scholarly journals IDENTIFICATION OF A MAJOR BASIC PROTEIN IN GUINEA PIG EOSINOPHIL GRANULES

1973 ◽  
Vol 137 (6) ◽  
pp. 1459-1471 ◽  
Author(s):  
Gerald J. Gleich ◽  
David A. Loegering ◽  
Jorge E. Maldonado
Keyword(s):  
Molecular Weight ◽  
Guinea Pig ◽  
Peroxidase Activity ◽  
Basic Protein ◽  
Low Molecular Weight ◽  
Major Basic Protein ◽  
Cationic Protein ◽  
Major Band ◽  
Electrophoretic Patterns ◽  
Eosinophil Granule

Elucidation of the functions of the eosinophil might be accomplished by analysis of the granule constituents. We have purified eosinophils (93% or greater) from the peritoneal cavity of the guinea pig and have investigated a variety of methods to disrupt cells and liberate intact granules. Lysis in 0.34 M sucrose gave the best yield of granules and these had the characteristic morphology of eosinophil granules when examined by electron microscopy. Granules were solubilized by a variety of treatments and the solutions analyzed by polyacrylamide electrophoresis at pH 3 in 6 M urea. Comparison of the electrophoretic patterns of solubilized eosinophil and neutrophil granules revealed a difference: a major portion (53±3%; x ±1 SE) of the protein from the eosinophil granule migrated as a single component. This major band protein has a molecular weight between 6,000 and 12,000 daltons and a pI of 10 or greater. Analysis of eosinophil granule constituents on Sephadex G-50 revealed two main peaks; peak 1 possessed peroxidase activity and peak 2 contained the major band protein. These studies indicate that eosinophil granules contain a cationic protein of low molecular weight which lacks peroxidase activity and which accounts for greater than 50% of granule protein.

scholarly journals Elevated serum levels in human pregnancy of a molecule immunochemically similar to eosinophil granule major basic protein.

1983 ◽  
Vol 158 (4) ◽  
pp. 1211-1226 ◽  
Author(s):  
D E Maddox ◽  
J H Butterfield ◽  
S J Ackerman ◽  
C B Coulam ◽  
G J Gleich
Keyword(s):  
Guinea Pig ◽  
Pregnant Women ◽  
Dose Response ◽  
Basic Protein ◽  
Serum Levels ◽  
Major Basic Protein ◽  
Response Curves ◽  
Double Antibody ◽  
Eosinophil Granule ◽  
In Pregnancy

We have shown that serum levels of a molecule immunochemically similar to eosinophil granule major basic protein (MBP) are elevated in pregnant women throughout gestation. MBP levels increase during gestation and plateau at approximately 7,500 ng/ml by the 20th wk (greater than 10-fold above normal). Levels return to normal after delivery, with a T1/2 of 13.7 d. The MBP in pregnancy serum is remarkably similar to the eosinophil granule MBP in that: (a) pregnancy MBP fully inhibits the binding of radiolabeled MBP standard in a double antibody radioimmunoassay; (b) this inhibition reaction is specific for human MBP because pregnancy serum produces no inhibition of the binding of radiolabeled guinea pig MBP in the guinea pig MBP radioimmunoassay; (c) in a two-site immunoradiometric assay for MBP, slopes of dose-response curves for pregnancy serum, purified MBP, and serum from a patient with hypereosinophilic syndrome are identical, and maximal binding is comparable; (d) reduction and alkylation of pregnancy sera increases measured MBP 100-fold, as previously shown for eosinophil granule MBP in serum; and (e) the MBP in pregnancy serum demonstrates the same pattern of heat lability as has been previously reported for MBP. Four observations have raised the possibility that the eosinophil is not the source of the MBP in pregnancy serum: (a) no correlation between serum MBP level and peripheral blood eosinophil count exists in pregnant women, in contrast to previous studies of patients with eosinophilia; (b) levels of three other eosinophil-associated proteins are normal or low in pregnancy sera, whereas the serum levels of these proteins are elevated in patients with eosinophilia; (c) the slopes of dose-response curves for pregnancy sera and MBP standards differ in the double antibody radioimmunoassay; and (d) the molecule in pregnancy serum elutes from Sephadex G-50 columns at the void volume, while eosinophil granule MBP and the MBP in serum of patients with eosinophilia elute at a volume consistent with the previously established molecular weight of 9,300. These findings suggest that the MBP in pregnancy serum is derived from a source other than the eosinophil.

