Glycogen metabolism in the capillary endothelium. Electron histochemical study of glycogen synthetase and phosphorylase in the pecten capillary of the chick

Glycogen synthetase and phosphorylase activities in the pecten capillary of the chick were studied electron histochemically. Polyglucose particles synthesized by glycogen synthetase or phosphorylase in the pecten capillary were demonstrated to be amorphous. They were located in the cytoplasmic matrix of the endothelium and expanded it widely. The endothelial cell of the pecten capillary plays a role in glycogenesis as well as glycogenolysis and may require substantial energy to its important transport functions.     

Electron microscopic histochemistry of ATPase and alkaline phosphatase activities in mouse digital corpuscles

Summary ATPase and alkaline phosphatase (ALPase) activities were histochemically examined in the sensory corpuscle of mouse digital pads at light and electron microscopic levels. ATPase activity was found on the plasma membranes of the lamellar cells. Moderate activity was present in the caveolae, but little activity could be demonstrated in the other portions of the plasma membrane. Slight ATPase activity was also demonstrated on the plasma membranes of nerve terminals. ALPase activity was found in the caveolae but not on other portions of the plasma membranes of the lamellar cells. ALPase activity was also observed on the nerve terminals. Neither ATPase nor ALPase activity was found on the pre-terminal portions of the nerve fibres.These findings indicate that the caveolae of the lamellar cells may be involved in a transport function and that the plasma membrane of the nerve terminals may have some permeability properties different from those of the pre-terminal portions of nerve fibres.     

Differentiation, growth and activity of rat bone marrow stromal cells on resorbable poly(L/DL-lactide) membranes

Nonporous and porous membranes produced from poly(L/DL-lactide) 80/20% were characterized using profilometry, contact-angle measurements, infra-red spectroscopy, X-ray photoemission spectroscopy and scanning electron microscopy, and used to culture bone marrow stromal cells isolated from the rat femora. The cells were cultured for 5, 10, 15 and 20 days. Cell growth and activity was estimated from the amounts of DNA, alkaline phosphatase activity and total protein amount present in the cell lysate and cell differentiation was assessed histochemically. Cell morphology was estimated from scanning electron microscopy. The cells fully expressed osteoblastic phenotype, revealed spindle-shaped, ellipsoidal morphology, developed podia, produced an abundant fibrillar extracellular matrix and mineral noduli. The number of cells on the membranes increased with time of culturing and was higher for the porous membranes than the nonporous membranes. Osteoblastic differentiation was most significant between 5 and 10 days of culture. The total amounts of DNA, alkaline phosphatase and proteins increased with time of culturing. The surface characteristics of the porous membranes were superior to the nonporous membranes.     

Ochratoxin A-induced mycotoxic porcine nephropathy: alterations in enzyme activity in tubular cells.

Mycotoxic porcine nephropathy was induced by p.o. administration of crystalline ochratoxin A for periods of 5 days, 3 months and 2 years. Enzyme activities of the renal tissue were studied histochemically. These were NADH-tetrazolium reductase, NADPH-tetrazolium reductase, lactate dehydrogenase, isocitrate dehydrogenase, succinate dehydrogenase, glucose-6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, unspecific acid phosphatase and unspecific alkaline phosphatase. The activity of NADH-tetrazolium reductase and succinate dehydrogenase was reduced in the proximal tubule of all nephrons after 5 days ochratoxin A exposure and remained reduced after 3 months and 2 years exposure. The effect of ochratoxin A on these enzymes would appear to cause the impairment of proximal tubular function and the morphological changes observed in the proximal tubule in ochratoxin A-induced mycotoxic porcine nephropathy. The localization of alterations in enzyme activity corresponds to the localization of ochratoxin A previously demonstrated in the kidney. The activities of NADPH-tetrazolium reductase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and unspecific alkaline phosphatase were reduced focally corresponding to the areas with focal tubular atrophy and the degree of reduction was roughly parallel to the degree of atrophy.     

