The precision of PiCCO® measurements in hypothermic post-cardiac arrest patients.

The aim of the present study was to determine the precision of the PiCCO(®) system for post-cardiac arrest patients who underwent therapeutic hypothermia. The precision of the measurements for cardiac output, global end-diastolic volume, extravascular lung water and the pulmonary vascular permeability index was assessed using the least significant change; this was regarded as precise when less than 15%. A total of 462 measurement sets were prospectively performed on 88 patients following successful resuscitation after cardiac arrest. Using the mean value of three injections for a measurement, the least significant change for the cardiac output, global end-diastolic volume, extravascular lung water and pulmonary vascular permeability index measurements were found to be 7.8%, 8.5%, 7.8% and 12.1%, respectively. No significant differences between hypothermia (n=150) and non-hypothermia (n=312) were found. The PiCCO-derived variables were found to be precise for post-cardiac arrest patients even under conditions of varying body temperature.

Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4.

This report shows that cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) plays a key role in T cell-mediated dominant immunologic self-tolerance. In vivo blockade of CTLA-4 for a limited period in normal mice leads to spontaneous development of chronic organ-specific autoimmune diseases, which are immunopathologically similar to human counterparts. In normal naive mice, CTLA-4 is constitutively expressed on CD25(+)CD4(+) T cells, which constitute 5-10% of peripheral CD4(+) T cells. When the CD25(+)CD4(+) T cells are stimulated via the T cell receptor in vitro, they potently suppress antigen-specific and polyclonal activation and proliferation of other T cells, including CTLA-4-deficient T cells, and blockade of CTLA-4 abrogates the suppression. CD28-deficient CD25(+)CD4(+) T cells can also suppress normal T cells, indicating that CD28 is dispensable for activation of the regulatory T cells. Thus, the CD25(+)CD4(+) regulatory T cell population engaged in dominant self-tolerance may require CTLA-4 but not CD28 as a costimulatory molecule for its functional activation. Furthermore, interference with this role of CTLA-4 suffices to elicit autoimmune disease in otherwise normal animals, presumably through affecting CD25(+)CD4(+) T cell-mediated control of self-reactive T cells. This unique function of CTLA-4 could be exploited to potentiate T cell-mediated immunoregulation, and thereby to induce immunologic tolerance or to control autoimmunity.

TSH receptor - autoantibody interactions.

TSH receptor (TSHR) autoantibodies (TRAbs)activate the TSHR cyclic AMP cascade (stimulating TRAbs) or act as TSHR antagonist (blocking TRAbs), and both types inhibit TSH binding to the TSHR. Isolation of human monoclonal TSHR autoantibodies (stimulating M22 and blocking 5C9) has been a key milestone in studies of the TSHR and TSHR autoimmunity. Comparison of M22 and TSH interactions with the TSHR at the atomic level reveal that M22 heavy and light chains mimic TSH alpha and beta chains, respectively, in the way they bind to the receptor, but the evolutionary forces which have caused this close molecular mimicry are as yet completely unknown. More recently two more human monoclonal antibodies to the TSHR (K1-18 with stimulating and K1-70 with blocking activities) have been isolated from a single blood sample collected from a patient with hypothyroidism who previously presented with hyperthyroidism. K1-18 and K1-70 were derived from different lymphocytes as shown by V region genes analysis. This provides, for the first time, clear proof that a patient can produce both blocking and stimulating TRAbs at the same time. Although it has been postulated that stimulating and blocking TRAbs bind to different regions on the TSHR, our studies showed that antibodies of both types bind well to the TSHR containing only N-terminal amino acids 22-260. Whether TRAbs make contact with other parts of the TSHR in order to produce their biological effects (stimulation or blocking) remains to be elucidated.

Antithrombin concentrate use in sepsis-associated disseminated intravascular coagulation: re-evaluation of a 'pendulum effect' drug using a nationwide database.

