scholarly journals 125I-Labelling of Erythropoietin without Loss of Biological Activity

1976 ◽  
Vol 159 (2) ◽  
pp. 287-292 ◽  
Author(s):  
M J Murphy
Keyword(s):  
Biological Activity ◽  
Glucose Oxidase ◽  
Catalytic Properties ◽  
Specific Radioactivity ◽  
Biological Integrity ◽  
High Specific Radioactivity

Erythropoietin, the hormone that regulates erythropoiesis in mammals, was 125I-labelled by using the catalytic properties of lactoperoxidase and the H2O2-generating properties of glucose oxidase. This methodology, both rapid and simple, not only produced hormone preparations with high specific radioactivity but also did not substantially alter the biological integrity of erythropoietin when it was assayed in vivo.

scholarly journals Reverse cholesterol transport in the isolated perfused rat spleen

1990 ◽  
Vol 268 (2) ◽  
pp. 499-505 ◽  
Author(s):  
M A Mindham ◽  
P A Mayes ◽  
N E Miller
Keyword(s):  
Whole Blood ◽  
Chemical Potential ◽  
Specific Radioactivity ◽  
Density Lipoprotein ◽  
Potential Gradient ◽  
Cholesterol Transport ◽  
Unesterified Cholesterol ◽  
Chemical Potential Gradient ◽  
High Specific Radioactivity

1. A method has been developed which enables the rat spleen to be loaded in vivo with [3H]cholesterol to a high specific radioactivity using cholesterol-labelled erythrocytes. The erythrocytes were shown to be rapidly degraded by the spleen and not released intact during subsequent perfusion. 2. When labelled spleens were perfused with whole blood or serum, lipoproteins in the high-density lipoprotein (HDL) range were shown to be the principal lipoprotein vehicles for the removal of cholesterol, the specific radioactivity of cholesterol being much greater in the HDL fractions than in other lipoproteins, particularly in the d 1.175-1.210 fraction. 3. The formation of [3H]cholesteryl ester was restricted to the major HDL fractions. 4. Experiments utilizing individual HDL fractions added to a basal perfusate indicated that HDL1 (d 1.050-1.085) was of less importance in the removal of cholesterol from the spleen than HDL subfractions of higher density. Also, a decrease in density of the lipoproteins was observed during perfusion, concurrent with uptake of cholesterol, especially in the d 1.085-1.125 subfraction. 5. When [3H]cholesterol-labelled spleens were perfused with whole blood, about half of the radioactivity released was detected in erythrocytes, indicating a rapid exchange or transport of cholesterol. Thus erythrocytes could play an important role in the transfer of unesterified cholesterol when the chemical potential gradient is favourable.

Novel Double-Isotope Technique for Enzymatic Assay of Catecholamines, Permitting High Precision, Sensitivity and Plasma Sample Capacity

1981 ◽  
Vol 61 (5) ◽  
pp. 591-598 ◽  
Author(s):  
M. J. Brown ◽  
D. A. Jenner
Keyword(s):  
Plasma Sample ◽  
Specific Radioactivity ◽  
High Specific Radioactivity ◽  
Coefficients Of Variation ◽  
Isotope Technique ◽  
Noradrenaline And Adrenaline ◽  
Radioenzymatic Assays ◽  
Double Isotope ◽  
Catecholamine Concentration

1. A novel use of a double-isotope method is described which allows radioenzymatic assays to combine precision and sensitivity. 2. In the catechol O-methyltransferase assay separate portions of each plasma sample are incubated with either S-[3H]- or S-[14C]-adenosyl-l-methionine. Standards of noradrenaline and adrenaline are added to the latter portions and are thus converted into standards of [14C]metadrenalines. These are added to the 3H-labelled portions after the incubation, where they function as tracers. 3. The final recovery of 14C radioactivity corrects for (a) the efficiency of methylation in the plasma sample concerned and (b) the recovery of metadrenalines during the extraction procedures. 4. The 3H/14C ratio is constant in each assay for a given catecholamine concentration and is determined for samples to which standards of noradrenaline and adrenaline are added to the 3H- (as well as the I4C-) labelled portions before the initial incubation. 5. The sensitivity of the assay is increased by using high specific radioactivity S-[3H]adenosyl-l-methionine (60-85 Ci/mmol), and low backgrounds are maintained by catecholamine depletion in vivo in the rats used for enzyme preparation. 6. Both catecholamines (1.5 pg/ml; 10 pmol/l) may be detected; the coefficients of variation are 3.0 and 3.2% for noradrenaline and adrenaline respectively (intra-assay) and 4.6 and 5.0% (inter-assay).

