Mutation of the gene encoding human TTF-2 associated with thyroid agenesis, cleft palate and choanal atresia.

Congenital hypothyroidism occurs in one of every three to four thousand newborns, owing to complete or partial failure of thyroid gland development. Although thyroid hypoplasia has recently been associated with mutations in the thyrotropin (TSH) receptor, the cause of thyroid agenesis is unknown. Proteins including thyroid transcription factors 1 (TTF-1; refs 4,5) and 2 (TTF-2; refs 6,7) and Pax8 (refs 8,9) are abundant in the developing mouse thyroid and are known to regulate genes expressed during its differentiation (for example, thyroid peroxidase and thyroglobulin genes). TTF-2 is a member of the forkhead/winged-helix domain transcription factor family, many of which are key regulators of embryogenesis. Here we report that the transcription factor FKHL15 (ref. 11) is the human homologue of mouse TTF-2 (encoded by the Titf2 gene) and that two siblings with thyroid agenesis, cleft palate and choanal atresia are homozygous for a missense mutation (Ala65Val) within its forkhead domain. The mutant protein exhibits impaired DNA binding and loss of transcriptional function. Our observations represent the first description of a genetic cause for thyroid agenesis.

Rapidly dissolving repaglinide powders produced by the ultra-rapid freezing process.

The objective of the study was to produce rapidly dissolving formulations of the poorly water-soluble drug repaglinide using an innovative new technology, ultra-rapid freezing (URF), and to investigate the influence of excipient type on repaglinide stability. Repaglinide compositions containing different types and levels of excipients and different drug potencies (50%-86%) were produced by the URF technology. Repaglinide/excipient solutions were frozen on a cryogenic substrate, collected, and lyophilized to form a dry powder. Surfactants, including sodium dodecyl sulfate, and alkalizing agents such as diethanolamine (DEA) and tromethamine (TRIS) were incorporated into the compositions. Forced degradation of repaglinide was conducted under stressed conditions (eg, elevated temperature, exposure to peroxide) to determine the stability of the drug in such environments. The solubility of repaglinide increased as a function of increasing pH; therefore, incorporation of an alkalizing agent into the URF formulations increased the drug's solubility. Drug instability resulted when the drug was exposed to pH values above 9.0. URF formulations containing alkalizing agents showed no degradation or spontaneous recrystallization in the formulation, indicating that increased stability was afforded by processing. URF processing created nanostructured drug/excipient particles with higher dissolution rates than were achieved for unprocessed drug. Alkalizing agents such as TRIS and DEA, present at levels of 25% to 33% wt/wt in the formulations, did not cause degradation of the drug when processed using URF. URF processing, therefore, yielded fast-dissolving formulations that were physically and chemically stable, resistant to alkali degradation or spontaneous recrystallization in the formulation.

Contrasting effects of activating mutations of GalphaS and the thyrotropin receptor on proliferation and differentiation of thyroid follicular cells.

The cyclic AMP pathway is a major regulator of thyrocyte function and proliferation and, predictably, its inappropriate activation is associated with a sub-set of human thyroid tumours. Activating mutations are, however, more common in the thyrotropin receptor (TSHR) than in its downstream transducer, Galphas. To investigate whether this reflects an inherent difference in their oncogenic potency, we compared the effects of retrovirally-transduced mutant (A623I) TSHR or (Q227L) Galphas (GSP), using the rat thyroid cell line FRTL5 and primary human thyrocytes. In FRTL5, expression of GSP or mutant (m) TSHR induced a 2 - 3-fold increase in basal levels of cAMP. This was associated with TSH-independent proliferation (assessed by both cell number and DNA synthesis) and function (as shown by increased expression of thyroglobulin (Tg) and the sodium/iodide symporter). In primary cultures, expression of mTSHR, but not GSP, consistently induced formation of colonies with epithelial morphology and thyroglobulin expression, capable of 10 - 15 population doublings (PD) compared to less than three in controls. Thus, while mTSHR and GSP exert similar effects in FRTL5, use of primary cultures reveals a major difference in their ability to induce sustained proliferation in normal human thyrocytes, and provides the first direct evidence that mTSHR is sufficient to initiate thyroid tumorigenesis.

Initial clinical trial of a selective retinoid X receptor ligand, LGD1069.

