Cyclic nucleotide phosphodiesterase was demonstrated in rat and hamster hippocampus by a histochemical method. This method is based on a cerium precipitation technique. The enzymatic reaction was specific and demonstrated the distribution of cyclic nucleotide phosphodiesterase in the hippocampal fields of rats and hamsters. There were no differences between cAMP and cGMP as substrates. There were very strong reaction in the pyramidal layer, in the neuropil of the molecular layer and in the mossy fibre layer. Strong enzymatic activity was localized in pyramidal cells in their dendrites, filial cells and capillaries.
Cyclic nucleotide phosphodiesterase was demonstrated in the hippocampus of the hamster by an immunohistochemical method. The enzyme was localized in the somata of pyramidal and granular cells and their dendrites. Furthermore, capillaries and the neuropil were strongly stained. Glial cells could be detected in fibre layers (corpus callosum). The antiserum was produced against bovine heart Ca2+/calmodulin phosphodiesterase and cross-reacted with brain phophodiesterase.
The incorporation patterns of [14C]adenine ([14C]A) and, as control, [3H]thymidine ([3H]TdR) by proliferative cells in the intestinal mucosa of rats and mice were studied by the autoradiographic technique. The animals received 3 daily, subcutaneous injections of the isotopic markers (1μCi/g body weight each) and examinations were made 3hr after the last injection. The most striking finding was a strong labeling with [14C]A of a portion of non-lymphoid lamina propria cells (LPC) in the core of jejunal villi, in contrast to weak labeling of the epithelial cells with this isotopic marker. Furthermore, the labeling of epithelial cells decreased in intensity to a large extent following RNase treatment. In contrast, the silver grains over the LPC did not markedly decrease in number after RNase treatment and the cells still remained heavily labeled. Following extraction of DNA with DNase treatment, the heavy labeling of LPC vanished completely. This provides evidence for the incorporation of [14C]A into DNA of the LPC to a great extent. The overall patterns of [3H]TdR labeling of mucosal cells, on the other hand, was characterized by fairly uniform labeling of proliferative cells and no great difference in the labeling intensity was recognized between the epithelial cells and the LPC of jejunal villi. The results mentioned above were discussed in relation to the peculiar adenine metabolism of the LPC. It was suggested that the LPC have a limited capacity for de novo adenine synthesis and because of this they were labeled with [14C]A to a greater degree. The LPC which exhibited heaviest labeling with [14C]A had the same morphological characteristics as those of the primitive reticular (or reticulum) cells, which have been described by Maximow (9) and Marshall (8) as undifferentiated mesenchymal cells. The similarity of the former cells to the latter cells was discussed.
The incorporation patterns of [14C]adenine ([14C]A) by reticulum cells in the lymphoid tissues of young adult rats were studied by the autoradiographic technique. The animals received 3 daily subcutaneous injections of [14C]A (1μCi/g body weight each) and examinations were made 3hr after the last injection. It was found that, in the lymphoid tissues, non-lymphoid cells such as proliferative reticulum cells incorporated [14C]A to a greater extent, following RNase treatment in particular, than did lymphoid cells. The most striking finding was the occurrence of proliferative reticulum cells which exhibited especially strong labeling with [14C]A even after RNase treatment. By treatment with DNase and by extraction of both DNA and RNA with hot perchloric acid treatment, it was confirmed that the especially strong labeling of these cells with [14C]A following RNase treatment represents DNA labeling. The reticulum cells exhibiting especially strong DNA labeling showed the appearance of blast cells; namely, they had a large, round or ovoid, pale-staining nucleus and scanty, indistinct cytoplasm. Therefore, such cells were designated as “blast-like” reticulum cells (BLRC). In this study, the BLRC were easily distinguishable from lymphoblasts because of weak DNA labeling with [14C]A of the latter cells. In addition to BLRC, a portion of interdigitating reticulum cells in the mesenteric lymph node also exhibiting DNA labeling with [14C]A, but to a lesser extent than did the BLRC. It was suggested, therefore, that the BLRC might be the stem cells of interdigitating reticulum cells. The BLRC exhibiting the heaviest labeling with [14C]A had the same morphological features as those of the primitive reticular (or reticulum) cells, which have been described by Maximow (8) and Marshall (7) as the stem cells of not only reticulum cells but also various types of blood cells. The relationship of the former cells to the latter cells is discussed.
The ultracytochemical localizations of adenosine nucleotidase activities were investigated in the human term placenta. The enzymes studied were as follows: 5′-nucleotidase (5′-N), Ca++-activated adenosine triphosphatase (Ca++ ATPase), Mg++-activated adenosine triphosphatase (Mg++ ATPase) and nucleotide diphosphatase (NDPase). 5′-N activity was demonstrated using not only the lead nitrate method of Wachstein and Meisel but also the recently developed cerium and neodymium method as well. The reaction products for 5′-N activity were found on the external surface of the microvillous plasma membrane of the syncytiotrophoblast. Alpha-beta-methylene adenosine diphosphate (AOPCP), at a concentration of 2.0mM, effectively inhibited 5′-N activity. Ca++ATPase and Mg++ATPase activity were observed strongly on the microvillous membrane of the syncytiotrophoblast and weakly on the basal plasma membrane of the syncytiotrophoblast. NDPase, using ADP as a substrate, was localized on the microvillous membrane. These observations suggest that the syncytiotrophoblast is active in the nucleotide metabolism and that microvillous surface of the syncytium may play an important role in regulating the feto-placental-maternal microcirculation in the human term placenta.
