Akira Kawaoi

PEROXIDASE-LABELED ANTIBODY A NEW METHOD OF CONJUGATION'

A new method of conjugating horseradish peroxidase with proteins was developed. The carbohydrate moiety of fluorodinitrobenzene-blocked peroxidase was oxidized with sodium periodate to form aldehyde groups. The peroxidase-aldehyde was then bound to free amino groups of proteins unidirectionally at high efficiencies. Peroxidase-labeled immunoglobulin retained its immunologic as well as enzymatic activities.

Expression of thyroid transcription factor-1 in normal and neoplastic lung tissues.

The expression of thyroid transcription factor-1 in normal and neoplastic tissues and cell lines of the human lung was investigated using immunohistochemistry and in situ hybridization in conjunction with reverse transcription polymerase chain reaction. In normal lung tissues, immunoproducts of thyroid transcription factor-1 were observed in the nuclei of alveolar cells and bronchiolar cells. Interestingly, in distal bronchioles, immunohistochemistry and in situ hybridization revealed that thyroid transcription factor-1 was present not only in nonciliated cells (Clara cells) but also in ciliated cells and basal cells. In neoplastic tissues, thyroid transcription factor-1 was demonstrated in adenocarcinomas and small cell lung carcinomas with high frequency: 96% and 89% of cases, respectively. Thyroid transcription factor-1 was not detected in squamous cell carcinomas and large cell carcinomas. The strong immunoreactivity of thyroid transcription factor-1 or simultaneous expressions of thyroid transcription factor-1 and surfactant protein A tended to correlate with the differentiation phenotypes in adenocarcinomas; they were more frequently present in the well-differentiated type than were moderately and/or poorly differentiated types. By reverse transcription polymerase chain reaction, expression of thyroid transcription factor-1 messenger RNA was observed in squamous cell carcinomas in addition to in adenocarcinomas and small cell lung carcinomas, and this finding was confirmed in the cell lines from squamous cell carcinomas. Only one case of 99 adenocarcinomas that originated in various organs other than lung and thyroid immunohistochemically expressed thyroid transcription factor-1. Our results suggest that thyroid transcription factor-1 can play an important role for the maintenance and/or differentiation process in bronchiolar and alveolar cells. Thyroid transcription factor-1 expression associates with histologic types and/or differentiation of lung cancers and can be a valuable marker for the better understanding of their biological nature and pathological behavior.

Expression of thyroid transcription factor-1 (TTF-1) in human C cells and medullary thyroid carcinomas

Thyroid transcription factor-1 (TTF-1) has been known to regulate the transcriptional activity of thyroid-specific genes in thyroid follicular cells. We recently identified TTF-1 mRNA expression in rat thyroid C cells. The current study was undertaken to elucidate how TTF-1 is expressed in human C cells and medullary thyroid carcinomas (MTCs), and how this expression influences the functions and clinical behavior of these cells. By immunohistochemistry, the nuclei of normal and hyperplastic C cells distinctively reacted with antibody against TTF-1, whereas the immunostaining intensity in C cells was rather weak and heterogeneous in comparison with that in follicular cells. Identical TTF-1 immunoreactivity was observed in all 15 MTC specimens examined. The reaction intensity did not depend on tumor patterns or cell features. In nonisotopic in situ hybridization, an antisense riboprobe clearly hybridized the cytoplasms of C cells and MTC cells, which concurrently showed immunohistochemical positivity for calcitonin. Northern blot analysis indicated a marked hybridization with TTF-1 mRNA of approximately 2.3 kb in an MTC specimen. Furthermore, the presence of TTF-1 mRNA was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in the human MTC cell line, TT. Our results suggest that human thyroid C cells and MTC cells express TTF-1 in connection with their functional ability. Therefore, TTF-1 expression can be a functional marker not only for follicular cells and follicular cell tumors but also for C cells and medullary C cell carcinomas.

