Incorporation Of 125I Research Articles

Isolation and Characterization of Surface Antigens from Schistosoma mansoni. I. Evaluation of Techniques for Radioisotope Labeling of Surface Proteins from Adult Worms

Radiolabeled surface proteins of adult Schistosoma mansoni were prepared by in vitro labeling of whole worms, and by labeling freeze-thaw surface membrane extracts. Incorporation of 125I into surface proteins was attempted using the lactoperoxidase, chloramine-T, iodosulfanilic acid, and Bolton-Hunter methods. Radiolabeling of whole worms with lactoperoxidase, chloramine-T and iodosulfanilic acid yielded a single protein peak (mol wt greater than 100,000) on SDS-PAGE, and showed considerable incorporation of label in the lipid fraction. Bolton-Hunter labeling of whole worms yielded four major peaks with molecular weights of 100,000, 60,000, 30,000 and 21,000, and minor peaks with molecular weights of 26,000, 36,000, 43,000, 68,000 and 78,000; three of the four major peaks corresponded to prominent bands in Coomassie blue-stained gels. Although carbohydrate-labeling techniques were not successful, a single carbohydrate band, molecular weight greater than 100,000, was detected was PAS staining. Radiolabeling of freeze-thaw extracts yielded results similar to those obtained with whole worms. Electron microscopy revealed the tegument to be left intact and undamaged after labeling with the Bolton-Hunter reagent.

Effects of Thyrotropin on Thyroglobulin Exocytosis and Iodination in the Rat Thyroid Gland*

Rats, pretreated with thyroxine for 2 days, were given one or two iv injections of 500 mU of TSH; in some groups the second TSH dose was replaced by 0.75 micronmol isoproternol. The effects of the thyroid stimulators on the following parameters were studied: the number of exocytotic vesicles in the follicle cells; the incorporation of 125I into thyroid proteins, measured over periods of 5 min; and the thyroidal cAMP contents. At 2 h after TSH administration, a second dose of TSH failed to stimulate iodination while at 8 h the iodination response was "normal". Two hours after TSH the follicle cells contained practically no exocytotic vesicles but at 8 h they had a full supply of vesicles, and this was emptied by the second TSH injection. THE CAMP content was less increased by the second TSH injection than by the first one, but the stimulatory effect of the second TSH dose on cAMP was the same at 2 h and at 8 h; this indicates that the lack of iodination response at 2 h was not simply due to blocking of TSH receptors. Isoproternol, which acts on other receptors than does TSH, cause a similar cAMP increase incontrols and at 2 h and 8 h after TSH, but stimulated iodination only in controls and at 8 h after TSH; this supported the conclusion that the lack of iodination response to a second TSH dose at 2 h was not due to impairment of the adenylate cyclase-cAMP system. These observations taken together strongly indicate that a rapid iodination response to TSH depends on stimulated exocytosis which, in turn, requires a pool of exocytotic vesicles in the follicle cells. Such a coupling between exocytosis and iodination seems appropriate since by exocytosis uniodinated thyroglobulin and membrane, showing peroxidase activity histochemically, are delivered to the site of iodination, the apical cell surface.

Iodinated phospholipids and the in vitro iodination of proteins of dog thyroid gland.

Slices of dog thyroid gland were incubated with liposomes consisting of (125)I-labelled phosphatidylcholine (the iodine was covalently linked to unsaturated fatty acyl chains). The (125)I label of (125)I-labelled liposomes was incorporated into thyroid protein and/or thyroglobulin at a higher rate than was the (131)I label of either Na(131)I or (131)I(2). The iodine was shown to be protein-bound by the co-migration of the labelled iodine with protein under conditions where free iodine, iodide and lipid-bound iodine were removed from protein. The uptake of iodine from the iodinated phospholipid was probably due to phospholipid exchange between the iodinated liposomes and the thyroid cell membrane, since (a) (14)C-labelled phospholipid was metabolized to (14)CO(2) and (b) many lipids in the tissue slice became (14)C-labelled. A very strong inhibition of iodide ;uptake' from Na(131)I, caused by thiosulphate, produced only a minor inhibition of the incorporation of (125)I from (125)I-labelled liposomes into thyroid protein and/or thyroglobulin. This implies that free iodide may not necessarily be formed from the iodinated phospholipids before their entrance or utilization in the cell. Synthetic polytyrosine polypeptide suspensions showed some iodination by (131)I-labelled liposomes. In tissues with low tyrosine contents, such as liver and kidney, only a trace uptake was observed. Salivary gland showed some uptake. Endoplasmic reticulum of thyroid gland showed a higher iodine uptake than that of the corresponding plasma membranes. These experiments, together with the demonstration of the diet-dependent presence of iodinated phospholipids in dog thyroid, leads us to suggest that iodination of the membrane phospholipids of thyroid cells may be directly or indirectly involved at some stage in the synthesis of thyroglobulin, or exists as a scavenger mechanism, to re-utilize and/or recover released iodine from unstable compounds inside the thyroid cell.

