scholarly journals Assignment of Sp alpha I/74 hereditary elliptocytosis to the alpha- or beta-chain of spectrin through in vitro dimer reconstitution

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 2061-2069
Author(s):  
B Pothier ◽  
N Alloisio ◽  
J Marechal ◽  
L Morle ◽  
MT Ducluzeau ◽  
...  
Keyword(s):  
Beta Chain ◽  
Experimental Conditions ◽  
White Families ◽  
Hereditary Elliptocytosis ◽  
Partial Digestion ◽  
Gene Level ◽  
Self Association ◽  
Definition Of ◽  
A Minor

Partial digestion of spectrin dimers in vitro has allowed the definition of domains. For example, the portions of the dimers that are involved in spectrin self-association are represented by the alpha I and the beta I domains. The alpha I domain (80 Kd) is further cleaved into a minor 78 Kd fragment and, more substantially, into a 74 Kd fragment. The intensity of the latter, which we expressed as the 74:(80 + 78 + 74) ratio, or the 74:alpha I ratio, is variable depending on the experimental conditions, eg, in fine, on the conformation of the alpha I domain. A number of cases of hereditary elliptocytosis (HE) are associated with an increase of the 74:alpha I ratio, also referred to as the Sp alpha I/74 abnormality. Several lines of evidence have suggested that the causal mutations may lie in the alpha- or the beta- chain, a point of importance before one undertakes studies at the gene level. In order to address this question, we reconstituted spectrin dimers in vitro, combining alpha- and beta-chains of various origins, and then carried out partial digestion and assayed the Sp alpha I/74 abnormality. The patterns obtained with reconstituted dimers were nearly identical to those of native dimers. We applied the assay to three spectrin variants that cause Sp alpha I/74 HE: (1) a variant that we previously designated spectrin Nice and whose beta-chain lacks a 4 Kd fragment in its C-terminal region; and two distinct variants that we found in two unrelated white families and that we provisionally designated spectrin Lyon and spectrin Culoz. The Sp alpha I/74 abnormality appeared in all kinds of dimers that harbored the beta- chain of spectrin Nice, or the alpha-chain of spectrin Lyon or spectrin Culoz, respectively. Therefore, we confirmed that spectrin Nice is a (alpha I/74) beta-variant, and established that both spectrin Lyon and spectrin Culoz are (alpha I/74) alpha-variants. The present assay may be extended to any spectrin variant displaying the Sp alpha I/74 abnormality.

scholarly journals Assignment of Sp alpha I/74 hereditary elliptocytosis to the alpha- or beta-chain of spectrin through in vitro dimer reconstitution

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 2061-2069 ◽  
Author(s):  
B Pothier ◽  
N Alloisio ◽  
J Marechal ◽  
L Morle ◽  
MT Ducluzeau ◽  
...  
Keyword(s):  
Beta Chain ◽  
Experimental Conditions ◽  
White Families ◽  
Hereditary Elliptocytosis ◽  
Partial Digestion ◽  
Gene Level ◽  
Self Association ◽  
Definition Of ◽  
A Minor

Abstract Partial digestion of spectrin dimers in vitro has allowed the definition of domains. For example, the portions of the dimers that are involved in spectrin self-association are represented by the alpha I and the beta I domains. The alpha I domain (80 Kd) is further cleaved into a minor 78 Kd fragment and, more substantially, into a 74 Kd fragment. The intensity of the latter, which we expressed as the 74:(80 + 78 + 74) ratio, or the 74:alpha I ratio, is variable depending on the experimental conditions, eg, in fine, on the conformation of the alpha I domain. A number of cases of hereditary elliptocytosis (HE) are associated with an increase of the 74:alpha I ratio, also referred to as the Sp alpha I/74 abnormality. Several lines of evidence have suggested that the causal mutations may lie in the alpha- or the beta- chain, a point of importance before one undertakes studies at the gene level. In order to address this question, we reconstituted spectrin dimers in vitro, combining alpha- and beta-chains of various origins, and then carried out partial digestion and assayed the Sp alpha I/74 abnormality. The patterns obtained with reconstituted dimers were nearly identical to those of native dimers. We applied the assay to three spectrin variants that cause Sp alpha I/74 HE: (1) a variant that we previously designated spectrin Nice and whose beta-chain lacks a 4 Kd fragment in its C-terminal region; and two distinct variants that we found in two unrelated white families and that we provisionally designated spectrin Lyon and spectrin Culoz. The Sp alpha I/74 abnormality appeared in all kinds of dimers that harbored the beta- chain of spectrin Nice, or the alpha-chain of spectrin Lyon or spectrin Culoz, respectively. Therefore, we confirmed that spectrin Nice is a (alpha I/74) beta-variant, and established that both spectrin Lyon and spectrin Culoz are (alpha I/74) alpha-variants. The present assay may be extended to any spectrin variant displaying the Sp alpha I/74 abnormality.