scholarly journals The separation of intracellular serum albumin from rat liver

1971 ◽  
Vol 123 (4) ◽  
pp. 643-648 ◽  
Author(s):  
J. D. Judah ◽  
Marion R. Nicholls
Keyword(s):  
Serum Albumin ◽  
Rat Liver ◽  
Specific Radioactivity ◽  
Exchange Chromatography ◽  
Rat Serum ◽  
Radioactive Impurity ◽  
Simple Method ◽  
High Specific Radioactivity ◽  
Chromatographic Procedure

1. Antibody precipitation of serum albumin from rat liver extracts yields impure preparations of the protein. 2. When rat liver is labelled with l-[1-14C]leucine, antibody precipitation of albumin leads to material that is contaminated with a protein or proteins of very high specific radioactivity. Only 10–25% of the radioactivity of the antibody precipitate is associated with serum albumin. 3. A chromatographic procedure is described that can be used to separate radiochemically pure serum albumin from antibody precipitates obtained from extracts of rat liver. 4. Extracellular albumin secreted by liver slices yields a precipitate with antibody which contains much less radioactive impurity. About 70–90% of the radioactivity is associated with serum albumin. Serum albumin separated by antibody precipitation from rat serum labelled in vivo was not contaminated with the radiochemical impurities associated with intracellular albumin. 5. A simple method is described of obtaining the content of serum albumin in rat liver extracts by the technique of isotope dilution and ion-exchange chromatography.

Glycosylation of plasma proteins with [U-14C]gIucose in vivo

1982 ◽  
Vol 60 (10) ◽  
pp. 942-951
Author(s):  
Rudolf Vrba ◽  
Stephen P. Adams
Keyword(s):  
Molecular Weight ◽  
Body Weight ◽  
Sialic Acid ◽  
Plasma Proteins ◽  
Gel Filtration ◽  
Specific Radioactivity ◽  
Apparent Molecular Weight ◽  
Covalent Bonds ◽  
High Specific Radioactivity

Within 1–12 h after subcutaneous injection of [U-14C]glucose, significant amounts of 14C attached by stable (probably covalent) bonds were found in plasma proteins of rats. More than 30% of 14C of [U-14C]glucose injected per 1 g body weight was incorporated into proteins contained in 1 mL of plasma. Less than 5% of 14C of the [14C]glucose-labelled plasma proteins was soluble in cold dilute perchloric acid, whereas more than 80% of 14C in the [14C]glucose-labelled plasma proteins was soluble in 50% saturated solutions of ammonium sulfate. From [U-14C]glucose-labelled plasma proteins, approximately 50% of the incorporated 14C was recovered in carbohydrate moieties (sialic acid, 8–12%; mannose and galactose, 15–31%; hexosamines, 8–14%) and the rest of the 14C (42–64%) was recovered from protein residue. Gel-filtration and electrophoresis profiles of distribution of 14C in [U-14C]leucine-labelled plasma proteins were very similar to those of [U-14C]glucose-labelled plasma proteins; a relatively high specific radioactivity was observed in fractions having an apparent molecular weight of 105 × 103 or its multiples (220 × 103 and 520 × 103). However, about 99% of the incorporated 14C was recovered from the protein residue of [14C]leucine-labelled plasma proteins, whereas only traces of 14C were found in the carbohydrate moieties. [U-14C]Alanine is also a relatively poor precursor of 14C for incorporation into carbohydrate moieties of plasma proteins as compared with [U-14C]glucose.

scholarly journals An alternative procedure for incorporating radiolabelled cholesteryl ester into human plasma lipoproteins in vitro

1985 ◽  
Vol 226 (1) ◽  
pp. 319-322 ◽  
Author(s):  
D C K Roberts ◽  
N E Miller ◽  
S G L Price ◽  
D Crook ◽  
C Cortese ◽  
...  
Keyword(s):  
Cholesteryl Ester ◽  
Human Plasma ◽  
Specific Radioactivity ◽  
Density Lipoprotein ◽  
Plasma Lipoproteins ◽  
Cholesteryl Esters ◽  
Simple Method ◽  
High Specific Radioactivity

A simple method has been developed for labelling human plasma lipoproteins to high specific radioactivity with radioactive cholesteryl esters in vitro. After isolation by preparative ultracentrifugation, the selected lipoprotein was incubated for 30 min at 4 degrees C in human serum (d greater than 1.215) that had been prelabelled with [4-14C]cholesteryl oleate or [1,2-3H]cholesteryl linoleate, and was then re-isolated by ultracentrifugation. All major lipoprotein classes were labelled by the procedure. Specific radioactivities of up to 18 d.p.m. pmol-1 (46 d.p.m. ng-1) were achieved. When radiolabelled high-density lipoprotein was infused intravenously, the radioactive cholesteryl ester behaved in vivo indistinguishably from endogenous cholesteryl esters produced by the lecithin (phosphatidylcholine): cholesterol acyltransferase reaction.

