Kalervo Hiltunen

Cloning and Characterization of a Novel Human Lysyl Hydroxylase Isoform Highly Expressed in Pancreas and Muscle

We report the isolation and characterization of cDNA clones for a novel isoform of lysyl hydroxylase (lysyl hydroxylase 2), a posttranslational enzyme of collagen biosynthesis. The open reading frame predicted a protein of 737 amino acids, including an amino-terminal signal peptide. The amino acid sequence has overall similarity of over 75% to the lysyl hydroxylase (lysyl hydroxylase 1) characterized earlier. This similarity is even higher in the carboxyl-terminal end of the molecules. Lysyl hydroxylase 2 contains nine cysteine residues, which are conserved in lysyl hydroxylase 1. Furthermore, the conserved histidines and aspartate residues required for lysyl hydroxylase activity are present in the sequence. Northern analysis identified a transcript of 4.2 kilobases, which was highly expressed in pancreas and muscle tissues. Expression of cDNA in insect cells using a baculovirus vector yielded proteins with lysyl hydroxylase activity and an antiserum against a synthetic peptide of the deduced amino acid sequence recognized proteins with molecular weights of 88 and 97 kDa in homogenates of the transfected cells.

Respiratory control in isolated perfused rat heart. Role of the equilibrium relations between the mitochondrial electron carriers and the adenylate system

The effects of KCl-induced cardiac arrest on the redox state of the fluorescent flavoproteins and nicotinamide nucleotides and on that of cytochromes c and a were studied by surface fluorometric and reflectance spectrophotometric methods. These changes were compared with measurements of the concentrations of the adenylate system, creatine phosphate, some intermediates of the tricarboxylic acid cycle and reactants of the glutamate dehydrogenase system. KCl-induced cardiac arrest caused reduction of the fluorescent flavoproteins and nicotinamide nucleotides, oxidation of cytochromes c and a, inhibition of oxygen consumption and an increase in the ATP/(ADP X Pi) ratio. The increase in the latter was due mainly to a decrease in the concentration of Pi and an equivalent increase in creatine phosphate. The cytochromes c and a were maintained at equal redox potential and changed in parallel. When the redox state of the mitochondrial NAD couple was calculated from the glutamate dehydrogenase equilibrium, the free energy change (deltaG) corresponding to the potential difference between the NAD couple and cytochrome c was 115.8 kj/mol in the beating heart and 122.2 kj/mol in the arrested heart. The deltaG values of ATP hydrolysis calculated from the concentrations of ATP, Pi and ADP, corrected for bound ADP, were 111.1 kj/2 mol and 115.4 kj/2 mol in the beating and arrested heart respectively. The accumulation of citrate and the direction of the redox changes in the respiratory carriers indicate that the tricarboxylic acid cycle flux is controlled by the respiratory chain. The data also show a near equilibrium between the electron carriers and the adenylate system and suggest that the equilibrium hypothesis of mitochondrial respiratory control is applicable to intact myocardial tissue.

Candida tropicalis Etr1p and Saccharomyces cerevisiae Ybr026p (Mrf1'p), 2-enoyl thioester reductases essential for mitochondrial respiratory competence.

ABSTRACT We report here on the identification and characterization of novel 2-enoyl thioester reductases of fatty acid metabolism, Etr1p fromCandida tropicalis and its homolog Ybr026p (Mrf1′p) fromSaccharomyces cerevisiae. Overexpression of these proteins in S. cerevisiae led to the development of significantly enlarged mitochondria, whereas deletion of the S. cerevisiae YBR026c gene resulted in rudimentary mitochondria with decreased contents of cytochromes and a respiration-deficient phenotype. Immunolocalization and in vivo targeting experiments showed these proteins to be predominantly mitochondrial. Mitochondrial targeting was essential for complementation of the mutant phenotype, since targeting of the reductases to other subcellular locations failed to reestablish respiratory growth. The mutant phenotype was also complemented by a mitochondrially targeted FabI protein from Escherichia coli. FabI represents a nonhomologous 2-enoyl-acyl carrier protein reductase that participates in the last step of the type II fatty acid synthesis. This indicated that 2-enoyl thioester reductase activity was critical for the mitochondrial function. We conclude that Etr1p and Ybr026p are novel 2-enoyl thioester reductases required for respiration and the maintenance of the mitochondrial compartment, putatively acting in mitochondrial synthesis of fatty acids.

Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance

Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1−/− mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1−/− only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.

Ethanol-induced changes in gastric mucosal content of cyclic AMP and ATP in the rat

The stomach of urethane-anaesthetised rats was perfused with 10% (v/v) ethanol. At 40 min, the secretion of acid was strongly inhibited and the contents of cyclic AMP and ATP were lowered in the superficial mucosa, but not in whole gastric mucosa. After discontinuation of the ethanol perfusion, the rate of gastric acid output as well as the cyclic AMP and ATP levels recovered. Most of the acid-secreting parietal cells were found in the superficial mucosa where the changes of cyclic AMP and ATP took place. The results suggest that lowering of the mucosal contents of cyclic AMP and ATP may be involved in the ethanol-induced inhibition of the gastric acid output.