Results of an open label feasibility study of sodium valproate in people with McArdle disease.

McArdle disease results from a lack of muscle glycogen phosphorylase in skeletal muscle tissue. Regenerating skeletal muscle fibres can express the brain glycogen phosphorylase isoenzyme. Stimulating expression of this enzyme could be a therapeutic strategy. Animal model studies indicate that sodium valproate (VPA) can increase expression of phosphorylase in skeletal muscle affected with McArdle disease. This study was designed to assess whether VPA can modify expression of brain phosphorylase isoenzyme in people with McArdle disease. This phase II, open label, feasibility pilot study to assess efficacy of six months treatment with VPA (20 mg/kg/day) included 16 people with McArdle disease. Primary outcome assessed changes in VO2peak during an incremental cycle test. Secondary outcomes included: phosphorylase enzyme expression in post-treatment muscle biopsy, total distance walked in 12 min, plasma lactate change (forearm exercise test) and quality of life (SF36). Safety parameters. 14 participants completed the trial, VPA treatment was well tolerated; weight gain was the most frequently reported drug-related adverse event. There was no clinically meaningful change in any of the primary or secondary outcome measures including: VO2peak, 12 min walk test and muscle biopsy to look for a change in the number of phosphorylase positive fibres between baseline and 6 months of treatment. Although this was a small open label feasibility study, it suggests that a larger randomised controlled study of VPA, may not be worthwhile.

Homology modelling of pyrophosphosrylase, enzyme involved in chitin pathway of Moniliophthora perniciosa.

Moniliophthora perniciosa (Sthael) (Singer) Phillips-Mora is the causal agent of witches' broom disease, which can infect Theobroma cacao decreasing the production of cocoa about 60%. M. perniciosa has a set of potential enzymes that can be useful targets for design of new inhibitors. After the release of the aminoacid sequence of pyrophosphorylase of M. perniciosa, a comparative modelling approach was carried out to obtain the 3D structure of this target. This model can be useful to develop new inhibitors against witches' broom disease.

A glycosyltransferase with a length-controlling activity as a mechanism to regulate the size of polysaccharides.

Cyclic beta-1,2-glucans (CbetaG) are osmolyte homopolysaccharides with a cyclic beta-1,2-backbone of 17-25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the CbetaG synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked beta-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a beta-1,2-glucan phosphorylase present at the CbetaG synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the beta-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this "length-controlling" phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides.

Synthesis and mobilization of glycogen during metamorphosis of the medfly Ceratitis capitata.

There are no recent reports focusing on insect glycogen metabolism that take the advances made in mammalian and yeast systems into account. Moreover, little is known about glycogen synthesis and degradation during insect metamorphosis. The biosynthesis and mobilization of insect glycogen were measured during the larva to adult transition in the Medfly, Ceratitis capitata. The glycogen accumulated by larva decreased to reach almost undetectable levels at the beginning of the pupation process. Histological preparations of 40 h muscles and fat body confirmed a low glycogen content, in contrast with high glycogen images in third larva tissues. After 40 h, glycogen was synthesized de novo and accumulates up to adult ecdysis. We obtained the metamorphosis-dependent profiles of phosphorylase, glycogen synthase, and a glycogenin-like protein. This novel insect glycogen initiator protein (the first measured in an arthropod) appeared to be similar to the homologous enzymes from vertebrates and yeast. We have correlated these results with other biochemical events and anatomical landmarks to understand the use of storage carbohydrates during the sequence of metamorphosis events.

Distribution of glycogen phosphorylase and cytochrome oxidase in the central nervous system of the turtle Trachemys dorbigni.

