Immunohistochemical demonstration of glycogen phosphorylase in rat brain slices

Paraffin-embedded sections from paraformaldehyde-fixed rat brain were stained immunocytochemically for glycogen phosphorylase brain isozyme BB, using a monoclonal mouse antibody and the biotin-strept-avidin method, with either horseradish peroxidase or beta-galactosidase as marker enzymes. Two cell types showed strong glycogen phosphorylase-immunoreactivity: Astrocytes and ependymal cells. Most intensive staining was observed in the cerebellar cortex, the neocortex and the hippocampus. Astrocytes in the cerebellar white matter stained positively. The choroid plexus cells stained poorly or not at all. Neurons throughout the brain were negative, as well as oligodendrocytes and bundles of myelinated nerve fibers. These data are consistent with the immunocytochemical localization of glycogen phosphorylase in astroglia-rich primary cultures derived from rat brain.     

Immunohistochemical demonstration of glycogen phosphorylase in rat brain slices

Summary Paraffin-embedded sections from paraformaldehyde-fixed rat brain were stained immunocytochemically for glycogen phosphorylase brain isozyme BB, using a monoclonal mouse antibody and the biotin-streptavidin method, with either horseradish peroxidase or -galactosidase as marker enzymes. Two cell types showed strong glycogen phosphorylase-immunoreactivity: Astrocytes and ependymal cells. Most intensive staining was observed in the cerebellar cortex, the neocortex and the hippocampus. Astrocytes in the cerebellar white matter stained positively. The choroid plexus cells stained poorly or not at all. Neurons throughout the brain were negative, as well as oligodendrocytes and bundles of myelinated nerve fibers. These data are consistent with the immunocytochemical localization of glycogen phosphorylase in astroglia-rich primary cultures derived from rat brain.     

Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-metal salt methods

The present study has ultrastructurally applied the tannic acid-ferric chloride (TA-Fe) and the TA-uranyl acetate (TA-UA) methods to thin sections of glutaraldehyde-fixed, unosmicated embedded epiphyseal cartilage from rat tibiae to demonstrate complex carbohydrates. The strongest TA-Fe and TA-UA staining was observed after fixation of the specimens in glutaraldehyde containing TA. TA-Fe (pH 1.5) strongly stained matrix granules presumed to be proteoglycan monomers and chondrocyte secretory granules at various maturational stages but did not stain collagen fibrils and glycogen. TA-UA (pH 4.2) strongly stained matrix granules, intracellular glycogen, and chondrocyte secretory granules, and moderately stained collagen fibrils in the cartilage matrix. Ribosomes and nuclei were not stained above background staining with UA alone. In alpha-amylase-digested specimens, all TA-UA-reactive cytoplasmic glycogen was selectively removed. Testicular hyaluronidase digestion of specimens selectively removed TA-UA staining in matrix granules and all TA-Fe staining. When the pH of the UA solution was reduced to 1.5, TA-UA staining of glycogen and collagen was markedly decreased or absent, whereas staining of anionic sites was unaltered and significantly greater than with UA staining alone. Thus the TA-metal salt methods are pH dependent and allow differential intracellular and extracellular localization of complex carbohydrates in cartilage tissues at the electron microscope level.     

Electron staining of the cell surface coat by osmium-low ferrocyanide

In aldehyde-fixed liver and renal cortex of rat and mouse several variations of postfixation with osmium tetroxide plus potassium ferrocyanide ( FeII ) were tried. Depending on the ferrocyanide concentration different staining patterns were observed in TEM. -Osmium-High Ferrocyanide [40 mM (approximately 1%) OsO4 + 36 mM (approximately 1.5%) FeII , pH 10.4], stains membranes and glycogen. Cytoplasmic ground substance, mitochondrial matrices and chromatin are partially extracted, cell surface coats remain unstained. Membrane contrast, but extraction too, are higher with solutions containing cacodylate- than phosphate-buffer. -Osmium-Low Ferrocyanide [40 mM (approximately 1%) OsO4 + 2 mM (approximately 0.08%) FeII , pH 7.4], stains cell surface coats and basal laminae, but not glycogen, except for special cases. The trilaminar structure of membranes is poorly delineated. Signs of cytoplasmic extraction are not visible. The surface coat staining is stronger and more widespread with solutions containing phosphate- instead of cacodylate-buffer; it is enhanced by section staining with lead citrate. The cell surface coat stain does not traverse tight junctions nor permeate membranes.     

