Histochemical studies on the olfactory glomeruli of squirrel monkey

Summary Detailed histochemical studies on the distribution of various oxidative and dephosphorylating groups of enzymes have been made in the olfactory glomeruli of the squirrel monkey. The olfactory glomeruli showed strongly positive activity for succinic dehydrogenase, lactic dehydrogenase, monoamine oxidase, alkaline phosphatase, adenosinetriphosphatase and simple esterase. They showed moderately positive activity for cytochrome oxidase, specific cholinesterase, 5'nucleotidase; mildly positive activity for acid phosphatase; and negligible activity for nonspecific cholinesterase and glucose-6-phosphatase. The glomeruli did not show the presence of any thiamine pyrophosphatase-positive Golgi apparatus. The blood vessels surrounding the glomeruli were strongly positive for the nonspecific cholinesterase test. The significance of these results are discussed briefly.     

Histochemical observations on the exoerythrocytic malaria parasite Plasmodium berghei in rat liver

Enzyme histochemical methods were performed on sporozoite infected liver tissue of rats in order to gain insight into the nutrition and metabolism of exoerythrocytic forms of Plasmodium berghei. The following enzymes were demonstrated in the hepatocytic stages of the parasites, obtained 41 and 48 h after inoculation of sporozoites: acid phosphatase, cytochrome oxidase, NADH-tetrazolium reductase, succinate dehydrogenase, NAD+ and NADP+ dependent isocitrate dehydrogenase, NADP+-dependent malate dehydrogenase, lactate dehydrogenases, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenases and alpha-glycerol-phosphate dehydrogenase. The results suggest that a conventional Embden-Meyerhoff pathway, pentose phosphate pathway and Krebs' citric acid cycle may in part be present in these exoerythrocytic parasites. Alkaline phosphatase, nucleoside polyphosphatase, 5' nucleotidase, glucose-6-phosphatase, alpha-glucan phosphorylase, NAD+ dependent malate dehydrogenase, amino-peptidase M and non-specific esterases were not detected by our techniques in the parasite. The enzyme distribution of this intrahepatocytic malaria parasite revealed by histochemistry is compared with the enzyme distribution in the other phases of the parasite's life cycle.     

The influence of freezing and freeze-drying of tissue specimens on enzyme activity

Summary In the presented study the influence of freezing and freeze-drying on enzyme activity is described. Attention is paid to 16 enzymes which can be used for quantitative enzyme histochemical techniques.With the exception of succinate dehydrogenase only, no significant inactivation during freezing and freeze-drying procedures could be demonstrated with lactate dehydrogenase, malate dehydrogenase (NAD⁺), malate dehydrogenase (decarboxylating) (NADP⁺), isocitrate dehydrogenase (NADP⁺), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADH-oxydoreductase, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome c oxidase, phosphoglucomutase, glucosephosphate isomerase, glucose-6-phosphatase, acid phosphatase, -glucuronidase and non specific aryl esterase. Therefore the results supply a sound foundation for those quantitative enzyme histochemical techniques in which tissue specimens are frozen or frozen-dried before enzyme estimations are performed.     

The serum activity of glucose-6phosphatase and 5'-nucleotidase during human pregnancy.

An attempt has been made to show that the increase in enzyme activities in sera of pregnant women found with glucose-6-phosphate and adenosine 5'-monophosphate as substrates (described as glucose-6-phosphatase and 5'-nucleotidase) was due to the increase in alkaline phosphatase. The three enzyme activities has pH optima and heat stability characteristics of alkaline phosphatase. The response to the action of inhibitors and activators was typical for alkaline phosphatase. There was an identical increase in all three enzyme activities during pregnancy. As a control similar investigations were made with liver and placental tissue extracts.     

