Cathepsins B, H, and L, representative lysosomal cysteine proteinases, have been shown to be involved in the degradation of proteins, generation of bioactive proteins, antigen processing, etc. Recent biochemical as well as Immunohisto/cytochemical studies have demonstrated that these enzymes are transferred from the trans Golgi network to lysosomes of cells, and in some cases, to secretory granules of certain endocrine cells, or they are secreted from cells. Localization of these enzymes in lysosomes differs depending on cell types even in the same tissues, suggesting that expression of these enzymes is regulated corresponding to cell specialization. Cathepsins B and H are localized in secretory granules of some peptide hormone-producing cells; particularly, cathepsin B is co-localized with renin in various endocrine cells producing active renin, indicating that cathepsin B is a major candidate of the renin converting enzyme. Moreover, these cysteine proteinases are secreted as their pro- or active forms from various tissue cells. In the resorption lacuna facing osteoclasts, secreted cathepsin L has been suggested to play a major role in the degradation of organic bone constituents, particularly collagen. Thus cathepsins B, H, and L act as biomodulators in various cells and tissues. In the present review, we introduce the precise localization of cathepsins B, H, and L and discuss their possible roles in cells and tissues.
Parvalbumin was purified from the skeletal muscle of the Mongolian gerbil (Meriones unguiculatus) and used to raise a polyclonal antiserum in a rabbit. The affinity-purified antibodies were used to study the distribution of parvalbumin in the brain of adult and developing gerbil and compare it with the distribution of cytochrome oxidase activity. In the adult brain, immunoreactivity was found in distinct subpopulations of neurons. There were densely stained areas, most of which contained immunopositive cell somata and their processes in the diffusely stained neuropil. Some highly stained areas were filled with immunopositive dot-like structures or fiber segments. Comparison of these parvalbumin-rich areas with cytochrome oxidase-rich areas revealed a close spatial correlation. In the developing brain, the spatial coincidence was still observed. In the developing cerebral cortex, parvalbumin-positive cells first appeared preferentially in the area which had had high cytochrome oxidase activity in the neuropil. At the cellular level, cytochrome oxidase staining often condensed in a part of the cell body before whole cell bodies became stained. Thus, the adult pattern of spatial coincidence appears, in at least some areas, to be generated in several stages.
A type of perivascular cell (fluorescent granular perithelial cell; FGP cell) found around the blood vessels of the rat brain contains granules which show auto-fluorescence in the cytoplasm. In this study, the localization of lipids and low-density lipoprotein (LDL) in these FGP cells was investigated cytochemically and immunocytochemically in order to determine the functions of FGP cells. The cerebellar cortexes of male Wistar rats, aged 8 weeks to 30 months, were used. Triglyceride (TG), phospholipid (PL) and cholesteryl ester (CE) were revealed in FGP cells by using enzymic digestive methods. Endocytotic vesicles containing TG, PL and CE were located inside the FGP cells and were fused with granules. G1, G3, G4 and foamy G5 granules had TG, PL, CE and LDL in and around their membranes. G2 granules, with lower electron density than G1 granules, contained TG, PL, CE and LDL in their heterogeneous matrices. Lipids appeared to occur in granules as a lipoprotein. The results suggest that FGP cells take up TG, PL and CE by endocytosis and internalize them into these granules as components of LDL, playing the role of a scavenger macrophage in the perivascular space of the central nervous system.
The localization of estrogen receptor in normal human pituitary glands and in human pituitary adenomas was investigated using immunoreactivity to polyclonal antibodies against human estrogen receptor in five normal pituitary samples and 45 specimens of pituitary adenoma. The relationship between the expression of estrogen receptor and the production of pituitary hormones was clarified using double immunohistochemical staining. Estrogen receptor was present in both cells of normal pituitary glands and pituitary adenomas. In normal pituitary glands, adenohypophysial cells were immunoreactive for estrogen receptor irrespective of hormone production. In pituitary adenomas, 35 of 45 specimens showed positive immunoreactivity to estrogen receptor. All cases of lactotroph cell adenoma, corticotroph cell adenoma, gonadotroph cell adenoma, and plurihormonal adenoma with clinical signs of acromegaly showed positive immunoreactivity to estrogen receptor. The present study demonstrates the intracellular localization of estrogen receptor in normal adenohypophysial cells of human pituitary glands and in human pituitary adenomas.
