in aqueous solutions of human haemoglobins. The method useci, proton magnetic relaxation, yields information about the accessibility of solvent pl.\u27otons towards the haem-iron. No differences in the relaxation rates were detected by this method between the unstripped carbonmonoxyhaemoglobin and the phosphate-stripped sample in the presence and absence of IHP. There are considerable changes in those relaxation rate·s due to the paramagnetic haem-iron of aquomethaemoglobin when IHP is added to the stripped adult haemoglobin, but none is observed for the foetal haemoglobin, although a similar shift in the spin-state equilibrium ts expected for both haemoglobins on addition of ,!HP. Neither was there any change with IHP in solutions of adult fluoromethaemoglobin. It is concluded thart there is no tightening of the haem-pockets upon addition of IHP to solutions of any of the three haemoglobin samples. An increase in the accessibility of the haem-pockets is probable only for the aquometfom1 of the adult haemoglobin. It is suggested that the structural aspect of ligand affinity, i.e. the haem-pocket conformation, is not as decisive in altering the affinity by IHP as is possibly the change in the haem-iron spin-state induced by !HP-binding

Solvent Proton Magnetic Relaxation in Solution of Rabbit Liver Cytochrome P450. On the Corfelation Time for the Electron Proton Dipole-Dipole Interaction

Structural parameters can be derived from PMR measurements if the correlation time fdr the spin interactions is known. The relaxation rates induced by the ferric haem-iron of cyt P450 were found to be frequency independent from 10 to 37 MHz. The possible limits for the correlation time according to Solomon\u27s theory are discussed with regard to the possible existence of a water molecule at the sixth coordination site of the haem-iron. No definite conclusion could be reached in this respect, but the results are definetely in favour of a haem environment which can accomodate several water molecules exchanging quickly with the bulk of solvent

Solvent Proton Magnetic Relaxation in Solution of Rabbit Liver Cytochrome P450. On the Corfelation Time for the Electron Proton Dipole-Dipole Interaction

Structural parameters can be derived from PMR measurements if the correlation time fdr the spin interactions is known. The relaxation rates induced by the ferric haem-iron of cyt P450 were found to be frequency independent from 10 to 37 MHz. The possible limits for the correlation time according to Solomon\u27s theory are discussed with regard to the possible existence of a water molecule at the sixth coordination site of the haem-iron. No definite conclusion could be reached in this respect, but the results are definetely in favour of a haem environment which can accomodate several water molecules exchanging quickly with the bulk of solvent

Predicting Outcomes of Prostate Cancer Immunotherapy by Personalized Mathematical Models

Therapeutic vaccination against disseminated prostate cancer (PCa) is partially effective in some PCa patients. We hypothesized that the efficacy of treatment will be enhanced by individualized vaccination regimens tailored by simple mathematical models.We developed a general mathematical model encompassing the basic interactions of a vaccine, immune system and PCa cells, and validated it by the results of a clinical trial testing an allogeneic PCa whole-cell vaccine. For model validation in the absence of any other pertinent marker, we used the clinically measured changes in prostate-specific antigen (PSA) levels as a correlate of tumor burden. Up to 26 PSA levels measured per patient were divided into each patient's training set and his validation set. The training set, used for model personalization, contained the patient's initial sequence of PSA levels; the validation set contained his subsequent PSA data points. Personalized models were simulated to predict changes in tumor burden and PSA levels and predictions were compared to the validation set. The model accurately predicted PSA levels over the entire measured period in 12 of the 15 vaccination-responsive patients (the coefficient of determination between the predicted and observed PSA values was R(2) = 0.972). The model could not account for the inconsistent changes in PSA levels in 3 of the 15 responsive patients at the end of treatment. Each validated personalized model was simulated under many hypothetical immunotherapy protocols to suggest alternative vaccination regimens. Personalized regimens predicted to enhance the effects of therapy differed among the patients.Using a few initial measurements, we constructed robust patient-specific models of PCa immunotherapy, which were retrospectively validated by clinical trial results. Our results emphasize the potential value and feasibility of individualized model-suggested immunotherapy protocols

Age-dependent response of murine female bone marrow cells to hyperbaric oxygen

Consequences of age on the effects of hyperbaric oxygen (HBO) on bone marrow (BM) derived stem cells and progenitors (SCPs) are largely unknown. We treated 2- and 18-month old C57BL/6 female mice by HBO. Hematopoietic stem cells and progenitors, enumerated as colony-forming units in culture, were doubled only in peripheral leukocytes and BM cells of young mice receiving HBO. In old mice colony-forming unit fibroblast numbers, a measure of mesenchymal stromal cells (MSCs) from BM, were high but unaffected by HBO. To further explore this finding, in BM-MSCs we quantified the transcripts of adipocyte early-differentiation genes peroxisome proliferator-activated receptor-γ, CCAAT/enhancer binding protein-β and fatty-acid binding protein 4; these transcripts were not affected by age or HBO. However, osteoblast gene transcripts runt-related transcription factor 2, osterix (OSX) and alkaline phosphatase (AP) were twofold to 20-fold more abundant in MSCs from old control mice relative to those of young control mice. HBO affected expression of osteoblast markers only in old MSCs (OSX gene expression was reduced by twofold and AP expression was increased threefold). Our data demonstrate the impact of aging on the response of BM SCPs to HBO and indicate the potentially different age-related benefit of HBO in wound healing and tissue remodeling