The localization of enzymes in the cotyledons of Pisum arvense L. during germination

Summary Enzyme activity is not uniformly distributed through the cotyledon of Pisum arvense. Initially the peripheral region, certain scattered cells of the storage tissue and the procambium show a high level of activity of succinic dehydrogenase, cytochrome oxidase, acid phosphatase and esterase. Activity of acid phosphatase declines sharply after the first day of germination; activity of the other enzymes declines after about three days. In the storage tissue, where activity is lower initially, it declines after about five days and is correlated with the disappearance of the reserves. The pattern of alkaline phosphatase activity is similar except that activity is lower in the procambium but increases in the sieve-elements during differentiation of the phloem. 5-nucleotidase and glucose-6-phosphatase activity is low throughout the cotyledon but it also increases to a significant level in the sieve-elements. Activity of starch synthesizing enzymes is high in the parenchymatous bundle sheath, where they may be involved in the pathway from lipids to soluble carbohydrates.     

Placenta of discordant twins: lack of change in histochemically detectable enzyme activities.

We localised three important enzymes histochemically in placental trophoblasts from women who gave birth to dichorionic discordant twins, in which the co-twin was affected by foetal growth restriction (FGR). The enzymes studied were adenosine diphosphate-degrading enzyme (ADP-degrading enzyme, plasma membrane enzyme), cytochrome c oxidase (mitochondrial enzyme), and glucose-6-phosphatase (endoplasmic reticular enzyme). We compared these enzyme activities and their distribution patterns among placentas of the smaller (FGR) co-twin, larger co-twin, pre-eclamptic singleton with FGR, and normal singletons with birth weight of appropriate for their gestational ages. In FGR co-twin placentas, the intensity and localisation pattern of these three enzymes did not differ from those seen in the larger co-twin and normal singleton placentas. Decreased ADP-degrading activity and cytochrome c oxidase negative mitochondria, which were characteristic features of pre-eclamptic trophoblasts, were not observed in FGR co-twin placentas. These observations indicated that, in the FGR co-twin, enzyme-histochemically detectable trophoblastic cell dysfunction may be absent, or if present, less prominent, compared with pre-eclamptic FGR. We previously reported that placental trophoblasts from singleton idiopathic FGR also showed no reduction in these enzyme activities. In mechanism and pathophysiology, FGR in dichorionic discordant twins may be quite different from pre-eclamptic FGR, but somewhat resembles idiopathic FGR, though all three disorders lead to placental insufficiency, resulting in limited foetal growth.     

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.     

Histochemical localization of acid and alkaline phosphatases and glucose-6-phosphatase of the hepatopancreas of the crab, Scylla serrata (Forskål)

Simultaneous histochemical localization of non-specific monophosphate esterases, acid and alkaline phosphatases, as well as a specific monophosphate esterase, glucose-6-phosphatase has been made on the hepatopancreas of the marine crab, Scylla serrata (Forskål). Maximum activity of the 3 enzymes was observed in the juvenile and mature absorptive cells. Lining cells of the main hepatopancreatic duct exhibited a moderate activity of the 3 enzymes whereas the embryonic and fibrillar cells and connective tissue of the gland showed negative reactions for the 3 enzymes. The secretory cells showed a positive reaction for these enzymes only at the brush border.Bilateral eyestalk removed evoked a rise in the activity of the 3 enzymes within 2–4 h. The same effect was observed after injection of eyestalk extract to both normal and eyestalkless animals followed by restoration to the normal level after 24 h.The present observations indicate that glucose-6-phosphatase and acid phosphatase may be under the direct influence of eyestalk hormone(s) while alkaline phosphatase activity appears to be related to changes in the substrate. The physiological significance of the various cell types and enzymes is discussed.     

