The age of Calluna stands moderates post‐fire regeneration rate and trends in northern Calluna heathlands

Questions: Does stand age influence the direction and rate of post‐fire successional dynamics in coastal Calluna heaths and can old degraded heath vegetation be restored through reintroduction of fire? Location: Coastal heaths in the Tarva archipelago, central Norway. Methods: We investigated revegetation dynamics after experimental fires set in young (8 years since last fire) and old (>50 years since last fire) grazed heath stands. A repeated measures design was used, with floristic data recorded in permanent plots in the post‐fire successions (n=12) over a 7‐year period. The data were analysed using multivariate ordination techniques (PCA, RDA and PRC) and mixed effects models. Results: The age of Calluna stands strongly influenced post‐fire succession, different trends due to age explained 10.4% of variation in floristic data. Young heath showed faster succession towards pre‐fire community composition than old heath, and this could partially be explained by succession‐related factors: young heath had lower cover of mosses and lichens in the pre‐burned vegetation, and lower cover of litter early in succession. Young heath had a less pronounced overall community response to fire than old heath. Vegetative regeneration of C. vulgaris was absent in both old and young heath, but Calluna still re‐established as the dominant species within 5–7 years in both young and old stands. Regeneration dynamics were also affected by habitat conditions, different trends due to habitat explained 6% of variation. Conclusions: Our study demonstrates that old stands do develop characteristic heathland vegetation and structure after fire, and while potential invasives into the system such as trees and rhizomatous species are present, they do not impair Calluna regeneration or vegetation development towards the target heathland community composition and structure. Further, as our young stands are only in their second fire rotation after restoration, we suggest that characteristic dynamics of managed heathlands can re‐establish relatively rapidly, even in severely degenerated sites (>50 years since last fire). Site‐specific factors also need to be considered. We conclude that there is restoration potential in old heaths, despite slow dynamics in the first rotation.     

Electrical pulse stimulation of cultured human skeletal muscle cells as an in vitro model of exercise

Background and Aims

Physical exercise leads to substantial adaptive responses in skeletal muscles and plays a central role in a healthy life style. Since exercise induces major systemic responses, underlying cellular mechanisms are difficult to study in vivo. It was therefore desirable to develop an in vitro model that would resemble training in cultured human myotubes.

Methods

Electrical pulse stimulation (EPS) was applied to adherent human myotubes. Cellular contents of ATP, phosphocreatine (PCr) and lactate were determined. Glucose and oleic acid metabolism were studied using radio-labeled substrates, and gene expression was analyzed using real-time RT-PCR. Mitochondrial content and function were measured by live imaging and determination of citrate synthase activity, respectively. Protein expression was assessed by electrophoresis and immunoblotting.

Results

High-frequency, acute EPS increased deoxyglucose uptake and lactate production, while cell contents of both ATP and PCr decreased. Chronic, low-frequency EPS increased oxidative capacity of cultured myotubes by increasing glucose metabolism (uptake and oxidation) and complete fatty acid oxidation. mRNA expression level of pyruvate dehydrogenase complex 4 (PDK4) was significantly increased in EPS-treated cells, while mRNA expressions of interleukin 6 (IL-6), cytochrome C and carnitin palmitoyl transferase b (CPT1b) also tended to increase. Intensity of MitoTracker®Red FM was doubled after 48 h of chronic, low-frequency EPS. Protein expression of a slow fiber type marker (MHCI) was increased in EPS-treated cells.

Conclusions

Our results imply that in vitro EPS (acute, high-frequent as well as chronic, low-frequent) of human myotubes may be used to study effects of exercise.     

Conditional cold avoidance drives between-population variation in germination behaviour in Calluna vulgaris

Background and Aims

Across their range, widely distributed species are exposed to a variety of climatic and other environmental conditions, and accordingly may display variation in life history strategies. For seed germination in cold climates, two contrasting responses to variation in winter temperature have been documented: first, an increased ability to germinate at low temperatures (cold tolerance) as winter temperatures decrease, and secondly a reduced ability to germinate at low temperatures (cold avoidance) that concentrates germination towards the warmer parts of the season.

Methods

Germination responses were tested for Calluna vulgaris, the dominant species of European heathlands, from ten populations collected along broad-scale bioclimatic gradients (latitude, altitude) in Norway, covering a substantial fraction of the species'' climatic range. Incubation treatments varied from 10 to 25 °C, and germination performance across populations was analysed in relation to temperature conditions at the seed collection locations.

