Cytochemical markers of bladder carcinogenesis

Summary Enzyme cytochemical and immunocytochemical techniques at the light and electron microscope levels were used to study the distribution of potential markers of chemical transformation in rodent bladders. In rat tumours induced byin vivo treatment with methylnitrosourea, alkaline phosphatase localization was normal on the external surface of the plasma membranes of some cells but abnormal in others where reaction product was seen only on intracellular membranes. 5-Nucleotidase localization was abnormal in all cells, being seen on endoplasmic reticulum and nuclear membranes only, while in normal bladders only ectoenzyme localization was seen. Heterogeneity of alkaline phosphatase and 5-nucleotidase localization was seen on the plasma membranes of these tumours after 15 days in organ culture. Some cells produced enzyme and others did not; in other cells only parts of the membrane reacted heavily, while other regions were negative.In transformed cell cultures and tumours of mouse bladder derived byin vitro treatment of explants with dimethylbenz (a) anthracene, a bimodal pattern of alkaline phosphatase localization was seen. Cells had either normal ectoenzyme reaction product or abnormal intracellular membrane reaction product. 5-Nucleotidase and ADPase were lost after transformation while cAMP-phosphodiesterase was retained as an ectoenzyme. Mg.ATPase and a cAMP-independent, calcium-insensitive protein phosphatase were induced in transformed cell cultures. An epithelial antigen was detected in the cytoplasm of both normal and transformed cells associated with reticular cytoplasmic ground substance, plasma membrane vesicles and cytoskeletal elements.     

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.     

Cytochemical and biochemical identification of lysosomes in cryptococcus neoformans

Normaski optics, fluorescence and electron microscopy were employed to demonstrate the occurrence of lysosomes in capsulated, enzymatically decapsulated, and dewalled cells of a human isolate ofCryptococcus neoformans. Fluorescent studies, using acridine orange as a lysosomal indicator, revealed the presence of variously sized, spherical, reddish-orange fluorescing bodies. Electron microscopy studies demonstrated the presence of acid phosphatase (AP), a lysosome marker enzyme, in single-membrane bound organelles. Lysosomes were removed from dewalled cells and separated by differential centrifugation on ficoll gradients. That fraction indicating the highest assay for AP was centrifuged at high speed, and the resulting pellet was fixed for electron microscopy and stained by the Gomori procedure for AP. Sections of the pellets revealed AP stained vesicles of the same size range as those within intact cells.     

Cytochemical and biochemical determination of acid phosphatase activity in yeasts]

Optimal conditions of the cytochemical assay for acid phosphatase in protoplasts and whole cells of S. cerevisiae have been described. Dimethyl sulfoxide was used to increase the permeability of the yeast cell envelope. In the yeast cells, grown up to the end of the exponential phase, acid phosphatase is shown to be located mainly in the central vacuole and on the cell envelope surface. A considerable activity of acid phosphatase is demonstrable on the surface of the plasma membrane and within adjacent vesicles that represent, presumably, part of the endoplasmic reticulum. Acid phosphatase can be considered as a marker enzyme for yeast cell vacuoles.     

Cytochemical and X-ray microanalysis studies of intracellular calcium pools in scale-bearing cells of the coccolithophoridEmiliania huxleyi

Summary Emiliania huxleyi is a coccolithophorid with a life cycle including a stage characterized by the occurrence of a scale-bearing cell type. The scales are composed of organic material and are produced in the cisternae of the Golgi apparatus. The present report deals with the ultrastructural calcium localization in scale-bearing cells using cation-precipitating agents. Cations were precipitated either with potassium pyroantimonate alone or according to a combined procedure in which cells are treated first with potassium oxalate, or potassium carbonate, or potassium phosphate, and then with potassium pyroantimonate. The distribution of electron-opaque deposits was the same when visualized by all four techniques. The most extensive deposits occurred in the Golgi apparatus, the peripheral space (a cellular compartment totally encompassing the protoplast), the multivesicular bodies, and the cell vacuole. X-ray microanalysis revealed that calcium was a constituent of the electron-opaque deposits. The uptake and transport of calcium, as universal functions of the Golgi apparatus, are discussed.     

Cytochemical and biochemical demonstration of an ATPase in membranes of human peroxisomes.