There are four systematic reviews and meta-analyses of trials of antithrombin use for sepsis or critically ill patients published to date with conflicting results. The two studies that showed positive results used data only from septic patients who were also diagnosed with disseminated intravascular coagulation (DIC), whereas the two studies showing negative results included data from all septic and/or critically ill patients in their analyses. We believe that the underlying diseases of the study population must be as homogeneous as possible when evaluating treatment efficacy for sepsis-associated DIC. We published two large-scale antithrombin studies of sepsis-associated DIC using a Japanese nationwide database. The above-mentioned DIC studies reported significant associations between antithrombin use and better 28-day mortality in both populations (DIC-associated with severe pneumonia, n = 9075; and with severe abdominal sepsis, n = 2164). Now is the time to initiate multinational antithrombin trials exclusively among sepsis-associated DIC patients.

Nuclear receptor corepressors activate rather than suppress basal transcription of genes that are negatively regulated by thyroid hormone.

A group of transcriptional cofactors referred to as corepressors (CoRs) were recently shown to play a central role in basal silencing of genes that contain positive triiodothyronine (T3) response elements. In a reciprocal manner, negatively regulated genes are stimulated by unliganded thyroid hormone receptor (TR) and repressed upon the addition of T3. We used a TR beta mutant, called P214R, which fails to interact with CoRs, to examine whether CoRs also play a role in the control of genes that are negatively regulated in response to T3. In studies of three negatively regulated genes (the pituitary thyroid-stimulating hormone alpha-subunit [TSH alpha], TSH beta, and hypothalamic thyrotropin-releasing hormone [TRH] genes), stimulation of basal promoter activity by unliganded TR beta was impaired by introducing the P214R CoR mutation. Coexpression of each of the CoRs SMRT (silencing mediator for retinoid receptors and TRs) and NCoR (nuclear receptor CoR) enhanced basal stimulation of the negatively regulated promoters in a TR-dependent manner, but this effect was not seen with the P214R TR mutant. The mechanism of CoR effects on negatively regulated promoters was explored further with a series of GAL4-TR chimeric receptors and mutants that allowed TR effects to be assessed independently of receptor interactions with DNA. These experiments revealed that, like the negative regulation of genes by wild-type TR, basal activation occurred with GAL4-TR, but not with the GAL4-P214R mutant, and was reversed by the addition of T3. These results suggest that TR interactions with negatively regulated genes may be driven through protein-protein interactions. We conclude that a subset of negatively regulated genes are controlled by a novel mechanism that involves TR-mediated recruitment and basal activation by SMRT and NCoR. Addition of T3 reverses basal activation, perhaps by dissociation of CoRs.

A pediatric occurrence of crescentic glomerulonephritis associated with antineutrophil cytoplasmic antibodies and mesangial IgA deposits.

Antineutrophil cytoplasmic antibody-(ANCA) associated glomerulonephritis usually shows histopathologic features of pauciimmune crescentic glomerulonephritis and occurs late in life. We report a 14-year-old Japanese girl presenting with proteinuria, hematuria and mildly elevated serum creatinine. A renal biopsy specimen demonstrated crescentic glomerulonephritis, immunofluorescence showed mesangial IgA staining. Electron microscopic examination disclosed paramesangial deposits. Serum ANCA against myeloperoxidase (MPO) were detected at high titers. Myeloperoxidase-ANCA-related nephritis accompanied by IgA nephropathy is considered rare in childhood and teen years. Yet, if ANCA assays and detailed electron microscopic examination of renal specimens were performed routinely in patients with rapidly progressive glomerulonephritis, the diagnosis might be more frequent in young patients.

Migration and proliferation of primordial germ cells in the early chicken embryo.

