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Background: Schools are socially dense environments, and school-based outbreaks often predate and fuel community-wide transmission of seasonal and pandemic influenza. While preemptive school closures can effectively reduce influenza transmission, they are disruptive and currently recommended only for pandemics. We assessed the feasibility of implementing other social distancing practices in K-12 schools as a first step in seeking an alternative to preemptive school closures., Methods: We conducted 36 focus groups with education and public health officials across the United States. We identified and characterized themes and compared feasibility of practices by primary versus secondary school and region of the United States., Results: Participants discussed 29 school practices (25 within-school practices implemented as part of the school day and 4 reduced-schedule practices that impact school hours). Participants reported that elementary schools commonly implement several within-school practices as part of routine operations such as homeroom stay, restriction of hall movement, and staggering of recess times. Because of routine implementation and limited use of individualized schedules within elementary schools, within-school practices were generally felt to be more feasible for elementary schools than secondary schools. Of reduced-schedule practices, shortening the school week and the school day was considered the most feasible; however, reduced-schedule practices were generally perceived to be less feasible than within-school practices for all grade levels., Conclusions: Our findings suggest that schools have many options to increase social distance other than closing. Future research should evaluate which of these seemingly feasible practices are effective in reducing influenza transmission in schools and surrounding communities.

The objective of this qualitative study was to explore the perspectives of school and preparedness officials on the feasibility of implementing a range of social distancing practices to reduce influenza transmission during a pandemic. In the summer of 2017, we conducted 36 focus groups by teleconference and webinar lasting 90 min with school and preparedness stakeholders from across the United States. We identified and characterized 11 themes arising from the focus group protocol's domains as well as unanticipated emergent themes. These themes were: the need for effective stakeholder communication, the importance of partnering for buy-in, the role of social distancing in heightening anxiety, ensuring student safety, how practices work in combination, challenges with enforcement, lack of funding for school nurses, differing views about schools' role in protecting public health, the need for education and community engagement to ensure consistent implementation, the need for collaborative decision-making, and tension between standardizing public health guidance and adapting to local contexts. Addressing several crosscutting considerations can increase the likelihood that social distancing practices will be feasible and acceptable to school stakeholders.

Background: During an evolving influenza pandemic, community mitigation strategies, such as social distancing, can slow down virus transmission in schools and surrounding communities. To date, research on school practices to promote social distancing in primary and secondary schools has focused on prolonged school closure, with little attention paid to the identification and feasibility of other more sustainable interventions. To develop a list and typology of school practices that have been proposed and/or implemented in an influenza pandemic and to uncover any barriers identified, lessons learned from their use, and documented impacts., Methods: We conducted a review of the peer-reviewed and grey literature on social distancing interventions in schools other than school closure. We also collected state government guidance documents directed to local education agencies or schools to assess state policies regarding social distancing. We collected standardized information from each document using an abstraction form and generated descriptive statistics on common plan elements., Results: The document review revealed limited literature on school practices to promote social distancing, as well as limited incorporation of school practices to promote social distancing into state government guidance documents. Among the 38 states that had guidance documents that met inclusion criteria, fewer than half (42%) mentioned a single school practice to promote social distancing, and none provided any substantive detail about the policies or practices needed to enact them. The most frequently identified school practices were cancelling or postponing after-school activities, canceling classes or activities with a high rate of mixing/contact that occur within the school day, and reducing mixing during transport., Conclusion: Little information is available to schools to develop policies and procedures on social distancing. Additional research and guidance are needed to assess the feasibility and effectiveness of school practices to promote social distancing.

This study sought to examine whether: (1) the health composition of the social networks of children living in subsidized housing within market rate developments (among higher-income neighbors) differs from the social network composition of children living in public housing developments (among lower-income neighbors); and (2) children's social network composition is associated with children's own health. We found no significant differences in the health characteristics of the social networks of children living in these different types of public housing. However, social network composition was significantly associated with several aspects of children's own health, suggesting the potential importance of social networks for the health of vulnerable populations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Autoantibodies to 65 kDa glutamic acid decarboxylase (GAD65) are produced in many patients with autoimmune polyendocrine syndrome type II (APS-II) or stiff-man syndrome (SMS) and are heterogeneous in their epitope specificities, recognizing both conformational and linear determinants. Major linear epitopes of GAD, which are recognized by autoantibodies in a minority of these patients, occur in the N-terminal and C-terminal regions. We have investigated antibody recognition of the N- and C-termini of GAD65 in relation to their structural features as an approach to understanding what modifications to the native GAD structure may occur that facilitate the generation of antibodies specific to linear epitopes in these regions during the autoimmune pathogenesis. A monoclonal antibody specific to the N-terminus of GAD65 bound both native and denatured GAD in ELISA, whereas monoclonal and polyclonal antibodies specific to the C-terminus of GAD bound only denatured GAD. These antibodies were epitope mapped using random peptide phage-display libraries and the epitopes related to a previously proposed structural model of GAD65. This has led us to propose that the alpha-helical secondary structure of the C-terminus of GAD65 must be denatured to generate linear epitopes. In contrast, the N-terminus is both surface exposed and linear in the native structure, but may be masked by membrane interactions, which must be broken to facilitate recognition by B cells.

Previous studies have shown that, in patients with primary alterations in thyroid hormone secretion, the level of the natural logarithm of serum TSH (lnTSH) is negatively related to the level of free T4. Because such patients can generally be assumed to exhibit normal tissue responsivity to thyroid hormone, we were interested in determining whether the lnTSH/free T4 index (FTI) relationship in patients with established thyroid hormone resistance (THR) exhibit a lower slope than patients with normal tissue sensitivity to thyroid hormone. We have therefore analyzed the relationship between the lnTSH and the FTI in members of three families with documented THR. In these patients, a given dose of T4 was maintained for a 1- to 2-month period, to achieve hormonal equilibration. Two of the families, though not related, exhibited the same mutation, E460K. The third was identified as A317T. As anticipated, the slope of the lnTSH/FTI ratio was significantly lower in the patients with THR than in T4-treated patients who were presumed to have normal sensitivity to thyroid hormone. The slope of the lnTSH/FTI relationship seemed to be characteristic of the specific mutation involved in the three genotypes (wild-type and two mutations) examined. Further, the in vivo slope of the lnTSH/FTI relationship seemed to be linearly related to the T3 association constant of the in vitro translated receptor. These findings support the potential usefulness of measuring the slope of lnTSH, as a function of the FTI, in quantitating pituitary THR.

