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M Garbarz

Publications and source records attributed to M Garbarz.

At least 55 records · Page 3Linked to original sources
PubMed1986

Hereditary elliptocytosis: clinical, morphological and biochemical studies of 38 cases.

We report clinical, morphological and biochemical studies performed on 38 cases of hereditary elliptocytosis (HE). The major determinant of membrane shape and stability is a proteinaceous meshwork named membrane skeleton, composed mainly of spectrin, actin, protein 4.1 and ankyrin. Spectrin is a heterodimer composed of two chains alpha and beta. Two spectrin dimers associate head to head to form a tetramer. Spectrin tetramers are cross-linked by actin and protein 4.1 to form the skeletal meshwork. We observed two types of membrane defects in the 38 patients studied: 24 patients (13 kindreds) exhibited spectrin self-association defect (type I HE) and 14 patients (6 kindreds) displayed deficiency in protein 4.1. A mutation in the spectrin chain was mostly found in the cases of type I HE. These mutations were depicted on tryptic digest patterns of spectrin. Three pathological variants were thus identified and characterized by the appearance of an abnormal peptide, with a molecular weight of either 74,000 or 65,000, or 46,000 daltons. In one family, the spectrin self-association defect was related to a shortened spectrin beta chain. Deficiency in protein 4.1 was found in 14 patients by means of polyacrylamide gel electrophoresis of red cell membranes. In 12 heterozygous cases of HE, the decrease in the amount of protein band 4.1 was between 40% and 50%. In 2 homozygous HE cases, protein band 4.1 was totally absent. Immunoelectrotransfer blots of red cell membrane proteins using a monoclonal antibody against protein 4.1 allowed characterization of additional bands in two families. In some cases variations in the amount of glycophorin C were noticed. Comparative studies of the two types of membrane abnormalities in HE clearly showed the absence of correlation between clinical, morphological phenotypes, and specific molecular etiology. However, all HE patients with protein 4.1 deficiency were caucasian and most of the type I HE were of black extraction. A study of red cell deformability using an ektacytometer revealed that the cell deformability under isotonic conditions was decreased in all HE patients. When the deformability was studied as a function of the osmolality of the suspending medium, the curve obtained had a trapezoid shape. This typical profile appeared to be constant in type I HE. We showed that the molecular abnormalities of the spectrin alpha chain, found in most type I HE correlated well with the functional spectrin defect.(ABSTRACT TRUNCATED AT 400 WORDS)

Black People↗
PubMed1985

Pathologic and nonpathologic variants of the spectrin molecule in two black families with hereditary elliptocytosis.

Five patients with hereditary elliptocytosis (HE) from two unrelated black families were studied. The patients had prominent elliptocytosis and a decreased erythrocyte resistance to heat treatment. In one infant blood smears showed elliptocytosis and poikilocytosis; his erythrocytes fagmented at a lower temperature than those of his mother and sister, both having typical mild HE. Defective dimer-dimer association was present in all patients. Limited tryptic digestion of spectrin and subsequent analysis by one- and two-dimensional electrophoresis revealed a similar and reproducible decrease in the 80,000-dalton peptide (alpha I domain) and the concomitant appearance of a 46,000-dalton peptide. All the patients had the polymorphism of the spectrin alpha II domain commonly observed in black populations. In addition, modifications relative to the alpha III domain were detected; similar variants were found in one black control subject out of 136 and are likely related to a genetic polymorphism of the alpha III domain. No differences were observed between the peptide patterns in the infant with poikilocytosis and those of his HE sister and mother.

Adult↗
PubMed1985

Characterization of rat hepatocyte plasma membrane domains by monoclonal antibodies.

