Functional diversity of FGF-2 isoforms by intracellular sorting

Regulation of the subcellular localization of certain proteins is a mechanism for the regulation of their biological activities. FGF-2 can be produced as distinct isoforms by alternative initiation of translation on a single mRNA and the isoforms are differently sorted in cells. High molecular weight FGF-2 isoforms are not secreted from the cell, but are transported to the nucleus where they regulate cell growth or behavior in an intracrine fashion. 18 kDa FGF-2 can be secreted to the extracellular medium where it acts as a conventional growth factor by binding to and activation of cell-surface receptors. Furthermore, following receptor-mediated endocytosis, the exogenous FGF-2 can be transported to the nuclei of target cells, and this is of importance for the transmittance of a mitogenic signal. The growth factor is able to interact with several intracellular proteins. Here, the mode of action and biological role of intracellular FGF-2 are discussed.     

Traits help explain species' performance away from their climate niche centre

Aim

Climate change impacts on biota are variable across sites, among species and throughout individual species' ranges. Niche theory predicts that population performance should decline as site climate becomes increasingly different from the species' climate niche centre, though studies find significant variation from these predictions. Here, we propose that predictions about climate responses can be improved by incorporating species' trait information.

Location

Europe.

Methods

We used observations of plant species abundance change over time to assess variation in climate difference sensitivity (CDS), defined as how species performance (colonization, extinction and abundance change) relates to the difference of site climate from the mean temperature and precipitation of each species' range. We then investigated if leaf economics, plant size and seed mass traits were associated with the species' CDS.

Results

Species that performed better (e.g. increased in abundance) towards sites progressively cooler than their niche centre were shorter and had more resource-acquisitive leaves (i.e. lower leaf dry matter content or LDMC) relative to species with zero or the opposite pattern of temperature difference sensitivity. This result supports the hypothesis that if sites cooler than niche centres are more stressful for a species, then shorter stature is advantageous compared with taller species. The LDMC result suggests the environment selects for more resource-acquisitive leaf strategies towards relatively cooler climates with shorter growing seasons, counter to expectations that conservative strategies would be favoured in such environments. We found few consistent relationships between precipitation difference sensitivities and traits.

Main Conclusions

The results supported key a priori foundations on how trait-based plant strategies dictate species responses to climate variation away from their niche centre. Furthermore, plant height emerged as the most consistent trait that varied with species CDS, suggesting height will be key for theory development around species response to climate change.     

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.     

DNA adduct levels in fish from pristine areas are not detectable or low when analysed using the nuclease P1 version of the 32P-postlabelling technique

In order to understand and apply DNA adduct formation in fish liver as a biomarker for aquatic pollution, information concerning the natural background levels in non-contaminated organisms, caused by endogenous compounds, is of fundamental importance. In this study, DNA adducts were analysed in liver of 11 fish species from arctic and sub-arctic areas in the northern Atlantic using the nuclease P1 version of the ³²P-postlabelling technique. The collected fish were assumed not to have been influenced by anthropogenic pollution apart from possible long-range transported pollutants. As polycyclic aromatic hydrocarbons (PAHs) are thought to be fundamental in forming the type of DNA adducts detected by the method used, biliary PAH metabolite levels were measured in a selection of the investigated species. In all investigated individuals, the levels of PAH metabolites were undetectable. Controlled on-site exposure experiments with benzo[a]pyrene (polar cod) and laboratory experiments with crude oil (polar cod and Atlantic cod) were conducted. DNA adducts were formed in both these species. The field-sampled fish showed undetectable levels of DNA adducts or levels just above the detection limit. The present study supports the assumption that when DNA adducts are detected by the nuclease P1 version of the ³²P-postlabelling method in fish liver, it can be interpreted as DNA damage caused by pollutants.     

Type IV Pilus Assembly Proficiency and Dynamics Influence Pilin Subunit Phospho-Form Macro- and Microheterogeneity in Neisseria gonorrhoeae

The PilE pilin subunit protein of the gonococcal Type IV pilus (Tfp) colonization factor undergoes multisite, covalent modification with the zwitterionic phospho-form modification phosphoethanolamine (PE). In a mutant lacking the pilin-like PilV protein however, PilE is modified with a mixture of PE and phosphocholine (PC). Moreover, intrastrain variation of PilE PC modification levels have been observed in backgrounds that constitutively express PptA (the protein phospho-form transferase A) required for both PE and PC modification. The molecular basis underlying phospho-form microheterogeneity in these instances remains poorly defined. Here, we examined the effects of mutations at numerous loci that disrupt or perturb Tfp assembly and observed that these mutants phenocopy the pilV mutant vis a vis phospho-form modification status. Thus, PC modification appears to be directly or indirectly responsive to the efficacy of pilin subunit interactions. Despite the complexity of contributing factors identified here, the data favor a model in which increased retention in the inner membrane may act as a key signal in altering phospho-form modification. These results also provide an alternative explanation for the variation in PilE PC levels observed previously and that has been assumed to be due to phase variation of pptA. Moreover, mass spectrometry revealed evidence for mono- and di-methylated forms of PE attached to PilE in mutants deficient in pilus assembly, directly implicating a methyltransferase-based pathway for PC synthesis in N. gonorrhoeae.     