The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmunity affecting its major sites of expression, GABA-ergic neurons and pancreatic beta-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specific amino acid residues which form autoreactive B-cell epitopes in this molecule. Detailed mapping of 13 conformational epitopes, recognized by human monoclonal antibodies derived from patients, together with two and three-dimensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5'-phosphate-binding middle domain (residues 201-460) and to dialkylglycine decarboxylase in the C-terminal domain (residues 461-585). Six distinct conformational and one linear epitopes cluster on the hydrophilic face of three amphipathic alpha-helices in exons 14-16 in the C-terminal domain. Two of those epitopes also require amino acids in exon 4 in the N-terminal domain. Two distinct epitopes reside entirely in the N-terminal domain. In the middle domain, four distinct conformational epitopes cluster on a charged patch formed by amino acids from three alpha-helices away from the active site, and a fifth epitope resides at the back of the pyridoxal 5'-phosphate binding site and involves amino acid residues in exons 6 and 11-12. The epitopes localize to multiple hydrophilic patches, several of which also harbor DR*0401-restricted T-cell epitopes, and cover most of the surface of the protein. The results reveal a remarkable spectrum of human autoreactivity to GAD65, targeting almost the entire surface, and suggest that native folded GAD65 is the immunogen for autoreactive B-cells., (Copyright 1998 Academic Press.)

The cerebellar Purkinje cell-specific PCP-2 gene is transcriptionally activated by thyroid hormone during the 2nd and 3rd weeks of postnatal life in the rat. In contrast, thyroid hormone has no detectable effects on PCP-2 expression in the fetal rat. We now present data that suggest that the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor (COUP-TF) represses triiodothyronine (T3)-dependent transcriptional activation of PCP-2 in the immature Purkinje cell. Gel shift assays show that the PCP-2 A1TRE and adjoining sequences (-295/-199 region) bind to rat and mouse brain nucleoproteins in a developmentally regulated fashion and that one of these nucleoproteins could be the orphan nucleoprotein COUP-TF. In support of this hypothesis, in vitro translated COUP-TF binds to the -295/-199 region and COUP-TF represses T3-dependent activation of the PCP-2 promoter in transient transfection analyses. Finally, immunohistochemical studies reveal that COUP-TF is specifically expressed in the immature fetal and early neonatal Purkinje cell and that this expression diminishes coincident with thyroid hormone induction of PCP-2 expression. Our findings are consistent with the hypothesis that the presence or absence of inhibitory proteins bound to the thyroid hormone response element of T3-responsive genes governs the responsivity of these genes to thyroid hormone during brain development.

In rat pups, thyroid hormone dependent brain development coincides with the appearance of the thyroid hormone receptor (TR)beta1 isoform. This finding led to the suggestion that TRbeta1 plays an essential role in brain development. The recent availability of a mouse TRbeta knockout strain allowed us to test this possibility by determining whether TRbeta is essential for the normal developmental pattern of expression of two thyroid hormone regulated brain genes, myelin basic protein (MBP), and Purkinje cell protein 2 (Pcp-2). Northern analysis of total mRNA from the brains of wild-type mice established that, as in the rat pup, the initial rate of rise of the MBP and Pcp-2 mRNA is slowed in the hypothyroid state. Supporting the effectiveness of TRbeta gene deletion was the finding that the thiiodothyronine (T3) nuclear binding capacity in the livers and brains of knockout animals was consistent with the fractional contribution of TRbeta1 to total binding capacity in the wild-type tissues. Further, no TRbeta1 could be detected by isoform-specific immunoprecipitation of nuclear receptor extracts. However, deletion of the functional TRbeta in the TRbeta knockout mice did not affect the normal ontogeny of expression of the Pcp-2 and MBP genes in the postnatal pup. We conclude that TRbeta is not essential for the normal developmental expression of these T3 dependent brain genes.

Studies were undertaken to test whether alterations in fetal brain thyroid hormone levels during the final week of gestation can prematurely induce gene expression in brain or affect cerebellar morphogenesis. Pregnant dams were treated either by administration of 0.025% methimazole (MMI) in the drinking water from day 14 post conception (PC14) or administration of 2.5 mg T4/100 g BW on PC15. On PC21, treatment with MMI resulted in a 53% fall in fetal brain T3 levels and excess T4 resulted in a 2- to 3-fold increase to concentrations observed in adult brains. Neither excess nor reduced levels of T3 caused alterations in the expression of the myelin basic protein, Pcp-2 or calmodulin kinase IV genes. Cerebella of control brains showed early evidence of foliation and the presence of a several cell thick Purkinje cell layer and an external granule layer. No treatment induced effects were evident. Thus, at the late fetal stage in the rat, the developing brain appears to be unresponsive to thyroid hormone despite the presence of thyroid hormone receptors. We infer the presence of as yet unidentified factors that suppress precocious response to thyroid hormone or the absence of cofactors essential for such a response.

Previous studies in our laboratory show that triiodothyronine upregulates expression of the cerebellar Purkinje cell-specific gene Pcp-2 during the first 2 weeks of rat neonatal life. A specific thyroid hormone response element, the A1 TRE, mediates this regulation. The finding that the contiguous 68 bases (-267/ -199) of the Pcp-2 promoter 3' to the A1 TRE repressed T3 response in transactivation studies suggested that this sequence could play a role in preventing premature T3-dependent activation of Pcp-2 in the fetus. We now show that deletion of this region resulted in enhanced T3-dependent activation of the native Pcp-2 promoter. The sequence is not a generalized silencer since it does not alter basal activity of mouse mammary tumor virus (MMTV) or thymidine kinase (TK) promoters. Deletion and linker scanning studies indicate that the 5' 30 bases of the -267/ -199 region mediate most of the response silencing activity. The -267/ -199 region also attenuates T3-induced transactivation mediated by other TREs. Gel shift analysis reveals that nuclear proteins from fetal but not adult brains complex with the -267/ -199 region, supporting the hypothesis that this region binds proteins that suppress Pcp-2 expression early in brain development.

We compared the regulation of myelin basic protein (MBP) gene expression by T3 in differentiating oligodendrocytes in culture with that previously observed by us in the neonatal rat brain. As in intact brain, expression of the T3R alpha gene preceded that of the T3R beta gene. Although the absence of T3 retarded the rate of accumulation of MBP messenger RNA, the level ultimately attained was similar to that reached in the presence of T3. This relationship mirrored the pattern observed in the neonatal brain. Transient transfection experiments showed that T3 regulates MBP expression at the transcriptional level, but only for a limited period during differentiation. These observations imply that the early rise of MBP messenger RNA is T3 dependent, whereas the terminal levels are maintained independently of T3. Both the T3-dependent and, surprisingly, the T3-independent expression of MBP require the presence of an intact T3 response element. T3 receptor may regulate MBP expression in a ligand-independent manner, or a nuclear factor other than T3 receptor may bind to the T3 response element of MBP to regulate terminal gene expression. These findings support the use of differentiating oligodendrocytes as a model of T3-induced brain development.