The early events of fatty acid absorption in the small intestine were ultracytochemically investigated by the PFP reaction, which stained unsaturated lipids. Five min after administration of the unsaturated long chain fatty acid, docosahexaenoic acid (C22:6), ordinary electron staining did not cause any morphological changes in the apical cytoplasm except for the electron dense droplets which appeared in the ER below the terminal web, but a PFP positive particulate or amorphous substance appeared in the microvilli, the terminal web and around the ER below the terminal web. It seemed to be formed by aggregation of small particles. Sometimes the particles appeared separately. The PFP positive substances did not seem to be enclosed by a membrane, and could be free fatty acid diffusing down to the ER. Fatty acid may penetrate the microvillus membranes in the form of a small particle, diffuse down the microvilli to the terminal web and be transformed to droplets by enzyme at the ER.
Tissue and organ distribution of radioactive carbon from 14C-labeled tryptophan in the mouse was studied by whole-body autoradiography and biochemical analysis. The mice injected intravenously with L-[side chain-3-14C]tryptophan were sacrificed at various intervals. Examination of autoradiographs disclosed that the injected 14C-tryptophan was rapidly taken up from the blood by the organs. The radioactivity in the pancreas was the highest and predominant throughout the intervals after injection in this investigation. The comparative values among radioactivity in various organs estimated by a liquid scintillation counter were consistent with those obtained from whole-body autoradiographs. Radioactivity in the acid-insoluble fractions was increased with time in all organs examined. High-performance liquid chromatography of the acid-soluble fractions disclosed radioactive tryptophan and alanine in the blood, liver, kidney and small intestine, while radioactive alanine was not detected in the brain.
Elemental distributions throughout the root radius in the absorption zone were measured by x-ray microanalysis of frozen hydrated bulk Plantago maritime root samples. Relative elemental concentrations in the cytoplasm were measured in freeze-substituted sections of undifferentiated cells and xylem parenchyma cells in the absorption zone. When grown in the presence of 200mM NaCl, relative levels of Na+ and K+ reaching the stele are adjusted from those in the culture solution principally by an initial enhancement of the K+ level in the epidermis, and a subsequent reciprocal decrease in Na+ and increase in K+ levels at the endodermis. Mean Cl- levels do not balance those of Na+ in any of the analysed cells. Together with differences observed between control and salt grown roots in the Mg and Ca levels in the inner cortex, the regulation of Na+ and K+ reaching the xylem vessels is discussed in terms of ion pumps and ATPases, and related to the overall salinity tolerance of P. maritima. The mean relative cytoplasmic concentration ratio of Na+: K+ was 0.6:1.0 in the undifferentiated cells and 1.1:1.0 in the xylem parenchyma; in the latter the mean relative concentrations of Na+, K+ and Cl- were appreciably higher than in the former. The absolute ion concentrations in the cytoplasm, and the extents of variations therein, rather than the Na+:K+ ratio, may be correlated with the overall salinity tolerance of the plant.
Specimens from patients with Bowen's disease were studied for keratin and involucrin distribution as determined by antibodies polyclonal (TK), and monoclonal (KL1 and PKK1) against keratin, and polyclonal antibodies against involucrin, respectively. Squamous cells in normal skin were positive for TK detectable keratins throughout the epidermis, while the spinous cells were strongly positive for KL1 detectable ones. Basal cells were negative for KL1, but characteristically strongly positive for PKK1. Normal epidermis showed marked staining for involucrin in upper spinous and granular cell layers. Specimens from patients with Bowen's disease exhibited irregular expression of antigen by the three types of anti-keratin antibodies with a loss of the regular or zonal distribution pattern as found in the normal epidermis. The lesions also showed negative staining for involucrin. Thus, affected epithelial cells in Bowen's disease showed marked differences in stainability with respect to present four immunoreagents.
The side effects of mitomycin C (MMC) on liver cells and the protective effectiveness were investigated morphologically, biochemically and histo-cytochemically. Coenzyme Q10 (CoQ10) prevented the cells from inflation, mitochondria swelling and vacuolation of endoplasmic reticulum, which were caused by the administration of MMC. This might be due to CoQ10 preventing the cells from decreasing energy metabolism. The biochemical determination of enzyme activities indicated significant difference between the treatments in 5′-nucleotidase (5′-Nase) activity and in contrast, Mg++-adenosine triphosphatase (Mg++-ATPase) was not found statistically different. The histo-cytochemical observation showed that the activity of 5′-Nase and Mg++-ATPase were decreased by being treated with MMC. But the localization pattern of the enzyme activities was not different from the control when both CoQ10 and MMC were administrated. The present results suggested that CoQ10 could prevent damage caused by antineoplastic agents.