scholarly journals An insertional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin nice (beta 220/216)

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 517-523
Author(s):  
WT Tse ◽  
PG Gallagher ◽  
B Pothier ◽  
FF Costa ◽  
A Scarpa ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Frameshift Mutation ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
De Novo Mutation ◽  
Molecular Defect ◽  
Beta Chain ◽  
Alpha Chain ◽  
Oligonucleotide Hybridization

Spectrin Nice (beta 220/216) is a spectrin variant associated with a shortened beta chain found in a patient with elliptocytosis. The shortened beta chain (beta' chain) appeared as an additional band of approximately 216 Kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was defective in its ability to be phosphorylated. There were increased amounts of spectrin dimers in crude spectrin extracts from the propositus and the association constant of spectrin dimer self-association was decreased. There was an associated increase of the alpha I 74-Kd fragment from the alpha chain after partial trypic digestion of spectrin. To identify the underlying molecular defect, we analyzed cDNA for beta spectrin obtained by polymerase chain reaction amplification of reverse-transcribed reticulocyte messenger RNA from peripheral blood of the propositus. DNA sequencing of individual as well as pooled subclones showed that two extra bases (GA) are inserted in codon no. 2046 in one allele of the beta-spectrin gene. The insertion results in a frameshift mutation and generates an aberrant C- terminus truncated by about 4 Kd, consistent with the estimated size of the beta' chain observed. By allele-specific oligonucleotide hybridization, the insertion was shown to be present in the propositus and absent in his parents, confirming a previous proposal that it is a de novo mutation. The determination of the location of the mutation in spectrin Nice points to specific regions of the beta-spectrin chain where phosphorylation may occur. A model is proposed to describe the interaction between the alpha- and beta-spectrin chains and to explain the effects of the mutation found in spectrin Nice on the trypsin digestion pattern of its associated alpha chain.

scholarly journals An insertional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin nice (beta 220/216)

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 517-523 ◽  
Author(s):  
WT Tse ◽  
PG Gallagher ◽  
B Pothier ◽  
FF Costa ◽  
A Scarpa ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Frameshift Mutation ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
De Novo Mutation ◽  
Molecular Defect ◽  
Beta Chain ◽  
Alpha Chain ◽  
Oligonucleotide Hybridization

Abstract Spectrin Nice (beta 220/216) is a spectrin variant associated with a shortened beta chain found in a patient with elliptocytosis. The shortened beta chain (beta' chain) appeared as an additional band of approximately 216 Kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was defective in its ability to be phosphorylated. There were increased amounts of spectrin dimers in crude spectrin extracts from the propositus and the association constant of spectrin dimer self-association was decreased. There was an associated increase of the alpha I 74-Kd fragment from the alpha chain after partial trypic digestion of spectrin. To identify the underlying molecular defect, we analyzed cDNA for beta spectrin obtained by polymerase chain reaction amplification of reverse-transcribed reticulocyte messenger RNA from peripheral blood of the propositus. DNA sequencing of individual as well as pooled subclones showed that two extra bases (GA) are inserted in codon no. 2046 in one allele of the beta-spectrin gene. The insertion results in a frameshift mutation and generates an aberrant C- terminus truncated by about 4 Kd, consistent with the estimated size of the beta' chain observed. By allele-specific oligonucleotide hybridization, the insertion was shown to be present in the propositus and absent in his parents, confirming a previous proposal that it is a de novo mutation. The determination of the location of the mutation in spectrin Nice points to specific regions of the beta-spectrin chain where phosphorylation may occur. A model is proposed to describe the interaction between the alpha- and beta-spectrin chains and to explain the effects of the mutation found in spectrin Nice on the trypsin digestion pattern of its associated alpha chain.

