A high efficient protocol for soybean root transformation by Agrobacterium rhizogenes and most stable reference genes for RT-qPCR analysis.

KEY MESSAGE: A 55% transformation efficiency was obtained by our optimized protocol; and we showed that GmELF1 - ß and GmELF1 - α are the most stable reference genes for expression analyses under this specific condition. Gene functional analyses are essential to the validation of results obtained from in silico and/or gene-prospecting studies. Genetic transformation methods that yield tissues of transient expression quickly have been of considerable interest to researchers. Agrobacterium rhizogenes-mediated transformation methods, which are employed to generate plants with transformed roots, have proven useful for the study of stress caused by root phytopathogens via gene overexpression and/or silencing. While some protocols have been adapted to soybean plants, transformation efficiencies remain limited; thus, few viable plants are available for performing bioassays. Furthermore, mRNA analyses that employ reverse transcription quantitative polymerase chain reactions (RT-qPCR) require the use of reference genes with stable expression levels across different organs, development steps and treatments. In the present study, an A. rhizogenes-mediated soybean root transformation approach was optimized. The method delivers significantly higher transformation efficiency levels and rates of transformed plant recovery, thus enhancing studies of soybean abiotic conditions or interactions between phytopathogens, such as nematodes. A 55% transformation efficiency was obtained following the addition of an acclimation step that involves hydroponics and different selection processes. The present study also validated the reference genes GmELF1-ß and GmELF1-α as the most stable to be used in RT-qPCR analysis in composite plants, mainly under nematode infection.

Restriction of dietary Iodine does not ameliorate the early effect of anti-thyroid drug therapy for Graves' disease in an area of excessive iodine intake.

The close relationship between iodine intake and the effects of anti-thyroid drugs (ATD) for Graves' disease (GD) has been well established. However, it remains unknown whether restriction of dietary iodine improves the effect of ATD. This study aimed to clarify this issue in Japanese patients with GD who routinely ingest large amounts of dietary iodine. We performed a prospective clinical study in 81 patients with newly diagnosed GD who were divided into an iodine restricted group and a control group. Urinary iodine, thyroid hormones and TSH receptor antibody were measured during the first 8 weeks of ATD therapy. Urinary iodine concentrations in the iodine restricted group were significantly lower than in the control group (p=0.043). However, there were no significant differences in the decrease of thyroid hormones and TSH receptor antibody between the two groups. Restriction of dietary iodine does not ameliorate the effect of ATD therapy for GD in an area of excessive iodine intake.

Expression of S100A2 and S100A6 in thyroid carcinomas.

AIMS: S100 calcium-binding proteins are known to play multiple roles in carcinoma development. In this study, we focused on two kinds of these proteins, S100A2 and S100A6, and investigated their expression in thyroid neoplasms. METHODS AND RESULTS: We investigated S100A2 and S100A6 expression in 141 thyroid neoplasms by immunohistochemistry. S100A2 was not expressed in normal follicles or follicular tumours, with one exception. Although 89.5% of papillary carcinoma were positive for S100A2, the expression was heterogeneous except in two cases. In anaplastic carcinoma, 78.5% of cases expressed S100A2 diffusely, while the remaining cases were negative. In normal follicles, S100A6 expression was always low, while 8.3% of follicular adenomas and 39.5% of follicular carcinomas showed increased expression. In papillary carcinomas, S100A6 expression was increased in 75% of cases, but in anaplastic carcinomas it was decreased, with only 14.3% showing high expression. CONCLUSIONS: The expression patterns of S100A2 and S100A6 in thyroid neoplasms are unique compared with those of other carcinomas, suggesting that: (i) S100A2 and S100A6 contribute to certain events in papillary carcinoma progression, and (ii) S100A2 expression is one of the biological characteristics of anaplastic carcinoma.

High-throughput differential screening of mRNAs by serial analysis of gene expression: decreased expression of trefoil factor 3 mRNA in thyroid follicular carcinomas.

