Ctla4 and multiple sclerosis in the Italian population.

CTLA4 protein is a receptor molecule that plays a critical role as a negative regulator of the immune response. Therefore, genetic variations in CTLA4 may confer susceptibility to autoimmune diseases such as multiple sclerosis (MS). In order to investigate the association of two CTLA4 polymorphisms (+49 A/G and -318 C/T) with multiple sclerosis, sporadic MS patients and healthy controls from Italy were genotyped through direct DNA sequencing. Considering single-loci variations, no differences in the allelic and genotypic frequencies between patients and controls were found. However, considering a putative interaction at the two loci, the T/G combination was more frequently observed in patients than in controls. This result suggests that this allelic combination of the CTLA4 polymorphisms may be involved in the susceptibility to MS in the Italian population.

Time-resolved DNA-microarray reading by an intensified CCD for ultimate sensitivity.

We describe a novel technique for DNA-microarray reading based on time-resolved fluorescence measurements. We used an intensified CCD camera with picosecond resolution to acquire a set of time-delayed fluorescence images from a mutation DNA microarray marked with cyanine 3. We measured the fluorescence lifetimes of the marker and the background separately, and we used this information to calculate the amplitude map of the marker, starting from the time-delayed images. This procedure allowed us to identify hybridized spots that are not visible in fluorescence images acquired with continuous-wave detection.

Bohr effect in hemoglobin deoxy/cyanomet intermediates.

The Bohr protons released by oxygen exposure of the unliganded subunits of intermediates (alpha +CN-beta) (alpha +CN-beta) and (alpha beta +CN-) (alpha beta +CN-) were obtained by titrations of concentrated solutions of these species. The Bohr protons released by oxygen exposure of the other intermediates were obtained from titrations of equilibrium mixtures of two parental species, (alpha beta) (alpha beta), (alpha +CN-beta) (alpha +CN-beta), (alpha beta +CN-) (alpha beta +CN-), and (alpha +CN-beta +CN-) (alpha +CN-beta +CN-), in which the concentration of the hybrid intermediate was determined by cryogenic electrophoretic techniques. The Bohr effect of the intermediates was calculated by subtracting the Bohr protons released by oxygen exposure of the intermediates from the total Bohr protons of deoxyhemoglobin at the same pH. The Bohr effects of intermediates (alpha +CN-beta) (alpha beta) and (alpha beta +CN-) (alpha beta) were similar and vanished at pH 8 where the total Bohr effect of deoxyhemoglobin is still significant. This suggests that the Bohr effect in these intermediates is tertiary in the quaternary T structure. The curve of the Bohr effect of intermediate (alpha +CN-beta +CN-) (alpha beta), which was close to the curve obtained by adding the Bohr effects of the two monoliganded intermediates at acidic and physiological pH values, was significantly different from the curve obtained by adding the Bohr effects of one liganded subunit of intermediate (alpha +CN-beta) (alpha +CN-beta) and one liganded subunit of intermediate (alpha beta +CN-) (alpha beta +CN-).(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of the oxidation reaction of deoxyhemoglobin as studied by isolation of the intermediates suggests tertiary structure dependent cooperativity.

The intermediates in the oxidation of deoxyhemoglobin by ferricyanide in 0.1 M KCl, at 20 degrees C and three pH values, were studied by cryogenic techniques. Data analysis was carried out according to a simple four rate constant model, ignoring the functional heterogeneity of the subunits, to simulate the time courses of the oxidation reaction, as studied by the stopped-flow technique [Antonini et al., (1965) Biochemistry 4, 345], which show anticooperativity at neutral pH and cooperativity at alkaline pH. Data analysis according to a 12 rate constant model indicated that the rate of oxidation of the beta subunit in the first oxidation reaction was 4 times faster than the rate of oxidation of the alpha subunit at pH 6.2 and 12 times faster at pH 8.5. The reactions involving the alpha subunit were noncooperative except for the last oxidation step at acid and neutral pH, but were cooperative at alkaline pH. The reactions involving the beta subunit were partly noncooperative and partly anticooperative. These complex mechanistic patterns suggest that a simple two-state model requiring the concerted transition of the tertiary structures of the subunits from the T to the R conformation is not adequate to interpret the oxidation reaction and that tertiary structures contribute, positively and negatively, to cooperativity. A structural hypothesis is suggested to explain the difference in the reactivities of the alpha and beta subunits.

The association reaction between hemoglobin and carbon monoxide as studied by the isolation of the intermediates. Implications on the mechanism of cooperativity.