Oxidation of tissue polysaccharides by periodic acid in dimethyl sulfoxide and its anhydrous and aqueous mixtures.

Periodic acid (1% w/v) solvated by anhydrous dimethyl sulfoxide (DMSO) readily induced a strong Schiff reaction in a variety of structures containing polysaccharides, but not glycogen. With the increasing amounts of water added to DMSO, glycogen was also oxidized, while the selective localization of other polysaccharides remained unimpaired. Periodate, solvated in the anhydrous acetic acid-DMSO mixture, rapidly induced concomitant oxidation of nucin and glycogen-containing structures. Sodium bisulfite addition derivatives of carbonyls, induced by periodate oxidation in DMSO, were stained meta- and orthochromatically with toluidine blue at controlled pH. Certain metachromatic tissue components were strongly birefringent in polarized light in contrast to the identical structures oxidized by aqueous periodate. Marked differences in staining reactions elicited in identical structures by periodate in DMSO as compared with aqueous periodate suggest that DMSO-periodate method considerably enhances the range of histochemical oxidations by periodate.     

A simple method for cloning genes involved in glucan biosynthesis: isolation of structural and regulatory genes for glycogen synthesis in Escherichia coli.

A simple and widely applicable method for cloning genes involved in glucan biosynthesis is described. An Escherichia coli genomic library was prepared in the low-copy plasmid, pLG339, and E. coli transformants from this library were screened by staining with iodine vapor. Colonies that stained darker than the control were isolated and characterized. The three classes of clones that were identified included: (i) plasmids encoding E. coli glycogen biosynthetic (glg) structural genes, (ii) clones that resulted in elevated glycogen levels, but did not encode glg structural genes or enhance the level of the first enzyme of the pathway, ADPglucose pyrophosphorylase (AGPP), and (iii) clones that enhanced the level of AGPP, but did not encode this enzyme. Two clones from the latter class also enhanced glgC'-'lacZ-encoded beta-galactosidase activity, and may encode factors that regulate the expression of glg structural genes. It should be possible to readily clone glycogen biosynthetic genes from other bacterial species via this method. The method could be made specific for a desired glg gene by using a recipient strain that is defective in the gene of interest.     

Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue

Cardiac conduction fibers fixed either in glutaraldehyde and OsO4 or treated additionally en bloc with uranyl acetate were studied in order to demonstrate the structure of glycosomes (protein-glycogen complex). Sections were stained histochemically by periodic acid-thiosemicarbazide-silver proteinate (PA--TSC--SP) for glycogen followed by uranyl acetate and lead citrate (U-Pb) for protein. In control sections periodic acid was replaced by hydrogen peroxide (H2O2). Glycogen appeared in all sections stained by PA-TSC-SP. Protein was poorly contrasted in periodic acid treated histochemical sections taken from fixed in glutaraldehyde and OsO4. Simultaneous staining of glycogen and protein was achieved in sections of tissue treated en bloc with uranyl acetate. This treatment revealed two classes of glycosomes: 1) glycosomes deposited freely in the cytoplasm whose structure was disintegrated after treatment with uranyl acetate: 2) glycosomes associated with other cellular structures that remained intact. Staining of glycogen and protein in the same section demonstrated for the first time the structure of intact glycosomes.     

Glycogen granules in resting and inflammatory rainbow trout phagocytes--an ultrastructural study

The ultrastructural image of glycogen granules in the cytoplasm of rainbow trout phagocytes in sections stained by the conventional lead or uranyl-lead stains is highly dependent on fixation conditions, the granules being visible only when adequate fixation protocols are used. Morphometry of samples processed for the detection of peroxidase or esterase activities (to specifically label neutrophils and macrophages, respectively), and simultaneously stained for the specific detection of glycogen, showed that inflammatory peritoneal neutrophils were richer in glycogen granules than resting neutrophils. This increase in glycogen content occurs after the migration from the haematopoietic tissues and peripheral blood to the inflamed foci. Glycogen granules could not be found in resting peritoneal macrophages but were found in inflammatory macrophages. The macrophage granules occurred in smaller amounts than in neutrophils, and consisted of granules identical to those of neutrophils together with significantly smaller granules. No evidence for the utilization of glycogen by neutrophils phagocytosing bacteria within the peritoneal cavity was found.     

