High levels of DNA polymerase ß mRNA corresponding with the high activity in Graves' thyroid tissue.

INTRODUCTION: High DNA polymerase ß activity has been observed in the thyroid tissue of patients with Graves' disease (Nagasaka et al. in Metabolism 37:1051-1054, 1988). This fact aroused our interest in whether the alteration of DNA polymerase ß activity depends on DNA polymerase ß (DNA poly ß) mRNA levels, which may be modulated by thyroid-stimulating hormone (TSH) or thyroid-stimulating substances, i.e. TSH receptor antibody (TRAb). RESULT: Addition of TSH or TRAb to primary cultures of Graves' disease thyroid cells for 4 h led to no increase in DNA poly ß mRNA levels. In contrast, thyroid hormone synthesizing enzyme, peroxidase, mRNA levels increased fivefold after coculture with TSH and TRAb, even though DNA poly ß activity and mRNA levels are already significantly higher in Graves' disease thyroid tissues, compared with normal thyroid tissue. DISCUSSION: These results indicate that DNA poly ß expression in Graves' disease thyroid cells may be maximally activated or plateau in response to thyroid-stimulating immunoglobulins, or that the activation of to poly ß expression may occur via pathways other than the G protein and cyclic AMP system.

Unconventional magnetic and thermodynamic properties of S=1/2 spin ladder with ferromagnetic legs.

We have succeeded in synthesizing single crystals of a new organic radical 3-Cl-4-F-V [3-(3-chloro-4-fluorophenyl)-1,5-diphenylverdazyl]. Through the ab initio molecular orbital calculation and the analysis of the magnetic properties, this compound was confirmed to be the first experimental realization of an S=1/2 spin-ladder system with ferromagnetic leg interactions. The field-temperature phase diagram indicated that the ground state is situated very close to the quantum critical point. Furthermore, we found an unexpected field-induced successive phase transition, which possibly originates from the interplay of low dimensionality and frustration.

Insulinoma may mask the existence of Type 1 diabetes.

BACKGROUND: Insulinoma is a tumour of insulin-producing cells of the pancreas and is known to be one of the causes of hypoglycaemia. Usually, appropriate removal of the insulinoma results in normalization of blood glucose levels. However, we found novel cases of insulinoma, in which hyperglycaemia developed soon after resection of the insulinoma. CASE REPORT: We encountered two patients with repeated hypoglycaemia caused by insulinoma. Following removal of the insulinoma, unanticipated hyperglycaemia was observed in both patients. Thereafter, their blood tests revealed low levels of serum C-peptide and high titres of anti-glutamic acid decarboxylase antibody, indicating concomitant Type 1 diabetes. Indeed, histological examination of the resected specimen revealed that one patient showed insulitis in non-tumorous pancreatic tissue in which ß-cells had already disappeared. Moreover, inflammatory cells infiltrated the insulinoma, as if it were insulitis of Type 1 diabetes, suggesting the existence of anti-islet autoimmunity. CONCLUSION: These are first cases of insulinoma associated with underlying Type 1 diabetes. Physicians should be aware of the possibility that insulinoma may mask Type 1 diabetes, and measurement of anti-islet autoantibodies may be helpful to find underlying Type 1 diabetes, such as in these cases. It is pathologically interesting that the immune cell infiltration into insulinoma may be suggestive of anti-islet autoimmunity.

Association of symptomless TMJ sounds with occlusal force and masticatory performance in older adults.

This study investigated associations between temporomandibular joint (TMJ) sounds and occlusal force or masticatory performance stratified by posterior occlusal supports in older Japanese adults. The subjects consisted of 1646 independently living people over 60 years. Masticatory performance, occlusal force, TMJ sounds and maximal mouth opening were examined. Posterior occlusal supports were classified by the Eichner Index. The prevalence of TMJ sounds was 27.7%, limitation of mouth opening (< 40 mm) was 7.9% and TMJ pain was only 1.5%. In the Eichner C group, TMJ sounds were significantly associated with lower occlusal force (OR = 3.20, P = 0.046) and lower masticatory performance (OR = 3.18, P = 0.041) after controlling for gender and age. These associations were not found in the Eichner A and B groups. Within the limitations of this study, the presence of TMJ sounds, even if they were symptomless, was associated with impairment of masticatory function in older adults with reduced occlusal support.

