Deletion of thyrotropin receptor residue Asp403 in a hyperfunctioning thyroid nodule provides insight into the role of the ectodomain in ligand-induced receptor activation.

Somatic mutations of the TSH receptor (TSHR) gene are the main cause of autonomously functioning thyroid nodules. Except for mutations in ectodomain residue S281, all of the numerous reported activating mutations are in the TSHR membrane-spanning region. Here, we describe a patient with a toxic adenoma with a novel heterozygous somatic mutation caused by deletion of ectodomain residue Asp403 (Del-D403). Subsequent in vitro functional studies of the Del-D403 TSHR mutation demonstrated greatly increased ligand-independent constitutive activity, 8-fold above that of the wild-type TSHR. TSH stimulation had little further effect, indicating that the mutation produced near maximal activation of the receptor. In summary, we report only the second TSHR ectodomain activating mutation (and the first ectodomain deletion mutation) responsible for development of a thyroid toxic adenoma. Because Del-D403 causes near maximal activation, our finding provides novel insight into TSHR structure and function; residue D403 is more likely to be involved in the ligand-mediated activating pathway than in the ectodomain inverse agonist property.

Extent of hypoechogenic area in the thyroid is related with thyroid dysfunction after subacute thyroiditis.

OBJECTIVE: To gain an insight into risk factors for hypothyroidism after subacute thyroiditis (SAT), we examined the correlation between initial laboratory and ultrasonographic findings and sequential thyroid dysfunction among treatment modalities. PATIENTS: We reviewed retrospectively the medical records of 252 patients (26 men and 226 women) with SAT who consecutively visited our thyroid clinic at Kuma Hospital for at least 6 months from 1996 through 2004. RESULTS: Throughout the course, 135 patients (53.6%) developed transient or permanent hypothyroidism. Levels of TSH were most often elevated (greater than 5 IU/ml) 2 months after SAT onset regardless of treatment, and 97.0% of patients who showed transient or permanent hypothyroidism clustered within 6 months from onset. During follow-up, patients treated with prednisone (PSL) were more likely to have normal thyroid function than patients not treated or those receiving anti-inflammatory drug therapy. In patients who developed hypothyroidism with PSL treatment or without treatment, the rates of bilateral hypoechogenic areas (HEA) were 6-fold higher than those of unilateral HEA. Moreover, permanent hypothyroidism occurred in 5.9% of patients, and all patients with permanent hypothyroidism presented initially with bilateral HEA and had consequently small thyroid size with or without abnormal autoimmunity. CONCLUSIONS: The rates of thyroid dysfunction after SAT were significantly lower in patients receiving PSL. Extent of HEA in the thyroid, but not laboratory findings, may be a possible marker for developing thyroid dysfunction after SAT.

Enhanced efficacy of transcriptionally targeted suicide gene/prodrug therapy for thyroid carcinoma with the Cre-loxP system.

Our recent study demonstrates the feasibility of the thyroglobulin (TG) promoter in transcriptionally targeted gene therapy for thyroid carcinomas expressing TG, albeit less effectively than the constitutive viral promoter. The present study was, therefore, designed to enhance the activity of the TG promoter with the Cre-loxP system. Our data demonstrate that the in vitro cytotoxic effect of herpes simplex virus thymidine kinase/ganciclovir obtained with the TG promoter and the Cre-loxP system is approximately 5-10-fold higher than that with the TG promoter alone. Enhanced tumor growth inhibition was also observed in in vivo tumor models. These data indicate the usefulness of the Cre-loxP system to enhance the activity of a tissue (or tumor)-specific promoter in transcriptionally targeted cancer gene therapy.

Retrovirus-mediated herpes simplex virus thymidine kinase gene transduction renders human thyroid carcinoma cell lines sensitive to ganciclovir and radiation in vitro and in vivo.

