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L Baronciani

Publications and source records attributed to L Baronciani.

35 records · Page 2Linked to original sources
PubMed1994

Goat immunoglobulin purification on phosphocellulose and DEAE Affi-Gel blue.

We describe a method for the efficient purification of immunoglobulins G (IgG) to near homogeneity from goat serum. This was achieved by performing first an AS-40 fractionation on goat serum, followed by chromatography on phosphocellulose (P11) equilibrated in citrate buffer at pH 5.7. Peak I, eluted at V0 from P11, contained all IgG and the other serum proteins, except beta-globulins and most of the alpha-2-globulins, which are eluted in a second peak with 0.24 M K-phosphate in citrate buffer at pH 6.0. Peak I, concentrated and dialyzed in 20 mM K-phosphate buffer pH 8.0, was then applied onto a DEAE Affi-Gel Blue column equilibrated in the same buffer. Two peaks were obtained from this column: peak I, eluted at V0 contained a pure IgG fraction, while the other serum proteins were in peak II. We conclude that the P11 step, performed under the conditions we report here, is very useful to retain the alpha-2 and beta-globulins, which contaminate the IgG when only the DEAE Affi-Gel Blue purification step is used.

Animals↗
PubMed1993

Analysis of pyruvate kinase-deficiency mutations that produce nonspherocytic hemolytic anemia.

The intron sequences of the human L-type pyruvate kinase gene (PKLR) were determined by using primers selected from the known cDNA sequence. Oligonucleotide primers for these determined intron sequences were used to sequence the exons. When this technique was applied to the DNA of 10 unrelated patients with pyruvate kinase deficiency, the following eight different mutations in the coding region were detected: del391-393, A401, C464, G721, A1076, T1456, T1484, A1529. The A1529 mutation was found repeatedly in unrelated individuals, even in the homozygous state. The context with respect to a polymorphism at nt 1705 was compatible with a single origin for this mutation, and it may represent a balanced polymorphism. In normal subjects, five differences from the published cDNA sequence were documented.

Amino Acid Sequence↗
PubMed1993

Molecular analysis of G6PD variants in northern Italy: a study on the population from the Ferrara district.

In the Ferrara district, an area south of the Po delta, four different variants of glucose-6-phosphate dehydrogenase (G6PD;E.C.1.1.49) have been described as a result of biochemical characterization of the enzyme protein: one was G6PD Mediterranean (G6PD Med) and three were local variants named Ferrara I, II, and III. The Ferrara I variant was recently analysed at the DNA level and shown to correspond to G6PD A376G/202A, while the mutations causing the variants II and III, still remain unknown. We analysed the G6PD coding region of 18 apparently unrelated G6PD deficient subjects, whose families have lived in the Ferrara district for at least three generations: 12 subjects had G6PD Med563T/1311T, 3, G6PD Santamaria376G/542T and 2, G6PD A-376G/202A. In one subject we found a new mutation, a G-->A transition at nucleotide 242 causing an Arg-->His amino-acid replacement at position 81. We named this new variant G6PD Lagosanto242 A. Phenotypically the enzyme has nearly normal kinetic properties and appears different from the variants Ferrara II and III.

Child↗
PubMed1992

New glucose-6-phosphate dehydrogenase mutations from various ethnic groups.

Seven new mutations that produce glucose 6 phosphate dehydrogenase (G6PD) deficiency are described. Three are in variants that were biochemically characterized and described previously, while four were found in samples that had not been characterized biochemically. Several of the mutations affect the amino acids that are mutated in other G6PD variants. As had been noted previously, variants that are associated with nonspherocytic anemia are located either near the glucose 6 phosphate or the NADP binding sites. Variants more distant from these sites are not associated with chronic hemolysis.

Amino Acid Sequence↗
PubMed1992

Acetaldehyde, ethanol and acetone concentrations in blood of alcohol-treated mice receiving aldehyde dehydrogenase-loaded erythrocytes.