Although the role of the three functional thyroid hormone receptor isoforms (TR beta 1, TR beta 2, and TR alpha 1) remains unclear, studies by Hodin and Lazar et al. have suggested that restriction of TR beta 2 messenger RNA (mRNA) to rat pituitary could reflect a specific regulatory role in the pituitary. Supporting their hypothesis was a significant fall in pituitary TR beta 2 mRNA after T3 administration. These observations prompted us to assess the effect of thyroidal state on the level of TR beta 2 protein, as inferred by immunoprecipitation of TR beta 2 nuclear binding activity. In contrast to the behavior of the mRNA, we noted surprising stability in the levels of total nuclear TR binding capacity and TR isoform distribution in the transition from hypo- to hyperthyroid states. Calculations based on these and previous data from this laboratory (7) show that the average cellular content of TR beta 2 mRNA in pituitary is 0.6 molecules, whereas the content of TR beta 2 mRNA molecules in extrapituitary tissues is less than 0.007 molecule/cell. A high TR beta 2 protein/mRNA ratio in extrapituitary tissues thus could reflect a rapid turnover of TR beta 2 mRNA compared to TR beta 2 protein. This would explain the widespread distribution of TR beta 2 protein and the scarcity of mRNA in extrapituitary tissues.

Two recent studies have claimed that thyroid hormone administration accelerates malic enzyme gene expression in the neonatal brain in contrast to the well-documented lack of effect of triiodothyronine on malic enzyme gene expression in the adult brain. Since these observations conflict with earlier observations in our laboratory, we reinvestigated the effect of thyroid hormone status on the ontogeny of malic enzyme gene expression in the neonatal rat. Neither hypothyroidism nor hyperthyroidism influenced the ontogenesis of malic enzyme activity in neonatal brain whereas the patterns of gene expression and enzyme activity in liver were markedly affected. Our results suggest that tissue-specific factors in brain block thyroid hormone-induced gene expression by thyroid hormone.

Immunohistochemical studies previously reported from this laboratory showed that astrocytes in adult rat brain appear devoid of all thyroid hormone receptor (TR) isoforms. These findings, however, contrast with reports of measurable nuclear T3 binding in astrocytes in cell culture. To address this discrepancy, TR protein and messenger RNA (mRNA) content of type 1 and type 2 astrocytes in culture were assayed. Type 1 cells represent astrocytes present in brain in vivo. Type 2 astrocytes differentiate in culture from bipotential progenitor O-2A cells in the presence of serum. Under serum-free conditions, these progenitor cells differentiate into oligodendroglia. Total nuclear T3 binding capacity in both type 1 and type 2 astrocytes was approximately 3000 sites/cell. Northern blots showed the presence of mRNA for TRbeta1, TRalpha1, and TRalpha2 in type 2 cells but failed to reveal the presence of these mRNAs in type 1 astrocytes. Moreover, Northern blots also failed to reveal TRbeta2 mRNA in both type 1 and type 2 astrocytes. These findings, therefore, raised a question as to which receptor isoform was responsible for the nuclear binding capacity observed in type 1 astrocytes. As anticipated, immunocytochemical analysis demonstrated prominent nuclear signals for TRbeta1, TRalpha1, and TRalpha2 mRNA in type 2 astrocytes but failed to demonstrate TRbeta1, TRalpha1, or TRalpha2 in type 2 astrocytes. Application of RT-PCR, however, revealed the presence of low levels of TRbeta2 mRNA in type 1 astrocytes. When stained with a specific anti-TRbeta2 antiserum, both type 1 and type 2 astrocytes showed a strong fluorescent signal concentrated in the nucleus. These data indicate that under the special conditions of cell culture, expression of the TRbeta2 isoform in type 1 accounts for the measured nuclear T3 binding capacity.

The three currently recognized T3 binding thyroid hormone receptor (TR) isoforms, TR alpha 1, TR beta 1, and TR beta 2, arise from two distinct genes (alpha and beta), whereas two closely related non-T3-binding receptor variants, collectively designated TR alpha 2, arise from alternate splicing of the alpha gene transcript. Using a panel of specific antisera to these isoforms we have assessed the presence or absence of TRs in oligodendrocytes and astrocytes of rat cerebrum and cerebellum. Inferences as to colocalization of the receptor isoforms and cell-specific marker proteins were based on immunohistochemical analysis of the differential emissions of paired immunofluorescent probes. Antisera against myelin basic protein (MBP) identified oligodendroglia, and glial fibrillary acidic protein identified astrocytes. MBP-positive oligodendrocytes displayed positive fluorescent signals with each of the three TR isoform-specific antisera and the antiserum to the receptor variants. These findings are consistent with the concept that the MBP gene is a direct target for thyroid hormone action. TR immunoreactivity appeared to localize primarily to the nuclei of these cells. In contrast, we observed no immunofluorescent signals for any of the TR isoforms in glial fibrillary acidic protein-positive astrocytes. These findings raise the possibility that any effect of thyroid hormone on astrocyte function and structure is mediated indirectly as a result of interaction of thyroid hormone with receptors situated in nonastrocyte cells or as a result of nonnuclear mechanisms.

Messenger RNA for thyroid hormone receptor (TR) isoforms alpha 1 and beta 1 are widely distributed in rat tissues. Until recently, TR beta 2 mRNA was believed to be limited to the pituitary and the assumption was made that TR beta 2 protein was similarly restricted. We determined the distribution of TR beta 2 protein in selected adult and fetal rat tissues using three anti-TR beta 2 antisera directed to different amino acid sequences of the distinctive A/B domain of TR beta 2. The proportion of total nuclear binding capacity cleared by each antiserum was determined by saturation analysis. 10-20% of total binding capacity in adult brain, liver, kidney, and heart was immunoprecipitated by each antiserum. Use of specific antibodies to TR beta 1 and TR alpha 1 showed these isoforms accounted for the remainder of total T3 binding. Fetal liver and brain, however, contained only TR alpha 1. Immunohistochemical analysis of the adult tissues showed TR beta 2 present in nuclei. Reverse transcription polymerase chain reaction detected low levels of TR beta 2 mRNA in the adult tissues. We infer that TR beta 2 accounts for a significant fraction of TR in adult rat tissues despite the low levels of its mRNA.

Early in ontogeny thymocytes have a surface marker phenotype that resembles activated mature T cells but they lack expression of the T cell receptor (TCR) complex. We have made preparations of day 14/15 triple negative fetal thymocytes that exhibit the activated T lymphocyte markers CD25, intercellular adhesion molecule 1, Ly-6A/E, CD44, and heat stable antigen and are rapidly proliferating as evidenced by flow cytometric examination of BrdU incorporation. We found that binding activities of the gene regulators nuclear factor (NF)-kappa B, the NF-kappa B p50 homodimer complex, nuclear factor of activated T cells (NF-AT), oct-1, oct-2, activator protein 1 (AP-1), and serum response factor (SRF), are all present in these early thymocytes. Whereas the octamer factors and SRF persist during ontogeny, NF-kappa B, NF-AT, and AP-1 decrease and are undetectable in the adult thymus. Transfection of disaggregated thymocytes by electroporation or intact thymic lobes by gold-particle bombardment revealed that reporter constructs for NF-kappa B, NF-AT, AP-1, octamer factors and, to a small extent, the TCR-alpha enhancer were active in early thymocyte development. We rigorously eliminated the possibility that these transcriptional events were due to minor populations of TCR+ cells by showing that these reporter constructs were also active in recombinase activating gene (RAG)-/- thymocytes that are incapable of completing TCR gene rearrangement, and predominantly contain cells that have an activated phenotype. Thus, transcriptional events that are usually triggered by antigen stimulation in mature T cells take place early in thymic ontogeny in the absence of the TCR. Our analysis suggests that there are striking regulatory similarities but also important differences between the activation processes that take place in antigen-stimulated mature T cells and thymic progenitor cells.