Characteristics of the morphofunctional status of lung macrophages in the phagocytosis of particles of varying cytotoxic action

The cytotoxic effect of quartz and coal on phagocytizing lung macrophages was studied by transmissive and scanning electron microscopy and histochemically (the detection of acid phosphatase). The level of phagocytic activity, the activity and localization of acid phosphatase, the number and state of organelles, and the type of macrophage surface processes were shown to represent the objective criteria of the morphofunctional state of macrophages and the cytotoxic effect of the particles phagocytized.     

Pecten of the chick eye demonstrated by vascular casts

Vascular casts demonstrated the capillary network and characteristic features of the inner surface of the capillaries in the chick pecten oculi. Pecten capillaries anastomosed free with each other to form a complicated network of many layers of capillaries. The capillary diameter varied from capillary to capillary, and sometimes ampulla-like features were seen. Numerous pores in the casts of the capillaries corresponded to endothelial processes. Pits represented of nuclear imprints. These scanning electron microscopic findings in vascular casts were not incompatible with those seen by transmission electron microscopy.     

Demonstration of the intralobular lymphatics in the guinea pig pancreas by an enzyme-histochemical method

Intralobular lymphatics in the guinea pig pancreas were demonstrated enzyme-histochemically showing the extent, distribution and fine structure by combined light and transmission electron microscopy. 5'-nucleotidase(5'-Nase)-positive lymphatic vessels were present throughout the pancreas. Intralobular lymphatics among the acini were comparatively rare and generally independent of the blood capillaries, pancreatic ducts and acini. These lymphatics revealed the usual structural features, such as typical intercellular junctions and very tenuous vascular walls without continuous basal laminae. Fine precipitates of the cerium-based reaction product for 5'-Nase activity were found to be associated with cell membranes of the lymphatic endothelium and pinocytotic vesicles. Lymphatics were not closely related to the endocrine islets, although alkaline phosphatase(ALPase)-positive blood capillaries were well developed. Collecting lymphatic vessels with valves with weaker 5'-Nase activity were also detected in the interlobular connective tissue. ALPase activity, absent in the lymphatics, was positive in the blood capillaries, suggesting that it is also a useful way of demonstrating, histochemically, the blood capillaries in the guinea pig pancreas.     

Acid and alkaline phosphatase activity in migrating primordial germ cells of the early chick embryo

Little information is available concerning enzyme activity in primordial germ cells (PGCs) of the early chick embryo. The present study is designed to examine the disposition of alkaline and acid phosphatase activity in the PGCs during their migration into the developing gonads of the early chick embryo. White Leghorn chick embryos were sacrificed at daily intervals from 1 to 6 days of incubation. Following sacrifice the embryos were fixed, dehydrated, and embedded in glycol methacrylate (GMA). Alkaline and acid phosphatases were demonstrated by the simultaneous diazo-coupling method. The embryonic tissues at the different ages were examined for PGCs and the histochemical reactions for alkaline and acid phosphatases in these cells evaluated. Acid phosphatase activity did not appear within PGCs until 3 days of incubation, and then in only a few PGCs in the active phase of their migration in the dorsal mesentery, suggesting that there is no large wave of degeneration of these cells during migration. Alkaline phosphatase activity was observed as early as 2 days of incubation in PGCs during the passive phase of their migration in extraembryonic blood vessels. Alkaline phosphatase-positive PGCs in the active phase of migration were also found in the dorsal mesentery; however, the cellular localization of this enzyme differed from that observed in the passively migrating PGCs, indicating that there are alterations in the metabolic activities of these cells during the active and passive phases of migration.     

Phosphatase activities of rat intestinal enterocytes and their relation to diverse luminal pH, with special references to the possible localization of phytase along the brush border membrane