PURPOSE: The retinoid response is mediated by nuclear receptors, including retinoic acid receptors (RARs) and retinoid "X" receptors (RXRs). All-trans retinoic acid (RA) binds only RARs, while 9-cis RA is an agonist for both RARs and RXRs. Recently, LGD1069 was identified as a highly selective RXR agonist with low affinity for RARs. We undertook a dose-ranging study to examine the safety, clinical tolerance, and pharmacokinetics of LGD1069 in patients with advanced cancer. PATIENTS AND METHODS: Fifty-two patients received. LGD1069 administered orally once daily at doses that ranged from 5 to 500 mg/m2 for 1 to 41 weeks. Treatment proceeded from a starting dose of 5 mg/m2. Pharmacokinetic sampling was performed on selected patients on days 1, 15, and 29. RESULTS: Reversible, asymptomatic increases in liver biochemical tests were the most common dose-limiting adverse effect. Less prominent reactions included leukopenia, hypertriglyceridemia, and hypercalcemia. Characteristic retinoid toxicities, such as cheilitis, headache, and myalgias/arthralgias, were mild or absent. Two patients with cutaneous T-cell lymphoma experienced major antitumor responses. Pharmacokinetic studies obtained in 27 patients at eight dose levels showed that the day 1 area under the plasma concentration-times-time curves (AUCs) were proportional to dose. At all doses studied, the day 1 AUCs were similar to those on days 15 and 29, indicating a lack of induced metabolism. CONCLUSION: LGD1069 is a unique compound that exploits a newly identified pathway of retinoid receptor biology that may be relevant to tumor-cell proliferation and apoptosis. Further investigation of this drug is warranted. Based on the results of this study, a dose of 300 mg/m2 is recommended for single-agent trials.

Clinical study of 9-cis retinoic acid (LGD1057) in acute promyelocytic leukemia.

The use of all-trans retinoic acid (RA) for remission induction markedly increases survival of patients with acute promyelocytic leukemia (APL) compared to patients treated solely with cytotoxic chemotherapy. However, clinical resistance to this agent develops rapidly, which has been associated with a progressive decline in plasma drug concentrations. Previous studies suggested that 9-cis RA, a retinoid receptor 'pan agonist' did not induce its own catabolism to the same extent as all-trans RA. Therefore, we conducted a dose-ranging study of this compound in patients with both relapsed and newly diagnosed APL. We treated 18 patients with morphologically diagnosed APL (13 relapsed, five newly diagnosed). The daily dose of 9-cis RA ranged from 30 to 230 mg/m2/day given as a single oral dose. Four of 12 (33%) relapsed patients (three of whom were previously treated with all-trans RA) and four of five (80%) newly diagnosed patients achieved complete remission. The sole failure in the newly diagnosed group died early from an intracranial hemorrhage. One other patient with t(9;12) translocation had substantial hematologic improvement. The drug was generally well tolerated; headache and dry skin were the most common adverse reactions. Three patients were treated with corticosteroids for signs of incipient 'RA syndrome.' These preliminary data suggest that 9-cis RA is an effective agent for remission induction and deserves further investigation in patients with retinoid-sensitive APL.

Key innovation or adaptive change? A test of leaf traits using Triodiinae in Australia.

The evolution of novel traits ("key innovations") allows some lineages to move into new environments or adapt to changing climates, whereas other lineages may track suitable habitat or go extinct. We test whether, and how, trait shifts are linked to environmental change using Triodiinae, C4 grasses that form the dominant understory over about 30% of Australia. Using phylogenetic and relaxed molecular clock estimates, we assess the Australian biogeographic origins of Triodiinae and reconstruct the evolution of stomatal and vascular bundle positioning. Triodiinae diversified from the mid-Miocene, coincident with the aridification of Australia. Subsequent niche shifts have been mostly from the Eremaean biome to the savannah, coincident with the expansion of the latter. Biome shifts are correlated with changes in leaf anatomy and radiations within Triodiinae are largely regional. Symplectrodia and Monodia are nested within Triodia. Rather than enabling biome shifts, convergent changes in leaf anatomy have probably occurred after taxa moved into the savannah biome-they are likely to have been subsequent adaptions rather than key innovations. Our study highlights the importance of testing the timing and origin of traits assumed to be phenotypic innovations that enabled ecological shifts.

Thyrotropin receptor transcripts in human adipose tissue.

Thyroid associated ophthalmopathy (TAO) is generally considered to have an autoimmune pathogenesis but the target antigen has yet to be identified. It is most frequently associated with Graves' disease and there is some logic in assuming that the same antigen, the thyrotropin receptor (TSHR), is the common link. Previous studies, mostly PCR based, aimed at investigating TSHR transcripts in the orbit, have yielded conflicting results, although there is circumstantial evidence for their presence in orbital fat. In this study, we have examined adult human adipose and muscle tissues from various locations, initially by PCR and subsequently by northern blot. We obtained the expected 610bp product in normal intestinal and orbital fat but not skeletal muscle, following two rounds of PCR amplification but only when reverse transcription used a TSHR specific primer. In northern blots, despite loading all of the RNA obtained from total normal orbital fat contents, TSHR transcripts were at the limit of detection and similarly for large samples of intestinal fat. The exception was RNA obtained from TAO orbital fat, in which TSHR transcripts of 4.6 and 1.7kb were clearly visible, as in the thyroid. We conclude that normal adult adipose tissues contain low levels of TSHR transcripts. In TAO, TSHR transcripts are elevated probably due to an increased number of cells, in particular of preadipocytes in orbital adipose tissue.

