UDP-N-acetylglucosamine 4'-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4'-epimerase activity

The protozoan parasite Giardia intestinalis has a simple life cycle consisting of an intestinal trophozoite stage and an environmentally resistant cyst stage. The cyst is formed when a trophozoite encases itself within an external filamentous covering, the cyst wall, which is crucial to the cyst's survival outside of the host. The filaments in the cyst wall consist mainly of a beta (1-3) polymer of N-acetylgalactosamine. Its precursor, UDP-N-acetylgalactosamine, is synthesized from fructose 6-phosphate by a pathway of five inducible enzymes. The fifth, UDP-N-acetylglucosamine 4'-epimerase, epimerizes UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine reversibly. The epimerase of G. intestinalis lacks UDP-glucose/UDP-galactose 4'-epimerase activity and shows characteristic amino acyl residues to allow binding of only the larger UDP-N-acetylhexosamines. While the Giardia epimerase catalyzes the reversible epimerization of UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine, the reverse reaction apparently is favored. The enzyme has a higher Vmax and a smaller Km in this direction. Therefore, an excess of UDP-N-acetylglucosamine is required to drive the reaction towards the synthesis of UDP-N-acetylgalactosamine, when it is needed for cyst wall formation. This forms the ultimate regulatory step in cyst wall biosynthesis.     

Co-infection of Giardia intestinalis and Cyclospora cayetanensis in an immunocompetent patient with prolonged diarrhea: case report

Cyclospora cayetanensis is an agent of emerging infectious disease, and a recognized cause of diarrhea in some patients. Also, the flagellated protozoan, Giardia intestinalis, induces a diarrheal illness of the small intestine. Cases of cyclosporiasis are frequently missed, primarily due to the fact that the parasite can be quite difficult to detect in human fecal samples, despite an increasing amount of data regarding this parasite. On the other hand, G. intestinalis can be readily recognized via the microscopic visualization of its trophozoite or cyst forms in stained preparations or unstained wet mounts. In this report, we describe an uncommon case of co-infection with G. intestinalis and C. cayetanensis in an immunocompetent patient with prolonged diarrhea, living in a non-tropical region of Turkey.     

Immunocytochemical localization of glycogen phosphorylase kinase in rat brain sections and in glial and neuronal primary cultures

Summary. The physiological function of brain glycogen and the role of phosphorylase kinase as a regulatory enzyme in the cascade of reactions associated with glycogenolysis in the brain have not been fully elucidated. As a first step toward elucidating such a function, we studied the localization of phosphorylase kinase in glial and neuronal primary cell cultures, and in adult rat brain slices, using a rabbit polyclonal antibody against skeletal muscle glycogen phosphorylase kinase. Immunocytochemical examination of rat astroglia-rich primary cultures revealed that a large number of cells were positive for glycogen phosphorylase kinase immunoreactivity. These cells were also positive for vimentin, a marker for immature glia, while they were negative for glial fibrillary acidic protein, a marker for mature astroglia, and for galactocerebroside, an oligodendroglial marker. Neurons in rat neuron-rich primary cultures did not show any kinase-positive staining. In paraformaldehyde-fixed adult rat brain sections, phosphorylase kinase immunoreactivity was detected in glial-like cells throughout the brain, with relatively high staining found in the cerebral cortex, the cerebellum, and the medulla oblongata. Phosphorylase kinase immunoreactivity could not be detected in neurons, with the exception of a group of large neurons in the brain stem, most likely belonging to the mesencephalic trigeminal nucleus. Phosphorylase kinase was also localized in the choroid plexus and to a lesser degree in the ependymal cells lining the ventricles. Phosphorylase kinase thus appears to have the same cellular distribution in nervous tissue as its substrates, i.e. glycogen phosphorylase and glycogen, which suggests that the physiological role of brain phosphorylase kinase is the mobilization of glycogen stores to fuel the increased metabolic demands of neurons and astrocytes.     

Identification and partial characterization of excretory/secretory products with proteolytic activity in Giardia intestinalis

This report examines the presence of proteolytic activity detected in media collected from in vitro cultures of Giardia intestinalis, and the partial characterization by gelatin-substrate polyacrylamide gel electrophoresis and inhibition studies. Gelatin-substrate polyacrylamide gel electrophoresis revealed 6 bands with proteolytic activity, with estimated molecular weights of 36, 59, 63, 72, 103, and 175 kDa. These bands were not present in the control medium. On the other hand, G. intestinalis trophozoite lysates showed proteolytic bands at 16, 20, 66, 82, 108, and 120 kDa, thus indicating that intracellular proteases could be different from the excretory/secretory (E/S) products. Based on inhibition studies, 2 bands of 59 and 63 kDa were inhibited by iodoacetic acid, indicating the presence of cysteine proteases. Partial inhibition of a band of 36 kDa was found with EDTA, a metal-chelating agent, suggesting the possible presence of metalloproteases. The presence of aspartic and serine proteases were not detected under the assay conditions used. As G. intestinalis E/S may be involved in differentiation mechanisms of the parasite and also be responsible for the mucosal alterations that occur in giardiasis, the characterization of these proteases may facilitate their evaluation as targets in the therapy of the disease.     