Mannitol in six autotrophic stramenopiles and Micromonas

Mannitol plays a central role in brown algal physiology since it represents an important pathway used to store photoassimilate. Several specific enzymes are directly involved in the synthesis and recycling of mannitol, altogether forming the mannitol cycle. The recent analysis of algal genomes has allowed tracing back the origin of this cycle in brown seaweeds to a horizontal gene transfer from bacteria, and furthermore suggested a subsequent transfer to the green micro-alga Micromonas. Interestingly, genes of the mannitol cycle were not found in any of the currently sequenced diatoms, but were recently discovered in pelagophytes and dictyochophytes. In this study, we quantified the mannitol content in a number of ochrophytes (autotrophic stramenopiles) from different classes, as well as in Micromonas. Our results show that, in accordance with recent observations from EST libraries and genome analyses, this polyol is produced by most ochrophytes, as well as the green alga tested, although it was found at a wide range of concentrations. Thus, the mannitol cycle was probably acquired by a common ancestor of most ochrophytes, possibly after the separation from diatoms, and may play different physiological roles in different classes.Key words: algae, stramenopiles, mannitol cycle, primary metabolism, osmotic stress, evolutionBrown algae produce mannitol directly from the photoassimilate fructose-6-phosphate. Its metabolism occurs through the mannitol cycle, which involves four enzymatic reactions: (1) the reduction of fructose-6-phosphate (F6P) to mannitol-1-phosphate (M1P) via the activity of an M1P dehydrogenase (M1PDH); (2) the production of mannitol from M1P via an M1P phosphatase (M1Pase); (3) the oxidation of mannitol via the activity of a mannitol-2-dehydrogenase (M2DH) yielding fructose; and (4) the phosphorylation of fructose yielding F6P and involving a hexokinase (HK).^1,2 The first completed draft of a brown algal genome enabled the identification of candidate genes for each of these steps.³ As these genes were not found in the genomes of the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum, mannitol metabolism in stramenopiles was considered a trait typical for brown algae. The corresponding genes were thought to have been acquired horizontally from bacteria and subsequently transferred to some green algae.⁴ Recently, however, homologs of several genes of the cycle were also found in the genome of the pelagophyte Aureococcus anophagefferens⁵ and an EST library produced for the dictyochophyte Pseudochattonella farcimen (Dittami et al. personal communication). These observations prompted us to examine the presence of mannitol in a range of strains covering different classes of autotrophic stramenopiles (ochrophytes). In addition, because of the identification of genes encoding enzymes for the production of mannitol through the mannitol cycle in the green alga Micromonas, one strain of this genus was also included in our analysis.     

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.     

Benfotiamine increases glucose oxidation and downregulates NADPH oxidase 4 expression in cultured human myotubes exposed to both normal and high glucose concentrations

The aim of the present work was to study the effects of benfotiamine (S-benzoylthiamine O-monophosphate) on glucose and lipid metabolism and gene expression in differentiated human skeletal muscle cells (myotubes) incubated for 4 days under normal (5.5 mM glucose) and hyperglycemic (20 mM glucose) conditions. Myotubes established from lean, healthy volunteers were treated with benfotiamine for 4 days. Glucose and lipid metabolism were studied with labeled precursors. Gene expression was measured using real-time polymerase chain reaction (qPCR) and microarray technology. Benfotiamine significantly increased glucose oxidation under normoglycemic (35 and 49% increase at 100 and 200 μM benfotiamine, respectively) as well as hyperglycemic conditions (70% increase at 200 μM benfotiamine). Benfotiamine also increased glucose uptake. In comparison, thiamine (200 μM) increased overall glucose metabolism but did not change glucose oxidation. In contrast to glucose, mitochondrial lipid oxidation and overall lipid metabolism were unchanged by benfotiamine. The expression of NADPH oxidase 4 (NOX4) was significantly downregulated by benfotiamine treatment under both normo- and hyperglycemic conditions. Gene set enrichment analysis (GSEA) showed that befotiamine increased peroxisomal lipid oxidation and organelle (mitochondrial) membrane function. In conclusion, benfotiamine increases mitochondrial glucose oxidation in myotubes and downregulates NOX4 expression. These findings may be of relevance to type 2 diabetes where reversal of reduced glucose oxidation and mitochondrial capacity is a desirable goal.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-011-0252-8) contains supplementary material, which is available to authorized users.