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Biomedical subjects

A Fazi

Publications and source records attributed to A Fazi.

At least 19 recordsLinked to original sources
PubMed1992

Glucose 1,6-bisphosphate-overloaded erythrocytes: a strategy to investigate the metabolic role of the bisphosphate in red blood cells.

Human erythrocytes overloaded with glucose 1,6-bisphosphate were prepared in order to establish the metabolic significance of this phosphorylated sugar in the intact red cell. The intracellular glucose 1,6-bisphosphate concentration was increased six- and twofold over the normal level by encapsulating (i) the commercially available compound and (ii) the glucose 1,6-bisphosphate synthase obtained from rabbit skeletal muscle, respectively. In both experimental conditions, a reduction of glucose utilization by the loaded cells was observed after reequilibration to the steady state. At the steady state, the concentrations of the glycolytic intermediates and of the adenine nucleotides appeared substantially unmodified when compared with those of controls, with the exception of a 50% reduction of glucose and fructose 6-phosphate measured in erythrocytes encapsulated with exogenous glucose 1,6-bisphosphate. Under the considered experimental conditions, the elevated intracellular glucose 1,6-bisphosphate appears to display an inhibitory effect on hexokinase that overcomes the possible activation of phosphofructokinase or pyruvate kinase.

2,3-Diphosphoglycerate↗
PubMed1992

[Risk stratification in patients with a first myocardial infarct based on simple clinico-instrumental variables].

UNLABELLED: It is an acknowledged fact that the prognosis for patients with a first myocardial infarction depends mainly on the degree of residual left ventricle function. We wanted to evaluate the importance that certain simple clinical and instrumental variables can have in stratifying post-infarction cardiovascular risk with particular emphasis on chronic obstructive lung disease (COLD). We selected 97 out of the 512 patients treated in the coronary intensive care unit (CICU) from February 1, 1988 to October 31, 1990 according to the following criteria: First myocardial infarction; no cardiogenic shock; no serious concomitant diseases with considered negative prognosis within 6 months. The following variables were considered for all the patients: age; sex; positive family history for ischemic heart disease; history of diabetes mellitus; arterial hypertension; previous cerebrovascular incident; history of obstructive arteriopathy of the lower limbs, of angor and COLD. The following tests were performed on all the patients: echocardiogram prior to discharge form the CICU; angiocardioscintigraphy with Tc-99 between the 20th and 30th day following the acute event; bicycle ergometer stress test on the 30th day. END POINTS: general mortality; cardiac mortality; non-fatal reinfarction; residual angina at 3 months. All the patients were treated with aspirin (325 mg/die) and/or heparin (12,500 units subcutaneously). All 97 patients were monitored for a mean follow-up time of 19.8 months. General mortality was 2.08% (for reinfarction) 24 (24.7%) non-fatal cardiac events.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1992

Red blood cells as an antigen-delivery system.

The use of adjuvants is usually required to induce strong immunological responses to protein antigens. However, in many cases these adjuvants cannot be extensively applied in human and veterinary vaccinations because of associated inflammatory reactions or granuloma formation. We show here that protein antigens (bovine serum albumin, hog liver uricase, and yeast hexokinase), coupled to autologous red blood cells by way of a biotin-avidin-biotin bridge, elicit an immunological response in mice similar to or higher than that obtained by the use of Freund's adjuvant. Quantities as low as 0.5 micrograms/mouse are high enough to generate these immunological responses. Furthermore, splenocytes of mice immunized by red blood cell-coupled antigens can be used to generate hybridomas secreting monoclonal antibodies. Thus, the delivery of antigens by autologous red blood cells is an effective way to avoid the use of adjuvants for producing anti-peptide antibodies and possibly to generate peptide vaccines.

Animals↗
PubMed1991

Cell age dependent decay of human erythrocytes glutathione S-transferase.

Human red blood cells contain both glutathione S-transferase sigma (GST sigma) and glutathione S-transferase rho (GST rho). While the first isozyme does not change in red blood cell fractions of different mean density (age), GST rho, the main isozyme, shows a pronounced cell age dependent decay. Ion-exchange chromatographic experiments show that GST rho consists of only one isozymic form in young erythrocytes but is present in two components, with different electric charge, in mature and old cells. The "secondary" GST rho isozyme is more heat stable than the "primary" GST rho isozyme with the result that the total GST activity shows an apparent increase in heat stability during cell aging due to the formation of "secondary" isozymes. The kinetic properties and specificity of this enzyme do not show appreciable modifications during cell ageing. The data reported in this paper suggest that red blood cell aging is associated with a reduced detoxifying ability due to GST rho decay.

