Previously, we perfused the lungs of several mammals (rat, mouse, guinea pig and hamster) with the detergent-containing medium and found that abundant filaments were present in apical caps of nonciliated bronchiolar epithelial (Clara) cells. These filaments were bound to the plasma membrane of the apical cap at one end and ran into the interior of the cytoplasm at the other end. In the present study, we used heavy meromyosin (HMM) and an immunohistochemical method to identify these filaments in rat Clara cell. Most filaments in apical caps bound HMM, but some filaments were not decorated with HMM. To examine the latter filaments, paraffin sections were stained immuno-histochemically utilizing the antibodies against intermediate filament proteins. Apical caps were positive for cytokeratin but were negative for other intermediate filament proteins, vimentin, desmin, neurofilament protein and glial fibrillary acidic protein. These results suggested that filaments in apical cap of nonciliated bronchiolar epithelial (Clara) cells were composed of actin and cytokeratin.
The precise localization of aromatase, the estrogen synthesizing enzyme, in the ovary of golden hamster, guinea pig and cow were immunocytochemically studied using anti-human placental aromatase cytochrome P-450 antisera. The positive reaction for aromatase was detected mainly in the granulosa cells of large preovulatory follicles of all species studied. In addition, some cells of corpora lutea showed weak positive reaction. The granulosa cells of smaller follicles, theca interna cells, oocytes and peritoneal epithelial cells were entirely negative to the immunostaining. The intensity of the immunoreaction to the anti-aromatase antibody was stronger in the hamster ovary than in the ovaries of the guinea pig and cow. Electron microscopic examination revealed that the positively stained granulosa cells of hamster had well developed smooth endoplasmic reticulum. The present results and our previous data obtained in rats and mice ovaries suggest that the appearance of aromatase in the granulosa cell of the preovulatory follicle is a general phenomenon in many species of mammals.
D-galactosamine was injected i. p. into rats at a single dose level of 250mg/kg. Basophilic hepatocytes and their mitotic forms were observed 48hr and 72hr after the administration of the amino sugar. At these stages, the biochemical activities of alkaline phosphatase (ALPase) and γ-glutamyl transpeptidase (γ-GTP) in rat liver homogenate increased significantly in comparison to those in the control rats. In the histochemical analysis, 48hr and 72hr after administration of the amino sugar, a high level of activity of both ALPase and γ-GTP was seen along the cell borders between adjacent hepatocytes and along the entire hepatocyte surface. At the level of electron microscopy, the reaction products showing ALPase activity were observed in abundance along the complex interdigitation of the plasma membranes, which were present on the cell borders between adjacent hepatocytes.
We prepared monoclonal antibodies (McAbs) against secretory granule membrane of the rabbit parotid gland. Two McAbs, IgG2a (McAb-116) and IgM (McAb-64), which were directed to the glycoprotein of 55-60Kd which contained N-linked carbohydrate, were obtained. It was suggested that this glycoprotein was an integral membrane protein and localized on the luminal side of secretory granules. The following cells were immunostained with the McAbs: a) among exocrine cells, acinar cells of the parotid and submandibular glands, chief cells of the stomach and pancreatic exocrine cells; and b) among endocrine cells, parafollicular cells of the thyroid gland, adenohypophyseal cells, enterochromaffin cells of the digestive tract, adrenal medullary cells and juxtaglomerular cells of the kindney. These findings suggested that secretory granule specific antigen (SGSA), existed commonly in the secretory granules of both exocrine and endocrine cells. Immunoelectoron microscopy revealed that SGSA was localized exclusively along the membranes of secretory granules in the parotid acinar cells, exocrine pancreatic cells and adenohypophyseal cells. In parotid gland, SGSA was also recognized on the membranes of the Golgi lamellae and vacuoles and apical cell membranes. It is suggested that SGSA may play an important role in secretory granule formation and exocytosis in secretory cells. The two McAbs may provide a useful tool for investigating the formation, translocation and recycling of secretary granule membrane proteins.