Phosphatase activities associated with the intestinal brush border membrane (BBM) of the rat were examined histochemically in relation to the characteristic environment of the intestine, where luminal pH fluctuates drastically between alkaline and acid pH ranges. Special attention was given to intestinal alkaline phosphatase (IALP) and phytase on the BBM. Whole body fresh-frozen sections of young rats and their rapidly frozen and freeze-substituted small intestines, embedded in Technovit 7100, were processed for the histochemical demonstration of phosphatase activity at three different pH values (9.2, 7.3, and 5.2), representing the deviation of luminal pH in vivo. Either an azo-dye method or lead-salt method was employed using naphthol AS-MX phosphate and ATP as substrate, respectively. With the azo-dye method, intense phosphatase reactions were demonstrated along the BBM at all three pH ranges. Phosphatase reactions of the BBM at pH 9.2 and 7.3 were abolished by L(+)-phenylalanine, heat pre-treatment, and EDTA chelation although some reaction remained at pH 7.3 after the treatment with EDTA or L(+)-phenylalanine. Phosphatase reactions of the BBM at pH 5.2 were resistant to L(+)-phenylalanine, L(+)-tartrate, PCMB and EDTA chelation, implying that the characteristics of the enzyme responsible for phosphohydrolysis at acid pH values differed from those at higher pH values. The lead-salt method in which ATP was used as substrate revealed intense reactions--which were dependent on Mg++ and stimulated by Ca++ and resistant to L(+)phenylalanine--to be localized along the BBM at alkaline and neutral pH values, but not at acid pH values. In vitro experiments showed progressive hydrolysis of naphthol AS-MX phosphate by purified phytase at pH 5.2, in a dose-dependent manner, and suggested the possible involvement of phytase in the phosphatase reactions of the BBM at acid pH. These data indicate that the phosphatase reactions at alkaline and neutral pH values, associated with the BBM of the rat intestine, represent IALP and Mg++/ Ca++-ATPase, while those at acid pH appear to correspond to phytase activity, something which has not been demonstrated by histochemical methods despite the availability of extensive data based on biochemical analyses.     

Effect of age on intestinal calcium absorption and adaptation to dietary calcium.

To study the reported decline in intestinal calcium absorption with age, calcium active transport, immunoreactive calcium protein (CaBP) content, and alkaline phosphatase activity were measured in the intestine of two strains of rats aged 3-wk--20 mo. Calcium active transport, as measured by everted gut sacs from Sprague-Dawley rats, was greatest at 3 wk, but it declined rapidly with no active transport demonstrable at 3 mo or thereafter. CaBP content closely paralleled the decline in active transport, but alkaline phosphatase activity increased as active transport decreased. Intestinal adaptation to dietary calcium was studied by feeding high- and low-calcium diets to Fischer 344 rats aged 1.5--12 mo. In 1.5-mo-old rats fed a low-calcium diet, there was an increase in calcium active transport, CaBP content, and alkaline phosphatase activity relative to animals fed a high-calcium diet. However, the magnitude of this intestinal adaptation decreased with age until there was only marginal adaptation by 12 mo. The observed changes in calcium active transport with age and diet may be explained by the parallel changes in the vitamin D-dependent CaBP content of the intestine.     

Enzyme activity in mice with transplantable leukemia

Activity of cobalt activated acylase, gamma-glutamyltransferase, leucylaminopeptidase and alanylaminopeptidase in serum and liver of mice with transplantable leukemias (L1210, L1210/ara-C, L1210/CH3-G, AKSL-4, plasmacytoma ADJ-PC-5) were determined. Adenosinotriphosphatase, 5'-nucleotidase and alkaline phosphatase were histochemically localized in lymphatic nodes and spleen. Among the investigated enzymes the rise in serum activity of cobalt activated acylase and gamma-glutamyltransferase was demonstrated. A substantial increase of leucylaminopeptidase and alanylaminopeptidase was shown in the liver. A decrease in the histochemical reactions of all the studied enzymes was observed.     

Histochemical demonstration of the heterogeneity of the epithelium of proximal tubules in the chick mesonephros

Proximal tubules (PT) in 7-10-day old chick mesonephros were examined histochemically to evaluate their structural and functional properties related to the absorption capacity of the epithelium and its possible alterations leading to cystically dilated tubules (CDT). Alkaline phosphatase activity at the apical cell membrane was demonstrated in varying intensities in large PT. A similar heterogeneity was also detected in the expression of proteoglycans (Lewis(x) antigen) localized in the apical part of the epithelial cell membrane but not in the basolateral membrane parts (beta-catenin, Na(+),K(+)-ATPase). In analogy, the ability to accumulate trypan blue was found in the same cell population. We hypothesize that epithelial cells in proximal tubules of nephrons with a defective apical cell membrane cause reduced fluid absorption and subsequent overfilling and dilation of the tubules.     