Key Results

Seeds from all populations germinated rapidly and to high final percentages under the warmer incubation temperatures. Under low incubation temperatures, cold-climate populations had significantly lower germination rates and percentages than warm-climate populations. While germination rates and percentages also increased with seed mass, seed mass did not vary along the climatic gradients, and therefore did not explain the variation in germination responses.

Conclusions

Variation in germination responses among Calluna populations was consistent with increased temperature requirements for germination towards colder climates, indicating a cold-avoidance germination strategy conditional on the temperature at the seeds'' origin. Along a gradient of increasing temperatures this suggests a shift in selection pressures on germination from climatic adversity (i.e. low temperatures and potential frost risk in early or late season) to competitive performance and better exploitation of the entire growing season.     

Remodeling of Oxidative Energy Metabolism by Galactose Improves Glucose Handling and Metabolic Switching in Human Skeletal Muscle Cells

Cultured human myotubes have a low mitochondrial oxidative potential. This study aims to remodel energy metabolism in myotubes by replacing glucose with galactose during growth and differentiation to ultimately examine the consequences for fatty acid and glucose metabolism. Exposure to galactose showed an increased [¹⁴C]oleic acid oxidation, whereas cellular uptake of oleic acid uptake was unchanged. On the other hand, both cellular uptake and oxidation of [¹⁴C]glucose increased in myotubes exposed to galactose. In the presence of the mitochondrial uncoupler carbonylcyanide p-trifluormethoxy-phenylhydrazone (FCCP) the reserve capacity for glucose oxidation was increased in cells grown with galactose. Staining and live imaging of the cells showed that myotubes exposed to galactose had a significant increase in mitochondrial and neutral lipid content. Suppressibility of fatty acid oxidation by acute addition of glucose was increased compared to cells grown in presence of glucose. In summary, we show that cells grown in galactose were more oxidative, had increased oxidative capacity and higher mitochondrial content, and showed an increased glucose handling. Interestingly, cells exposed to galactose showed an increased suppressibility of fatty acid metabolism. Thus, galactose improved glucose metabolism and metabolic switching of myotubes, representing a cell model that may be valuable for metabolic studies related to insulin resistance and disorders involving mitochondrial impairments.     

Temperature and precipitation,but not geographic distance,explain genetic relatedness among populations in the perennial grass Festuca rubra

Aims Knowledge of genetic structure of natural populations and its determinants may provide key insights into the ability of species to adapt to novel environments. In many genetic studies, the effects of climate could not be disentangled from the effects of geographic proximity. We aimed to understand the effects of temperature and moisture on genetic diversity of populations and separate these effects from the effects of geographic distance. We also wanted to explore the patterns of distribution of genetic diversity in the system and assess the degree of clonality within the populations. We also checked for possible genome size variation in the system.     

Substantial Downregulation of Myogenic Transcripts in Skeletal Muscle of Atlantic Cod during the Spawning Period

Gonadal maturation is an extremely energy consuming process for batch spawners and it is associated with a significant decrease in growth and seasonal deterioration in flesh quality. Our knowledge about the molecular mechanisms linking sexual maturation and muscle growth is still limited. In the present study, we performed RNA-Seq using 454 GS-FLX pyrosequencing in fast skeletal muscle sampled from two-year-old Atlantic cod (Gadus morhua) at representative time points throughout the reproductive cycle (August, March and May). In total, 126,937 good quality reads were obtained, with 546 nucleotide length and 52% GC content on average. RNA-Seq analysis using the CLC Genomics Workbench with the Atlantic cod reference UniGene cDNA data revealed 59,581 (46.9%) uniquely annotated reads. Pairwise comparison for expression levels identified 153 differentially expressed UniGenes between time points. Notably, we found a significant suppression of myh13 and myofibrillar gene isoforms in fast skeletal muscle during the spawning season. This study uncovered a large number of differentially expressed genes that may be influenced by gonadal maturation, thus representing a significant contribution to our limited understanding of the molecular mechanisms regulating muscle wasting and regeneration in batch spawners during their reproductive cycle.     