Oxidative Metabolism of the Human Eosinophil

We have compared the oxidative metabolism of human eosinophils (80%-90% purity) to that of neutrophils. Hexose monophosphate (HMP) shunt activity of eosinophils was higher than that of neutrophils under either resting or phagocytizing conditions. Eosinophil HMP shunt activity also was stimulated by phorbol myristate acetate, a membrane-active agent. Eosinophils showed a marked incorporation of 125I into trichloroacetic acid-insoluble material under resting conditions, which increased markedly during phagocytosis. Eosinophils likewise showed a greater reduction of nitroblue tetrazolium dye during phagocytosis than did neutrophils. Measurement of other parameters of oxidative metabolism indicated that eosinophils generated superoxide anion following phagocytosis and also elicited a burst of chemiluminescence similar to that observed during phagocytosis by neutrophils. Measurement of NADPH oxidase activity demonstrated that this enzyme was 3-6 times more active in fractions isolated from eosinophils than in corresponding fractions isolated from neutrophils; this was observed over a range of substrate concentrations. The eosinophil enzyme sedimented differently than the neutrophil enzyme with differential centrifugation; neither showed sedimentation characteristics of peroxidase. These data indicate that eosinophils possess a similar, although in some ways more potent, oxidative burst than neutrophils and are consistent with a role for NADPH oxidase in the initiation of that burst.

Oxidative metabolism of the human eosinophil

We have compared the oxidative metabolism of human eosinophils (80%-90% purity) to that of neutrophils. Hexose monophosphate (HMP) shunt activity of eosinophils was higher than that of neutrophils under either resting or phagocytizing conditions. Eosinophil HMP shunt activity also was stimulated by phorbol myristate acetate, a membrane- active agent. Eosinophils showed a marked incorporation of 125I into trichloroacetic acid-insoluble material under resting conditions, which increased markedly during phagocytosis. Eosinophils likewise showed a greater reduction of nitroblue tetrazolium dye during phagocytosis than did neutrophils. Measurement of other parameters of oxidative metabolism indicated that eosinophils generated superoxide anion following phagocytosis and also elicited a burst of chemiluminescence similar to that observed during phagocytosis by neutrophils. Measurement of NADPH oxidase activity demonstrated that this enzyme was 3–6 times more active in fractions isolated from eosinophils than in corresponding fractions isolated from neutrophils; this was observed over a range of substrate concentrations. The eosinophil enzyme sedimented differently than the neutrophil enzyme with differential centrifugation; neither showed sedimentation characteristics of peroxidase. These data indicate that eosinophils possess a similar, although in some ways more potent, oxidative burst than neutrophils and are consistent with a role for NADPH oxidase in the initiation of that burst.

Alterations of red cell membranes from phenylhydrazine-treated rabbits

Reticulocytosis was induced in rabbits by two methods: phlebotomy and injection of phenylhydrazine. Normal erythrocytes, reticulocytes from bed rabbits, reticulocytes from phenylhydrazine-treated rabbits, and erythrocytes treated in vitro with phenylhydrazine were compared with respect to their plasma membrane labeling by galactose oxidase and NaB 3H 4, and lactoperoxidase-catalyzed incorporation of 125I. Normal erythrocyte membranes and membranes from reticulocytes of bled rabbits showed almost identical labeling patterns, the presence of 2–3 glycoproteins with moderate to low mobilities on dodecyl sulfate acrylamide gel electrophoresis. Labeling in the absence of enzyme was negligible. In contrast, the reticulocytes from phenylhydrazine-treated rabbits exhibited a large incorporation of tritium without prior treatment with galactose oxidase. Even after prereduction with unlabeled NaBH 4 to remove this nonspecific labeling, the labeled glycoprotein components found in normal erythrocytes were not detectable. Normal erythrocytes treated in vitro with phenylhydrazine, washed, and labeled with galactose oxidase had labeling patterns, including high nonspecific incorporation of 3H, similar to those observed with in vivo phenylhydrazine treatment. Solubilization of membranes with lithium diiososalicylate followed by partitioning with phenol showed that the same glycoproteins were presented in normal or phenylhydrazine membranes, although only the former extract was labeled by galactose oxidase. Individual carbohydrates from the membranes were analyzed by gas-liquid chromatography and, in the case of hexosamines, on the amino acid analyzer. The results of these analyses indicated a slight decline in galactose content with phenylhydrazine treatment. Reticulocyte membranes from bled rabbits also showed a decrease in galactose content, although it was less pronounced. Most of the label incorporated by nonspecific borohydride labeling of membranes from phenylhydrazine-treated animals was found associated with protein. The modified amino acids from labeled proteins are similar to those formed in reactions of oxidized lipids and proteins in model systems.

