AICAR inhibits oxygen consumption by intact skeletal muscle cells in culture

Activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) with aminoimidazole carboxamide ribonucleotide (AICAR) increases skeletal muscle glucose uptake and fatty acid oxidation. The purpose of these experiments was to utilize AICAR to enhance palmitate consumption by mitochondria in cultured skeletal muscle cells. In these experiments, we treated C2C12 myotubes or adult single skeletal muscle fibers with varying concentrations of AICAR for different lengths of time. Surprisingly, acute AICAR exposure at most concentrations (0.25–1.5 mM), but not all (0.1 mM), modestly inhibited oxygen consumption even though AICAR increased AMPK phosphorylation. The data suggest that AICAR inhibited oxygen consumption by the cultured muscle in a non-specific manner. The results of these experiments are expected to provide valuable information to investigators interested in using AICAR in cell culture studies.     

Peach fruit ripening: A proteomic comparative analysis of the mesocarp of two cultivars with different flesh firmness at two ripening stages

A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the “unripe” to the “ripe” phase.The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; ‘Oro A’) and a melting flesh (MF; ‘Bolero’) cultivar, at “unripe” and “ripe” stages as defined by some parameters typical of ripening, were then analyzed.The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage (“unripe” versus “ripe”) and/or the genetic background of the cultivar (‘Oro A’ versus ‘Bolero’).Thirty out of the 53 differently abundant spots were identified by LC-ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress.Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric (“unripe”) to the climacteric (“ripe”) phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status.The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics.     

A photobioreactor with pH control: demonstration by growth of the marine diatom Skeletonema costatum

A photobioreactor with pH control for cultivation of algae isdescribed. The magnetically stirred culture flask is connectedto separate reservoirs for medium and for acid and base (dilutedHCl and NaOH, respectively). A pH electrode is inserted intothe culture flask and coupled to a pH controller, which activatesacid and base titration at set points of pH 8.1 and 7.8, respectively.Illumination is provided by light tubes with a diel light :dark cycle. The use of the photobioreactor in batch mode isillustrated by showing pH curves in different growth phasesof the marine diatom Skeletonema costatum. The photobioreactorcan also be run as a semicontinuous or continuous reactor withslight modifications.     

Study protocol: Brief intervention for medication overuse headache - A double-blinded cluster randomised parallel controlled trial in primary care

ABSTRACT: BACKGROUND: Chronic headache (headache [GREATER-THAN OR EQUAL TO] 15 days/month for at least 3 months) affects 2--5% of the general population. Medication overuse contributes to the problem. Medication-overuse headache (MOH) can be identified by using the Severity of Dependence Scale (SDS). A "brief intervention" scheme (BI) has previously been used for detoxification from drug and alcohol overuse in other settings. Short, unstructured, individualised simple information may also be enough to detoxify a large portion of those with MOH. We have adapted the structured (BI) scheme to be used for MOH in primary care. METHODS: A double-blinded cluster randomised parallel controlled trial (RCT) of BI vs. business as usual. Intervention will be performed in primary care by GPs trained in BI. Patients with MOH will be identified through a simple screening questionnaire sent to patients on the GPs lists. The BI method involves an approach for identifying patients with high likelihood of MOH using simple questions about headache frequency and the SDS score. Feedback is given to the individual patient on his/her score and consequences this might have regarding the individual risk of medication overuse contributing to their headache. Finally, advice is given regarding measures to be taken, how the patient should proceed and the possible gains for the patient. The participating patients complete a headache diary and receive a clinical interview and neurological examination by a GP experienced in headache diagnostics three months after the intervention. Primary outcomes are number of headache days and number of medication days per month at 3 months. Secondary outcomes include proportions with 25 and 50% improvement at 3 months and maintenance of improvement and quality of life after 12 months. DISCUSSION: There is a need for evidence-based and cost-effective strategies for treatment of MOH but so far no consensus has been reached regarding an optimal medication withdrawal method. To our knowledge this is the first RCT of structured non-pharmacological MOH treatment in primary care. Results may hold the potential of offering an instrument for treating MOH patients in the general population by GPs. Trial registration ClinicalTrials.gov identifier: NCT01314768.     

