Optimization of enzymatic preparation of glucose 1-phosphate by response surface methodology / 生物工程学报

With glucose as substrate, sodium tripolyphosphate as the phosphorus acylating agent, and phosphorylase of Solanum tuberosum as the catalyst, glucose 1-phosphate was synthesized. Based on a three-level, three-variable Box-Behnken experimental design, response surface methodology was used to evaluate the effects of temperature, molar ratio of glucose to sodium tripolyphosphate and time on the production. The structure of the product was confirmed by 1H NMR spectra. The results show that the optimum conditions were as follows: temperature 35 degrees C, molar ratio of glucose to sodium tripolyphosphate 1.35:1 and time 19 h.

Role of pectin from cucumber (Cucumis sativus) in modulation of protein kinase C activity and regulation of glycogen metabolism in rats.

The regulatory role of protein kinase C (PKC) in glycogen metabolism in pectin fed rats was investigated. Administration of pectin (5 g/kg body wt/day) from cucumber (Cucumis sativius L.) led to inhibitory effects on PKC activity in the liver of rats. In the brain and pancreas, PKC activity was significantly higher in pectin-treated rats as compared to the control group. Level of blood glucose was significantly lowered and the level of glycogen in the liver was significantly increased in pectin-administered rats. Glycogen synthase activity was enhanced, while glycogen phosphorylase enzyme showed inhibition in pectin-treated rats. Results indicated that pectin administration might have caused an increase in the secretion of the insulin, which, in turn, had a stimulatory effect on the PKC activity in the pancreas. The decreased PKC activity in the liver and increased PKC activity in the brain and pancreas on pectin administration indicated enhanced glycogenesis and reduced glycogenolysis.

O efeito do 1-metilciclopropeno o metabolismo de carboidratos de bananas (Musa acuminata AAA cv. Nanicão) / The effect of 1-methylcyclopropene on bananas (Musa acuminata AAA cv. Nanicão

A banana é um fruto climatérico que apresenta alta taxa respiratória e alta produção de etileno após a colheita, o que a torna altamente perecível. Acredita-se que o 1-MCP é capaz de ligar-se ao receptor do hormônio etileno, bloqueando sua ação e, conseqüentemente, retardando o amadurecimento do fruto. Bananas (Musa acuminata AAA cv. Nanicão) com aproximadamente 110 dias pós-antese foram armazenadas em condições controladas de umidade e temperatura. Parte da amostra foi tratada com 1-MCP (100 nl/L, outra parte foi tratada com etileno (100 ppm – 7L/min), e, uma terceira parte, foi mantida como controle. Os frutos foram caracterizados, durante o período de amadurecimento, em relação à produção de etileno e C`O IND. 2’ (por cromatografia à gás) , à concentração de amido (pelo método enzimático descrito por Cordenunsi e Lajolo 1995) e açúcares (glicose, frutose, sacarose e maltose – por HPLC-PAD). Também foram analisados os comportamentos das enzimas α e ß – amilases, fosforilase, DPE 1 e DPE 2 por atividade enzimática in vitro ou por P.A.G.E. nativo e, quando possível, foram avaliados os comportamentos destas enzimas frente a tradução (Western blotting) e transcrição protéica (Northern blotting). A degradação de amido, assim como a respiração, a produção de etileno e síntese de açúcares foram retardadas nos frutos tratados com o 1-MCP. Como conseqüência destas mudanças, também houve uma alteração nos perfis das atividades enzimáticas...

metabolic activity of the adult rabbit choroid plexus: an enzymatic histochemical approach

To investigate the metabolic activity of the adult rabbit choroid plexus, using succinate dehydrogenase, phosphorylase and alpha-naphthylacetate esterase as histochemical markers of, the aerobic, glycolytic, and lipolytic pathways, respectively. Coverslip-mounted choroid plexus sections of adult rabbits were stained histochemically for the above enzymes. To characterize the esterase isoform[s], sections were incubated with various esterase modifiers before identification of the esterase activity. Sections of liver and kidney [controls] were simultaneously treated as for choroid plexus sections. Strong reactivity of the choroidal epithelium for both succinate dehydrogenase and esterase was readily detectable, while phosphorylase activity was virtually absent. In contrast to the B-isoform of esterase characteristically dominated the controls, the choroidal esterase activity was attributed mainly to C-isoform. The results suggest that the energy required for CSF formation by the adult choroid plexus is derived almost exclusively from aerobic oxidation, including fat metabolism. The high esterase activity in the choroid plexus, and in particular the unique pattern of the choroidal esterase versus the esterase of the controls, were interpreted to offer a potential target for future inhibitors of the energy of fat metabolism and thereby for CSF reduction

