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The expression patterns of three microtubule-associated proteins (MAP1A, MAP1B, and MAP2A&B) were investigated in the developing optic tectum. Expression of MAP1B and middle-molecular-weight peptide of neurofilament (NF-M) was first observed in the same mesencephalic cells on day 3 of incubation, indicating that neuroblasts had been produced. At day 5, MAP1A and MAP2A&B expression appeared in the cellular layer containing the first neuroblasts that differentiate into large multipolar cells. The NF-M+ neurites in the striatum album centrale (SAC) and the striatum opticum (SO) were MAP1B+ up to day 19, but the intensity of MAP1B immunoreactivity decreased with development. All three MAPs were expressed in large multipolar neurons in the developing stratum griseum centrale from the beginning of maturation. Stratum griseum et fibrosum centrale cellular layers, containing radially arranged piriform neurons, were MAP1A−/MAP2A&B− on day 11 but became MAP1A+/MAP2A&B+ during later stages. These results suggest that the timing of MAP expression in neuronal maturation of large multipolar cells differs from that of piriform cells. The expression of MAPs has revealed specific cellular events in the developing optic tectum. Based on our observations, the development of the optic tectum can be divided into four periods.

Granulysin has been identified as an effector molecule co-localized with perforin in the cytotoxic granules of cytotoxic T lymphocytes and natural killer (NK) cells, and has been reported to kill intracellular pathogens in infected cells in the presence of perforin and to induce a cytotoxic effect against tumor cells. The aim of the present study was to elucidate whether intracellular expression of granulysin and perforin by NK cells might be associated with progression of cancer. Flow cytometric analysis demonstrated high levels of perforin and granulysin expression by CD3(-) CD16(+) cells in healthy controls. In contrast, cancer patients exhibited significantly decreased levels of granulysin expression ( P

In eight groups of subjects operating various hand-held vibrating tools and aged from 30 to 59 years, the prevalence rates of vibration-induced white finger (VWF) and numbness, pain, or stiffness in the upper and lower extremities were investigated. Hand-transmitted vibration levels (HTVLs) were measured on the back of the hand, by means of unidirectional (x-axis) vibration dosimeters, and the frequency-weighted acceleration levels [(Lh,w)eq,t] were determined as the vibration levels. The prevalence rates of VWF and numbness of the hands in these subjects were compared to the prevalence rates of Raynaud's phenomenon (RP) and numbness of the hands in 1027 males and 1301 females not occupationally exposed to vibration (age range: 30-59 years). It was observed that in subjects exposed to HTVLs of between 1.1 and 2.5 m/s2, the prevalence of VWF was between 0.0% and 4.8%. The prevalence of VWF reached 9.6% in a group of workers exposed to HTVLs of 2.7-5.1 m/s2. The latter group showed a significant difference (P < 0.05) in the prevalence of VWF compared to the 2.7% prevalence of RP in male subjects of the general population. The prevalence of VWF in female subjects exposed to vibration (4.3%) was not significantly different from the prevalence of RP in females of the general population (3.4%). The prevalence rates of numbness of the hands were in the range of 6.5%-30.4% in the exposed groups and in the range of 13.4%-29.5% in the general population. Among the subjective symptoms, only VWF showed a significant positive correlation with HTVLs (R2 = 0.5, P < 0.05). It was concluded that in decisions concerning quantitative recommendations for vibration exposure, the prevalence of VWF should be employed. With a view to decreasing the risk of developing VWF, estimated vibration safety values for 4 h and 2 h daily exposures are discussed.

Thorough irradiation on specimens with strong green light before or after pararosaniline Feulgen staining destroys specifically the primary-fluorescent substances in the background. By this treatment of pre- or post-irradiation, accuracy of DNA cytofluorometry is markedly improved and the Feulgen specific fluorescence is stabilized. Selecting proper wavelength, this technique will universally be useful in microfluorometry of any fluorochromes for reducing background fluorescence.