Localization of an alkyl-acetyl-glycerol-CDP-choline: cholinephosphotransferase activity in submitochondrial fractions of Tetrahymena pyriformis

Tetrahymena pyriformis contains platelet-activating factor (PAF) as a minor lipid, which is biosynthesized de novo. A dithiothreitol-insensitive CDP-choline:cholinephosphotransferase (AAG-CPT), which utilizes alkyl-acetyl-glycerol as a substrate, had been detected in both the mitochondrial and microsomal fractions of the protozoan. In the present report, localization of this enzyme in submitochondrial fractions was studied. Cell fractionation was evaluated with enzyme and morphological markers. In this respect, succinate dehydrogenase, NADPH:cytochrome c reductase, glucose-6-phosphatase, alkaline phosphatase, monoaminoxidase, and cytochrome c oxidase activities were investigated. In the presence of antimycin A, mitochondrial activity of NADPH-cytochrome c reductase, was increased, while the microsomal one was reduced. Cardiolipin was distributed in the inner mitochondrial membrane. Alkaline phosphatase was found exclusively in the cytosol of the protozoan. The main portion of the dithiothreitol-insensitive AAG-CPT was localized in the inner mitochondrial membrane. Our data indicate that mitochondria are able to produce PAF, which might be associated with their function.     

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5''-nucleotidase.

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum an nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

Morphology of the mitochondria and endoplasmic reticula of chorion laeve cytotrophoblasts: their resemblance to villous syncytiotrophoblasts rather than villous cytotrophoblasts

We examined the morphological features of the mitochondria and endoplasmic reticula of chorion laeve cytotrophoblasts from term human fetal membranes, and compared them with those of syncytiotrophoblasts and cytotrophoblasts from human placental villi. Ultrastructural enzyme histochemistry of cytochrome c oxidase and glucose-6-phosphatase were used as cytochemical markers for these intracellular organelles. Chorion laeve cytotrophoblasts possessed abundant endoplasmic reticula, and small mitochondria with a few cristae, which were characteristic of villous syncytiotrophoblasts rather than villous cytotrophoblasts. As for these organellar structures, statistical analysis confirmed similarities between chorion laeve cytotrophoblasts and villous syncytiotrophoblasts, but significant differences between laeve cytotrophoblasts and villous cytotrophoblasts. Though these two cytotrophoblasts originated from one common cell in early placental development, they exhibited quite different organellar morphology during placental/chorioamniotic differentiation. Considering previous data, we concluded that chorion laeve cytotrophoblasts were metabolically active cells, similar to villous syncytiotrophoblasts, performing many functions in fetal membrane physiology.     

Ultrastructural localization of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium of rat

The effect of ovarian hormones on the activities of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium was studied in immature and ovariectomized rats, using ultracytochemical techniques. Comparative studies were done on normal rats at the luteal phase and on day 14 of pregnancy. Various vaginal cells show different degrees of response to progesterone and diethylstilbestrol (DES) with regard to glucose-6-phosphatase activity. Intense glucose-6-phosphatase activity was observed in the cisternae of granular endoplasmic reticulum (rER), Golgi saccules and vesicles, and nuclear envelope of both basal cells and stromal cells of progesterone treated rats, whereas in the basal cells and stromal cells of DES-treated and control animals the enzyme was totally lacking. Detectable glucose-6-phosphatase activity was also observed, however, in the rER cisternae and Golgi complex of keratohyalin-secreting squamous intermediate cells of the vaginal epithelium of DES-treated rats. Alkaline phosphatase was also found on the limiting membranes of secretory granules of mucocytes in animals at the luteal phase and during pregnancy. DES and progesterone in the doses used did not affect alkaline phosphatase activity in the rat vagina. Overall, progesterone enhances glucose-6-phosphatase activity in basal cells of the rat vagina prior to completion of mucification. Alkaline phosphatase was found in all cells involved in mucin secretion.     

Studies on the pathophysiology of acute renal failure. II. A histochemical study of the proximal tubule of the rat following administration of mercuric chloride.