The hepatocyte plasma membrane consists of three morphologically and functionally distinct domains, the sinusoidal, the lateral and the canalicular. To study the distribution of antigenic determinants among these domains, we prepared monoclonal antibodies by immunizing mice with a crude, plasma membrane-enriched liver fraction. Four monoclonal antibodies were obtained that recognized various parts of the rat hepatocyte plasma membrane when tested by indirect immunofluorescence and immunoperoxidase assay performed on formaldehyde-fixed liver tissue. Each antibody gave a different staining pattern when analyzed by light and electron microscopy. A59 exclusively labelled the part of the sinusoidal membrane facing the sinusoids. A39 mainly labelled the sinusoidal membrane. B1 mainly labelled the lateral membrane, while the labelling by B10 was almost completely limited to the canalicular membrane. Immunoblotting showed that the antibody B1 recognized an antigen of approximately 100 kilodaltons and that B10 recognized an antigen of approximately 125 to 130 kilodaltons. These antibodies allow us to distinguish the three domains of the hepatocyte plasma membrane.

Animals↗
PubMed1985

The characterization of protein 4.1 Presles, a shortened variant of RBC membrane protein 4.1.

In a previous report (Blood 60:265, 1982), we described a family with an abnormal RBC membrane protein doublet, which we considered a shortened protein 4.1 on the basis of biochemical and genetic data. Using an anti-4.1 monoclonal antibody, we confirm here that the shortened protein derives from protein 4.1. One of the members of the family contemporaneously displayed the 4.1 (-) trait, eg, the heterozygous state of this variety of hereditary elliptocytosis that lacks protein 4.1. The 4.1a/4.1b ratio was low whenever the 4.1 trait was present, regardless of the type of protein 4.1 involved. The RBCs of the compound heterozygote, containing only the shortened species of protein 4.1, made it possible to analyze without interference the contact between shortened protein 4.1 and sialoglycoprotein beta, or glycoconnectin. Shortened protein 4.1 did not alter the amount of glycoconnectin in the ghosts nor did it change its extractability into the Triton shells. Limited proteolysis of shortened polypeptides 4.1a and 4.1b showed that they are sequence related. It is conflicting that the persons carrying the shortened protein 4.1 are devoid of specific clinical and morphological abnormalities, apart from those pertaining to the 4.1- trait, when the latter is present.

Antibodies, Monoclonal↗
PubMed1984

Hereditary elliptocytosis with a spectrin molecular defect in a white patient.

According to recent works, hereditary elliptocytosis (HE) appears to be related in some instances, to a defective self-association of spectrin (type I HE). We report a new case of type I HE observed in a white patient. Study of limited tryptic digestion of a spectrin dimer showed modification of a peptide involved in the dimer self-association process.

Adult↗
PubMed1984

Molecular defect of spectrin in the family of a child with congenital hemolytic poikilocytic anemia.

We present the study of a black family in which the proband suffered from a severe neonatal hemolytic anemia with poikilocytosis. Both the parents, sister's, and brother's proband were clinically normal. The presence of poikilocytes in proband led to a search for a red cell membrane skeleton defect. Owing to recent improvements in the erythrocyte membrane knowledge, it is now possible to approach the diagnosis by means of biochemical evaluation of both parents, even if they are asymptomatic. So, the first time discovery of a spectrin self-association defect in both parents allowed us to suspect double inheritance of this abnormality in the proband. A complete morphological and biochemical evaluation of the family allowed us to propound the diagnosis of heterozygous type I hereditary elliptocytosis (HE) for both parents and the sister and the diagnosis of homozygous type I HE for the proband owing to the following reasons: slight ovalocytosis was present in both parents and the sister; cell deformability ektacytometric studies gave the same profiles of curve as those observed in patients with HE. Defective spectrin dimer self-association found in both parents was also observed in the sister and proband, associated with the same abnormal spectrin digest pattern, namely a decrease in the amount of a 80,000-dalton peptide and a corresponding increase in a 74,000-dalton peptide. However, clinical presentation of the proband was consistent either with hereditary pyropoikilocytosis or homozygous hereditary elliptocytosis; erythrocyte thermal sensitivity studies in the proband could not be conclusive because of the presence of transfused cells. Both these diagnoses are discussed in detail.(ABSTRACT TRUNCATED AT 250 WORDS)

Anemia, Hemolytic, Congenital↗
PubMed1984

A variant of erythrocyte membrane skeletal protein band 4.1 associated with hereditary elliptocytosis.