On histochemical enzyme changes in association with canalicular activity of alkaline phosphatase in human liver.

56 human liver biopsy specimens with insignificant or no histological changes, but with abnormally strong canalicular alkaline phosphatase activity, were studied histochemically for other enzyme changes. In comparison with normal specimens, more extensive and increased canalicular activity of gamma-glutamyl transferase, and increase of canalicular leucine aminopeptidase, was found, while the sinusoidal activity of the latter enzyme was decreased. Staining for adenosine triphosphatase regularly desclosed the normal pattern of sinusoidal and canalicular activity. The lysosomal enzymes, acid phosphatase and beta-glucuronidase, stained more intensely than ordinarily, while the reactions for enzymes present in the cytosol (lactic dehydrogenase), in the mitochondria (succinic dehydrogenase, imonoamine oxidase) and in the endoplasmic reticulum (glucose-6-phosphatase) were normal.     

Microvillus inclusion disease: specific diagnostic features shown by alkaline phosphatase histochemistry.

A technique using alkaline phosphatase histochemistry on routine sections of four jejunal biopsy specimens and one necropsy sample was applied to show that alkaline phosphatase activity, normally present in the brush border, occurs in the enterocytes of patients with microvillus inclusion disease. Sections were cut at 5 micron, mounted on to glass slides, and dried overnight at 37 degrees C before staining for alkaline phosphatase activity by the indoxyl phosphatase nitro blue tetrazolium method. Incubation periods amounted to 10 minutes for biopsy specimens and 30 minutes to one hour for necropsy samples. The demonstration of alkaline phosphatase activity in routinely processed biopsy specimens provides an effective, quick, and definitive test in the diagnosis of microvillus inclusion disease without recourse to electron microscopy.     

Enzyme histochemical reactions at the demarcation line in frostbite: an experimental study on rabbits.

Eleven frostbites were induced on the ears of seven New Zealand White rabbits and specimens were taken from the lesion after 1, 4 and 8 hours, and from ten further frostbites on the ears of six rabbits for examination 1, 3 and 7 days later. The specimens were taken at the border between the frozen and non-frozen skin. NADH-diaphorase, alkaline phosphatase and esterase were demonstrated histochemically in the sample, which was also studied by haematoxylin and eosin staining. Five ears served as controls. Some granulocytes could be seen accumulating in the vessels and in the dermis at the border of the frostbite area after only 1 hour, and other enzyme rich cells (macrophages) also began to appear. After 4 hours the inflammation was quite obvious with the enzyme reactions clearly observable in the sections. After 8 hours there was no marked difference compared with the 4-hour picture. It was only after 3 days that the line of demarcation between the normal and frostbite tissue could be seen clearly. This was oblique in some specimens and vertical in others. The degeneration in the lesion could best be demonstrated by the NADH-diaphorase and esterase reactions and the early inflammation by the alkaline phosphatase reaction.     

Electron microscopic findings of polyglucose reacting with iodine

The reaction product of iodine and native glycogen or polyglucose synthesized histochemically by glycogen synthetase or phosphorylase with branching enzyme was studied in the paraboloid of the chick retina by light and electron microscopy. Native glycogen in the paraboloid stained brown, while histochemically synthesized polyglucose stained brown or purple. Electron microscopy revealed that the paraboloid in all experimental groups appeared to have many vacant spaces at the sites of polyglucose particles, as after the amylase digestion test. Native and histochemically synthesized polyglucose particles themselves looked like fine granules or appeared to have various densities in electron micrographs. These findings suggest that native or histochemically synthesized polyglucose particles are partly masked by the iodine atoms involved in the amylase channel in electron micrographs.     

