scholarly journals Antibody-dependent lymphocyte-mediated granulocyte cytotoxicity in man

Blood ◽  
1978 ◽  
Vol 51 (1) ◽  
pp. 97-108 ◽  
Author(s):  
GL Logue ◽  
R Kurlander ◽  
P Pepe ◽  
W Davis ◽  
H Silberman
Keyword(s):  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Normal Subjects ◽  
Exchange Chromatography ◽  
Phagocytic Cells ◽  
Igg Antibody ◽  
Microtiter Plates ◽  
Granulocyte Antibodies

Abstract Sera from two patients with granulocytopenia associated with collagen vascular disease caused the destruction of normal human granulocytes by autologous lymphocytes in vitro. Granulocyte cytotoxicity was measured by the release of 51Cr during incubation with test sera and lymphocytes in microtiter plates. Between 8% and 46% granulocytoxicity was produced in granulocytes from 8 normal donors by the sera from these two patients. Less than 6% granulocytotoxicity was seen with the sera from 14 normal subjects and 29 patient controls. Treatment of lymphocyte preparations with carbonyl iron and magnetic separation to remove phagocytic cells or treatment with complement-coated red cells followed by repeated gradient centrifugation to remove complement receptor- bearing lymphocytes did not reduce the granulocytotoxicity. There was a dose-response relationship between the concentration of positive sera and granulocytotoxicity. When these sera were fractionated by Sephadex G-200 gel filtration and by ion-exchange chromatography with DEAE- cellulose, the active component appeared in the IgG-containing fractions. Thus, IgG antibody-dependent, lymphocyte-mediated granulocyte cytotoxicity represents a means of detecting human granulocyte antibodies and is a possible mechanism of autoimmune neutropenia in these two patients.

scholarly journals Antibody-dependent lymphocyte-mediated granulocyte cytotoxicity in man

Blood ◽  
1978 ◽  
Vol 51 (1) ◽  
pp. 97-108
Author(s):  
GL Logue ◽  
R Kurlander ◽  
P Pepe ◽  
W Davis ◽  
H Silberman
Keyword(s):  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Normal Subjects ◽  
Exchange Chromatography ◽  
Phagocytic Cells ◽  
Igg Antibody ◽  
Microtiter Plates ◽  
Granulocyte Antibodies

Sera from two patients with granulocytopenia associated with collagen vascular disease caused the destruction of normal human granulocytes by autologous lymphocytes in vitro. Granulocyte cytotoxicity was measured by the release of 51Cr during incubation with test sera and lymphocytes in microtiter plates. Between 8% and 46% granulocytoxicity was produced in granulocytes from 8 normal donors by the sera from these two patients. Less than 6% granulocytotoxicity was seen with the sera from 14 normal subjects and 29 patient controls. Treatment of lymphocyte preparations with carbonyl iron and magnetic separation to remove phagocytic cells or treatment with complement-coated red cells followed by repeated gradient centrifugation to remove complement receptor- bearing lymphocytes did not reduce the granulocytotoxicity. There was a dose-response relationship between the concentration of positive sera and granulocytotoxicity. When these sera were fractionated by Sephadex G-200 gel filtration and by ion-exchange chromatography with DEAE- cellulose, the active component appeared in the IgG-containing fractions. Thus, IgG antibody-dependent, lymphocyte-mediated granulocyte cytotoxicity represents a means of detecting human granulocyte antibodies and is a possible mechanism of autoimmune neutropenia in these two patients.

Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

1987 ◽  
Vol 114 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Chohei Shigeno ◽  
Itsuo Yamamoto ◽  
Shegiharu Dokoh ◽  
Megumu Hino ◽  
Jun Aoki ◽  
...  
Keyword(s):  
Bone Resorption ◽  
High Performance ◽  
Basic Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Factor ◽  
Human Tumours ◽  
Acid Stable ◽  
Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

Identification of N-Monoacetylcystine in Uraemic Plasma

1981 ◽  
Vol 60 (3) ◽  
pp. 331-334 ◽  
Author(s):  
F. Gejyo ◽  
G. Ito ◽  
Y. Kinoshita
Keyword(s):  
Plasma Levels ◽  
Gel Filtration ◽  
Normal Subjects ◽  
Exchange Chromatography ◽  
Cysteic Acid ◽  
Homocysteic Acid ◽  
Normal Limits ◽  
Acidic Nature ◽  
Sulphur Amino Acids

1. An unidentified ninhydrin-positive substance of an acidic nature was detected in the plasma of uraemic patients. This substance was isolated from haemodialysate by ion-exchange chromatography and gel filtration, and identified as a sulphur-containing amino acid: N-monoacetylcystine. 2. The quantitative determination of sulphur amino acids in plasma revealed that the plasma levels of cysteic acid, homocysteic acid, taurine, cystine and cystathionine as well as N-monoacetylcystine in uraemic patients were markedly higher than in normal subjects (P < 0.001 for each). However, the plasma levels of methionine in uraemic patients were within normal limits.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

scholarly journals The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

1972 ◽  
Vol 130 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Colin H. Self ◽  
P. David J. Weitzman
Keyword(s):  
Molecular Weight ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Low Concentrations ◽  
Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

Purification and characterization of prostate-specific antigen (PSA) complexed to alpha 1-antichymotrypsin: potential reference material for international standardization of PSA immunoassays

1995 ◽  
Vol 41 (9) ◽  
pp. 1273-1282 ◽  
Author(s):  
Z Chen ◽  
A Prestigiacomo ◽  
T A Stamey
Keyword(s):  
Molecular Mass ◽  
Prostate Specific Antigen ◽  
Specific Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Free Psa ◽  
Apparent Homogeneity ◽  
International Standardization