scholarly journals PHYSIOCHEMICAL AND BIOLOGICAL PROPERTIES OF THE MAJOR BASIC PROTEIN FROM GUINEA PIG EOSINOPHIL GRANULES

1974 ◽  
Vol 140 (2) ◽  
pp. 313-332 ◽  
Author(s):  
Gerald J. Gleich ◽  
David A. Loegering ◽  
Friedrich Kueppers ◽  
Satya P. Bajaj ◽  
Kenneth G. Mann
Keyword(s):  
Guinea Pig ◽  
Disulfide Bonds ◽  
Basic Protein ◽  
Biological Properties ◽  
Low Molecular Weight ◽  
Major Basic Protein ◽  
Guanidinium Chloride ◽  
E Coli ◽  
Single Polypeptide Chain ◽  
Single Polypeptide

Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.

Parallel Reduction of Plasma Levels of High and Low Molecular Weight Kininogen in Patients with Cirrhosis

1999 ◽  
Vol 82 (11) ◽  
pp. 1428-1432 ◽  
Author(s):  
Cheryl Scott ◽  
Francesco Salerno ◽  
Elettra Lorenzano ◽  
Werner Müller-Esterl ◽  
Angelo Agostoni ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Liver Disease ◽  
Liver Function ◽  
Plasma Levels ◽  
Plasma Concentrations ◽  
Normal Subjects ◽  
Low Molecular Weight ◽  
Major Band ◽  
Sds Page ◽  
Normal Controls

SummaryLittle is known about the regulation of high-molecular-weight-kininogen (HK) and low-molecular-weight-kininogen (LK) or the relationship of each to the degree of liver function impairment in patients with cirrhosis. In this study, we evaluated HK and LK quantitatively by a recently described particle concentration fluorescence immunoassay (PCFIA) and qualitatively by SDS PAGE and immunoblotting analyses in plasma from 33 patients with cirrhosis presenting various degrees of impairment of liver function. Thirty-three healthy subjects served as normal controls. Patients with cirrhosis had significantly lower plasma levels of HK (median 49 μg/ml [range 22-99 μg/ml]) and LK (58 μg/ml [15-100 μg/ml]) than normal subjects (HK 83 μg/ml [65-115 μg/ml]; LK 80 μg/ml [45-120 μg/ml]) (p < 0.0001). The plasma concentrations of HK and LK were directly related to plasma levels of cholinesterase (P < 0.0001) and albumin (P < 0.0001 and P < 0.001) and inversely to the Child-Pugh score (P < 0.0001) and to prothrombin time ratio (P < 0.0001) (reflecting the clinical and laboratory abnormalities in liver disease). Similar to normal individuals, in patients with cirrhosis, plasma HK and LK levels paralleled one another, suggesting that a coordinate regulation of those proteins persists in liver disease. SDS PAGE and immunoblotting analyses of kininogens in cirrhotic plasma showed a pattern similar to that observed in normal controls for LK (a single band at 66 kDa) with some lower molecular weight forms noted in cirrhotic plasma. A slight increase of cleavage of HK (a major band at 130 kDa and a faint but increased band at 107 kDa) was evident. The increased cleavage of HK was confirmed by the lower cleaved kininogen index (CKI), as compared to normal controls. These data suggest a defect in hepatic synthesis as well as increased destructive cleavage of both kininogens in plasma from patients with cirrhosis. The decrease of important regulatory proteins like kininogens may contribute to the imbalance in coagulation and fibrinolytic systems, which frequently occurs in cirrhotic patients.

scholarly journals Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein.

1988 ◽  
Vol 263 (25) ◽  
pp. 12559-12563
Author(s):  
T L Wasmoen ◽  
M P Bell ◽  
D A Loegering ◽  
G J Gleich ◽  
F G Prendergast ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Basic Protein ◽  
Major Basic Protein ◽  
Amino Acid Sequence Analysis ◽  
Human Eosinophil ◽  
Eosinophil Granule

scholarly journals A Novel and Highly Divergent Homolog of Human Eosinophil Granule Major Basic Protein

1999 ◽  
Vol 274 (20) ◽  
pp. 14464-14473 ◽  
Author(s):  
Douglas A. Plager ◽  
David A. Loegering ◽  
Deborah A. Weiler ◽  
James L. Checkel ◽  
Jill M. Wagner ◽  
...  
Keyword(s):  
Basic Protein ◽  
Major Basic Protein ◽  
Human Eosinophil ◽  
Eosinophil Granule

