Monoclonal antibodies were prepared to branched-chain amino acid transferase (BAT) type I isozyme purified from rat kidney. Two stable clones producing specific antibodies to the enzyme were obtained. Specificity was confirmed by immunoblots of purified BAT after polyacrylamide gel electrophoresis and testing enzyme inhibition by the antibodies. In the immunohistochemical study, type I BAT was localized in the mitochondria of the proximal tubular cells of the rat kidney. In the liver, reaction was observed in the mitochondria of parenchymal cells. The cells surrounding the central veins exhibited strong immunoreaction. Immunoelectron microscopy using a rapid freezing and substitution technique revealed type I BAT localization in the mitochondria matrices of rat liver parenchymal cell. A faint reaction was present in the cytoplasm. Immunoblot analysis of mitochondrial and post-microsomal fractions from rat liver and kidney also indicated that the enzyme is chiefly present in the mitochondria.
The monoclonal antibodies we prepared may provide a useful tool for studying changes in both isozyme pattern and localization of type I isozyme in the prenatal and neonatal rat and in tumor cells.

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The distribution of immunoreactive (ir-) atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was studied immunohistochemically and immunoelectronmicroscopically in the heart of the newt, Cynops pyrrhogaster. The difference in immunostaining for ANP and BNP indicated that the heart of the newt contains two peptides, and that they are immunologically related to mammalian ANP and BNP, respectively. Immunoreactivity for ANP was stronger than that for BNP in the atrium, postcaval vein and sinus venosus of all specimens, while immunoreactivity for BNP was similar to or stronger than that for ANP in the ventricle of most specimens. Ir-ANP and ir-BNP appeared to be colocalized in many cardiocytes in the atrial wall and in some of ventricular cardiocytes. The double protein A-gold labeling technique revealed the granules showing immunoreactivity only for ANP and those showing only for BNP in the same cardiocyte of the atrium and ventricle. In addition, there were some granules showing coexistence of ir-ANP and ir-BNP.

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The activity of cytochrome oxidase in photoreceptor inner segments of copper deficient rats was investigated by the diaminobenzidine method with electron microscopy. The deposits of reactive products resulting from oxidation in the mitochondrial intermembrane-intracristal space were measured in the computed image analyzer. Copper deficient rats showed neurological disorders and significantly low levels of serum copper throughout the experiment. The light microscopic findings showed that stainability for cytochrome oxidase in the retinal photoreceptor inner segment was less in the copper deficient rats than in the control rats. Ultrastructurally, DAB-OsO₄ reaction products were diffusely located on the inner membranes and in the intracristal spaces of mitochondria in the control rats. Depositions of DAB in the copper deficient rats were insignificant and occupied a narrow area of the intracristal space, although the mitochondria showed no morphological changes. It was concluded that copper deficiency reduced cytochrome oxidase activity in the photoreceptor inner segments without morphological changes of the mitochondria.

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The short-term axotomy induced changes in both neurofilament triplet protein (NFP) and neuronspecific enolase (NSE) immunoreactivites (IR) were studied in the rat lumber (L_4>-L₆) dorsal root ganglia (DRG) 1, 3, 7 and 14 days after sciatic nerve transection. Control DRG showed NFP IR only in the axons whereas perikarya were unlabeled. The axotomy causes the presence of NFP IR in a subpopulation (mainly large and intermediate) of primary sensory neurons and increase in a time-dependent manner in both the number (1.3% at 3 days to 11.8% at 14 days) and the intensity of IR (188.8±26.4 at 3 days to 153.9±33.8 at 14 days) for NFP IR. However, no morphological evidence of chromatolysis was observed in NFP IR perikarya. On the other hand, all the primary sensory neurons from control DRG were NSE IR with a variable intensity of staining which was not related to the neuron size. No significant changes were observed in NSE IR of the large and intermediate sized neurons, and a non significant decrease in IR intensity of the small sized ones was noticeable for all the axotomized DRG. These results suggest that short-term axotomy induces perikaryal accumulation of NFP in a subpopulation of primary sensory neurons (mainly large and intermediate in size), while it did not modify apparently the NSE-IR.