In avian species, primordial germ cells (PGC) use the vascular system as a vehicle to transport them to the future gonadal region. The aim of this study was to elucidate the details of migration system and size of the PGC population in the early chicken embryo. We analyzed whole chicken embryos during stages X and 2 to 17 by immunohistochemical staining using specific antibody raised against chicken vasa homolog. At stage X, PGC were dense in the central zone of the area pellucida. Following the formation of the primitive streak, PGC moved anteriorly to the edge of the extraembryonic region. The size of the PGC population increased gradually during stages X (130.4 +/- 31.9) to 10 (439.3 +/- 93.6). At stage 10, PGC began to accumulate in the region anterior to the head, and then we could observe that PGC invaded into the vascular system in this region. At stage 11, the number of PGC decreased in the region anterior to the head (129.8 +/- 42.5 to 46.7 +/- 4.2) and increased in the blood vessels (194.0 +/- 41.6 to 285.0 +/- 7.5). No PGC could be recognized in the intermediate mesoderm, the future gonadal region, until stage 14, but they first appeared there at stage 15. The number of PGC recognized in the intermediate mesoderm increased from stage 15 to 17. Interestingly, the number of PGC between the left and right sides of this region was consistently and significantly different (P < 0.05) in females and males. The present study mainly clarified that chicken PGC continue to proliferate throughout early development, many PGC invaded into the vascular system from the region anterior to the head in stage 11, and PGC actively left the blood vessels and migrated to the intermediate mesoderm from stage 15.

A novel natural mutation in the thyroid hormone receptor defines a dual functional domain that exchanges nuclear receptor corepressors and coactivators.

In a patient with severe resistance to thyroid hormone (RTH), we found a novel mutation (leucine to serine in codon 454, L454S) of the thyroid hormone receptor beta. This mutation is in the ligand-dependent transactivation domain that has been shown to interact with transcriptional coactivators (CoAs). The mutant protein binds T3, but its ability to activate transcription of a positively regulated gene (TRE-tk-Luc), and to repress a negatively regulated gene (TSHalpha-Luc), is markedly impaired. As anticipated from its location, the L454S mutant interacts weakly with CoAs, such as SRC1 and glucocorticoid receptor interacting protein 1 (GRIP1) in gel mobility shift assays and in mammalian two-hybrid assays, even in the presence of the maximal dose of T3. In contrast, in the absence of T3, the L454S mutant interacts much more strongly with nuclear receptor corepressor (NCoR) than does the wild-type receptor, and the T3-dependent release of NCoR is markedly impaired. By comparison, the NCoR interaction and T3-dependent dissociation of an adjacent AF-2 domain mutant (E457A) are normal. These findings reveal that the Leu 454 is involved directly, or indirectly, in the release of corepressors (CoRs) as well as in the recruitment of CoAs. The strong interaction with NCoR at a physiological concentration of T3 results in constitutive activation of the TSH genes as well as constitutive silencing of positively regulated genes. When the dominant negative effect was examined among various mutants, it correlated surprisingly well with the potency of NCoR binding but not with the degree of impairment in CoA binding. These findings suggest that the defective release of NCoRs, along with retained dimerization and DNA binding, are critical features for the inhibitory action of mutant thyroid hormone receptors. These studies also suggest that helix 12 of the thyroid hormone receptor acts as a dual functional domain. After the binding of T3, its conformation changes, causing the disruption of CoR binding and the recruitment of CoAs.

A fusion protein of the estrogen receptor (ER) and nuclear receptor corepressor (NCoR) strongly inhibits estrogen-dependent responses in breast cancer cells.

