Calcineurin-induced energy wasting in a transgenic mouse model of heart failure

Overexpression of calcineurin (CLN) in the mouse heart induces severe hypertrophy that progresses to heart failure, providing an opportunity to define the relationship between energetics and contractile performance in the severely failing mouse heart. Contractile performance was studied in isolated hearts at different pacing frequencies and during dobutamine challenge. Energetics were assessed by 31P-NMR spectroscopy as ATP and phosphocreatine concentrations ([ATP] and [PCr]) and free energy of ATP hydrolysis (|Delta G( approximately ATP)|). Mitochondrial and glycolytic enzyme activities, myocardial O2 consumption, and myocyte ultrastructure were determined. In transgenic (TG) hearts at all levels of work, indexes of systolic performance were reduced and [ATP] and capacity for ATP synthesis were lower than in non-TG hearts. This is the first report showing that myocardial [ATP] is lower in a TG mouse model of heart failure. [PCr] was also lower, despite an unexpected increase in the total creatine pool. Because Pi concentration remained low, despite lower [ATP] and [PCr], |Delta G( approximately ATP)| was normal; however, chemical energy did not translate to systolic performance. This was most apparent with beta-adrenergic stimulation of TG hearts, during which, for similar changes in |Delta G( approximately ATP)|, systolic pressure decreased, rather than increased. Structural abnormalities observed for sarcomeres and mitochondria likely contribute to decreased contractile performance. On the basis of the increases in enzyme activities of proteins important for ATP supply observed after treatment with the CLN inhibitor cyclosporin A, we also conclude that CLN directed inhibition of ATP-producing pathways in non-TG and TG hearts.     

1alpha,25(OH)2D3 regulates chondrocyte matrix vesicle protein kinase C (PKC) directly via G-protein-dependent mechanisms and indirectly via incorporation of PKC during matrix vesicle biogenesis.

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1alpha,25(OH)(2)D(3). Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1alpha,25(OH)(2)D(3) in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1alpha,25(OH)(2)D(3) during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1alpha,25(OH)(2)D(3), PKCzeta in matrix vesicles was inhibited, while PKCalpha in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCzeta in matrix vesicles was increased, while PKCalpha in plasma membranes was unchanged. The effect of 1alpha,25(OH)(2)D(3) was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1alpha,25(OH)(2)D(3) membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1alpha,25(OH)(2)D(3)-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1alpha,25(OH)(2)D(3) in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or G(i)/G(s) had no effect. However, inhibition of G(q) blocked the effect of 1alpha,25(OH)(2)D(3). The rapid effect of 1alpha,25(OH)(2)D(3) also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1alpha,25(OH)(2)D(3) at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid.     

Biosynthesis of riboflavin in archaea. 6,7-dimethyl-8-ribityllumazine synthase of Methanococcus jannaschii.

Heterologous expression of the putative open reading frame MJ0303 of Methanococcus jannaschii provided a recombinant protein catalysing the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Steady state kinetic analysis at 37 degrees C and pH 7.0 indicated a catalytic rate of 11 nmol.mg-1.min-1; Km values for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxybutanone 4-phosphate were 12.5 and 52 micro m, respectively. The enzyme sediments at an apparent velocity of about 12 S. Sedimentation equilibrium analysis indicated a molecular mass around 1 MDa but was hampered by nonideal solute behaviour. Negative-stained electron micrographs showed predominantly spherical particles with a diameter of about 150 A. The data suggest that the enzyme from M. jannaschii can form capsids with icosahedral 532 symmetry consisting of 60 subunits.     

Nitrogen vs. phosphorus limitation across an ecotonal gradient in a mangrove forest

Mangrove forests are characterized by distinctive tree-height gradientsthat reflect complex spatial, within-stand differences in environmentalfactors,including nutrient dynamics, salinity, and tidal inundation, across narrowgradients. To determine patterns of nutrient limitation and the effects ofnutrient availability on plant growth and within-stand nutrient dynamics, weused a factorial experiment with three nutrient treatment levels (control, N,P)and three zones along a tree-height gradient (fringe, transition, dwarf) onoffshore islands in Belize. Transects were laid out perpendicular to theshoreline across a mangrove forest from a fringe stand along the seaward edge,through a stand of intermediate height, into a dwarf stand in the interior ofthe island. At three sites, three trees were fertilized per zone for 2yr. Although there was spatial variability in response, growth byR. mangle was generally nitrogen (N) -limited in thefringe zone;phosphorus (P) -limited in the dwarf zone; and, N- and/or P-limited in thetransition zone. Phosphorus-resorption efficiency decreased in all three zones,and N-resorption efficiency increased in the dwarf zone in response to Penrichment. The addition of N had no effect on either P or N resorptionefficiencies. Belowground decomposition was increased by P enrichment in allzones, whereas N enrichment had no effect. This study demonstrated thatessential nutrients are not uniformly distributed within mangrove ecosystems;that soil fertility can switch from conditions of N to P limitation acrossnarrow ecotonal gradients; and, that not all ecological processes respondsimilarly to, or are limited by, the same nutrient.     