The modulation of the human sodium iodide symporter activity by Graves' disease sera.

The transport of iodide into the thyroid, catalyzed by the Na+/I- symporter (NIS), is the initial and rate-limiting step in the formation of thyroid hormones. To study the basic characteristics of the human (h) NIS, we have established a Chinese hamster ovary cell line stably expressing the hNIS (CHO-NIS9). In agreement with previous work on the rat NIS, iodide uptake in these cells was initiated within 2 min of the addition of 131I, reaching a plateau after 30 min. Both perchlorate and thiocyanate inhibited iodide uptake in a dose-dependent manner, with inhibition evident at concentrations of 0.01 and 0.1 micromol/L, respectively, and reaching complete inhibition at 20 micromol/L and 500 micromol/L, respectively. Ouabain, which blocks the activity of the Na+/K+ adenosine triphosphatase, also inhibited iodide uptake in a dose-dependent manner, starting at concentrations of 100 micromol/L and reaching maximum inhibition at 1600 micromol/L, indicating that iodide uptake in these cells is sodium dependent. CHO-NIS9 cells were further used to study 88 sera from patients with Graves' disease, for iodide uptake inhibitory activity, which were compared with sera from 31 controls. Significant iodide uptake inhibition was taken as any inhibition in excess of the mean + 3 SD of the results with the control sera. On this basis, 27 (30.7%) of the Graves' sera, but none of the controls, inhibited iodide uptake in CHO-NIS9. IgGs from these patients also inhibited iodide uptake, indicating that this inhibitory activity was antibody mediated. In summary, we have established a CHO cell line stably expressing the hNIS and shown that antibodies in GD sera can inhibit iodide uptake in these cells. This further emphasizes the role of NIS as a novel autoantigen in thyroid immunity.

Adipogenesis in thyroid eye disease.

PURPOSE: Adipogenesis contributes to the pathogenesis of thyroid eye disease (TED). Thyrotropin receptor (TSHR) transcripts are present in orbital fat. This study was conducted to determine whether they are expressed as functional protein, and if so, whether this is restricted to TED orbits or to a particular stage in adipocyte differentiation. METHODS: Samples of fat were obtained from 18 TED-affected orbits and 4 normal orbits, and 9 were obtained from nonorbital locations. Frozen sections were examined by immunocytochemistry using monoclonal antibodies specific for the human TSHR. Samples were disaggregated and the preadipocytes separated from the mature by differential centrifugation and cultured in serum-free or DM and examined for morphologic changes, oil red O and TSHR staining, and TSH-induced cyclic adenosine monophosphate (cAMP) production. RESULTS: Marked immunoreactivity was observed in frozen sections from all three TED samples and faint staining in both normal orbital fat samples. In vitro, 1% to 5% of preadipocytes displayed TSHR immunoreactivity in five of six TED and two of three normal orbital samples and in three of five nonorbital samples. Differentiation, was induced in all 14 orbital samples. Three of four nonorbital samples contained occasional differentiated cells. Fifty percent to 70% of differentiating cells demonstrated receptor immunoreactivity. Two of three TED and four of four nonorbital preadipocytes in DM and/or mature adipocytes displayed a TSH-mediated increase in cAMP. CONCLUSIONS: The results indicate that orbital fat TSHR transcripts are expressed as protein, which can be functional. This is not aberrant in TED orbits, although expression may be upregulated. The majority of preadipocytes undergoing differentiation express the receptor, indicating a key role for this population in one mechanism for increasing orbital volume.

The sodium iodide symporter gene and its regulation by cytokines found in autoimmunity.