Erythrocyte Aging↗
PubMed1991

Specificity of glucose 1,6-bisphosphate synthesis in rabbit skeletal muscle.

1. To compare glucose 1,6-bisphosphate synthesis in different types of cells, we partially purified (2000-fold) a glycerate 1,3 P2-dependent glucose 1,6-bisphosphate synthase from rabbit skeletal muscle. 2. In agreement with the results reported by others for mouse brain and pig skeletal muscle, the enzyme can be separated from bulk phosphoglucomutase (PGM) activity by DEAE-cellulose chromatography of crude cellular extract. This cannot be achieved on human hemolysates where glycerate 1,3-P2-dependent glucose 1,2-bisphosphate synthesis is displayed only by multifunctional PGM2 isoenzymes. 3. The Km values for glycerate 1,3-P2 (0.50 microM), glucose 1-phosphate (90 microM), Mg2+ (0.22 mM), and also pH optimum (7.8) and mol. wt (70,000) of the rabbit skeletal muscle enzyme are similar to those of the enzymes from mouse brain and human red blood cells, but they differ from those reported for the pig skeletal muscle enzyme.

Animals↗
PubMed1991

Purine nucleoside phosphorylase from human erythrocytes: a kinetic study of the fully separated isoenzymes.

Human red cell lysates contain at least seven electrophoretically distinct isoenzymes of purine-nucleoside phosphorylase (PNPase); the proportion of more anodal bands increases as the erythrocyte ages, suggesting that the native enzyme is subjected to progressive post-translational modifications. The age dependent electrophoretic changes observed in the hemolysate are associated with the downward curvature of the Lineweaver-Burk double reciprocal plot at high inosine-substrate concentrations unlike the single-banded PNPase from tissue cultures of rapidly dividing cells. Thanks to the high resolution power of the ion-exchange HPLC technique utilized we have been able to fully separate all the seven isoenzymes and correlate structural to functional modifications in PNPase from human erythrocytes. Our results indicate that the downward curvature of Lineweaver-Burk plot is not due to a mixture of isoforms with low and high Km for inosine but that the allosteric activation by the inosine substrate is the direct consequence of structural modification(s) on the "primary" form of the enzyme.

Adult↗
PubMed1991

Human red blood cells as bioreactors for the inactivation of harmful xenobiotics.

Human red blood cells are able to inactivate lipophilic electrophiles by conjugation with reduced glutathione. This metabolic ability was found to be limited by the rate of permeation of the xenobiotic into erythrocytes and by the amount of available reduced glutathione. By a procedure of hypotonic dialysis, isotonic resealing and reannealing human red blood cells were overloaded with increasing amounts of reduced glutathione up to three- to fourfold the normal level without modification of their metabolic functions or of their energetic state. These overloaded erythrocytes were able to conjugate increasing amounts of xenobiotics and to export the resulting conjugates from the cells. These properties of glutathione overloaded erythrocytes are significant for the use of carrier erythrocytes in cases of acute intoxication by lipophilic electrophiles.

Biotechnology↗
PubMed1990

Purification and partial characterization of the phosphoglucomutase isozymes from human placenta.

We have developed a simple procedure for the purification of phosphoglucomutase (PGM) isozymes from human placenta of healthy women. The technique involves the ammonium sulfate fractionation, ion-exchange and dye-ligand chromatographies. By this method we obtained homogeneous isozyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci. The final specific activities were 1134.6-1441.8 units/mg for PGM1 forms and 40.2-46.5 units/mg for PGM2 forms. On SDS-polyacrylamide gel electrophoresis analysis, the final preparations gave a single protein band of 58,500 and 69,000 Mr for PGM1 and PGM2 isozymes, respectively. These forms have the same kinetic properties, but from the substrate specificity experiments we have found that PGM2 forms are more effective for catalyzing the phosphoribomutase and glucose 1,6-bisphosphate synthase reaction than PGM1 forms. All these properties are shared by the same isozymes previously isolated from human erythrocytes but in this procedure the use of human placenta for the PGM isozymes purification takes advantage of high specific activity of PGM in the extracts of this tissue as well as obtaining highly homogeneous protein suitable for studies at molecular level.

Adult↗
PubMed1990

Immunological studies on erythrocyte phosphoglucomutase isozymes.

Human erythrocytes (phenotype PGM1 a1 or PGM1 a3) contain two sets of phosphoglucomutase isozymes, produced by the expression of the PGM1 and and PGM2 loci. The two sets are constituted each by two forms, of which that called "secondary" is thought to derive from the post-translational modification of that called "primary". Cross-reactivities of these isozymes were studied by means of monospecific rabbit antibodies against purified human red cell PGM1 and PGM2 "primary" isozymes. The results show that the PGM1 and PGM2 forms are not immunologically related and provide further proof of the post-synthetic origin of "secondary" isozymes and of the multifunctionality of PGM2 phosphoglucomutases.