In an attempt to develop gene therapy for thyroid carcinomas, the present studies were undertaken to evaluate in vitro and in vivo therapeutic efficacy and toxicity of herpes simplex virus thymidine kinase (HSV-tk) gene and ganciclovir (GCV) treatment, a widely used prodrug/suicide gene therapy, in human thyroid carcinoma cell lines, FRO and WRO cells, using a means of retrovirus-mediated gene transduction. In vitro experiments demonstrated dose- and time-dependent cell killing by transduction of the HSV-tk gene followed by GCV treatment. The IC50 (the concentration required to elicit 50% growth inhibition) shifted from 250 to 0.5 mg/liter in FRO cells, and from 3,000 to 0.09 mg/liter in WRO cells with therapeutic indexes of 500 and 33,000, respectively. Treatment with 30 mg/liter GCV for 4 days led to complete cell death in HSV-tk tumor cells. Nontransduced cells mixed with transduced cells were also effectively killed by GCV (bystander effect). Low concentrations of GCV, which alone showed little cytotoxicity, enhanced radiation-induced cytotoxicity (radiosensitization). In vivo sc FRO-tk tumor models in nude mice also showed dose- and time-dependent tumor regression. The IC50 was less than 2 mg/kg, and treatment with 100 mg/kg GCV for 2 weeks completely eradicated all tumors. The bystander effect and radiosensitization were also obtained in vivo. These results suggest that the HSV-tk/GCV approach to human thyroid carcinoma cells appears to be very efficacious, with a wide therapeutic range, and exerts a bystander effect and radiosensitization both in vitro and in vivo. Thus, HSV-tk/GCV system, alone or in combination with radiotherapy, may be a promising suicide gene therapy for thyroid carcinomas.

Ontogenetic changes in the expression of estrogen receptor alpha and beta in rat pituitary gland detected by immunohistochemistry.

The physiological effects of estrogen on the pituitary, including cellular proliferation and regulation of hormone synthesis, are mediated by the nuclear estrogen receptor (ER). The purpose of this study was to determine ontogenetic expression of two types of ERs (ERalpha and ERbeta) in the pituitary using specific antibodies, monoclonal antibody (1D5) for ERalpha and polyclonal antibody generated against ERbeta. First, we confirmed the detection of 66- and 55-kDa bands for ERalpha and ERbeta, respectively, in the rat pituitary extract by Western blotting. Then immunostaining with these antibodies was performed using fetal and adult Wistar rat tissues, combined with PRL or LHbeta immunohistochemistry. Intense ERbeta signal was detected throughout the pituitary from day 12 of gestation. However, staining for ERalpha only became detectable from day 17 of gestation. In contrast with the fetal period, nuclei stained for ERalpha were widely distributed in the anterior lobe in the adult rat, whereas ERbeta-positive cells were restricted in the anterior lobe. LHbeta, but not PRL, was colocalized in ERbeta-positive cells. Our results indicated that the major population of ER subtypes in the rat pituitary gland has changed around the day of birth and that the expression of ERbeta may be involved in the differentiation of pituitary cell function to synthesize a specific hormone.

Palmitoylation of human thyrotropin receptor: slower intracellular trafficking of the palmitoylation-defective mutant.

We here show that the epitope-tagged human TSH receptor (TSHRmyc) is covalently modified with palmitic acid by thioesterification. Side-directed mutagenesis identified Cys699 in the C-terminal cytoplasmic tail of the receptor as the putative palmitoylation site. Mutation of Cys699 to Ala results in the nonpalmitoylated receptor (TSHRmycC699A) in which high affinity TSH binding, Gs coupling, homologous desensitization and TSH-induced internalization are unaffected. In contrast, abolition of palmitoylation appears to decrease the rate of the intracellular trafficking of the receptor. However, since most of TSHRmycC699A seems to be fully processed finally and the receptor number of TSHRmycC699A on the cell surface is comparable to that of TSHRmyc, our results suggest that abolition of palmitoylation delays the cell surface expression of TSHR, but does not trap the receptor intracellularly, although another possibility for proteolytic degradation of either the 95 kDa or the 100 kDa mutant receptor can not be excluded. Thus, post-translational modification of TSHR by palmitoylation may provide a novel mechanism of enhancing the rate of intracellular trafficking of the receptor.