Fibrinolytic activity without fibrinogenolysis during long-distance racing in horses

1981 ◽  
Vol 50 (2) ◽  
pp. 245-249 ◽  
Author(s):  
E. W. Ferguson ◽  
L. L. Bernier ◽  
G. P. Shaughness ◽  
J. H. Boucher
Keyword(s):  
Gel Electrophoresis ◽  
Fibrinolytic Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Clot Lysis ◽  
Beta Chain ◽  
Long Distance ◽  
Alpha Chain ◽  
Molecular Weights ◽  
Lysis Time

Fourteen horses were studied during a 157-km endurance ride. Two humans who ran the 157 km were also evaluated at the finish. Fibrin monomer samples were examined by two-dimensional gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two major species of horse Beta-chain with higher molecular weights and different isoelectric mobilities than human beta-chain were observed. Horse alpha-chains had higher molecular weights than human alpha-chains but similar alpha-chain heterogeneities. Mean euglobulin lysis time (ELT) in the horses was accelerated to similar levels throughout the ride (52% of control at 44 km, P less than 0.01), but mean plasma clot lysis time (PCLT) decreased progressively during the ride (30% of control at finish, P less than 0.005). Similar values for ELTs and PCLTs were noted in the runners and horses at the finish. Although fibrinolytic activity was accelerated for an extended period by the strenuous activity of this long-distance race, no evidence of increased carboxyl terminal degradation of the A alpha-chain was observed. This study suggests that prolonged physiological stimulation of the fibrinolytic enzyme (plasmin) system is not responsible for fibrinogen A alpha-chain heterogeneities observed in horses and humans. It further demonstrates the usefulness of horses as models for the study of exercise-induced fibrinolysis.

scholarly journals Characterization of patients with an increased susceptibility to bacterial infections and a genetic deficiency of leukocyte membrane complement receptor type 3 and the related membrane antigen LFA-1

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 882-890 ◽  
Author(s):  
GD Ross ◽  
RA Thompson ◽  
MJ Walport ◽  
TA Springer ◽  
JV Watson ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Bacterial Infections ◽  
Nk Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Beta Chain ◽  
Alpha Chain ◽  
The Common ◽  
Respiratory Responses ◽  
Increased Susceptibility

Abstract Three children from two unrelated families had a history of recurrent bacterial infections, and their neutrophils were shown to have deficient phagocytic and respiratory responses and possible deficiencies in chemotaxis or adherence. Their neutrophils were strikingly deficient in the ability to ingest or give a respiratory burst in response to unopsonized bakers' yeast or zymosan (Z). Tests for neutrophil and monocyte CR1 (C3b/iC3b receptor) and CR3 (iC3b receptor) demonstrated rosettes with both EC3b and EC3bi. However, EC3bi were bound only to CR1, and not to CR3, because EC3bi rosettes were inhibited completely by anti-CR1. Neutrophils, monocytes, and natural killer (NK) cells also did not fluorescence stain with monoclonal antibodies specific for the alpha-chain of CR3 (anti-Mac-1, anti-Mol, OKM1, and MN-41). Quantitation of C receptors with 125I monoclonal anti-CR1 and anti-CR3 indicated that neutrophils from each patient expressed normal amounts of CR1 per cell but less than 10% of the normal amount of CR3. Examination of neutrophils by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that a normal glycoprotein of approximately 165,000 daltons was missing. Immunoblotting of these gels indicated that the missing band was the alpha-chain of CR3. Subsequent analysis of all three patients' cells also demonstrated a deficiency of LFA-1 alpha-chain and the common beta- chain that is shared by the CR3/LFA-1/p150,95 membrane antigen family. The deficiency of LFA-1 probably explained the absent NK cell function, as normal NK cell activity is inhibited by anti-LFA-1 but not by anti- CR3. The reduced phagocytic and respiratory responses to Z were probably due to CR3 deficiency, because treatment of normal neutrophils with anti-CR3, but not anti-FLA-1, inhibits responses to Z by 80% to 90%. Ingestion of Staphylococcus epidermidis by normal neutrophils was shown to be partially inhibited by monoclonal antibodies to the alpha- chain of either CR3 or LFA-1, and monoclonal antibody to the common beta-chain inhibited ingestion by 75%. Thus, both CR3 and LFA-1 may have previously unrecognized functions as phagocyte receptors for bacteria. The absence of this type of nonimmune recognition of bacteria by these children's neutrophils may be one of the reasons for their increased susceptibility to bacterial infections.