As an alternative method to 3H-thymidine radioautography, immunohistochemical staining against incorporated bromodeoxyuridine (BrdU), an analogue of thymidine, was performed on rat stomach and small intestine organs embedded in Lowicryl K4M resin. After the administration of BrdU to animals intraperitoneally, organ tissues were removed and processed in a routine manner for resin embedding procedures. Semithin and ultrathin serial sections were cut and incubated in mouse anti-BrdU monoclonal antibody and stained with anti-mouse IgG or protein A coupled with gold colloid. Semithin sections were further intensified by physical development using silver acetate as an ion donor. In the electron microscopic labeling, pretreatment with hydrogen peroxide increased the labeling intensity. The findings from both light and electron microscopic observation were compared and a good correlation was obtained. Various labeling patterns were found in S-phase cell nuclei from stomach and small intestines in vivo. Labeling patterns of nuclei matrix were divided into five subtypes according to labeling patterns at the electron microscopic level.
Epitopes of the Na, K-ATPase α subunit were mapped on isolated, unfixed plasma membranes by double immunolabeling electron microscopy with protein A gold and negative staining. In this study we have defined several factors, which influence the labeling of the epitopes with sequence-specific antibodies. The results demonstrate that simultaneous labeling with low and high affinity antibodies can be achieved with a labeling sequence consisting of low affinity antibody-small gold probe-high affinity antibody-large gold probe, and that incubation with free protein A is essential for masking cross-contamination caused by attachment of the second probe to unoccupied Fc portion of the first antibody. Equal labeling was obtained on membranes without fixation and after fixation with 4% formaldehyde. An estimate of labeling efficiency showed that 1.3% of the total α subunit N-termini in p21 Na, K-ATPase membrane crystals could be detected with a 5nm gold probe. The present results define optimal conditions for the application of double immunonegative staining to isolated membranes.
A 40KDa zinc-binding protein (ZBP), ZBPP-2, was purified to homogeneity from rat intestinal mucosa by a combination of zinc-chelating affinity column, hydroxyapatite column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electroelution. This protein was deleted in intestinal crypts after dithizone-treatment of rats, as we reported previously for ZBPP-1, a 90KDa ZBP. Four independent mouse monoclonal antibodies (mAbs) to purified 40KDa ZBP (all μκ isotype) were prepared. In addition to 40KDa ZBP, these mAbs detected several higher molecular size ZBP in Western blot. Although non-zinc binding protein fraction seemed to contain abundant antigens reactive to anti-ZBPP-2 mAbs, immunohistochemistry of rat intestine with these mAbs selectively stained Paneth cells. Moreover, the staining disappeared 6hr after dithizone-treatment and resumed after 3 days as Paneth cells regenerated. Accordingly, the ZBPP-2 was another unique marker for Paneth cells.
The atrophic effects of two synthetic steroidal anti-androgens, chlormadinone acetate (CMA) and TZP-4238, on the adrenal gland of rats were investigated by histopathological and immunocytochemical procedures. Male Sprague-Dawley rats were divided into four experimental groups. Group 1 consisted of intact controls. Groups 2 and 3 received TZP-4238 10mg/kg/day and CMA 50mg/kg/day p. o., respectively, for 3 weeks. Group 4 received distilled water containing 2% Tween 80 instead of TZP-4238 or CMA. CMA caused marked atrophy of the adrenal gland. Histopathologically, the remarkable atrophy was observed in the adrenal cortical cells of zonae fasciculata and reticularis. Intracellular localization of glutathione-peroxidase (GSH-PO) which effectively reduces the lipid peroxides, was mainly observed in the cytoplasmic matrix (cytosol GSH-PO) near the mitochondria or lipid droplets. In ddition, immunoreactivity of intramitochondrial GSH-PO (mitochondrial GSH-PO) was less than that in the controls. Androgen receptor (AR) was localized exclusively in the nuclei of the adrenal cortical cells. In CMA-treated rats, AR was localized also in the nuclei of the adrenal cortical cells of the atrophic cortical zones. In contrast, TZP-4238 exerted no effect on the adrenal gland under the present experimental conditions. Therefore, it is suggested that TZP-4238 have a less inhibitory influence than CMA on the pituitary-adrenal axis. We further speculated that adrenal cortical cells of the atrophic zones induced by CMA treatment was decreased in corticosterone production and increased or remained in adrenal androgen production.