Vibratome sections of hippocampus of adult rabbits were stained by a modified Hortega's silver-carbonate method. Impregnated materials were examined by electron microscopy to decide fine-structural characteristics of the resting microglia. Comparing their characteristics with those of macrophages, we came to the following conclusions: (1) Impregnated resting microglia in the hippocampus of adult rabbits can be identified as cells having distinct fine structures. (2) Resting microglia are morphologically different from macrophages or their precursor cells, and, therefore, seem not to be hematogenous cells sojourning in the normal brain parenchyma.

Rat brain was obtained on the 4th day after its damage by a stab wound. The injured and the normal control brain tissues were stained by the immunofluorescence technique using anti-granulomonocytic rabbit serum. After the fluorescence observation the same tissues were further stained by hematoxylin and eosin (HE) and studied comparatively by light microscopy. The following results were obtained: (1) The normal adult rat brain lacks the cells which react with the antiserum, thus the resting microglia occurring in the normal adult brain are antigenically different from the cells of the monocyte-macrophage system. (2) In the injured brain tissues monocytes extravasate, enter-brain parenchyma, and take ameboid forms or become macrophages. (3) Among the reactive cells in the injured brain, all of the brain macrophages and most of the ameboid cells were reactive with the antiserum thereby indicating their monocytic origin.

The healing process of cysteamine-induced duodenal ulcer was studied by [3H]thymidine autoradiography. After the development of ulcer in the duodenum, cell proliferation was markedly activated not only in the crypts but also in the Brunner's glands near the ulcer. In the initial stages of ulcer healing, they both contributed to form the surface covering regenerating epithelium. Granulation tissue also proliferated at the base of the ulcer. In later stages of ulcer healing, new crypts were formed in the floor of the ulcer. New villi regenerated from these crypts and Brunner's glands regenerated by proliferation in situ. The ulcer base then was completely covered with new villi and granulation tissue was replaced by dense fibrous connective tissue. The present study suggested that the Brunner's glands, together with the crypts of Lieberkühn, play an important role in the healing process of cysteamine-induced duodenal ulcer.

Feulgen nuclear staining with pararosanilin-SO2 was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell. The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality between the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by Böhm et al. (1968) and a reaction with ninhydrin at 37 degrees C for 10 h. The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure. Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.

Signet ring cell carcinoma was induced in canine stomachs by N-ethyl-N'-nitro-N-nitrosoguanidine, and modes of cell proliferation and turnover in the carcinoma were studied by 3H-thymidine autoradiography in conjunction with morphometric analysis. From 2 to 15 months after the cessation of 8 months carcinogen treatment, carcinomas in an early stage were obtained. Most of the cancer tissues confined to the lamina propria showed a layered structure. This comprised three layers; the superficial and the deep layer were composed of signet ring cells, and the middle layer was composed of small round cells. The dogs were labeled with 3H-thymidine by s.c. injection and by local infusion of the celiac artery. Flash-labeled autoradiographs revealed that most 3H-thymidine incorporating cancer cells were located around the middle layer, with a small amount of mucin. Using a pulse labeling experiment, those labeled carcinoma cells were shown to migrate from the middle layer towards the surface. Morphometric analysis of the autoradiographs showed that the small cells in the middle layer migrated upwards and produced mucin to become full-blown signet ring cells by 5.5 days. In 15 days, most labeled cancer cells in the superficial layer had disappeared. This mode of cellular turnover appeared to mimic a cell renewal system of the normal gastric mucosa. If the cancer cells turn over in this way, the tumor must grow slowly, remaining as an intramucosal cancer for a relatively long period.

