Ultrastructural and ultracytochemical investigation of human gastric carcinomas

Summary We describe the ultrastructure of various types of gastric carcinoma cells as well as their histochemical properties as visualized at the electron-microscope level. The histochemical properties of tumour cells were compared with those of homologous normal epithelial cells. The localization and activity of ATPase, IDPase, acidic phosphatase and alkaline phosphatase as well as of the oxidoreductases (cytochrome oxidase, succinate dehydrogenase and NADH-dehydrogenase) were studied. Our findings demonstrated that, in tumour cells, a complicated process of structural-functional restructuring takes place. It seems that a number of ultracytochemical properties may be preserved or may disappear altogether, also, such properties may become enhanced or weaker. This heterogeneity of the histochemical properties of tumour cells is discussed with regard to the role of the stem (polypotent) cell in the process of the histogenesis (cytogenesis) of human gastric carcinomas.In honour of Prof. P. van Duijn     

Histochemical studies of mucosubstances in metaplastic epithelium of the stomach,with special reference to the development of intestinal metaplasia

Summary Processes in the development of intestinal metaplasia of the stomach were investigated from the morphological and histochemical approaches using light and electron microscopic techniques. The specimens taken from 38 gastric carcinomas and 15 gastric and/or duodenal ulcers were subjected to this study. Morphological appearances of the intestinal metaplasia observed in routine examination with hematoxylin and eosin staining was able to be divided into complete and incomplete metaplasia by the light and electron microscopic histochemical stainings of the mucosubstances. The columnar cells at the area of the incomplete metaplasia had both the properties of the intestinal epithelia and the gastric foveolar epithelia. The incomplete as well as the complete metaplasia arose from the generative cells at the isthmus of the gland. The generative cells, however, sometimes gradually transformed to produce the complete metaplastic cells. The two processes of the development of the intestinal metaplasia were proposed and discussed.     

Histochemical studies of developing human fetal small intestine

Summary The histochemical characteristics of absorptive and mucus-producing cells have been described in 16 specimens of small intestine from human fetuses between 7 and 22 weeks of age. In the youngest specimens a slight to moderate reaction was found for all enzymes examined which included those known from biochemical or ultrastructural studies to be located predominantly in Golgi apparatus (thiamine pyrophosphatase), surface membrane (alkaline phosphatase, adenosine triphosphatase, leucine aminopeptidase), lysosomes (acid phosphatase), and mitochondria (succinic dehydrogenase). Mucus granules of somehat unusual location and staining properties were also found at this time. A progressive intensification of a majority of these histochemical reactions was observed between 3 and 6 fetal months.These findings indicate that the histochemical apparatus for degradation and absorption of nutrients and for elaboration of protein and mucus is already well established in the human fetus by the end of this 6 month period. It is not known, however, whether these activities occur in utero or whether they contribute significantly to fetal nutrition.     

Light microscopic demonstration of myoid material in nuclei

Summary In the rabbit and bovine cornea the activity of alkaline phosphatase using histochemical as well as biochemical methods was investigated. Biochemically the enzyme activity was studied in separated corneal layers. In the histochemical investigation the best results were obtained in cryostat sections using the azocoupling method with naphthol AS-MX phosphate and Variamine Blue RT Salt. The enzyme activity was found not only in the epithelium and endothelium (as was described previously) but even in keratocytes. The mutual relation of activities in the epithelium and in keratocytes differed in both species. The overall activity found by histochemical methods is in good agreement with the biochemical determination of alkaline phosphatase (p-nitrophenyl phosphate as the substrate). Besides the histochemical approach shows an uneven distribution of alkaline phosphatase activity in individual cells which cannot be assessed by the biochemical determination.     

Inactivation of rat gastric mucosal histidine decarboxylase by phosphatase

Histidine decarboxylase of supernatants as well as of purified preparations from rat gastric mucosa is inactivated by a non-specific phosphatase in the absence of pyridoxal 5'-phosphate. The inactivation is a time and concentration-dependent process. Pyridoxal 5'-phosphate, but not histidine, protects the enzyme against phosphatase action. The inactivation is reversible, only pyridoxal 5'-phosphate reactivates the inactivated enzyme. Pyridoxamine 5'-phosphate is ineffective for histidine decarboxylase, but is converted into an active coenzyme only in gastric supernatant. Evidence for the occurrence of an active phosphatase in gastric tissue is also presented; its properties are those of an acid phosphatase and are similar to those of phosphatases hydrolyzing pyridoxal 5'-phosphate in other tissues. The data indicate that phosphatase promotes apoenzyme formation and may play a role in the regulation of histamine synthesis.     