scholarly journals Involvement of the interleukin 2 pathway in the rearrangement and expression of both alpha/beta and gamma/delta T cell receptor genes in human T cell precursors.

1988 ◽  
Vol 168 (6) ◽  
pp. 2231-2249 ◽  
Author(s):  
M L Toribio ◽  
A de la Hera ◽  
J Borst ◽  
M A Marcos ◽  
C Márquez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Beta Chain ◽  
Gamma Delta ◽  
Human T Cell ◽  
Alpha Beta ◽  
A Minor

In this report, we have undertaken the phenotypic, functional and molecular characterization of a minor (less than 5%) subpopulation of adult thymocytes regarded as the earliest intrathymic T-cell precursors. Pro-T cells were immunoselected and shown to express different hematopoietic cell markers (CD45, CD38, CD7, CD5) and some activation-related molecules (4F2, Tr, HLA class II), but lack conventional T cell antigens (CD2-1-3-4-8-). TCR-gamma RNA messages are already expressed at this early ontogenic stage, while alpha and beta chain TCR genes remain in germline configuration. In vitro analyses of the growth requirements of pro-T cells demonstrated the involvement of the IL-2 pathway in promoting their proliferation and differentiation into CD3+ CD4+ or CD8+ mature thymocytes. Moreover, during the IL-2-mediated maturation process rearrangements and expression of both alpha and beta chain TCR genes occurred, and resulted in the acquisition of alpha/beta as well as gamma/delta (either disulphide-linked or non-disulphide-linked) heterodimeric TCR among the pro-T cell progeny.

scholarly journals A new abnormal variant of spectrin in black patients with hereditary elliptocytosis

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1208-1217 ◽  
Author(s):  
MC Lecomte ◽  
D Dhermy ◽  
C Solis ◽  
A Ester ◽  
C Feo ◽  
...  
Keyword(s):  
Thermal Sensitivity ◽  
Kinetic Studies ◽  
Tryptic Digestion ◽  
Interaction Domain ◽  
Normal Value ◽  
Alpha Chain ◽  
Hereditary Elliptocytosis ◽  
Black Patients ◽  
Self Association ◽  
A Minor

Abstract Seven black patients with mild hereditary elliptocytosis (HE) from five unrelated families were studied. The erythrocytes of these patients exhibited an abnormal thermal sensitivity (between 45 degrees C and 47 degrees C instead of 49 degrees C). An important defect of spectrin dimer self-association was detected in two ways: (1) the proportions of spectrin dimer (SpD) extracted from membranes at 4 degrees C under low ionic strength conditions were increased between 25% and 56% (normal value 15% +/- 2%); (2) the spectrin dimer----tetramer conversion in solution were defective with an association constant value between 0.4 and 2.4 X 10(5) M-1 for a normal value of 6 +/- 0.4 X 10(5) M-1. Spectrin (Sp) from HE patients and normal volunteers (32 black and 22 white subjects) was submitted to limited tryptic digestion, followed by one- or two-dimensional separation of the peptides. Peptide patterns of crude Sp from all seven HE patients exhibited a marked and reproducible decrease in 80,000-dalton peptide (previously identified as the dimer- dimer interaction domain of the alpha-chain) and a concomitant appearance of a novel 65,000-dalton peptide. A minor fragment at 28,000 daltons was also decreased. Tryptic digestion of HE spectrin dimer and tetramer (SpT), isolated after the SpD self-association procedure in solution, revealed modifications (decrease in the 80,000-dalton peptide and presence of a 65,000-dalton peptide) predominantly in HE SpD when peptide patterns of HE SpT were quite similar to control SpT patterns. Immunoblots with anti-alpha-chain antibodies revealed that the 65,000- dalton peptide derived from the alpha-chain. Kinetic studies of Sp digestion showed that the 65,000-dalton peptide did not result from further digestion of a 74,000 intermediate and was not a precursor of 46,000- to 50,000-dalton peptides. These results show a new structural defect of Sp-alpha-chain, associated with a defective Sp dimer self- association in HE.