At present, it appears abundantly clear that thyroid hormone exerts its major action at the nuclear level by regulating the level of mRNAs of specific genes. There are at least three TR isoforms that mediate hormonal effects at the tissue level. Characterization of the functional domains of these receptor isoforms is as yet incomplete, and the possibility that these receptors could have ligand-independent functions is a matter under current investigation. TRs are now recognized as members of a large superfamily of transactivating proteins involved in the regulation of gene expression. Recent studies have shown an unexpected degree of complexity in the nature of the association of the T3 receptors and the DNA of target genes. They have vividly pointed out the multiple interactions possible between the T3-receptor complex and other proteins participating in the process of gene regulation. These insights have provided a solid base for understanding differences in the gradation of thyroid hormone effect from one tissue to another. The microdissection of the molecular process that has occurred in the past 20 years has proceeded in part through the application of relatively artificial in vitro systems and assays. Whereas such approaches have undoubtedly reaped rich rewards in pointing out potential or possible mechanisms, they do not define the actual workings in the animal. Additional studies designed to examine at the molecular level the operation in vivo of physiologic networks influenced by thyroid hormones appear as an essential next step in understanding the biology of the hormone system. The application of transgenic models should materially assist such efforts.

The thyroid hormone receptors (TR) are nuclear proteins that include TR alpha and TR beta subtypes, each encoded by a separate gene. Both TR alpha and TR beta give rise to several isoforms of which three, TR alpha 1, TR beta 1, and TR beta 2 bind T3 and mediate the action of thyroid hormone. Although TR beta 2 was initially thought to be confined to the anterior pituitary, we recently observed small quantities of TR beta 2 messenger RNA (mRNA) by polymerase chain reaction analysis of discrete hypothalamic regions. To further examine the distribution of TR beta 2 in the brain, we performed immunocytochemical studies using a highly specific antiserum to TR beta 2, raised against a unique amino acid sequence (TR beta 2[131-145]) that is not present in the other known TRs. This antiserum immunoprecipitated TR beta 2 but not TR alpha 1 or TR beta 1. Immunoreactive TR beta 2 was widely distributed throughout the brain and primarily localized to the cell nucleus. Particularly intense immunostaining was present in the cerebral cortex, cerebellum, and hypothalamus, including regions where TR beta 2 mRNA had not previously been identified. In addition, immunoprecipitation of nuclear extracts with anti-TR beta 2 reduced total T3 binding capacity by approximately 20%, suggesting that immunoreactive TR beta 2 comprises a substantial portion of the total content of nuclear thyroid hormone binding proteins. These studies demonstrate that immunoreactive TR beta 2 is more widely represented in the central nervous system than previously suspected and may play an important role in mediating the action of T3 in many different regions of the brain. The finding of TR beta 2-like material could be due to a disproportionately high ratio of the TR beta 2 translation product and its mRNA in certain regions of the brain, or could indicate the existence of a novel TR beta 2-related protein that is important for T3 binding.

We have determined the contribution of the thyroid hormone receptor (TR) isoforms TR alpha 1 and TR beta 1 to the postnatal rise in rat hepatic nuclear T3-binding capacity. In agreement with previous studies, total hepatic nuclear binding capacity rose by about 8-fold from the 19th day of gestation to young adulthood at 2 months of age (0.10 +/- 0.03 to 0.86 +/- 0.17 pmol/mg DNA). The levels of specific TR species were measured by immunoprecipitation of T3-binding activity from hepatic extracts using a panel of antisera directed against specific regions of the TR isoforms. The difference between receptor immunoprecipitated with antibody against TR beta 1 and that precipitated with an antibody against an identical region in both TR beta 1 and TR alpha 1 was tentatively assumed to represent TR alpha 1. TR alpha 1 accounted for virtually all T3-binding activity in fetal liver on gestational day 19 (G19), increased by 2-fold shortly after birth, and remained constant thereafter. TR alpha 1 mRNA, on the other hand, was highest in concentration on G16 and fell by 50-75% in the adult. TR beta 1 was undetectable by immunoprecipitation of hepatic extracts from fetuses on G19. However, Northern analysis showed the presence of TR beta 1 mRNA in the fetal liver, which rose in concentration by 3- to 4-fold in late gestation and then remained constant. The contribution of TR beta 1 to total binding capacity rose to 33% and 40% on postnatal days 15 and 30, respectively, and to 80% in the adult liver. Immunohistochemical analyses of hepatic sections confirmed the presence of very low levels of TR beta 1 in fetal liver as early as G16 and G19, and a sharp rise in TR beta 1 protein concentration in the postnatal period. This indicated that the increase in TR beta 1-binding capacity results from increased TR beta 1 mass. The increase in TR beta 1-binding capacity, thus, is due to increased translational efficiency of the beta 1 mRNA or stabilization of the TR beta 1 protein. The prominence of TR alpha 1 in both rat fetal liver and fetal brain, as previously demonstrated in our laboratory, raises the possibility that this receptor isoform may carry out specialized functions in the fetus and that TR beta 1 subserves still other functions at later stages of development.(ABSTRACT TRUNCATED AT 400 WORDS)

Everyone seems to believe Americans are entitled to the best health care, regardless of cost. But how to curb soaring health expenses and provide care to the 37 million uninsured in our country is a perplexing problem, one that policy makers and providers are trying to solve with a wide variety of health reform proposals.

A panel of anti-thyroid hormone receptor (TR) antisera were generated to allow direct assay of the concentrations of the alpha 1 and beta 1 receptor isoforms in nuclear extracts from adult rat liver, kidney, brain and heart, and fetal brain. An antiserum, immunoglobulin G (IgG)-beta 1, raised against amino acid sequence 62-92 of the rat TR-beta 1 specifically precipitated only TR-beta 1 in vitro translation products. A second antiserum, IgG-alpha 1/beta, generated against a sequence that is identical in the ligand binding region of rat TR-alpha 1 and TR-beta isoforms immunoprecipitated both TR-alpha 1 and -beta 1 translation products. These IgG preparations were used to specifically immunoprecipitate thyroid hormone receptor binding activity from nuclear extracts. IgG-beta 1 cleared almost 80%, and the IgG-alpha 1/beta immunoprecipitated nearly all binding from hepatic nuclear extracts. This distribution of TR protein, 80% beta 1 and 20% alpha 1, is the same as previously reported for their respective mRNAs in liver. In heart, kidney, and brain IgG-beta 1 cleared 45, 43, and 28% of total binding, respectively, and IgG-alpha 1/beta cleared all T3 binding activity from these tissues. In agreement with an earlier study, marked variations in specific protein/mRNA ratios were noted among these tissues. Consistent with our earlier report of the presence of only very low levels of TR-beta 1 mRNA in fetal brain, IgG-beta 1 cleared just 5% of binding in this tissue. Studies using an antiserum (IgG-ch) generated against homologous segments of the hinge region in both TR-alpha 1 and -beta 1 yielded results which contrasted sharply with those of IgG-alpha 1/beta. Whereas IgG-ch could also immunoprecipitate virtually all binding from hepatic extracts it cleared only 40-50% of binding from the other tissues, including fetal brain in which TR-alpha 1 accounts for greater than 90% of binding protein. The data suggest the presence of posttranslational modification of the TR-alpha 1 protein in the hinge region, consistent with the presence in this segment of potential phosphorylation sites.