Antibodies↗
PubMed1989

Cell age dependent decay of human erythrocytes glucose-6-phosphate isomerase.

Glucose-6-phosphate isomerase shows a biphasic decay pattern during red blood cell aging, which is very fast during the first part of cell's life span in circulation. This decay is not due to accumulation of inactive enzyme molecules, as shown by immunological studies, but is accompanied by the formation of secondary isozymes (i.e., chemically modified forms). Electrophoretic and ion-exchange chromatographic experiments show that glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) consists of only one isozymic form in young erythrocytes but is present in two components, with different electric charge, in mature and old cells. This secondary isozyme is more stable to heat treatment and is inactivated by IgG anti-glucose-6-phosphate isomerase with a lower affinity than the native isozyme. In vitro incubation of homogeneous human glucose-6-phosphate isomerase under conditions known to produce enzyme deamination does not reproduce the isozymic pattern found in erythrocytes, suggesting that one or more mechanisms other than those previously described to explain glucose-6-phosphate isomerase microheterogeneity occur in the human erythrocyte.

Chromatography↗
PubMed1989

Isoenzymes of phosphoglucomutase from human red blood cells: isolation and kinetic properties.

A procedure has been developed for the purification of phosphoglucomutase from human red cell (phenotype PGM1 a1 or a3) lysates. It yields homogeneous isoenzyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci with distinctive pI (from 6.07 to 5.29). There are substantial differences between PGM1 and PGM2 isoenzymes, having single polypeptide chains of 58,500 and 69,000 Mr respectively and showing different thermostability. The kinetic properties of all the isoenzymes for the phosphoglucomutase reaction are essentially the same (apart from the specific activity of 1089-1263 units/mg for PGM1 forms vs 37-42 units/mg for PGM2 forms), but there are striking differences in substrate specificity. In fact the products of PGM1 locus are "true" phosphoglucomutases, being specific to mutate glucose monophosphates, whereas the PGM2 forms also display phosphoribomutase and glucose 1,6-bisphosphate synthetic activities. Some kinetic properties of these "side activities" are also reported.

Chromatography, DEAE-Cellulose↗
PubMed1988

Decrease in frequency of anginal episodes by control of thrombin generation with low-dose heparin: a controlled cross-over randomized study.

Increased thrombin generation is frequently associated with an increase in anginal activity. A cross-over, single-blind, completely randomized study was planned in order to evaluate whether the control of thrombin generation affected the increase in anginal activity. After discharge from the hospital, 24 patients (18 men and 6 women, aged 40 to 69 years) suffering from spontaneous angina were followed up to 12 months and were alternatively treated during two consecutive 6-month periods with calcium heparin, 12,500 IU by the subcutaneous route, or with placebo by the intramuscular route, in addition to the usual antianginal medications. Thrombin generation and clinical activity of angina were assessed every 15 days by measuring fibrinopeptide A (FPA) plasma levels and by grading in three classes (symptomless, mildly symptomatic, and severely symptomatic) the anginal activity on the basis of the number and the time concentration of the ischemic attacks and ECG changes. Low-dose heparin treatment significantly reduced both the FPA plasma level (from 4.1 +/- 3.7 to 2.3 +/- 1.8 ng/ml, p less than 0.001) and the clinical activity of angina. During heparin treatment, the frequency of the observations in the severely and mildly symptomatic classes decreased, respectively, by 53% and by 30%, whereas that in the symptomless class increased by 23% (p less than 0.001) in comparison with the period on placebo. Present results indicate that the control of thrombin generation obtained by low-dose heparin treatment favorably affects the degree of anginal activity in patients with spontaneous angina.

Aged↗
PubMed1988

Simultaneous preparation from human placenta of several enzymes of glucose metabolism.

A procedure for the simultaneous purification to homogeneity of hexokinase, phosphoglucomutase 1 and 2, aldolase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase from human origin has been developed. Human placenta homogenate was first chromatographed on DE-52 column which retains hexokinase and glucose-6-phosphate dehydrogenase while the other enzymes are recovered in the unabsorbed protein fraction. The other steps in the purification involve Matrex gel and specific affinity chromatography for the DE-52 retained enzymes and phosphocellulose and Matrex gel chromatography for the other enzymes. All the enzymes mentioned were obtained in one week, with recoveries from 14 percent for glucose-6-phosphate dehydrogenase to 75 percent for hexokinase. Thus, the procedures utilized seem to be useful in obtaining large amounts of enzymes in a a homogeneous form from an easily available human tissue.

Chromatography, Affinity↗