Mild hypothermia reduces cardiac post-ischemic reactive hyperemia

BACKGROUND: In experimentally induced myocardial infarction, mild hypothermia (33–35°C) is beneficial if applied prior to ischemia or reperfusion. Hypothermia, when applied after reperfusion seems to confer little or no benefit. The mechanism by which hypothermia exerts its cell-protective effect during cardiac ischemia remains unclear. It has been hypothesized that hypothermia reduces the reperfusion damage; the additional damage incurred upon the myocardium during reperfusion. Reperfusion results in a massive increase in blood flow, reactive hyperemia, which may contribute to reperfusion damage. We postulated that hypothermia could attenuate the post-ischemic reactive hyperemia. METHODS: Sixteen 25–30 kg pigs, in a closed chest model, were anesthetized and temperature was established in all pigs at 37°C using an intravascular cooling catheter. The 16 pigs were then randomized to hypothermia (34°C) or control (37°C). The left main coronary artery was then catheterized with a PCI guiding catheter. A Doppler flow wire was placed in the mid part of the LAD and a PCI balloon was then positioned proximal to the Doppler wire but distal to the first diagonal branch. The LAD was then occluded for ten minutes in all pigs. Coronary blood flow was measured before, during and after ischemia/reperfusion. RESULTS: The peak flow seen during post-ischemic reactive hyperemia (during the first minutes of reperfusion) was significantly reduced by 43 % (p < 0.01) in hypothermic pigs compared to controls. CONCLUSION: Mild hypothermia significantly reduces post-ischemic hyperemia in a closed chest pig model. The reduction of reactive hyperemia during reperfusion may have an impact on cardiac reperfusion injury

Sialylation processes in mitochondria: evidence for two distinct sialyltransferases located in the outer membrane.

International audiencePrevious studies have shown that purified mitochondrial outer membrane is able to catalyze the transfer of sialic acid from CMP-Neu5Ac to an exogenous asialoglycoprotein acceptor, asialofetuin. Considering the heterogeneity of the glycan chains borne by this glycoprotein, an investigation of mitochondrial sialyltransferase activities was undertaken. Our data provide evidence for the existence of two distinct sialyltransferases in purified mitochondrial outer membranes. The use of different acceptor substrates, the temperature dependence of these enzymes, and their different sensitivity towards a sulfhydryl reagent, p-CMB, allowed us to discriminate between a galactoside alpha(2-3) sialyltransferase and a galactoside alpha(2-6) sialyltransferase presumably involved in the sialylation of O- and N-glycan chains of glycoprotein, respectively. These results are discussed in terms of mitochondrial autonomy for post-translational events.Previous studies have shown that purified mitochondrial outer membrane is able to catalyze the transfer of sialic acid from CMP-Neu5Ac to an exogenous asialoglycoprotein acceptor, asialofetuin. Considering the heterogeneity of the glycan chains borne by this glycoprotein, an investigation of mitochondrial sialyltransferase activities was undertaken. Our data provide evidence for the existence of two distinct sialyltransferases in purified mitochondrial outer membranes. The use of different acceptor substrates, the temperature dependence of these enzymes, and their different sensitivity towards a sulfhydryl reagent, p-CMB, allowed us to discriminate between a galactoside alpha(2-3) sialyltransferase and a galactoside alpha(2-6) sialyltransferase presumably involved in the sialylation of O- and N-glycan chains of glycoprotein, respectively. These results are discussed in terms of mitochondrial autonomy for post-translational events

Organization of mitochondrial UDP-glucose:dolichylmonophosphate glucosyltransferase in the outer membrane.

International audiencePrevious studies have shown the existence of an autonomous mitochondrial UDP-glucose: dolichylmonophosphate glucosyltransferase, located in mitochondrial outer membrane of liver cells. To improve our knowledge about the topographical aspects of glycosylation in mitochondria, we have investigated the organization of this enzyme in intact mitochondria, using controlled proteolysis with trypsin and sensitivity towards amino-acid specific reagents. Our data provides evidence: --for a mitochondrial glucosyltransferase facing the cytoplasmic side of the outer membrane --and for the involvement of histidine and tryptophan residues as well as sulfhydryl groups in the catalytic activity of the enzyme.Previous studies have shown the existence of an autonomous mitochondrial UDP-glucose: dolichylmonophosphate glucosyltransferase, located in mitochondrial outer membrane of liver cells. To improve our knowledge about the topographical aspects of glycosylation in mitochondria, we have investigated the organization of this enzyme in intact mitochondria, using controlled proteolysis with trypsin and sensitivity towards amino-acid specific reagents. Our data provides evidence: --for a mitochondrial glucosyltransferase facing the cytoplasmic side of the outer membrane --and for the involvement of histidine and tryptophan residues as well as sulfhydryl groups in the catalytic activity of the enzyme

Use of Percoll gradients for isolation of human placenta mitochondria suitable for investigating outer membrane proteins.