Acute renal failure was induced in male rats by the subcutaneous injectioon of 4 mg HgC12 per kg body weight. Enzyme activities of the proximal tubule were studied histochemically at six time intervals from 15 min to 24 h. The enzyme studied were alkaline phosphatase, 5'-nucleotidase, acid phosphatase, alpha-glycerophosphate dehydrogenase (NAD-independent), malic dehydrogenase, succinic dehydrogenase, latic dehydrogenase, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase. Decreases in activity were observed for alkaline phosphatase and 5'-nucleotidase after 15 min. Acid phosphatase was decreased after 30 min. These three enzymes returned to control levels after 3 h, but malic dehydrogenase and alpha-glycerophosphate dehydrogenase were decreased at this time interval. Succinic dehydrogenase was first decreased after 6 h. The earliest morphological changes detectable by light microscopy were observed in pars recta tubules in the medullary rays after 6 h, a time when all enzymes studied showed widespread decreased activity throughout the proximal tubule. After 24 h, the pars convoluta appeared morphologically normal but the pars recta was necrotic and exhibited calcification, whereas enzyme activity was decreased (absent in some cases) in both pars convoluta and pars recta. These results support the hypothesis that Hg++, when given in a sublethal dose, is associated with early histochemical changes in the brush border of the proximal tubule, which may be related to early changes in sodium reabsorption and to the subsequent development of acute renal failure. The observation that changes in plasma membrane-associated enzymes occur early and prior to alterations in enzymes of mitochondria and the endoplasmic reticulum suggests that Hg++ interacts initially with the plasma membrane.     

Subcellular fractionation of epithelial cells from toad urinary bladder. 1. Assay of marker enzymes and differential centrifugation

We have undertaken the analytical fractionation of epithelial cells from toad urinary bladder, a tissue extensively used to study epithelial transport of ions and water. In an attempt to establish markers for the main subcellular organelles, a number of enzymes were assayed in cell homogenates. The nearly ubiquitous plasma membrane marker 5'-nucleotidase, and the transferases that donate N-acetylglucosaminyl, galactosyl, and sialyl residues to glycoproteins and glycolipids in the Golgi complex were not detectable. Glucose-6-phosphatase activity was low in relation to that of nonspecific phosphatases and, therefore, not suitable for identifying the endoplasmic reticulum. Like the cytosolic enzyme lactate, dehydrogenase, catalase was essentially found in the high-speed supernatant, with a noteworthy part of aminopeptidase (substrate, leucyl-beta-naphthylamide) and NAD glycohydrolase. Other enzymes, including cytochrome c oxidase, acid phosphatase, acid N-acetyl-beta-glucosaminidase, alkaline phosphatase, alkaline phosphodiesterase I, nucleoside diphosphatase (substrate ADP), oligomycin-resistant Mg++-ATPase, and mannosyltransferase (acceptor, dolichylphosphate) were fairly active and largely sedimentable. After differential centrifugation, cytochrome oxidase, acid phosphatase, and acid N-acetyl-beta-glucosaminidase were typically associated with the large granule fraction, whereas the other sedimentable enzymes exhibited a broad distribution profile overlapping the nuclear, large granule, and microsome fractions. Their behavior in density equilibrium centrifugation is examined in a companion paper.     

Enzyme histochemical studies on rat lungs 2. Normal enzyme distribution in the alveolar tissue of the mature lung

Synopsis The activity and distribution of the following eighteen oxidative and hydrolytic enzyme systems have been investigated in the lung of the adult rat: reduced NAD dehydrogenase, reduced NADP dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase, glutamate dehydrogenase, -hydroxybutyrate dehydrogenase, acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, adenosine triphosphatase, 5-nucleotidase, non-specific esterase, cytochrome oxidase and -glucuronidase.The low concentration of cells in sections of inflated lung may have made histochemical demonstration of some enzymes impossible because the enzyme concentration was below that detectable by the method employed.The carboxylic acid cycle and the hexose monophosphate shunt were potentially active but fatty acid metabolism was not indicated.The granular reaction sometimes encountered in alveolar cell cytoplasm may be useful for differentiating alveolar cell types, but further cytochemical studies are required to resolve the possible metabolic differences of alveolar cells.     

Preparation of subcellular fractions from rat liver: comparison of the Polytron with the Dounce homogenizer.