Iodide concentration by the thyroid gland, an essential step for thyroid hormone synthesis, is mediated by the Na+/I- symporter (NIS). To identify factors that may regulate this process, we have studied NIS gene expression in the Fisher rat thyroid cell line (FRTL-5) by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. Increasing concentrations of bovine TSH (0.1, 1, 10, 50 and 100 mU/l), with or without tumour necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) or interleukin-1 alpha (IL-1 alpha) were added to FRTL-5 cells previously deprived of TSH for a minimum of 5 days. RNA was extracted and samples were studied for NIS expression. TSH enhanced NIS mRNA expression in a dose-dependent manner, with induction evident at 0.1 mU/l, reaching a peak at 50 mU/l, an effect detected after 6 h of stimulation, but not in the first 2 h. Both TNF alpha and, to a lesser extent, IL-1 alpha inhibited basal and TSH-induced NIS expression. High concentrations of IFN gamma also downregulated TSH-stimulated NIS mRNA expression. Using the same technique, we also investigated NIS mRNA tissue distribution in two male and one female Wistar rats. High levels of NIS expression were detected in the thyroid, stomach, and mammary gland, lower levels were found in the intestine, adipose tissue and liver, borderline levels were expressed in the salivary gland, and no expression was detected in the kidneys. In summary, we have shown that TSH upregulates rat NIS gene expression in vitro, and this induction can be modulated by cytokines. Analysis of the distribution of rat NIS mRNA ex vivo demonstrated variable levels of NIS transcription in different tissue samples.

DNA banking: the effects of storage of blood and isolated DNA on the integrity of DNA.

Long-term storage of DNA is required for a number of genetic studies; prior to extraction, blood samples may be subject to elevated temperatures for variable intervals. We have studied the effect of temperatures ranging from -70 degrees C to +65 degrees C on human blood and on DNA extracted from it. DNA in solution stored at ambient temperatures up to 37 degrees C for 6 months was digestible by three different restriction endonucleases, whereas storage at 45 degrees C is deleterious after 6-7 weeks. DNA can be extracted from blood samples stored at -70 degrees C for at least 2 months or at 23 degrees C for a week or more, but blood stored at these temperatures may yield less high-molecular-weight DNA. Cell pellets from which plasma has been removed also can serve as a source of DNA. Isolated DNA stored dry for years (up to 30) is difficult to dissolve and may appear degraded, but a sample stored dry for 13 years and then in solution at -20 degrees C for 7 years appeared to be intact.

Family with Pelizaeus-Merzbacher disease/X-linked spastic paraplegia and a nonsense mutation in exon 6 of the proteolipid protein gene.

We report on a C-to-T transition in exon 6 of the PLP gene in a male with Pelizaeus-Merzbacher disease/X-linked spastic paraplegia. The transition changes a glutamine at amino acid residue 233 to a termination codon. This premature stop codon probably results in a truncated protein that is not functional. Six other relatives were analyzed for the mutation and two female carriers were identified. Autopsy data on one male are presented.

Sialic acid residues contribute to the heterogeneity of human serum ribonuclease: demonstration by isoelectric focusing and neuraminidase treatment of serum.

Ribonuclease (RNase) activity from human serum appears as multiple zones of activity following isoelectric focusing in thin layer polyacrylamide gel. At least one but not all of these zones is cross reactive with rabbit antibovine pancreatic RNase A antiserum. Treatment of serum or partially purified serum RNase with neuraminidase reduces the complexity of the serum RNase banding pattern to a major band which focuses at a pH of 9.5 or greater and a minor zone of activity which focuses at about pH 6.0-6.2. Trypsin does not affect the pattern. Thus, sialic acid residues account for a large portion of the heterogeneity of human serum RNase. Neuraminidase treatment is requisite for evaluating RNase from serum and certain other sources.

Tissue specificity of human phosphodiesterase. I. Blood serum.

Human blood serum was found to contain two enzymes which hydrolyze various phosphate diester and phosphonate ester bonds. The enzymes were isolated by butanol extraction, ammonium sulfate precipitation, column chromatography and gel electrophoresis. Zymograms showed that one of these enzymes is serum alkaline phosphatase and the other is a 'true' phosphodiesterase I. Serums of 25 persons showed no polymorphisms for either activity. Alkaline phosphatase hydrolyzes phenolic thymidine 5'-nucleotide esters readily, but phenylphosphonate esters very poorly. Serum phosphodiesterase I prefers phenylphosphonate esters to nucleotide diesters, and has no detectable monoesterase activity.

Tissue specificity of human phosphodiesterase. II. Intestinal mucosa.

Extracts of human intestinal mucosa were examined for their ability to hydrolyze various phosphodiester, phosphomonoester and phenylphosphonate ester linkages. Enzymes carrying out these reactions were partially purified by butanol extraction, ammonium sulfate precipitation and DEAE-cellulose chromatography, and examined for polymorphism on polyacrylamide gels. Two species of alkaline phosphatase and at least five species of PDE I were identified. Antibodies to purified bovine intestinal phosphatase and phosphodiesterase were found specific for the respective human enzymes.