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The distribution pattern of epidermal growth factor receptor (EGFR) was investigated in paraffin sections of squamous epithelial lesions, including benign and malignant lesions in the oral mucosa and skin. Normal oral epithelium showed a distribution of cell membrane positive type of EGFR staining; whereas, keratinized cells were negative. Benign lesions, papilloma, condyloma, and keratoacanthoma, also showed similar distribution of cell membrane positive type. Leukoplakia in the oral mucosa, senile keratosis and Bowen's disease displayed a mixed pattern with cell membrane positive type (CMPT) and focal cell membrane positive type (FCMPT). Paget's cells in Paget's disease showed whole plasma positive type distribution, as non cell membrane positive type. Squamous-cell carcinoma in the oral cavity indicated a reduction of EGFR staining. Distribution profiles were divided into 3 patterns: CMPT, FCMPT, and non cell membrane positive type (NCMPT), and occasionally, mixing of 2 or 3 patterns was found in the same squamouscell foci. Histologic, keratinized or malignancy grades in squamous cell carcinomas were not directly related to EGFR expression and distribution.

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The presence of laminin and type-IV collagen in the basement membrane of the rat incisor enamel organ at different stages of odontogenesis was demonstrated by means of pre-embedding immunoperoxidase method.
Immunoreaction for laminin and type-IV collagen was observed in the basement membrane and in the fibrillar layer attached to the membrane demarcating the inner enamel epithelium from the dental papilla. Occasional reaction products for laminin and type-IV collagen localized in the rough endoplasmic reticulum cisternae in cells of the inner enamel epithelium and dental papilla indicated cooperative production of basement membrane components. The basement membrane of differentiating ameloblasts showed reactions for laminin and type-IV collagen, though the intensity was consistently lower than that of reactions in the inner enamel epithelium. These reactions gradually became indistinct and discontinuous and disappeared entirely prior to dentin formation. At this time, positive reaction products were observed in the deep, irregular invaginations and coated pits in the distal ends of differentiating ameloblasts, indicating resorption of basement membrane components. The basement membrane-like structure appearing between the maturing ameloblasts and the enamel surface reacted positively for laminin. Occasional occurrence of reaction products in rough endoplasmic reticulum of maturing ameloblasts indicated laminin production. No reaction products for type-IV collagen, however, occurred in the basement membrane-like structure. Positive immunoreactions were observed in the basement membrane demarcating the outer enamel epithelium and the papillary layer from the dental sac. Basement membranes of blood capillaries that had penetrated into the papillary layer also showed intense immunoreaction. Reaction products were frequently seen in the perinuclear spaces and the rough endoplasmic reticulum cisternae of outer enamel epithelium cells but not of papillary layer cells.

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Lansoprazole (AG-1749) is a potent inhibitor of (H⁺K⁺) -ATPase and suppresses gastric acid secretion in the rat. The present study was conducted to examine the localization of lansoprazole in the gastric mucosa in the rat. Rats were given³H-labeled lansoprazole orally and sacrificed 1, 8 and 24 hr later. The distribution of the compound in the gastric mucosa was then chronologically examined by microradioautography. With light microscopy, the silver grains due to ³H were observed exclusively over the parietal cells in the gastric mucosa. Most of the grains were found over the cytoplasmic canaliculi in the parietal cells. There was no distinctive deposition of silver grains over other components such as chief cells, capillary endothelial cells or cells in lamina propria in the gastric mucosa. The number of silver grains over the parietal cells was greatest in the rat sacrificed 8 hr after administration, and was reduced 24 hr after administration. Electron microscopic observations revealed most of the silver grains localized over the microvilli of intracellular secretory canaliculi in the parietal cells. These results suggest that orally-administered lansoprazole binds to secretory membrane, possibly (H⁺K⁺) -ATPase in the parietal cells and remains there for more than 24 hr, which may explain the fact that lansoprazole shows long-lasting inhibition of gastric acid secretion in rats.