scholarly journals Spectrin Nice (beta 220/216): a shortened beta-chain variant associated with an increase of the alpha I/74 fragment in a case of elliptocytosis

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1759-1765
Author(s):  
B Pothier ◽  
L Morle ◽  
N Alloisio ◽  
MT Ducluzeau ◽  
C Caldani ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Association Constant ◽  
De Novo ◽  
Sodium Dodecyl ◽  
De Novo Mutation ◽  
Beta Chain ◽  
Additional Band ◽  
Self Association ◽  
Spleen Enlargement ◽  
Chain Variant

We describe a new spectrin variant with a truncated beta-chain. It was discovered in a 17-year-old white boy presenting with intermittent jaundice and spleen enlargement. He also displayed numerous smooth elliptocytes. On sodium dodecyl sulfate-polyacrylamide gel, the truncated beta-chain (beta'-chain) appeared as an additional band of approximately 216 kilodaltons, migrating between spectrin beta-chain and ankyrin. It represented 30% of total beta-chain. The beta'-chain reacted with an antispectrin beta-chain monoclonal antibody. It failed to become phosphorylated when ghosts were incubated in the presence of [gamma-32P] adenosine triphosphate. Whole spectrin tetramerization was defective since the amount of spectrin dimer was increased in spectrin crude extract and the association constant of the spectrin dimer self- association was decreased. Spectrin whole tetramer isolated from spectrin crude extracts contained small quantities of beta'-chain. Spectrin tryptic peptides showed an increase of the 74,000-dalton fragment at the expense of the 80,000-dalton fragment. So far, the latter abnormality has been used to characterize a number of cases of hereditary elliptocytosis or pyropoikilocytosis with no other apparent change. In the present case, we consider that the abnormality is a consequence of the beta-chain alteration. The parents seemed asymptomatic. As a result, we regard this new spectrin variant as deriving from a de novo mutation.

scholarly journals Spectrin Nice (beta 220/216): a shortened beta-chain variant associated with an increase of the alpha I/74 fragment in a case of elliptocytosis

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1759-1765 ◽  
Author(s):  
B Pothier ◽  
L Morle ◽  
N Alloisio ◽  
MT Ducluzeau ◽  
C Caldani ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Association Constant ◽  
De Novo ◽  
Sodium Dodecyl ◽  
De Novo Mutation ◽  
Beta Chain ◽  
Additional Band ◽  
Self Association ◽  
Spleen Enlargement ◽  
Chain Variant

Abstract We describe a new spectrin variant with a truncated beta-chain. It was discovered in a 17-year-old white boy presenting with intermittent jaundice and spleen enlargement. He also displayed numerous smooth elliptocytes. On sodium dodecyl sulfate-polyacrylamide gel, the truncated beta-chain (beta'-chain) appeared as an additional band of approximately 216 kilodaltons, migrating between spectrin beta-chain and ankyrin. It represented 30% of total beta-chain. The beta'-chain reacted with an antispectrin beta-chain monoclonal antibody. It failed to become phosphorylated when ghosts were incubated in the presence of [gamma-32P] adenosine triphosphate. Whole spectrin tetramerization was defective since the amount of spectrin dimer was increased in spectrin crude extract and the association constant of the spectrin dimer self- association was decreased. Spectrin whole tetramer isolated from spectrin crude extracts contained small quantities of beta'-chain. Spectrin tryptic peptides showed an increase of the 74,000-dalton fragment at the expense of the 80,000-dalton fragment. So far, the latter abnormality has been used to characterize a number of cases of hereditary elliptocytosis or pyropoikilocytosis with no other apparent change. In the present case, we consider that the abnormality is a consequence of the beta-chain alteration. The parents seemed asymptomatic. As a result, we regard this new spectrin variant as deriving from a de novo mutation.