The level of hepatic nuclear T3-binding capacity falls in rats subjected to fasting. To define the mechanism underlying these changes, we have assayed in liver the concentration of the mRNA coding for the beta 1-receptor (beta 1-TR) isoform, the total nuclear T3-binding capacity, and the fraction of the total binding capacity that can be specifically immunoprecipitated with an anti-beta 1-TR immunoglobulin G preparation. Although no changes in beta 1-TR mRNA concentration were noted, we observed a 60% fall in total binding capacity. beta 1-TR mRNA levels were preserved despite a 50% fall in total poly(A)+ RNA. The fall in beta 1-TR protein, however, was consistent with a generalized decrease in total hepatic protein content. This study provides yet another instance in which measurement of receptor mRNA is not consonant with the behavior of the nuclear T3 receptor protein.

Rat c-erbA beta 1 mRNA rises in cerebrum during the first 10 days of life, coincident with an increase in tissue triiodothyronine (T3) levels and T3-dependent brain development. These data suggest that the beta 1 receptor may mediate the T3 effect. However, in cerebellum c-erbA beta 1 mRNA levels were very low. Since cerebellar development, including dendritic arborization of Purkinje cells, is a T3-sensitive process, we assessed the levels of the beta 1 receptor protein in cerebellum during development. Antisera to unique peptide regions of beta 1 were raised. Their specificity was demonstrated by specific immunoprecipitation of the in vitro translated product, 85% immunoprecipitation of the T3 binding activity in hepatic nuclear extracts, and Western blot analysis of tissue extracts. Immunohistochemical studies using anti-beta 1 antiserum stained liver nuclei but not testis nuclei, which contain no T3 binding activity or beta 1 mRNA. In cerebellar Purkinje cells, an immunofluorescent signal, localized to the nucleus and more intense than that seen in the liver, was observed. A positive but weaker signal was also present in the granule cells. Thus, we may infer that the cerebellum contains significant concentrations of beta 1 receptor protein despite the low beta 1 mRNA content. Both the intensity of staining in Purkinje cell nuclei and immunoprecipitable beta 1 receptor binding capacity rose in the neonatal period. Antiserum to the non-T3 binding alpha 2 variant protein was also prepared and a distinctive pattern of fluorescence was observed. Strong fluorescence was seen in the nuclei of granule cells, but none was seen in Purkinje cells. The alpha 2 fluorescence in testis was high, consistent with the high levels of alpha 2 mRNA in this tissue. The fluorescent signal appeared to originate primarily in dividing spermatogonia. Our findings support the concept that the beta 1 receptor plays a central role in T3-induced brain development and strongly suggest that the Purkinje cell is a direct target for T3.

Metabolic balance studies were carried out to determine the interrelationships of thyroid hormone-induced lipogenesis, lipolysis, and energy balance in the free-living rat. Intraperitoneal doses of 15 micrograms triiodothyronine (T3)/100 g body wt per d caused an increase in caloric intake from 26.5 +/- 1.7 (mean +/- SEM) kcal/100 g per d to 38.1 +/- 1.5 kcal/100 g per d. Food intake, however, rose only after 4-6 d of treatment and was maximal by the 8th day. In contrast, total body basal oxygen consumption rose by 24 h and reached a maximum by 4 d. Since total urinary nitrogen excretion and hepatic phosphoenolpyruvate carboxykinase mRNA did not rise, gluconeogenesis from protein sources did not supply the needed substrate for the early increase in calorigenesis. Total body fat stores fell approximately 50% by the 6th day of treatment and could account for the entire increase in caloric expenditure during the initial period of T3 treatment. Total body lipogenesis increased within 1 d and reached a plateau 4-5 d after the start of T3 treatment. 15-19% of the increased caloric intake was channeled through lipogenesis, assuming glucose to be the sole substrate for lipogenesis. The metabolic cost of the increased lipogenesis, however, accounted for only 3-4% of the T3-induced increase in calorigenesis. These results suggest that fatty acids derived from adipose tissue are the primary source of substrate for thyroid hormone-induced calorigenesis and that the early increase in lipogenesis serves simply to maintain fat stores. Since the mRNAs coding for lipogenic enzymes rise many hours before oxygen consumption and lipolysis, these results suggest that T3 acts at least in part by an early coordinate induction of the genes responsible for these processes.

We have quantitated in adult and developing rat tissues the molar concentrations of c-erbA alpha 1- and beta 1-mRNAs, which code for nuclear T3-binding proteins, and c-erbA alpha 2-mRNA, which is generated by alternate splicing of the alpha gene transcript and codes for a receptor variant that does not bind T3. Comparison of the concentrations of c-erbA alpha 1-mRNA, beta 1-mRNA, or their sum to the T3 nuclear binding capacity per mg of DNA in adult liver, kidney, heart, cerebrum, and cerebellum and during the ontogeny of liver and brain shows that the T3 binding capacity/c-erbA mRNA ratio is tissue-specific and related to developmental state. Administration of T3 resulted in a 40-50% fall in the alpha 1 signal of adult liver, kidney, and heart without changing either the beta 1 signal or T3 binding capacity. A 40-fold increase in rat brain beta 1-mRNA occurred in the transition between the 19-day gestational fetus and the 10-day-old neonate. This corresponds to the period during which the T3 content rises in brain and during which T3 is known to influence central nervous system development. Our findings indicate that important translational or post-translational factors influence nuclear binding capacity and raise the possibility that c-erbA beta 1 may play a primary role in mediating T3 effects in developing and adult animals.

