The sites of thyroid hormone formation in rabbit thyroglobulin

Rabbit thyroglobulin (Tg) was labeled in vivo with 125I and purified by gel filtration. Separation by high performance liquid chromatography (HPLC) of tryptic digests of S-cyanoethylated Tg yielded four major iodothyronine-containing peaks, designated A, B, C, and D. These were further purified on HPLC and sequenced for identification of amino acid residues and for location of the iodothyronine by 125I counting. The published primary structure for bovine Tg, derived from cDNA sequencing of the Tg gene (Mercken, L., Simons, M.J., Swillens, S., Massaer, M., and Vassart, G. (1985) Nature 316, 647-651), permitted tentative location of the rabbit hormonogenic peptides within the Tg polypeptide chain. Site A, corresponding to bovine residue 5, contained 44% of Tgs [125I]T4 (thyroxine) and 25% of its [125I]T3 (triiodothyronine); its specific activity of iodine was higher than that for other sites, indicating priority of iodination. Site B, containing 24% of Tgs [125I]T4 and 18% of its [125I]T3, corresponded to bovine residue 2555. Site C, at the third residue from the C terminus (bovine residue 2748), was the major T3 site, accounting for over 50% of Tgs [125I]T3. The amino acid sequence around this site shows less homology among different animal species than do those flanking the other hormonogenic sites. Site D accounted for 17% of Tgs [125I]T4 and corresponded to bovine Tyr-1291, in the midportion of Tgs polypeptide chain. The three major T4-forming sites had the sequence Asp-Tyr (sites B and D) or Glu-Tyr (site A), while the sequence Ser-Tyr-Ser appeared to favor T3 synthesis (site C), suggesting an important influence of primary structure on hormonogenesis. We conclude that site A is the major T4-forming site and site C the major T3-forming one, but others are available and offer the opportunity for flexibility in meeting different demands for hormone formation.     

Structural differences around hormonogenic sites on thyroglobulins from different species detected by monoclonal antibodies

Thyroxine remains attached to its synthetic site in thyroglobulin until it is released by proteolysis. Strong homology in the primary sequence surrounding thyroxine-forming residues in thyroglobulins from various species suggests a unique three-dimensional structure at hormonogenic sites. To examine this, two thyroxine-binding mouse anti-(chicken thyroglobulin) monoclonal antibodies, 1A10 and 5F6, were used as probes for this region in an enzyme-linked immunosorbent inhibition assay. The thyroxine content of thyroglobulins had a marked positive influence on the monoclonal antibody binding: when the thyroxine content of human thyroglobulin rose by 6.6-fold, cross-reactivities rose 25-fold for the 1A10 monoclonal antibody and 17.6-fold for the 5F6 monoclonal antibody. However, interspecies comparison of thyroglobulin preparations with similar thyroxine content showed lower than expected cross-reactivities for human, pig and sheep thyroglobulins when compared with chicken thyroglobulin. Only when the thyroxine content of heterologous thyroglobulin preparations was two or three times higher did the cross-reactivities equal or surpass that of chicken thyroglobulin. It is concluded that in thyroglobulin there are structural differences in the different animal species near the thyroxine-forming sites bound by these monoclonal antibodies. The known primary sequence similarity does not seem to result, therefore, in identical three-dimensional structures about this site. These differences may reflect species-specific variations in distant regions brought close as a result of chain folding to form the hormonogenic site, such as those around the donor diiodotyrosine residue or in polysaccharide structures. These monoclonal antibodies provide information about the structure of thyroglobulin, which cannot be obtained from knowledge of the amino acid sequence alone.     

