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Biomedical subjects

E Goldwasser

Publications and source records attributed to E Goldwasser.

At least 127 records · Page 7Linked to original sources
PubMed1979

Radioimmunoassay of erythropoietin.

A radioimmunoassay for erythropoietin has been developed using 125I labeled pure human erythropoietin and an anti-erythropoietin antiserum produced in a rabbit immunized with human erythropoietin. Two techniques are presented for labeling erythropoietin, both resulting in an immunologically reactive labeled reactant. One method involves the use of lactoperoxidase and the other a reagent known as IODO-GEN. The second International Reference Preparation of human erythropoietin is used as a standard and a double antibody scheme is used for the separation of the free and antibody bound labeled hormone. The radioimmunoassay is sensitive to an absolute amount of erythropoietin equivalent to 0.4 milliunits. Bioassays for erythropoietin require approximately 100 times this amount. The use of pure erythropoietin as the labeled reactant has removed certain discrepancies seen in previous attempts to develop radioimmunoassays for this hormone, e.g., sera from patients without kidneys do not give the high values previously seen. Sera from anemic individuals not only give rise to high radioimmunoassay values but also show a parallel relationship with the erythropoietin standard when halving dilutions are analyzed. Desialated erythropoietin is also reactive with the same parallelism. Bleeding of a normal individual increases the serum erythropoietin level and transfusion decreases it. Erythropoietin from a variety of laboratory animals is also reactive in the radioimmunoassay, with very high values being observed in hypoxic animals.

Anemia↗
PubMed1979

A radioimmunoassay for erythropoietin.

We describe the development of a radioimmunoassay (RIA) for erythropoietin. Antisera were raised in rabbits with an impure human urinary erythropoietin preparation used as immunogen, but with pure human erytropoietin serving as the labeled antigen in the RIA and as a primary standard. The immunoreactivity of erythropoietin is not altered significantly by the mode of labeling with radioiodine, even though the biologic activity is lost. With this method, it is possible to detect 2--3 mU of erythropoietin in a volume of 0.1--0.3 ml. Therefore, the method can be used for detection of normal and subnormal serum titers as well as elevated titers. RIA for erythropoietin does not distinguish between the native (active in vivo) and the asialo form (inactive in vivo) and cannot yet be used for routine monitoring of crude fractions during purification.

Animals↗
PubMed1978

Hodgkin disease and red cell aplasia.

A teenage boy with mixed-cellularity Hodgkin disease presented with severe anemia secondary to pure red cell aplasia of marrow without evidence of lymphomatous infiltration or hemolysis. In vitro studies of the patient's serum demonstrated an inhibitor of erythropoietin activity which appeared to be an IgG but which did not directly bind erythropoietin. The patient's anemia resolved and the inhibitor disappeared following chemotherapy for Hodgkin disease. Presumably, the inhibitor was directed at a very early stage of red blood cell production. This phenomenon may be related to other autoimmune manifestations occasionally seen in patients with lymphomas. The case is presented to bring attention to the unusual occurrence of red cell aplasia in Hodgkin disease. Several hypotheses concerning significance and etiology of the anemia are detailed.

Adolescent↗
PubMed1978

Extraction of erythropoietin from normal kidneys.

Significant amounts of active erythropoietin were extracted from the kidneys of normal rats, cattle, dogs, and rabbits by homogenization of the organs in 0.1 M phosphate buffer. The mean erythropoietin activities of the extracts, as determined by the starved-rat assay, were 0.26 U/g beef kidney, 0.41 U/g dog kidney, and 0.11 U/g rat kidney. The dog kidney extracts had a mean activity of 0.35 U/g, as measured by stimulation of hemoglobin synthesis in cultured bone marrow cells (in vitro assay) and produced a dose-dependent stimulation of 59Fe incorporation into circulating red cells when assayed in polycythemic mice. Extracts of rabbit kidney cortices had a mean activity of 2.12 U/g, as measured by stimulation of hemoglobin synthesis in cultured bone marrow cells. When the dog kidney homogenate was fractionated on DEAE-cellulose, all of the erythropoietin activity was adsorbed to the exchanger in the presence of 0.01 M acetate buffer, pH 4.5, and was completely eluted by 0.1 M Na2HPO4-0.5 M NaCl, pH 8. An antibody made against human urinary erythropoietin completely inactivated the erythropoietic factor in the dog kidney extract. Serum from a donor dog had no erythropoietin activity when assayed in the starved rat, suggesting that the factor in the extracts is intracellular erythropoietin rather than that contained in plasma trapped in the renal vasculature. The complete inactivation of the erythropoietic factor in these kidney homogenates by antierythropoietin and its behavior on DEAE-cellulose indicate that this factor is structurally similar to native plasma erythropoietin. The extracts are completely active without being incubated in the presence of serum.

Animals↗
PubMed1977

Simultaneous effects of erythropoietin and colony-stimulating factor on bone marrow cells.

