Among a total of 60 fluorochromes synthesized, 2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate (HNPP) was found to be the most suitable for detection of membrane-bound DNA; Hind III-fragments of lambda DNA were electrophoresed, transferred to nylon membrane, hybridized with digoxigenin-labeled probes, treated with phosphatase-labeled antibody and then incubated with HNPP as a substrate. The fluorescent reaction product is insoluble in water and precipitates in situ. Under UV illumination (302nm), bands containing as small as 70fg (2.5×10^-20mol) of DNA were detected after 2hr of incubation. A higher sensitivity level of 10fg was achieved in spot tests. This enables us to detect single copy genes with less than 1μg of genomic DNA in southern blot hybridization. The reaction product is soluble in dimethylformamide, so that reprobing is possible after 1min of immersion of membranes in dimethylformamide at room temperature. The HNPP method thus appears to be useful for detection of subpicogram quantities of DNA.

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Immunohistochemical features of mucoepidermoid carcinoma (19 cases) in human salivary glands were studied by means of the detection of polymorphic epithelial mucin (PEM) assessed with the MAM-3 antigen using MoAbs 67D11 and 115G3, and the MAM-6 antigen using MoAbs 115D8 and 115F5. Immunohistochemically, the distribution of PEM was divided into three types; 1) cell membrane positive type, 2) luminal surface positive type, and 3) tumor cell cytoplasm positive type. Epidermoid tumor cells exhibited the distribution of the cell membrane positive type for MoAb 115G3 staining, however, lacked such a distribution for MoAb 67D11. Intermediate tumor cells showed a staining of luminal surface positive type with MoAb 115F5 and 115D8, negative staining for MoAb 67D11, and an irregular positive staining for MoAb 115G3. Mucin producing cells lacked PEM staining, but reacted strongly with the PAS reagent. Tumor cells of high grade malignancy displayed a heterogeneity for PEM expressions, in particular for MoAb 115G3 staining. The highest intensity of MoAb 115G3 staining appeared in tumor cells intermingled with negative tumor foci. The identification of the MAM-6 antigen showed a distribution of the irregular tumor cell cytoplasm positive type. The histogenesis of mucoepidermoid carcinoma in human salivary glands was implicated in terms of the immunohistochemical assessment of PEM detection.

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Creatine kinase (CK) is a dimer composed of two immunologically distinct subunits, CK-M and CK-B, and takes part in the production of adenosine triphosphate. In the present study, the distribution of CK in the muscles and the nervous system was almost identical to that reported in older fetuses. However, in the epithelia of trachea and esophagus, CK-B immunoreactivity was apparent only in young embryos and decreased with the age of the embryo, while CK-M immunoreactivity was absent or weak in early stages and increased thereafter. In the surface ectoderm, CK-B was not detected immunohistochemically while CK-M was evident in early embryos and decreased as the embryonic stage advanced. Among the ectodermal derivatives, the lens placode was immunoreactive only to CK-M antibody at stage 13. This immunoreactivity decreased and finally disappeared along with the formation of the lens vesicle. In the urogenital system, mesonephric tubules were immunoreactive to both CK-B and CK-M antibodies, while mesonephric ducts were immunoreactive only to the CK-M antibody. Mesonephric glomeruli were not immunostained with these antibodies. These results in the urogenital system were found in all embryos examined. The present results suggest that the distribution of CK subunits in several organs of earlier embryos differs not only from that in adults, but also from that in older embryos and fetuses.

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To examine the effects of superoxide dismutase on embryonic development after the implantation stage, mouse blastocysts were cultured under low-(5%), standard- (20%), and high-oxygen (40%) conditions with or without superoxide dismutase (SOD), which is a potent scavenger of superoxide radicals. Under 5% O₂ conditions, the rates of formation of two germ cell layers and egg cylinder were 61.5% and 43.6%, respectively. These rates did not differ significantly from values determined in embryos cultured under 20% O₂ conditions (69.7% and 51.3%, respectively). However, under 40% O₂ conditions, the respective two germ cell layers and egg cylinder rates of formation were significantly lower than those under 5% and 20% O₂ conditions (31.7% and 27.2%). Addition of 5μg/ml SOD resulted in no significant difference in formation rates of two germ cell layers (71.3%) and egg cylinder (52.9%) from those of the SOD-free group (61.3% and 45.2%). However, both rates were significantly decreased at SOD concentrations of 10μg/ml or greater. Microscopic examination of the morphology of cultured embryos showed that the degeneration of the inner cell mass (ICM) occurred follwing its migration from the trophetoderm and direct exposure to medium supplemented with 500μg/ml SOD. These results indicate that excessive oxidative stress exerts adverse effects on embryonic development after implantation. Embryos which are exposed to such stress after the implantation stage are in turns more resistant to these effects than those observed at the pronuclear stage, which is known to proliferate under 5% O₂ conditions. Moreover, it is suggested that higher concentrations of SOD in the medium may diminish the concentration of superoxide anion radicals in ICM during differentiation, thus causing the degeneration of ICM.

