Formiminoglutamate excretion in rat urine after whole body irradiation, histidine administration, starvation and stress.

Formiminoglutamate (FIGLU) urinary excretion was studied in rats subjected to whole body 60Co irradiation with doses of 450, 650 and 850 R. During the first post-irradiation days, FIGLU excretion doubled after both lower doses. From day 3, 450 R led to a decrease, whereas 650 and 850 R were followed by a still enhanced FIGLU excretion. No correlation to the radiation dose was found. A daily intraperitoneal administration of histidine in a dose of 200 mg/kg led to a constant 5-fold increase of FIGLU output and to more distinct differences in post-irradiation FIGLU excretion. Two days starvation or ACTH administration, followed by doubled urinary total 17-hydroxycorticoids, did not interfere with FIGLU excretion.     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm     

Hyperthyroid hearts display a phenotype of cardioprotection against ischemic stress: a possible involvement of heat shock protein 70.

Hyperthyroid hearts are shown to display a phenotype of cardioprotection against ischemic stress, but the underlying signaling mechanisms remain largely unknown. The present study investigated the possible relation of HSP70 to the thyroid hormone induced cardioprotection. HSP70 is a redox-regulated molecular chaperone, and enhances cell survival under stress. Thyroxin (25 microg/100 g body weight) was administered to Wistar male rats for four days (THYR-4d) and two weeks (THYR-14d), respectively, while untreated animals served as controls (CON-4d, CON-14d). Isolated hearts from control and thyroxin treated rats were subjected to 20 min zero-flow ischemia followed by 45 min of reperfusion (I/R). The amount of HSP70 in the myocardium for THYR-14d was 1.85 times the levels of CON-14d (p < 0.05). The levels of HSP70 expression were no different between THYR-4d and CON-4d, p > 0.05. This was only accompanied by an increase in MDA levels (used as an index of oxidative stress) in THYR-14d compared to untreated hearts. These changes corresponded to a differential response of the heart to I/R; post-ischemic recovery of function was significantly increased in THYR-14d compared to CON-14d, and was no different between the THYR-4d and CON-4d hearts. In conclusion, long-term thyroxin administration results in increased tolerance of the myocardium to I/R and enhances the expression of HSP70 which may, at least in part, account for this response.     

Indomethacin inhibits thrombin-, but not thyroxin-stimulated resorption of fetal rat limb bones

The bone-resorbing effects of thrombin and thyroxin, two agents that stimulate resorption in neonatal mouse calvaria by prostaglandin-dependent mechanisms, were examined in cultures of fetal rat limb bones. Thrombin produced maximal resorption in the limb bone cultures at a concentration of 100 U/ml when bones were cultured in BGJ supplemented with 1 mg/ml bovine serum albumin. The effects of thrombin were partially inhibited by 0.5 and 10 uM indomethacin. Thrombin failed to elicit resorption when the limb bones were cultured in DMEM with 15% horse serum. Thyroxin stimulated the resorption of limb bones in both BGJ-albumin and DMEM-serum media. Resorption was elicited by thyroxin concentrations of 10 nM-10 uM. 30 uM thyroxin failed to stimulate resorption. The dose-response curve to thyroxin was shallow, and the agent did not produce maximal resorption. The bone-resorbing effects of thyroxin were not affected by 0.5 or 10 uM indomethacin.     

Indomethacin inhibits thrombin-, but not thyroxin- stimulated resorption of fetal rat limb bones

The bone-resorbing effects of thrombin and thyroxin, two agents that stimulate resorption in neonatal mouse calvaria by prostaglandin-dependent mechanisms, were examined in cultures of fetal rat limb bones. Thrombin produced maximal resorption in the limb bone cultures at a concentration of 100 U/ml when bones were cultured in BGJ supplemented with 1 mg/ml bovine serum albumin. The effects of thrombin were partially inhibited by 0.5 and 10 uM indomethacin. Thrombin failed to elicit resorption when the limb bones were cultured in DMEM with 15% horse serum.Thyroxin stimulated the resorption of limb bones in both BGJ-albumin and DMEM-serum media. Resorption was elicited by thyroxin concentrations of 10 nM − 10 uM. 30 uM thyroxin failed to stimulate resorption. The dose-response curve to thyroxin was shallow, and the agent did not produce maximal resorption. The bone-resorbing effects of thyroxin were not affected by 0.5 or 10uM indomethacin.     

