The energy metabolism of the brain has been measured in cat model using high performance liquid chromatography(HPLC). The experimental groups were divided into three according to the duration of ischemia. In 1- and 3-hour ischemia groups, recirculation had increased the ATP, UTP and GTP significantly to 39-49%, 53-57% and 39-62% of the sham control value respectively. Also in these groups, recirculation had increased adenylate energy charge(E.C.) to 75-82% of sham control value. Whereas there were slight increase in adenylate E.C. after recirculation in 5-hr ischemia group, with the remainders not increasing significantly. The Na+, K+-ATPase activities were not significant statistically among the groups. These results suggest that in order to prevent from the irreversible ischemic brain damage, restoration of blood flow must be accomplished within 3 hours from the onset of the acute focal ischemia in cat.
Adenosine Triphosphate ; Animals ; Brain ; Cats ; Chromatography, High Pressure Liquid ; Energy Metabolism* ; Guanosine Triphosphate ; Ischemia ; Uridine Triphosphate

The study was designed to examine the effects of pretreatment with mannitol, methyl prednisolone and nimodipine on the acute focal cerebral ischemia in the cats of occlusion of the proximal part of the middle cerebral artery via the postorbital approach. The energy metabolisms of the brain was measured utilizing the high liquid performance chromatography in the brain tissues of cats. The experimental animals were seperated into 3 groups. group I: the sham control group. group II: the recirculation group. group III: the treatment group. There were significant increase in the ATP, GTP, UTP and E.C. levels in focal ischemic cerebral tissues of the treatment group when compared with the recirculation group. It is suggested that pretreatment with the combination of these drugs may prevent the ischemic damage from the acute focal cerebral ischemia by the maintenance of high energy metabolites. However further studies should determine the synergistic pharmacologic mechanisms in this therapeutic strategy.
Adenosine Triphosphate ; Animals ; Brain ; Brain Ischemia* ; Cats ; Chromatography ; Energy Metabolism ; Guanosine Triphosphate ; Mannitol ; Middle Cerebral Artery ; Nimodipine ; Prednisolone ; Uridine Triphosphate

RalA GTPase, a member of Ras superfamily proteins, shows alternative forms between the active GTP-binding and the inactive GDP-binding states. Ral-specific guanine nucleotide exchange factor such as RalGDS interacts with activated Ras and cooperates with Ras indicating that Ral can be activated through Ras signaling pathway. Another activation path for Ral are through Ca2+-dependent but Ras-independent manner. In this study, studies were carried out to examine possible effects of Ca2+ and calmodulin, Ca2+-binding protein, directly on the GTP/GDP-binding state to recombinant unprenylated GST-RalA proteins. The results showed that Ca2+ stimulated the binding of GTP to RalA, whereas it reduced the binding of GDP to RalA. However, it does not involve a high affinity association of Ca2+ with RalA. Ca2+/calmodulin stimulated the GTPase activity of RalA. These results indicate that Ca2+ alone activates RalA by stimulating GTP-binding to RalA and Ca2+/calmodulin inactivates RalA by increasing the activity of RalGTPase.
Animal ; Brain/metabolism ; Calcium/*metabolism ; Calmodulin/*metabolism ; GTP Phosphohydrolases/*metabolism ; Guanosine Diphosphate/metabolism ; Guanosine Triphosphate/*metabolism ; Rats ; Support, Non-U.S. Gov't ; Synaptosomes/metabolism

The author studied the effect of the recirculation on the energy metabolism in acute focal cerebral ischemia. Acute focal cerebral ischemia was produced by transorbital occlusion of the left middle cerebral artery with a Heifetz clip under the operating microscope. The animals were divided into three groups according to the duration of the ischemia. Each group was reperfused for 1, 4 and 7 hour by removing the clip from the artery. In 1-hour ischemic group ATP and GTP was reduced to 38% and IMP to 40% of the sham control. After 7-hour recirculation in this group ATP was resynthesized to 85%, GTP to 85%, and IMP to 180% of the sham control. In 3-hour ischemic group ATP was reduced to 19%, GTP to 23%, and IMP to 29% of the sham control. In this group ATP, GTP and IMP rose to 43%, 49% and 84%, respectively after 7-hour recirculation ATP, GTP and IMP were markedly reduced in 5-hour ischemic group, and despite recirculation these substances continued to decrease and none of them were detected after 7-hours of recirculation, It was suggested that the restoration of the circulation must be accomplished within 1-hour or at least within 3-hour of the ischemic insult of the cats to prevent the irreversible brain infarction.
Adenosine Triphosphate ; Animals ; Arteries ; Brain Infarction ; Brain Ischemia ; Cats ; Edema* ; Energy Metabolism* ; Guanosine Triphosphate ; Ischemia ; Middle Cerebral Artery