Consensus sequences for early iodination and hormonogenesis in human thyroglobulin

Thyroglobulin from a human goiter, containing four atoms of iodine/molecule (660,000 daltons), was iodinated with Na 125I and KI in vitro to achieve a net addition of either 2 or 7.8 atoms of iodine/molecule. After fractionation by high performance liquid chromatography, iodinated tryptic peptides from S-cyanoethylated 125I-thyroglobulin were purified, sequenced, characterized by [125I]iodoamino acid distribution, and localized within thyroglobulins primary structure based upon the published cDNA sequence, (Malthiery, Y., and Lissitsky, S. (1987) Eur. J. Biochem. 165, 491-498). The addition of 2 atoms of iodine/molecule of thyroglobulin produced iodotyrosyls at five principal sites, with no 125I-hormone formation. The addition of 7.8 atoms iodinated the same sites more heavily, produced iodotyrosyls at 10 additional sites, and formed iodothyronines at 5 sites. After addition of 2 atoms of iodine, tyrosyl 24 and 11% of thyroglobulins 125I, while tyrosyl 2572 had 24%, but with 7.8 added atoms of iodine, tyrosyl 24 had more of the thyroglobulins [125I]iodothyronine (36 versus 26%). Since tyrosyls 149, 866, and 1466 were iodinated early but did not form the inner rings of iodothyronines, they are attractive candidates for donors of outer iodothyronyl rings. The sequences around the iodotyrosyls fall into three consensus groups, as follows: 1) Glu/Asp-Tyr, associated with synthesis of thyroxine (residues 24, 2572, and 1309), or iodotyrosine (residues 2586 and 991); 2) Ser/Thr-Tyr-Ser, associated with synthesis of iodothyronine (residue 2765) and iodotyrosine (1466 and 883); and 3) Glu-X-Tyr, 7 of the remaining 8 iodotyrosyls occur in this sequence, and we found iodine incorporation at each place this sequence appears in the thyroglobulin molecule. Iodine has been found at homologues of most of these sites in thyroglobulins of other species. We conclude that the primary structure of thyroglobulin, and particularly these consensus sequences, have a major role in the formation of thyroid hormones and their iodinated precursors.     

Thyroxine transport in choroid plexus

The role of the choroid plexus in thyroid hormone transport between body and brain, suggested by strong synthesis and secretion of transthyretin in this tissue, was investigated in in vitro and in vivo systems. Rat choroid plexus pieces incubated in vitro were found to accumulate thyroid hormones from surrounding medium in a non-saturable process. At equilibrium, the ratio of thyroid hormone concentration in choroid plexus pieces to that in medium decreased upon increasing the concentration of transthyretin in the medium. Fluorescence quenching of fluorophores located at different depths in liposome membranes showed maximal hormone accumulation in the middle of the phospholipid bilayer. Partition coefficients of thyroxine and triiodothyronine between lipid and aqueous phase were about 20,000. After intravenous injection of 125I-labeled thyroid hormones, choroid plexus and parts of the brain steadily accumulated 125I-thyroxine, but not [125I]triiodothyronine, for many hours. The accumulation of 125I-thyroxine in choroid plexus preceded that in brain. The amount of 125I-thyroxine in non-brain tissues and the [125I]triiodothyronine content of all tissues decreased steadily beginning immediately after injection. A model is proposed for thyroxine transport from the bloodstream into cerebrospinal fluid based on partitioning of thyroxine between choroid plexus and surrounding fluids and binding of thyroxine to transthyretin newly synthesized and secreted by choroid plexus.     

Thyroglobulin and the biosynthesis of thyroid hormones

Thyroglobulin (mol. wt. 660 kDa) is the specific protein of the thyroid gland in which are synthesized and stored the thyroid hormones (thyroxine and 3,5,3'-triiodothyronine). It is formed of equal-sized subunits (330 kDa) containing each identical polypeptide chains to which are associated two types of oligosaccharide units representing 8 to 10% by weight of the protein. The studies reported in this paper describe the presence in thyroglobulin of discrete hormonogenic sites. After chemical (CNBr) and enzymatic (trypsin and protease V8 of S. aureus) treatments of the protein, four different hormone-containing peptide segments have been isolated, purified and sequenced. They correspond to the hormonogenic tyrosine-containing sites of the protein. One tyrosine is located at 4 amino acid residues from the N-terminal asparagine of the chain and is a major site for thyroxine synthesis. Another one which represents the triiodothyronine site is situated 2 amino acids before the C-terminal lysine. Finally, two other sites, one of low affinity and the other of high affinity for iodine and thyroxine formation, are equally located in the C-terminal part of the chain. The hormone-forming regions localized at the opposite far ends of the thyroglobulin chain(s) likely represent zones more accessible to iodination and with a conformation suited for the coupling of iodotyrosine into iodothyronine residues and ultimately protease attack to release the free hormones into the circulation. The presence of hormonogenic sites of different affinities for iodine allows thyroglobulin to modulate adaptively its hormonogenic capacity to external iodine supply. The molecular mechanism of this process is still unknown.     