Efeito do ácido indol-3-acético no metabolismo amido-sacarose durante o amadurecimento da banana (Musa spp) / Indole-3-acetic acid effect on starch-to-sucrose metabolism during banana ripening

Dentre os vários processos que concorrem para o amadurecimento da banana, a degradação do amido e sua conversão em açucares solúveis, principalmente sacarose, são dois dos processos nais relevantes para a obtenção o sabor doce característico do fruto maduro. Embora venha sendo estudado há anos, ainda não foram esclarecidos quais os mecanismos regulátorios e os possíveis sinais hormonais envolvidos no controle da degradação do amido e na síntese da sacarose. O presente estudo objetivou avaliar o efeito do ácido indol-3-acético (AIA), um hormônio da classe das auxinas com reconhecido efeito retardador do amadurecimento, sobre o metabolismo amido-sacarose e algumas enzimas correlacionadas, em bananas...

Effect of acute administration of isoproterenol on submandibular salivary gland of female rat.

The functions of salivary glands are under the regulation of both sympathetic as well as parasympathetic nerve fibers. Further, it has also been demonstrated that chronic administration of a beta-adrenergic agonist isoproterenol (IPR) results in hypertrophy and hyperplasia of submandibular gland [Schneyer C A, Am J Physiol, 203 (1962) 232]. Specific purpose of the present attempt was to look for metabolic responses of submandibular gland of oestrous female rats at very short intervals after 10 min of administration of 5, 10 and 15 micrograms of IPR to females in oestrous condition; pharmacological action and clearance time being only 8 min. The results indicated significant reduction in case of enzymic activities of phosphorylase, total ATPase and Na(+)-K+ ATPase. Cyclic AMP-specific phosphodiesterase and succinate dehydrogenase activities were suppressed only with 5 micrograms dose, but with rising dose levels the effect was not so apparent. Protein content of the gland was reduced slightly by administration of IPR. Hence, it became clear that submandibular gland responds rapidly to IPR administration. Implications of these observations are discussed.

Heat shock proteins and their role in early mammalian development

Elevated body temperature can result from many agents in the natural environment, such as fever, hot weather and heavy exercise. In our modern living conditions additional sources of induced hyperthermia including hot baths, saunas, drugs, electromagnetic radiation and ultrasound have been introduced. Hyperthermia during pregnancy has been shown to cause a wide spectrun of effects in art species studied, including humans, the outcome depending on the dose of heat absorbed by the mother and embryo and the stage of enbryonic or fetal development when exposed. The dose of heat is the product of the elevation of temperature above normal and the duration of the elevation. In relatively uncontrolled natural environmental exposures, embryonic death and resorption or abortion are probably the most common outcome. In less severe exposures (smaller doses) major or minor developmental defects can result and the central nervous system appears to be a major target for its effects. Heat damage to embryos appears to be by apoptotic and other forms of cell death in organs at critical stages of development. Over many millennia all living orgaisms appear to have developed protective mechanisms against excess heat, known collectively as the heat shock response. This response has been studied intensively over the last 20 years and its mechanisms of protection are now becoming more clearly defined. Exposures to heat (and a number of other toxic agents) trigger the heat shock response which is characterized by abrupt suspension in the normal protein synthesis and the concurrent induction of heat shock genes (hsp) and the synthesis of a set of protein families known collectively as the heat shock proteins (HSP). The hsp ape known to be involved in the response in embryos, each has at least two copies, one which appears to have functions in the normal embryonic development (cognate) and another which is induced at a certain dose of heat (induced) and which can offer some protection against damage for some time after the initiating dose. Most cognate HSP can normally be found in embryos at all stages of development. At certain critical, early stages of organ formation increased activity of one or more of the hsp families can be identified at the site of the organ analogue. The inducible HSP are usually undetectable during normal development and generally become inducible at these critical inductive stages of organ development, implying a protective function for that process. Excess heat is known to cause denaturation of proteins. Each of the known HSP families appears to protect cells through their chaperone functions in which they bind to adhesive sites on newly synthesized or heat damaged and partially unfolded structural and functional proteins. This prevents the formation of function-less aggregates. The damaged proteins are then either presented for degradation or are reconstituted by orderly disengagement from the chaperone protein. The molecular mechanisms of initiating and regulating the response are now becoming more clearly defined. Trigger mechanisms include release of prostaglandin Al which can be modulated by glucocorticoids and nonsteroidal anti-inflammatory agents. A heat shock factor (HSF) binds to the heat shock element (hse) on the gene sequence and initiates the hsp response. The signal induction pathway involves mitogen activated proteins (MAP) and stress activated proteins (SAP) which are regulated by phosphorylation. Signals are amplified by kinase cascades while they are being transmitted to the nucleus. Activated MAP and SAP kinases regulate the process by phosphorylation of proteins including transcription factors, HSP, other protein kinases and phosphorylases, growth factor receptors and cytoskeletal proteins. Although this research has defined some pathways indicating how and why heat can cause some defects, a means of preventing them has not yet emerged.