Relationship between glycogen accumulation and the laforin dual specificity phosphatase

Laforin, encoded by the EPM2A gene, is a dual specificity protein phosphatase that has a functional glycogen-binding domain. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons and other tissues. We examined the level of laforin protein in several mouse models in which muscle glycogen accumulation has been altered genetically. Mice with elevated muscle glycogen have increased laforin as judged by Western analysis. Mice completely lacking muscle glycogen or with 10% normal muscle glycogen had reduced laforin. Mice defective in the GAA gene encoding lysosomal alpha-glucosidase (acid maltase) overaccumulate glycogen in the lysosome but did not have elevated laforin. We propose, therefore, that laforin senses cytosolic glycogen accumulation which in turn determines the level of laforin protein.     

Photoaffinity labeling of concanavalin A. Preparation of a concanavalin A derivative with reduced valence.

Concanavalin A (Con A) was labeled with p-azidophenyl alpha-D-mannopyranoside under ultraviolet irradiation and the reaction products were separated by affinity chromatography on Sephadex G-100 at pH 5. One of the Con A derivatives thus obtained was characterized as a monovalent dimer at pH 5 and a divalent tetramer at pH 7 by sedimentation equilibrium and equilibrium dialysis, indicating that this photoaffinity labeling did not alter the quaternary structure of Con A. In agreement with these results, the labeled Con A did not show the capacity to precipitate glycogen at pH 5, but it formed precipitates with glycogen at pH 7. Although its hemagglutinating activity was found to be weaker than that of the native Con A, the dose-response cure of the labeled Con A in the mitogenic stimulation of human peripheral lymphocytes was almost identical to that of the native con A.     

Multicomponent phenol hydroxylase-catalysed formation of hydroxyindoles and dyestuffs from indole and its derivatives

AIMS: To establish multicomponent phenol hydroxylases (mPHs) as novel biocatalysts for producing dyestuffs and hydroxyindoles such as 7-hydroxyindole (7-HI) from indole and its derivatives. METHODS AND RESULTS: We have isolated Pseudomonas sp. KL33, which possesses a phenol degradation pathway similar to that found in Pseudomonas sp. CF600. Pseudomonas sp. KL28 is a strain that can grow on n-alkylphenols as a carbon and energy source. Escherichia coli strains expressing mPH from strain KL28 (mPH(KL28)) and strain KL33 (mPH(KL33)) catalysed the formation of indigo and 7-HI, respectively, from indole. In addition, both mPHs catalysed the production of dyestuffs and hydroxyindoles from indole derivatives. The mPH(KL28) has proved to be one of the most versatile biocatalysts that can accommodate a wide range of indole derivatives for catalysing the formation of dyestuffs. CONCLUSIONS: The present work provides a new approach in producing various dyestuffs and hydroxyindoles from indole and its derivatives by mPHs. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that mPHs may serve as potential agents for organic syntheses as well as bioremediation.     

Effects of exogenous glucose on survival and infectivity of Schistosoma mansoni cercariae

The effects of exogenous glucose in artificial spring water (ASW) were studied on the survival and infectivity of Schistosoma mansoni cercariae. The mean percent survival of cercariae maintained in 1% glucose in ASW for 36 and 48 hr was significantly greater than that of cercariae maintained identically in ASW. Cercariae maintained in ASW with or without glucose for 24 hr, fixed in neutral buffered formalin, and stained in Oil Red O, showed an accumulation of neutral lipid in the tail. Cercariae maintained as described above and stained in periodic acid-Schiff exhibited depleted glycogen, mainly from the tail. Cercariae maintained in ASW with glucose for 24 hr did not resynthesize glycogen. Cercariae maintained in ASW with glucose for 24 hr were as capable of infecting male FVBN202 mice as were freshly emerged cercariae, and increased the percent of worm recovery. Exogeneous glucose added to ASW prolonged the survival of S. mansoni cercariae and increased infectivity in terms of worm recovery.     