Characterization of the cocaine binding site on the sigma-1 receptor

The cocaine photoaffinity label 3-iodo-4-azidococaine ([125I]IACoc) binds to the sigma-1 receptor with an affinity that is 2-3 orders of magnitude higher than the parent compound cocaine [Kahoun, J. R., and Ruoho, A. E. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. In the present study, the binding properties of several cocaine derivatives to the guinea pig liver sigma-1 receptor were determined. The results from assessing the affinity of various derivatives of cocaine which were substituted on the phenyl ring indicated that an important determinant of binding to the guinea pig sigma-1 receptor binding site may be the development of a dipole in the ring in which the pi electron density of the phenyl ring is reduced. This implies that an electron-rich source is present in the sigma-1 receptor binding site, such as the pi system of an aromatic ring or other electron-rich side chains, which interact with the phenyl ring of cocaine. The precise [125I]IACoc derivatization site in the guinea pig sigma-1 receptor was identified using chemical cleavage and purification of the resulting labeled peptides. Cyanogen bromide cleavage of the [125I]IACoc photolabeled sigma-1 receptor followed by radiosequencing identified Asp188, which is located in the putative steroid binding domain-like II (SBDL II) near the carboxyl terminus, as the site of [125I]IACoc insertion. Systematic truncation of the C-terminus indicated the requirement for the last 15 amino acid residues of the receptor for [125I]IACoc photolabeling.     

The importance of thyroglobulin structure for thyroid hormone biosynthesis.

Thyroglobulin (Tg) is the most important protein in the thyroid because it provides the matrix for thyroid hormone biosynthesis. Here we review experimental work, principally from our laboratory, on the relationship between Tg structure and hormonogenesis. Early work showed that Tg's most important hormonogenic site was located in a fragment of approximately 26 kDa obtained on chemical reduction. With the establishment of the cDNA sequence of Tg, this and other major sites could be localized within Tg's polypeptide chain. The four major hormonogenic sites, designated A, B, C, and D, are located respectively at tyrosyls 5, 2553, 2746, and 1290. In most species, site A accounts for about 40% of Tg's hormone, and site B for about 25%. Site C is associated with increased T3, at least in some species. Site D is prominent in guinea pigs and rabbits, and TSH favors hormonogenesis at it in these species. Sequential iodination of low iodine human Tg shows three consensus sequences associated with early iodination and with T4 formation. Recent work has identified Tyr130 in beef Tg as donor of an outer iodothyronine ring, most likely to Tyr5, the most important hormonogenic site. In addition to its biochemical importance, Tg has clinical interest in familial goiter and autoimmune thyroid disease. Further elucidation of Tg structure and its relation to thyroid hormone synthesis will contribute to thyroid physiology and to its clinical application.     

Endotoxin-induced reduction of beta-adrenergic binding sites on splenic lymphocytes in vivo and in vitro: its modulation by anterior hypothalamic lesions