The concentrations of the intermediates in the association reaction between human hemoglobin and CO at 20 degrees C, pH 7, under conditions of negligible dissociation of the ligand, were measured by cryogenic techniques. The monoligated species were predominant at all values of overall ligand bound studied. The analysis of the experimental data assuming a scheme of four consecutive reactions indicated that the binding rates increased in a continuous fashion. A significant acceleration after the binding of the second molecule of ligand occurred in the presence of 0.1 M KCl, but not with the addition of an excess of inositol hexaphosphate, indicating that major functional, and possibly structural, transitions occur at the diligated state. Differences in the concentrations of the intermediates in the same state of ligation were observed under all conditions. The analyses of the data on the basis of schemes of multiple pathways of reaction indicated that the beta subunits reacted about 1.5 times faster than the alpha subunits in the first ligation reaction. After the addition of inositol hexaphosphate, the alpha subunits reacted about 1.5 times faster than the beta subunits in the first ligation step, but the overall rate of the first CO binding step was unchanged.

What the intermediate compounds in ligand binding to hemoglobin tell about the mechanism of cooperativity.

The populations of the intermediates in concentrated solutions of hemoglobin A0 equilibrated at various PCO values, pH 7.0, 0.1 M KCl, and 20 degrees C, have been determined using cryogenic methods. Data on CO saturations and distributions of intermediates were analysed in terms of the free energies of dimer-tetramer assembly of the intermediates (G.K. Ackers and F.R. Smith, Annu. Rev. Biophys. Chem. 16 (1987) 583). The cooperative free energy value of the singly ligated species was approximately one-half the total cooperative energy. The cooperative free energy value of the doubly ligated species was not significantly different from that of carboxyhemoglobin. Because of experimental error, the observed difference in concentrations among the populations of the doubly ligated species cannot be taken as indicative of their functional heterogeneity. Additional studies on some NO intermediates have emphasized that (alpha 1 beta 1)(alpha 2 beta 2)X, a key intermediate in the formulation of the 'third-state' hypothesis in the deoxy/cyanomethemoglobin system, has a free energy value for dimer-tetramer assembly which is critically dependent on the nature of the ligand X as suggested by Ackers and Smith (reference as cited above).

The dissociation of carbon monoxide from hemoglobin intermediate.

To investigate the mechanism of allosteric switching in human hemoglobin, we have studied the dissociation of the ligand (CO) from several intermediate ligation states by a stopped-flow kinetic technique that utilizes competitive binding of CO by microperoxidase. The hemoglobin species investigated include Hb(CO)4, the diliganded symmetrical species (alpha beta-CO)2 and (alpha-CO beta)2, and the di- and monoliganded asymmetrical species (alpha-CO beta-CO)(alpha beta), (alpha-CO beta)(alpha beta-CO), (alpha beta-CO) (alpha beta), and (alpha-CO beta)(alpha beta). They were obtained by rapid reduction with dithionite of the corresponding valence intermediates that in turn were obtained by chromatography or by hybridization. The nature and concentration of the intermediates were determined by isoelectric focusing at -25 degrees C. The study was performed at varying hemoglobin concentrations (0.1, 0.02, and 0.001 mM [heme]), pH (6.0, 7.0, 8.0), with and without inositol hexaphosphate. The results indicate that: (a) hemoglobin concentration in the 0.1-0.02 mM range does not significantly affect the kinetic rates; (b) the alpha chains dissociate CO faster than the beta chains; (c) the symmetrical diliganded intermediates show cooperativity with respect to ligand dissociation that disappears in the presence of inositol hexaphosphate; (d) the monoliganded intermediates dissociate CO faster than the diliganded intermediates; (e) the asymmetrical diliganded intermediates are functionally different from the symmetrical species.

The intermediate compounds between human hemoglobin and carbon monoxide at equilibrium and during approach to equilibrium.

The procedure of Perrella et al. (Perrella, M., Benazzi, L., Cremonesi, L., Vesely, S., Viggiano, G., and Rossi-Bernardi, L. (1983) J. Biol. Chem. 258, 4511-4517) for trapping the intermediate compounds between human hemoglobin and carbon monoxide was validated by quantitatively determining during the approach to equilibrium all the species present in a solution containing large amounts of intermediates. An accurate estimate of the intermediate compounds at 50% carbon monoxide saturation in 0.1 M KCl, pH 7, at 22 degrees C, allowed the calculation, according to Adair's scheme, of the four equilibrium constants. At 50% ligand saturation, the pool of intermediate species was about 12% of the total. A slightly greater concentration of tri-liganded than mono-liganded species was found. Carbon monoxide bound to beta chains in slightly greater excess with respect to alpha chains in both the mono- and tri-liganded species. The symmetrical bi-liganded intermediates, alpha 2 beta CO2 and alpha 2CO beta 2, were absent. The nature of the bi-liganded intermediate found to be present in detectable amounts by our technique has yet to be clarified: it could be either the asymmetrical species (alpha beta) (alpha CO beta CO) and (alpha beta CO) (alpha CO beta) or both of them. Such a finding on the functional heterogeneity among the four possible bi-liganded intermediates is consistent with hypotheses of the existence of more than two quaternary structures in the course of ligand binding to hemoglobin.