We have compared the affinities for T3 and the T3 analog binding characteristics of the in vitro translational products of seven c-erbA cDNAs (chicken c-erbA alpha; human placental c-erbA beta; rat c-erbA beta-1; rat c-erbA alpha-1; rat c-erbA alpha-2; human testis c-erbA alpha-2; and human kidney c-erbA alpha-2). Four of these (chicken c-erbA alpha, human placental c-erbA beta, rat c-erbA beta-1, rat c-erbA alpha-1) bound T3 with high affinity as previously described. When compared under identical conditions of synthesis and [125I]T3 binding, there was no significant difference between the affinity of the chicken c-erb A alpha-1 and the human c-erbA beta but in a more limited series the affinity of rat c-erbA beta-1 for T3 was 4.6-fold higher than that of the rat c-erbA alpha-1. In vitro translational products of the beta-probes showed a characteristic 2.2-fold higher triiodothyroacetic acid/T3 ratio than did the products of the alpha-probes, regardless of the species of origin of the probe. As previously established, the rat c-erbA alpha-2 product did not bind T3. However, in contrast to two published reports, the human testis and kidney alpha-2 probe products also failed to bind T3. These findings indicate that highly conserved C-terminal 37-40 residues are important for high affinity T3 binding by proteins encoded by the c-erb A family of genes.

We have studied the hepatic messenger RNA (mRNA) activity profile in chronically azotemic rats and sought to determine whether the observed changes could be mediated either by reduced food intake or diminished thyroid function at the tissue level. mRNA activity profiles were produced by two-dimensional gel electrophoretic separation of radioactively labeled products of an in vitro reticulocyte lysate system which had been programmed by hepatic RNA. Of the approximately 240 translational products identified in this system, seven sequences were consistently altered in azotemia. In pair-fed animals six of these also decreased, but the alterations in three were depressed to a significantly lesser extent in the pair-fed group. Moreover, analysis of covariance suggested that food intake could account for the differences in only one sequence. The possibility that the mRNA activity profile in azotemia could represent the effects of diminished thyroid function was minimized by the finding that the reductions in plasma thyroxine (T4) and triiodothyronine (T3) levels observed were due largely to reduced plasma protein binding, with maintenance of the mean free T4 and free T3 concentrations within the normal range. The changes in only one mRNA sequence could be related to free T3 levels alone. Our findings, therefore, indicate that although diminished food intake and reduced thyroid function may contribute to some of the observed changes in the mRNA activity profiles, the bulk of alterations in azotemia appear to be mediated by other mechanisms. The striking overlap between the sequences affected by azotemia and pair-feeding raises the speculation that altered gene expression in azotemia may reflect an impaired hepatic response at the pretranslational level to metabolic signals associated with food intake.

Recent studies have described a DNase I hypersensitive site in the 5'-flanking region of the rat hepatic S14 gene that is closely associated with its expression. A 111-base pair subfragment (-389 to -279) of this region interacts specifically in a gel shift assay with a protein present in hepatic nuclear protein extracts. This protein, designated P1, was not present in extracts of other tissues, even those in which the gene is expressed and hormonally regulated. The binding activity of P1 is exceedingly low in extracts from hypothyroid rats and is markedly increased by administration of thyroid hormone. However, the slow accumulation of P1 after thyroid hormone administration indicates that increased levels of P1 are not necessary for the acute hormonal induction of S14 gene expression. The level of P1 binding activity increases in the evening, synchronous with circadian variation of hepatic mRNA S14. Since neither P1 binding activity nor circadian variation in mRNA-S14 levels are observed in the other tissues expressing the S14 gene, P1 may function to modulate the circadian rhythm observed in hepatic S14 gene expression. DNase I footprinting analysis revealed that P1 binds to a defined nucleotide sequence, 5'-AAAAGAGCTATTGATTGCCTGCA-3', located between -310 and -288 in the S14 gene.

We have examined the role of rapidly turning over proteins in the T3 regulation of multiple rat hepatic genes. T3 induction of the rapidly responsive mRNA-S14 was markedly inhibited by cycloheximide (1 mg/100 g BW) or emetine (3 mg/100 g) injected ip 30 min before T3 (mRNA-S14 concentration was only 35% of that in T3-treated controls 8.5 h after administration of either protein synthesis inhibitor, P less than 0.01). Cycloheximide exhibited a similar effect on each of five other more slowly responsive T3 regulated genes. When cycloheximide was given 10 h after T3, the expected T3-induced rise of mRNA-S7 activity was completely prevented, and for mRNA-S4 activity the anticipated rise was blunted to 40% of T3-treated control (P less than 0.05). Cycloheximide caused sharp declines in the activity of two other mRNAs, S6 and S8, which because of shorter lag times of response to T3, had already risen when the drug was given. Values for both these mRNAs returned to the baseline hypothyroid level within 6 h of injection of the drug and remained low for a further 8 h (P less than 0.05). The expected deinduction of mRNA-S10 by T3 was also markedly modified. T3 lowered this mRNA to 11% of the hypothyroid control after 8 h, whereas cycloheximide given 30 min before the hormone blunted this fall to only 72% of control (P less than 0.01). Thus there appeared to be a 70% reduction in the rate of T3 induced fall of mRNA-S10. We did not find that cycloheximide caused a generalized decrease in poly (A)+ RNA mass.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies from this laboratory have shown that nuclear displacement of triiodothyronine (T3) is more rapidly dissipated after iv injection of triiodothyroacetic acid (triac) than after equimolar doses of T3. This suggested that the discrepancy between the strong nuclear binding of triac and its relatively weak thyromimetic effect could be explained by a more rapid fractional rate of triac metabolism. To test this hypothesis, tracer studies with radioactively labeled T3 and triac were carried out in normal male Sprague-Dawley rats. Noncompartmental analysis showed that the average residence time of tracer triac in the exchangeable compartment was 5.5 h compared with 10.9 h for T3. The metabolic clearance rate of triac was 14.4 ml/h/100 g BW and of T3 17.6 ml/h/100 g. The average distribution space of triac was 78.2 ml/100 g and of T3 190.7 ml/100 g. The fraction of isotope excreted via the fecal route was 0.46 for triac and 0.41 for T3. Triac was approximately 16 times as firmly bound to plasma proteins as was T3. Isotopic studies suggested that a rapid exchange of tracer triac occurred between plasma, cytosol, and nuclei, similar to previously observed relationships for T3. Based on the daily dose of T3 and triac required to maintain a normal concentration of alpha-glycero-phosphate dehydrogenase in thyroidectomized animals, the T3:triac potency ratio was estimated to be 3.0. Our observations support the concept that a shorter duration of nuclear occupancy observed in previous experiments can be attributed to a more rapid fractional metabolism of triac and may explain the lesser hormonal effect of this compound. Since labeled triac could not be identified in nuclear extracts after the injection of isotopically labeled T3, it appears unlikely that the initiation of hormonal effects by T3 at the nuclear level is dependent on its prior conversion to triac.