Chronic hyperglycemia reduces substrate oxidation and impairs metabolic switching of human myotubes

Skeletal muscle of insulin resistant individuals is characterized by lower fasting lipid oxidation and reduced ability to switch between lipid and glucose oxidation. The purpose of the present study was to examine if chronic hyperglycemia would impair metabolic switching of myotubes. Human myotubes were treated with or without chronic hyperglycemia (20 mmol/l glucose for 4 days), and metabolism of [¹⁴C]oleic acid (OA) and [¹⁴C]glucose was studied. Myotubes exposed to chronic hyperglycemia showed a significantly reduced OA uptake and oxidation to CO₂, whereas acid-soluble metabolites were increased compared to normoglycemic cells (5.5 mmol/l glucose). Glucose suppressibility, the ability of acute glucose (5 mmol/l) to suppress lipid oxidation, was 50% in normoglycemic cells and reduced to 21% by hyperglycemia. Adaptability, the capacity to increase lipid oxidation with increasing fatty acid availability, was not affected by hyperglycemia. Glucose uptake and oxidation were reduced by about 40% after hyperglycemia, and oxidation of glucose in presence of mitochondrial uncouplers showed that net and maximal oxidative capacities were significantly reduced. Hyperglycemia also abolished insulin-stimulated glucose uptake. Moreover, ATP concentration was reduced by 25% after hyperglycemia. However, none of the measured mitochondrial genes were downregulated nor was mitochondrial DNA content. Microarray and real-time RT-PCR showed that no genes were significantly regulated by chronic hyperglycemia. Addition of chronic lactate reduced both glucose and OA oxidation to the same extent as hyperglycemia. In conclusion, chronic hyperglycemia reduced substrate oxidation in skeletal muscle cells and impaired metabolic switching. The effect is most likely due to an induced mitochondrial dysfunction.     

Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene-maturity onset diabetes of the young (CEL-MODY): a protein misfolding disease

CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.     

A nuclear export sequence located on a beta-strand in fibroblast growth factor-1

Receptor-bound and endocytosed fibroblast growth factor-1 (FGF-1) is able to cross the vesicle membrane and translocate to cytosol and nucleus. This suggests an intracellular role of FGF-1, which also signals by activating transmembrane FGF receptors. Phosphorylation of internalized FGF-1 by nuclear protein kinase C delta induces rapid export from the nuclei by a leptomycin B-sensitive pathway. In the present work, we have searched for and identified a Leu-rich nuclear export sequence (NES) at the C terminus of FGF-1 required for its nuclear export and able to confer nuclear export activity to a reporter protein in an in vivo system. Mutants where hydrophobic amino acids within the NES were exchanged for alanine exhibited reduced or abolished nuclear export. As demonstrated in co-immunoprecipitation experiments, a complex containing FGF-1, exportin-1, and its co-factor Ran-GTP, was formed in vitro. Formation of this complex in vivo was demonstrated by a peroxisomal targeting assay. Formation of the FGF-1-exportin-1-Ran-GTP complex in vitro as well as nuclear export of FGF-1 in vivo was dependent on phosphorylation of FGF-1, and it was abolished by leptomycin B. The FGF-1 NES was found to be situated along a beta-strand, which has not been reported before, since NESs usually are alpha-helical.     

Phosphorylation of fibroblast growth factor (FGF) receptor 1 at Ser777 by p38 mitogen-activated protein kinase regulates translocation of exogenous FGF1 to the cytosol and nucleus

Exogenous fibroblast growth factor 1 (FGF1) signals through activation of transmembrane FGF receptors (FGFRs) but may also regulate cellular processes after translocation to the cytosol and nucleus of target cells. Translocation of FGF1 occurs across the limiting membrane of intracellular vesicles and is a regulated process that depends on the C-terminal tail of the FGFR. Here, we report that translocation of FGF1 requires activity of the alpha isoform of p38 mitogen-activated protein kinase (MAPK). FGF1 translocation was inhibited after chemical inhibition of p38 MAPK or after small interfering RNA knockdown of p38alpha. Translocation was increased after stimulation of p38 MAPK with anisomycin, mannitol, or H2O2. The activity level of p38 MAPK was not found to affect endocytosis or intracellular sorting of FGF1/FGFR1. Instead, we found that p38 MAPK regulates FGF1 translocation by phosphorylation of FGFR1 at Ser777. The FGFR1 mutation S777A abolished FGF1 translocation, while phospho-mimetic mutations of Ser777 to Asp or Glu allowed translocation to take place and bypassed the requirement for active p38 MAPK. Ser777 in FGFR1 was directly phosphorylated by p38alpha in a cell-free system. These data demonstrate a crucial role for p38alpha MAPK in the regulated translocation of exogenous FGF1 into the cytosol/nucleus, and they reveal a specific role for p38alpha MAPK-mediated serine phosphorylation of FGFR1.