Erythropoietin or colony-stimulating factor, or both, were added to rat or mouse marrow cell cultures, and the responses to each inducer were measured. Colony-stimulating factor caused the suppression of erythropoietin-stimulated hemoglobin synthesis, and erythropoietin caused the suppression of the granulocyte-macrophage colony formation that is dependent on colony-stimulating factor. The extent of suppression by each inducer was dose-dependent. Marrow cells from plethoric rats were more sensitive to suppression of erythropoietin action by colony-stimulating factor than were normal marrow cells. These findings suggest that either (i) the receptors for erythropoietin and for colony-stimulating factor have overlapping specificities and that the "wrong" inducer may bind without having an inductive effect, or (ii) the target cells for erythropoietin and colony-stimulating factor are very closely related or are the same.

Animals↗
PubMed1977

Purification of human erythropoietin.

Human erythropoietin, derived from urine of patients with aplastic anemia, has been purified to apparent homogeneity. The seven-step procedure, which included ion exchange chromatography, ethanol precipitation, gel filtration, and adsorption chromatography, yielded a preparation with a potency of 70,400 units/mg of protein in 21% yield. This represents a purification factor of 930. The purified hormone has a single electrophoretic component in polyacrylamide gels at pH 9, in the presence of sodium dodecylsulfate at pH 7, and in the presence of Triton X-100 at pH 6. Two fractions of the same potency and molecular size, by sodium dodecyl sulfate gel electrophoresis, but differing slightly in mobility at pH 9, were obtained at the last step of fractionation. The nature of the difference between these two components is not yet understood.

Anemia, Aplastic↗
PubMed1977

The effects of erythropoietin in vitro on spleen colony-forming cells.

Erythropoietin (epo) added to liquid cultures of mouse bone marrow cells effected both the numbers of spleen colony-forming cells (CFU) in the cultures and the types of spleen colonies formed from these cells in irradiated hosts. Epo caused an increase in the number of CFU detected in cultures on the second day; this effect persisted through day 10, with the maximal increase occurring on the seventh day. The magnitude of the rise on day 7 was proportional to the amount of epo added. The increase in spleen colonies found with cells cultured in the presence of epo was due solely to erythroid colonies. After seven days in culture without epo, there was a peak of cells that formed non-erythroid colonies. This peak did not appear when the cells were cultured in the presence of epo.

Animals↗
PubMed1976

Erythropoietin.

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Cell Differentiation↗
PubMed1976

Erythropoietin production by human renal carcinoma cells in culture.

Cells from human renal tumors were grown in monolayer cultures, and the media obtained at each medium change were assayed for erythropoietin activity. The medium from carcinoma I (a granular cell tumor) contained a high level of activity initially. The concentration of erythropoietin activity decreased with time in culture, but was significantly higher than that in controls after four months in vitro. There was , in addition, evidence of an inhibitory material present in the culture media. The activity formed by the tumor cells could be neutralized by an antibody to human urinary erythropoietin. The difference between activity measured in marrow cell cultures and that found by in vitro assay, and the chromatographic properties of the active preparation, suggest that the tumor-derived activity may be largely asialoerthropoietin. Two other renal carcinomas, of a different cellular type, produced significant erythropoietic activity.

Animals↗
PubMed1976

On the mechanism of erythropoietin-induced differentiation. XIV. The apparent effect of etiocholanolone on initiation of erythropoiesis.

Etiocholanolone, when tested in normal mice or in mice that have been out of an hypoxic atmosphere for only a few days, stimulates erythropoiesis, but appears to have no effect on erythropoiesis when tested in plethoric mice that have very low residual red cell formation. The steroid, similarly, stimulates hemoglobin synthesis by marrow cells in culture if they are from normal mice, but has no effect on cells from transfused mice. Out data suggest that 5 beta-H androstane and pregnane derivatives do not have a primary action on the induction of erythropoiesis, but act on cells that are already differentiated.

Animals↗
PubMed1975

An assay for erythropoietin in vitro at the milliunit level.

A method is described for the assay of erythropoietin using primary cultures of adult rat bone marrow cells. Either total labeled iron uptake by the cells or hematin synthesis from labeled iron may be used as the measure of erythropoietin action. The method is useful in the range 0.001 to 0.010 U, where the log response is linear with the log dose, and can be carried out in 2 working days. This method has the disadvantage of detecting asialoerythropoietin which has no activity in vivo.

Animals↗
PubMed1975

Erythropoietin and the differentiation of red blood cells.

Erythropoietin is the primary factor regulating red blood cell formation in mammals and some other animals. It has been purified from plasma derived from anemic sheep and from the urine of anemic patients and is a glycoprotein. The sheep hormone has a molecular weight of 46,000 and appears to consist of a single chain. The carbohydrate and amino acid compositions where known, are summarized, as are the known structural requirements for biological activity. Its mode of action as the inducer of red cell differentiation has been studied in marrow cell and fetal liver cell cultures. Erythropoietin interacts first with a protein receptor on its target cell causing the appearance of a cytoplasmic protein thought to be a mediator that, in turn, causes an increased rate of transcription in the target cell nuclei. There are several different species of RNA synthesized before the cells initiate hemoglobin synthesis. The effect on transcription is only indirectly dependent on DNA synthesis. Some properties of the primitive erythropoietin-responsive cell are discussed and a cellular model for blood cell differentiation is presented.

Amino Acids↗