The thyrotropin receptor in thyroid eye disease.

Thyroid eye disease (TED) has an autoimmune etiology, but the nature of the autoantigen that is the target of the initiating event remains unknown. A number of candidates have been proposed based on Western blotting, library screening, and deduction from sequence similarity. A strong favorite is the thyrotropin receptor (TSHR), which is the target of the thyroid stimulating antibodies (TSAB) of Graves' disease (GD). We have recently demonstrated TSHR transcripts in orbital adipose tissue from a patient with TED by Northern blot, transcripts in normal adipose tissue being at the limit of detection. We have shown that the transcripts are translated into protein by immunohistochemical analysis using two monoclonal antibodies to the TSHR generated by genetic immunization. TSHR immunoreactivity is associated with elongated cells with the appearance of a fibroblast, often adjacent to clusters of adipocytes, in orbital biopsies from patients with TED but not in strabismus or pseudotumor biopsies. In animal studies, we have transferred thyroiditis to naive BALBc and NOD mice, using T cells primed to the human TSHR, either using the receptor expressed as a bacterial fusion protein or by genetic immunization. The BALBc develop a Th2-type response to the receptor, but the NOD a Th1-type with thyrocyte destruction. Orbital pathology, edema, infiltration by mast cells and lymphocytes, and adipose accumulation was also induced in 68% of the BALBc but none of the NOD mice. Together these data indicate that the preadipocyte expresses the TSHR and that a Th2 autoimmune response to the receptor may be an initiating event in TED.

Transcription of thyroid autoantigens in non-expressing tissues.

Reverse transcriptase- polymerase chain reaction (RT-PCR) enhances the probability of detecting rare transcripts in complex mixtures of mRNA. Using thyroid autoantigens and the controversy about the role of the TSH-receptor (TSH-R) in thyroid-associated ophthalmopathy as an example, this study demonstrates the problems of interpreting RT-PCR results in typically non-expressing tissues resulting from the extremely high sensitivity of the method. Unexpected transcripts for thyroperoxidase, thyroglobulin, TSH-R (exon 1-4, 354 bp), FSH-receptor, or insulin fragments were demonstrated in a number of thyroid or orbit-derived as well as unrelated tissues or cell types. Unexpected transcripts were most prevalent in fibroblasts, irrespective of the tissue of origin and most likely caused by ectopic transcription. To establish a physiological significance of rare transcripts such as the TSH-R in orbital tissues, demonstration of the protein in addition to the positive RT-PCR results is needed.

A non-organic and non-enzymatic extraction method gives higher yields of genomic DNA from whole-blood samples than do nine other methods tested.

We compared ten methods for extraction of DNA from whole blood. Nine methods require incubation with either enzymes or treatment of organic solvents or both. The 'Rapid Method' (RM) (Method 10) avoids the use of organic solvents (phenol/chloroform) and eliminates completely the use of proteinase K. Thus, the time and cost of DNA extraction are reduced significantly. This is accomplished by salting out and precipitation of the cellular proteins in saturated sodium chloride. This method takes less than an hour to completion, without compromising the yield or the quality of DNA. Using RM, we can make DNA from 0.1 ml of whole blood and as little as 0.5 ml of blood yields DNA sufficient to run a few Southern blots. The RM can also be applied to packed cells. The DNA is free of RNA, protein and degrading enzymes. The uncut DNA runs as a typical slow-migrating, high-molecular-weight and undegraded species in an agarose gel. The DNA is suitable for digestion by various restriction endonucleases. This procedure works equally well with fresh blood samples and with those that are stored at 4 degrees C and -70 degrees C. To our knowledge the RM reported here is the safest, fastest and most quantitative and economical method for preparation of DNA from whole blood and cells.

On the ultrastructure and permeability of taste buds of the marine teleost Ciliata mustela.

The abundant taste buds of the barbels and free fin rays of the five bearded rockling, Ciliata mustela contain an average of 100-150 cells, falling into two types. Tubule-containing cells ('t-cells'), tentatively identified as receptor cells, and each surrounded by fibril-containing cells ('f-cells') in the central part of the bud. t-Cells also occur in two concentric shells separated by indifferent epithelial cells at the periphery of the bud. f-Cells are characterized by their concentrations of fine fibrils, and by granules or vesicles of 180-190 mmu diameter. The 100 or so receptor cells in a taste bud are innervated by some 250 axons. Lanthanum penetrates more deeply into the extracellular space of taste buds than into the extracellular space of the general epithelium, perhaps indicating that a greater area than the mere protruding tip of receptor cells may be accessible to chemical stimulation. Degenerating cells may provide an important route of entry for such external agents.