Blood levels of acetaldehyde (ACh), ethanol and acetone were investigated in mice treated with ethanol for 6 months and receiving compatible erythrocytes (RBCs) overloaded with aldehyde dehydrogenase (AlDH). Following an acute dose of ethanol, ACh levels were significantly lower in these animals than in alcohol-treated mice receiving AlDH-unloaded RBCs, and were similar to the ACh levels of normal mice. The peak ethanol concentration was higher in normal mice than in both groups of alcohol-treated animals, while acetone concentrations were not significantly different in the three groups of animals.

Acetaldehyde↗
PubMed1991

Glucose-6-phosphate dehydrogenase activity and protein turnover in erythroblasts separated by velocity sedimentation at unit gravity and Percoll gradient centrifugation.

This work was undertaken to improve a separation method for preparation of large amounts of erythroid cells of different age with homogeneous and minimal contamination of myeloid cells. Our method was suitably employed in the study of the decay mechanism of glucose-6-phosphate dehydrogenase (G6PDH) during the erythroid cell maturation. Twenty fractions of erythroid cells at different advancing stages of maturation were prepared by fractionating, at unit gravity, bone marrow cells from anaemic rabbit. The specific activity of the G6PDH was assayed and plotted vs the fraction number and the typical sigmoid curve of the activity decay was drawn. The separated cells were then grouped in three sets of fractions following the three phases of the sigmoid curve and the fractions of each set were combined. From the cytochemical analysis of the three main fractions so obtained, we found a 25-30% myeloid cell contamination in the first fraction, while in the other two fractions the myeloid contamination was 10% or less. For this reason we performed a rapid separation of the first fraction on a discontinuous percoll gradient. By this method, the myeloid cell contamination of the first fraction was levelled down to the other two. The fractions, so obtained, (I, II and III in order of increasing cell maturation) showed a four fold decrease of glucose-6-phosphate dehydrogenase activity expressed both per cell number and on protein base. On the contrary the concentration of the total soluble proteins did not change significantly in the three fractions. The three purified cellular populations were used to provide information on the protein turnover of the erythroid cells during their development. We measured, in intact cells, the rate of synthesis and degradation of total proteins and then, in cell lysates, we determined the rate of degradation of G6PDH, purified from rabbit RBC and radiolabeled by reductive methylation with C14-formaldehyde. The rates of proteolysis obtained with total proteins and methyl-G6PDH clearly indicate that the proteolytic machinery of the erythroblasts reduces its activity during the cell maturation.

Anemia↗
PubMed1991

Glucose-6-phosphate dehydrogenase activity in dorsal root ganglia of vitamin E-deficient rats.

The effect of dietary vitamin E on the activity of glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) was studied in the dorsal root ganglia of rat. One-month-old male Sprague-Dawley rats were randomly assigned to two dietary treatment groups for 2 months. The first received a standard diet supplemented with vitamin E, the second was fed a basal vitamin E-deficient diet. The activity of G6PD was markedly decreased in ganglia of the deficient animals with respect to the controls. On the other hand, the activity of the 6PGD was not significantly altered in the deficient animals. In the red cells the two enzyme activities presented a similar situation and the level of the reduced glutathione in the red cells was not significantly altered by the status of dietary vitamin E. Kinetic analysis with crude extracts of ganglia or partially purified G6PD demonstrated that there was no direct modulatory effect of the vitamin on the enzyme activity. Moreover, nondenaturing gel electrophoresis performed in this study revealed that none of the three G6PD activity bands which appeared on the acrylamide gel were significantly altered in the deficient animals. At present, the mechanism linking the G6PD activity with the status of dietary vitamin E remains unknown. Our results suggest, however, that a reduced NADPH generation produced by a decay of G6PD activity may limit the glutathione peroxidase, a very active enzyme in detoxifying peroxides, and may predispose the nervous tissue to oxidant injury.