A microfluorometer is applied for simultaneous measurement of DNA content and grain count on a Feulgen stained cell nucleus in an autoradiograph. The autoradiograph is made on a coverslip and mounted on a slide glass with the emulsion side facing downwards. DNA cytofluorometry by incident light excitation can be carried out without interference from the silver grains. Switch-over from DNA cytofluorometry to grain counting is performed instantaneously by changing excitation filters to a red interference filter. The amount of red light reflected by the silver grains is measured by the microfluorometer. The photometric reading gives a value proportional to the grain count. The measurement of DNA content together with grain counting on a cell is completed within a few seconds.

In the human heart, it is well known that the polyploidization of working heart-muscle cells increases in proportion to increases in heart weight, but there has been no investigation of the process of polyploidization in the specialized heart-muscle cells of the cardiac conduction system which have a nerve-like function. In order to investigate the process of polyploidization in these cells, the nuclear DNA content of atrioventricular nodal cells was measured using cytofluorometry. Tissue samples taken from autopsied hearts without arrhythmias were embedded in paraffin blocks after Carnoy fixation. Blocks containing the atrioventricular conduction system were cut according to the serial sectioning method of Lev et al. The compact atrioventricular nodes were removed from thick paraffin sections (150 micron) under a stereomicroscope. The cells were then isolated by enzyme digestion and ultrasonic treatment. Smears of the isolated cells were double stained with azocarmin-G and acriflavine-Feulgen. Cytofluorometric DNA determinations of the DNA content of atrioventricular nodal cells were performed. Atrioventricular nodes were found to be composed of a large number of diploid cells and a small number of tetraploid cells. No octaploid cells were found. These findings reveal that the process of polyploidization in atrioventricular nodal cells is different from that found in working heart-muscle cells.

DNA-DAPI complexes emit strong bluish white fluorescence when excited by ultraviolet light so that even very small amounts of DNA such as those in mitochondria, chloroplasts, and virus particles can be visualized. Moreover, the staining procedure with DAPI is very simple and requires no hydrolysis. However, DAPI staining was considered unsuitable for quantitative purpose; nonspecific cytoplasmic fluorescence, scattering of strong emission light, and fading of the fluorescence under UV excitation were major problems of DAPI staining in quantitative cytofluorometry. We found that (1) nonspecific cytoplasmic fluorescence could be eliminated by reducing the DAPI concentration to 50 ng/ml, (2) fluorescence decay was markedly decreased by adding electron donors and molecules containing SH radicals in the mounting media, and (3) light scattering became negligible after reducing the intensity of the excitation light. Thus satisfactory precision could be obtained in DNA quantification by epifluorescent cytophotometry on DAPI stained specimens.

To examine reliability of DNA cytofluorometry with conventional Feulgen staining, measurement was carried out on smears of small and large neurons of human cerebellum, using a Digital-Microfluorometer with incident green light excitation. Nuclear fluorescence of the inner granule neuron and the Purkinje cell came to good agreement with each other when the staining was reduced so that the maximal extinction of the excitation beam nowhere exceeded the value of 0.1. The reduction of the feulgen staining is realized with unimpaired proportionality by treatment with 0.05% Schiff's reagent (pH 2.3) at 7–10° C for 10 min. It is concluded that this staining condition assures accurate determination of DNA content irrespective of size of the nucleus and of pattern of the chromatin distribution.

The incorporation of35SO4 and Glucose-3H has been autoradiographically investigated in human gastric mucosa, especially in intestinal metaplasia, applying in vivo local labelling method during the operation of gastro-intestinal tract. The fact that sulfated mucin is actively secreted in gastric mucosa of various animals is already known, but little about the mucin metabolism of human stomach itself. In intestinal metaplasia of human gastric mucosa, markedly high metabolism of sulfated mucin have been observed compared with its low metabolism in normal gastric mucosa. In detail observation of intestinal metaplasia, columnar cells, which were considered to be absorptive cells, play an active and primary roll in the production of mucin. The goblet cells are to be considered rather as retentive cells with a slow turnover of mucin. Intestinal metaplasia occuring in the pyloric gland are, is suggestive of adaptation ability of gastric mucin for the disturbed mucin metabolism of pyloric glands to protect the gastric mucosa.