Insulin versus oral agents in the management of Cystic Fibrosis Related Diabetes: a case based study

Background

Although thyroxin therapy clearly is beneficial to children with frank hypothyroidism there is little data on the effects of thyroxin in children with compensated or subclinical hypothyroidism. The objective of this study was to determine the effect of thyroxin therapy on cognitive function in children with compensated hypothyroidism. The hypothesis was that thyroxin therapy would change neuropsychological function.

Methods

Eleven patients with a history of sub clinical hypothyroidism entered the study. At the start of the study, six out of the 11 were on thyroxin therapy, while 5 were off therapy. All patients underwent a battery of neuropsychological testing and thyroid function tests at the start of study. Based on the results of thyroid function tests, two of the 5 patients who were off thyroxin were started back on thyroxin. All of the 6 patients who were on thyroxin were taken off thyroxin. All patients then underwent repeat neuropsychological testing and thyroid functions after an average of 91 days.

Results

Thyroxin therapy could not be shown to have an effect on neuropsychological function in this short term study. Our patients had attention problems as compared to the normal population. No significant differences were found between our subjects and normal population standards in verbal processing, visual processing, motor speed/coordination and achievement.

Conclusion

In this small, short term study, thyroxin therapy could not be shown to affect neuropsychological function in children with compensated hypothyroidism. These children may have attention problems but appear to have normal verbal and visual processing, motor speed/coordination and achievement.     

Stress-induced changes in the area ratio or the quantitative ratio of myocardial mitochondria and myofibrils and their correction with thyroid hormones

An experimental study performed on 30 male white rats has demonstrated that immobilization stress was associated with a decrease in mitochondrial to myofibril area ratio (Smch/Smf) in the left ventricle and interventricular septum by 34.6% and 46.9%, respectively. Pretreatment with small doses of thyroid hormones which did not influence body weight, heart rhythm or serum thyroxin level prevented the decrease and caused Smch/Smf increase in the left ventricle and interventricular septum by 68.7% and 45.8%, respectively. The doses of thyroid hormones applied caused a more marked increase of mitochondrial to myofibril area ratio in the control rats.     

Alpha-fetoprotein and serum hormone levels following liver intoxication with carbon tetrachloride

Rats were submitted to carbon tetrachloride intoxication at two different doses. Serum level of estradiol, progesterone, cortisol and thyroxin were measured by radioimmunoassays and correlated with the histological evidences of liver regeneration and serum alpha-fetoprotein levels. A two fold increase of progesterone was observed 48hrs after CCl₄ administration. Cortisol levels were moderately increased at both doses of CCl₄. Despite the five fold increase of alpha-fetoprotein (which is the major estradiol binding protein in these sera) no changes in estradiol levels were observed. Thyroxin levels showed a two fold increase after 72hrs. This result contrasts with the drop of this hormone after partial hepatectomy which has been previously published. These experiments show that a new hormonal imbalance (directly or indirectly due to the toxic) is involved both in liver regeneration and alpha-feto-protein synthesis.     

Fluorescent-Antibody Targeting of Insulin-Like Growth Factor-1 Receptor Visualizes Metastatic Human Colon Cancer in Orthotopic Mouse Models

Fluorescent-antibody targeting of metastatic cancer has been demonstrated by our laboratory to enable tumor visualization and effective fluorescence-guided surgery. The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of metastatic colon cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24–31) was conjugated with 550 nm, 650 nm or PEGylated 650 nm fluorophores. Subcutaneous, orthotopic, and liver metastasis models of colon cancer in nude mice were targeted with the fluorescent IGF-1R antibodies. Western blotting confirmed the expression of IGF-1R in HT-29 and HCT 116 human colon cancer cell lines, both expressing green fluorescent protein (GFP). Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of colon cancer cells. Subcutaneously- and orthotopically-transplanted HT-29-GFP and HCT 116-GFP tumors brightly fluoresced at the longer wavelengths after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted HCT 116-GFP tumors were brightly labeled by fluorescent IGF-1R antibodies such that they could be imaged non-invasively at the longer wavelengths. In an experimental liver metastasis model, IGF-1R antibodies conjugated with PEGylated 650 nm fluorophores selectively highlighted the liver metastases, which could then be non-invasively imaged. The IGF-1R fluorescent-antibody labeled liver metastases were very bright compared to the normal liver and the fluorescent-antibody label co-located with green fluorescent protein (GFP) expression of the colon cancer cells. The present study thus demonstrates that fluorophore-conjugated IGF-1R antibodies selectively visualize metastatic colon cancer and have clinical potential for improved diagnosis and fluorescence-guided surgery.     