scholarly journals A deletional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin Tokyo (beta 220/216)

Blood ◽  
1992 ◽  
Vol 80 (8) ◽  
pp. 2115-2121 ◽  
Author(s):  
A Kanzaki ◽  
M Rabodonirina ◽  
Y Yawata ◽  
R Wilmotte ◽  
H Wada ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genomic Dna ◽  
Frameshift Mutation ◽  
Stop Codon ◽  
Phosphorylation Site ◽  
Beta Chain ◽  
Potential Phosphorylation Site ◽  
Self Association ◽  
Beta Gene

Abstract A novel spectrin variant carrying a truncated beta-chain and designated Spectrin Tokyo (beta 220/216) is presented. It was associated with elliptocytosis and moderate uncompensated hemolysis. The dimer self- association was reduced. An increase of the alpha I 74-Kd fragment was detected upon partial trypsin digestion. Analysis of cDNA and genomic DNA showed a 1-base deletion in codon 2059 (GCC AGC-->GCA GCT; Ala-Ser-- >Ala-Ala) that belongs to exon X of spectrin beta-gene. A missense sequence extended down to (new) codon 2075. Serine 2060, a potential phosphorylation site, was replaced by alanine. The shortened beta-chain failed to undergo phosphorylation in vitro. Spectrin Tokyo shared the same stop codon, overlapping normal codons 2076 and 2077 (CTG AAA), as Spectrin Nice (beta 220/216), which is caused by a dinucleotide insertion in codon 2046 and contains 2076 amino acids. However, for some reason, Spectrin Tokyo had a lower incorporation level into the membrane than Spectrin Nice.

scholarly journals Characterization of Specific Protein-RNA Complexes Associated with the Coupling of Polyadenylation and Last-Intron Removal

2002 ◽  
Vol 22 (13) ◽  
pp. 4579-4586 ◽  
Author(s):  
Charles Cooke ◽  
James C. Alwine
Keyword(s):  
Specific Protein ◽  
Polyadenylation Signal ◽  
Site Formation ◽  
Coupling Mechanism ◽  
Experimental Conditions ◽  
Specific Complex ◽  
Intron Removal ◽  
Definition Of ◽  
Rna Complexes

ABSTRACT Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (AC′, AC, and BC) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The AC′ complex is formed by 30 s after mixing, the AC complex is formed between 1 and 2 min after mixing, and the BC complex is formed by 2 to 3 min after mixing. AC′ is a precursor of AC, and the AC′ and/or AC complex is a precursor of BC. Of the three complexes, BC appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the AC′ complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the AC′ complex is dependent on the polypyrimidine tract. The transition from AC′ to AC appears to require an intact 3′-splice site. Formation of the BC complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the AC′ complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the BC complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.

scholarly journals Toxicological Assessment of ITER-Like Tungsten Nanoparticles Using an In Vitro 3D Human Airway Epithelium Model

Nanomaterials ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 1374
Author(s):  
Isabelle George ◽  
Chiara Uboldi ◽  
Elodie Bernard ◽  
Marcos Sobrido ◽  
Sarah Dine ◽  
...  
Keyword(s):  
Metabolic Activity ◽  
Airway Epithelium ◽  
Cell Model ◽  
Experimental Conditions ◽  
Toxicological Assessment ◽  
Human Airway ◽  
Human Airway Epithelium ◽  
A Minor ◽  
Tungsten Nanoparticles

The International Thermonuclear Experimental Reactor (ITER) is an international project aimed at the production of carbon-free energy through the use of thermonuclear fusion. During ITER operation, in case of a loss-of-vacuum-accident, tungsten nanoparticles (W-NPs) could potentially be released into the environment and induce occupational exposure via inhalation. W-NPs toxicity was evaluated on MucilAir™, a 3D in vitro cell model of the human airway epithelium. MucilAir™ was exposed for 24 h to metallic ITER-like milled W-NPs, tungstate (WO42−) and tungsten carbide cobalt particles alloy (WC-Co). Cytotoxicity and its reversibility were assessed using a kinetic mode up to 28 days after exposure. Epithelial tightness, metabolic activity and interleukin-8 release were also evaluated. Electron microscopy was performed to determine any morphological modification, while mass spectrometry allowed the quantification of W-NPs internalization and of W transfer through the MucilAir™. Our results underlined a decrease in barrier integrity, no effect on metabolic activity or cell viability and a transient increase in IL-8 secretion after exposure to ITER-like milled W-NPs. These effects were associated with W-transfer through the epithelium, but not with intracellular accumulation. We have shown that, under our experimental conditions, ITER-like milled W-NPs have a minor impact on the MucilAir™ in vitro model.