Role of maltose enzymes in glycogen synthesis by Escherichia coli

Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ΔmalP (encoding maltodextrin phosphorylase), ΔmalQ (encoding amylomaltase), ΔglgA (encoding glycogen synthase), and ΔglgA ΔmalP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ΔmalP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ΔmalP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ΔmalQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ΔglgA and ΔglgA ΔmalP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase.     

A comparison of the toxicity of certain dyestuffs to the conidia of Fusarium culmorum

The toxicity of a number of dyestuffs to the spores of Fusarium culmorum and Cercosporella herpotrichoides was determined by the slide-germination technique. No attempt was made to distinguish between fungistatic and fungicidal activity.
The toxicity of basic dyestuffs was unaffected by the acid radicle associated with the dye base.
The high toxicity to Fusarium culmorum of malachite green dye base was reduced weight for weight and mole for mole by substitution of ethyl, propyl or butyl groups for methyl groups.
The reduction of malachite green to malachite green leuco base removed toxicity.
The substitution of amino groups and alkylated amino groups in benzene nuclei of triphenyl methane increased toxicity, whereas acid groups reduced toxicity. Sulphonation and carboxylation reduced toxicity to vanishing point.
Alkylation of amino groups increased, but alkylation of benzene nuclei did not affect toxicity appreciably.
When the central carbon atom of the triphenyl methane dyestuffs was replaced by nitrogen (e.g. Bindschedler's green) the diphenyl ammonium compounds were less toxic than the corresponding triphenyl-methane compounds.
The prevention of rotation of the aminated benzene rings by bridging, in the o -position to central atom, with O or N, and so obtaining a planar molecule only slightly affected toxicity.
Certain acid dyes stimulated fungal growth.
The toxicity of the basic dyestuffs seems to depend not on one specific part but on the molecule as a whole, and within certain limits the structure may be varied without pronounced changes in toxicity.     

The role of protein kinase C in the inactivation of hepatic glycogen synthase by calcium-mobilizing agonists.

The regulation of glycogen synthase by Ca2+-mobilizing hormones was studied by using rat liver parenchymal cells in primary culture. Long-term exposure of hepatocytes to 4 beta-phorbol 12-myristate 13-acetate (TPA) resulted in a decrease in vasopressin or ATP inhibition of glycogen synthesis and glycogen synthase activity, without any change in the activation of glycogen phosphorylase. In contrast, treatment with TPA did not diminish the effects of glucagon, isoprenaline or A23187 on glycogen synthase or phosphorylase. TPA treatment for 18 h did not change specific [3H]vasopressin binding, but abolished protein kinase C activity in a concentration-dependent manner. The effects of TPA to decrease protein kinase C activity and to reverse the inactivation of glycogen synthase by vasopressin were well correlated and were mimicked by mezerein, but not by 4 alpha-phorbol. However, 1 microM-TPA totally inhibited protein kinase C activity, but reversed only 60% of the vasopressin effect on glycogen synthase. It is therefore concluded that Ca2+-mobilizing hormones inhibit glycogen synthase partly, but not wholly, through a mechanism involving protein kinase C.     

Staining of rabbit eosinophil and pseudoeosinophil leukocytes.

Air-dried rabbit blood was stained by HE, PAS and a modification of the Undritz II method. Eosin stained granules red in the eosinophil leukocytes. PAS was negative and the modified Undritz method failed to give consistent results. Cells with eosinophilic granules appeared in the corneal stroma 1 h after removing the corneal epithelium. They were stained red consistently by both eosin and the modified Undritz II method. Electron micrographs failed to demonstrate crystalloids in the granules. Because of the staining characteristics and the lack of crystalloids in their granules these cells were classified as pseudoeosinophil leukocytes. The electron micrographs showed some glycogen 12 h after denuding the cornea, however, glycogen was not well stained by PAS until 18 h after denuding.     

Stimulation of glycogen synthesis and lipogenesis by glutamine in isolated rat hepatocytes.