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The patterns of 892 synapses with participating GABA elements were examined in the rat anterior cingulate cortex, using a preembedding immunohistochemical method. Somas and dendrites of pleomorphic GABA neurons received both GABA and unstained terminals at a ratio of 2 to 5, forming axo-somatic and axo-dendritic synapses. Some GABA terminals displayed axo-axonic contacts with either GABA or unstained terminals at an almost equal ratio. Occasionally, two GABA elements forming a synapse in turn showed further contacts with common or separate postsynaptic elements to constitute large synaptic complexes. Triadic arrangement of GABA terminals was noted adjacent to a nonGABA terminal forming an axo-dendritic synapse. The other GABA terminals formed synapses with nonGABA pyramidal cells on their apical dendrites, somas and axon initial segments. The majority of these GABA terminals, including small round vesicles, displayed symmetric contacts. A quarter of the examined synapses were found to be composed of contacts between two GABA elements. The high incidence of synapses between two GABA elements suggested the presence of a particular local network of GABA system in a restricted cortical area.

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The distribution of ecdysteroid binding sites in the hypodermal tissue of crayfish (Procambarus clarkii) was examined autoradiographically in correlation with the molting stage. The radiolabeled hormone analogue ponasterone A (25-deoxy-20-hydroxyecdysone) and thaw-mount autoradiographic techniques were used. These techniques enabled us to prevent the dislocation of soluble ponasterone A. Ecdysteroid binding sites were demonstrated only at certain molting stages, the small gastrolith period (Proecdysis D₀ to D₂ early). Ponasterone A binding sites appeared in nuclei and cytoplasm of epidermis, Leydig cells and pillar like cells through the stages. The notable appearance of nuclear ecdysteroid binding sites at proecdysis stage D₀ coincided with the induction of calcium reabsorption from the exoskeleton, suggesting a strong correlation of the two events. The findings also suggest that the cells involved in calcium transport are targets for ecdysteroids.

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We investigated the specific expression of protein kinase C (PKC) in primary human adrenal tumors resected surgically, using type-specific monoclonal antibodies MC-1a, -2a and -3a. Included were adrenocortical adenomas (Cushing's syndrome, Conn's syndrome and clinical nonfunctioning), pheochromocytoma and neuroblastoma. Immunoblotting experiments revealed that the adrenal cortex, medulla, adrenocortical adenomas and pheochromocytoma contained type III isozyme as the major subtype, whereas the neuroblastoma expressed both types II and III isozymes. PKC specific antibodies recognized two bands with a molecular mass of 79 and 81kDa. Immunoreactivity was mainly present in the cytosolic fraction of the adrenal cortex, medulla and adrenocortical adenomas. The translocation of PKC from cytosol to particulate membrane fractions was observed in cases of pheochromocytoma and neuroblastoma. Microscopically, strong immunostaining was present in the zona fasciculata to reticularis of the adrenal cortex, while the zona glomerulosa and medulla demonstrated minimal staining. The intermediate-type and compact-type cells of adrenocortical adenomas gave a strong positive reaction. The MC-2a positive cells of neuroblastoma were limited to the perivascular area. Immunoblotting and electron microscopic observations of the adrenocortical and adenoma cells showed immunoreactive products on the endoplasmic reticulum, mitochondria and plasma membrane, with a different intensity. In addition to the hypothesis that PKC may play an important role in the regulation of steroidogenesis and catecholamine secretion, our results indicate that the localization of PKC differs in tumors derived from adrenocortical and medullary cells. The possibility that the expression of type II isozyme in neuroblastoma may be associated with neural differentiation would have to be considered.

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Our objective was to evaluate a method that incorporates a freezing and thawing process to detect cytochrome c oxidase in normal mitochondria in myogenic cell lines in monolayer culture, without the loss of enzyme activity. We used electron microscopy to detect and investigate cytochrome c oxidase activity. When cell preparations were subjected to a freezing and thawing process after being fixed, 91.4% of mitochondria were positively stained. No positive staining was obtained in cells treated by conventional procedures without the freezing process. This method facilitates the positive staining of mitochondria and should be useful in analyzing pathological conditions of mitochondria in cultured cells.

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An immunohistochemical study was carried out to determine the localization of recoverin-like Ca²⁺-binding protein (P23k) in mouse brain. P23k was found in pyramidal neurons in the cerebral cortex and hippocampus, granule cells in the cerebellum, periglomerular cells in the olfactory bulb, and neuronal cells in the paraventricular nucleus of the hypothalamus and the posterior mamillary nucleus. In most cell types, P23k was localized mainly on the plasma membrane, and partly in the cytosol. Since P23k has a Ca²⁺-dependent membrane-binding property, the difference in the subcellular localization of P23k seems to be caused by the variety of intracellular Ca²⁺-levels in the positive cells. The wide range of positive cell types implies that P23k may have a wide range of physiological functions.

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