Expression of vascular endothelial growth factor (VEGF) in human thyroid neoplasms

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is an angiogenic factor that plays important roles in tumor growth. Angiogenesis studies on VEGF deal with various types of malignant tumors, but very little is known about the role or significance of VEGF in human thyroid neoplasms. Therefore, in the current study, we determined whether VEGF is found in normal and neoplastic thyroids and whether its expression is altered in different histological types of thyroid neoplasms. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that all specimens of thyroid tumors expressed bands corresponding to 121-, 165-, and 189-amino acid forms of VEGF. Northern blot analysis showed an increase in VEGF mRNA levels in neoplastic tissues in comparison with normal thyroid samples. By nonisotopic in situ hybridization, most of the tumor cells in follicular adenomas expressed VEGF mRNA, whereas VEGF mRNA expression was identified only in epithelium of isolated follicles in normal thyroid tissues. In papillary thyroid carcinomas, an intense labeling with VEGF probe was often found in overlying tumor cells of neoplastic papillae. VEGF expression was distinctly intensified in undifferentiated carcinoma cells that were immediately adjacent to necrotic foci. The immunohistochemical localizations of VEGF protein were comparable to the localization of VEGF mRNA. In conclusion, our results suggest that the histological types of thyroid tumor may determine the vascular pattern through a paracrine mechanism involving VEGF.

Thyroid Transcription Factor 1 Is Calcium Modulated and Coordinately Regulates Genes Involved in Calcium Homeostasis in C Cells

ABSTRACT Thyroid transcription factor 1 (TTF-1) was identified for its critical role in thyroid-specific gene expression; its level in the thyroid is regulated by thyrotropin-increased cyclic AMP levels. TTF-1 was subsequently found in lung tissue, where it regulates surfactant expression, and in certain neural tissues, where its function is unknown. Ligands or signals regulating TTF-1 levels in lung or neural tissue are unknown. We recently identified TTF-1 in rat parafollicular C cells and parathyroid cells. In this report, we show that TTF-1 is present in the parafollicular C cells of multiple species and that it interacts with specific elements on the 5′-flanking regions of the extracellular Ca2+-sensing receptor (CaSR), calmodulin, and calcitonin genes in C cells. When intracellular Ca2+ levels are increased or decreased in C cells, by the calcium ionophore A23187, by physiologic concentrations of the P2 purinergic receptor ligand ATP, or by changes in extracellular Ca2+ levels, the promoter activity, RNA levels, and binding of TTF-1 to these genes are, respectively, decreased or increased. The changes in TTF-1 inversely alter CaSR gene and calcitonin gene expression. We show, therefore, that TTF-1 is a Ca2+-modulated transcription factor that coordinately regulates the activity of genes critical for Ca2+homeostasis by parafollicular C cells. We hypothesize that TTF-1 similarly coordinates Ca2+-dependent gene expression in all cells in which TTF-1 and the CaSR are expressed, i.e., parathyroid cells, neural cells in the anterior pituitary or hippocampus, and keratinocytes.

Purification and immunoelectron microscopic localization of cellular glutathione peroxidase in rat hepatocytes: Quantitative analysis by postembedding method

To measure quantitatively the intracellular distribution of cellular glutathione peroxidase (GPX) in rat hepatocytes, ultrathin sections were stained by a postembedding immunogold technique. GPX had a specific activity of 1670 Units/mg protein, and was purified 2050-fold from rat liver by means of heat denaturation, ammonium sulfate fractionation, and a series of chromatographic procedures including thiol-Sepharose 4B. The purified GPX was shown to be electrophoretically pure, and was a homotetramer of 22 kDa subunits. Monospecific polyclonal antibodies were raised in rabbits by immunization. By immunoblot analysis, both the light mitochondrial the and cytosolic fractions of rat liver homogenate gave a single band with an identical mobility to that of the purified enzyme. Under the light microscope, hepatocytes showed nuclear staining and granular cytoplasmic staining, corresponding to certain intracellular structures. The labeling density (number of gold particles/μm2) for GPX obtained by immunoelectron microscopy was 11.9 in the nuclei, 19.6 in mitochondria, 3.32 in peroxisomes, 1.95 in lysosomes, and 9.81 in the cytoplasmic matrix. These results suggest that cellular GPX is present in various compartments of rat hepatocytes, and that the GPX occurs in relatively higher amounts in mitochondria.

Immunohistochemical localization of superoxide dismutases in fetal and neonatal rat tissues.