Glycogen phosphorylase (GP) and cytochrome oxidase (CO) activities were mapped histochemically in the brain of the turtle Trachemys dorbigni. In the telencephalon, both activities occurred in the olfactory bulb, in all cortical areas, in the dorsal ventricular ridge, striatum, primordium hippocampi and olfactory tubercle. In the diencephalon, they were identified in some areas of the hypothalamus, and in rotundus and geniculate nuclei. Both reactions were detected in the oculomotor, trochlear, mesencephalic trigeminal nuclei, the nucleus of the posterior commissure, torus semicircularis, substantia nigra and ruber and isthmic nuclei of the mesencephalon. In all layers of the optic tectum GP activity was found, but CO only labelled the stratum griseum centrale. In the medulla oblonga both enzymes appear in the reticular, raphe and vestibular nuclei, locus coeruleus and nuclei of cranial nerves. In the cerebellum, the granular and molecular layers, and the deep cerebellar nuclei were positive for both enzymes. The Purkinje cells were only reactive for CO. In the spinal cord, motor and commissural neurones exhibited a positive reaction for the two enzymes. However, CO also occurred in the marginal nucleus and in the lateral funiculus. These results may be useful as a basis for subsequent studies on turtle brain metabolism.

Glycogen brain branching enzyme.

Rat brain glycogen branching enzyme was partially purified in order to elucidate its mechanism of action. The alpha1,4-alpha1,6-glucan polysaccharide was synthesized using rat brain branching enzyme under two different elongation conditions: Glc-1-P and phosphorylase or UDP-Glc and glycogen synthase. The products obtained demonstrated that the cpolysaccharides synthesized (pattern of the spectra obtained in the presence of Krisman's reagent, lambda max, parameter A and R, % beta-amylolysis and degree of branching) under different incubation times are nearly constant. These results imply that the degree of branching of a polysaccharide depends only on the enzyme specificity.

Analysis of primer independent phosphorylase activity in potato plants: high levels of activity in sink organs and sucrose-dependent activity in cultured stem explants.

One isoform of potato (Solanum tuberosum L., cv. Spunta), type L phosphorylase (EC 2.4.1.1), exhibiting primer independent activity appears to be tuber-specific. However, this activity can also be modulated by exogenous sucrose in storage as well as in non-storage organs. Primer independent phosphorylase (PIPh) activity in microtubers and shoots of in vitro plantlets was found to be much higher than in tubers and shoots of soil-grown plants. Detached leaves of soil-grown plants showed an increase in PIPh activity as well when incubated in sucrose-containing Murashige-Skoog (MS) medium. This increase was always accompanied by a rise in starch content. The presence of metabolizable carbohydrates in the growth or incubation medium are likely to be responsible for the observed rise in PIPh activity. In vitro microtubers and micropropagated plantlet organs (shoots and roots) exhibited a correlation between measurable PIPh activity and presence of enzyme protein, as judged by Western blot analysis using anti-potato tuber type L phosphorylase antibody. Therefore, in addition, to be developmentally regulated (tuber-specific accumulation), PIPh activity associated with the tuber type L isoform might be under a form of metabolic regulation.

Biochemistry below 0 degrees C: nature's frozen vertebrates.

Although alien to man, the ability to endure the freezing of extracellular body fluids during the winter has developed in several species of terrestrially hibernating frogs and turtles as well as in many species of insects and other invertebrates. Wood frogs, for example, can endure freezing for at least 2 weeks with no breathing, no heart beat or blood circulation, and with up to 65% of their total body water as ice. Our studies are providing a comprehensive view of the requirements for natural freezing survival and of the physical and metabolic protection that must be offered for effective cryopreservation of vertebrate organs. Molecular mechanisms of natural freeze tolerance in lower vertebrates include: 1) control over ice crystal growth in plasma by ice nucleating proteins, 2) the accumulation of low molecular weight cryoprotectants to minimize intracellular dehydration and stabilize macromolecular components, and 3) good ischemia tolerance by all organs that may include metabolic arrest mechanisms to reduce organ energy requirements while frozen. Cryomicroscopy of tissue slices and magnetic resonance imaging (MRI) of whole animals is revealing the natural mode of ice propagation through an organism. MRI has also revealed that thawing is non-uniform; core organs (with high cryoprotectant levels) melt first, facilitating the early resumption of heart beat and blood circulation. Studies of the production and actions of the natural cryoprotectant, glucose, in frogs have shown its importance in maintaining a critical minimum cell volume in frozen organs and new work on the metabolic effects of whole body dehydration in 3 species of frogs has indicated that adaptations supporting freeze tolerance grew out of mechanisms that deal with desiccation resistance in amphibians. Studies of the regulation of cryoprotectant glucose synthesis by wood frog liver have shown the role of protein kinases and of alpha and beta adrenergic receptors in regulating the glycemic response, and of changes in membrane glucose transporter proteins to facilitate cryoprotectant distribution.