A family comprising three patients (a mother and two children) with mild hereditary elliptocytosis was studied. Each patient had prominent elliptocytosis, reduced red cell deformability, and normal erythrocyte thermal sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the erythrocyte membranes in each patient showed decreased levels of band 4.1 (approximately half of the normal value) and the presence of an additional band migrating below protein band 4.2. This additional band was shown to derive from protein 4.1. Comparative partial proteolytic mapping of protein 4.1 and the additional band revealed a number of common peptides. Enzyme-linked immunoelectrotransfer blots of the patients' erythrocyte membranes using a monoclonal antibody to protein 4.1 revealed that, in addition to protein 4.1, two other bands below protein 4.2 were stained; one of these bands migrated in the same position as the additional band detected in the Coomassie Blue-stained gels. Immunoblotting of the patients' whole cells using the antibody to protein 4.1 revealed that this altered band 4.1 occurred as such in the intact red cell. SDS-PAGE of protein 4.1 purified from one patient showed the presence of two lower molecular weight bands below protein 4.1; the lower band migrated in the same position as the additional band found on SDS-PAGE of the patients' erythrocyte membranes. The patient's purified protein 4.1 displayed a decrease of about 40% in the binding activity with crude spectrin extracted from normal controls. Spectrin-spectrin interactions were normal in the three patients. The additional band present in the patients' red cell membranes probably represents a proteolytic degradation product. This alteration, present both in whole cells and isolated membranes, might affect the intact cells in vivo. We suggest that the patients' erythrocyte membrane instability may be related to the presence of an abnormal protein 4.1 whose modulatory influence on the spectrin-actin interaction in the skeleton is defective.

Adult↗
PubMed1983

[Congenital hemolytic anemia with erythrocyte thermal instability and defect of spectrin tetrameric polymerization. Study of erythrocyte deformability in a new case of hereditary pyropoikilocytosis using diffraction viscosimetry].

Hereditary pyropoikilocytosis (HPP) is a rare congenital hemolytic anaemia observed so far in patients of black extraction. In many cases, the severity of the anaemia has led to early splenectomy, which uniformly improved the hematological conditions. The disease is characterized by extreme anisocytosis and poikilocytosis with erythrocyte fragmentation. The pathognomonic feature is the abnormal thermal sensitivity of red cells which fragment in vitro at 45-46 degrees C (instead of 49 degrees C in normal subjects). In the case reported here, erythrocyte fragmentation (which appears at 43 degrees C) and increased osmotic fragility are studied using the Ektacytometer. Observation of different erythrocyte fractions isolated by differential centrifugation shows the extreme heterogeneity of the erythrocyte population. Studies of erythrocyte membrane proteins confirm that the disease is related to defective dimer-dimer association of spectrin which is the major membrane skeletal protein. This molecular spectrin abnormality in accounting for the membrane instability is recognized to a lesser degree in the asymptomatic mother. This may be of use in the diagnosis of HPP which is difficult to establish in a transfusion-dependent infant.

Anemia, Hemolytic, Congenital↗
PubMed1982

[Contribution of immunochemical methods to the study of human red-cell membrane proteins].

Together with the classical technics of biochemical analysis, immunological methods have led to differentiate, characterize the main red-cell membrane proteins and to better understand their organization. Immunological methods were particularly involved in the study of: 1) the membrane skeletal proteins, specially spectrin which localization in the membrane, structure and functions have been specified; 2) the principal integral proteins, glycophorin and protein band 3, the transmembrane orientation of which has been corroborated by topographic immunological mapping of their cytoplasmic domain; 3) the anchor chain protein, linking the membrane skeleton to the transmembrane proteins. These methods could further help to a better understanding of the membrane structure. Two kinds of work can be considered using monoclonal antibodies provided by the hybridoma method: 1) the purification by immunoabdsorbent technics, of quantitatively minor membrane proteins, would allow to study their structure and functions; 2) an immunological mapping with monoclonal antibodies specific against each of the skeleton proteins, of membrane preparations observed in electronic microscopy, would permit to visualize the real architecture of these different proteins in the red-cell membrane.