Comparison of time-action profiles of insulin glargine and NPH insulin in normal and diabetic dogs.

Intermediate insulin injections are commonly used for glycemic control in insulin dependent diabetic dogs acting as a replacement for natural insulin. Neutral Protamin Hagedorn (NPH) insulin and insulin glargine are two types of injectable insulin preparations commonly used in humans. In our study, we investigated the time-action profiles of both aforementioned insulin preparations in normal dogs in order to determine whether co-administration of NPH and glargine would be of benefit to insulin dependent diabetic dogs as it is for humans suffering from insulin dependent diabetes. Time-action profiles of NPH insulin and insulin glargine in normal dogs demonstrated a clear difference between both insulin preparations confirming that NPH insulin is an intermediate-acting preparation whereas insulin glargine is a long-lasting preparation. In addition, co-administration of NPH insulin and insulin glargine resulted in tight glycemic control as compared to NPH insulin alone in insulin dependent diabetic dogs. However, co-administration result in hypoglycemia at the dosages tested.

Cloning of cDNAs encoding argininosuccinate lyase and arginase from Rana catesbeiana liver and regulation of their mRNAs during spontaneous and thyroid hormone-induced metamorphosis.

Thyroid hormones are responsible for a change in the expression of many target genes during amphibian metamorphosis. In this study we cloned and sequenced cDNAs encoding two of the five urea cycle enzymes, argininosuccinate lyase and arginase, from adult liver of Rana catesbeiana. The cDNAs for the bullfrog argininosuccinate lyase and arginase encoded proteins of 467 and 321 amino acids with predicted molecular weights of 52,257 and 35,088, which were 72-75 and 64-68% identical to the mammalian enzymes, respectively. The accumulation of the mRNAs for argininosuccinate lyase and arginase in liver increased 26 and 4-times in a coordinated manner during spontaneous metamorphosis. Thyroid hormone-treatment induced about 5 and 10-times accumulation of mRNAs for argininosuccinate lyase and arginase in liver from premetamorphosing tadpoles within 4 days. These results suggest that the mRNA levels of the two enzymes in liver are upregulated by thyroid hormone during metamorphosis.

Effect of calmodulin inhibitors on thyroid hormone secretion.

The effect of calmodulin inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) and trifluoperazine, on TSH-induced thyroid hormone secretion from rat thyroid was examined in vivo and in vitro. The ip administration of 5 mg W-7 to the rat inhibited T4 and T3 secretion from rat thyroids at 2, 3, and 4 h after the ip injection of 2 IU TSH, and so did the ip injection of trifluoperazine at 3 and 4 h. However, the ip injection of N-(6-aminohexyl)-1-naphthalene sulfonamide as a control substance did not show any significant inhibition of T4 and T3 release. To identify the site of action of calmodulin, the effect of W-7 on (Bu)2cAMP-induced thyroid hormone secretion was tested in vitro. One hundred micromolar W-7 completely inhibited T4 release from the rat thyroid when it was enhanced by TSH or (Bu)2cAMP, suggesting that the inhibitory effect of W-7 is subsequent to cAMP formation. These results suggest that calmodulin may play a role in thyroid hormone secretion from the thyroid, acting beyond cAMP formation.

Enolase subunits in patients with neuroendocrine tumors.

Concentrations of the alpha-, beta-, and gamma-subunits of enolase in surgically resected tumors and serum obtained from patients with various endocrine tumors were measured by a sensitive enzyme immunoassay system. Tissue concentrations of the gamma-subunit were significantly elevated in patients with neuroendocrine tumors concurrent with high concentrations of the gamma-subunit in their serum, which fell to normal in 90% of those who underwent tumor resection. Immunohistochemical analysis revealed that considerable amounts of the gamma-subunit were contained specifically in these tumors. These results indicate that the gamma-subunit of enolase is a useful marker for diagnosing and monitoring patients with neuroendocrine tumors.