To assess the effect of starvation and to explore the potential interrelationship of starvation and thyroid status at the pretranslational level, we have analyzed by two-dimensional gel electrophoresis, the hepatic translational products of starved and fed euthyroid and hypothyroid rats. 5 d of starvation resulted in a statistically significant change in 27 of 240 products visualized, whereas hypothyroidism caused a change in 20, both in comparison with the fed euthyroid state. Of considerable interest was that 68% of all changing messenger (m)RNA sequences were common to the hypothyroid and starved groups and showed the same directional shift. Further, both starvation and hypothyroidism yielded comparable decreases in total hepatic cytoplasmic RNA content. Although it has been well established that the level of circulating triiodothyronine (T3) and the level of hepatic nuclear receptors fall in starvation, this reduction cannot account for the observed decrease of total hepatic RNA nor for all of the alterations in the concentrations of specific mRNA sequences. Thus, administration of T3 to starved animals in a dose designed to occupy all nuclear T3 receptors fails to prevent the fall in total RNA and the majority of starvation-induced changes in the level of mRNA sequences. Moreover, starvation of athyreotic animals results in a further decrease in total RNA and in a further change in the level of individual mRNA species. We conclude, therefore, that although the reduced levels of circulating T3 and the nuclear T3 receptors can contribute to the observed results of starvation, the starvation-induced changes are not exclusively mediated by this factor. The striking overlap in the genomic response between hypothyroid and starved animals raises the possibility that those biochemical mechanisms regulated at a pretranslational level by T3 are either not helpful or injurious to the starving animal. The reduction in circulating T3 and nuclear receptor sites together with T3-independent mechanisms initiated in the starved animal may constitute redundant processes designed to conserve energy and substrate in the nutritionally deprived organism.

A noncompartmental method is proposed for the calculation of the total distribution volume of exchangeable iodothyronine, the fractional irreversible removal rate from the exchangeable pool, and the metabolic clearance rate of labeled triiodothyronine (T3) and thyroxine (T4). The method depends on a graphic analysis and does not require the use of computers. The results of noncompartmental analysis of T3 and T4 distribution and metabolism are compared to single compartmental calculations in 12 subjects. Single compartmental analysis appeared to overestimate the distribution volume of T3 by a factor of 2.9, the metabolic clearance rate by a factor of 1.4, and underestimate the fractional removal rate by a factor of 0.48. Whereas single compartmental analysis did not introduce a significant error into the calculation of the metabolic clearance rate of T4, the distribution volume appeared to be overestimated by a factor of 1.4 and the fractional removal rate underestimated by a factor of 0.9.

Studies were undertaken in hypothyroid rats in an effort to define the kinetics of growth hormone (GH) accumulation in response to i.v. pulse injections of triiodothyronine (T(3)) and to calculate the relationship between nuclear occupancy by T(3) and the instantaneous rate of accumulation of pituitary GH. Results were contrasted to the findings in previous studies of the induction of hepatic mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and malic enzyme (ME) by T(3). The dose of T(3) required to achieve half-maximal accumulation of GH in 24 h was 0.6 mug/100 g body wt, a value 15-fold less than the half-maximal dose for alpha-GPD and ME induction at a comparable time after injection. Although significant increase in pituitary GH were evident as early as 3 h after injection of maximally effective doses of T(3), the rate of increase became linear only 12 h after injection. After achievement of peak values, the pituitary content of GH decayed with a similar terminal t((1/2)) of 3.9 days and 4.1 days in two groups of animals injected with a single dose of 1.0 and 50 mug T(3)/100 g body wt, respectively. In vivo isotopic displacement studies carried out at the equilibrium time point indicated that the pituitary nuclear binding capacity was 5.5 ng T(3)/g tissue and that the plasma concentration at which one-half of the nuclear sites are occupied is 1.0 ng/ml. Nuclear occupancy as a function of time was calculated from the estimated plasma T(3) concentration after injection of the dose and the half-occupancy plasma concentration. These data were then analyzed by application of the mathematical model previously developed to ascertain the relationship between nuclear occupancy and the rate of hepatic enzyme induction. Results indicated that the pituitary nuclear occupancy-response relationship was generally linear, in marked contrast to the highly amplified relationship between nuclear occupancy and the response of ME and alpha-GPD to T(3) in the liver. In supplementary experiments, euthyroid rats received daily injections of 200 mug of T(3) for 7 days to keep nuclear sites nearly saturated for the duration of the experiment. No significant increase in the pituitary GH content above euthyroid base-line levels was noted. This also contrasts with the marked increase above euthyroid levels in alpha-GPD and ME observed in previous studies. Our findings suggest the existence of major differences between the specific mechanisms which lead to the induction of pituitary GH and the hepatic enzymes by T(3).

Alpha-amanitin was shown to inhibit triiodothyronine (T3)-induced increases in mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytoplasmic malic enzyme activity in the livers of male Sprague-Dawley rats. A 3-fold increase in alpha-GPD observed 24 h after the iv injection of 3 microngT3/100 g BW was completely inhibited by administration of alpha-amanitin at 0 and 8 h. Similarly, alpha-amanitin blocked a two- to four-fold increase in malic enzyme 24 h following iv injection of 3 mg T3/100 g BW into euthyroid rats. After the initial inhibition of enzyme induction by alpha-amanitin was dissipated, however, a delayed but striking increase in enzyme activity occurred. In hypothyroid animals, alpha-GPD activity rose after the initial 24 h inhibition and reached levels at 72 h equal to those observed in hypothyroid rats treated with T3 only. In euthyroid animals treated with T3 and alpha-amanitin, a delayed increase in malic enzyme activity was observed at 72 h and attained values at 96 h similar to those in euthyroid animals injected with T3 only. The delayed rise in enzyme response is most easily explained by the formation of a long-lived intermediate during the exposure of the nuclear sites to T3.

We have analyzed the factors responsible for the circadian variation in rat hepatic mRNA-S14. Regulation of this sequence, which is found in lipogenic tissues and encodes a protein (S14) believed to be associated with fatty acid synthesis, is an excellent model of the interaction of thyroid hormone and dietary factors at the hepatocellular level. The mRNA exhibits a 3-fold diurnal variation (peak, approximately 2000 h; nadir, 0800 h) in ad libitum feeding rats on a 12-h light, 12-h dark photoschedule. We studied the effects of the photoschedule, periodic food intake, hypophysectomy, and induction by thyroid hormone (T3) on the mRNA-S14 rhythm. Adaptation to feeding restricted to either light or dark periods for 15 days did not greatly affect the diurnal rhythm. Photoreversal resulted in a 180 degrees phase shift, whereas the rhythm persisted in the presence of constant light. Oscillation continued around a higher baseline after a receptor-saturating dose of T3 in both normal and hypophysectomized rats. Our results indicate primary entrainment of the mRNA-S14 diurnal rhythm to the photoperiod, rather than to periodic food intake. Moreover, the circadian regulatory signal, which probably originates in the central nervous system, appears capable of antagonizing a maximal T3-inductive stimulus and does not originate in the pituitary gland. Persistence of the oscillation in constant light rules out circulating melatonin as the mediator. Synchronization of the rhythm by the photoschedule suggests that neuroendocrine factors are important determinants of rhythmic changes in hepatic gene expression.