Biochemistry below 0§C: nature's frozen vertebrates

Although alien to man, the ability to endure the freezing of extracellular body fluids during the winter has developed in several species of terrestrially hibernating frogs and turtles as well as in many species of insects and other invertebrates. Wood frogs, for example, can endure freezing for at least 2 weeks with no breathing, no heart beat or blood circulation, and with up to 65 per cent of their total body water as ice. Our studies are providing a comprehensive view of the requirements for natural freezing survival and of the physical and metabolic protection that must be offered for effective cryopreservation of vertebrate organs. Molecular mechanisms of natural freeze tolerance in lower vertebrates include: 1) control over ice crystal growth in plasma by ice nucleating proteins, 2) the accumulation of low molecular weight cryoprotectants to minimize intracellular dehydration and stabilize macromolecular components, and 3) good ischemia tolerance by all organs that may include metabolic arrest mechanisms to reduce organ energy requirements while frozen. Cryomicroscopy of tissue slices and magnetic resonance imaging (MRI) of whole animals is revealing the natural mode of ice propagation through an organism. MRI has also revealed that thawing is non-uniform; core organs (with high cryoprotectant levels) melt first, facilitating the early resumption of heart beat and blood circulation. Studies of the production and actions of the natural cryoprotectant, glucose, in frogs have shown its importance in maintaining a critical minimum cell volume in frozen organs and new work on the metabolic effects of whole body dehydration in 3 species of frogs has indicated that adaptations supporting freeze tolerance grew out of mechanisms that deal with desiccation resistance in amphibians. Studies of the regulation of cryoprotectant glucose synthesis by wood frog liver have shown the role of protein kinases and of (alpha and beta adrenergic receptors in regulating the glycemic response, and of changes in membrane glucose transporter proteins to facilitate cryoprotectant distribution.

Effects of temperature shifts on the activities of Neurospora crassa glycogen synthase, glycogen phosphorylase and trehalose-6-phosphate synthase.

Conidiospore germlings of Neurospora crassa submitted to a heat shock at 45 degrees C accumulate trehalose and degrade glycogen. The opposite occurs upon reincubation at a physiologic temperature (30 degrees C). These observations suggest a temperature-dependent mechanism for the preferential synthesis of one or the other sugar reserve. Here we show that concomitant with these shifts of temperature, occurred reversible changes in the activities of glycogen synthase and phosphorylase. Glycogen synthase was inactivated at 45 degrees C while phosphorylase was activated. The reverse was true when the cells were shifted back to 30 degrees C. Addition of cycloheximide did not prevent the reversible enzymatic changes, which remained stable after gel filtration. Apparently, the effects of temperature shifts occurred at the level of reversible covalent enzymatic modifications. Trehalose-6-phosphate synthase properties were also affected by temperature. For instance, the enzyme was less sensitive to in vitro inhibition by inorganic phosphate at 50 degrees C than at 30 degrees C. Fructose-6-phosphate partially relieved the inhibitory effect of phosphate at 30 degrees C but not at 50 degrees C. These effects of the assay temperature, inorganic phosphate, and fructose-6-phosphate, on trehalose-6-phosphate synthase activity, were more evident for crude extracts obtained from heat-shocked cells. Altogether, these results may contribute to explain the preferential accumulation of trehalose 45 degrees C, or that of glycogen at 30 degrees C.

Insulin counters the glycogenolytic effect of arginine vasotocin in liver pieces from the axolotl, Ambystoma mexicanum, cultured in vitro.

In organ cultures of liver tissue from the axolotl, Ambystoma mexicanum, 1 nmol/l arginine vasotocin (AVT) increased tissue cyclic AMP (cAMP) concentration, activated glycogen phosphorylase, and caused glycogen breakdown and glucose release. Addition of 10 nmol/l insulin had no effect on any of these parameters. Addition of glucagon together with AVT caused a further increase in tissue cAMP but not in glucose release. Ten nanomoles per liter of insulin added to the cultures 5 min before 1 nmol/liter AVT inhibited all the above actions of AVT. This inhibitory action of insulin was not apparent in the presence of the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), which indicates that insulin activates cAMP phosphodiesterase and so reduces the concentration of cAMP in the tissue. This cannot occur in the presence of IBMX. These findings confirm previous reports that AVT causes hepatic glycogenolysis in the axolotl via an increase in tissue cAMP level.