Hormonal control of endometrial glycogen metabolism in the Macaca arctoides

Endometrial biopsies obtained throughout the menstrual cycle of the Macaca arctoides show the glycogen content paralleling the serum progesterone fluctuations which occur during the menstrual cycle. Secretory phase samples contained a three-fold higher concentration of glycogen when compared to follicular phase tissue. Changes in the activity levels of the glycogen metabolizing enzymes, glycogen phosphorylase and glycogen synthetase, during various stages of the menstrual cycle are in accord with the concept that the post-ovulatory increase in endometrial metabolism is a function of progesterone influence on this tissue. Endometrial glycogen synthetase activity remains low during the early proliferative phase of the cycle and becomes significantly elevated (two-to three-fold) during the early secretory phase of the cycle. Glycogen phosphorylase shows a similar cyclicity later in the luteal phase, reaching maximal activity between the seventeenth to nineteenth day of the cycle and remaining elevated through the twenty-sixth day of the cycle. The coincident nature of the rise in peripheral progesterone to increases in uterine glycogen metabolism suggest that progesterone may be the prime modulator of uterine endometrial metabolism during the post-ovulatory phase.     

Modifications of the activities of key enzymes and intracellular levels of cyclic nucleotides, in correlation with the glyogen deposition in a cultured hepatoma cell line.

Glycogen accumulation in growing cultures of ZHC cells (originally derived from the Zajdela ascitic hepatoma) is accompanied by an increase in glycogen synthetase (E.C. 2.4.1.11) and phosphorylase (E.C. 2.4.1.1) activities. Essentially the synthetase b and the phosphorylase a are involved in this process. The glycogen accumulation in ZHC cells us preceeded by a noticeable peak of cAMP, whereas cGMP rises early after replating and then decreases simultaneously with the growth rate. The present results suggest that these cultured hepatoma cells undergo throughout every passage an induction process involved in glycogen synthesis storage. Since the original ascites cells growing in vivo (which lack glycogen) and the cultured ZHC cells exhibit similar glycogen synthetase and phosphorylase activities, the resurgence of the glycogenic function (Staedel and Beck, 1978) in the in vitro cultureed cells does not seem related to a change in these two enzymes. By contrast, the high cyclic nucleotide levels in the cultured cells, as compared to those in the ascites cells, offer a possible explanation.     

Wine yeast strains engineered for glycogen overproduction display enhanced viability under glucose deprivation conditions

We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions. Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity. All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth. Strains lacking GPH1, which encodes glycogen phosphorylase, are unable to mobilize glycogen. Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses GSY2, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity. This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts. Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters. The engineered strain might better resist some stages of nutrient depletion during industrial use.     

Effects of denervation on the glycogen content and on the activities of enzymes of glucose and glycogen metabolism in rat diaphragm muscle

1. Changes in the content and concentration of glycogen and in the activity of a number of enzymes involved in glucose and glycogen metabolism were studied in the rat hemidiaphragm after unilateral denervation. 2. After nerve section the tissue hypertrophies; this hypertrophy is said to be confined to the smaller red fibres and not to the white. 3. The total hexokinase activity increases, whereas that of total glycogen phosphorylase decreases. The specific activity of phosphorylase a, determined after Halothane anaesthesia, remains fairly constant. 4. In fed animals the denervated tissue stores less glycogen, but in the early stages its glycogen content does not fall on starvation. 5. The effect of denervation on the specific activities of several other characteristically white-fibre enzymes are not consistent with the response of glycogen phosphorylase; the increase in content of glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase is thought to be related to proliferation of the sarcoplasmic reticulum. 6. The ratio of lactate dehydrogenase M/H subunits increases at the height of the hypertrophy, but then declines as the mass of the tissue falls. 7. The chronology of these changes in enzyme activities suggests a multiplicity of distinct responses after nerve section not consistent with any one model, either specific fibre development or reversion to de-differentiated, foetal-type metabolism.     

Wine Yeast Strains Engineered for Glycogen Overproduction Display Enhanced Viability under Glucose Deprivation Conditions

We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions. Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity. All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth. Strains lacking GPH1, which encodes glycogen phosphorylase, are unable to mobilize glycogen. Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses GSY2, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity. This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts. Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters. The engineered strain might better resist some stages of nutrient depletion during industrial use.     