Glutamine stimulated glycogen synthesis and lactate production in hepatocytes from overnight-fasted normal and diabetic rats. The effect, which was half-maximal with about 3 mM-glutamine, depended on glucose concentration and was maximal below 10 mM-glucose. beta-2-Aminobicyclo[2.2.1.]heptane-2-carboxylic acid, an analogue of leucine, stimulated glutaminase flux, but inhibited the stimulation of glycogen synthesis by glutamine. Various purine analogues and inhibitors of purine synthesis were found to inhibit glycogen synthesis from glucose, but they did not abolish the stimulatory effect of glutamine on glycogen synthesis. The correlation between the rate of glycogen synthesis and synthase activity suggested that the stimulation of glycogen synthesis by glutamine depended solely on the activation of glycogen synthase. This activation of synthase was not due to a change in total synthase, nor was it caused by a faster inactivation of glycogen phosphorylase, as was the case after glucose. It could, however, result from a stimulation of synthase phosphatase, since, after the addition of 1 nM-glucagon or 10 nM-vasopressin, glutamine did not interfere with the inactivation of synthase, but did promote its subsequent re-activation. Glutamine was also found to inhibit ketone-body production and to stimulate lipogenesis.     

STUDIES CONCERNING THE NATURE OF THE SECRETORY ACTIVITY OF THE ISOLATED RINGER-PERFUSED FROG LIVER : I. THE DIFFERENTIAL SECRETION OF PAIRS OF DYESTUFFS

The isolated Ringer-perfused frog liver is able to concentrate in its secretion several hundred or even thousand times a great number of dyestuffs. When two dyestuffs are perfused simultaneously, the liver separates them to a lower or higher degree, some times to such an extent that the secretion of one of them is entirely suppressed by the other. Which one of the two dyestuffs appears prevailingly in the secretion, does not depend upon its diffusibility or lipoid solubility, but perhaps upon its adsorbability. This is concluded from the fact that dyestuffs provided with several (4 to 5) strongly hydrophilic sulfonate groups in their molecule, are not permitted to pass the gland.     

Abnormal metabolism of glycogen phosphate as a cause for Lafora disease

Lafora disease is a progressive myoclonus epilepsy with onset in the teenage years followed by neurodegeneration and death within 10 years. A characteristic is the widespread formation of poorly branched, insoluble glycogen-like polymers (polyglucosan) known as Lafora bodies, which accumulate in neurons, muscle, liver, and other tissues. Approximately half of the cases of Lafora disease result from mutations in the EPM2A gene, which encodes laforin, a member of the dual specificity protein phosphatase family that is able to release the small amount of covalent phosphate normally present in glycogen. In studies of Epm2a(-/-) mice that lack laforin, we observed a progressive change in the properties and structure of glycogen that paralleled the formation of Lafora bodies. At three months, glycogen metabolism remained essentially normal, even though the phosphorylation of glycogen has increased 4-fold and causes altered physical properties of the polysaccharide. By 9 months, the glycogen has overaccumulated by 3-fold, has become somewhat more phosphorylated, but, more notably, is now poorly branched, is insoluble in water, and has acquired an abnormal morphology visible by electron microscopy. These glycogen molecules have a tendency to aggregate and can be recovered in the pellet after low speed centrifugation of tissue extracts. The aggregation requires the phosphorylation of glycogen. The aggregrated glycogen sequesters glycogen synthase but not other glycogen metabolizing enzymes. We propose that laforin functions to suppress excessive glycogen phosphorylation and is an essential component of the metabolism of normally structured glycogen.     

Aminopeptidase activity of elastotic fibres--a histochemical artifact?

Synopsis Elastotic fibres of human skin and of elastofibroma were found to be stained when sections of these tissues had been incubated for aminopeptidase using leucine naphthylamide as substrate and Fast Blue B as the coupling agent. Sections that had been inactivated (with formalin or mercuric chloride) and partially covered with an intact kidney section were stained identically. A dialysing membrane placed between the inactivated skin section and the intact kidney section did not prevent staining of the elastotic fibres. The fibres were also stained when sections were incubated in an aqueous mixture of naphthylamine and an excess of Fast Blue B. It is concluded that the staining of elastotic fibres in histochemical amino-peptidase reactions may be due to adsorption of coloured azo derivatives and may not indicate enzyme activity in the fibres.