The gene for hepatocyte nuclear factor (HNF)-4alpha is activated by glucocorticoids and glucagon, and repressed by insulin in rat liver.

The gene for a transcription factor hepatocyte nuclear factor-4alpha (HNF-4alpha) is responsible for maturity-onset diabetes of the young, type 1. We examined hormonal regulation of the HNF-4alpha gene in the liver. Stimulation of primary-cultured rat hepatocytes with dexamethasone or glucagon led to induction of HNF-4alpha mRNA, being antagonized by insulin. In the liver of streptozotocin-induced diabetic rat, mRNA and protein levels for HNF-4alpha were elevated, and were normalized by insulin treatment. Therefore, HNF-4alpha in the liver is likely to be involved in the regulation of glucose metabolism in response to these hormones.

CCAAT/enhancer-binding protein beta is required for activation of genes for ornithine cycle enzymes by glucocorticoids and glucagon in primary-cultured hepatocytes.

Transcription of genes for enzymes of the ornithine cycle is activated by hormones such as glucocorticoids and glucagon. Promoters and enhancers of several genes for the enzymes interact with the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, and C/EBPbeta has been suggested to mediate glucocorticoid response of the gene for arginase, the last enzyme of the cycle. To determine the contribution of C/EBPbeta to hormonal regulation of genes for ornithine cycle enzymes, we examined mice with targeted disruption of the C/EBPbeta gene. Induction of genes for the enzymes by intraperitoneal injection of dexamethasone and glucagon was almost intact in the liver of C/EBPbeta-deficient mice. On the other hand, in primary-cultured hepatocytes derived from C/EBPbeta-deficient mice, induction of genes for the first enzyme carbamylphosphate synthetase, as well as for arginase, in response to dexamethasone and/or glucagon was severely impaired. Therefore, C/EBPbeta is required for hormonal induction of the genes for ornithine cycle enzymes in primary-cultured hepatocytes, while the deficiency of C/EBPbeta is compensated for in vivo.

NADPH-diaphorase in the developing rat: lower brainstem and cervical spinal cord, with special reference to the trigemino-solitary complex.

A previous study indicated that in adult rat, a distinctive neuronal group in the dorsomedial division of the subnucleus oralis of the spinal trigeminal nucleus (SpVo) and the rostrolateral part of the nucleus of the solitary tract (Sn) is stained for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), and suggested that the labeled structures are involved with sensorimotor reflexive functions. This study aimed to characterize the developmental expression of NADPH-d in SpVo and Sn, including other areas of the lower brainstem and cervical spinal cord, by means of the enzyme histochemical staining technique, from the prenatal through the postnatal period. On embryonic day 12 (E12), no neurons in the brain were stained for NADPH-d, whereas blood vessels were stained. Labeling in the vessels was consistently present throughout pre- and postnatal periods but decreased with development. On E15, labeled neurons appeared in the dorsomedial part of SpVo and the rostrolateral part of Sn, but not in the other nuclei. The labeled neurons in both nuclei increased in numbers drastically to E17. Postnatally, they tended to increase gradually in Sn, but to decrease slightly in SpVo. The cell size of labeled neurons reached a plateau at E17 in SpVo, but at postnatal day 4 (P4) in Sn. In other nuclei on E17, labeling appeared in the lateral paragigantocellular reticular, intermediate reticular, medullary reticular, pedunculopontine tegmental, and spinal vestibular nuclei, and laminae V, VI, and X of the cervical spinal cord. On E20 and P0, labeling appeared in the dorsal column, laterodorsal tegmental, raphe obscurus, parvocellular reticular, ventral gigantocellular reticular, and parahypoglossal nuclei, and laminae IX of the cervical spinal cord. On P4 labeling appeared in the parabrachial and median raphe nuclei, medial and caudolateral Sn, the magnocellular zone of subnucleus caudalis of the spinal trigeminal nucleus (SpVc), and laminae III/IV of the cervical spinal cord. On P10, labeling appeared in the paratrigeminal and dorsal raphe nuclei, the superficial zone of SpVc, and laminae I/II of the cervical spinal cord. No newly labeled neurons appeared in any nuclei after P14. The very early appearance of NADPH-d staining in SpVo and Sn, which precedes the appearance of NADPH-d elsewhere in the brainstem, suggests that the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) system has an important role for primitive orofacial sensorimotor reflexive functions. Furthermore, the pattern of developmental expression of NADPH-d in SpVo and Sn suggests that the NO/cGMP system is organized in a distinct manner in different nuclei.

