Serial renal biopsies were performed to examine the age-related morphological changes of murine lupus nephritis in NZW / NZB (NZB / W) F1mice. Renal biopsies were performed at 3, 5, 7 and 9 months of age in the same mouse. The earliest histological change was slight mesangial thickening. Mesangial hypercellularity was seen in one-third of all the cases at 7 months. With age, the incidence of mesangial thickening was progressively increased, and capillary wall thickening appeared simultaneously. The incidence of interstitial cell infiltration was found in 100% at 11 months. Significant correlations among the capillary wall thickening, crescent formation and proteinuria were observed. In an immunofluorescent study, a significant correlation between immunofluorescent deposition in the capillary wall and proteinuria was observed. The pattern of immunoglobulin deposition changed from mesangial type to capillary type with age. However, the pattern of gp 70 (retroviral antigen) deposition in glomeruli varied considerably, and in some mice the gp 70 deposition decreased with age. Furthermore, a significant correlation between the titer of circulating gp 70 immune complex (IC) and gp 70 deposits in glomeruli was found. These results suggest that the age-related morpholigical changes are due to multifarious characters of IC and due to numerous immunological disorders.
The fine structural localization of acid phosphatase activity was studied by Gomori's method in the four cases of giant cell tumors of bone. In the stromal cells, the final reaction product was deposited over lysosomes. And in the multinucleated giant cells, the final product was deposited over lysosome-like organelles and also over the digestive vacuoles of autophagic or of a little heterophagic origin. These vacuoles were suspected to be secondary lysosome-like organelles in various stages. No deposits of reaction product were observed in giant cells in media containing sodium fluoride, L- (+) -tartrate, or those lacking the substrate. Degenerating giant cells containing autophagic secondary lysosome-like organelles were sometimes observed. It is suspected that their ultimate fates are cell death or autolysis and that acid phosphatase is excreted in these occasions from the cytoplasm to the extracellular medium.
Membrane structure of phagosomes in the process of fusion with each other or lysosomes was investigated mainly by an ultracytochemical method. It was noted that the glycocalyx-like structure extruding to the luminal sides of phagosomes was absent in the apposed regions of membranes during phagosomal fusion, although it was remarkable in the remainders of phagosomal membranes. Furthermore, reaction products of both ALPase and ACLase activities used as markers representing proteins were not demonstrated there, either. On the other hand, PFP reaction, which has been said to be positive for carbohydrates and unsaturated lipids, caused electron density in the total length of the membranes of phagosomes during fusion. The electron density in apposed membrane regions seemed to be attributed to unsaturated lipid, because glycocalyx-like structure consisting of carbohydrates was absent there. These findings suggest that membranes which are fusing are devoid of most, if not all, carbohydrates and proteins and rich in unsaturated lipids.
Immunohisto-cytochemical localization and biochemical activity of glutathione-peroxidase (GSH-PO) in rat testes were studied in rats under the following experimental conditions, i. e., 1) normal untreated, 2) 1 week after hypophysectomy, 3) gonadotropin administration to hypophysectomy, in order to clarify the role of GSH-PO. In the normal untreated group, GSH-PO was immunohisto-cytochemically localized in cytoplasm in close relation with the smooth endoplasmic reticulum of Leydig cells. In the hypophysectomized group, the Leydig cells were markedly atrophic and GSH-PO was localized in the cytoplasm around the swollen mitochondria or vesiculated smooth endoplasmic reticulum. By gonadotropin administration to the hypophysectomized group, the Leydig cells became markedly hypertrophic and contained well developed smooth endoplasmic reticulum. Immunohisto-cytochemically, GSH-PO was observed in cytoplasm near the well developed smooth endoplasmic reticulum of the Leydig cells. Intensity of GSH-PO staining was stronger than that of the other experimental group. The biochemical GSH-PO activity of the isolated Leydig cells was also increased as compared to the other groups. These findings strongly suggest that a very close relationship lies between the testicular GSH-PO and membrane metabolism including reduction of lipid peroxides of smooth endoplasmic reticulum.
We demonstrated α-fetoprotein (AFP) -positive cells in the late as well as in the early stage of 3′-methyl-4-dimethylaminoazobenzen (3′-Me-DAB) hepatocarcinogenesis, using immunocytochemical procedures with improved fixation and processing of tissue sections. The superior value of periodate-lysine-4% paraformaldehyde (PLP) as the fixative in the demonstration of AFP-positive cells and the usefulness of cryostat sections in floating immunoreactive processing are discussed. It is suggested that AFP-positive cells are new cell populations in the evolution of hepatocellular carcinoma.
Ultrastructural changes of splenic macrophages of rats after γ-ray irradiation were investigated, especially focused on lysosomal behaviors together with the cytochemical demonstration of acid phosphatase (AcPase) activity. γ-ray irradiation (total 800 rads) caused extensive phagocytosis in splenic macrophages. Damaged lymphocytes and erythrocytes were massively phagocytosed by these macrophages. The large number of heterophagosomes thus formed within macrophages subsequently acquired AcPase activity and converted to large heterophagolysosomes (HPL). Then, some portions of large HPL started to show constrictions which resulted in the separation and formation of many small lysosomes. Some of large HPL further became flattened and projected pseudopod-like processes which surrounded some cytoplasmic area or organelles of macrophage, especially small lysosomes, forming autophagolysosomes. These lysosomal behaviors may be related to the regulation or self-control of the lysosomal system within macrophages.
The localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity, a part of the reaction of the Na+-K+-ATPase complex, was ultracytochemically investigated in the rat hippocampal formation called substantia gelatinosa cerebri. Microslicer sections of the hippocampal formation fixed with a mixture of 0.25% glutaraldehyde and 1% paraformaldehyde for 30 min were incubated in the medium for K+-NPPase. In the light microscopic observations, the most abundant reaction products were present in the stratum moleculare of the hippocampal formation. Electron microscopically, K+-NPPase activity was demonstrated on the axolemma, neurofilamentous structures in the axoplasm and synaptic plasma membrane. These activities were distinctly decreased with addition of 10 mM ouabain or substitution of Na+ for K+. Additionally, ouabain-insensitive, K+-independent p-NPPase activity was observed on the mitochondria, nuclear envelope and endoplasmic reticulum. Neurotubules did not show any K+-NPPase activity.
The effect of the bile, an inductive factor of duodenal alkaline phosphatase (Alpase) in rats with surgically produced bile duct ligation and in organ cultures, was investigated by biochemical and cytochemical methods. Surgical bile duct ligation induced Alpase activity on the duodenal cells and also rats injected with bile from rats with bile duct ligation showed a great increase in activity. Duodenum from 1-month-old rats could best be maintained for 24 hr in a modified millipore filter roller tube using DM-153 medium without serum. Results in organ culture confirmed the observation in vivo and showed the induction of Alpase activity in the duodenum after administration of the bile and the depression of the rate of Alpase induction by the bile under tunicamycin presence. These results suggest that the bile from rats with bile duct ligation contains an inductive factor of Alpase in the duodenal cells. Organ culture is a very useful method and good model for the analysis of inductive factor of some substances in cell or tissues. Our findings are discussed in this paper.
The development of flow cytometry by Van Dilla et al. (21), Hulett et al. (10), Dittrich and Göhde (8) and Sprenger et al. (19) has added a new dimension to the field of cytochemistry. Flow cytometry is the rapid automated measurement of cells and cellular constituents using a device known as a flow cytometer and sorter. The flow cytometer performs fluorescence analysis on a single cell at the rate of 1000 cells per second and has the capability to sort cells from the main population. In this report I will summarize the present staining techniques and illustrate how they may be used for cell cycle and chromatin structure analyses of the cultured cells.
Biochemical and immunocytochemical examinations of histone H1 in G1 arrested and senescent human fibroblasts were performed to pursuit the mechanisms of human cell aging in vitro. Examinations of histone synthesis in serum depleted or contact inhibited cell cultures indicated that the ratio of histone synthesis over DNA synthesis and molar synthesis of histone H1 to nucleosomal histones did not decline with accumulation of resting cell populations, while senescent cells had a lower ratio of histone H1 synthesis than young cells. Histone H1 contents plotted as a function of DNA synthesis activity revealed that senescent cultures had a lower content of histone H1 than young cultures at any stage of cell proliferation. Immunocytological study using antiserum against histone H1 on cocultivated young and senescent cells showed that immunofluorescence intensity was very low in senescent nuclei in comparison with young cell nuclei. Thus, we conclude that a decline in content and synthesis of histone H1 relative to nucleosomal histones with passage number was not simply due to the passage-related accumulation of resting cells, but actually reflects age-specific changes in cycling cells or whole cell populations in the senescent cultures. The depletion of histone H1 in chromatin was discussed as a possible cause of DNA strand break and relaxation of gene repression.
The cellular localization of cancer-associated alkaline phosphatase is studied in three cultured human cancer cell lines, which are established as monophenotypic to early placental in KMK-2, Kasahara in HeLa S3-5, and Regan isozymes in HeLa S3-10 respectively. First, it is confirmed that the optimal conditions of the cell fixation, the substrate concentration and the cultured method, as factors which influenced on the electron cytochemical demonstration of alkaline phosphatase should be thoroughly studied cell-line to cell-line in order to accurately interpret the localization of each isozyme concerned. Second, it is shown that all of the enzyme activities of the early placental, Kasahara and Regan isozymes are located on the whole plasma membrane, particularly the outer lamella and the spaces between the outer and inner lamellae. However, it is less certain that the reaction products in some intracytoplasmic organelles of KMK-2 and HeLa S3-5 are the specific sites of the early placental isozyme or the Kasahara isozyme. Effects of some activators and inhibitors to each isozyme on the enzyme activity and the cellular localization are also described.
Comparative studies on changes in glycolytic and hydrolytic enzyme activities and cyclic nucleotide contents in bovine vascular smooth muscle cells were carried out in an attempt to elucidate the metabolic changes which occur in vascular cells, under condition of hyperlipidemia. Decreased glycolytic enzymes and increased acid phosphatase activities were found in smooth muscle cells cultured with 5% hyperlipidemic LDL which contained many sudanophilic droplets in their cytoplasma. Statistically significant low levels of cAMP and high levels of cGMP were confirmed in these cells cultured in hyperlipidemic media, as compared with those in control cells. This data suggests that decrease in cyclic AMP content and glycolytic enzyme activities accelerates lipid phagocytosis and cell division, both important phenomena in the course of atherosclerosis.