We demonstrated a neutral Mg-ATPase activity in human peroxisomal membranes. To establish the precise experimental conditions for detection of this ATPase, both cytochemical and biochemical characterizations were first carried out in liver peroxisomes from control and cipofibrate-treated rats. The results demonstrated an Mg-ATPase reaction in both normal and proliferated peroxisomes. The nucleotidase activity, with marked preference for ATP, was sensitive to the inhibitors N-ethylmaleimide and 7-chloro-4-nitro-benzo-2-oxadiazole (NBDCl). An ultrastructural cytochemical analysis was developed to evaluate the peroxisomal localization, which localized the reaction product to the peroxisomal membrane. These characteristics can help to differentiate the peroxisomal ATPase from the activity found in mitochondria and endoplasmic reticulum. The conditions established for detecting the rat peroxisomal ATPase were then applied to human peroxisomes isolated from liver and skin fibroblasts in culture. A similar Mg-ATPase activity was readily shown, both cytochemically and biochemically, in the membranes of human peroxisomes. These results, together with previous evidence, strongly support the presence of a specific ATPase in the human peroxisomal membrane. This ATPase may play a crucial role in peroxisome biogenesis.     

Cytochemical and biochemical observations on the cell wall of the spore ofGlomus epigaeum

Summary The cell wall of the spore ofGlomus epigaeum Daniels and Trappe, which has fibrillar subunits regularly arranged in arcs, was studied ultrastructurally and biochemically.The periodic acid/thiocarbohydrazide/silver proteinate (PATAg) reaction for polysaccharide location (Thiéry 1967) and the silver methenamine reaction for protein location (Swift 1968) were performed on whole spores, progressively alkaline-extracted and autoclaved spores, and untreated and alkaline-extracted cell wall fractions. The cytochemical results and those obtained from frozen sections indicated that the fibrils forming the main structure of the outer and inner wall consist of chitin. Quantitative determinations showed that chitin is the most important component (47%) of the alkali-insoluble residue and represents 27.2% of the whole cell wall fraction. It occurs predominantly as the acetylated form. Cytochemical and biochemical observations showed that the matrix surrounding the fibrils is made of alkali-soluble, PATAg positive polysaccharides (4.98% of the whole cell wall fraction). Monomers were identified by gas liquid chromatography as being -lactone of glucuronic acid, and glucose, rhamnose and mannose. Alkali-soluble proteins are an important part of the matrix, being spread mostly throughout the inner wall and constituting a large portion (55.1 %) of the alkali-soluble fraction.From the results we derive a model in which the chemical components are interconnected to build up a macromolecular network, in agreement with electron-microscopic observations.     

Energy depletion and autophagy. Cytochemical and biochemical studies in isolated rat hepatocytes

Summary In this paper, data dealing with the sensitivity of autophagy towards partial ATP depletion in isolated rat hepatocytes are reviewed. Partial reduction of intracellular ATP causes: (1) a decrease of proteolytic flux; (2) a decrease in uptake of cytosolic components into the autophagic-lysosomal compartment; (3) either a decrease or no change in the ratio between volume densities of autophagosomes and lysosomes, depending on whether or not the cytosolic phosphate potential is affected; and (4) impairment of the lysosomal proton pump. It is concluded that the consecutive steps of autophagy all respond to relatively small changes of intracellular ATP concentration.     

Cytochemical localization and biochemical characterization of dipeptidyl aminopeptidase II in macrophages and mast cells

Dipeptidyl aminopeptidase II (DAP II) was demonstrated cytochemically at light and electron microscope levels in rat macrophages and mast cells using Lys-Ala-4-methoxy-2-naphthylamide as a specific substrate. The enzyme which was found to be lysosomal in both cell types, was analyzed biochemically in extracts by measuring fluorometrically the liberated naphthylamine, and was visualized in sections microscopically using azo-coupling methods. DAP II was further characterized by isoelectric focusing techniques. Macrophage DAP II was found to be typical of that found in other rat tissues in terms of its structural latency, substrate specificity, inhibitor sensitivities, and pH activator requirements. Addition DAP II isozymes, not previously recognized, were observed.     

Cytochemical and biochemical studies on neutrophil alkaline phosphatase in parents of trisomy 21 children

Summary A study of karyotypes and neutrophil alkaline phosphatase (NAP) was carried out for 66 parents (33 couples) of trisomy 21 children and for 60 control parents (30 couples). Enzyme activity was determined simultaneously by biochemical and cytochemical techniques. In the mothers of trisomy 21 children we found: (a) by biochemical techniques, a significant increase of NAP activity in polymorphonuclear leukocyte (PMN) homogenates (P<0.01) and a lower supernatant/pellet ratio (P<0.01); (b) by cytochemical techniques (with or without thermal and urea treatments) NAP activity was significantly higher (P<0.001) than in control mothers; in the fathers, the two techniques gave normal NAP activity results.     