Adenovirus-mediated tumor suppressor p53 gene therapy for anaplastic thyroid carcinoma in vitro and in vivo.

The present study was designed to evaluate the therapeutic efficacy of adenovirus-mediated wild-type (wt) tumor suppressor p53 expression in four human thyroid carcinoma cell lines harboring p53 mutations (ARO, FRO, NPA, and WRO) and normal human thyroid follicular cells with wt-p53 in vitro and in vivo. In vitro infection of replication-deficient recombinant adenovirus vector expressing wt-p53 led to a dose-dependent cell killing in both normal and carcinoma cells. In contrast, adenovirus expressing Escherichia coli beta-galactosidase showed little effect. The sensitivity to p53-mediated cell killing varied among the cells used. It was, at least partly, dependent on their adenovirus infectivity in carcinoma cells, whereas normal thyroid cells were relatively resistant to p53-mediated cell death despite its highest adenovirus infectivity. The mechanism of cell killing by wt-p53 was shown, by flow cytometric analysis, to be apoptosis. Furthermore, wt-p53 expression renders two out of four carcinoma cell lines (FRO and NPA) more sensitive to doxorubicin and one (FRO) to 5-fluorouracil, independent of treatment schedule. In vivo experiments, using FRO and NPA cells, showed that growth of sc tumors in nude mice was nearly completely inhibited by direct injection of adenovirus expressing wt-p53 [1 x 10(9) plaque-forming units/tumor]. This effect was augmented by its combination with doxorubicin treatment (4 mg/kg, thrice a week), which led to tumor regression. Our results therefore indicate that adenovirus-mediated wt-p53 gene introduction seems to be a potential clinical utility in gene therapy for anaplastic thyroid carcinomas, particularly when combined with chemotherapy.

Targeting the replication of adenovirus to p53-defective thyroid carcinoma with a p53-regulated Cre/loxP system.

In this article, we evaluated the feasibility of the restricted replication-competent adenoviruses for treatment of anaplastic thyroid carcinomas (ATCs), which are very aggressive and difficult to treat. Because ATCs very often harbor p53 mutations, we used wt-p53 as a regulatory factor to restrict virus replication and cytopathic effect to p53-mutated cells. The recently reported "gene inactivation strategy" using p53-regulated Cre/loxP system was employed; this system consists of two recombinant adenoviruses. One has an expression unit of the synthetic p53 - responsive promoter and the Cre recombinase gene (Axyp53RECre), and another contains two expression units; the first consists of E1A gene flanked by a pair of loxP sites downstream of the constitutive CAG promoter and the second E1B19K gene under the control of the CMV promoter (AdCALE1AL). We expected that coinfection of these two adenoviruses into the cells with wt-p53 would lead to expression of the Cre, which excises E1A gene and switches off E1A expression resulting in no virus replication, whereas in the cells with mutant p53 E1A could be expressed that leads to virus replication and cell lysis. Our in vitro data demonstrate that although infection of AdCALE1AL alone led to E1A expression, viral replication and cytolysis in all the thyroid cells examined irrespective of their p53 status, the double infection did so in FRO cells (p53-null ATC) but not in FRO cells stably expressing wt-p53 and normal thyroid cells with wt-p53. These data indicate that our double infection method may have a potential for treatment of ATC and probably also other p53-defective cancer cells.

Expression of copper trafficking genes in the mouse brain.

Copper homeostasis in the brain must be strictly maintained, since copper is an essential trace element and is potentially toxic. To understand the mechanism of copper homeostasis in the brain, we cloned several mouse homologues of copper trafficking genes and performed in situ hybridization histochemistry. mCTR1, mATX1, and mATP7a were highly expressed in the choroid plexus, indicating that the choroid plexus uses the trafficking pathway from uptake to efflux to transport copper to the cerebrospinal fluids. We suggest that these genes may regulate copper concentration in the brain through the choroid plexus.