Neutrophils play a major role in host defense against microbial infection. There are some clues indicate that neutrophils may also play a role in the pathophysiology of the airway obstruction in chronic asthma. We studied the roles of intracellular calcium and GTP gamma S in the regulation of neutrophils exocytosis using pipette perfusion and membrane capacitance measurement technique in whole cell patch clamp configuration. The results showed that the membrane capacitance increase induced by calcium revealed a biphasic process. The first phase occurred when the calcium level was between 0.2-14 micromol/L with a plateau amplitude of 1.23 pF and a calcium EC50 of 1.1 micromol/L. This phase might correspond to the release of the tertiary granules. The second phase occurred when the calcium concentration was between 20-70 micromol/L with a plateau increment of 6.36 pF, the calcium EC50 being about 33 micromol/L. This phase might represent the release of the primary and secondary granules. Intracellular calcium also simultaneously increased the exocytotic rate and the eventual extent in neutrophils. On the other hand, GTP gamma S can increase the exocytotic rate in a dose-dependent manner but had no effect on the eventual extent of membrane capacitance increment (>6 pF) if the cell was stimulated for a long period (>20 min). GTP gamma S (ranging from 20 to 100 micromol/L) induced the neutrophils to release all four types of the granules at very low intracellular calcium level.
Calcium ; metabolism ; Cell Degranulation ; drug effects ; Exocytosis ; drug effects ; GTP-Binding Proteins ; metabolism ; physiology ; Guanosine Triphosphate ; analogs & derivatives ; pharmacology ; Humans ; Neutrophils ; metabolism ; physiology ; Patch-Clamp Techniques

Objective To investigate the effect of Echinococcus granulosus cyst fluid(EgCF) on the cytoskeletal rearrangement and phagocytosis and the migration of macrophages induced by lipopolysaccharide(LPS). Methods Peritoneal macrophages of C57BL/6 mice were isolated and cultured in vitro, and divided into control group and LPS group and LPS combined with EgCF group. After 48 hours of treatment, filamentous actin (F-actin) changes were observed with rhodamine-labelled phalloidin staining and fluorescence microscopy; Transwell^TM chamber was used to test cell migration ability and flow cytometry to test cell phagocytosis. After 1 hour of treatment, PI3K and AKT, phosphorylated AKT (p-AKT), Rac1, guanosine triphospho-Rac1 (GTP-Rac1), WASP and Arp2 protein expressions were detected with Western blot analysis. Results Compared with the control group, after LPS stimulation, macrophages were deformed significantly; pseudopodia increased; actin cytoskeleton increased and was more distributed in pseudopodia; the ability of migration and phagocytosis were significantly improved, and the expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 proteins significantly increased. EgCF treatment caused cell shrinkage and disappearance of pseudopodia protrusions of LPS-activated cells, and led to the reduced phagocytic and migratory of cells; the protein expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 decreased significantly compared with the LPS group. Conclusion LPS induces the migration and enhances phagocytosis of macrophages while EgCF inhibits these effects, which is related to actin cytoskeleton rearrangement.
Mice ; Animals ; Lipopolysaccharides/pharmacology* ; Echinococcus granulosus/metabolism* ; Proto-Oncogene Proteins c-akt ; Cyst Fluid/metabolism* ; Mice, Inbred C57BL ; Macrophages/metabolism* ; Phagocytosis ; Actins/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Guanosine Triphosphate/pharmacology*

Platelet is activated through signal transduction, that mainly includes phospholipase-beta (PLCbeta) pathway, protein tyrosine kinases (PTK) pathway, phosphatidylinositol3-kinase (PI3-K) pathway, mitogen-activated protein kinases (MAPK) pathway, cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway and phospholipase A2 (PLA2) pathway. This article focuses on the relationship between signal transduction and platelet activation.
Calcium ; metabolism ; Enzyme Precursors ; metabolism ; Guanosine Triphosphate ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; Phosphatidylinositol 3-Kinases ; physiology ; Platelet Activation ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction ; Syk Kinase ; von Willebrand Factor ; physiology