C/EBPβ and GATA-1 Synergistically Regulate Activity of the Eosinophil Granule Major Basic Protein Promoter: Implication for C/EBPβ Activity in Eosinophil Gene Expression

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1429-1439 ◽  
Author(s):  
Yuji Yamaguchi ◽  
Hitoshi Nishio ◽  
Kenji Kishi ◽  
Steven J. Ackerman ◽  
Toshio Suda
Keyword(s):  
Binding Site ◽  
Cell Line ◽  
Binding Sites ◽  
Basic Protein ◽  
Regulatory Role ◽  
Expression Vectors ◽  
Regulatory Function ◽  
Dependent Manner ◽  
Major Basic Protein ◽  
Eosinophil Granule

Abstract Eosinophil granule major basic protein (MBP) is expressed exclusively in eosinophils and basophils in hematopoietic cells. In our previous study, we demonstrated a major positive regulatory role for GATA-1 and a negative regulatory role for GATA-2 in MBP gene transcription. Further analysis of the MBP promoter region identified a C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp upstream of the functional GATA-binding site in the MBP gene. In the cell line HT93A, which is capable of differentiating towards both the eosinophil and neutrophil lineages in response to retinoic acid (RA), C/EBP mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPβ expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPβ protein could bind to the potential C/EBP-binding site (bp −90 to −82) in the MBP promoter. Furthermore, we have demonstrated that both C/EBPβ and GATA-1 can bind simultaneously to the C/EBP- and GATA-binding sites in the MBP promoter. To determine the functionality of both the C/EBP- and GATA-binding sites, we analyzed whether C/EBPβ and GATA-1 can stimulate the MBP promoter in the C/EBPβ and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPβ and GATA-1 expression vectors produced a 5-fold increase compared with cotransfection with the C/EBPβ or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPβ. Expression of FOG (F̲riendo̲fG̲ATA), which binds to GATA-1 and acts as a cofactor for GATA-binding proteins, decreased transactivation activity of GATA-1 for the MBP promoter in a dose-dependent manner. Our results provide the first evidence that both GATA-1 and C/EBPβ synergistically transactivate the promoter of an eosinophil-specific granule protein gene and that FOG may act as a negative cofactor for the eosinophil lineage, unlike its positively regulatory function for the erythroid and megakaryocyte lineages.

Clinical And Experimental Studies On FBG Heterogeneity And Fibrinogenolysis —Especially In DIC And UK Treatment

1981 ◽  
Author(s):  
M Yamauchi ◽  
H Takei ◽  
T Seya ◽  
Y Oguma ◽  
T Murakoshi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Fibrinolytic Activity ◽  
Experimental Studies ◽  
Experimental Models ◽  
Low Molecular Weight ◽  
Electrophoretic Patterns ◽  
Sds Page ◽  
Cirrhotic Patients ◽  
Partial Degradation

ABy means of SDS-PAGE (3.3% gel), Fbg heterogeneity originated from partial degradation of Aα chain was studied. Comparison of electrophoretic patterns of plasma and corresponding serum made it possible to identify 2 major Fbg bands designated as high-molecular-weight Fbg (HMW, MW 350,000) and low-molecular-weight Fbg (LMW, MW 310,000). LMW comprised 28×2% (mean×S.D) of total Fbg (HMW+LMW) in healty subjects. The elevation of fibrinolytic activity did not accompany the increase of percentages of LMW in various diseases, even in cirrhotic patients whose levels of α2;PI were low. In DIC patients percentages of LMW were decreased extremely (12×6%, mean×SD). Samples from animal experimental models of DIC exhibited the same pattern of Fbg heterogeneity as that of DIC patients.UK was added to the purified Fbg in vitro. On the earliest stage of the fibrinogenolysis. 2 bands appeared newly on SDS-PAGE, while the bands of HMW and LMW were decreased. One of these new bands (Band 1) corresponded with a major compornent of Fraction 1-9 of Mosesson. It was located in the slightly anodal position (MW 300,000) from LMW band. Another band (MW 270,000) migrated between Band 1 and the band of Frag X. The same pattern of Fbg heterogeneity was observed in patients recieving large dose of UK. After cessation of UK treatment these new bands disappeared, while the bands of HMW was increased extremelThese findings suggest that HMW is a freshly synthesized Fbg and that unknown mechanism without plasmin may present for the conversion HMW to LMW.

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