International audienceHuman mitochondria were isolated from placenta by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation as assessed by marker enzyme analysis and electron microscopy. The advantages over previous methods are the rapidity of the procedure and the excellent resolution of mitochondria and lysosomes. Moreover, the high extent of intactness of the mitochondria so obtained made them particularly well suited for investigating outer membrane proteins. Taking advantage of this method, we have purified human mitochondrial porin. The purified protein consists of a single unglycosylated polypeptide of molecular mass 33 kDa.Human mitochondria were isolated from placenta by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation as assessed by marker enzyme analysis and electron microscopy. The advantages over previous methods are the rapidity of the procedure and the excellent resolution of mitochondria and lysosomes. Moreover, the high extent of intactness of the mitochondria so obtained made them particularly well suited for investigating outer membrane proteins. Taking advantage of this method, we have purified human mitochondrial porin. The purified protein consists of a single unglycosylated polypeptide of molecular mass 33 kDa

Mitochondrial dolichyl-phosphate mannose synthase. Purification and immunogold localization by electron microscopy.

International audienceMitochondrial dolichyl-phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step. The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band. A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30-kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl-phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria.Mitochondrial dolichyl-phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step. The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band. A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30-kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl-phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria

Further characterization of mitochondrial outer membrane: evidence for the presence of two endogenous sialylated glycoproteins.

International audienceThe distribution of sialic acid-containing glycoproteins was investigated in highly purified mitochondrial membranes using labeled Sambucus nigra agglutinin as a detection system. Two sialylated glycoproteins were shown to be true components of the mitochondrial outer membrane. Relative to monoamine oxidase activity, these glycoproteins were found to be preferentially located in the "free" outer membrane fraction. As sialic acid is thought to be involved in molecular recognition, a role for these glycoproteins in mediating the interactions between mitochondria and other sub-cellular organelles is considered.The distribution of sialic acid-containing glycoproteins was investigated in highly purified mitochondrial membranes using labeled Sambucus nigra agglutinin as a detection system. Two sialylated glycoproteins were shown to be true components of the mitochondrial outer membrane. Relative to monoamine oxidase activity, these glycoproteins were found to be preferentially located in the "free" outer membrane fraction. As sialic acid is thought to be involved in molecular recognition, a role for these glycoproteins in mediating the interactions between mitochondria and other sub-cellular organelles is considered

Investigation of glycosylation processes in mitochondria and microsomal membranes from human skeletal muscle.

International audienceGlycoconjugates are directly involved in major skeletal muscle functions. As little is known about glycosylation processes in muscle, we investigated glycoconjugate synthesis in subcellular fractions from human skeletal muscle tissue. Mitochondria and microsomal membranes were prepared from muscle biopsies by thorough mechanical disruption and differential centrifugations. This procedure resulted in the isolation of intact mitochondria (1 mg protein/g muscle) and of a microsomal fraction (1.5 mg protein/g muscle). Glycosyltransferases were studied in both subcellular fractions using either dolichylmonophosphate as a polyprenic acceptor or chemically modified fetuin as a glycoprotein substrate. Our results provide evidence for high rates of glycosylation in muscle. The highest activities were obtained with GDP-mannose: dilichylmonophosphate mannosyltransferase, a key enzyme in glycosylation process (220 pmol/mg per h in mitochondria and 1,550 pmol/mg per h in microsomal membranes). Substantial individual variations were observed for dolichol pathway glycosyltransferases but low individual variations were found for glycosyltransferases involved in maturation of glycoproteins. The role which glycosylation defects may play in muscle dysfunction has yet to be defined.Glycoconjugates are directly involved in major skeletal muscle functions. As little is known about glycosylation processes in muscle, we investigated glycoconjugate synthesis in subcellular fractions from human skeletal muscle tissue. Mitochondria and microsomal membranes were prepared from muscle biopsies by thorough mechanical disruption and differential centrifugations. This procedure resulted in the isolation of intact mitochondria (1 mg protein/g muscle) and of a microsomal fraction (1.5 mg protein/g muscle). Glycosyltransferases were studied in both subcellular fractions using either dolichylmonophosphate as a polyprenic acceptor or chemically modified fetuin as a glycoprotein substrate. Our results provide evidence for high rates of glycosylation in muscle. The highest activities were obtained with GDP-mannose: dilichylmonophosphate mannosyltransferase, a key enzyme in glycosylation process (220 pmol/mg per h in mitochondria and 1,550 pmol/mg per h in microsomal membranes). Substantial individual variations were observed for dolichol pathway glycosyltransferases but low individual variations were found for glycosyltransferases involved in maturation of glycoproteins. The role which glycosylation defects may play in muscle dysfunction has yet to be defined