Nuclear receptor corepressor (NCoR) mediates repression (silencing) of basal gene transcription by nuclear receptors for thyroid hormone and retinoic acid. The goal of this study was to create novel estrogen receptor (ER) mutants by fusing transferable repressor domains from the N-terminal region of NCoR to a functional ER fragment. Three chimeric NCoR-ER proteins were created and shown to lack transcriptional activity. These fusion proteins silenced basal transcription of the ERE2-tk-Luc reporter gene and inhibited the activity of co-transfected wild-type ER (wtER), indicating that they possess dominant negative activity. One of the fusion proteins (CDE-RD1), containing the ER DNA-binding and ligand-binding domains linked to the NCoR repressor domain (RD1), was selected for detailed examination. Its hormone affinity, intracellular localization, and level of expression in transfected cells were similar to wtER, and it bound to the estrogen response element (ERE) DNA in gel shift assays. Glutathione-S-transferase pull-down assays showed that CDE-RD1 retains the ability to bind to steroid receptor coactivator-1. Introduction of a DNA-binding domain mutation into the CDE-RD1 fusion protein eliminated silencing and dominant negative activity. Thus, the RD1 repressor domain prevents transcriptional activation despite the apparent ability of CDE-RD1 to bind DNA, ligand, and coactivators. Transcriptional silencing was incompletely reversed by trichostatin A, suggesting a histone deacetylase-independent mechanism for repression. CDE-RD1 inhibited ER-mediated transcription in T47D and MDA-MB-231 breast cancer cells and repressed the growth of T47D cells when delivered to the cells by a retroviral vector. These ER-NCoR fusion proteins provide a novel means for inhibiting ER-mediated cellular responses, and analogous strategies could be used to create dominant negative mutants of other transcription factors.

Recombinant human soluble thrombomodulin and mortality in severe pneumonia patients with sepsis-associated disseminated intravascular coagulation: an observational nationwide study.

BACKGROUND: The association between recombinant human soluble thrombomodulin (rhTM) use and mortality in patients with sepsis-associated disseminated intravascular coagulation (DIC) remains controversial. OBJECTIVES: To examine the hypothesis that rhTM could be effective in the treatment of patients with sepsis-associated DIC following severe pneumonia. METHODS: Propensity score and instrumental variable analyses using a nationwide administrative database, the Japanese Diagnosis Procedure Combination inpatient database, were used. The main outcome was 28-day in-hospital all-cause mortality. RESULTS: Eligible patients (n = 6342) from 936 hospitals were categorized into the rhTM group (n = 1280) or control group (n = 5062). Propensity score matching created a matched cohort of 1140 pairs with and without rhTM. No significant difference in 28-day mortality was documented between the two groups in the unmatched analysis (rhTM vs. control, 37.0%, 474/1280 vs. 36.9%, 1866/5062; odds ratio [OR], 1.00; 95%CI, 0.98-1.03), nor in the propensity-matched analysis (37.6%, 429/1140 vs. 37.0%, 886/1140; OR, 1.01; 95%CI, 0.93-1.10). The logistic regression analysis did not show a significant association between the use of rhTM and 28-day mortality in propensity-matched patients (OR, 1.00; 95%CI, 0.87-1.22). An analysis using the hospital rhTM-prescribing rate as an instrumental variable found that receipt of rhTM was not associated with reduction in mortality at 28 days (risk difference, 0.008; 95% CI, -0.08-0.98). CONCLUSIONS: This large retrospective nationwide study demonstrated that there might be little association between the use of rhTM and mortality in severe pneumonia patients with sepsis-associated DIC. A multinational randomized trial is required to confirm this.

Nuclear corepressors enhance the dominant negative activity of mutant receptors that cause resistance to thyroid hormone.