Failure of X-rays to mutate class II histocompatibility loci in balb/c mouse spermatogonia

Adult Balb/c Kh male mice were irradiated (pelvic region, 250 kVcp X-rays, 60 rad per min) and three months later were mated to untreated C57BL/6 Kh females. Their B6C F₁ progeny were screened for mutations at the Class II histocompatibility loci, i.e. those that carry similar alleles in the parental lines and are therefore homozygous in the F₁ progeny. The treatment groups were: single doses of 0, 350, 500, 650 and 800 rad; split doses 1 day apart, totalling 500, 650 and 800 rat; split doses averaging 52 days apart, totalling 650 and 800 rad. Thirty-six mutants were identified in 13,614 progeny. Twelve of them occurred in five clusters of two or three, presumably owing to five gonadal mosaics among 940 parents. Irradiation did not increase the spermatogonial mutation rate. The only effect of exposure appeared to be a decrease in the mutation rate of the 1-day split dose-groups compared to those with the same total doses in a single exposure or in two fractions, 52 days apart.     

Effect of thyroxine and prednisolone and the exogenous RNA induced by them on the proliferation of cells

As demonstrate experiments performed on cells of the primary culture of the newborn rat kidney, injection of thyroxin stimulates, and addition of prednisolone inhibits the proliferative activity of the cells in the culture. At a combined and simultaneous administration of these two hormones, as well as at injection of thyroxin 2 h before prednisolone, the thyroxin program, stimulating the cell proliferation is expressed. When thyroxin is administered 2 h after prednisolone, during first 12 h the prednisolone program is expressed, and then the proliferative activity of the culture returns to the initial level. At the cooperative action of oppositely directed factors for realization of the cell proliferative program, an essential role play the time factor and lag-period duration for each of the hormones interacting. Exogenic RNAs, obtained from the kidneys of the rats, to whom thyroxin or prednisolone have been injected, when they (RNAs) are injected into the culture, they produce effects, similar to those that are noted at injecting these hormones into the incubation medium. Thus, injection of the hormones to rats, results in formation, by the cells of the organ, induced RNAs, capable to transfer a hormonal signal and produce a hormone-like effect at regulation of the proliferative activity of the cell culture in the obtained exogenic RNA from the organ-donor.     

THE EFFECTS OF HYDROGEN ION CONCENTRATION UPON THE METAMORPHIC PATTERN OF THYROXIN- AND IODINE-TREATED TADPOLES

1. It has been shown quantitatively that the degree of response of the hind limbs of tadpoles to the action of thyroxin is dependent upon the lengths of the limbs at the beginning of treatment. 2. Both the potency of the inducing substance and the rate of penetration of the substance into the animal might be involved in the effects of hydrogen ion concentration on induced development. 3. Changes in hydrogen ion concentration affect the inducing power of thyroxin and iodine differently. With thyroxin, it is the rate of penetration of the molecule which determines the amount of growth, but with iodine it is the chemical form in which the substance has entered the animal which is of prime importance. 4. The hydrogen ion concentration of thyroxin solutions does not affect their potency when they are injected into tadpoles. 5. Change in hydrogen ion concentration of the environment does not affect the potency of thyroxin injected into tadpoles. 6. When thyroxin is administered in the environmental solution its effects, as measured by increase in hind limb length are greater at higher than at lower hydrogen ion concentrations in the range tested. 7. Since the potency of thyroxin is unaffected by change in hydrogen ion concentration when the thyroxin solution is injected, the above fact (point 6) seems explicable only on the basis of differences in the rate of penetration of thyroxin into the animals at the different hydrogen ion concentrations. 8. These differences in penetration of the thyroxin at different hydrogen ion concentrations may be the result of a differential effect of hydrogen ion concentration upon the rate of metabolism of the animal. The metabolic rate is significantly greater when the tadpoles are kept in solutions of higher hydrogen ion concentration than when they are kept in solutions of low hydrogen ion concentration. It is postulated that the rate of metabolism, since it controls the rate of intake of the environmental fluid and therefore of dissolved thyroxin, also controls the amount of thyroxin-induced development. 9. Change in hydrogen ion concentration of iodine solutions affects their potency when injected into tadpoles. A peak of effectiveness is reached at about the neutral point, with a lowered efficiency as the hydrogen ion concentration is either increased or decreased from this point. 10. Change in hydrogen ion concentration of the environment affects the potency of iodine injected into tadpoles. The effect is similar to that noted in point 9. 11. The hydrogen ion concentration of the environment seems to affect the chemical nature of the iodine in solution in the environment. If this is so, it is possible that the differences in the metamorphic effects of iodine at different hydrogen ion concentrations are dependent upon the chemical form of iodine present. 12. The effect of hydrogen ion concentration on normal development is similar to that on thyroxin-induced development; an effect on the rate of metabolism of the animal causes increased growth in more acid solutions.     