Actins↗
PubMed1982

[Human red cell cytoskeleton: structure, functions, abnormalities (author's transl)].

Associated with the red cell membrane cytoplasmic surface, the cytoskeleton is composed of a protein complex. This structure is a major element of membrane stability, shape and flexibility. Among the constitutive proteins, spectrin, red-cell actin and protein band 4.1 are quantitatively and structurally the most important. Some elements, relative to structure, stoichiometry and interactions between these proteins begin to be understood, specially owing to in vitro studies of reassembling cytoskeleton structures from purified constitutive elements. Besides, the nature of some interactions between the cytoskeleton and the red cell membrane has been specified. Nevertheless, it is not possible at the present time to establish an architectural picture of the cytoskeleton anatomy, and the factors actually regulating this structure physiology are not characterized. Understanding of the normal cytoskeleton will perhaps be helped by studies of abnormal models which cytoskeleton protein structure and function could be defective but this has not so far been formally demonstrated.

Actins↗
PubMed1982

Spectrin beta-chain variant associated with hereditary elliptocytosis.

An electrophoretically fast-moving variant of the spectrin beta-chain was discovered in the erythrocyte membranes of a woman and her father who both exhibited elliptocytosis and mild hemolytic anemia. This abnormal beta'-subunit (Mr = 214,000) co-existed with a decreased normal beta-chain and represented about half of the total beta-chains in the membrane. In contrast to the spectrin beta-chain, the beta'-chain was phosphorylated neither in the membrane by endogenous protein kinases nor in solution by pure membrane casein kinase whether or not the spectrin was dephosphorylated by erythrocyte cytosolic spectrin phosphatase. The presence of the beta'-chain was associated with a defective self-association of spectrin dimer to form tetramer as manifested by: (a) an excess of spectrin dimer in the 4 degrees C spectrin crude extract, (b) a defective self-association of the spectrin dimer in the 37 degrees C crude spectrin extracts. Gel electrophoretic analysis of the tetramer and dimer species isolated from the proband's 4 degrees C extract showed that the tetramer contained trace amounts of the beta'-chain, whereas in contrast, a large proportion of beta'-chain was present in the dimer. These results demonstrated the responsibility of the beta'-chain for the defective reassociation of spectrin dimer into tetramer. The study of this abnormal spectrin confirms the participation of spectrin beta-chain in dimer-dimer association and strongly suggests that the phosphorylation sites of the normal beta-chain are located at the end of the molecule involved in the dimer-dimer interactions.

Adult↗
PubMed1982

[Contribution of immunochemical methods to the study of human red-cell membrane proteins (author's transl)].

Together with the classical technics of biochemical analysis, immunological methods have led to differentiate, characterize the main red-cell membrane. proteins and to better understand their organization. Immunological methods were particularly involved in the study of: 1) the membrane skeletal proteins, specially spectrin which localization in the membrane, structure and functions have been specified; 2) the principal integral proteins, glycophorin and protein band 3, the transmembrane orientation of which has been corroborated by topographic immunological mapping of their cytoplasmic domain; 3) the anchor chain protein, linking membrane skeleton to the transmembrane proteins. These methods could further help to a better understanding of the membrane structure. Two kinds of work can be considered using monoclonal antibodies provided by the hybridoma method : 1) the purification by immunoadsorbent technics, of quantitatively minor membrane proteins, would allow to study their structure and functions ; 2) an immunological mapping with monoclonal antibodies specific against each of the skeleton proteins, of membrane preparations observed in electronic microscopy, would permit to visualize the real architecture of these different proteins in the red-cell membrane

Ankyrins↗
PubMed1981

In vitro phosphorylation of the red blood cell cytoskeleton complex by cyclic AMP-dependent protein kinase from erythrocyte membrane.