Cytochemical and biochemical characterization of testicular peritubular myoid cells

Testicular peritubular myoid cells secrete a paracrine factor that is a potent modulator of Sertoli cell functions involved in the maintenance of spermatogenesis. These cells also play an integral role in maintaining the structural integrity of the seminiferous tubule. To better understand this important testicular cell type, studies were initiated to characterize cultured peritubular cells using biochemical and histochemical techniques. The electrophoretic pattern of radiolabeled secreted proteins was similar for primary and subcultured peritubular cells and was unique from that of Sertoli cells. Morphologic differences between Sertoli cells and peritubular cells were noted and extended with histochemical staining techniques. Desmin cytoskeletal filaments were demonstrated immunocytochemically in peritubular cells, both in culture and in tissue sections, but were not detected in Sertoli cells. Desmin is proposed to be a marker for peritubular cell differentiation as well as a marker for peritubular cell contamination in Sertoli cell cultures. Peritubular cells and Sertoli cells were also stained histochemically for the presence of alkaline phosphatase. Staining for the alkaline phosphatase enzyme was associated with peritubular cells but not with Sertoli cells. Alkaline phosphatase is therefore an additional histochemical marker for peritubular cells. Biochemical characterization of peritubular cells relied on cell-specific enzymatic activities. Creatine phosphokinase activity, a marker for contractile cells, was found to be associated with peritubular cells, while negligible activity was associated with Sertoli cells. Alkaline phosphatase activity assayed spectrophotometrically was found to be a useful biochemical marker for peritubular cell function and was utilized to determine the responsiveness of primary and subcultured cells to regulatory agents. Testosterone stimulated alkaline phosphatase activity associated with primary cultures of peritubular cells, thus supporting the observation that peritubular cells provide a site of androgen action in the testis. Retinol increased alkaline phosphatase activity in subcultured peritubular cells. Alkaline phosphatase activity increased in response to dibutyryl cyclic adenosine monophosphate (AMP) in both primary and subcultured peritubular cell cultures. Observations indicate that the ability of androgens and retinoids to regulate testicular function may be mediated, in part, through their effects on peritubular cells. This provides additional support for the proposal that the mesenchymal-epithelial cell interactions between peritubular cells and Sertoli cells are important for the maintenance and control of testicular function. Results imply that the endocrine regulation of tissue function may be mediated in part through alterations in mesenchymal-epithelial cell interactions.     

Histochemical findings in the rat gastric mucosa during starvation

Summary The influence of starving on the activity of enzymes of the rat gastric mucosa was investigated by selected histochemical methods. Beside the conventional methods of enzymatic histochemistry the technique of semipermeable membranes was used in the proof of lysosomal enzymes. Dehydrogenases were proved in aqueous and also in gel media with PMS.During the starvation in the parietal cells a marked increase took place in the activity of acid phosphatase, E-600 resistant esterase, less in -glucuronidase. High activity of the lysosomal enzymes in macrophages did not change during starvation. Nor did any changes took place in the activity of alkaline phosphatase in the endothelium of the capillaries. The chief cells in the control and starving animals, in contrast to the human gastric mucosa, did not contain any non-specific esterase. Concerning dehydrogenase, parietal cells with a different activity of these enzymes were observed both in starved and control animals.In the rat gastric mucosa starving induced changes in the activity of the enzymes which mark important organelles of the cells. Thus it is possible to consider the observed histochemical changes as a functional manifestation of morphological damage of cellular structures which are affected during starvation.     