scholarly journals Characterization of patients with an increased susceptibility to bacterial infections and a genetic deficiency of leukocyte membrane complement receptor type 3 and the related membrane antigen LFA-1

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 882-890
Author(s):  
GD Ross ◽  
RA Thompson ◽  
MJ Walport ◽  
TA Springer ◽  
JV Watson ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Bacterial Infections ◽  
Nk Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Beta Chain ◽  
Alpha Chain ◽  
The Common ◽  
Respiratory Responses ◽  
Increased Susceptibility

Three children from two unrelated families had a history of recurrent bacterial infections, and their neutrophils were shown to have deficient phagocytic and respiratory responses and possible deficiencies in chemotaxis or adherence. Their neutrophils were strikingly deficient in the ability to ingest or give a respiratory burst in response to unopsonized bakers' yeast or zymosan (Z). Tests for neutrophil and monocyte CR1 (C3b/iC3b receptor) and CR3 (iC3b receptor) demonstrated rosettes with both EC3b and EC3bi. However, EC3bi were bound only to CR1, and not to CR3, because EC3bi rosettes were inhibited completely by anti-CR1. Neutrophils, monocytes, and natural killer (NK) cells also did not fluorescence stain with monoclonal antibodies specific for the alpha-chain of CR3 (anti-Mac-1, anti-Mol, OKM1, and MN-41). Quantitation of C receptors with 125I monoclonal anti-CR1 and anti-CR3 indicated that neutrophils from each patient expressed normal amounts of CR1 per cell but less than 10% of the normal amount of CR3. Examination of neutrophils by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that a normal glycoprotein of approximately 165,000 daltons was missing. Immunoblotting of these gels indicated that the missing band was the alpha-chain of CR3. Subsequent analysis of all three patients' cells also demonstrated a deficiency of LFA-1 alpha-chain and the common beta- chain that is shared by the CR3/LFA-1/p150,95 membrane antigen family. The deficiency of LFA-1 probably explained the absent NK cell function, as normal NK cell activity is inhibited by anti-LFA-1 but not by anti- CR3. The reduced phagocytic and respiratory responses to Z were probably due to CR3 deficiency, because treatment of normal neutrophils with anti-CR3, but not anti-FLA-1, inhibits responses to Z by 80% to 90%. Ingestion of Staphylococcus epidermidis by normal neutrophils was shown to be partially inhibited by monoclonal antibodies to the alpha- chain of either CR3 or LFA-1, and monoclonal antibody to the common beta-chain inhibited ingestion by 75%. Thus, both CR3 and LFA-1 may have previously unrecognized functions as phagocyte receptors for bacteria. The absence of this type of nonimmune recognition of bacteria by these children's neutrophils may be one of the reasons for their increased susceptibility to bacterial infections.

scholarly journals Mapping of the properdin-binding site in the third component of complement

1984 ◽  
Vol 217 (1) ◽  
pp. 323-326 ◽  
Author(s):  
J D Lambris ◽  
J Alsenz ◽  
T F Schulz ◽  
M P Dierich
Keyword(s):  
Binding Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Fragment ◽  
Alpha Chain ◽  
Factor I ◽  
Assay Procedure ◽  
Complement Component C3 ◽  
Third Component Of Complement

