In order to confirm the relationship between glutathione-peroxidase (GSH-PO) localization and biological significance on steroid hormone-producing organs, immunocytochemical localization of GSH-PO in adrenal gland, testis, ovary and prostate of the rat was investigated. GSH-PO was well stained in specific cells of the tissues examined, but staining intensity was altered by the func tional state of the cells. Furthermore, GSH-PO staining was modified by lipoperoxidative damage in tissues or cells. Therefore, we proposed that the pattern of GSH-PO staining should be a more sensitive and specific indicator of oxidative damage in tissues. Thus, the staining pattern of GSH-PO is thought to be a useful marker for lipid peroxidation in the cell.
I examined the localization of gamma-glutamyltranspeptidase (GGT) and alkaline phosphatase (ALP) in the rat hepatoma cell line, McA-RH 7777, using enzyme cytochemical and immunocytochemical means. Both enzymes were confined to the cytoplasm around the nuclei. Golgi markers, namely Golgi 58K protein and a substance that reacts to wheat germ agglutinin (WGA), were also localized in the same sites. When McA-RH 7777 cells were cultured in a medium supplemented with dexamethasone, GGT activity was found in the borders between adjacent cells in addition to the cytoplasm around the nuclei. Moreover, the number of microtubular fibers in the cytoplasm increased remarkably. When McA-RH 7777 cells were synchronously cultured with excess thymidine and hydroxyurea, GGT and ALP were localized during the S~G2 period in the cell cycle at the borders of adjacent cells and were also found in the cytoplasm around the nuclei. Most McA-RH 7777 cells during this period assumed a flat form with numerous cell to cell contacts. This indicates that contact between adjacent McA-RH 7777 cells promotes the translocation of GGT or ALP from the Golgi apparatus to the plasma membrane.
In this study, we observed and analyzed the sub-micron motion of interphase chromosomes in living cells, labeled with the fluorescent thymidine analogue, Texas-Red dUTP. Our approach has an advantage in that chromosomes can be analyzed with regard to the nuclear architecture. We calibrated the observed motion of fluorescence-labeled chromatin by eliminating the rotational and translational movement of living nuclei that could significantly affect chromatin motion. Mathematical analyses of chromatin motion showed that: (1) interphase chromatin in living nuclei moves randomly, and the motion is limited within a small sub-region; (2) chromatin near the nuclear envelope moves in a more limited area than does centrally located chromatin; and (3) closely situated chromatin domains move independently of each other. Random and constrained chromatin motion in living nuclei supports the concept that interphase chromatin fibers are loose, flexible and floating in the nuclear matrix, and that chromatin anchors to the backbone of chromosomes. Moreover, that the random motions of DNA domains are independent of each other suggests that interphase chromatin arranges without structurally rigid continuity. This active motion of chromatin is consistent with dynamic biological processes, requiring chromosome motility and interactions. Additionally, the dynamic properties of interphase chromosomes may be significant in the interpretation of acquired chromosomal aberrations.
Bromodeoxyuridine (BrdU) is known to cause base mispairs and DNA strand breaks during DNA synthesis, culminating in growth suppression. To know whether P53 gene plays any role in the BrdU-induced growth suppression, we continuously gave BrdU (20 μM) to the synchronized culture of human gastric cancer cell lines, MKN-45 (P53-wild type) and MKN-28 (P53-mutant), shortly after release from the G1/S block by hydroxyurea. In comparison with the control culture, the growth of MKN-28 was not suppressed until 48 hr of BrdU exposure, while that of MKN-45 was already suppressed at the 24 hr point. Continuous exposure to BrdU caused a S-phase delay and G2 arrest of around 6 hr each in MKN-45 and a delay only of the second S phase in MKN-28 in comparison with synchronized control cultures of matched cell cycle phase. BrdU-exposed MKN-45 cells showed a significantly higher incidence of apoptosis after the cells passed through the first G2 phase and the second S/G2 phases, but MKN-28 did not. It thus appears that the delays of S/G2 phases and an increased incidence of apoptosis in the first cell cycle are p53-dependent. The growth suppression in the second S/G2 phase or later observed in both of the cell lines may be p53-independent.
The function of the tuft (brush) cells is still obscure. From the morphological similarities between tuft cells and chloride cells in the fish gill, which secrete or absorb NaCl, it was hypothesized that mammalian tuft cells may share functional roles similar to chloride cells. To test this hypothesis, the following points were studied on rat tuft cells. 1) The ultrastructural changes of bile duct tuft cells after secretin stimulation showed a moderate increase of basolateral membrane infoldings and a decrease of tubulovesicles in the apical cytoplasm. 2) The ultrastructural changes of tuft cells in the gastric groove of the rats on a high NaCl diet for 4 weeks, which were directly exposed to a high NaCl diet, and showed a marked shortening of microvilli and an appearance of numerous large vacuoles in the cytoplasm. 3) Cytochemical demonstrations of chloride and sodium ions were analyzed by energy-dispersive X-ray micro analysis (EDX). Massive reaction products of chloride were found in the apical cytoplasm of some tuft cells of fasting rats 5 min after secretin stimulation, but they were virtually absent in the tuft cells without stimulation. Reaction products of sodium were predominantly found along the basolateral membranes of tuft cells 5 min after secretin stimulation, but they were absent along those of tuft cells without stimulation. 4) Immunohistochemical demonstration of Na+/K+-ATPase, which is highly reactive in chloride cells of the fish gill, was positive in the tuft cell cytoplasm. These findings indicate that the mammalian tuft cells have properties similar to chloride cells in other vertebrates. By monitoring the electrolyte concentration with their long microvilli, tuft cells may regulate concentrations of electrolytes, probably NaHCO3, in the secretory fluid of hollow organs.
Spa-1-like protein (SPAL) is a newly-identified GTPase-activating protein (GAP) family protein. To verify subcellular localization of SPAL, we performed fluorescence immunohistochemistry in Mardin-Darby canine kidney (MDCK) cells and rat esophagus, pancreas and colon using a cell adhesion-related protein β-catenin as a marker of cell-cell contact sites. SPAL and β -catenin were colocalized along the lateral cell-cell contact sites of MDCK cells, and along the entire cell-cell contact sites of the stratified epithelial cells of the esophagus. In the pancreatic exocrine acinus, SPAL was detected only at the apical end of the β-catenin-immunostained lateral cell-cell contact sites. In the colon, SPAL was localized not only to the apical end of the lateral cell-cell contact sites but also to the apical cell surface. Taken together, SPAL was localized to the entire cell-cell contact sites of the non-polarized cells while in the polarized cells preferentially to the lateral cell-cell contact sites with some variations depending on the cell types. These results suggest that SPAL may be involved in regulation of the epithelial cell-adhesion.
The tumor suppressor gene adenomatous polyposis coli (Apc) is mutated in familial adenomatous polyposis (FAP) and sporadic colorectal tumors. The product of the Apc gene (APC) has been found to bind to β-catenin and human homolog of discs large (hDLG), and to be involved in the Wnt/Wingless signaling pathway. In this study, we examined distribution and subcellular localization of APC and its colocalization with β-catenin and hDLG in the mouse kidney by immunohistochemistry and immunoelectron microscopy. APC was highly expressed throughout the medullary region of the kidney, and localized mainly in the basal cytoplasm of epithelial cells of the thin portion of Henle's loop. It was found that APC was colocalized with β-catenin and hDLG by double-immunolabelling at both light- and electron-microscopic levels. These results suggest that APC complexed with β-catenin and hDLG may be involved in some signaling pathways regulating cellular functions of Henle's loop epithelial cells.