Abstract We describe for the first time a protocol to purify to apparent homogeneity an in vitro-prepared complex of prostate-specific antigen (PSA) and alpha 1-antichymotrypsin (ACT) by using a combination of gel filtration and ion-exchange chromatography. The purity of the PSA-ACT complex was confirmed by gel electrophoresis and Western blot. The PSA-ACT complex was stable in the pH range 6.0 to 7.8; it was also stable in various matrices, temperatures, and high concentrations of salt. Purification of the PSA-ACT complex was highly reproducible. An absorptivity of 0.99 L x g-1 x cm-1 at 280 nm was assigned to the PSA-ACT complex, based on amino acid analysis. Because PSA and ACT bind in a 1:1 molar ratio, we determined the molecular mass of the PSA-ACT complex as the mass encoded by the cDNA of ACT (plus 26% carbohydrate) plus the molecular mass of PSA (28,430 Da), which totals 89,280 Da. Using this material, we made two common calibrators, one of 100% PSA-ACT complex and one of 90% PSA-ACT complex plus 10% free PSA by volume (90:10 calibrator). Substitution of these calibrators for the manufacturers' calibrators in nine commercial immunoassays substantially reduced differences between immunoassays, especially for serum PSA values between 4 and 10 micrograms/L. The 90:10 calibrator is recommended as a universal calibrator for international standardization of PSA immunoassays.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

scholarly journals Proteoglycan synthesis in two murine bone marrow stromal cell lines

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1777-1783 ◽  
Author(s):  
SL Kirby ◽  
SA Bentley
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Dermatan Sulfate ◽  
Cell Layer ◽  
Gradient Centrifugation ◽  
Exchange Chromatography ◽  
Proteoglycan Synthesis ◽  
Murine Bone Marrow

There is evidence indicating that stromal proteoglycans are an important functional component of the hematopoietic microenvironment. Proteoglycan synthesis was therefore investigated in the MS3–2A and D2XRII hematopoietic stromal cell lines. These lines differ in their capacity to support hematopoiesis in vitro, D2XRII supporting in vitro hematopoiesis, whereas MS3–2A does not. Cells were labeled with 35S- sulfate as precursor, and 4 mol/L guanidine HCl extracts of cells and media were analyzed by ion-exchange chromatography, cesium chloride density gradient centrifugation, and molecular sieve chromatography. Proteoglycans were further examined by enzymatic and chemical digestions. MS3–2A cells produced at least three proteoglycan species. Two chondroitin/dermatan sulfate (CS/DS) proteoglycans, Kav = 0.40 and Kav = 0.68 on Sepharose CL-2B, were present primarily in the medium. The respective glycosaminoglycan molecular weight (mol wt) values were 38 kd and 40 kd. A heparan sulfate (HS) proteoglycan of Kav = 0.58 and glycosaminoglycan mol wt 36 kd was present primarily in the cell layer extract. D2XRII cells synthesized two HS proteoglycans. The larger (Kav = 0.45; glycosaminoglycan mol wt, 30 kd) was of low density on gradient centrifugation and more prominent in the cell layer extracts, whereas the smaller (Kav = 0.68; glycosaminoglycan mol wt, 38 kd) was dense and present mainly in the culture medium. A single CS/DS proteoglycan species of Kav 0.78 and average glycosaminoglycan of mol wt 18 kd was present in roughly equal amounts in the medium and in the cell layer. MS3–2A and D2XRII thus appear phenotypically distinct with respect to proteoglycan synthesis. These differences are discussed in relation to the microenvironmental function of bone marrow stromal elements.

gamma-Tubulin in mammalian cells: the centrosomal and the cytosolic forms

1996 ◽  
Vol 109 (4) ◽  
pp. 875-887 ◽  
Author(s):  
M. Moudjou ◽  
N. Bordes ◽  
M. Paintrand ◽  
M. Bornens
Keyword(s):  
Mammalian Cells ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Soluble Form ◽  
Cellular Distribution ◽  
Cell Fractionation ◽  
Two Dimensional Gel Electrophoresis ◽  
Gamma Tubulin

The centrosome is one of the cellular organelles for which the mechanism by which it operates still remains to be unlavelled. The finding of the association with the centrosome of gamma-tubulin, a protein which belongs to the tubulin superfamily, has provided a long sought after biochemical tool with which to address centrosome function. We have generated a specific anti-gamma-tubulin polyclonal antibody to study the biochemical properties and the cellular distribution of the human lymphoblastic gamma-tubulin. Using cell fractionation and mass isolation of centrosomes, we observed that in contrast to the figures suggested by immunofluorescence, a minimum figure of 80% of total gamma-tubulin exists as a cytosolic form. The centrosomal form, for which at least half is not strongly associated with the centrosome, behaves in two-dimensional gel electrophoresis identically to the soluble form (as at least two spots of a pI of around 6). Post-embedding immunolocalization reveals that gamma-tubulin is distributed in the pericentriolar matrix but is also closely associated with centrioles. Using a combination of gel filtration, ion exchange chromatography, equilibrium sucrose gradient centrifugation and immunoprecipitation, we show that the major part of cytosolic gamma-tubulin might be involved in complexes heavier than the Tcp1 particle. We further demonstrate, by co-immunoprecipitation of gamma-tubulin and Tcp1 with either anti-Tcp1 or anti-gamma-tubulin antibodies, that a small part of gamma-tubulin participates in Tcp1-gamma-tubulin particles. Interestingly, the soluble form of gamma-tubulin co-purifies with taxol-stabilized microtubules and its association with microtubules resisted salt, ATP and GTP treatments. The existence of a centrosomal form and a large pool of cytosolic gamma-tubulin-containing complexes in somatic cells suggests that the overall gamma-tubulin cellular distribution does not seem to be as straightforward as it was drawn earlier.

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