Treatment of thyroid carcinoma cells with four different suicide gene/prodrug combinations in vitro.

To develop a suitable suicide gene/prodrug therapy for the treatment of thyroid carcinomas, the relative therapeutic efficacy of four different suicide gene/prodrug combinations was compared in thyroid carcinomas in vitro. Herpes simplex virus thymidine kinase and ganciclovir (HSV-TK/GCV), Escherichia coli cytosine deaminase and 5-fluorocytosine (CD/5FC), E coli nitroreductase and CB1954 (NTR/CB1954), and human deoxycytidine kinase and cytosine arabinoside (dCK/AraC) were employed. The suicide genes were transduced into two thyroid carcinoma cell lines with retroviral vectors in which all the suicide genes were under the control of the same promoter. When the relative efficacy of four suicide gene/prodrugs was compared with therapeutic index and degree of bystander effect, we found a clear dissociation between these two parameters. Thus, HSV-TKIGCV demonstrated the widest therapeutic index, while CD/5FC and NTR/CB1954 showed the stronger bystander effect than HSV-TK/GCV. dCK/AraC had little efficacy. Advantages and limitations of each suicide gene/prodrug combinations are discussed.

Preliminary time-flow study: comparison of interpretation times between PACS workstations and films.

Toshiba Hospital installed a PACS and RIS in the diagnostic imaging department along with a hospital-wide HIS in May 1993. Our PACS includes three diagnostic workstations each of which is provided with six monochrome CRT monitors. The diagnostic workstations have been used as the primary tools for interpretation of almost all radiographic images. The actual image interpretation time was measured for diagnostic workstations (237 examinations) and for conventional hard-copy films (219 examinations) for CR, CT, and MRI. The difference in interpretation times between diagnostic workstations and films was not significant. With regard to image interpretation time, diagnostic workstations are thought to be acceptable for practical image reading.

Clinical evaluation of newly developed CRT viewing station: CT reading and observer's performance.

The clinical performance of the new viewing station with six CRT monitors (17-inch, 1,024 x 1,280) was evaluated. In the primary interpretation of CT images, time measurements were carried out for eight radiologists. No significant differences in reading time existed between CRT and film in 3 of 4 readers in head CT series, and in 2 of 6 readers in body CT series. Compared with the previous system, the new prototype system achieved an approximately 30% decrease in reading time in both head and body CT studies and could reduce mental and eye fatigue.

Display method can affect interobserver agreement: comparison of 'zoom-and-pan' and 'browse-and-paste' for primary CT interpretation.

Eight radiologists interpreted body CT images of 30 cases using a viewing station (six 17-in. monitors, 1024x1280). Using two different display methods, 'zoom-and-pan' and 'browse-and-paste', the readers described the presence or absence of liver tumors using a five-point rating scale and temporal changes between the current and previous studies using a seven-point rating scale. There was no significant difference in kappa values for tumor detection between the two display modes. However, in describing temporal changes, the kappa value of the browse-and-paste was significantly lower than that of zoom-and-pan (p<0.01). Browse-and-paste may have the disadvantage of greater interobserver variation.

Increased expression levels of tensin3 mRNA in thyroid functional adenomas as compared to non-functioning adenomas.

Tensin3 is a member of tensin family which is localized in focal adhesion. In our previous study, a high level of tensin3 mRNA expression was observed in the thyroid but not in other tissues, thus, tensin3 gene was regarded as a novel thyroid-specific gene. The high expression level of tensin3 mRNA in normal thyroid tissue suggests some fundamental roles in thyroid functions. In fact, the expression level of tensin3 mRNA was low in most thyroid carcinomas and non-functioning thyroid follicular adenomas which do not produce thyroid hormone. In the present study, we measured the expression levels of tensin3 mRNA in twelve functional adenomas and compared the results with those in normal thyroid tissues, adenomatous goiters and non-functioning thyroid follicular adenomas. Tensin3 mRNA was expressed abundantly in all twelve functional adenomas at almost the same level as in normal thyroid tissues and adenomatous goiters, while its expression was significantly lower in non-functioning follicular adenomas. Considering these facts, an abundant expression of tensin3 mRNA is observed in tissues that produce thyroid hormone, which suggests some fundamental roles in basic thyroid functions.