Tumour-cell-induced production of tumour necrosis factor by monocytes of gastric cancer patients receiving BCG immunotherapy

Summary Human peripheral blood monocytes cocultured with tumour cells were used as an in vitro model of in situ interactions between tumour-infiltrating macrophages and the tumour. Tumour cells stimulated de novo expression of the human tumour necrosis factor (TNF) gene in monocytes and caused the release of TNF into the culture supernatant. A group of 14 patients with stage IVA gastric cancer receiving adjuvant chemotherapy (5-FU, Adriamycin, mitomycin C: FAM) or immunochemotherapy (BCG+FAM) was investigated for the ability of monocytes to produce TNF in vitro upon stimulation with tumour cells or purified protein derivative of tuberculin (PPD). Patients were followed at biweekly intervals, i.e. before each instillation of BCG epicutaneously over a period of 10 weeks. It was found that monocytes of some patients receiving BCG at the end of the observation period had an enhanced ability to produce TNF following stimulation with tumour cells. In contrast, such production was not substantially altered during the study period in patients on chemotherapy. PPD-induced TNF production was much weaker and was not significantly changed during this observation time. We infer that BCG immunotherapy may induce the subtle changes in some cancer patients that lead to an increased interaction between monocytes and tumour cells and result in enhanced production of cytokine(s) with antitumour properties.     

Histochemical distribution of four types of enzymes and mucous cells in the gastrointestinal tract of reared half‐smooth tongue sole Cynoglossus semilaevis

The histochemical distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non‐specific esterase (NSE), peroxidase (POD) and mucous‐cell types was evaluated in the gastrointestinal tract of the half‐smooth tongue sole Cynoglossus semilaevis. The enzymes were detected in the entire stretch of the gastrointestinal tract. ACP activity was found in the supranuclear region of enterocytes and the lamina propria of the intestine, as well as the cytoplasm of epithelial cells of the stomach. The staining intensity of ACP in the anterior and posterior intestines was stronger than in the stomach. ALP activity was detected in the striated border of enterocytes and muscularis of the whole intestine, lamina propria and supranuclear cytoplasm of the enterocytes in the anterior intestine, as well as in the blood vessels of the stomach. The staining intensity for ALP in the anterior intestine was stronger than in the posterior segment and the latter was stronger than in the stomach. NSE activity was detected in the cytoplasm of the epithelial cells in the entire gastrointestinal tract, with the anterior intestine showing stronger intensity than the stomach. POD activity was located in the blood cells of the lamina propria of the gastrointestinal tract and the levels in the stomach were similar to the anterior and posterior intestines. Alcian blue (pH 2·5) periodic acid Schiff (AB‐PAS) histochemical results revealed three types of mucous cells in the gastrointestinal tract. Type I cells (PAS+AB‐) were observed among the gastric mucosa columnar cells in the stomach and enterocytes in the basal region of the villi and in the middle and top regions of the intestinal villi. Type II cells (PAS‐AB+) and type III cells (PAS+AB+) were not detected in the stomach but were distributed ubiquitously among enterocytes in the middle and top regions of the intestinal villi.     

Electron microscope histochemical localization of alkaline phosphatase(s) in Bacillus licheniformis.

Sites of alkaline phosphatase (APase) activity in a facultative thermophilic strain of Bacillus licheniformis MC14 have been localized by electron microscope histochemistry, using a lead capture method. The effects of 3% glutaraldehyde and 3.0 mM lead on APase activity were investigated, and these compounds were found to significantly inhibit enzyme activity, 68 and 18%, respectively. A number of parameters were varied in studies to localize APase activity, including: growth temperature (55 and 37 degrees C); substrate concentration in the histochemical mixture (0.06, 0.15, 0.30, 1.00 mM); fixatives; protoplast preparations and whole cells; phosphate-repressed and -derepressed cells; and age of vegetative cells (mid-log and late log). These variations affected the number but not the location of lead phosphate deposits, which appeared at discrete sites along the inner side of the cytoplasmic membrane. Control cells incubated in histochemical mixtures lacking substrate, lead, or both exhibited no lead phosphate depositis. The histochemical localization at membrane sites correlated well with biochemical localization data, which indicated that greater than 80% of the APase activity was associated with the membrane fraction in logarithmically growing cells.     