scholarly journals A deletional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin Tokyo (beta 220/216)

Blood ◽  
1992 ◽  
Vol 80 (8) ◽  
pp. 2115-2121
Author(s):  
A Kanzaki ◽  
M Rabodonirina ◽  
Y Yawata ◽  
R Wilmotte ◽  
H Wada ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genomic Dna ◽  
Frameshift Mutation ◽  
Stop Codon ◽  
Phosphorylation Site ◽  
Beta Chain ◽  
Potential Phosphorylation Site ◽  
Self Association ◽  
Beta Gene

A novel spectrin variant carrying a truncated beta-chain and designated Spectrin Tokyo (beta 220/216) is presented. It was associated with elliptocytosis and moderate uncompensated hemolysis. The dimer self- association was reduced. An increase of the alpha I 74-Kd fragment was detected upon partial trypsin digestion. Analysis of cDNA and genomic DNA showed a 1-base deletion in codon 2059 (GCC AGC-->GCA GCT; Ala-Ser-- >Ala-Ala) that belongs to exon X of spectrin beta-gene. A missense sequence extended down to (new) codon 2075. Serine 2060, a potential phosphorylation site, was replaced by alanine. The shortened beta-chain failed to undergo phosphorylation in vitro. Spectrin Tokyo shared the same stop codon, overlapping normal codons 2076 and 2077 (CTG AAA), as Spectrin Nice (beta 220/216), which is caused by a dinucleotide insertion in codon 2046 and contains 2076 amino acids. However, for some reason, Spectrin Tokyo had a lower incorporation level into the membrane than Spectrin Nice.

scholarly journals Abnormal spectrin in hereditary elliptocytosis

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 141-151
Author(s):  
SL Marchesi ◽  
WJ Knowles ◽  
JS Morrow ◽  
M Bologna ◽  
VT Marchesi
Keyword(s):  
Hemolytic Anemia ◽  
Alpha Subunit ◽  
Tryptic Digestion ◽  
Clinical Expression ◽  
Hereditary Elliptocytosis ◽  
Oligomer Formation ◽  
Conformational Alteration ◽  
Self Association

An abnormal alpha subunit of erythrocyte spectrin has been described in hereditary pyropoikilocytosis (HPP), a rare hemolytic anemia characterized by erythrocyte budding and fragmentation. In HPP spectrin, the N terminal domain of the alpha subunit (alpha I T80) shows increased susceptibility to tryptic digestion, resulting in cleavage to a 50,000-d peptide, presumably due to a change in primary structure of the alpha I domain which alters conformation and generates the new cleavage site. The functional result of this conformational alteration is marked impairment of spectrin oligomer formation in vitro, consistent with the established role of alpha I T80 in spectrin self-association. In the present study, we demonstrate an abnormal spectrin alpha subunit in two kindreds with hereditary elliptocytosis (HE) that is qualitatively identical to HPP spectrin. Clinical expression of HE in these families ranges from mild elliptocytosis without hemolysis to severe poikilocytic hemolytic anemia clinically resembling HPP. In all affected individuals, a fraction of alpha I T80 is abnormal, as shown by its cleavage during mild tryptic digestion to the 50 kd peptide described in HPP; the fraction of alpha I T80 affected is directly proportional to the severity of clinical expression of HE. Spectrin oligomer formation is likewise impaired to a degree which correlates with hematologic disease. One of the HE kindreds studied demonstrated polymorphism in the spectrin alpha II domain, previously described as a frequent occurrence in blacks. This family also demonstrates a variant alpha III domain in spectrin that has not previously been described. We conclude that the abnormality in the alpha I domain originally described in HPP spectrin is shared by a subset of patients with HE; the severity of clinical expression, ranging from mild nonhemolytic HE to poikilocytic hemolytic anemia, is related to the fractional quantity of the alpha subunit that is affected.