A simple and inexpensive apparatus and techniques are described for recording fluorographs of whole-body cryosections of mice injected with a fluorescent compound. The apparatus was constructed with commercial fluorescent tubes which emit UV light of 300-400nm and a single-lens reflex camera equipped with UV cut and Kodak Wratten filters. Autofluorescence of the whole-body section observed by using this technique was discussed and the effects of lapse of time after cryosectioning, fixation, and pH on the autofluorescence were also investigated. Autofluorescence of the skeletal muscle, myocardium, smooth muscle, brain, kidney cortex and pancreas was intensified with time after cryosectioning, but that of the Harderian gland was gradually faded. In other tissues, autofluorescence remained unchanged up to 2 weeks after cryosectioning. All fixatives used in this experiment reduced the autofluorescence of tissues and organs. Among the fixatives, 100% acetone had the least effect on autofluorescence. At pH4, autofluorescence of all tissues was considerably increased in comparison with pH7. With increasing pH, autofluorescence of most tissues was gradually reduced except for the bones and the Harderian gland.
A synthetic 39-mer anti-sense strand oligodeoxynucleotide complementary to the region of human cellular yes gene (c-yes-1) mRNA that codes for a polypeptide of 543 amino acids (Mr: 60, 801) and the corresponding sense strand 39-mer oligodeoxynucleotide were chemically synthesized by β-cyanoethyl phospho-ramidite method. The probes thymine-thymine (T-T) dimerized by UV irradiation were used for in situ hybridization studies. Various cells were fixed with ethanol-acetic acid (3:1) at room temperature for 20min and hybridization was carried out in 50% formamide for 36hr at 37°C. The good signal was obtained by utilizing anti-T-T dimer serum (IgG frafction), peroxidase-labeled goat anti-rabbit IgG antibody, and diaminobenzidine-4-HCl and H2O2 as substrates. The anti-sense strand synthetic probe hybridized with cellular RNA of cultured cells with c-yes-1 mRNA, while the sense strand synthetic probe did not. The c-yes-1 mRNA was clearly detected in the cytoplasm of c-yes-1 gene product-expressing cultured cells by in situ hybridization using a T-T dimerized synthetic probe. Synthetic T-T dimerized oligodeoxynucleotides appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.
Immunoperoxidase staining patterns with a monoclonal antibody against human breast cancer cell membrane, 125B4, were investigated in frozen sections of normal tissues from adult rats of both sexes and of various mammary tumors from 7, 12-dimethylbenz (α) anthracence (DMBA)-treated female rats. In normal tissues, 125B4 preferentially recognizes myoepithelial layers of the mammary, sweat and salivary glands, basally situated epithelial cells of the skin, vagina, esophagus, urethra, urinary bladder, seminal vesicle and epididymis, and endothelial cells of blood vessels. It does not react with luminal cells of the abovementioned organs, epithelial cells of the stomach, intestine, liver, pancreas and uterus, and mesenchymal cells. The reactivity in benign mammary tumors is comparable to that in normal mammary tissue, but it is observed on some adenoma cells scattered spottedly in proliferating fibroadenoma. In malignant mammary tumors, the reactivities are not restricted to the banally localized carcinoma cells and distributed abundantly in the carcinoma foci of squamous cells carcinoma, adenocarcinoma and carcinosarcoma. Neoplastic mesenchymal cells cannot be stained with 125B4, which stains endothelial cells within the tumor stroma. Except for the endothelium, therefore, our data indicate that this antibody may react to the antigens present in basal and myoepithelial cells of rat mammary parenchyma.
Protein kinase A (PKA) was localized in the rat hippocampus by an immunohistochemcal technique. The antibody was raised against the catalytic subunit of bovine heart PKA and cross-reacted with the brain enzyme, as could be revealed by immunoblotting. The strongest reaction was detected in the pyramidal and granular cell layers and in the lacunoso-molecular layer of the hippocampus formation. The reaction in the radial layer was restricted to the dendrites of the pyramidal cells. In the other regions mainly filial cells were stained.
The protein kinase A was demonstrated at the electron microscopic level in the rat hippocampus by an immunocytochemical method. The enzyme was localized in neurons as well as in filial cells. Immunocytochemical reactions were found in the cytoplasm of the cells, on the endoplasmic reticulum and in nuclei. Strong staining was detected in synapses, in the postsynaptic thickenings and on the microtubules of dendrites. No staining was found on axonal microtubules and in oligodendrocytes. Astroglial cells were regularly stained, somata as well as processes contained reaction product.