Metabolic effects of proglycosyn.

Proglycosyn, a phenylacyl imidazolium compound that lowers blood glucose levels, was demonstrated previously to promote hepatic glycogen synthesis, stabilize hepatic glycogen stores, activate glycogen synthase, inactivate glycogen phosphorylase, and inhibit glycolysis. In the present study proglycosyn was found to inhibit fatty acid synthesis, stimulate fatty acid oxidation, and lower fructose 2,6-bisphosphate levels, but to have no significant effects on cell swelling and the levels of cAMP in hepatocytes prepared from fed rats. Verapamil and atropine blocked the effects of proglycosyn on glycogen metabolism, but these compounds inhibit proglycosyn accumulation by hepatocytes. Proglycosyn stimulated phosphoprotein phosphatase activity in postmitochondrial extracts, as measured by dephosphorylation of phosphorylase a and glycogen synthase D, but this action required a very high concentration of the compound, making it unlikely to be the actual mechanism involved. It is proposed that a metabolite of proglycosyn is responsible for its metabolic effects.     

Control of phosphorylase kinase in the isolated glycogen particle by Ca2+-Mg2+ synergistic activation and cAMP-dependent phosphorylation

The isolated glycogen particle provides a means to examine the regulation of glycogen metabolism with the components organized in a functional cellular complex. With this system, we have studied the control of phosphorylase kinase activation by Ca2+ and cAMP. Contrary to a previous report (Heilmeyer, L. M. G., Jr., Meyer, F., Haschke, R. H., and Fisher, E. H. (1980) J. Biol. Chem. 245, 6649-6656), phosphorylase kinase became activated during incubation of the glycogen particle with MgATP2- and Ca2+. Part of this activation could be attributed to the action of the cAMP-dependent protein kinase; however, it was not possible to quantitatively correlate activation with phosphorylation in the presence of Ca2+ and Mg2+ due to a large, but uncertain, contribution of synergistic activation caused by these ions. This latter activation had properties similar to those described by King and Carlson (King, M. M., and Carlson, G. M. (1980) Arch. Biochem. Biophys. 209, 517-523) with the purified enzyme, and its occurrence also explains why phosphorylase kinase activation in the glycogen particle was not observed previously. The cAMP-dependent activation of phosphorylase kinase in the glycogen particle has been characterized. It occurred in a similar manner when either the cAMP-dependent protein kinase or cAMP was added, thus indicating that the phosphorylation sites of phosphorylase kinase complexed in the glycogen particle were accessible to endogenous or exogenous enzyme. In the glycogen particle, both the alpha and beta subunits were phosphorylated by the cAMP-dependent protein kinase, but the alpha subunit dephosphorylation appeared to be preferentially regulated by Ca2+. The activity of phosphorylase kinase in the glycogen particle is regulated by the phosphorylation of both the alpha and beta subunits.     

Purification of Glycogen Phosphorylase from Bovine Brain and Immunocytochemical Examination of Rat Glial Primary Cultures Using Monoclonal Antibodies Raised Against This Enzyme

The physiological function in brain of glycogen and the enzyme catalyzing the rate-limiting step in glycogenolysis, glycogen phosphorylase (EC 2.4.1.1), is unknown. As a first step toward elucidating such a function, we have purified bovine brain glycogen phosphorylase isozyme BB 1,700-fold to a specific activity of 24 units/mg protein. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining, a single major protein band corresponding to an apparent molecular mass of 97 kDa was observed. Mouse monoclonal antibodies raised against the enzyme were purified and shown to be monospecific as indicated by immunoblotting. Immunocytochemical examination of astroglia-rich primary cultures of rat brain cells revealed a colocalization of glycogen phosphorylase with the astroglial marker glial fibrillary acidic protein in many cells. The staining for the enzyme appeared at two levels of intensity. There were other cells in the culture showing no specific staining under the experimental conditions employed. Neurons in neuron-rich primary cultures did not show positive staining. The data suggest that glycogen phosphorylase may be predominantly an astroglial enzyme and that astroglia cells play an important role in the energy metabolism of the brain.     

The behavior of hepatic phosphorylase b kinase, phosphorylase a and b after administration of glucagon to patients with glycogen storage disease type VIa

In the patients with glycogen storage disease (GSD) type VIa and different serum glucose response to glucagon, the activities of hepatic phosphorylase b kinase, phosphorylase a and b were estimated before and after the intravenous administration of glucagon. 3 min after the administration of glucagon an increase in the activities of phosphorylase b kinase and phosphorylase a was found in liver tissue of all patients except one. These enzymatic activities, however, did not exceed the values of these enzymes in the control liver biopsies without glucagon loading. After the intravenous administration of glucagon an unsuspected increase of phosphorylase b activity was observed in the control liver tissues and in patients with GSD type VIa, except one. In vitro investigations revealed that an increase of hepatic phosphorylase b activity occurs during its conversion to phosphorylase a. We suppose that this phosphorylase b represents a partially phosphorylated form of this enzyme (an intermediate form) that is due to the action of the active phosphorylase b kinase. The correlations between the activities of phosphorylase b kinase, phosphorylase a and an intermediate form of phosphorylase b and hepatic glycogen degradation after administration of glucagon has been discussed.     