Animals↗
PubMed1991

Glucose-6-phosphate dehydrogenase Lodi844C: a study on its expression in blood cells and muscle.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency was found in erythrocytes, lymphocytes and muscle of an Italian male, whose family has lived for at least three generations in Lodi (Lombardy, northern Italy). The subject was hospitalized for myalgia and dark urine after intense physical exercise, but no sign of anemia and chronic hemolysis were present at rest. Family studies revealed that the mother and the maternal aunt had the same enzymopathy. The enzyme-specific activity in red blood cells was 15% of control and the kinetic properties were the following: slower electrophoretic mobility; biphasic pH activity curve; slightly reduced thermal stability, and increased utilization of the substrate analogs. The analysis of our patient's DNA showed a G----C mutation at nucleotide 844 which causes an Asp----His amino acid change in position 282. This is the same mutation found by De Vita et al. in the G6PD Seattle-like variant. However, by following a new convention, we labelled our variant as G6PD Lodi844C. As far as the muscle is concerned, we found that the enzyme-specific activity in this tissue was 14% of control values, but cultured myotubes and myoblasts revealed a normal level of G6PD as well as skin fibroblasts. On the contrary in the same type of cultured cells obtained from G6PD Mediterranean subjects, the G6PD activity was about 20% of normal. Our results complete the characterization of this mutant enzyme, demonstrate the expression of the deficit in muscle and describe the enzyme behaviour in cultured cells.

Adult↗
PubMed1991

[Purification from goat antiserum of immunoglobulins against G6PD from rabbits].

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.

Animals↗
PubMed1990

Rapid purification of glucose-6-phosphate dehydrogenase from mammal's erythrocytes.

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.

Acetates↗
PubMed1990

[Hepatic hematopoiesis in phenylhydrazine-induced hemolytic anemia].

Ten adult rabbits were divided into two groups: the control rabbits, which received subcutaneous injections of 0.9% NaCl in three days; the experimental animals which received 3 mg/Kg body weight of phenylhydrazine (PHZ) subcutaneously also in three days. On the 8th day from the initial treatment the control and experimental animals were sacrificed, blood was collected to determine hematological parameters and livers were cut into small pieces. Sections were prepared by pressing the pieces onto slides which were stained with the Giemsa stain. The hematocrit and the reticulocytosis of experimental animals were 25 + 3%, and 70 + 5% respectively. In the liver sections of the PHZ treated animals we found a very rich population of immature erythroblasts. In fact proerythroblasts and basophilic erythroblasts were 19%, polychromatic and orthochromatic erythroblasts were 22% and 13% respectively. On the contrary, these cells were absent in the control livers. The lymphocyte and lymphoblast population, on the other hand, was very rich in control animals with a value of 38.8% compared to 1.62% in the anemic animals. The results clearly indicate the hematopoietic function of the liver in the anemic animals although the low percentage of orthochromatic erythroblasts with respect to their precursors suggests the ineffectiveness of the process.

Anemia, Hemolytic↗
PubMed1990

[Progression of the peripheral blood profile in phenylhydrazine-induced hemolytic anemia].

Male rabbits were made anemic by subcutaneous injections of 3 mg/Kg body weight of phenylhydrazine (PHZ) for the duration of three days. Blood samples were collected prior to the experiment, on the 2nd and 5th day from the end of the treatment. Hematocrit, reticulocyte count, MCV, osmotic resistance, RBC volume distribution curve and morphological analysis were performed on each sample in order to obtain a picture of the progressive changes of the parameters following the PHZ administration. Hematocrit values changed from 49 to 27%, whereas the reticulocytosis increased from 0.6 to 73%. A significant elevation of MCV was detected and the cell volume distribution curve, which presented the typical bell-shape profile in the normal animals became bimodal on the 5th day. The osmotic resistance of RBC of anemic animals, showed a marked deviation from the normal pattern. In fact initial haemolysis started at 0.75-0.70% NaCl concentration in the 5th day samples, while it was between 0.55-0.50% before the PHZ treatment. Finally, morphological analysis revealed a progressive increase of RBC with Heinz bodies, of macro-megalocytes and of immature erythroblasts thus indicating that the cell population, produced during recovery from PHZ induced anemia, is widely heterogeneous.

Anemia, Hemolytic↗
PubMed1990

[Effect of ATP on glucose-6-phosphate dehydrogenase during erythroblast maturation in the rabbit].