The interaction between walker tumour cells and mucosa cells in the lamina propria of gastric mucosa in rats

One series of 12 rats was exposed to X-irradiation (1500 R) of the stomach 19 days before implantation of Walker tumour cells in the gastric mucosa, and the frequency of tumour take and the extent of tumour growth after 10 days were compared with a second series with the same tumour implantation, but without X-ray exposure. In a third series simple gastric ulcers without tumour were produced by clamping the gastric wall with a heated (80 degrees) surgical needle holder, and the animals were killed 5-7 day later. All the rats were given injections of vinblastine sulfate 3 hours and of 3H-TDR 1 hour before sacrifice. In viewfields with diameter 180 mu the vinblastine-arrested mitoses and labelled cells on the tumour side of the tumour/mucosa border were calculated as percentages of all tumour cells. In the mucosa the total number of proliferating cells was counted at various distances from the border of the tumour or ulcer. No clear differences in the frequency of tumour take and the extent of tumour growth were found between the X-irradiated and the normal rat stomachs, and it is concluded that the X-ray exposure 3 weeks prior to tumour implantation did not reduce the normal mucosal resistance to tumour growth. The percentage of arrested mitoses and labelled cells in the tumour decreased one view field away from the mucosal border, and the number of proliferating cells in the mucosa bordering on the tumours showed a gradual fall with increasing distance up to 0.8-1.0 mm from the tumour border; within these distances, however, the numbers were much higher than at corresponding distances from edges of the ulcers. The Walker tumour thus seems to stimulate cell proliferation in mucosa to a much greater extent than a simple ulcer does. The causes of this phenomenon and the possible roles of "chalones" or "anti-chalones" are discussed.     

Selectable plasmid vectors with alternative and ultrasensitive histochemical marker genes

Three different histochemical marker genes--E. coli beta-galactosidase gene (lacZ), Drosophila alcohol dehydrogenase gene (ADH) and human placenta alkaline phosphatase gene (ALP)--were cloned into a eukaryotic expression vector also containing the neomycin resistance gene. After calcium phosphate transfection and G418 sulfate selection of recipient BALB/c 3T3 cells, stable transfectants were pooled for histochemical staining. The lacZ-bearing cells produce aqua blue staining for beta-galactosidase; ADH-bearing cells, blue-black staining for alcohol dehydrogenase; and ALP-bearing cells, red staining for alkaline phosphatase. Cells carrying different marker genes can be easily differentiated by double-staining protocols. In addition, various photographic films can be used to enhance the colors of specific histochemically tagged cell classes. These plasmid vectors, providing selectability with the neomycin resistance gene and ultrasensitivity of alternative histochemical marker genes, will be very effective in virtually any biological system requiring analyses of multiple cell clones or classes in culture model systems or in situ.     

Histochemical analysis of molluscan stomach and intestinal alkaline phosphatase: A sialoglycoprotein

Summary Though sialoprotein nature of alkaline phosphatase of certain mammalian organs has been suggested by biochemical investigations, no histochemical techniques have yet been applied to elucidate this concept. With this view, the alkaline phosphatase of stomach and intestine of a mollusc—Semperula maculata—was analysed histochemically to elucidate its sialoglycoprotein nature. The localisation of alkaline phosphatase and sialic acid was investigated by employing well known and standard histochemical techniques.Alkaline phosphatase was localised selectively in the brush border of the mucosa of stomach and intestine, it was Mg⁺⁺ nonsensitive but showed a structure-linked sensitivity to phenylalanine. The sialomucins were selectively localised in the brush border, whereas the goblet cells contained both the sialomucins and sulfomucins, and the connective tissue of lamina propria contained sulfomucins. The localisation of alkaline phosphatase and sialomucins in the brush border uniquely coincided with each other. The alkaline phosphatase activity in the brush border was completely lost after neuraminidase treatment at 37.5° C for 16 h. Such effect of neuraminidase on alkaline phosphatase activity was pH dependent and controlled by velocity of reaction. Heat-inactivated neuraminidase showed no effect on alkaline phosphatase activity.These histochemical results have been interpreted as suggesting a sialoglycoprotein nature of alkaline phosphatase in the brush border, and sialic acid somehow seems to be essential for enzyme activity. These results, thus, indicate necessity of visualising some of the sialo-glycoproteins as macromolecules with catalytic activity.     