To find mRNAs whose expression differs between thyroid follicular adenomas and carcinomas, a high-throughput analysis of mRNAs in these two tumours was performed. This method, named high-throughput differential screening by serial analysis of gene expression (HDSS), combines a modified method of serial analysis of gene expression (SAGE) and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). A total of 40 candidate tag sequences that showed extremely different expression levels between a follicular carcinoma and a follicular adenoma in the SAGE analysis were analysed by real-time quantitative RT-PCR, using RNAs from an additional four typical follicular carcinomas and adenomas. One sequence tag that represents trefoil factor 3 (TFF3) mRNA showed a clear difference in its expression level between adenomas and carcinomas. The expression levels of TFF3 mRNA in 48 follicular adenomas and 29 follicular carcinomas were measured by real-time quantitative RT-PCR using a specific probe for TFF3. They were significantly decreased in follicular carcinomas, especially in widely invasive types and those with evident metastases. These results indicate that the decreased expression of TFF3 mRNA is a marker of follicular carcinomas, especially those with a high risk of invasion or metastasis.

Tie-1 tyrosine kinase expression in human thyroid neoplasms.

AIMS: To investigate tie-1 expression in human thyroid neoplasms. Recent studies have demonstrated that receptor-type tyrosine kinases (RTKs) contribute to carcinoma progression. Tie-1 is one of the RTKs and plays a role in angiogenesis, although its pathophysiological significance in human carcinoma is still to be elucidated. METHODS AND RESULTS: Immunohistochemical expression of tie-1 was studied in various thyroid neoplasms. Tie-1 immunoreactivity was only occasionally observed in normal follicular cells. In papillary carcinoma, tie-1 was classified as positive in carcinoma cells in 55.7% of the cases and was more frequently expressed in those of smaller size with an absence of a poorly differentiated lesion. In contrast, tie-1 was positive in only 8.3% of anaplastic carcinoma and no cases of follicular carcinoma or adenoma were positive. CONCLUSIONS: These results suggest that tie-1 has a role in thyroid tumorigenesis, especially in the early phase of papillary carcinoma, but it is not important in the progression of anaplastic carcinoma or follicular tumour.

PGP9.5 mRNA could contribute to the molecular-based diagnosis of medullary thyroid carcinoma.

The protein gene product 9.5 (PGP9.5) is a ubiquitin hydrolase that is widely expressed in neuronal tissues at all stages of neuronal differentiation and is a known neuroendocrine marker. Medullary thyroid carcinoma (MTC) arises from parafollicular cells and is reported to overexpress several mRNAs such as RET, calcitonin, and CEA. These markers are thought to be useful in determining a molecular-based diagnosis of MTC. We examined the expression levels of PGP9.5 mRNA in 80 thyroid tissues using real-time quantitative reverse transcription (RT-PCR) and found that PGP9.5 mRNA was overexpressed in all 11 MTCs examined, both hereditary and sporadic, but not in other histological tumour types. Furthermore, by RT-PCR, PGP9.5 mRNA was detected only in aspirates from three medullary carcinomas, and not in aspirates from other tumour types. These results demonstrate that, in addition to the expression of RET, calcitonin and CEA, PGP9.5 mRNA expression may contribute to the molecular-based diagnosis of MTCs.

Polo-like kinase 1 overexpression is an early event in the progression of papillary carcinoma.