scholarly journals A new abnormal variant of spectrin in black patients with hereditary elliptocytosis

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1208-1217
Author(s):  
MC Lecomte ◽  
D Dhermy ◽  
C Solis ◽  
A Ester ◽  
C Feo ◽  
...  
Keyword(s):  
Thermal Sensitivity ◽  
Kinetic Studies ◽  
Tryptic Digestion ◽  
Interaction Domain ◽  
Normal Value ◽  
Alpha Chain ◽  
Hereditary Elliptocytosis ◽  
Black Patients ◽  
Self Association ◽  
A Minor

Seven black patients with mild hereditary elliptocytosis (HE) from five unrelated families were studied. The erythrocytes of these patients exhibited an abnormal thermal sensitivity (between 45 degrees C and 47 degrees C instead of 49 degrees C). An important defect of spectrin dimer self-association was detected in two ways: (1) the proportions of spectrin dimer (SpD) extracted from membranes at 4 degrees C under low ionic strength conditions were increased between 25% and 56% (normal value 15% +/- 2%); (2) the spectrin dimer----tetramer conversion in solution were defective with an association constant value between 0.4 and 2.4 X 10(5) M-1 for a normal value of 6 +/- 0.4 X 10(5) M-1. Spectrin (Sp) from HE patients and normal volunteers (32 black and 22 white subjects) was submitted to limited tryptic digestion, followed by one- or two-dimensional separation of the peptides. Peptide patterns of crude Sp from all seven HE patients exhibited a marked and reproducible decrease in 80,000-dalton peptide (previously identified as the dimer- dimer interaction domain of the alpha-chain) and a concomitant appearance of a novel 65,000-dalton peptide. A minor fragment at 28,000 daltons was also decreased. Tryptic digestion of HE spectrin dimer and tetramer (SpT), isolated after the SpD self-association procedure in solution, revealed modifications (decrease in the 80,000-dalton peptide and presence of a 65,000-dalton peptide) predominantly in HE SpD when peptide patterns of HE SpT were quite similar to control SpT patterns. Immunoblots with anti-alpha-chain antibodies revealed that the 65,000- dalton peptide derived from the alpha-chain. Kinetic studies of Sp digestion showed that the 65,000-dalton peptide did not result from further digestion of a 74,000 intermediate and was not a precursor of 46,000- to 50,000-dalton peptides. These results show a new structural defect of Sp-alpha-chain, associated with a defective Sp dimer self- association in HE.

Contractile properties of the shortening rat diaphragm in vitro

1987 ◽  
Vol 62 (3) ◽  
pp. 1111-1116 ◽  
Author(s):  
I. A. Mardini ◽  
R. J. McCarter
Keyword(s):  
Isometric Tension ◽  
Contractile Properties ◽  
Rat Diaphragm ◽  
Experimental Conditions ◽  
Diaphragmatic Fatigue ◽  
Definition Of ◽  
Diaphragm In Vitro ◽  
The Relationship ◽  
Frequency Of Stimulation

Diaphragmatic fatigue has been defined in terms of the failure of the muscle to continue to generate a given level of tension. Appropriate shortening of the diaphragm is, however, just as important for adequate ventilation. In this study we have examined in vitro the contractile properties of the rat diaphragm under afterloaded isotonic conditions and the effect of fatigue on the ability of the diaphragm to shorten. Shortening of the muscle strips was found to depend on size of afterload, frequency of stimulation, duration of stimulation, and initial length of the muscle. The afterloaded isotonic length-tension relationship coincided with the relationship between length and active isometric tension only for relatively small afterloads. Fatigue of the muscle strips, induced by isometric or afterloaded isotonic contractions, was associated with a decline in the extent of shortening as well as a decrease in active isometric tension. Ability to shorten and ability to develop isometric tension did not decrease to the same extent under all conditions. We conclude that active shortening, as well as active isometric tension, is decreased by muscular fatigue and that changes in these properties can be different depending on experimental conditions. The results suggest that the definition of diaphragmatic fatigue should be expanded to include the ability of the muscle to shorten by an appropriate amount. The results also suggest that measurement of isometric performance may not provide a complete estimate of the overall performance of the fatigued diaphragm.

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