The properdin-binding site in the human third complement component (C3) was mapped by using isolated C3b, C3c, alpha- and beta-chains of C3 and C3 polypeptide fragments and an enzyme-linked-immunosorbent-assay procedure. The C3 chains and the polypeptide fragments were purified to homogeneity by preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The alpha-chain polypeptides included a 68 kDa and a 43 kDa polypeptide, which were generated by cleavage of C3b with factors I and H, and a 40 kDa, 33 kDa (C3d) and 27 kDa polypeptide, which were generated by cleavage of C3b with porcine elastase. It was shown that properdin binds to C3b, C3c, alpha-chain, and to the 43 kDa (factor-I + H-derived), as well as to 40 kDa (elastase-derived) alpha-chain fragment, but not to the beta-chain 68 kDa, 33 kDa (C3d) and 27 kDa alpha-chain fragments. Thus the binding site for properdin resides on the 40-43 kDa C-terminal alpha-chain fragment of C3.

Identification of the Poly(C) Binding Protein in the Complex Associated With the 3′ Untranslated Region of Erythropoietin Messenger RNA

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 2111-2120 ◽  
Author(s):  
Maria F. Czyzyk-Krzeska ◽  
Amy C. Bendixen
Keyword(s):  
Mrna Stability ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mobility Shift ◽  
Protein Factor ◽  
Ribonucleoprotein Complexes ◽  
Sds Page

Hypoxia regulates expression of erythropoietin (EPO), a glycoprotein that stimulates erythrocytosis, at the level of transcription and also possibly at the level of messenger RNA (mRNA) stability. A pyrimidine-rich region within the EPO mRNA 3′ untranslated region was implicated in regulation of EPO mRNA stability element and shown to bind protein factors. In the present study we wished to identify the protein factor binding to the pyrimidine-rich sequence in the EPO mRNA stability element. Using mobility shift assays, ultraviolet light cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroelution of protein factors from the gel slices corresponding to the ribonucleoprotein complexes, we found that two isoforms of a 40 kD poly(C) binding protein (PCBP, also known as CP or hnRNPE), PCBP1, and PCBP2 are present in that complex. In Hep3B or HepG2 cells hypoxia induces neither expression of PCBP nor formation of the ribonucleoprotein complex associated with EPO mRNA that involves PCBP.

scholarly journals Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1202-1206 ◽  
Author(s):  
MG Bolyard ◽  
ST Lord
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Dependent Manner ◽  
Beta Chain ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
E Coli ◽  
Sds Page

Abstract The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.

The basophil activation marker defined by antibody 97A6 is identical to the ectonucleotide pyrophosphatase/phosphodiesterase 3

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3303-3305 ◽  
Author(s):  
Hans-Jörg Bühring ◽  
Martina Seiffert ◽  
Christina Giesert ◽  
Anke Marxer ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Messenger Rna ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Type Ii ◽  
Partial Amino Acid Sequence ◽  
Phosphodiesterase 3 ◽  
293 Cells ◽  
Basophil Cell

Abstract It has recently been shown that monoclonal antibody (MoAb) 97A6 detects a surface antigen expressed on basophils and their CD34+ precursor cells, as well as the basophil cell line KU-812. In this report the partial amino acid sequence of affinity chromatography– and sodium dodecyl sulfate–polyacrylamide gel electrophoresis–separated 97A6 antigen(s) from KU-812 lysates was determined. Sequence alignment of high-performance liquid chromatography–selected tryptic peptides from the resulting 130- and 150-kd bands revealed a 100% identity with amino acids 393 to 405 of ectonucleotide pyrophosphatase/phosphodiesterase-3 (E-NPP3; CD203c) but not of the related ectoenzyme PC-1 (E-NPP1). Moreover, MoAb 97A6 selectively recognized 293 cells transfected with human E-NPP3, but did not react with cells transfected with PC-1 or parental 293 cells. In addition, E-NPP3 messenger RNA expression was detected in basophils but not other peripheral blood cells. Finally, MoAb 97A6 immunoprecipitated phosphodiesterase activity from KU-812 cells and peripheral blood basophils, but not from other cell populations. These data demonstrate that MoAb 97A6 recognizes the functionally active type II transmembrane ectoenzyme E-NPP3.

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