The syndrome of resistance to thyroid hormone (RTH) is caused by multiple distinct mutations in the ligand-binding domain of the thyroid hormone receptor-beta (TRbeta). Although the mutant receptors are transcriptionally inactive, they inhibit normal receptor function in a dominant negative manner to cause hormone resistance. Recently, a group of transcriptional cofactors, referred to as corepressors (CoRs), was shown to induce ligand-independent silencing of genes that contain positive T3 response elements. CoRs also play a role in the ligand-independent basal activation of genes that are negatively regulated in response to T3. We hypothesized that CoR might play a role in the dominant negative inhibition by TRbeta mutants that cause RTH. In gel mobility shift assays, RTH mutants retained interactions with CoRs even in the presence of T3, whereas the ligand dissociated CoR from wild-type TRbeta. Using Gal4-TR chimeric receptors and a VP16-CoR fusion protein in an interaction assay, a strong positive correlation was found between mutant receptor interactions with CoR and transcriptional silencing activity. A mutation (P214R) that impairs CoR interactions with TR was introduced into the RTH mutants to assess the role of CoR in dominant negative activity. In transient transfection assays, introduction of the P214R CoR mutation decreased RTH mutant silencing of positively regulated genes and basal activation of negatively regulated genes. The dominant negative activity of several different RTH mutants, studied by cotransfection with wild-type receptor, was greatly diminished by the CoR mutation, and this effect was seen with both positively and negatively regulated genes. These results suggest that CoR interactions play a critical role in the dominant negative effect of RTH mutants and support the idea that these proteins are involved in the regulation of genes that are positively as well as negatively regulated by T3.

Characterization of interaction between nuclear T3 receptors and antiserum against cellular-erb A peptide.

In our previous study, we raised a polyclonal antiserum against a synthetic 15 amino acid peptide encoding the putative hormone binding domain of c-erb A, which not only inhibited T3-binding to rat liver nuclear T3 receptor (NT3R) but also immunoprecipitated [125I]T3-NT3R complex. In the present study, interactions between this antiserum (4 BII-immunoglobulin G (IgG] and NT3R were further characterized. 4BII-IgG interefered T3 from binding to rat liver NT3R both in the nuclear extract and in isolated nuclei in a similar manner. Preincubation with 4BII-IgG decreased the association constant value with no effect on maximal binding capacity of NT3R. Lineweaver-Burk plot revealed competitive inhibition of T3-NT3R binding. Both the inhibition of T3 binding and immunoprecipitation of NT3R by the antiserum were similarly observed with rat liver, kidney, brain, and testis, and human kidney nuclear extract. Since the polypeptide sequence which was used for immunization is highly homologous between c-erb A alpha 1 and beta, it is likely that 4BII-IgG recognizes both c-erb A alpha 1 and beta in various tissues. On the contrary, 4BII-IgG did not interfere with T3 binding to rat liver cytosol T3-binding protein and thyroxine-binding globulin (TBG) prepared from human serum, nor immunoprecipitated these binding proteins labeled with [125I]T3. The binding protein for reverse T3 (NrT3BP) in the rat liver nuclear extract was neither immunoprecipitated nor influenced in its rT3-binding activity by 4BII-IgG. When the immune complex between the nuclear extract and 4BII-IgG was removed by antirabbit IgG goat serum, the remaining T3-binding activity was reduced by 87%, while no rT3-binding activity was immunodepleted, suggesting that NrT3BP is a protein different from NT3R. The data show that the antiserum against c-erb A peptide recognizes NT3R specifically with no distinction between difference in tissues and species. Carboxyl-terminal region of c-erb A/NT3R is critical for T3-binding.

Estimation of the protein content of thyroid hormone receptor alpha 1 and beta 1 in rat tissues by western blotting.

Recent studies of the expression of c-erbA/thyroid hormone receptor (TR) mRNAs have revealed a dissociation between T3-binding activity and the behavior of the mRNAs that code the functional TRs in some tissues. Compared with T3-binding activity, TR(alpha 1 + beta 1) mRNA is disproportionally high in the brain and low in the liver. Using anti-TR antiserum, 4BII, which recognizes TR alpha 1 and beta 1, but not the alpha 2-variant, we measured TR protein content in rat tissues by Western blotting. Two protein bands of 47 and 55 kilodaltons (kDa) were specifically identified as TR proteins. The positions of the in vitro transcription/translation products of c-erbA/TR alpha 1 and beta 1 cDNA on the gel were consistent with those of the 47- and 55-kDa bands, respectively. The 47- and 55-kDa proteins in nuclear proteins extracted with 0.4 M KCl from rat tissues were analyzed by Western blotting, and the intensity of TR protein bands in each tissue was measured by a densitometer. The relative TR protein concentration was highest in liver, followed by brain, kidney, and testis. We compared the TR protein level measured by Western blotting with the maximal T3-binding capacity (Cmax) in the same aliquot of samples from liver and brain. Both the TR protein level and the Cmax in the brain were about 40% of those in the liver, suggesting that the Cmax per receptor molecule is constant in these two tissues, and an abundant amount of functional TR proteins exists in the liver, corresponding to the high level of T3-binding activity.