Cytochemical localization and biochemical analysis of the enzyme markers in human hepatoma cell lines

Three enzyme makers, glucose-6-phosphatase, ATPase and gamma-glutamyl transpeptidase, have been used in studying carcinogenesis of hepatocellular carcinoma. They have been investigated in animal models and human hepatocellular carcinoma in vivo and in vitro. But the inconsistent levels of these three enzymes associated with this type of carcinoma raised the possibility that the carcinoma cells might have derived from the cells originating from different stages of differentiation. To evaluate this possibility, three human cell lines, Hep G2, Hep 3B, and HA 22T, all thought to be arrested in different stages of differentiation based on their biochemical and morphological characteristics, were used as models. The three enzyme markers glucose-6-phosphatase, ATPase and gamma-glutamyl transpeptidase were examined cytochemically and biochemically. Our results showed that there was no correlation between the ATPase levels and the stages of the cell line's differentiation. But both glucose-6-phosphatase and gamma-glutamyl-transpeptidase were higher in cells that were more differentiated.     

Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor

Biochemical and histochemical studies have shown the presence of various carbohydrates in enamel. Using lectin-gold cytochemistry, we have examined the distribution of glycoconjugates containing N-acetyl-D-galactosamine (GalNAc) and/or N-acetyl-glucosamine (GlcNAc)/N-acetyl-neuraminic acid (NeuNAc) residues in rat incisor ameloblasts and in forming and maturing enamel embedded in Lowicryl K4M, LR Gold, and LR White resins. The enamel proteins that contain these carbohydrate moieties were further characterized by lectin blotting. All three resins allowed, albeit to a variable degree, detection of the binding sites for Helix pomatia agglutinin (HPA) and wheat germ agglutinin (WGA) GalNAc, and GlcNAc/NeuNAc, respectively. In general, Lowicryl K4M permitted more intense reactions with both lectins. Lectin binding was observed over the rough endoplasmic reticulum (weak labeling with WGA), the Golgi apparatus, lysosomes, secretory granules, and the enamel matrix. These compartments were shown by double labeling with WGA and anti-amelogenin antibody, and by previous immunocytochemical studies, to contain enamel proteins. Furthermore, WGA binding was more concentrated at the growth sites of enamel. Lectin blotting showed that several proteins in the amelogenin group were glycosylated and contained the sugars GalNAc and GlcNAc/NeuNAc. Fewer proteins were stained by HPA than by WGA, and the staining pattern suggested that the extracellular proteins recognized by these two lectins are processed differently. The HPA-reactive proteins were lost by or during the early maturation stage, whereas many of the WGA-reactive proteins persisted into the mid maturation stage. The heterogeneous staining of certain protein bands observed with WGA suggests that they contain more than one component. Two distinct glycoproteins containing GlcNAc/NeuNAc also appeared during the maturation stage. These results are consistent with the notion that ameloblasts produce an extracellular matrix composed mainly of glycosylated amelogenins which are differently processed throughout amelogenesis.     

Cytochemical and biochemical observations on the alveolar guanylate cyclase of golden hamster lung.

Particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing] has been cytochemically evidentiated in the cells which make-up the lung air-blood barrier. The cytochemical procedure utilized demonstrates the presence of membrane-bound guanylate cyclase activity through precipitation of lead pyrophosphate in tissues incubated with GTP or with guanylyl imidodiphosphate. Electron microscopic examination reveals that guanylate cyclase (GC) is localized, as micropinocytic vesicles, within endothelial components of small blood vessels, in basal lamina and in the flat alveolar cells. The secretory alveolar cells also exhibit the positive GC reactivity in their peripheric cytoplasm and in their microvilli. The observations support that GC and cGMP are involved in cellular transport phenomena. The enzyme might play a role in the secretion process of surface active material. Positive staining has been found also in other types of cells, namely alveolar macrophages and fibroblasts. A biochemical evaluation of GC activity shows that about 30-40% of this activity is associated with the particulate fraction, which justifies its abundance in the cytochemical reports shown in the paper.     