Bacterial endotoxin induced a 38% decrease in the number of beta-adrenergic binding sites (Bmax) on splenic lymphocytes, four days after intraperitoneal administration to guinea pigs. No change in the affinity (Kd) for [125-I]-cyanopindolol ([125-I]-CYP) binding was observed. Incubation of guinea pig splenocytes in vitro with different concentrations of bacterial endotoxin for 24 hours resulted in an increased incorporation of [3H]-thymidine, a parameter for lymphocyte activation. Activation of splenic lymphocytes with the optimal endotoxin concentration of 100 micrograms/ml for 24 hours induced a 27% decrease in the Bmax whereas the Kd for [125-I]-CYP binding was not changed. Based on these findings, we speculate that activation of lymphocytes with endotoxin in vitro and in vivo is associated with a reduction in the number of beta-adrenergic binding sites on these cells. Anterior hypothalamic (AHA) lesions protected against the endotoxin-induced reduction in the number of beta-adrenergic binding sites on lymphocytes. The protective effect of these lesions could not be related to alterations in the plasma levels of cortisol, triiodothyronine (T3), thyroxine (T4), adrenaline and noradrenaline or to splenic noradrenaline content. Since AHA lesions have been shown to inhibit several lymphocyte functions, it is suggested that these lesions prevent lymphocyte activation after in vivo endotoxin administration and through this abrogate the reduction of the beta-adrenergic binding sites.     

In vitro studies of the thyroglobulin degradation pathway: endocytosis and delivery of thyroglobulin to lysosomes, release of thyroglobulin cleavage products--iodotyrosines and iodothyronines

Iodinated thyroglobulin stored in the thyroid follicular lumen is subjected to an internalization process and thought to be transferred into the lysosomal compartment for proteolytic cleavage and thyroid hormone release. In the present study, we have designed in vitro models to study: 1) the transfer of endocytosed thyroglobulin into lysosomes, and 2) the intracellular fate of free thyroid hormones and iodinated precursors generated by intralysosomal proteolysis of thyroglobulin. Open follicles prepared from pig thyroid tissue by collagenase treatment were used to probe the delivery of exogenous thyroglobulin to lysosomes via the differentiated apical cell membrane. Open follicles were incubated with pure [125I]thyroglobulin with or without unlabeled thyroglobulin in the presence or in the absence of chloroquine. Subcellular fractionation on a Percoll gradient showed that [125I]thyroglobulin was internalized and present in low (for the major part) and high density thyroid vesicles. In chloroquine-treated open follicles, we observed the appearance of a definite fraction of [125I]thyroglobulin in a lysosome subpopulation having the expected properties of phagolysosomes or secondary lysosomes. In contrast, in control open follicles, the amount of [125I]thyroglobulin or degradation products found in high density vesicles was lower and associated with the bulk of lysosomes, i.e., primary lysosomes. The content in thyroglobulin and degradation products of lysosomes at steady-state was analyzed by Western blot using polyclonal anti-pig thyroglobulin antibodies. Under reducing conditions, immunoreactive thyroglobulin species correspond to polypeptides with molecular weights ranging from 130,000 to less than 20,000. The presence of free thyroid hormones and iodotyrosines inside lysosomes and their intracellular fate was studied in dispersed thyroid cells labeled with [125I]iodide. Neo-iodinated [125I]thyroglobulin gave rise to free [125I]T4 which was secreted into the medium. In addition to released [125I]T4, a fraction of free [125I]T4 was identified inside the cells. Lysosomes isolated from dispersed thyroid cells did not contain significant amounts of free [125I]T4. The free intracellular [125I]T4 fraction seems to represent an intermediate 'hormonal pool' between thyroglobulin-bound T4 and secreted T4. Evidence for such a precursor-product relationship was obtained from pulse-chase experiments. In conclusion: 1) open thyroid follicles have the ability to internalize thyroglobulin by a mechanism of limited capacity and to address the endocytosed ligand to lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical and Pharmacological Characterization of Serotonin-O-Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5-HT1B and 5-HT1D Binding Sites

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.     

Enhanced expression of cellular receptors for human interferon α on peripheral lymphocytes from patients with down''s syndrome

The sequence of 370 bases at the 5′-end of bovine thyroglobulin mRNA has been determine. A41 base untranslated segment was found preceeding the ATG initiator codon. It is followed by an open reading frame providing the first data on thyroglobulin primary structure. Analysis of the amino acid sequence demonstrated the presence of an 18 residue hydrophobic segment representing a putative signal peptide. Comparison of the amino terminal sequence of thyroglobulin with that of peptides known to contain thyroid hormones [7,8] demonstrated that the first tyrosine in native thyroglobulin is mainly found as thyroxine in the mature iodinated protein [8]. Our results clearly identify the amino-terminal region of thyroglobulin as an important hormonogenic domain of the protein.     