Demonstration of nuclear 3,5,3'-triiodothyronine receptor proteins in gonadotrophs and corticotrophs in rat anterior pituitary by double immunohistochemical staining.

Previously we raised an antiserum (4BII) against nuclear T3 receptor (NT3R), which recognizes c-erb A/NT3R alpha 1 and beta, but not alpha 2. An immunohistochemical study using 4BII revealed that among the tissues examined, the cerebral cortex, anterior pituitary, and thyroid gland exhibited strong immunostaining in the nuclei. In the present study we examined the localization of NT3R proteins in individual cell types of rat anterior pituitary, using the technique of double immunostaining with 4BII and antibodies against pituitary hormones. The cryostat sections and paraffin sections of rat pituitary were incubated with 4BII at 4 C overnight. The NT3R proteins were visualized as brown in the nuclei by avidin-biotin peroxidase staining. The control sections incubated with an antiserum which had previously absorbed with c-erb A peptide or an inactive antiserum showed very poor nuclear staining under the same condition. After being washed with 0.1 M glycine-HCl buffer (pH 2.2) for 2 h to remove 4BII, the sections were incubated with antiserum against individual anterior pituitary hormones at 4 C overnight. Using the method of alkali phosphatase/naphthol/Fast blue staining, the peptide hormones in the cytosol were stained blue. Double staining with 4BII and anti-FSH beta or anti-LH beta anti-serum clearly demonstrated that gonadotrophs contained the NT3R proteins. Similarly, the NT3R proteins in corticotrophs were demonstrated by the immunostaining with 4BII and anti-ACTH antiserum. As expected, the NT3R proteins were present in somatotrophs, thyrotrophs, and lactotrophs. On the other hand, folliculo-stellate cells, which are nonhormone secreting cells and are identified by their S-100 protein immunoreactivity, were stained by 4BII only faintly. The present study demonstrated the existence of NT3R proteins not only in somatotrophs, thyrotrophs and lactotrophs but also gonadotrophs and corticotrophs, suggesting some action of thyroid hormone through the receptor in these cells.

The thyroid hormone receptor variant alpha2 is a weak antagonist because it is deficient in interactions with nuclear receptor corepressors.

The thyroid hormone receptor splice variant, alpha2, is unable to bind thyroid hormone (T3) and has been proposed to function as an endogenous inhibitor of T3 action. In this report, we examined further the DNA sequence requirements for alpha2 binding to thyroid hormone response elements (TREs) in an attempt to identify response elements that mediate potent inhibition by alpha2. Heterodimers of alpha2 and retinoid X receptor were found to bind to a subset of TREs (DR4, direct repeats spaced by 4 bp) in which selected flanking and spacer sequences enhanced interactions with the AGGTCA core binding sequence. Despite the optimization of the TRE-binding sites, alpha2 remained a weak dominant negative inhibitor of TRE-driven transcription. A promoter interference assay was also developed for testing inhibition by alpha2. In these studies, alpha2 blocked gene transcription, but it required cotransfected retinoid X receptor, and it was not as potent as unliganded thyroid hormone receptors. These results led to the hypothesis that alpha2 might be deficient in interactions with nuclear receptor corepressors. Consistent with this view, alpha2 did not silence basal transcription in its native form or when linked to Gal4. Alpha2 also failed to interact with corepressors (NCoR and SMRT) in both gel shift assays and mammalian two-hybrid assays. We conclude that alpha2 is a weak antagonist of thyroid hormone action because it binds weakly to a limited repertoire of response elements, and it does not interact with corepressors. Thus, alpha2 may be able to compete with thyroid hormone receptors for binding to a limited group of target sites, but it is not able to actively inhibit transcription.