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Localization of basic fetoprotein (BFP) as well as its dynamics in malignant, normal tissue and precancerous lesions of the female genital organs were investigated. For light microscopy, conventionally processed paraffin sections were stained by the avidin-biotin peroxidase complex method. For electron microscopy, 4% paraformaldehyde fixed frozen sections were subjected to an indirect enzyme antibody method. As the results, BFP positive cases were 74% in ovarian carcinomas, 77% in endometrial carcinomas and 100% in cervical carcinomas. With respect to the localization of BFP in malignant, normal and precancerous cells, diffuse granular localization was observed in the cytoplasm of BFP positive cells. BFP was localized in the ribosomes of cells with well-developed organella, electron microscopically, and figures of dialyzing secretion were observed. Based on these findings, BFP was found in dedifferentiated cells still retaining the original structure and/or properties. This suggests BFP is a functional protein secreted by dialyzing pattern, and may become a good tumor marker of gynecological malignancies.

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To examine whether or not a part of human parathyroid adenomas have a malignant potential, 15 parathyroid tumors including 13 adenomas, 1 hyperplasia and 1 adenocarcinoma were studied for the amplification of the c-erbB2 proto-oncogene. Eleven adenomas of these tumors were also evaluated for the immunohistochemical expression of the c-erbB2 gene product. The amplification of the c-erbB2 proto-oncogene (2-fold or more greater than control) was recognized in 3 adenomas (23.1% of all adenomas) and 1 adenocarcinoma as compared to a pooled human normal placenta, which serves as control. Six out of 11 parathyroid adenomas (54.5%) revealed the immunohistochemical expression of the c-erbB2 gene product on the cell membrane and in the cytoplasm. Three adenomas (27.2%) showed the strongly positive (_??_) staining and the other 3 adenoma (27.2%) showed the weakly positive (+) staining. The 2 cases having the amplification of the c-erbB2 proto-oncogene also revealed the strongly positive staining. The c-erbB2 proto-oncogene, which has been reported to be amplified or expressed in some adenocarcinomas, might be associated with the initiation and progression of some parathyroid adenomas. The present findings might suggest that some parathyroid adenomas could have a malignant potential.

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Immunohistochemical expression of Interleukin-2 receptors (IL2R) was examined by an APAAP method in the peripheral blood mononuclear cells (PBMC) of 16 patients with chronic active hepatitis (CAH), 10 patients with chronic persistent hepatitis (CPH), and 15 normal controls, treated in vitro with HBsAg, ConA, or PHA for 48 hr. The percentage of IL2R-positive cells was significantly lower in the PBMC of CAH and CPH subjects treated with HBsAg or ConA, but not so with PHA, than that in controls (p<0.01). No significant differences were observed between CAH and CPH subjects. IL2R-Positive cells were surrounded with many (7-12) IL2R-negative mononuclear cells following treatment with HBsAg, and this observed phenomenon was termed an IL2R-positive cell “rosette”. The prevalence of rosette formation was much higher in controls than that in CAH and CPH subjects (p<0.01). Typical rosette was not observed following treatment with ConA or PHA. The abnormal IL2R expression observed suggested a connection with the poor immune response of patients with chronic hepetitis B.

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In this study, the presence of elongated thread- like lysosomes (nematolysosomes) was demonstrated cytochemically in large pyramidal cell of cerebrum, Purkinje cell of cerebellum and motor neuron in anterior horn of spinal cord when these cells were fixed with low concentrated glutaraldehyde fixatives in PIPES buffer. The acid phosphatase (AcPase; one of lysosomal marker enzymes) activity was mainly distributed in condensed vacuoles and stacks of well-developed Golgi apparatus and in lysosomes in the neurons. However, we have also found that AcPase activity was present not only on ordinary round lysosomes but also on the nematolysosomes. The nematolysosomes with positive AcPase activity were observed in the cytoplasm of the neurons of the central nervous system. The motor neurons have more nematolysosomes which may extend into the processes. In these cells there were many microtubules and microfilaments. This may suggest that the nematolysosomes are related to the AcPase transport in the neurons.

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