We have investigated the relationship between the administration of triiodothyronine (T3) and a high carbohydrate (CHO) fat-free diet in the induction of lipogenic enzymes in two rat tissues, liver, and fat. Male thyroidectomized rats were treated with graded daily doses of T3 and either supplemented with a high CHO diet or left on a regular diet. Enzymes studied included malic enzyme (ME), fatty acid synthetase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. In the liver, all four lipogenic enzymes showed a synergistic response between T3 administration and high CHO feeding. In fat, ME also responded synergistically. The interaction was reflected in an increased sensitivity to T3. The dose of T3 required to achieve 50% maximal response was reduced three- to seven-fold by the high CHO diet. This phenomenon could not be attributed to a dietary-induced alteration either in T3 metabolism or in number or affinity of the T3-nuclear receptors. Moreover, studies of the relative rate of synthesis of ME suggested a simultaneous time of onset in the induction of ME, within 2 h after the application of either T3 or CHO. Thus, it is unlikely that either stimulus is secondary to the other. Since parallel experiments from this laboratory (Towle, Mariash, and Oppenheimer,1980. Changes in hepatic levels of messenger ribonucleic acid for malic enzyme during induction by thyroid hormone or diet. Biochemistry. 19: 579-585.) show that ME induction both by CHO and T3 is mediated by an increase in specific messenger RNA for ME, the interaction of T3 and the dietary factor occurs at a pretanslational level.

We have recently reported evidence for the presence of stereospecific energy-dependent transport processes for T3 in rat tissues. These processes were responsible for maintenance of concentration differences of free L- and D-T3 across the cellular plasma and nuclear membranes. In rat liver, the free L-T3 concentration in cytosol was almost 3 times higher than that in plasma, and nuclear free L-T3 was 58-fold that in cytosol. In the present studies, freshly isolated hepatocytes were used to study these processes in vitro. Kinetic experiments demonstrated that equilibrium of [125I]T3 between cells and medium was rapid and complete within 5 min. Neither the rate of cellular accumulation nor the equilibrium distribution of T3 between cells and medium was influenced by the addition of up to 2 X 10(-7) M T3. Equilibrium of T3 between the nuclear and extranuclear fractions of the hepatocytes was reached more slowly, only after 45-60 min of incubation. The nuclear free T3 concentration was calculated from mass action principles with knowledge of the association constant (Ka) of the nuclear T3-binding sites under in vitro conditions and the fractional occupancy of the sites. Cytosolic free T3 was determined from measurements of the fraction of cellular [125I]T3 associated with cytosol (pc), and the binding power of cytosol was determined by equilibrium dialysis (bc). The cytosol to plasma free T3 ratio in these cells was near unity, suggesting an absence of the concentration difference previously observed in liver in situ. The nuclear to cytosol free T3 ratio was 7.9, approximately 7 times less than that in vivo. The addition of 2 mM KCN caused a further 23% reduction in the nuclear to cytosol ratio. As previously reported for liver in situ, uptake of T3 by hepatocytes is stereospecific. Cellular uptake of D-T3 was greater than that for L-T3. However, nuclear transport favored L-T3. The nuclear to cell ratio for L-T3 was almost 4 times greater than that for D-T3 (mean +/- SEM, 0.020 +/- 0.0005 vs. 0.0085 +/- 0.0005; P less than 0.001). Our studies indicate the presence in the isolated hepatocyte of a nuclear transport process for T3 similar to that observed in vivo, but operating with a markedly reduced efficiency.

It has been estimated that approximately one third to one half of persons undergoing total laryngectomy do not obtain a satisfactory voice. These patients remain aphonic or use artificial larynges to facilitate their communication. A multidisciplinary group at Thomas Jefferson University has developed a miniaturized artificial larynx that fits on a dental prosthesis or dental plate which has over a 100-dB sound pressure level output at the source and is powered by two hearing aid batteries with a life expectancy of over 100 hours of continuous use. Clinical trials have shown that persons using other artificial devices quickly adapt to this new artificial larynx, and the first person who began using the device, an Italian, commented that he could now use both hands; and felt that this was a great help because of his ethnic background.

Further studies have been performed to define the kinetic characteristics of nuclear triiodothyronine (T(3)) binding sites in rat liver (J. Clin. Endocrinol. Metab. 1972. 35: 330). Sequential determination of labeled T(3) associated with nuclei and cytoplasm over a 4-h period allowed analysis of the relationship of T(3) in nuclear and cytoplasmic compartments. A rapid interchange of hormone between nuclei and cytoplasm was demonstrated, and in vitro incubation experiments with nuclei yielded no evidence favoring metabolic transformation of T(3) by the nuclei. In vivo displacement experiments were performed by subcellular fractionation of liver (1/2) h after injection of [(125)I]T(3) with increasing quantities of unlabeled T(3). The nuclear binding capacity for T(3) could be defined (0.52 ng/mg DNA). Analysis of these experiments also allowed an estimation of the association constant of nuclear sites for T(3) (4.7 x 10(11)M(-1)). The affinity of these sites for T(3) was estimated to be 20-40 fold greater than for thyroxine (T(4)). Chromatographic analysis of the nuclear radioactivity after injection of labeled T(4) indicated that the binding of T(4) by the nucleus could not be attributed to in vivo conversion of T(4) to T(3) but reflected intrinsic cross-reactivity of the two iodothyronines at the nuclear binding sites.

We have investigated the transport of L- and D-triiodothyronine (T3) from plasma to cellular cytoplasm and from cytoplasm to nucleus by estimating the concentration of free hormone in these compartments in rat liver, kidney, brain, and heart. We assessed the distribution of T3 in various tissues and its metabolism by standard isotopic techniques and measured plasma and cytosolic tissue T3 by radioimmunoassay. In addition, we determined the fraction of radiosensitive T3 associated with the cytosol in individual tissues and estimated the cytosolic volume per gram of tissue. Equilibrium dialysis allowed us to determine the binding power of cytosols and plasma, and in vitro saturation techniques provided values for the affinity (ka) for L- and D-T3 of isolated nuclei in aqueous solution at 37 degrees C. We calculated the free cytosolic hormone from the product of cytosolic T3 and the binding power of cytosol for T3, and the free intranuclear T3 from the ka and previously determined ratio of occupied-to-unoccupied binding sites under steady state conditions in euthyroid animals. Our results showed that the free cytosolic/free plasma concentrations for L-T3 and D-T3, respectively, were: liver 2.8, 21.6; kidney 1.17, 63.3; heart 1.31, 1.58; brain 0.86, 0.24. The free nuclear/free cytosolic ratios for L-T3 and D-T3, respectively, were: liver 58.2, 3.70; kidney 55.9, 1.54; heart 80.6, 24.9; and brain 251, 108.6. Our findings suggest that stereospecific transport occurs both from plasma to cytosol and from cytosol to nucleus. The high gradients from cytosol to nucleus imply that there is an energy-dependent process and appear to account for the differences in the nuclear association constant determined in vivo and in vitro.