Schistosoma haematobium: histochemistry of glycogen, glycogen phosphorylase a and glycogen branching enzyme in niridazole-treated females.

The body posterior to the ovary of Schistosoma haematobium females was investigated. Glycogen, glycogen phosphorylase a (EC 2.4.1.1) and glycogen branching enzyme (EC 2.4.1.18) activities were detected in the subtegumental muscle system, parenchyma and mature vitelline cells, whereas no activities were detected in the tegument and immature vitelline cells of the parasite. Administration of a single niridazole dose of 250 mg kg-1 to the pouched mouse (Saccostomus camestris) produced the following changes in S. haematobium females: a relatively rapid depletion of glycogen stores due to disruption of the absorptive surface of the parasite, and to an increase in the activity of glycogen phosphorylase a; a reduction in the phosphorylase a to phosphorylase b-conversion capacity of glycogen phosphorylase phosphatase (EC 3.1.3.17); a decrease in glycogen branching enzyme activity; and a relatively rapid degeneration of parasite cells possibly due to their loss of endogenous energy reserves.     

Giardia intestinalis: transmission electron microscopy study of in vitro excystation]

The in vitro excystation process in Giardia intestinalis was studied by transmission electron microscopy (TEM). Untreated cysts served as controls. The excystation process was monitored by examination of organisms after the in vitro induction and at several times during the incubation phase. The control cyts had a thick wall, made of microfibrils, that appeared not to contain any weak areas. The peritrophic space extended between the cyst wall and the organism peripherally, the space was delimited by a thin cytoplasmic layer, "the outer cytoplasmic envelope" that subtended the cyst wall. During the in vitro incubation, the trophozoite cytoplasm retracted from the wall; thus, the peritrophic space became progressively larger. The outer cytoplasmic envelope detached from the cyst wall, then broke up forming numerous small vesicles lodged between the wall and the organism. The tight arrangement of the wall microfibirils was lost. Electron-dense vacuoles appeared in the peripheral cytoplasm of the trophozoite. The organism emerged through the posterior end of the cyst, leaving behind the empty husk. Emergence was followed by cell division. The possible interrelationships of biochemical and mechanical factors affecting the process of excystation are discussed in light of the present TEM findings.     

Cytophotometric analysis of glycogen, protein and DNA of a glycogen-storing rat hepatoma (N13) cell line.

This study examines the behavior of glycogen-storing rat hepatoma (N13) in vitro using cytophotometric techniques. A significant increase in glycogen is observed in these cells after 30 min incubation in a buffered solution containing 0.1 mM glucose, that is 80 times lower than the physiological glucose concentration in rat blood. N13 hepatoma cells grow exponentially in culture using RPMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. During the first day in culture these cells store a large amount of glycogen and this increase is also observed in serum-free cultures. In more prolonged cultures the amount of glycogen per cell gradually becomes lower, although the culturing conditions are maintained. Similar variations of protein are also observed during the initial period of culture. DNA distribution does not show significant changes, although in serum-free cultures an increase in the proportion of cells in S and G2/M phases is observed. The addition of glucagon, epinephrine and cyclic AMP derivatives to serum-free cultures does not impede the storage of glycogen. Nevertheless, addition of either 2 mM N6,O2'-dibutyryl cyclic AMP or 0.1 mM 8-(4-chlorophenylthio)-cyclic AMP blocks the cell cycle at G0/G1 and glycogen content does not decrease after the first day in culture. We believe that this cell line offers an appropriated model to study glycogen metabolism and its involvement in the neoplastic process.     

Determination of glycogen and enzymes of glycogen metabolism in human hair follicles

The skin epithelium and its organelles use glycogen as well as glucose as source of energy. Therefore the characterisation of glycogen metabolism and the enzymes involved is important in the study of mechanisms regulating the normal or abnormal differentiation of skin organelles such as sebaceous glands and hair follicles.The present paper describes fluorimetric methods for the determination of glycogen and for the measurements of phosphorylase and phosphorylase kinase activity in one and the same lysate of minute tissue samples. The methods were tested for their suitability on freshly isolated human hair follicles and cultured hair follicle cells. The possible use of these techniques for studies on the pathophysiology of acne and hirsutism is discussed.