The PolytronR and Dounce homogenizers have been evaluated for preparation of homogenates of rat liver prior to isolation of subcellular fractions by differential centrifugation. Marker enzymes used to evaluate the subcellular fractions included cytochrome oxidase, monoamine oxidase, D-amino acid oxidase, acid phosphatase, glucose-6-phosphatase, ethyl morphine demethylase, and lactate dehydrogenase. No significant difference in the distribution of enzymes (percent recovery or specific activity) was observed between the two methods of homogenization. In addition, there were no significant differences in the ultrastructural appearances and respiratory control ratios of the mitochondrial fractions prepared by the two methods of homogenization.     

Cytochemical model system for microsomal rat liver glucose-6-phosphate.

A method is described for the incorporation of a microsomal rat liver fraction into polyacrylamide films without significant loss of its glucose-6-phosphatase activity. The enzymatic activity was completely lost when the films were prepared with ammonium persulfate as initiator of the polymerization as previously described for alkaline phosphatase, but modification of this method showed that about 90% of the glucose-6-phosphatase activity could be retained. The enzyme in the films prepared with the new method was completely inhibited by alloxan, HgCl2, and preincubation in 0.05 M acetate buffer (pH 5.0) at 37 degrees C, as determined biochemically. Similar results were obtained for the enzyme in films determined histochemically according to the lead method of Wachstein and Meisel. In this respect the behavior of the incorporated enzyme is similar to that in suspension. Films fixed with 1.5% glutaraldehyde showed rapid inactivation of glucose-6-phosphatase. There was good correlation between the biochemical and histochemical activity determined after fixation. A method to embed polyacrylamide films in Epon for electron-microscopical investigation is also described. Dimethyl sulfoxide was used as the dehydrating agent instead of ethanol/acetone.     

Morphological, histochemical, and biochemical studies on the gut of Haemonchus contortus Rud., 1803).

A study has been made on the structure and chemical composition of the gut of Haemonchus contortus (Rud., 1803). The oesophagus has typically a triradiate, cuticle-lined lumen. The intestinal epithelium is provided with a well-developed brush border which contains periodic acid-Schiff-positive mucoproteins. The intestinal epithelium stores glycogen and lipids. It stains diffusely for phospholipids and general proteins and also for terminal-NH2 group. The presence of Fe2+ and Fe3+ containing pigments and activities of acid and alkaline phosphatases, glucose-6-phosphatase, and 5'-nucleotidase have been observed in the intestinal epithelium. Biochemically pH optimum for intestinal acid phosphatase has been found to be 4.8. The brush border shows positive reactions for acid phosphatase and glucose-6-phosphatase, and negative reactions for alkaline phosphatase and 5'-nucleotidase, and negative reactions for alkaline phosphatase and 5'-nucleotidase. The presence of enzymes in the brush border is related to extracellular digestion and absorption of nutrients.     

Enzyme histochemistry of the choroid plexus in rat and squirrel monkey

Summary The reactions given for various oxidative and hydrolytic enzymes by the choroid plexus of the squirrel monkey and the rat brain have been studied in detail. The lining cells show strong activity for citric acid cycle and glycolytic pathways enzymes. The stroma shows strong activity for adenosine triphosphatase, alkaline phosphatase, adenosine monophosphatase and glucose-6-phosphatase. The peripheral part or luminal borders of the cytoplasm of the choroidal cells show strong activity for alkaline phosphatase, adenosine monophosphatase and adenosine triphosphatase, and a well developed thiamine pyrophosphatase positive Golgi complex, indicating their participation in the formation and transport of secretory material. The nucleoli of the lining cells give a positive reaction for glucose-6-phosphatase and adenosine triphosphatase. Acid phosphatase like the thiamine pyrophosphatase positive Golgi material is found all over the cytoplasm. The functional significance of these findings is briefly discussed.This work has been carried out with the aid of Grant No. FR-00165 from the Animal Resources Branch, National Institutes of Health and NASA Grant NGR-11-001-016. T. R. Shanthaveerappa in previous publications.