Hepatic glycogen depletion in fed female rats induced by piroxicam

Se estudiaron en ratas hembras alimentadas normalmente los efectos de la administración intraperitoneal de piroxicam sobre los nivels hepáticos de glucógeno y la actividad de enzimas claves involucradas en el metabolismo de dicho homopolisacárido. El contenido de glucógeno en hígado disminuyó proporcionalmente al tiempo de tratamiento y a la dosis de piroxicam administrado. Dicho efecto persistió vários días después de suspender la administración de piroxicam. La administración de nadolol o de fenobarbital resultó ineficaz para prevenir el efecto depletorio provocado por piroxicam. En las ratas tratadas, la actividad de glucosa-6-fosfatasa, glucógeno fosforilasa y glucógeno sintetasa no cambió respecto a los controles. Tampoco se modificó significativamente la proporción de glucógeno fosforilasa en la forma activa (a), como consecuencia de sucesivas dosis diarias de piroxicam. En cambio, fue demostrada una reducción en la forma activa (I) de la glucógeno sintetasa. Esta reducción fue dependiente del tiempo de tratamiento con piroxicam. Además, la sobrecarga con glucosa resultó ineficiente para restabelecer la actividad del la glucógeno sintetasa y la síntesis de glucógeno en los animales tratados con piroxican. El efecto producido por piroxican sobre el metabolismo de glucógeno plantea la posibilidad de que el hígado llegue a resultar incapaz de mantener la homeostasis de la glucosa. Asimismo, la disminución en los niveles de glucógeno podría ocasionar un bloqueo en el metabolismo de drogas que fueren administradas conjuntamente con piroxicam, ya que la biotransformación de los xenobióticos es un proceso dependiente de las reservas de dicho polisacárido en las células hepáticas

Hepatic glycogen depletion in fed female rats induced by piroxicam

Se estudiaron en ratas hembras alimentadas normalmente los efectos de la administración intraperitoneal de piroxicam sobre los nivels hepáticos de glucógeno y la actividad de enzimas claves involucradas en el metabolismo de dicho homopolisacárido. El contenido de glucógeno en hígado disminuyó proporcionalmente al tiempo de tratamiento y a la dosis de piroxicam administrado. Dicho efecto persistió vários días después de suspender la administración de piroxicam. La administración de nadolol o de fenobarbital resultó ineficaz para prevenir el efecto depletorio provocado por piroxicam. En las ratas tratadas, la actividad de glucosa-6-fosfatasa, glucógeno fosforilasa y glucógeno sintetasa no cambió respecto a los controles. Tampoco se modificó significativamente la proporción de glucógeno fosforilasa en la forma activa (a), como consecuencia de sucesivas dosis diarias de piroxicam. En cambio, fue demostrada una reducción en la forma activa (I) de la glucógeno sintetasa. Esta reducción fue dependiente del tiempo de tratamiento con piroxicam. Además, la sobrecarga con glucosa resultó ineficiente para restabelecer la actividad del la glucógeno sintetasa y la síntesis de glucógeno en los animales tratados con piroxican. El efecto producido por piroxican sobre el metabolismo de glucógeno plantea la posibilidad de que el hígado llegue a resultar incapaz de mantener la homeostasis de la glucosa. Asimismo, la disminución en los niveles de glucógeno podría ocasionar un bloqueo en el metabolismo de drogas que fueren administradas conjuntamente con piroxicam, ya que la biotransformación de los xenobióticos es un proceso dependiente de las reservas de dicho polisacárido en las células hepáticas (AU)

Hepatic glycogen depletion in fed female rats induced by piroxicam.