The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor

Recurrent missense fibroblast growth factor receptor 3 (FGFR3) mutations have been ascribed to skeletal dysplasias of variable severity including the lethal neonatal thanatophoric dysplasia types I (TDI) and II (TDII). To elucidate the role of activating mutations causing TDI on receptor trafficking and endocytosis, a series of four mutants located in different domains of the receptor were generated and transiently expressed. The putatively elongated X807R receptor was identified as three isoforms. The fully glycosylated mature isoform was constitutively but mildly phosphorylated. Similarly, mutations affecting the extracellular domain (R248C and Y373C) induced moderate constitutive receptor phosphorylation. By contrast, the K650M mutation affecting the tyrosine kinase 2 (TK2) domain produced heavy phosphorylation of the nonglycosylated and mannose-rich isoforms that impaired receptor trafficking through the Golgi network. This resulted in defective expression of the mature isoform at the cell surface. Normal processing was rescued by tyrosine kinase inhibitor treatment. Internalization of the R248C and Y373C mutant receptors, which form stable disulfide-bonded dimers at the cell surface was less efficient than the wild-type, whereas ubiquitylation was markedly increased but apparently independent of the E3 ubiquitin-ligase casitas B-lineage lymphoma (c-Cbl). Constitutive phosphorylation of c-Cbl by the K650M mutant appeared to be related to the intracellular retention of the receptor. Therefore, although mutation K650M affecting the TK2 domain induces defective targeting of the overphosphorylated receptor, a different mechanism characterized by receptor retention at the plasma membrane, excessive ubiquitylation and reduced degradation results from mutations that affect the extracellular domain and the stop codon.     

Thyroxin specifically stimulates anuran larvae epidermal sodium pump during metamorphosis

1. Sodium pump, measured as K+-dependent, ouabain sensitive pNPPase, but not the non-specific, ouabain insensitive pNPPase, was stimulated by thyroxin in epidermis of tadpoles of Rana perezi. 2. Epidermal K+-pNPPase of thyroxin treated tadpoles was only stimulated in those stages already showing activity and reached levels similar to adult frogs in tadpoles at metamorphic climax.     

Effect of thyroxin on the functional state of the respiratory chain in liver mitochondria of the carp Cyprinus carpio during acclimatization to changes in water temperature

It has been demonstrated that oxidative phosphorylation in liver mitochondria of the carps which were kept within a week at 5 and 25 degrees C remains unaffected by the addition of 0.5 micron thyroxin to the incubation medium. Addition of thyroxin (0.5 micron) to mitochondrial suspension prepared from the liver of carps acclimated within a week at 20 and especially 25 degrees C, resulted in uncoupling of respiration and phosphorylation. Daily injections of thyroxin within a week (2 mu kg per 1 g of body weight) to carps at 20 and 25 degrees C resulted in the increase of the rate of non-phosphorylating oxidation and the decrease of oxidative phosphorylation in liver mitochondria. The increase of temperature of water from 5 to 30 degrees C decreases triiodthyronine content of the blood serum in the carp.     

Concanavalin A as a peripherally acting inhibitor of thyroxin-mediated metamorphosis in amphibians

Stimulated by reports that Concanavalin A (Con A), a plant protein and lectin from jack bean, has an inhibitory effect on thyroid activation induced by thyrotropin, we set out to test whether Con A inhibits thyroid action on hormone-sensitive target tissues in amphibians. We noted that premetamorphic tadpoles injected with 0.15 ml of thyroxin (T₄ 0.24 μM) responded by accelerating metamorphic change as indicated by precocious disappearance of the tail, and appreciable growth of the hind limbs and changes in mouth-part morphology. Tadpoles given an injection of thyroxin immediately followed by an injection of Con A (9.6 μM) showed no such metamorphic changes. In the second series of experiments tail fin discs obtained from premetamorphic tadpoles when placed in cultures supplemented with T₄ (0.24 μM) had completely shrunk within 96 hr. Tail fin discs that were raised in vitro in medium containing Con A as well as thyroxin failed to regress. In the third series of experiments tail discs were initially cultured in medium containing thyroxin and transferred within 48 hr to medium containing Con A. When Con A was added after this 48-hr exposure to thyroxin it was no longer effective in preventing tail fin disc resorption. We conclude tentatively (1) that Con A is a peripheral inhibitor of thyroxin and (2) this it somehow binds to the tissue or interacts with thyroxin rendering it ineffective before the hormone has a chance to act. The significance of finding a peripherally active inhibitor of thyroid hormone for studies of mechanism of action of this hormone on development and differentiation of hormone-sensitive target structures is obvious.     