Cytochemical, biochemical and molecular aspects of the process of keratinization in the epidermis of reptilian scales

The characteristics of scaled skin of reptiles is one of their main features that distinguish them from the other amniotes, birds and mammals. The different scale patterns observed in extant reptiles result from a long evolutive history that allowed each species to adapt to its specific environment. The present review deals with comparative aspects of epidermal keratinization in reptiles, chelonians (turtles and tortoises), lepidosaurian (lizards, snakes, sphenodontids), archosaurians (crocodilians). Initially the morphology and cytology of reptilian scales is outlined to show the diversity in the epidermis among different groups. The structural proteins (alpha-keratins and associated proteins), and enzymes utilized to form the corneous layer of the epidermis are presented. Aside cytokeratins (alpha-keratins), used for making the cytoskeleton, reptilian alpha-keratinocytes produce interkeratin (matrix) and corneous cell envelope proteins. Keratin bundles and degraded cell organelles constitute most of the corneous material of alpha-keratinocytes. Matrix, histidine-rich and sulfur-rich proteins are produced in the soft epidermis and accumulated in the cornified cell envelope. Main emphasis is given to the composition and to the evolution of the hard keratins (beta-keratins). Beta-keratins constitute the hard corneous material of scales. These small proteins are synthesized in beta-keratinocytes and are accumulated into small packets that rapidly merge into a compact corneous material and form densely cornified layers. Beta-keratins are smaller proteins (8-20 kDa) in comparison to alpha-keratins (40-70 kDa), and this size may determine their dense packing in corneocytes. Both glycine-sulfur-rich and glycine-proline-rich proteins have been so far sequenced in the corneous material of scales in few reptilian species. The latter keratins possess C- and N-amino terminal amino acid regions with sequence homology with those of mammalian hard keratins. Also, reptilian beta-keratins possess a central core with homology with avian scale/feather keratins. Multiple genes code for these proteins and their discovery and sequentiation is presently an active field of research. These initial findings however suggest that ancient reptiles already possessed some common genes that have later diversified to produce the specific keratin-associated proteins in their descendants: extant reptiles, birds and mammals. The evolution of these small proteins in lepidosaurians, chelonians and archosaurians represent the next step to understand the evolution of cornification in reptiles and derived amniotes (birds and mammals).     

Cytochemical and biochemical studies on the differentiation of microperoxisomes in the small intestine of the fetal mouse

The localization of catalase activity during the morphogenesis of duodenum and ileum has been studied in Swiss ICR mouse embryos from the 16th day of fetal life until birth. Catalase activity was also measured by a spectrophotometric method. Few diaminobenzidine-positive microperoxisomes are present at 15 days of gestation in undifferentiated cells of the stratified epithelium lining the lumen of duodenum and ileum. The number of microperoxisomes increases considerably in the duodenal enterocytes at 17 days; the highest concentration of microperoxisomes is attained at 18 days, after which time their number becomes stable until 4 weeks after birth. Biochemically, catalase activity is barely detected at 15 days in the first half of the small intestine, but afterwards it increases steadily up to 1 day after birth. In the ileum, the increase in microperoxisome number is far less important than in the duodenal enterocytes and reaches a maximum at 19 days of gestation, that is, immediately at birth. The level of catalase activity in the second half of the small intestine is also much lower than that measured in the first half. These results are discussed in relation to the biogenesis of microperoxisimes in the small intestine before birth.     

Programmed cell death. Cytochemical evidence for accumulation of calcium in mitochondria and its translocation into lysosomes: X-ray microanalysis in metamorphosing insect muscles

Summary The intersegmental muscles in the metamorphosing silkmothAntheraea polyphemus were examined by two electron cytochemical procedures for demonstration of calcium compartmentation during the two-day period of degeneration after emergence. Muscle fibres were treated with either oxalate—pyroantimonate, or phosphate—pyroantimonate procedures. The elemental composition of the reaction product arising from the oxalate procedure was determined with electron probe X-ray microanalysis of unstained thin sections by energy dispersive spectrometry and wavelength dispersive spectrometry. The wavelength dispersive data revealed high peaks of calcium and antimony in the electron-dense precipitates. No reaction was obtained in muscles after treatment with the phosphate—pyroantimonate method.Shortly after the emergence of the moth, very few calcium deposits were found in the mitochondria, which also contained amorphous matrix densities. During the rapid lytic phase (17 and 30 h after ecdysis), the mitochondria, autophagic vacuoles sequestering mitochondria, and lysosomal dense bodies issuing from the latter were highly reactive in each muscle fibre.These results demonstrate that the collapse of tracheae (hypoxic conditions) is correlated with the calcium overload of mitochondria when the cell calcium homeostasis is apparently lost. Such calcium overload of the mitochondria appears to cause irreversible damage to these organelles which are then sequestered in autophagic vacuoles. This mitochondrial autophagic process leads to calcium translocation into a lysosomal compartment. We suggest that the calcium lysosomal stores may have a transient function of cell detoxification and stimulation of calcium-dependent degradative processes prior to the final muscle collapse.