Measurement of lipase activity by a differential pH technique.

This is a new electrochemical method for determination of lipase activity in biological fluids, including serum, plasma, and duodenal juice. Advantages of turbidimetric methods--short reaction time, and small sample and reagent volumes--are combined with those of titrimetric methods: measurement of absolute activity (i.e., no standardization required), saturated substrate conditions, and direct measurement of reaction products. The proposed method is easy, inexpensive, and takes only 3 min. Precision is good: CV = 3.74% within day and 7.3% between days at the clinical-decision concentration, CV = 1.86% within day and 4.65% between days for above-normal lipase activities. The standard curve is linear up to 4500 U/L. Results (y) correlate well with those by turbidimetry (x): y = 0.9287x - 65.3 (r = 0.9719). Reference values are between 0 and 130 U/L.

The determination of ethanol in whole blood by differential pH measurements.

The application of a new technique based on the differential measurement of pH between two solutions to determine ethanol concentration in whole blood is reported. The ethanol is determined by measuring the change in pH following its enzymatic oxidation to acetaldehyde. The procedure correlates with the head space gas chromatographic method in the 0-108 mmol/l (0-5 g/l) ethanol whole blood concentration range according to the equation y = 1.344 + 1.013x (r = 0.997).

Urea, creatinine, and glucose determined in plasma and whole blood by a differential pH technique.

We report the conditions (buffer composition and enzyme activity) required for estimating three frequently determined analytes--urea, glucose, and creatinine--by use of an improved version of the differential pH apparatus previously described (Clin Chem 29: 80-85, 1983). For each analyte, we used only one specific enzyme, thus avoiding a chain of auxiliary and indicator reactions. The method requires about a minute for each determination in undiluted plasma or whole blood.

Isolation of intermediate compounds between hemoglobin and carbon monoxide.

A human hemoglobin solution partially saturated with carbon monoxide was rapidly quenched at -25 degrees C into a hydro-organic buffer containing ferricyanide. Under the experimental conditions of pH, ionic strength, and buffer composition used in this work, it was found that the deoxy hemes were rapidly transformed into their met form, whereas practically no carbon monoxide-bound hemes were oxidized before the separation of the mixture from the oxidizing agent. As a preliminary step to the analysis of the resulting solution, carbonylhemoglobin solutions partially oxidized with ferricyanide were studied by isoelectric focusing at -25 degrees C under identical conditions. The relative position in the gel of all nine possible valence hybrids was established as follows (going from the anodic to the cathodic side of the gel) alpha CO2 beta CO2, (alpha CO beta +)(alpha CO beta CO) (alpha CO beta CO), (alpha CO2 beta +2), (alpha + beta CO), (alpha + beta +)-(alpha CO beta CO), (alpha + beta +)(alpha CO beta +), (alpha +2 beta CO2), (alpha + beta +)(alpha + beta CO), alpha +2 beta +2. When carbonylhemoglobin and methemoglobin were mixed in equal proportion at -25 degrees C and then analyzed by isoelectric focusing at the same temperature, it was found that the contribution of valence hybrids other than alpha CO2 beta CO2 and alpha +2 beta +2 to the total amount of hemoglobin in the gel was no more than 6%. When carbonylhemoglobin and deoxyhemoglobin were mixed in the same proportion and incubated at 20 degrees C so to allow the redistribution of the carbon monoxide molecules between all possible binding sites to occur, a substantially higher amount of valence hybrids, derived from the oxidation of intermediate compounds of hemoglobin with carbon monoxide, was found. The isoelectric focusing separation indicated the presence in the original solution of intermediate species other than carbonylhemoglobin and deoxyhemoglobin at a concentration of about 10% of the total.

Simulation of continuous blood O2 equilibrium curve over physiological pH, DPG, and Pco2 range.