The effects of i.p. piroxicam administration on hepatic glycogen levels and enzymatic activities of key enzymes involved into glycogen metabolism in fed female rats were studied. Liver glycogen concentrations in treated rats decreased with increasing time of treatment and doses of piroxicam administered. The fall in glycogen caused by piroxicam persisted for several days after it was discontinued. Neither nadolol nor phenobarbital administration were able to prevent the depleting effect of piroxicam. In the treated rats, glucose-6-phosphatase, glycogen phosphorylase and glycogen synthase activities remained unchanged respect to control. Also, proportion of phosphorylase in the active (a) form was not significantly affected by successive piroxicam daily doses. In contrast, we demonstrated a decrease in the glycogen synthase in the active I form. This reduction was time-dependent on piroxicam treatment. Further, glucose loads were not capable to restore activity in the synthase enzyme and liver glycogen synthesis in animals treated with piroxicam. The impairment into glycogen metabolism produced by piroxicam administration suggests liver becomes unable to maintain glucose homeostasis. Furthermore, glycogen depletion might produce an impairment in the metabolism of drugs administered simultaneously with piroxicam, because biotransformation of xenobiotics is a process depending on glycogen storage in the liver cells.

Glycogen and enzymes of glycogen metabolism in rat embryos and fetal organs.

Glycogen content and the enzymes of glycogen metabolism have been measured in the postimplantation rat embryo over a period ranging from 9.5 to 18.5 days of gestation. The earliest periods studied were at days 9.5 and 10.5 of gestation, when the yolk sac becomes vascularized and heart beat is first established. The next intervals were at days 10.5-11.5 when vascular connections via the allantoic placenta are formed. At 14.5 and 18.5 days of development, 4 entire organs were analyzed; heart, liver, kidney and brain. The metabolic apparatus of glycogen metabolism was concentrated in the embryo at 10.5 days, then the heart region, and in the heart itself at later stages.

Energetic metabolism and fatigability in experimental myotonia.

Experimental myotonia was induced in rats by 2,4-dichloro-phenoxyacetic acid (2,4-D). After 4 to 24 h of treatment, the anterior tibialis muscles exhibited increased fatigue at low frequency (30 Hz) nerve stimulation, but they developed normal tension at high-frequency (100 Hz) stimulation. Glycogen content and the activities of glycogen phosphorylase, lactate dehydrogenase and malate dehydrogenase remained normal. The absence of correlation between fatigability and energetic metabolism in this experimental model of myotonia suggests a dysfunction in excitation-contraction coupling.

Evidence for the presence of glycogen in rat thymus.

Chemical and biochemical analysis of the polysaccharide, present in rat thymus, indicate that it consists of glucose units alpha-1,4 and alpha-1,6 linked. Electron microscopy reveals the presence of a polysaccharide, similar to the beta-glycogen particles observed in liver and muscle with an average diameter of 20-30 nm. They are located in the cytoplasmic area of T-cells from the cortical region of the thymus. Enzymatic analysis indicates that the beta-particles contain a highly branched glucan with short external chains. Some of the enzymes of glycogen metabolism: synthase, phosphorylase and branching were for the first time partially purified from rat thymus and some of their properties were studied. Therefore, glycogen appeared to be synthesized in rat thymus.

The degree of branching in (alpha 1,4)-(alpha 1,6)-linked glucopolysaccharides is dependent on intrinsic properties of the branching enzymes.

1. Branching enzymes from rat and rabbit liver, as well as from potato and maize were prepared. They were almost free from contaminating glucan-degrading enzymes. 2. In 'sweet corn' maize, two separate fractions with (alpha 1,4)glucan: (alpha 1,4)glucan alpha 6-glycosyltransferase activities were obtained. One of them synthesized amylopectin, the branched component of starch, in the presence of phosphorylase and Glc1P, while the other fraction synthesized phytoglycogen. Furthermore, in a maize variety which does not accumulate phytoglycogen, only one fraction of branching activity was found, that formed amylopectin under the above-mentioned conditions. 3. Comparative analyses performed with native (alpha 1,4)-(alpha 1,6)glucopolysaccharides, and those synthesized in vitro with the branching enzyme from the same tissue, demonstrated a close similarity between both glucans. 4. It may be concluded that the branching enzyme is responsible for the specific degree of (alpha 1,6) branch linkages found in the native polysaccharide.