We investigated the developmental profile of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in tissue sections obtained from fetal (Day 12 to 21 of gestation) and neonatal (Day 0 and 6) rats. Tissues were stained immunohistochemically with specific antisera against the respective rat SODs. There was a general trend towards richness of SODs in the epithelial linings and metabolically active sites, although differential distribution between the two SODs also existed. At Day 12 of gestation, immunoreactivity for both SODs was detected in the cardiomyocytes but not in other tissues. Hepatocytes expressed CuZnSOD at Day 14 and MnSOD at Day 17. By Day 18 CuZnSOD was detected in the epithelial cells of the gastrointestinal tract, respiratory tract, pancreatic islets, kidneys, and adrenals. These tissues exhibited MnSOD staining at Day 19. CuZnSOD occurred in the epithelia of the thyroid, thymus, and salivary glands at Day 19, while MnSOD was seen at Day 21. The increase in intensity of the staining for SODs occurred no later than postnatal Day 0, indicating that most tissues accumulated SODs during late gestation. Breathing atmospheric oxygen during early extrauterine life did not appreciably intensify the SOD staining. These results suggest that perinatal increase in SODs occurs as a general mechanism of preparation for birth.

Proliferating cell nuclear antigen (PCNA) immunostaining as an alternative to bromodeoxyuridine (BrdU) immunostaining for brain tumours in paraffin embedded sections

SummaryImmunohistochemical staining for proliferating cell nuclear antigen (PCNA) and BrdU using anti-PCNA and anti-BrdU monoclonal antibodies, respectively, was performed in 16 human brain tumours, including 3 glioblastomas multiforme, 2 anaplastic astrocytomas, 1 cerebellar astrocytoma, 2 recurrent meningiomas, 4 non-recurrent meningiomas, 3 neurinomas and 1 medulloblastoma. Patients with brain tumours received an injection of bromodeoxyuridine (BrdU) intravenously during surgery, and tumour specimens were fixed in 70% ethanol and embedded in paraffin. The percentage of positive cells for PCNA was compared with a BrdU labelling index using adjacent paraffin-embedded sections. The percentage of PCNA-positive cells was correlated with the BrdU labelling index and the histological malignancy of the brain tumours. The correlation coefficient was 0.84. This suggests that the immunohistochemical staining for PCNA in paraffin sections is a good alternative to the BrdU labelling index.

Immunohistochemical study of copper-zinc and manganese superoxide dismutases in the lungs of human fetuses and newborn infants: developmental profile and alterations in hyaline membrane disease and bronchopulmonary dysplasia

To determine the late gestational development of copper-zinc (CnZn) and manganese (Mn) superoxide dismutases (SOD) in human lung, immunohistochemical localization was performed for each SOD. The lung samples were taken from five aborted fetuses, four fetuses in which intrauterine death occurred, one full-term neonate, two premature infants with hyaline membrane disease and one premature infant with bronchopulmonary dysplasia (BPD). Morphometry was performed, and the percent area of positive staining was computed. The bronchial epithelium was intensely stained from the early stages of gestation (i.e. 17 weeks), while the staining intensity for both CuZnSOD and MnSOD in the peripheral airways increased gradually during lung development. The mean percent area of the staining for CuZnSOD and MnSOD from 16 to 38 weeks was increased 30-fold and 8-fold, respectively, and further increases were observed postnatally. CuZnSOD staining was markedly decreased in lungs with respiratory disorders. However, proliferating type II pneumocytes were intensely stained for MnSOD in the BPD lungs, making the staining area 3-fold larger than that in the control lungs. These results clearly depict age-related increases in staining for both CuZnSOD and MnSOD and an alteration in SOD distribution associated with neonatal respiratory disorders.

Primary leiomyosarcoma of the thyroid gland.

A primary leiomyosarcoma of the thyroid gland in a 72 year old Japanese woman is described. This is the second case reported in the English literature. The patient presented with a 7 month history of a gradually expanding tumor in the right neck. The surgical specimen taken by thyroid lobectomy revealed a relatively well demarcated tumor, 2X2X3 cm in size, confined to the right lobe. Histologically, the tumor showed a classical leiomyosarcomatous appearance of interlacing fascicles of spindle‐shaped cells with occasional blunt‐ended nuclei and a high frequency of mitotic figures. lmmunohistochemistry of the tumor cells clearly showed smooth muscle differentiation; the cells were positive for desmin, muscle‐specific action and vimentin and negative for cytokeratin, epithelial membrane antigen, carcinoembrionic antigen, thyroglobulin and calcitonin. The patient was free of disease for 3 years and 11 months without further treatment when evidence of multiple bone metastases appeared on bone scintigraphy. She died of pneumonia 4 years and 3 months after the lobectomy.