Hormonal regulation of DNA polymerase-beta activity in the rat thyroid gland.

Using hypophysectomized rats, it has been shown that DNA polymerase-beta activity in the adrenal gland and testis is largely influenced by pituitary trophic hormones. Sucrose gradient centrifugation of thyroid extracts revealed three peaks of DNA polymerase-beta activity sedimenting at 3.3S, 7.3S and 12S. Of these, hypophysectomy induced a decrease in the 3.3S DNA polymerase-beta, whereas other molecular forms were affected only slightly. DNA polymerase-alpha and -gamma activities were unaffected by hypophysectomy. These changes in DNA polymerase-beta caused by hypophysectomy were reversed by daily i.p. injection of TSH. Furthermore, stimulation of the thyroid by excess TSH induced by the administration of 1-methyl-2-mercaptoimidazole resulted in an increase of all forms of thyroid DNA polymerase-beta. These results show that the level of DNA polymerase is relatively constant after hypophysectomy but that DNA polymerase-beta in the rat thyroid gland is also modulated by TSH mainly through the change of activity of the polymerase-beta which sediments at 3.3S.

Effect of hypophysectomy on cyclic 3',5'-nucleotidemetabolizing enzymes in the rat thyroid gland.

Adenylate cyclase and cyclic AMP phosphodiesterase activities in the thyroid gland were significantly reduced after hypophysectomy, followed by a gradual restoration of the enzyme activities to the levels seen in sham-operated rats whereas a slight and persistent reduction was evident in guanylate cyclase and cyclic GMP phosphodiesterase activities in the same tissue. These changes in enzyme activities were restored by TSH administration but not by ACTH. The recovery of activity produced by TSH administration was inhibited by cycloheximide. Hypophysectomy, or TSH and cycloheximide administration, did not produce any significant changes in the concentrations of calmodulin, suggesting that the alteration of these enzyme activities is not induced by a decrease in the concentration of calmodulin. Since forskolin activation of adenylate cyclase did not restore the reduced activity in the hypophysectomized rat thyroid to the level found in the sham-operated control rat thyroid, we conclude that there is a reduction of the amount of enzyme after hypophysectomy rather than a change of the active site on adenylate cyclase. The spontaneous restoration of adenylate cyclase and cyclic AMP phosphodiesterase activities after hypophysectomy implies that cyclic AMP-metabolizing enzymes are responsive to an autoregulatory mechanism in thyroid follicular cells.

Changes of calmodulin concentration and cyclic 3',5'-nucleotide phosphodiesterase activities in cardiac muscle of hyper- and hypothyroid rats.

To investigate the effect of thyroid hormone on cardiac muscle dysfunction in hyper- and hypothyroid states, we evaluated cyclic 3',5'-nucleotide metabolism by measuring cyclic 3',5'-nucleotide phosphodiesterase activity and calmodulin concentrations in the cardiac muscles of hyper- and hypothyroid rats. Cyclic AMP (cAMP) concentration was significantly high in the cardiac muscle of hyperthyroid rats and low in that from hypothyroid rats compared with control rats. Cyclic AMP and cyclic GMP phosphodiesterase activities were significantly decreased in the soluble fraction of cardiac muscle from hyperthyroid rats and markedly increased in this fraction in hypothyroid rats compared with normal animals. Calmodulin concentration was high in hyperthyroid and low in hypothyroid rats. It was concluded from these findings that low cAMP-phosphodiesterase activity might, in part, bring about the high concentration of cAMP. Calmodulin was significantly high in the cardiac muscle of hyperthyroid rats and the reverse was the case in hypothyroid rats compared with normal rats. The implication is that, in hyper- and hypothyroid states, these changes may play an important role in cardiac function via their effect on cyclic nucleotide and Ca2+ metabolism.