Polo-like kinase 1 (PLK1) is one of the serine threonine kinases that contributes to cell mitosis and is regarded as a marker of cellular proliferation. However, its protein expression in human carcinoma has not been studied in depth. We investigated PLK1 expression in various thyroid neoplasms in order to elucidate its physiological significance in thyroid carcinoma. Normal follicular cells only occasionally expressed PLK1. In follicular tumours and anaplastic carcinoma, PLK1 overexpression was not a common event and only 5.9% of follicular adenoma, 7.1% of follicular carcinoma, and 11.8% of anaplastic carcinoma overexpressed this protein. However, 43.7% of papillary carcinoma overexpressed PLK1. Polo-like kinase 1 overexpression was more frequently observed in smaller papillary carcinoma lesions, and 62.5% of microcarcinoma (ranging from 4 mm to 1.0 cm) and even 66.7% of incidental carcinoma (less than 4 mm) overexpressed it, whereas this phenomenon could only be seen in 20.0% of lesions larger than 4.0 cm. Furthermore, PLK1 overexpression was not related to cell-proliferating activity evaluated by Ki-67 labelling index, but it was inversely linked to UICC stage, extrathyroidal invasion, and the presence of poorly differentiated lesion as proposed by Sakamoto et al. These findings strongly suggest that, unlike other carcinomas previously studied, PLK1 does not act as a cell cycle regulator but plays a constitutive role in papillary carcinoma especially in the early phase, and may contribute to the malignant transformation of this carcinoma.

Syndecan-1 expression in thyroid carcinoma: stromal expression followed by epithelial expression is significantly correlated with dedifferentiation.

AIM: To investigate the expression of syndecan-1 in thyroid neoplasia. Syndecan-1 is a proteoglycan regulating cell adhesion. Previous studies have demonstrated that decreased expression of syndecan-1 is linked to malignant progression. METHODS AND RESULTS: Syndecan-1 expression in thyroid neoplasia was studied immunohistochemically. Syndecan-1 was expressed in stromal cells as well as neoplastic epithelial cells. Stromal syndecan-1 expression was observed more frequently in papillary carcinomas larger than 10 mm in size than in microcarcinomas and in widely invasive than in minimally invasive follicular carcinomas. Furthermore, poorly differentiated carcinomas showed this phenomenon more than well-differentiated carcinomas, but the expression in undifferentiated carcinomas was similar to that of poorly differentiated carcinomas. Epithelial syndecan-1 expression was more frequently observed in anaplastic (undifferentiated) carcinomas than in papillary and follicular carcinomas. No significant difference in epithelial expression was found between well and poorly differentiated carcinomas, but undifferentiated carcinomas expressed epithelial syndecan-1 more frequently than did poorly differentiated carcinomas. CONCLUSIONS: These results are in contrast to those previously reported for carcinomas at other sites. It is suggested that the role of syndecan-1 in thyroid carcinomas might be unique. Stromal syndecan-1 expression followed by its epithelial expression is significantly related to progression, including dedifferentiation of thyroid carcinoma.

Cyclooxygenase-2 expression in thyroid neoplasms.

AIMS: Previous studies have demonstrated that cyclooxygenase-2 (COX-2) plays a role in carcinogenesis and carcinoma development. In this study, we investigated its expression in thyroid neoplasms in order to elucidate its role. METHODS AND RESULTS: COX-2 expression was studied immunohistochemically in 20 anaplastic (undifferentiated) carcinomas, 49 papillary carcinomas, 22 follicular carcinomas and 15 follicular adenomas. Positive staining was only occasionally seen in normal follicles or stromal cells. COX-2 over-expression was found in only 20.0% of follicular adenomas and 40.9% of follicular carcinomas. In papillary carcinomas, the incidence (81.3%) was significantly higher (P < 0.0001) than in follicular carcinomas, although COX-2 expression was reduced in cases with old age (P = 0.0190), large size (P = 0.0028), advanced stage (P = 0.0225), satellite tumours (P = 0.0363), and the presence of solid, scirrhous or trabecular growth patterns (P = 0.0018). Undifferentiated carcinomas less frequently over-expressed COX-2 (P = 0.0004), with an incidence of 40.0%. CONCLUSIONS: These results indicate that the up-regulation of COX-2 may contribute predominantly in the early phase of papillary carcinoma progression, whereas it plays a more adjuvant role in follicular carcinoma progression.

Expression of cdc25A and cdc25B proteins in thyroid neoplasms.