Using 3H-thymidine autoradiography and electron microscopy the authors studies the differentiation of the ependymal cells and glioblasts in the embryonic spinal cord of the chick. 1. Ependymal cells are differentiated from the matrix cells at 8 days of incubation. Signs of differentiation are increase in number of basal bodies (blepharoblasts), and appearance of immature cilia and rough-surfaced endoplasmic reticulum in the apical cytoplasm. 2. After 8 days of incubation remaining matrix cells lose the potency to produce neuroblasts and change into glioblasts. They lie beneath the immature ependymal layer, i.e., subependymal layer in sensu stricto. The fine structure of the immature glioblast is fairly similar to that of the matrix cell; characteristic are a small nucleus and numerous aggregates of free ribosomes throughout the cytoplasm. Some glioblasts migrate through the mantle layer toward the periphery and reach the neuropil region. At this stage the fine structure of this migrating glioblast is not remarkably different from that of glioblasts in the subependymal layer.

The origin and renewal of the argentaffin cells in the pyloric glands of hamsters were studied by flash, cumulative and pulse labelling autoradiography with 3H-thymidine. The argentaffin cells were identified by the Diazo Method using Fast Red B Salt. By flash labelling autoradiography, it was shown that the argentaffin cells located from the middle to the lower level of the pyloric mucosa were not labelled with 3H-thymidine, indicating that this cell type has no proliferative activity. On the 10th and the 20th day of cumulative labelling, 31% and 63% of the argentaffin cells in the gland were found to be labelled, respectively. The labelled argentaffin cells were concentrated in the upper part of the gland (around the region of the isthmus), and no label was found over nuclei of the cells at the lowermost level of the gland. These labelled cells were shown to undergo a downward migration in the days following pulse labelling. They were replaced by unlabelled (and weakly or very weakly labelled) cells which arose at the region of the isthmus. The argentaffin cells in the pyloric gland are thought to arise from epithelial precursor cells at the region of the isthmus. The labelled argentaffin cells in the gland were found to decrease in number almost exponentially after pulse labelling. This indicates that the life span of argentaffin cells is not fixed, but their renewal conforms to the "random loss system". The half time of turnover of this cell population was 15 days on average.

Ten hamsters received repeated injections of 3H-thymidine for 4 days and were allowed to survive for 7, 28, 42 and 100 days. Changes in spatial distribution of the labelled cells and in labelling indices of each cell line in the gastric glands were studied at various days after 3H-thymidine, the labelled parietal cells and the mucous neck cells were concentrated at the neck area. Starting from the neck area, they migrated an average of 3 micra downwards per day. By 42 days, they reached the middle level of the glands, where the labelled mucous neck cells decreased but the labelled chief cells increased in number. The differentiation of the chief cell then appears to take place at the middle level of the glands through transformation of the migratory mucous neck cells. After 4 days of the labelling, about 1.8% of the chief cells located in the lower part of the glands was found to undergo in situ replication. This indicates that the renewal of this cell type is partly assured by its own mitotic activity. The foveolar cell--the future surface epithelium--seems to migrate upwards along the long axis of the glandular tubule in the pipe line system, which means "first produced, first migrates". After migrating out from the neck area, the parietal cell and the mucous neck cell (the future chief cell) take an average of 200 days to reach the lower end of the glands. In the process of migration, however, the cells produced contemporaneously at the neck area became scatteringly spread from the neck towards the bottom of the gland. The time required for the newly-formed cells to reach the lower end of the gland varied between 100 and 300 days. In the gastric glands the cells first produced at the neck area do not first reach the lower end of the glands. This mode of random migration is referred to as the "stochastic flow system". As one of the probable factors which disturb the pipe line flow of downward cell migration, cellular movements perpendicular to the long axis of the glandular tubule were suggested to occur at random at any level of the gastric glands.