Comparison of the host ranges and antigenicity of Cryptosporidium parvum and Cryptosporidium wrairi from guinea pigs.

Oocysts of a Cryptosporidium isolate from guinea pigs were not infectious for adult mice, but were infectious for two of three newborn calves and for suckling mice. However, oocysts isolated from calves or mice infected with guinea pig Cryptosporidium were not infectious for guinea pigs. Four isolates of C. parvum from calves were incapable of infecting weanling guinea pigs. Microscopic examination of tissue from the colon and cecum of suckling guinea pigs inoculated with C. parvum revealed sparse infection of some pups. These host range studies and previously described differences in 125I-labeled oocyst surface protein profiles between Cryptosporidium sp. from guinea pigs and C. parvum suggest they are distinct species. We propose the name Cryptosporidium wrairi be retained. Studies with monoclonal antibodies indicate that C. wrairi and C. parvum are antigenically related.     

Thyroglobulin structure-function. The amino acid sequence surrounding thyroxine

A 19-residue, thyroxine (T4)-containing peptide, Tryp-T4, has been isolated from the tryptic digest of a low molecular weight, iodine-enriched fragment derived from 19S bovine thyroglobulin. This tryptic peptide represents the only site of significant iodination in the parent polypeptide fragment. The amino acid sequence of the tryptic peptide has been determined and is NH2-Asn-Ile-Phe-Glu-T4-Gln-Val-Asp-Ala-Gln-Pro-Leu-Arg-Pro-Cys-Glu-Leu-Gln- Arg-COOH. The carboxyl-terminal sequence of this peptide shows a high probability of a beta-turn. These findings establish the involvement of at least a single unique sequence within thyroglobulin in thyroxine biosynthesis and the general nature of a hormonogenic site within this protein. This sequence contains at least 30% of the thyroxine present in 19 S bovine thyroglobulin.     

Hormone-containing peptides from normal and goiter human thyroglobulins

A series of low iodine human thyroglobulin samples derived from colloid-rich goiter tissue was examined by HPLC mapping of tryptic digests and compared to normal human thyroglobulin. These samples ranged in iodine content from 2 to 8 gram-atoms of iodine (g.a. I) per mole and were not further iodinated in vitro. Peptides containing the principal hormonogenic sequence were detected using the long wavelength absorbance of the iodotyrosine derivatives at 325 nm. Two such peptides were isolated and sequenced. Their thyroxine content was confirmed by radioimmunoassay. The number of 325-nm-absorbing peaks was significantly lower in the normally iodinated human thyroglobulin than that observed the thyroglobulins of cattle and dog. This suggests a more restricted iodination in the human protein. Sodium dodecyl sulfate gel patterns of the reduced and alkylated proteins showed significant molecular size heterogeneity in all of the samples. Polypeptide fragments ranged in molecular size from approximately 330 to 45 kDa in the goiter derived material and from approximately 330 to 15 kDa in the normal human material. This difference between the proteins is consistent with earlier observations that peptides less than 45 kDa appear concomitantly with hormone formation. These data confirm that the human thyroglobulin molecule is capable of forming at least limited amounts of thyroid hormone at iodine levels as low as 4 g.a. I per mole. The hormone detected in this study was located at residue 5 near the amino terminus of the thyroglobulin molecule.     

Changes in the structure of thyroglobulin following the administration of thyroid-stimulating hormone.

