The ultrastructure of the central terminals of nociceptive primary afferent (Cl terminals) neurons in the mouse substantia gelatinosa was studied by the cytochemical method using fluoride-resistant acid phosphatase (FRAP). Small roundish, slender, sinuous and large scalloped terminals showed FRAP reactivity. The FRAP reaction was seen as electron dense granular deposits between spherical synaptic vesicles and around the membrane of the synaptic vesicles. Especially, dark, small sinuous and large scalloped terminals showed intense FRAP reactivity. From the previous studies, these terminals were in accord with the capsaicin-sensitive Cl terminals. Furthermore, larger, roundish terminals with less packed spherical synaptic vesicles, many mitochondria and a few dense cored synaptic vesicles showed FRAP reactivity. Our recent results that Cll terminals are also sensitive to capsaicin support the FRAP activity of the Cll terminals in the present study. Thus, various types of FRAP-positive Cl terminals and some Cll terminals are considered to be central endings of the capsaicin-sensitive nociceptive primary afferents.
This work presents evidence that the atrial specific granule (ASG) is one of the intracellular calcium stores (ICaSs). First, a high calcium (Ca) concentration up to 81 mmol/kg dry wt. in ASG was determined by using an electron microscopic x-ray microanalysis technique. Second, Ca2+-ATPase was observed on the membranes of ASG and of the sarcoplasmic reticulum (SR) by using the cytochemical technique. Third, the activities of the Ca2+-ATPase on both the ASG membrane and on the SR memebrane were inhibited by 0.1 mM quercetin, but not by 0.1mM oligomycin. Because SR is an established ICaS, the similarities found in the present work between ASG and SR led us to postulate that ASG may be an ICaS in atrial cardiocytes, and that the Ca2+-ATPase on ASG membrane may pump Ca2+ from cytosol into ASG and thus play a role in maintaining the high Ca concentration in it.
We examined the immunolocalization of stage-specific embryonic antigen-1 (SSEA-1) and related antigens in the developing trachea and lungs of fetal hamsters both in vitro and in vitro. On gestational days 10-16 (the day ot birth), fetal tracheas and lungs were fixed in phosphate-buffered paraform-aldehyde and frozen. Frozen sections were incubated with monoclonal antibodies against SSEA-1 (Lex hapten), sialyl SSEA-1 carrying i antigen and fucosyl SSEA-1 (LeYhapten). Some sections were treated with FITC-labeled secondary antibody for observation by confocal laser microscopy, and others were treated with colloidal gold-labeled secondary antibody for ultrastructural observation. In the developing tracheas, preferential staining in the ventral and lateral tracheal epithelium appeared for all of the antibodies by day 15, and ultrastructural study demonstrated that the regional difference in staining occurred regardless of cell type. In the developing lungs, positive sialyl SSEA-1-i antigen immunostaining was seen in the terminal portion of the epithelium by day 14, and the staining disappeared during the postnatal period. An in vitro study using explant organ culture of gestational day-11 lung with trachea revealed similar immuno-localization patterns. In the cultured trachea, the regional difference in immuno-staining was also seen with development of cartilage and smooth muscle, and in the cultured lung, the staining intensity became stronger in the terminal epithelium than in larger airway epithelium.
The localization of cathepsin L or type I collagen in the osteoclasts (rat femur) treated with or without E-64 (control) was examined immunocytochemically to investigate how E-64 affects the osteoclasts. Using a light microscope in the E-64-treated osteoclasts, the immunoreactivity for cathepsin L was extracellularly very weak compared with that in the control osteoclasts, but was strong intracellularly. The intracellular immuno-reactivity for type I collagen was found in the vacuoles in the E-64-treated osteoclasts but not in any vacuoles in the control osteo-clasts. On the other hand, the extracellular immunoreactivity along the resorption lacunae of the E-64-treated osteoclasts was somewhat weaker than that of the control osteoclasts. Using electron microscopy in the E-64-treated osteoclasts, only a small number of extracellular immunoreaction products for cathepsin L were seen along the resorption lacunae. In addition, intracellular cathepsin L was deposited in the endosome-lysosomal vacuoles which were well-developed by E-64. Gold particles indicating type I collagen appeared on the bone matrix, and they were also detected in the vacuoles and vesicles in the E-64-treated osteoclasts. However, they were not detected in the organelles of the control osteoclasts. Thus, in the E-64-treated osteoclasts, the extracellular release of cathepsin L was insufficient to suppress the degradation of collagen. In addition, the undegraded collagen seemed to be endocytosed. The above findings thus suggest that bone resorption is inhibited by this incomplete degradation of collagen.