Hydrocortisone influences developing collaterals

Summary The effect of chronic administration of hydrocortisone on the topographic distribution of several hydrolases has been studied in fully grown coronary arteries and in coronary collaterals of the dog.The response towards hydrocortisone appeared to be minimal in non-proliferating vessels, whereas significant enzyme adaptation was observed in growing vessels. New sites of activity were revealed for 5-nucleotidase and acid phosphatase. Strongly increased activity was noted in the vessel walls during the early stage of development for nucleoside diphosphatase and glucose 6-phosphatase. Well pronounced effects were observed equally for polyphosphatase and for thiamine pyrophosphatase. No modifications were induced in the case of alkaline phosphatase. An almost normal distribution pattern of these hydrolases was seen at the later stage of growth. The results were discussed in comparison with those obtained in untreated animals.     

Contribution to the knowledge of the fine structure of chondrosarcoma of bone. With a note on the localization of alkaline phosphatase and "ATPase".

Seven well differentiated chondrosarcomas of bone have been analyzed by electron microscopy, and the fine structural localization of adenosine triphosphatase and nonspecific alkaline phosphatase has been elucidated. On the basis of the fine structural appearance, two distinct cell types were shown to constitute the tumor tissue: chondrocyte-like cells and large "mitochondria-rich cells". Large, multinucleated cells in the tumor did not seem to correspond to osteoclasts but rather were likely to represent true neoplastic cells. Some chondrocyte-like cells appeared to be binucleated by virtue of deep, groove-like nuclear indentations. Adenosine triphosphatase and alkaline phosphatase were associated with the plasma membrane of both chondrocyte-like and mitochondria-rich cells suggesting that they might be of common origin. Normal chondroblasts and chondrocytes lack histochemically demonstrable adenosine triphosphatase on their plasma membrane. Presence of this enzyme in the tumor cells may indicate that they are histogenetically related to immature non-chondroid matrix forming cells (known to carry the enzymes).     

The effects of ovarian hormones on uterine phosphatases of the rhesus monkey (Macaca Mulatta)

Endometrial acid and alkaline phosphatases were studied histochemically in rhesus monkeys treated with various combinations of estrogen (E, 17β-estradiol and/or estriol), progesterone (P) and relaxin (R) or a low potent relaxin control preparation (NRF). In the cells of the uterine glands of the E-treated animal, the apical cytoplasm showed intense activity of both phosphatases. This estrogenic response was depressed in the stratum functionale by P and in the stratum basale by PR. With E, acid phosphatase-staining granules appeared in scattered stromal cells with eccentric nuclei. Addition of P or PR increased the number of acid phosphatase positive stromal cells, especially in the stratum functionale. With the exception of the sinus-like channels and superficial vessels of the stratum functionale of monkeys treated with ER and EPR, all endothelium of capillary and precapillary vessels was rich in alkaline phosphatase activity. Thus, acid alkaline phosphatases appear to be metabolically important components of the endometrium which undergo cyclic variation and reflect specific influences of the ovarian hormones; estrogens, progesterone and relaxin. The implications to human menstrual physiology are discussed.     

Morphological and Morphometric Study of the Pecten Oculi in the Budgerigar (Melopsittacus undulatus)

The pecten oculi is a highly vascular and pigmented organ placed in the vitreous body of the avian eye. As no data are currently available on the morphological organization of the pecten in the Psittaciformes, the pecten oculi of the budgerigar (Melopsittacus undulatus) was studied. The eyes from adult male budgerigars were examined by light, transmission, and scanning electron microscopy and a morphometric study on both light and transmission electron microscopy specimens was also performed in the different parts of the organ. In the budgerigar, the type of the pecten oculi was pleated. Its basal part had a cranio‐caudal and postero‐anterior course; its body consisted of 10–12‐folds joined apically by a densely pigmented bridge. The pecten showed many capillaries, whose wall was thick and formed by pericytes and endothelial cells. These latter had a large number of microfolds, rectilinear on their luminal surface and tortuous on their abluminal surface. Interstitial pigment cells were placed among the capillaries, filled with melanin granules and showed many cytoplasmic processes. The morphometric analysis demonstrated significant differences among the three parts of the organ relative to the length of the endothelial processes and to the number and size of the pigment granules. The morphological and morphometric analysis showed that the bridge of the budgerigar, different from the other birds, had a large number of capillaries, so that this part of the organ could also play a trophic role for the retina in addition to the choriocapillaris. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.