In each of three separate experiments, female guinea pigs in groups of 20 were given 4 units of thyroid-stimulating hormone (TSH) each day for 3 days, while controls were given saline. Na125I was injected on the 3rd day, and the animals were killed 22 hours later. The pooled throids of each group were homogenized, and thyroglobulin was purified by one of the following methods: gel filtration on Sephadex G-200 followed by density gradient ultracentrifugation, two sequential filtrations on 4 percent agarose, or filtration on 4 percent agarose followed by Sephadex G-200. TSH administration was associated with the folling changes in thyroglobulin: (1) an increase in the ratio of tri-iodothyronine to thyroxine; (2) a decrease in dissociation of the 19 S to the 12 S form; (3) an alteration in its pattern on gel electrophoresis in sodium dodecyl sulfate-urea; and (4) changes in its amino acid composition, with significant increases in the content of lysine (by 15 percent), isoleucine (by 15 percent), and methionine (by 7 percent) relative to leucine. Over-all, there were no significant changes in the content of iodine, fucose, hexosamine, or sialic acid. These data show that TSH can alter the composition of thyroglobulin independently of its effects on iodine content. We suggest that these changes may stem from alterations in the subunit composition of thyroglobulin. There were also small but significant variations in amino acid composition among the three preparations of thyroglobulin from saline-treated animals and among the three from the TSH-treated. This finding shows that thyroglobulin can be heterogeneous in its protein portion as well as in its iodine content.     

Synthesis and evaluation of radioiodinated cholesteryl ethers as lipoprotein probes.

Two novel cholesteryl ether derivatives were synthesized and radioiodinated: (1) [125I]cholesteryl m-iodobenzyl ether (125I-CIBE) and (2) [125I]cholesteryl 12-(m-iodophenyl)dodecyl ether (125I-CIDE). These radioiodinated ethers were incorporated into low-density lipoprotein (LDL) by incubating the compounds (solubilized in saline with Tween-20) with isolated LDL or with whole plasma. Such LDL preparations were taken up by cultured fibroblasts in a receptor-dependent manner similar to that of radioiodinated LDL. Upon injection into guinea pigs, 125I-CIBE-labeled guinea pig LDL cleared from the plasma similarly to radioiodinated guinea pig LDL. The primary sites of 125I-CIBE uptake were the adrenal and the liver, and the compound was stable to both hydrolysis and deiodination over 24 h. In summary, 125I-CIBE and 125I-CIDE, like previously described tritiated cholesteryl ethers, appear to be potentially useful tracers of cholesteryl ester uptake. Moreover, these radioiodinated probes have the advantage of being more easily quantitated in tissue samples as well as being detectable by noninvasive scintigraphic imaging.     

Identification and enzymatic deglycosylation of the myometrial oxytocin receptor using a radioiodinated photoreactive antagonist.

To identify and characterize oxytocin receptors, a 125I-labeled photoreactive oxytocin antagonist was synthesized. The specific oxytocin antagonist [1-(beta-mercapto-beta,beta- cyclopentamethylenepropionic acid), 2-O-methyltyrosine,4-threonine,8- ornithine,9-tyrosylamide]oxytocin ([Mca,Tyr(O-Me)2,Thr4,Orn8,Tyr9-NH2]oxytocin) described by Elands et al. (Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.-J., Bankowski, K., Manning, M., and Sawyer, W. H. (1987) Eur. J. Pharmacol. 147, 192-207) bound to the guinea pig uterine oxytocin receptor with high affinity (apparent Kd = 0.74 nM). The introduction of a 4-azidophenylamidino group at Orn8 resulted in the photoreactive ligand [Mca1,Tyr(O-Me)2,Thr4,Orn(4-azidophenylamidino)8,Tyr9- NH2]oxytocin, which retained the high binding affinity (Kd = 0.69 nM) of the parent compound. The photoreactive antagonist monoiodinated at Tyr9 had approximately double (Kd = 0.39 nM) the affinity of the photoreactive antagonist and several times that of oxytocin (Kd = 2.6 nM) for the guinea pig uterine oxytocin receptor. In photo-affinity labeling experiments using myometrial membranes obtained from guinea pigs during late pregnancy, the 125I-labeled photoreactive antagonist specifically labeled a protein with an apparent molecular mass of between 68 and 80 kDa: the labeling of this protein was completely suppressed by a 100-fold molar excess of oxytocin and oxytocin receptor-specific agonists, but not by vasopressin analogues specific for V1 or V2 receptors or by other peptide hormones. The ability of oxytocin to suppress labeling was decreased in the presence of guanosine 5'-O-(thiotriphosphate) or in the absence of Mn2+. Digestion of the photolabeled oxytocin receptor with endoglycosidase F gave rise to a protein with an apparent molecular mass of 38 +/- 2 kDa. The endoglycosidase F effect and the lack of endoglycosidase H action show that the myometrial oxytocin receptor is highly glycosylated with asparagine-linked complex oligosaccharide chains. Our results suggest that the radioiodinated photoreactive oxytocin antagonist could be a helpful tool in the isolation and further characterization of the oxytocin receptor.     

