Effects of pancreastatin on insulin and pancreatic polypeptide secretion in the dog

Porcine pancreastatin (1.19 nmol) was administered into the peripheral vein (i.v.) or the third cerebral ventricle (i.t.v.) of dogs and its effect on the secretion of insulin and pancreatic polypeptide (PP) studied. Neither means of administration had any effect on basal and glucose-induced insulin or PP secretion. However, i.v. pancreastatin did inhibit the i.v. CCK-8-induced insulin but not PP release. Pancreastatin may thus play a role in the regulation of insulin secretion in the canine pancreas.     

Coffee polyphenol caffeic acid but not chlorogenic acid increases 5'AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle

Chlorogenic acid is an ester of caffeic and quinic acids, and is one of the most widely consumed polyphenols because it is abundant in foods, especially coffee. We explored whether chlorogenic acid and its metabolite, caffeic acid, act directly on skeletal muscle to stimulate 5'-adenosine monophosphate-activated protein kinase (AMPK). Incubation of rat epitrochlearis muscles with Krebs buffer containing caffeic acid (≥0.1 mM, ≥30 min) but not chlorogenic acid increased the phosphorylation of AMPKα Thr(172), an essential step for kinase activation, and acetyl CoA carboxylase Ser(79), a downstream target of AMPK, in a dose- and time-dependent manner. Analysis of isoform-specific AMPK activity revealed that AMPKα2 activity increased significantly, whereas AMPKα1 activity did not change. This enzyme activation was associated with a reduction in phosphocreatine content and an increased rate of 3-O-methyl-d-glucose transport activity in the absence of insulin. These results suggest that caffeic acid but not chlorogenic acid acutely stimulates skeletal muscle AMPK activity and insulin-independent glucose transport with a reduction of the intracellular energy status.     

Expression of Arabidopsis plastidial phosphoglucomutase in tobacco stimulates photosynthetic carbon flow into starch synthesis

Phosphoglucomutase (PGM, EC 2.7.5.1) is one of the enzymes constituting the carbohydrate synthesis pathway in higher plants. It catalyzes the reversible conversion of glucose 6-phosphate (Glc6P) to glucose 1-phosphate (Glc1P). Previously, metabolic turnover analysis using (13)CO(2) in tobacco leaves demonstrated that conversion of Glc6P to Glc1P may limit carbon flow into carbohydrate synthesis. In order to assess the effects of PGM, Arabidopsis thaliana cytosolic or plastidial PGM was expressed under the control of cauliflower mosaic virus 35S promoter in tobacco plants (Nicotiana tabacum cv. Xanthi) and phenotypic analysis was performed. The transgenic plants expressing Arabidopsis plastidial PGM showed 3.5-8.2-fold higher PGM activity than that of wild-type, and leaf starch and sucrose contents increased 2.3-3.2-fold and 1.3-1.4-fold, respectively over wild-type levels. In vivo(13)C-labeling experiments indicated that photosynthetically fixed carbon in the transgenic plants could be converted faster to Glc1P and adenosine 5'-diphosphate glucose than in wild-type, suggesting that elevation of plastidial PGM activity should accelerate conversion of Glc6P to Glc1P in chloroplasts and increase carbon flow into starch. On the other hand, transgenic plants expressing Arabidopsis cytosolic PGM showed a 2.1-3.4-fold increase in PGM activity over wild-type and a decrease of leaf starch content, but no change in sucrose content. These results suggest that plastidial PGM limits photosynthetic carbon flow into starch.     

Copper(II) complexes of 1,10-phenanthroline-derived ligands: studies on DNA binding properties and nuclease activity

A series of copper(II) complexes of the type [Cu(L)]2+, where L = N,N'-dialkyl-1,10-phenanthroline-2,9-dimethanamine and R = methyl (L1), n-propyl (L2), isopropyl (L3), sec-butyl (L4), or tert-butyl (L5) group, have been synthesized. The interaction of the complexes with DNA has been studied by DNA fiber electron paramagnetic resonance (EPR) spectroscopy, emission, viscosity and electrochemical measurements and agarose gel electrophoresis. In the X-ray crystal structure of [Cu(HL2)Cl2]NO3, copper(II) is coordinated to two ring nitrogens and one of the two secondary amine nitrogens of the side chains and two chloride ions as well and the coordination geometry is best described as trigonal bipyramidal distorted square based pyramidal (TBDSBP). Electronic and EPR spectral studies reveal that all the complexes in aqueous solution around pH 7 possess CuN3O2 rather than CuN4O chromophore with one of the alkylamino side chain not involved in coordination. The structures of the complexes in aqueous solution around pH 7 change from distorted tetragonal to trigonal bipyramidal as the size of the alkyl group is increased. The observed changes in the physicochemical features of the complexes on binding to DNA suggest that the complexes, except [Cu(L5)]2+, bind to DNA with partial intercalation of the derivatised phen ring in between the DNA base pairs. Electrochemical studies reveal that the complexes prefer to bind to DNA in Cu(II) rather than Cu(I) oxidation state. Interestingly, [Cu(L5)]2+ shows the highest DNA cleavage activity among all the present copper(II) complexes suggesting that the bulky N-tert-butyl group plays an important role in modifying the coordination environment around the copper(II) center, the Cu(II)/Cu(I) redox potential and hence the formation of activated oxidant responsible for the cleavage. These results were compared with those for bis(1,10-phenanthroline)copper(II), [Cu(phen)2]2+.     