The guanine-nucleotide exchange factor (GEF) RalGPS1a activates small GTPase Ral proteins such as RalA and RalB by stimulating the exchange of Ral bound GDP to GTP, thus regulating various downstream cellular processes. RalGPS1a is composed of an Nterminal Cdc25-like catalytic domain, followed by a PXXP motif and a C-terminal pleckstrin homology (PH) domain. The Cdc25 domain of RalGPS1a, which shares about 30% sequence identity with other Cdc25-domain proteins, is thought to be directly engaged in binding and activating the substrate Ral protein. Here we report the crystal structure of the Cdc25 domain of RalGPS1a. The bowl shaped structure is homologous to the Cdc25 domains of SOS and RasGRF1. The most remarkable difference between these three Cdc25 domains lies in their active sites, referred to as the helical hairpin region. Consistent with previous enzymological studies, the helical hairpin of RalGPS1a adopts a conformation favorable for substrate binding. A modeled RalGPS1a-RalA complex structure reveals an extensive binding surface similar to that of the SOS-Ras complex. However, analysis of the electrostatic surface potential suggests an interaction mode between the RalGPS1a active site helical hairpin and the switch 1 region of substrate RalA distinct from that of the SOS-Ras complex.
Amino Acid Sequence ; Binding Sites ; Catalytic Domain ; Cloning, Molecular ; Crystallography, X-Ray ; Escherichia coli ; Guanosine Diphosphate ; metabolism ; Guanosine Triphosphate ; metabolism ; Humans ; Models, Molecular ; Molecular Conformation ; Molecular Sequence Data ; Plasmids ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; genetics ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; ral GTP-Binding Proteins ; chemistry ; genetics ; metabolism ; ral Guanine Nucleotide Exchange Factor ; chemistry ; genetics ; metabolism

As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.
Citrate (si)-Synthase ; chemistry ; Cloning, Molecular ; Conserved Sequence ; Escherichia coli ; cytology ; enzymology ; GTP Phosphohydrolases ; chemistry ; genetics ; isolation & purification ; metabolism ; Guanosine Diphosphate ; pharmacology ; Guanosine Triphosphate ; analogs & derivatives ; pharmacology ; Molecular Chaperones ; chemistry ; genetics ; isolation & purification ; metabolism ; Protein Denaturation ; drug effects ; Protein Renaturation ; drug effects ; Ribosomes ; metabolism ; alpha-Glucosidases ; chemistry

Guanylyl cyclase C (GC-C) is a member of a family of enzymes that metabolize GTP to cGMP and was first identified as a receptor for heat-stable enterotoxin. Guanylin (GNY) has since been identified as an endogenous ligand for GC-C in the intestine of several mammalian species. The GNY/GC-C system regulates ion transportation and pH in the mucosa. Recently, it was reported that GC-C and GNY are involved in lipid metabolism in rat mesenteric adipose tissue macrophages. To examine the role of GC-C and GNY in lipid metabolism in cattle, we used a bovine mesenteric adipocyte primary culture system and a coculture system for bovine adipocytes and GNY-/GC-C-expressing macrophages. Fat droplets were observed to accumulate in bovine mesenteric adipocytes cultured alone, whereas few fat droplets accumulated in adipocytes indirectly cocultured with macrophages. We also observed that GC-C was present in bovine mesenteric adipose tissue, and that fat droplet accumulation decreased after in vitro GNY administration. Expressions of mRNAs encoding lipogenic factors decreased significantly in adipocytes after either coculture or GNY administration. These results suggest that the GNY/GC-C system is part of the control system for lipid accumulation in bovine mesenteric adipose tissue.
Adipocytes ; Adipose Tissue* ; Animals ; Cattle* ; Coculture Techniques ; Enterotoxins ; Guanosine Triphosphate ; Guanylate Cyclase* ; Humans ; Hydrogen-Ion Concentration ; In Vitro Techniques ; Intestines ; Ion Transport ; Lipid Metabolism ; Macrophages ; Mucous Membrane ; Rats ; RNA, Messenger