A comparative study on the biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum (Apicomplexa).

Cryptosporidium sp. from guinea pigs and C. parvum were compared morphologically, electrophoretically, and for the ability to infect suckling mice. Oocysts from guinea pigs measured 5.4 x 4.6 (4.8-5.6 x 4.0-5.0) microns and had a shape index (length/width) of 1.17 (1.04-1.33). Oocysts of C. parvum were similar and measured 5.2 x 4.6 (4.8-5.6 x 4.2-4.8) microns with a shape index of 1.16 (1.04-1.33). All suckling mice inoculated with oocysts of C. parvum became infected, whereas most, but not all, mice fed oocysts of the guinea pig isolate also became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts by day 7 postinoculation than did mice infected with C. parvum, and the resulting infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins revealed striking differences between the two isolates. Cryptosporidium parvum had a wide molecular size range of 125I-labeled bands, whereas C. sp. from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with a smaller number of bands greater than 100 kDa.     

Total and free thyroxine and triiodothyronine in blood serum of mammals

1. Blood samples were obtained from seven species of mammals: horses, cattle, sheep, goats, pigs, guinea pigs and rats for determination of total and free thyroxine and triiodothyronine. Total thyroxine in the order listed above in ng/ml was: 15, 60, 79, 185, 53, 45 and 79. Free thyroxine in pg/ml was: 5.9, 10.0, 19.2, 32.1, 21.7, 6.7 and 51.3. 2. Total triiodothyronine in pg/ml was: 677, 1290, 979, 3170, 760, 317 and 1747. Free triiodothyronine in pg/ml was: 3.22, 4.40, 2.60, 6.74, 2.74, 2.42 and 10.88. 3. Percent free thyroxine was high in rats and low in guinea pigs, while percent free triiodothyronine was high in guinea pigs and low in goats. 4. Free thyroxine and percent free thyroxine were higher in some groups of horses, particularly stallions, than in other groups.     

Evidence for a secretion of thyroxine by isolated hog thyroid cells.

Isolated thyroid cells prepared from hog thyroid glands by tryptic dispersion were incubated with 131I- for 1--6 h. Free [131I]thyroxine was identified in the incubation medium by three chromatographic methods. Neither [131I]iodotyrosines nor [131I]triiodothyronine were detected. The [131I]thyroxine released in the medium by 100 mul of cells (packed cell volume) after a 6-h incubation period amounted to 1.16% (S.E. = +/- 0.39) of the total radioactivity. The medium [131I]thyroxine represented 15--25% of the total [131I]thyroxine synthesized during the 6 h of incubation. Thyrotropin, 1--60 munits/ml, increased the medium [131I]thyroxine content 2-4 fold. Dibutyryl cyclic AMP mimicked the effect of thyrotropin. The amount of medium [131]thyroxine was strictly related to the amount of incubated cells but was independent of the volume of the incubation medium. When prelabeled cells were incubated in the presence of methimazole the increase in medium [131I]thyroxine was quantitatively related to a decrease in the intracellular [131I]thyroxine. Addition of dinitrotyrosine, an inhibitor of the deiodinase activity, induced the release of iodotyrosines in the incubation medium. That the incubation supernatant of isolated thyroid cells did contain free thyroxine but not iodotyrosines suggests that the normal mechanisms of proteolysis of thyroglobulin and deiodination of iodotyrosines inside the cells are preserved. From these data, it was concluded that the thyroxine release by isolated cells represents a real secretion.