Immunohistochemical localization of nuclear 3,5,3'-triiodothyronine receptor proteins in rat tissues studied with antiserum against C-ERB A/T3 receptor.

We have previously raised an anti-c-erb A peptide antibody (designated 4B II) which immunoprecipitated in vitro transcription/translation products of c-erb A alpha 1 and beta. 4B II could recognize nuclear T3 receptor (NT3R) without distinction between difference in species and tissues. Using 4B II, we studied immunohistochemical localization of NT3R proteins in various tissues of the rat. Cryostat sections (4-6 microns) of selected rat tissues were incubated with 4B II at 4C overnight, followed by fluorescein-isothyocianate-conjugated anti-rabbit immunoglobulin G for 60 min at 25 C. The cellular localization of fluorescence in all tissues examined was exclusively nuclear. Under the same conditions, control sections stained with antiserum which had previously absorbed with c-erb A peptide or inactive serum showed no specific staining. In the brain the large nuclei, supposed to be neuronal, were strongly stained in the cerebral cortex and the granular layer of the cerebellum. In the kidney, cells in the glomerulus, the distal, but not the proximal, tubules, and the collecting ducts exhibited nuclear staining. Nuclear fluorescence was observed homogeneously in the heart and liver, but the intensity was much weaker in the latter. Less intense fluorescence was seen in the testis and spleen, although specific immunostaining was clearly observed in the nuclei of spermatocytes, Leydig cells, and the heads of the sperms in the testis, and many lymphocytes in the spleen. Nuclei of follicular cells of the thyroid exhibited very strong fluorescence, suggesting existence of plenty of NT3R proteins. The anterior pituitary showed strong immunostaining in most nuclei, and clear nuclear fluorescence was also detected in the intermediate lobe of the pituitary. The present study showed that NT3R distributes selectively in certain types of cells in many tissues and that the content of NT3R proteins seems to correlate with the concentration of c-erb A mRNA alpha 1 and beta among many organs.

Pituitary resistance to thyroid hormone associated with a base mutation in the hormone-binding domain of the human 3,5,3'-triiodothyronine receptor-beta.

Point mutations in the human T3 receptor-beta (TR beta) gene causing single amino acid substitutions have been identified in several different kindreds with generalized resistance to thyroid hormone. Until now, no study has been reported on the TR gene in cases of pituitary resistance (PRTH). In the present study, we analyzed the TR beta gene in a 30-yr-old Japanese female with PRTH. She exhibited clinical features of hyperthyroidism, elevated serum thyroid hormone levels accompanied by inappropriately increased secretion of TSH, mildly elevated basal metabolic rate, and increased urinary excretion of hydroxyproline. No pituitary tumor was detected. DNA fragments of exons 3-8 of the genomic TR beta gene were generated by the polymerase chain reaction and analyzed by a single stranded conformation polymorphism method. Exon 7 of the patient's TR beta gene showed an abnormal band, suggesting the existence of mutation(s). By subcloning and sequencing the DNA, a point mutation was identified in one allele at nucleotide 1297 (C to T), which altered the 333rd amino acid, arginine, to tryptophan. Neither of her apparently normal parents had any mutations of the TR beta gene. In vitro translation products of the mutant TR beta gene showed remarkably decreased T3-binding activity (Ka, 2.1 x 10(8) M-1; normal TR beta Ka, 1.1 x 10(10) M-1). Since the molecular defect detected in a patient with PRTH is similar to that seen in subjects with generalized resistance to thyroid hormone, both types of the syndrome may represent a continuous spectrum of the same etiological defect with variable tissue resistance to thyroid hormone.

Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans.

Ghrelin, an endogenous ligand for the GH secretagogue receptor, was isolated from rat stomach and is involved in a novel system for regulating GH release. Although previous studies in rodents suggest that ghrelin is also involved in energy homeostasis and that ghrelin secretion is influenced by feeding, little is known about plasma ghrelin in humans. To address this issue, we studied plasma ghrelin-like immunoreactivity levels and elucidated the source of circulating ghrelin and the effects of feeding state on plasma ghrelin-like immunoreactivity levels in humans. The plasma ghrelin-like immunoreactivity concentration in normal humans measured by a specific RIA was 166.0 +/- 10.1 fmol/ml. Northern blot analysis of various human tissues identified ghrelin mRNA found most abundantly in the stomach and plasma ghrelin-like immunoreactivity levels in totally gastrectomized patients were reduced to 35% of those in normal controls. Plasma ghrelin-like immunoreactivity levels were increased by 31% after 12-h fasting and reduced by 22% immediately after habitual feeding. In patients with anorexia nervosa, plasma ghrelin-like immunoreactivity levels were markedly elevated compared with those in normal controls (401.2 +/- 58.4 vs. 192.8 +/- 19.4 fmol/ml) and were negatively correlated with body mass indexes. We conclude that the stomach is a major source of circulating ghrelin and that plasma ghrelin-like immunoreactivity levels reflect acute and chronic feeding states in humans.

Dimerization properties of mutant thyroid hormone beta-receptors with auxiliary proteins.

Hormonal responsiveness in peripheral tissues is variable in patients with resistance to thyroid hormone (RTH). One cause of this may be differential interaction of RTH mutants of thyroid hormone receptor beta (TR beta) with TR auxiliary proteins (TRAPs). We used gel shift mobility assays to examine the interaction of wild-type and mutant TR beta s with retinoid X receptors (RXRs) and endogenous TRAPs. Some mutants showed reduced homodimerization but retained heterodimerization with recombinant RXRs. Wild-type TR beta formed heterodimeric complexes with multiple TRAPs in nuclear extracts of rat tissues, but RTH mutants showed variably altered heterodimerization with each TRAP. With liver nuclear extract, all mutants with impaired homodimerization also showed impaired TR beta-TRAP heterodimerization. Thus heterodimerizations with RXRs and TRAPs are differently affected by RTH mutations. Our results suggest that multiple TRAPs are expressed in tissue-specific patterns. The variability of TR beta heterodimerization with TRAPs may account, in part, for the variable tissue responsiveness in RTH.

The phytochemical lindleyin, isolated from Rhei rhizoma, mediates hormonal effects through estrogen receptors.

Some plant compounds or herb mixtures are popular alternatives to conventional therapies and contain organic compounds that bind to some nuclear receptors, such as the estrogen receptor (ER), to exert various biological effects. We studied the effect of various herbal extracts on ERalpha and ERbeta isoforms. One herbal extract, Rhei rhizoma (rhubarb), acts as an agonist to both ERalpha and ERbeta. The phytochemical lindleyin, a major component of rhubarb, might contribute to this estrogenic activity through ERalpha and ERbeta. 4-Hydroxytamoxifen, an ER antagonist, completely reversed the estrogenic activity of lindleyin. Lindleyin binds to ERalpha in vitro, as demonstrated using a fluorescent polarization assay. The in vivo effect of rhubarb extract was studied using a vitellogenin assay system in the freshwater fish, Japanese medaka (Oryzias latipes). There were marked increases in serum vitellogenin levels in male medaka exposed to rhubarb extract. We conclude that lindleyin, a component of some herbal medicines, is a novel phytoestrogen and might trigger many of the biological responses evoked by the physiological estrogens.