scholarly journals RNA dactyloscopy

2017 ◽  
Vol 63 (4) ◽  
Author(s):  
Marta Magdalena Gabryelska ◽  
Eliza Wyszko ◽  
Jan Barciszewski
Keyword(s):  
Biological Activity ◽  
Rna Secondary Structure ◽  
Tertiary Structure ◽  
Catalytic Properties ◽  
Hammerhead Ribozyme ◽  
Conformational Dynamics ◽  
Efficient Design ◽  
Rna Fingerprinting

Despite the wealth of data on RNA secondary structure, conformational dynamics and tertiary structure in vitro and in vivo, predicting RNA biological activity in cellular environments remains difficult. Here we describe a method of in silico RNA fingerprinting that allows efficient design of hammerhead ribozyme molecules with a high intracellular efficiency. Our method, which we call RNA dactyloscopy, is a reliable tool for assessing catalytic properties, modeling and design of RNA.

scholarly journals A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo

1983 ◽  
Vol 212 (3) ◽  
pp. 791-800 ◽  
Author(s):  
R C Pittman ◽  
T E Carew ◽  
C K Glass ◽  
S R Green ◽  
C A Taylor ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Specific Radioactivity ◽  
Apolipoprotein A1 ◽  
Intact Protein ◽  
Gut Contents ◽  
Tissue Samples ◽  
Cultured Fibroblasts ◽  
High Specific Radioactivity

We recently developed a general method for determining tissue sites of degradation of plasma proteins in vivo that made use of covalently attached radioactive sucrose. On degradation of the protein, the sucrose remained trapped in the cells as a cumulative marker of protein degradation. The method described here depends on the same principles, but uses an adduct of cellobiose and tyramine that is radioiodinated to high specific radioactivity and then covalently attached to protein. Use of the radioiodinated ligand increases the sensitivity of the method at least 100-fold and allows simplified tissue analysis. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and in vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was in all cases less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples.

Radiolabeling of Fibrinogen Using the Iodogen Technique

1981 ◽  
Vol 46 (03) ◽  
pp. 593-596 ◽  
Author(s):  
Linda C Knight ◽  
Andrei Z Budzynski ◽  
Stephanie A Olexa
Keyword(s):  
Specific Radioactivity ◽  
Oxidizing Agent ◽  
Iodine Monochloride ◽  
Human Fibrinogen

SummaryThe properties of human fibrinogen labeled with 125-Iodine using Iodogen (1, 3, 4, 6-tetrachloro-3α, 6α-diphenylglycoluril) as an oxidizing agent were compared with those of an iodine monochloride labeled counterpart. It was found that thrombin clottability, binding to staphylococci, the relative specific radioactivity of the Aα, Bβ, and γ chains and in vivo clearance from plasma in rabbits were the same in these two labeled fibrinogen preparations. Labeling efficiency was higher when iodogen was used. It is concluded that human fibrinogen labeled with radioiodine using the Iodogen technique is suitable for studies in vitro and in vivo.

STUDIES IN CONGENITAL GENERALIZED LIPODYSTROPHY

1973 ◽  
Vol 72 (3) ◽  
pp. 495-505 ◽  
Author(s):  
Oddmund Søvik ◽  
Svein Oseid
Keyword(s):  
Biological Activity ◽  
Insulin Response ◽  
Epididymal Adipose Tissue ◽  
Large Individual ◽  
Immunoreactive Insulin ◽  
List Type ◽  
Glucose Load ◽  
Congenital Generalized Lipodystrophy ◽  
The One

ABSTRACT The biological activity of plasma insulin from 4 cases of congenital generalized lipodystrophy has been studied, using rat diaphragm and epididymal adipose tissue in vivo. The results are compared with previous data on plasma immunoreactive insulin obtained in these patients. 2 of the 4 cases exhibited unusually high biological insulin activities during the fasting state as well as after an intravenous (iv) glucose load. In the fat pad assay activities as high as 10 000 μU insulin per ml were observed. During childhood the biological insulin activities were generally high, although there were large individual variations. However, in the one case studied after the age of puberty, the insulin response to a glucose load was negligible. Taken together, the biological and immunological activities observed strongly suggest the presence of pancreatic insulin in these patients. It appears that the circulating insulin has a fully biological activity. The decreasing insulin activities after cessation of growth are in agreement with the appearance of frank diabetes at this time.

Sign in / Sign up

Export Citation Format

Share Document