Cdc25B and cdc25A phosphates are prominent stimulators of cell cycle progression and recent studies have also suggested their oncogenic roles. To elucidate the role of these proteins in thyroid neoplasms, we immunohistochemically investigated their expression, and neither protein was expressed in normal follicular cells. Cdc25B was frequently overexpressed in follicular adenoma and minimally invasive follicular carcinoma, but the incidence was significantly lower in widely invasive follicular carcinoma. Furthermore, the cdc25B expression level significantly decreased with the dedifferentiation of thyroid carcinoma. Cdc25A overexpression was observed in high incidences in all types of thyroid neoplasms. These results suggest that cdc25B and cdc25A play oncogenic roles in thyroid follicules and that cdc25B works predominantly in the early phase of the progression of thyroid carcinoma, whereas cdc25A plays a fundamental role in the development of thyroid neoplasms.

Caveolin-1 overexpression is an early event in the progression of papillary carcinoma of the thyroid.

Caveolin-1 is a major structural component of caveolae, which are plasma membrane microdomains implicated in the regulation of intracellular signalling pathways. Previous in vitro and in vivo studies on the function of caveolin-1 in carcinoma showed controversial results, indicating that the physiological role of caveolin-1 varies according to the origin of carcinoma. In this study, we investigated caveolin-1 expression in thyroid neoplasms by means of immunohistochemistry using a rabbit polyclonal antibody against caveolin-1. Normal follicular cells did not express caveolin-1. In papillary carcinoma, caveolin-1 expression was observed in high incidence, and especially in microcancer (less than 1.0 cm in diameter), caveolin-1 was positive in all cases except one. In undifferentiated (anaplastic) carcinoma, its incidence was significantly reduced. On the other hand, all cases of follicular carcinoma and adenoma were classified as negative for caveolin-1. These results suggest that caveolin-1 may play a role predominantly in the early phase of papillary carcinoma, whereas it has little influence on follicular tumours.

Extremely reduced evolutionary rate of TATA-box binding protein in higher vertebrates and its evolutionary implications.

Previously we showed that the evolutionary rates of the Pax proteins are markedly reduced in higher vertebrates, as compared with those in the ancestral lineage of vertebrates, and we suggested that the reduced Pax protein evolution might be explained by increased functional constraints due to gene recruitment for other purposes or repeated expression in different developmental stages. To clarify the problem of whether the evolutionary rate variation found in the Pax proteins is an evolutionary feature generally recognized in most transcription factors, we have cloned and sequenced cDNAs encoding the TATA-box binding protein (TBP), a general transcription factor of eukaryotes, from Oryzias latipes, a Japanese medaka, Lampetra reissneri, a lamprey, and Ephydatia fluviatilis, a freshwater sponge. An evolutionary rate analysis of TBP has revealed that the evolutionary rate of TBP is extremely low in higher vertebrates, but not in the ancestral lineage of vertebrates, as found in the Pax proteins. In contrast, no marked reduction of the evolutionary rate in higher vertebrates is observed in the aldolase C, a house keeping enzyme. It is therefore likely that the increased functional constraint on TBP is responsible for the extremely low evolutionary rate in higher vertebrates. The temporal pattern of the evolutionary rate variation during vertebrate evolution was discussed.

Genetic algorithm-based maximum-likelihood analysis for molecular phylogeny.

A heuristic approach to search for the maximum-likelihood (ML) phylogenetic tree based on a genetic algorithm (GA) has been developed. It outputs the best tree as well as multiple alternative trees that are not significantly worse than the best one on the basis of the likelihood criterion. These near-optimum trees are subjected to further statistical tests. This approach enables ones to infer phylogenetic trees of over 20 taxa taking account of the rate heterogeneity among sites on practical time scales on a PC cluster. Computer simulations were conducted to compare the efficiency of the present approach with that of several likelihood-based methods and distance-based methods, using amino acid sequence data of relatively large (5-24) taxa. The superiority of the ML method over distance-based methods increases as the condition of simulations becomes more realistic (an incorrect model is assumed or many taxa are involved). This approach was applied to the inference of the universal tree based on the concatenated amino acid sequences of vertically descendent genes that are shared among all genomes whose complete sequences have been reported. The inferred tree strongly supports that Archaea is paraphyletic and Eukarya is specifically related to Crenarchaeota. Apart from the paraphyly of Archaea and some minor disagreements, the universal tree based on these genes is largely consistent with the universal tree based on SSU rRNA.