Changes in calmodulin concentration and cyclic 3',5'-nucleotide phosphodiesterase activity in skeletal muscle of hyper- and hypothyroid rats.

Hyper- and hypothyroid states occasionally induce skeletal muscle dysfunction i.e. periodic paralysis and thyroid myopathy. The etiology of these diseases remains unclear, but several findings suggest that the catecholamine-beta-receptor-cAMP system or other messenger systems are disturbed in these diseases. In this context, we evaluated changes in the cyclic 3',5'-nucleotide metabolic enzyme, cyclic 3',5'-nucleotide phosphodiesterase (PDE) and calmodulin concentrations in skeletal muscles of hyper- and hypothyroid rats. Activities of cyclic AMP-PDE were low in skeletal muscle both from hyper- and hypothyroid rats, and calmodulin concentration was high in hyperthyroid and low in hypothyroid rats, as compared with normal rats. DE-52 column chromatographic analysis showed that the cGMP hydrolytic activity in peak I and the cAMP hydrolytic activity in peak II were decreased in hypothyroid rats, whereas cAMP hydrolytic activity in peak III was unchanged. The cAMP hydrolytic activity in peak III was decreased in hyperthyroid rats, but the activities in peaks I and II were unchanged. These findings indicate that cAMP and calmodulin may have some role in skeletal muscle function in the hyperthyroid state, and that cAMP and calmodulin-dependent metabolism may be suppressed in the hypothyroid state.

Effect of eicosapentaenoic acid ethyl ester on hypothyroid function.

Thyroid hormones affect reactions in almost all pathways of lipid metabolism. It has been reported that plasma free fatty acid (FFA) concentration in hypothyroidism is generally within the normal range. In this study, however, we show that plasma FFA concentration in some hypothyroid patients is higher than the normal range. Symptoms of thyroid dysfunction in these individuals were less severe than those of patients with lower plasma FFA concentrations. From these findings we hypothesized that the change in FFA concentration must correlate with thyroid function. Using an animal model, we then examined the effect of highly purified eicosapentaenoic acid ethyl ester (EPA-E), a n-3 polyunsaturated fatty acid derived from fish oil, on thyroid function in 1-methyl-2-imidazolethiol (MMI)-induced hypothyroid rats. Oral administration of EPA-E inhibited reduction of thyroid hormone levels and the change of thyroid follicles in MMI-induced hypothyroid rats. These findings suggest that FFA may affect thyroid functions and EPA-E may prevent MMI-induced hypothyroidism.

Lipid peroxidation levels in rat cardiac muscle are affected by age and thyroid status.

Free radicals, hydroxyperoxides and H(2)O(2) are all known to damage cell components. This study was designed to compare the concentrations of hydroxyperoxide and free radical scavengers in the cardiac muscles of old rats in the hyper- or hypothyroid condition, to determine whether rates of peroxidation would differ with age, thyroid status, or both. Rats were rendered hyper- or hypothyroid by administration of l-thyroxine or methimazole for 4 weeks. Among the old rats, the lipid peroxide (LPO) concentrations, measured as thiobarbituric acid (TBA) reactants, were significantly greater in the hyperthyroid than in the euthyroid state and the LPO concentrations measured as TBA+Fe(3+) reactants, which may be precursors of LPO, were significantly greater in the hyperthyroid state, whereas in young rats, the LPO concentrations measured by TBA or TBA+Fe(3+) methods did not differ significantly in the hyperthyroid state. In the euthyroid state, the concentration of LPO measured as TBA+Fe(3+) reactants was significantly reduced with age. Xanthine oxidase (XOD) activity also was markedly increased with age, being more pronounced in the hyperthyroid than in the euthyroid state. The Mn and Cu/Zn superoxide dismutase activities were greater in the hyperthyroid than in the euthyroid state. Glutathione peroxidase activity decreased with age in the euthyroid and, particularly, in the hyperthyroid state. Catalase activity was not affected in the old rats. Concentrations of alpha-tocopherol in the old rats were high in the hyperthyroid state and low in the hypothyroid state, whereas the levels of beta- and gamma-tocopherols in these rats were unchanged in both conditions as compared with the euthyroid state findings. Data suggest that the site of free radical generation differs in older rats, with additional shifts in the location of intracellular lipid peroxidation being noted during hyperthyroidism. Thus, as rats age, the reduction of the free radical scavenger system and the increase in LPO and XOD activities might induce myocardial dysfunction.

