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.     

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.     

Effects of plant diversity, plant productivity and habitat parameters on arthropod abundance in montane European grasslands

Arthropod abundance has been hypothesized to be correlated with plant diversity but the results of previous studies have been equivocal. In contrast, plant productivity, vegetation structure, abiotic site conditions, and the physical disturbance of habitats, are factors that interact with plant diversity, and that have been shown to influence arthropod abundance. We studied the combined effect of plant species diversity, productivity and site characteristics on arthropod abundance in 71 managed grasslands in central Germany using multivariate statistics. For each site we determined plant species cover, plant community biomass (productivity), macro- and micronutrients in the soil, and characterized the location of sites with respect to orographic parameters as well as the current and historic management regimes. Arthropods were sampled using a suction sampler and classified a priori into functional groups (FGs). We found that arthropod abundance was not correlated with plant species richness, effective diversity or Camargo's evenness, even when influences of environmental variables were taken into account. In contrast, plant community composition was highly correlated with arthropod abundances. Plant community productivity influenced arthropod abundance but explained only a small proportion of the variance. The abundances of the different arthropod FGs were influenced differentially by agricultural management, soil characteristics, vegetation structure and by interactions between different FGs of arthropods. Herbivores, carnivores and detritivores reacted differently to variation in environmental variables in a manner consistent with their feeding mode. Our results show that in natural grassland systems arthropod abundance is not a simple function of plant species richness, and they emphasize the important role of plant community composition for the abundance patterns of the arthropod assemblages.     

Overriding the co-limiting import of carbon and energy into tuber amyloplasts increases the starch content and yield of transgenic potato plants

Transgenic potato (Solanum tuberosum) plants simultaneously over-expressing a pea (Pisum sativum) glucose-6-phosphate/phosphate translocator (GPT) and an Arabidopsis thaliana adenylate translocator (NTT1) in tubers were generated. Double transformants exhibited an enhanced tuber yield of up to 19%, concomitant with an additional increased starch content of up to 28%, compared with control plants. The total starch content produced in tubers per plant was calculated to be increased by up to 44% in double transformants relative to the wild-type. Single over-expression of either gene had no effect on tuber starch content or tuber yield, suggesting that starch formation within amyloplasts is co-limited by the import of energy and the supply of carbon skeletons. As total adenosine diphosphate-glucose pyrophosphorylase and starch synthase activities remained unchanged in double transformants relative to the wild-type, they cannot account for the increased starch content found in tubers of double transformants. Rather, an optimized supply of amyloplasts with adenosine triphosphate and glucose-6-phosphate seems to favour increased starch synthesis, resulting in plants with increased starch content and yield of tubers.