Wide tissue distribution of axolotl class II molecules occurs independently of thyroxin

 Unlike most salamanders, the Mexican axolotl (Ambystoma mexicanum) fails to produce enough thyroxin to undergo anatomical metamorphosis, although a “cryptic metamorphosis” involving a change from fetal to adult hemoglobins has been described. To understand to what extent the development of the axolotl hemopoietic system is linked to anatomical metamorphosis, we examined the appearance and thyroxin dependence of class II molecules on thymus, blood, and spleen cells, using both flow cytometry and biosynthetic labeling followed by immunoprecipitation. Class II molecules are present on B cells as early as 7 weeks after hatching, the first time analyzed. At this time, most thymocytes, all T cells, and all erythrocytes lack class II molecules, but first thymocytes at 17 weeks, then T cells at 22 weeks, and finally erythrocytes at 26–27 weeks virtually all bear class II molecules. Class II molecules and adult hemoglobin appear at roughly the same time in erythrocytes. These data are most easily explained by populations of class II-negative cells being replaced by populations of class II-positive cells, and they show that the hemopoietic system matures at a variety of times unrelated to the increase of thyroxin that drives anatomical metamorphosis. We found that administration of thyroxin during axolotl ontogeny does not accelerate or otherwise affect the acquisition of class II molecules, nor does administration of drugs that inhibit thyroxin (sodium perchlorate, thiourea, methimazole, and 1-methyl imidazole) retard or abolish this acquisition, suggesting that the programs for anatomical metamorphosis and some aspects of hemopoietic development are entirely separate. Received: 15 July 1997 / Revised: 28 October 1997     

On the mechanism of action of thyroxin, an amino acid analog of tyrosine

The concept of a hormone inevitably carries with it a set of specific connotations and prejudices which in the case of thyroxin may obscure rather than elucidate its mechanism of action. Viewed as a hormone, the very number and broad range of thyroxin effects defy resolution. Viewed as an amino acid analog, the behavior of this molecule becomes coherent, in that it resembles the behavior of amino acids in general and tyrosine in particular, with distinguishing characteristics appropriate to its own structure and its analog status.Thyroxin is only one of a number of amino acids appearing in the course of phylogeny, derived from modifications of aminoacyl residues in protein. Comparative physiologic studies suggest that the biogenesis of iodine-modified tyrosines long preceded their useful biological function. Eventually their presence in threatened aquatic forms may have provided survival advantage with regard to terrestrial adaptation.Both structure and function of the vertebrate thyroid gland reflect its foregut ancestry. Iodoproteins within the lumen of the thyroid follicle are subject to a process similar to gastric digestion of ordinary dietary proteins. Free iodoamino acids together with other amino acid constituents of the digest are then absorbed into the blood stream. As expected of an amino acid, thyroxin leaving the extracellular space enters into and influences the function of almost all cells and subcellular organelles, including the nucleus. Its analog properties are reflected in the fact that only small fractions of the available thyroxin pools participate at any one time in biological reactions.The diverse actions of thyroxin are compatible with the model set by the parent amino acid tyrosine, which is notably active at a number of branch points in its own metabolism. Thyroxin effects on growth and development conform with the known alterations in genetic response which occur in the presence of false protein amino acids and are also consistent with the rapid turnover and remodelling of protein systems which follow the incorporation of false amino acid residues into protein. Its effects on overall metabolic processes are consistent with the known propensity of tyrosine-like compounds to become converted to false transmitters in the adrenergic nervous system, which in the case of thyroxin, would result in the development of iodothyronine-derived adrenergic transmitter substances. Altered pigment metabolism in patients with thyroid disease and localization of iodoamino acids in pigment-bearing cells of many vertebrate species suggest that thyroxin may be an alternate substrate in the melanin biosynthetic pathway.Since thyroxin is required for normal body function, it may be classified as an indispensable amino acid along with the other biologically more ancient members of its class.