We analyzed 56 O2 equilibrium curves of fresh human blood, each from 0 to 150 Torr Po2. The data were collected over ranges of values for the 2,3-diphosphoglyceric acid-to-hemoglobin concentration ratio [DPG]/[Hb] of 0.2-2.7, for pH of 7.0-7.8, and for Pco2 of 7-70 Torr. Each curve was characterized according to the Adair scheme for the stepwise oxygenation of Hb, and the resulting constants (a1, a2, a3, a4) were analyzed to allow the simulation of the entire O2 equilibrium curve under any conditions of [DPG]/[Hb], pH, and Pco2 in the specified range. This analysis provides a powerful tool to study the affinity of Hb for O2 within the red blood cell and to predict the shape of the O2 equilibrium curve in various physiological and pathological states. Other attempts to predict blood O2 affinity have considered only P50 (the Po2 at one-half saturation with O2) or have provided too little data for continuous simulations.

Effect of temperature on the p50 value for human blood.

We investigated the effect of temperature (19, 30, 37, and 43 degrees C) on the p50 value for normal human blood at pco2 = 5.72 kPa (43 mmHg), at various pHs (range 7.0 to 7.6) and molar ratios of [2,3-diphosphoglycerate]/[Hb4] (range 0.4 to 2.4). The d(log p50)/d(pH) coefficient varied from 0.39 at 19 degrees C to 0.35 at 43 degrees C. The relationship between log p50 and 1/T (T = degrees Kelvin) was linear under the experimental conditions used, and the d(log p50)/d(1/T) coefficient varied between -2138 at pH 7.0 and -2162 at pH 7.6, independent of the concentration of 2,3-diphosphoglycerate. Assuming that the effect of pco2 on the p50 value is the same at 19, 30, and 43 degrees C as at 37 degrees C, one can use the reported coefficients to calculate the p50 value for normal human blood under conditions of temperature, pH, pco2, and 2,3-diphosphoglycerate concentrations prevailing under physiological and pathological conditions. The p50 value calculated by empirical equations, taking into account the effect of temperature, correlated well with the values for p50 determined experimentally (y = 0.9774x + 0.453; r = 0.998; n = 60), with an SD of 52 Pa (0.39 mmHg).

Measurement of glucose in plasma by a differential pH technique.

A new automatic apparatus based on the differential measurement of pH between two solutions has been developed. Two 25-microL (internal volume) glass capillary electrodes are used to measure the results of automated (under microcomputer control) chemical reactions that lead to the liberation or the uptake of hydrogen ions. The sensitivity of the differential pH measurements is better than +/- 0.0001 pH unit, and the change in H+ concentration that can be detected by such an apparatus is 1 mumol/L for plasma and 3 mumol/L for whole blood. The technique has been applied to the measurement of glucose in plasma, giving results in agreement with the specifications of the Food and Drug Administration reference method for quantitative determination of glucose (hexokinase/glucose-6-phosphate dehydrogenase method).

[Hemoglobin S as the cause of primary hyperviscosity of the blood]. / L'emoglobina S quale causa di iperviscosità primaria del sangue.

The hemoglobinemia S is a genetic defect due to a replacement of a single amino acid in the beta-chain of the human hemoglobin, leading to the most characteristic case of primary blood hyperviscosity. The difference in beta-chain of normal HbA compared to that of HbS is represented by the replacement of glutamic acid, normally found in the 6th position, with valine. The sickling of HbS-containing erythrocytes is due to the polymerization of deoxygenated HbS tetramers with formation of linear structures and to the parallel distribution of these fibers in the red blood cell. The full deoxygenation of HbS blood induces the characteristic morphological changes of red blood cell and increases the blood viscosity from 40 to 120% if compared to the viscosity of the same oxygenated blood. Any change in the viscosity of normal blood following deoxygenation was observed. Almost all the clinical symptoms found in patients carrying HbS can be directly or indirectly correlated to the increased blood viscosity following deoxygenation.

Isolation of intermediate valence hybrids between ferrous and methemoglobin at subzero temperatures.

Quenching a hemoglobin solution partially saturated with carbon monoxide into a hydro-organic solvent containing ferricyanide will produce under suitable conditions a population of partially oxidized and CO-bound hemoglobin molecules. Since each Fe3+ heme carries one extra charge, it should be possible, in theory, to resolve the spectrum of intermediate compounds between hemoglobin and carbon monoxide, which was originally present in solution. In this study we report: 1) the development of a simple and rapid method to quench aqueous hemoglobin solutions into a hydro-organic solvent at subzero temperatures; 2) the determination of suitable experimental conditions to isolate valence hybrids between carbonmonoxy- and methemoglobin by isoelectric focusing at temperatures as low as -25 degrees C; and 3) the identification and isolation of all valence hybrids of different charge between carbonmonoxy- and methemoglobin.