Evaluation of three commercial bovine ELISA kits for detection of antibodies against Alphaherpesviruses in reindeer (<Emphasis Type="Italic">Rangifer tarandus tarandus</Emphasis>)

Background  

The genus Varicellovirus (family Herpesviridae subfamily Alphaherpesvirinae) includes a group of viruses genetically and antigenically related to bovine herpesvirus 1 (BoHV-1) among which cervid herpesvirus 2 (CvHV-2) can be of importance in reindeer. These viruses are known to be responsible for different diseases in both wild and domestic animals. Reindeer are a keystone in the indigenous Saami culture and previous studies have reported the presence of antibodies against alphaherpesviruses in semi-domesticated reindeer in northern Norway. Mortality rates, especially in calves, can be very high in some herds and the abortion potential of alphaherpesvirus in reindeer, unlike in bovines, remains unknown.     

Epsin 1 is Involved in Recruitment of Ubiquitinated EGF Receptors into Clathrin-Coated Pits

Epsin consists of an epsin NH₂-terminal homology domain that promotes interaction with phospholipids, several AP-2-binding sites, two clathrin-binding sequences and several Eps15 homology domain-binding motifs. Epsin additionally possesses ubiquitin-interacting motifs (UIMs) and has been demonstrated to bind ubiquitinated cargo. We therefore investigated whether epsin promoted clathrin-mediated endocytosis of the ubiquitinated EGF receptor (EGFR). By immunoprecipitation, we found that epsin 1 interacted with ubiquitinated EGFR and that functional UIMs were essential for complex formation. Furthermore, RNA interference-mediated knockdown of epsin 1 was found to inhibit internalization of the EGFR, while having no effect on endocytosis of the transferrin receptor. Additionally, upon knockdown of epsin 1, translocation of the EGFR to central parts of clathrin-coated pits was inhibited. This supports the contention that epsin 1 promotes endocytosis of the ubiquitinated EGFR.     

Adaptive responses to iron-deficiency in callus cultures of two cultivars of Vitis spp

The correlation between iron chlorosis resistance and induction of adaptive mechanisms in grapevine calli belonging to cultivars with different susceptibility to iron chlorosis has been investigated. Fe(III)-chelate reductase was clearly linked to the Fe-efficiency status of the genotype. When growing on iron deprived medium (-Fe) calli of the Fe-efficient genotype "Cabernet sauvignon" showed a remarkable increase in enzyme activity, up to five times higher, with respect to +Fe cultures. Moreover, 31P-NMR revealed that in -Fe medium the increase of vacuolar Pi content of the Fe-efficient cultures was more pronounced than that recorded for the Fe-inefficient Vitis riparia. Furthermore, Fe starvation also enhanced the production of phenolic compounds in calli of "Cabernet sauvignon" with respect to those of Vitis riparia. The role of H(+)-ATPase as a marker of Fe-efficiency in tissue culture remains ambiguous in the case of grapevines.     

GSK-3beta negatively regulates skeletal myotube hypertrophy

Todetermine whether changes in glycogen synthase kinase-3 (GSK-3)phosphorylation contribute to muscle hypertrophy, we delineated theeffects of GSK-3 activity on C₂C₁₂ myotubesize. We also examined possible insulin-like growth factor I (IGF-I) signaling of NFAT (nuclear factors of activated T cells)-inducible geneactivity and possible modulation of NFAT activation by GSK-3. Application of IGF-I (250 ng/ml) or LiCl (10 mM) alone (i.e., bothinhibit GSK-3 activity) increased the area ofC₂C₁₂ myotubes by 80 and 85%, respectively.The application of IGF-I (250 ng/ml) elevated GSK-3 phosphorylationand reduced GSK-3 kinase activity by ~800% and ~25%,respectively. LY-294002 (100 µM) and wortmannin (150 µM), specificinhibitors of phosphatidylinositol 3'-kinase, attenuated IGF-I-inducedGSK-3 phosphorylation by 67 and 92%, respectively. IGF-I suppressedthe kinase activity of GSK-3. IGF-I (250 ng/ml), but not LiCl (10 mM), induced an increase in NFAT-activated luciferase reporteractivity. Cotransfection of a constitutively active GSK-3(cGSK-3) inhibited the induction by IGF-I of NFAT-inducible reporteractivity. LiCl, which inhibits GSK-3, removed the block by cGSK-3on IGF-I-inducible NFAT-responsive reporter gene activity. These datasuggest that the IGF-I-induced increase in skeletal myotube size issignaled, in part, through the inhibition of GSK-3.