Serum concentration of androstenediol and androstenediol sulfate in patients with hyperthyroidism and hypothyroidism.

Androstenediol (5-androsten-3beta, 17beta-diol, ADIOL) and androstenediol 3-sulfate (ADIOLS) are active metabolites of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), respectively, and have estrogenic activity and immunoregulatory function. We examined serum concentrations of ADIOL, ADIOLS, DHEA, DHEAS and pregnenolone sulfate (5-pregnen-3beta-ol-20-one sulfate, PREGS) in patients with Graves' thyrotoxicosis (male/female 9/14), hypothyroidism (11/20) and in normal controls (14/29). In hypothyroidism serum levels of all these steroids were significantly decreased in both genders. In hyperthyroidism, in contrast, serum levels of ADIOLS (male 1.49 +/- 0.69, female 0.64 +/- 0.31 micromol/l), DHEAS (male 7.43 +/- 3.91, female 5.13 +/- 2.03 micromol/l), and PREGS (male 1.13 +/- 0.58, female 1.07 +/- 0.85 micromol/l) were markedly increased, but serum concentrations of ADIOL and DEHA were not significantly different from controls (ADIOLS male 0.36 +/- 0.33, female 0.14 +/- 0.09 micromol/l; DHEAS male 2.88 +/- 1.70, female 1.86 +/- l1.03pmol/l; PREGS male 0.18 +/- 0.12, female 0.11 +/- 0.08 micromol/l; ADIOL male 3.76 +/- 1.35, female 1.91 +/- 1.17 nmol/l; DHEA male 9.23 +/- 3.49, female 13.5 +/- 10.8nmol/l). Serum concentrations of all these steroids correlated with the serum concentration of the thyroid hormones in these patients. Serum albumin and sex hormone-binding globulin concentrations were not related to these changes in the concentrations of steroids. These findings indicate that serum concentrations of ADIOLS, ADIOL, DHEAS, DHEA and PREGS were decreased in hypothyroidism, whereas serum ADIOLS, DHEAS and PREGS concentrations were increased but ADIOL and DHEA were normal in hyperthyroidism. Thyroid hormone may stimulate the synthesis of these steroids and sulfotransferase is speculated to be increased in hyperthyroidism. Increased ADIOLS might contribute to menstrual disturbances and gynecomastia in hyperthyroidism.

Identification of susceptibility loci for autoimmune thyroid disease to 5q31-q33 and Hashimoto's thyroiditis to 8q23-q24 by multipoint affected sib-pair linkage analysis in Japanese.

Autoimmune thyroid disease (AITD), including Graves' disease (GD) and Hashimoto's thyroiditis (HT), is caused by multiple genetic and environmental factors. The clinical and immunological features of GD and HT are distinct; however, there are multiplex families with both GD and HT, and cases in which GD evolves into HT. Thus, there may be specific susceptibility loci for GD or HT, and common loci controlling the susceptibility to both GD and HT may exist. A genome-wide analysis of data on 123 Japanese sib-pairs affected with AITD was made in which GD- or HT-affected sib-pairs (ASPs) were studied to detect GD- or HT-specific susceptibility loci, and all AITD-ASPs were used to detect AITD-common susceptibility loci. Our study revealed 19 regions on 14 chromosomes (1, 2, 3, 5, 6, 8, 9, 10, 11, 12, 13, 15, 18 and 22) where the multipoint maximum LOD score (MLS) was >1. Especially, chromosome 5q31-q33 yielded suggestive evidence for linkage to AITD as a whole, with an MLS of 3.14 at D5S436, and chromosome 8q23-q24 yielded suggestive evidence for linkage to HT, with an MLS of 3.77 at D8S272. These observations suggest the presence of an AITD susceptibility locus at 5q31-q33 and a HT susceptibility locus at 8q23-q24.