Dysregulation of intracellular copper trafficking pathway in a mouse model of mutant copper/zinc superoxide dismutase-linked familial amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are responsible for 20% of familial amyotrophic lateral sclerosis through a gain-of-toxic function. We have recently shown that ammonium tetrathiomolybdate, an intracellular copper-chelating reagent, has an excellent therapeutic benefit in a mouse model for amyotrophic lateral sclerosis. This finding suggests that mutant SOD1 might disrupt intracellular copper homeostasis. In this study, we investigated the effects of mutant SOD1 on the components of the copper trafficking pathway, which regulate intracellular copper homeostasis. We found that mutant, but not wild-type, SOD1 shifts intracellular copper homeostasis toward copper accumulation in the spinal cord during disease progression: copper influx increases, copper chaperones are up-regulated, and copper efflux decreases. This dysregulation was observed within spinal motor neurons and was proportionally associated with an age-dependent increase in spinal copper ion levels. We also found that a subset of the copper trafficking pathway constituents co-aggregated with mutant SOD1. These results indicate that the nature of mutant SOD1 toxicity might involve the dysregulation of the copper trafficking pathway, resulting in the disruption of intracellular copper homeostasis.     

A novel bifunctional molybdo-enzyme catalyzing both decarboxylation of indolepyruvate and oxidation of indoleacetaldehyde from a thermoacidophilic archaeon, Sulfolobus sp. strain 7

An enzyme, which catalyzes both decarboxylation of indolepyruvate and subsequent oxidation of indoleacetaldehyde into indoleacetate, was purified from a thermoacidophilic archaeon, Sulfolobus sp. strain 7. The enzyme showed a M_r of 280 kDa on gel filtration and was composed of three subunits (a, 89; b, 30; and c, 19 kDa), possibly in a stoichiometry of 2:2:2. Mo and Fe were detected. Thiamine pyrophosphate was absent. Biotin was suggested to bind to the b-subunit. The first step, the decarboxylation reaction, was specific for 2-oxoacids with an aromatic group, while in the second reaction, various aldehydes including glyceraldehyde, which is a glycolytic intermediate in the organism, were oxidized.     

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: mechanism of O2-inactivation of the enzyme and its stability in the aerobe

O2-inactivation of pyruvate:NADP+ oxidoreductase from mitochondria of Euglena gracilis was studied in vitro, and a mechanism which consists of two sequential stages was proposed. Initially, the enzyme is inactivated by the direct action of O2 in a process obeying second-order kinetics. Although the catalytic activity for pyruvate oxidation is lost by this initial inactivation, NADPH oxidation with artificial electron acceptors still occurs. Subsequently, a secondary, O2-independent inactivation occurs, rendering the enzyme completely inactive. Pyruvate stimulates the O2-inactivation while CoA and NADP+ protect the enzyme from O2. The O2-inactivation is accelerated by reduction of the enzyme with pyruvate and CoA. Reactivation of the O2-inactivated enzyme was studied in Ar by incubation with Fe2+ in the presence of some other reducing reagent such as dithiothreitol. The evidence obtained indicates that the partially inactivated enzyme, which retains catalytic activity for NADPH oxidation, can be reactivated, but the completely inactivated enzyme is not. When Euglena cells were exposed to 100% O2 the enzyme in the cells was inactivated by O2, but the rate was quite slow compared with that observed in vitro. The enzyme inactivated by O2 in the cells was almost completely reactivated in vitro by incubation with Fe2+ and other reducing reagents in Ar, suggesting that the secondary, O2-independent inactivation does not occur in situ. When the cells were returned to air, reactivation of the O2-inactivated enzyme in the cells began immediately. The enzyme, kept in isolated, intact mitochondria, was stable in air; however, the enzyme was inactivated by O2 when the mitochondria were incubated with a high concentration of pyruvate.     

Pyruvate:NADP+ oxidoreductase is stabilized by its cofactor,thiamin pyrophosphate,in mitochondria of Euglena gracilis

Pyruvate:NADP(+) oxidoreductase (PNO) is a thiamin pyrophosphate (TPP)-dependent enzyme that plays a central role in the respiratory metabolism of Euglena gracilis, which requires thiamin for growth. When thiamin was depleted in Euglena cells, PNO protein level was greatly reduced, but its mRNA level was barely changed. In addition, a large part of PNO occurred as an apoenzyme lacking TPP in the deficient cells. The PNO protein level increased rapidly, without changes in the mRNA level, after supplementation of thiamin into its deficient cells. In the deficient cells, in contrast to the sufficient ones, a steep decrease in the PNO protein level was induced when the cells were incubated with cycloheximide. Immunofluorescence microscopy indicated that most of the PNO localized in the mitochondria in either the sufficient or the deficient cells. These findings suggest that PNO is readily degraded when TPP is not provided in mitochondria, and consequently the PNO protein level is greatly reduced by thiamin deficiency in E. gracilis.     

Herpes Simplex Virus-Induced Expression of 70 kDa Heat Shock Protein (HSP70) Requires Early Protein Synthesis But Not Viral DNA Replication

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post-infection. HSP70 synthesis and accumulation increased in HSV-infected cells. Irradiation of HSV with UV-light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.