Intranuclear synthesized and native glycogen particles in human gastric cancer: Ultrastructure and histochemistry

Summary Ultrastructural and histochemical studies on human gastric cancer cells disclosed the presence of native and synthesized glycogen particles. The glycogen particles were investigated in the histochemical synthesis of glycogen particles from glucose 1-phosphate by the phosphorylase-branching glycosyltransferase system and non-incubated native glycogen in human gastric adenocarcinoma tubulare.It was observed that focal synthesis localized in the intracytoplasmic matrix and intranucleus. Intranuclear synthesized glycogen appeared as a rosette form ranging from 1100 to 1300 Å in diameter and free particles ranging from 325 to 900 Å in diameter. The synthesis of glycogen appeared in the nucleus as well as in the cytoplasm of the human gastric cancer cells, and the synthesized glycogen was observed as a group of particles. Newly formed glycogen particles appeared occasionally in the interchromatin area as a large macromolecular structure of rosette form.Native glycogen appeared as a free-particle (250–333 Å, medium=300 Å) and aggregated rosette from (694–1050 Å, medium=917 Å) in the autophagosome of gastric cancer cells. There was not, however, a native glycogen particle in the nuclei of gastric cancer cells.Under certain conditions the nuclei of gastric cancer cells can acquire the capacity to synthesize glycogen.     

Programmed cell death in the larval salivary glands of <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera,Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

Sodium butyrate induced alterations in lysosomal enzyme activity

Summary Treatment of cultured HeLa cells with 5 mM sodium butyrate causes an inhibition of growth as well as extensive chemical and morphological differentiation. Lysosomal enzyme activity changes have been associated with both normal and neoplastic growth as well as many aspects of the neoplastic process. The comparative ultrastructural results show that the butyrate-treated cells have a more extensive internal membraneous system than the untreated cells, whereas other organelles seem unaffected by the butyrate treatment. Methods for the histochemical localization of lysosomal acid phosphatase show a twofold increase in particulate reaction product in the butyrate-treated HeLa cells. Isolation of lysosomes followed by a comparative enzyme analysis shows a two to three fold increase in acid phosphatase activity per cell after 24 h of butyrate treatment, as well as three to four fold increase in β-glucuronidase activity. These increases reverse within 24 h of removal of the butyrate from the culture medium. These results as interpreted suggest that butyrate treatment may be preventing sublethal autolysis by arresting the leakage of the lysosomal enzymes from the lysosome into the cytosol and thus allowing the cell to chemically and morphologically differentiate. This work was supported by National Institute of Health Grant HD 14085-03.     

Enzyme cytochemical and immunocytochemical studies of flask cells in the amphibian epidermis

The localization of oxidoreductases and transport enzymes in flask cells of the amphibian epidermis was studied at the light-microscopic level. In these cells, the deposition of cytochemical reaction products was very similar to that found in fish epidermal ionocytes, thus demonstrating histochemical similarities between these two types of cells. The present histochemical results revealed high levels of activity of alkaline phosphatase (ALPase), potassium-dependent nitrophenylphosphatase (K+-p-NPPase) and carbonic-anhydrase isozymes (CA-I and CA-II) in the apical region of the flask cells, indicating that enzyme zonation may be the main site of the ion pumping.     

Cytochemical study of cells of primary and disseminated ascite Yoshida tumor cells

The different distribution of cytochemically demonstrable enzymes: lactate dehydrogenase (LDH, 1.1.1.27), succinate dehydrogenase (SDH, 1.3.99.1), dihydrofolate reductase (DHFR, 1.5.1.3), acid phosphatase (AcP, 3.1.3.2) and alkaline phosphatase (ALP, 3.1.3.1), has been documented in Yoshida ascites hepatoma cells in vivo or stored at 80 degrees C. The dehydrogenase activities (LDH, SDH, DHFR) show a strong reaction in all samples. An increased level of these enzyme activities has been observed in the malignant cells spreading through the organs of tumor bearing rats. On the contrary, in the same samples, acid and alkaline phosphatase activities are very low. The strong dehydrogenase activities observed in Yoshida ascite cells stress the rapid turnover of tumor cells. Our results indicate that the histochemical method may be a useful tool to detect the scattered tumor cells. Furthermore, the cytochemical methods allow the characterization of the metabolic pathways employed by the primary and disseminated tumor cells.