The glucose-6-phosphate dehydrogenase (G6PD) activity of erythroblasts, separated at different advancing stages of development, shows a marked decline of activity. A proteolytic mechanism, strictly controlled, is likely responsible of this decay, since a sufficient level of enzyme activity still remains in the circulating erythrocyte. In this report we suggest a model that could explain what triggers the mechanism of proteolytic degradation. HPLC analysis of the nucleotide content of erythroblasts and reticulocytes, showed a marked decline of adenine and pyridine nucleotides and of their catabolic products during the cell development. From thermostability tests, at fixed temperature, we have seen that ATP and NADP only, significantly protected the enzyme activity. In this light, we incubated 10 min at increasing temperatures, with and without ATP or NADP lysates of erythroblasts, separated at different stage of development and of reticulocytes. In the absence of nucleotides, we determined for all fractions a T degree break at 42 degrees C. In the presence of NADP all fractions were stabilized with no break point in the range 37-50 degrees C. On the contrary, the presence of ATP caused a progressive shift of the T degrees C break from the most immature erythroblasts (T degree break at 46 degrees C) to the reticulocytes (T degree break at 42 degrees C). Since ATP did not show any protective effect on the reticulocyte enzyme, we hypothesize the presence in these cells of a structurally modified G6PD. Furthermore, these data support our belief that the marked decline of ATP during cellular development, may represent the element responsible for the enzyme modification.

Adenosine Triphosphate↗
PubMed1990

[Splenic hematopoiesis in an experimental model of hemolytic anemia].

The splenic hemopoiesis of rabbits, made anemic with acetylphenylhydrazine, and of control animals was investigated. Pieces of spleen of both groups were fixed in formalin and embedded in paraffin. Paraffin sections, cut 5-7 microns in thickness, were stained with hematoxylin-eosin, Giemsa, Perls' method for tissue iron (hemosiderin), and Perls-Chayen's method for iron stored in the hemoglobin. The erythroid line in the anemic rabbits, showed a marked increase of proerythroblasts and basophilic erythroblasts, while the poli and orthochromatic erythroblasts were less than their precursors. In contrast these cells were more than their precursors in control animals. There was no notable quantitative difference in the mature elements of this line in the anemic animals and in the controls. Megaloblasts and macroblasts were frequently observed in anemic spleens but they were practically absent in the controls. Regarding to other cell lineages, we noted in the anemic spleens many macrophages containing Perls and Perls-Chayen positive material and some megakaryocytes. Our results indicate that the APH-induced anemia stimulate the erythropoietic activity of the spleen in the rabbit, but the reversion of the amplification phase of the differentiation steps reveals that the erythropoietic process is ineffective. The presence of megalo- and macroblasts provide morphological evidence of dyserythropoiesis and the megakaryocytes suggest that under the anemia condition also the platelet regenerating process is stimulated.

Anemia, Hemolytic↗
PubMed1990

[Glucose-6-phosphate dehydrogenase activity as a marker of resolution in the separation of rabbit erythroblasts according to their stage of maturation].

The glucose 6 phosphate dehydrogenase (G6PD) activity of erythroblasts, separated at different advancing stages of development by the velocity sedimentation technique at unit gravity, shows a characteristic sigmoidal curve. The G6PD activity is high and constant in the dividing compartment, steeply declines between the polychromatic and the orthochromatic stage, and returns almost constant during the development from the orthochromatic to the reticulocyte stage. This report focuses on the possibility to use the G6PD activity curve to indicate a loss of resolution in the fractionation process. For this purpose two parameters of the graph were used: (h) which represents the distance between the two constant phases of the curve and (s) the slope of the decreasing part of the graph. In this view we have fractionated on a 400 ml linear gradient of sucrose (1%-2% in PBS), increasing amounts of bone marrow cells from anaemic rabbit. Suspensions of 100, 200, 350 and 700 millions of cells (25 ml in PBS) were separated in different experiments and the obtained G6PD activity curves were compared. We have seen that the two parameters h and s remained constant up to 200 millions of cells, while they declined markedly when the number of cells loaded on the gradient increased to 350 and 700 millions. The separated cells were grouped into three sets of fractions: fraction I with cells belonging to the high and constant phase of the graph, fraction II to the decreasing phase and fraction III to the low and constant activity phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