Comparison of the metabolism of dodecanedioic acid in vivo in control, riboflavin-deficient and clofibrate-treated rats

Intravenous administration of dodecanedioate (or hexadecanedioate) to anaesthetized rats resulted in the urinary excretion of medium-chain dicarboxylic acids (adipic, suberic and sebacic acids). In control animals, the recovery of infused dodecanedioate in the form of urinary medium-chain dicarboxylic acids corresponded to 30% of the infused dose (22 mumol/100 g body mass). This excretion was markedly increased in riboflavin-deficient rats (75% of the infused dose) while it was severely decreased in clofibrate-treated animals (less than 5%). The initial velocity of this process was similar in both control and riboflavin-deficient rats. In control animals, halving the infused dose of dodecanedioate to 11 mumol/100 g body mass resulted in a halving of the initial rate of the urinary appearance of medium-chain dicarboxylates, while doubling the amount of dicarboxylate administered to 44 mumol/100 g body mass did not further modify this velocity, but rather prolonged the duration of the excretion of the resulting products. In riboflavin-deficient and clofibrate-treated rats, the hepatic peroxisomal dicarboxylyl-CoA beta-oxidation activity measured as dicarboxylyl-CoA H2O2-generating oxidase and cyanide-insensitive dicarboxylyl-CoA-dependent NAD+ reduction was increased about threefold and tenfold, respectively. Dicarboxylyl-CoA synthetase activity was normal in the clofibrate-treated rat livers but was increased more than tenfold in the livers from the riboflavin-deficient animals. This work provides evidence that in the rat both mitochondria and peroxisomes are involved in the catabolism of dicarboxylates.     

Palmitate Diet-induced Loss of Cardiac Caveolin-3: A Novel Mechanism for Lipid-induced Contractile Dysfunction

Obesity is associated with an increased risk of cardiomyopathy, and mechanisms linking the underlying risk and dietary factors are not well understood. We tested the hypothesis that dietary intake of saturated fat increases the levels of sphingolipids, namely ceramide and sphingomyelin in cardiac cell membranes that disrupt caveolae, specialized membrane micro-domains and important for cellular signaling. C57BL/6 mice were fed two high-fat diets: palmitate diet (21% total fat, 47% is palmitate), and MCT diet (21% medium-chain triglycerides, no palmitate). We established that high-palmitate feeding for 12 weeks leads to 40% and 50% increases in ceramide and sphingomyelin, respectively, in cellular membranes. Concomitant with sphingolipid accumulation, we observed a 40% reduction in systolic contractile performance. To explore the relationship of increased sphingolipids with caveolins, we analyzed caveolin protein levels and intracellular localization in isolated cardiomyocytes. In normal cardiomyocytes, caveolin-1 and caveolin-3 co-localize at the plasma membrane and the T-tubule system. However, mice maintained on palmitate lost 80% of caveolin-3, mainly from the T-tubule system. Mice maintained on MCT diet had a 90% reduction in caveolin-1. These data show that caveolin isoforms are sensitive to the lipid environment. These data are further supported by similar findings in human cardiac tissue samples from non-obese, obese, non-obese cardiomyopathic, and obese cardiomyopathic patients. To further elucidate the contractile dysfunction associated with the loss of caveolin-3, we determined the localization of the ryanodine receptor and found lower expression and loss of the striated appearance of this protein. We suggest that palmitate-induced loss of caveolin-3 results in cardiac contractile dysfunction via a defect in calcium-induced calcium release.     

Metabolic Maturation during Muscle Stem Cell Differentiation Is Achieved by miR-1/133a-Mediated Inhibition of the Dlk1-Dio3 Mega Gene Cluster

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Dealing with food shortage: larval dispersal behaviour and survival on non‐prey food of the hoverfly Episyrphus balteatus

1. Predatory larvae often have to face food shortages during their development, and thus the ability to disperse and find new feeding sites is crucial for survival. However, the dispersal capacity of predatory larvae, the host finding cues employed, and their use of alternative food sources are largely unknown. These aspects of the foraging behaviour of the aphidophagous hoverfly (Episyrphus balteatus De Geer) larvae were investigated in the present study. 2. It was shown that these hoverfly larvae do not leave a plant as long as there are aphids available, but that dispersing larvae are able to find other aphid colonies in the field. Dispersing hoverfly larvae accumulated on large aphid colonies, but did not distinguish between different pea aphid race–plant species combinations. Large aphid colonies might be easier to detect because of intensified searching by hoverfly larvae following the encounter of aphid cues like honeydew that accumulate around large colonies. 3. It was further shown that non‐prey food, such as diluted honey or pollen, was insufficient for hoverfly larvae to gain weight, but prolonged the survival of the larvae compared with unfed individuals. As soon as larvae were switched back to an aphid diet, they rapidly gained weight and some pupated after a few days. Although pupation and adult hatching rates were strongly reduced compared with hoverflies continuously fed with aphids, the consumption of non‐prey food most probably increases the probability that hoverfly larvae find an aphid colony and complete their development.     

Clearance of picoplankton-sized partides and formation of rapidly sinking aggregates by the pteropod, Limacina reiroversa

Findings from experiments showed that the web-feeding euthecosomatouspteropod, Limacina retroversa, can produce rapidly sinking,mucous aggregates. It is suggested that, by adhesion, theseaggregates scavenged picoplankton-sized particles, which werethus effectively cleared from the medium. In contrast, Calanusfinmarchicus was not able to clear these particles in our experiments.Sedimentation velocities of 10 aggregates measured in vivo wereup to 1000 m day^–1, with an average of {small tilde}300m day^–1 (not including two aggegates with neutral buoyancy).Mean velocities measured for feces of C.finmarchicus, Calanushyperboreus and Thyssnoessa sp. were consider ably lower. Wesuggest that the sedimentation of L retroversa aggregates wasthe source of mucous flocs collected in sediment traps (Bathmannet al., Deep-Sea Res., 38,1341–1360,1991) and at the seafloor at 1200 m depth in the southern Norwegian Sea. This processmay be an important mediator of sedimentation to the deep sea,when these pteropods are present in surface waters in largeabundance.