To evaluate the developmental changes of the nucleolar activity of neurons, we enumerated the number of argyrophilic nucleolar organizer regions (AgNOR) in neocortical neurons of embryonic and postnatal mouse brains. The number of AgNOR changed with time, peaking at late embryonic life and falling gradually through 12 weeks postnatally. Our observation gives indirect evidence that the number of AgNORs is a marker of ribosomal RNA transcriptional activity as well as of ribosomal protein synthesis activity in neurons but not of their proliferative activity, because they are post-mitotic. These findings indicate that the AgNOR staining technique could be a useful adjunct in assessing neuronal activity of the brain.
The effects of the Chinese traditional drug, Rikkunshito (TJ-43) to adriamycin (ADM) -induced gastric cytotoxicity were studied histo-and cytochemically. Single i. p. injection of ADM (1.0mg/100g b. w.) to rats decreased cytochemical activities of cyto-chrome C oxidase (COX) in mitochondria and Ca2+-ATPase in plasma membrane of parietal cells. Oral administration of TJ-43 (100 mg/100g b. w.) to rats for 5 days prior to ADM injection almost completely prevented a decrease of both the enzyme activities by ADM. Post-treatment of TJ-43 after ADM injection showed lower intensities of both the enzyme activities than in normal rats, but significantly higher than those of ADM-injected rats without TJ-43 treatment. Localization of lysosomal acid phosphatase (AcPase) appeared to be confined to lysosomes in normal and TJ-43-pre-treated rat parietal cells, but the reaction products in the ADM-treated rat with and without TJ-43 post-treatment tended to disperse into the cytoplasm of most parietal cells. Therefore, a destructive effect of ADM on lysosomal membrane was suggested in ADM-injected rats with and without TJ-43 post-treatment. Taken together, TJ-43 appeared to have preventive effect against ADM-induced cytotoxicity, whereas its post-treatment had partial, but significant curative effects on the parietal cells.
Injection of dithizone, a zinc chelating agent, induced selective killing of intestinal Paneth cells within a few min by forming toxic insoluble zinc-dithizonate complexes. However, regeneration of Paneth cells took place right after dithizone treatment and resumed the normal level as early as 72 hr with over-shooting persistent for 2 weeks. The process or regeneration in intestinal crypts was studied by means of morphology, uptake of the thymidine analog 5-bromo-2′-deoxy-uridine and immunohistochemical staining of Paneth cell differentiation markers such as lysozyme and zinc-binding proteins. It was found that Paneth cells regenerated without cell division and DNA synthesis apparently by direct transformation of non-Paneth crypt base columnar cells. Dithizone induced a transient wave of cell growth in all compartments of the intestinal crypt.
The evidence to support the luminal release of serotonin (5HT) from the enterochromaffin cells in rat embryo was demonstrated by immuno-electron microscopic study combined with HPLC measurement of 5HT levels in the lumen. A considerable amount of 5HT was detected in the lumen of embryonic gut. To examine the subcellular localization of 5HT in the EC cells, postembedding immunogold method was used. EC cells in embryonic gut showed a variety of characteristic features as follows. EC cells included secretory granules with high electron-dense cores at basal cytoplasm or both basal and apical cytoplasm. lmmunogold particles were densely packed throughout the cytoplasm rather than concentrating over cores of the secretory granules. In the apical cytoplasm of embryonic EC cells, immunogold particles were densely accumulated at the matrix and over the microvilli. The present findings suggest that 5HT is released into the intestinal lumen in a diacrine manner from embryonic EC cells.
In the present study, changes in calcium waves with alternation of diastolic [Ca2+] i were investigated in a single common rat neonatal cultured heart muscle cell without changes in extracellular conditions. Heart muscle cells were loaded with fluo-3 by the “whole cell clamp” technique. Rapid chan ges in diastolic [Ca2+] i by variations in the membrane potential clarified the influences on calcium waves. When the membrane potential was held at 0 mV, inter wave basal [Ca2+] i were maintained at a high level. The membrane-holding potential was then momentarily altered to -80 mV and the basal [Ca2+] i registered a lower level within 1 sec. During these events, spontaneous calcium waves were analyzed by confocal laser scanning microscopy (X-t scanning mode). The frequency and velocity of calcium waves at 0 mV significantly increased compared with those at-80 mV. Moreover, these changes were more pronounced with increasing extracellular [Ca2+]. These results suggest that diastolic [Ca2+] i; directly influences calcium waves.
Fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes allows cytogenetic information to be obtained rapidly and precisely from metaphase and interphase chromosomes. Multi-color FISH with two or more probes makes it feasible to delineate several chromosome aberrations simultaneously occurring in a nucleus. Here we present a simple and rapid method for multi-color FISH and its application to the cytogenetic investigation of tumor cells. In addition, the usefulness of this method is discussed.