New aspects of membrane dynamics of Amoeba proteus contractile vacuole revealed by vital staining with FM 4-64.

The contractile vacuole (CV) cycle of Amoeba proteus has been studied by phase contrast and electron microscopy. However, the understanding of membrane dynamics in this cycle is still poor. In this study, we used live imaging by fluorescence microscopy to obtain new insights. We succeeded in staining the CV with a styryl dye, FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide), and obtained the following results. (1) The CV membrane was directly stained with the dye in the external medium when the CV pore opened upon contraction. This indicates that transfer of plasma membrane to the CV does not occur. (2) The membrane dynamics during the CV cycle were elucidated. In particular, the fluorescent CV membrane was maintained as an aggregate just after contraction and the vacuole re-formed from the aggregate. Staining was maintained during continued contraction cycles. We conclude that the CV membrane is maintained during the CV cycle.

Implementation and evaluation of workflow based on hospital information system/radiology information system/picture archiving and communications system.

The purpose of this presentation is to review and evaluate computerized workflow of selected sites that have integrated systems of the hospital information system (HIS), radiology information system (RIS), and picture archiving and communications system (PACS). We then focus on some essential points of integration of those systems, such as avoiding multiple entries of patients demographic data, prefetching current and previous images to the correspondent workstations, and workflow management. To realize them by integrating multiple subsystems such as HIS/RIS/PACS integration, there must be exchange of the workflow control information, and consistency of the information between subsystems.

Control method for preloading with priority information in an integrated radiology information system/picture archiving and communication system.

More than 10 years has passed since the concept of picture archiving and communication systems (PACS) was first proposed. A great deal of effort has been expended to make PACS suitable for routine use in clinical settings, but only a few systems are currently used in this manner. A major reason is the lack of the assurance of throughput equivalent to that of a conventional system based on order sheets and analog films. In this report, two techniques to increase throughput have been introduced and studied. The first is the preloading of data elements from the various information systems and the PACS. The second is the use of the priority information to rank order the examinations placed on the list for interpretation. We have applied these techniques to an actual system and have measured the distribution of time for processing examinations. These two techniques appear to make PACS useful in routine practice because most of the urgent cases were interpreted within the target time of 40 minutes.

Identification of the sites of asparagine-linked glycosylation on the human thyrotropin receptor and studies on their role in receptor function and expression.

The amino-terminal ectodomain of human thyrotropin receptor (TSHR) contains six potential N-linked glycosylation sites (N-Xaa-S/T). This study was designed to evaluate the functional role of TSHR carbohydrates in detail. Because our previous mutagenesis study by Asn to Gln substitutions suggested the critical role of the first and third glycosylation sites (amino acids 77 and 113) for expression of the functional TSHR, we first constructed TSHR mutants having these two glycosylation sites to elucidate whether these two sites are sufficient for TSHR function and expression; this mutant however proved to be nonfunctional. Also the expression levels and function of TSHR mutants with a Ser/Thr to Ala substitution at the first or third glycosylation site were found to be intact. These data indicate that our previous data appear to result from amino acid substitution itself, not from disruption of glycosylation. The next series of the mutants was therefore constructed to identify at least how many glycosylation sites are necessary. Neither TSH binding nor cAMP response was detected in TSHR mutants with three glycosylation sites. However, the mutants with four glycosylation sites were fully functional in terms of TSH binding and cAMP production, although the expression levels were 30 to 40% of that in wild-type TSHR. Finally, Western blot revealed that all six glycosylation sites are actually glycosylated. These data indicate that 1) TSHR ectodomain contains six N-linked carbohydrates, and 2) glycosylation of at least four sites appears necessary for expression of the functional TSHR.