Hydrosoluble proteins extracted from human erythrocyte ghosts by dialysis at low ionic strength and alkaline pH contain a cyclic AMP-dependent protein kinase which phosphorylates in vitro the cytoskeleton components in crude extracts. Spectrin components 1 and 2, actin and protein band 4.1 undergo this cyclic AMP-dependent endogenous phosphorylation together with low molecular weight peptides solubilized with the cytoskeleton in hydrosoluble extract. However, pure spectrin and purified erythrocyte G-actin were not phosphorylated by purified cyclic AMP-dependent protein kinase from erythrocyte membrane. Purified G-actin when added free to crude extract does not undergo phosphorylation by the cyclic AMP-dependent protein kinase present in this extract. In contrast, purified cyclic AMP-dependent protein kinase added either to crude extract or to the purified cytoskeleton complex (spectrin, actin and protein band 4.1), phosphorylates spectrin, actin and protein band 4.1. We can conclude that (1) cyclic AMP-dependent phosphorylation of red cell cytoskeleton occurs in vitro only when the cytoskeleton components are in a complexed form; (2) red cell actin, like other cellular actins, may be phosphorylated by cyclic AMP-dependent protein kinase but only in the oligomeric form and not in the G form.

Actins↗
PubMed1981

Anti-spectrin in sera containing smooth muscle autoantibodies from patients with chronic active hepatitis.

Sera from patients with chronic active hepatitis containing anti-smooth muscle autoantibodies (SMA) react against rabbit muscle actin as well as human red cell spectrin. These anti-spectrin antibodies recognize the same antigen as rabbit anti-human spectrin antibodies and are not involved in the staining pattern given by SMA-containing sera tested with smooth muscle sections. These anti-spectrin antibodies probably recognize an antigenic structure common to both spectrin and another as yet undetermined molecule (which however is not likely to be myosin).

Actins↗
PubMed1981

[Human red cell cytoskeleton: structure, functions, abnormalities (author's transl)].

Associated with the red cell membrane cytoplasmic surface, the cytoskeleton is composed of a protein complex. This structure is a major element of membrane stability, shape and flexibility. Among the constitutive proteins, spectrin, red-cell actin and protein band 4.1 are quantitatively and structurally the most important. Some elements, relative to structure, stoichiometry and interactions between these proteins begin to be understood, specially owing to in vitro studies of reassembling cytoskeleton structures from purified constitutive elements. Besides, the nature of some interactions between the cytoskeleton and the red cell membrane has been specified. Nevertheless, it is not possible at the present time to establish an architectural picture of the cytoskeleton anatomy, and the factors actually regulating this structure physiology are not characterized. Understanding of the normal cytoskeleton will perhaps to helped by studies of abnormal models which cytoskeleton protein structure and function could be defective but this has not so far been formally demonstrated.

Actins↗
PubMed1980

[Familial pyridoxine-responsive sideroblastic anaemia. One case (author's transl)].

In a 24-year old man presenting with hypochromic microcytic anaemia, low reticulocyte count, increased serum iron and bone marrow erythroid hyperplasia with numerous ringed sideroblasts, the diagnosis of sideroblastic anaemia was confirmed by radioisotope study and bone marrow electron microscopy. The hereditary nature of the disease was demonstrated by the presence of microcytosis and increased serum iron in the mother and two sisters of the patient. One of the two sisters also had anaemia and abnormal ringed sideroblasts in her bone marrow. Her haemoglobin values and those of the propositus returned to normal under pyridoxine treatment, but hypochromia and abnormal sideroblasts persisted. This case of familial pyridoxine-responsive sideroblastic anaemia is consisted with X-chromosome-mediated transmission. The incomplete effect of pyridoxine suggests a congenital deficiency of some pyridoxine metabolism enzyme or another, as yet unidentified mechanism.

Adult↗
PubMed1980

[Interactions of cytosol enzymes with the red blood cell membrane].

It is not dubious that the regulation of erythrocyte metabolism occurs across the membrane and that accordingly interactions between cytosolic enzymes and membrane components necessarily exist. Several aspects of such relationships were reviewed. The results of some experiments carried out in non-physiological conditions should be carefully interpreted. However it can be accepted that some enzymes undergo reciprocal translocations between cytosol and membrane and that very probably these transfers play a role in the control of metabolic regulation in the red cell.

Binding Sites↗