Quantitative measurement of thyroglobulin mRNA in peripheral blood of patients after total thyroidectomy.

Previous studies have reported the clinical usefulness of reverse transcription-polymerase chain reaction (RT-PCR) detection of thyroglobulin (TG) mRNA in the peripheral blood of patients with differentiated thyroid carcinoma. To evaluate this usefulness, we measured TG mRNA in the peripheral blood of patients diagnosed with thyroid carcinoma after total thyroidectomy by real-time quantitative RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. Surprisingly, we detected TG mRNA in all samples obtained after total thyroidectomy, including those from 4 medullary carcinomas. Further, there was no statistical difference in expression levels of TG mRNA in the patients with or without metastasis, and no significant correlation was found between serum TG concentrations and the expression levels of TG mRNA. These results give rise to a question regarding the clinical applications of not only RT-PCR detection but also quantitative measurement of TG mRNA in peripheral blood.

[Extremely high level of serum thyroglobulin after surgery in a patient with follicular thyroid carcinoma. Recurrence, distant metastasis, or other etiology?].

A 67-year-old female patient with a tumor of the thyroid underwent right lobectomy of the thyroid. The tumor was histologically diagnosed as a follicular carcinoma of the thyroid. Serum studies revealed very high (> 8,000 ng/ml) levels of thyroglobulin after surgery. We suspected distant metastases from follicular carcinoma or recurrence in the left lobe of the thyroid. She therefore underwent left lobectomy of the thyroid. No recurrence of follicular carcinoma was recognized in the resected thyroid tissue, but severe autoimmune thyroiditis was diagnosed histologically. Total-body 131I scintigraphy did not show abnormal distant uptake. Serum studies revealed very low levels of thyroglobulin (< 2.0 ng/ml) after the second surgery. We speculate that silent thyroiditis might have occurred after the first surgery, resulting in the high levels of serum thyroglobulin. Silent thyroiditis should be considered as a possible cause of high serum thyroglobulin levels.

Large-scale analysis of mutations in RET exon 16 in sporadic medullary thyroid carcinomas in Japan.

Germline mutations in the RET proto-oncogene are the cause of multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Some cases of sporadic medullary thyroid carcinoma (MTC) have also been reported to have mutations in the RET gene. However, two previous reports have given discrepant results on the frequency of the mutations in RET in sporadic MTCs in Japan. To clarify this problem, we analyzed mutations in RET exon 16 in 72 sporadic MTCs by means of the two methods used in the previous studies, direct sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Mutations in exon 16 were detected in only 2 of 72 cases of sporadic MTC. These results suggest that when a MTC has a mutation in RET exon 16, it is more likely to be a hereditary MTC than a sporadic one in Japan.

Overexpression of kalpha1 tubulin mRNA in thyroid anaplastic carcinoma.

The expression levels of (k)alpha1 tubulin in 84 benign and malignant thyroid tissues were measured by means of real-time quantitative reverse transcription-polymerase chain reaction. An increased expression of (k)alpha1 tubulin mRNA was observed in all of five anaplastic carcinomas and some of the papillary carcinomas. Expression levels of (k)alpha1 tubulin relative to thyroglobulin mRNA were slightly increased in papillary carcinomas and greatly increased in anaplastic carcinomas. Chemotherapeutic agents which are targeted to microtubules may be considered as an alternative choice for the treatment of anaplastic carcinomas and some differentiated carcinomas in which increased expression of (k)alpha1 mRNA is observed.