Use of Radioimmunoassays to Determine the Concentration of Streptococcal Group-Specific Antibodies in Rabbit Antisera

A radioimmunoassay was developed for the quantitation of antibodies to the Group A, A-variant, and C carbohydrates in rabbit streptococcal antisera. The assay employs radiolabeled purified Group carbohydrate which has been tyrosylated to allow for incorporation of 125-I. This assay has an advantage over the intitative precipitin test because it measures non-precipitating antibody in addition to precipitating antibody. Furthermore, 7S anti-IgG in rabbit antisera give a falsely elevated value for the antibody concentration in the quantitative precipitin test. This did not occur with the radioimmunoassay. The assay described is reproducible, sensitive, and uses little antisera.

Enzymic iodination of the histidyl residue of secretin: A radioimmunoassay of the hormone

Lactoperoxidase (EC 1.11.1.7) was a more efficient catalyst than chloramine-T for the incorporation of 125I into secretin. Satisfactory radioimmunoassays were performed using the labelled product. Total hydrolysis and Edman degradation of the iodinated secretin showed that only the N-terminal histidyl residue of the hormone had been iodinated. Poly( l-histidine) was also iodinated using lactoperoxidase.

Enzymic iodination of polypeptide hormones for radioimmunoassay

The use of lactoperoxidase (EC 1.11.1.7) and chloramine-T as reagents for the incorporation of 125I into polypeptide hormones (glucagon, gastrin, insulin, B-chain of insulin) for radioimmunoassay, was compared. In general, results were more reproducible when the enzyme was used; 70–80% of the label present was incorporated into the polypeptides and the iodinated hormones had higher affinity for antibody (over 80% was bound by excess antibody) resulting in better standard curves in radioimmunoassay. The enzymic method appears to give rise to fewer side-reactions.

Lipid peroxidation during enzymatic iodination of rat liver endoplasmic reticulum

During enzymatic iodination of rat liver endoplasmic reticulum the peroxidation of endogenous lipids and the loss of cytochrome P 450 has been observed. This lipid peroxidation can be inhibited by the inclusion of a low concentration of butylated hydroxytoluene in the iodination mixture. This results in increased incorporation of 125I − and stabilization of cytochrome P 450.

Suppressor T Cells

Abstract The antigen-induced DNA synthetic response of a number of different thymocyte populations was studied in the spleens of lethally irradiated recipients by incorporation of 125I 5-iodo-2-deoxyuridine. The response of certain cell combinations was not only less than the sum of the two responding alone but less than one of the individual cell populations in the combination. The following combinations had this effect: 1) “Educated” and normal thymocytes < “educated” alone in the response to sheep red cells (SRBC); 2) parental and F1 thymocytes < parental alone in F1 hosts; 3) cortisone resistant (in a low dose) and normal thymocytes < normal alone in the response to SRBC. Thus, thymocytes are capable of suppressing the antigen-induced response of other thymocytes without the mediation of B cells or their product (i.e., antibody).

Onset of Function in the Human Fetal Thyroid: Biochemical and Radioautographic Studies from Organ Culture1

ABSTRACT Nineteen human fetal thyroids obtained in fresh condition were studied by the use of short-term organ culture. The incorporation of 125I added to the culture medium was investigated by means of biochemical and histologic techniques both in the gland and in a suitable control tissue. The crown-rump length of the fetuses ranged from 22 to 142 mm, which is equivalent to an estimated gestational period of 45–112 days. In the 7 fetuses of less than 68 mm no organic iodine, central colloid cavity or tissuebound 125I were found. At the 68 mm stage (74 gestational days) and at all later stages a full spectrum of organically bound, iodinated products was found (MIT, DIT, T4, and occasionally T3) and the tissue evidenced 125I binding in radioautographs. Sequential steps of biochemical maturation as reported in experimental animals were not identified in these tests. The control tissue contained only radioiodide. Sections of epon-embedded thyroid tissue, 1 μ in thickness, permitted identification of intrace...

METABOLIC STUDIES ON EXPERIMENTAL ALLERGIC THYROIDITIS.

The relationship between the patterns of incorporation of 125I in the thyroid and serum and the pathological changes in the gland is described in experimental allergic thyroiditis, 3–6 weeks after a single immunizing dose of homologous thyroid extract in Freund's adjuvant. At 3–4 weeks, reduced thyroid 125I uptake, low or normal labeled serum iodothyronine and normal serum 125Iiodoalbumin levels were found. Pathological changes at this time showed intense mononuclear infiltration with acinar invasion and disruption and focal hyperplasia, while the residual uninvolved acini appeared inactive. At 5–6 weeks after immunization, thyroid 125I uptake, labeled serum iodothyronine and 126I-iodoalbumin were elevated, and free labeled DIT was present at times in the serum and thyroid. 125I incorporation into the microsomal fraction was elevated at the expense of the supernatant fraction of the thyroid. The pathological appearance continued to be extensive inflammatory infiltration, but all acini showed hyperplasia. ...