Marcadores bioquímicos en el diagnóstico temprano del infarto agudo de miocardio / Biochemical markers in the early diagnosis of acute myocardial infarctaion

Con el transcurso del tiempo surgen mejores alternativas terapéuticas para los pacientes que sufren de infarto agudo de miocardio (IAM), enfermedad considerada en nuestro país como una de los principales causas de muerte; esto hace que el Laboratorio deba evolucionar permanentemente hacia la utilización de nuevas prácticas que sean cada vez más sensibles y específicas para poder realizar un diagnóstico precoz. En el presente trabajo se pretende realizar una revisión sobre los análisis de laboratorio históricamente más frecuentemente utilizados, así como también efectuar una actualización sobre los nuevos parámetros en estudio para el diagnóstico de IAM

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.

Extraction and evaluation of crude glucan obtained from Saccharomyces cerevisae cells

Células de uma cepa de Saccharomyces cerevisae muito comuns na produçäo de álcool no Brasil foram usadas para a obtençäo de glucano bruto. O produto tem características muito similares a outras preparaçöes já descritas e demonstrou-se útil como um espessante. O rendimento (glucano bruto seco/células secas de leveduras) foi de cerca de 12 por cento)

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

Comparative studies on the properties of glycogen phosphorylases from the foot muscles of two benthic bivalves, Sunetta scripta and Villorita cyprenoides, having different habitats.

Glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate-alpha-D-glucosyl transferase, EC 2.4.1.1) was partially purified from two bivalves found in different habitats, viz. Villorita cyprenoides, an estuarine bivalve, and Sunetta scripta, a marine bivalve, and their properties compared with other animal phosphorylases. While the kinetic mechanism was same as that of phosphorylases from other animal sources, it differed in the control mechanism from other phosphorylases. The observed differences support the earlier finding that the control mechanism adopted by different animals is dependent on the evolutionary status and energy needs.

Predicted secondary structure of glycogen phosphorylase from Escherichia coli as deduced using Chou-Fasman analysis.

Secondary structure of glycogen phosphorylase from Escherichia coli has been deduced using Chou-Fasman analysis. Out of 809 amino acid residues, 244 residues showed formation of alpha-helix (30%), 218 residues beta-pleated sheet (27%) and 192 residues (24%) showed formation of reverse beta turn, distributed all over the sequence. There are total 27 alpha-helix and 31 beta-pleated sheets distributed all over the molecule. A structure consisting of three consecutive strands of beta-pleated sheets and two joining alpha-helix is predicted for the stretch of the primary sequence from residues 325 to 372, thus showing the presence of a Rossman fold super secondary structure. There is a tyrosine at position 350 in the super secondary structure, in the area to contain a reverse beta turn. Several amino acids pairs are present in the sequence having Rossman fold super secondary structure.

Glycogen content & structure & some enzymes of glycogen metabolism in human foetal organs.

The glycogen content, and its structure and the enzymes involved in glycogenolysis in human foetal organs were studied at different periods of gestation. Of all the tissues studied glycogen content was found to be the highest in cardiac muscle. Very little glycogen was present in the foetal liver at 9-12 wk of gestation, this increased progressively to nearly 2 per cent at 24 wk. Glycogen content of placenta was lower than that of skeletal muscle and liver. The level of glycogen in adipose tissue, placenta and cerebrum was not high enough to play any role in glucose homeostasis of the foetus. Human foetal liver and skeletal muscle glycogen showed the normal branched structure while the liver glycogen was found to be unusually stable. Glycogen phosphorylase activity in the foetal liver and muscle was found to be low, i.e., about a fifth and a fourth of adult liver and muscle activity respectively. The stability of foetal liver glycogen and phosphorolytic activity in the liver and muscle indicate negligible glycogenolysis during foetal development. Glucose-6-phosphatase activity in foetal liver was undetectable below 12 wk of gestation, the activity increasing progressively up to 24 wk.

Glucose release by the liver under conditions of reduced activity of glucose 6-phosphatase

An inhibitor of glucose 6-phosphatase, isosteviol, was used in liver perfusion experiments to obtain a rough estimate of the control strength of the enzyme. Isosteviol only inhibited glucose release at high concentrations (1 mM), well above that needed for half-maximal action (70 micrnM). The decrease in glucose release was followed by an increase in the intracellular glucose 6-phosphate concentration. It was concluded that the control strength of glucose-6-phosphatase is relatively low