Utility of measuring serum parathyroid hormone-related protein concentration in leukemic patients with hypercalcemia for assessing disease status.

OBJECTIVE: To evaluate serum parathyroid hormone-related protein (PTHrP) as a marker of hypercalcemia in leukemic patients. DESIGN AND METHODS: We measured the serum levels of PTHrP, lactate dehydrogenase (LDH) and calcium in three patients with hypercalcemia due to leukemia. RESULTS: Serum levels of PTHrP, LDH and calcium were elevated at admission in all patients, and these levels were reduced to within the normal range after chemotherapy. However, normalization of serum PTHrP concentration occurred more rapidly than normalization of serum LDH levels after chemotherapy. The increase in serum PTHrP concentration accompanied leukemic cell proliferation and preceded the increases in serum LDH and calcium. Serum LDH concentration increased, but serum PTHrP concentration did not after administration of granulocyte colony-stimulating factor. CONCLUSION: These findings suggest that serum PTHrP may be a more useful marker than serum LDH or calcium in assessing the status of leukemic patients with hypercalcemia.

Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization.

Regional distribution of neurons expressing neuronal nitric oxide synthase mRNA in the rat brain was examined by non-radioisotopic in situ hybridization, using digoxigenin-labeled complementary RNA probes. Clustering of intensely positive neurons was observed in discrete areas including the main and accessory olfactory bulbs, the islands of Calleja, the amygdala, the paraventricular nucleus of the thalamus, several hypothalamic nuclei, the lateral geniculate nucleus, the magnocellular nucleus of the posterior commissure, the superior and inferior colliculi, the laterodorsal and pedunculopontine tegmental nuclei, the nucleus of the trapezoid body, the nucleus of the solitary tract and the cerebellum. Strongly-stained isolated neurons were scattered mainly in the cerebral cortex, the basal ganglia and the brain stem, especially the medulla reticular formation. In the hippocampus, an almost uniform distribution of moderately stained neurons was observed in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the Ammon's horn, while more intensely stained isolated neurons were scattered over the entire hippocampal region. These observations can serve as a good basis for studies on function and gene regulation of neuronal nitric oxide synthase.

Induction of the C/EBP beta gene by dexamethasone and glucagon in primary-cultured rat hepatocytes.

The synthetic glucocorticoid, dexamethasone, and glucagon cooperatively elevated the level of mRNA for the transcription factor CCAAT/enhancer binding protein beta (C/EBP beta) in primary-cultured rat hepatocytes. In response to dexamethasone and/or glucagon, C/EBP beta mRNA started to increase as early as 30 min, reached a maximum within 2 h, and then gradually decreased. The administration of cycloheximide, a protein synthesis inhibitor, led rather to an increase in C/EBP beta mRNA, which suggested that a labile negative protein factor(s) is involved in regulation of the C/EBP beta mRNA level. Cycloheximide further augmented the increases in C/EBP beta mRNA by dexamethasone and/or glucagon. Therefore, C/EBP beta mRNA accumulation in response to these hormones is apparently independent of ongoing protein synthesis. The elevation of the C/EBP beta mRNA level by these hormones was accounted for by increases in the rate of transcription of the C/EBP beta gene, as deduced on nuclear run-on analysis. Gel mobility shift analysis revealed that the DNA-binding activity of C/EBP beta was increased cooperatively by dexamethasone and glucagon. These results suggest that the C/EBP beta gene is primarily induced by glucocorticoids and/or glucagon and that the accumulated C/EBP beta protein is then involved in secondary activation of target genes in response to these hormones in the liver.