Proteomic signatures of in vivo muscle oxidative capacity in healthy adults

Adequate support of energy for biological activities and during fluctuation of energetic demand is crucial for healthy aging; however, mechanisms for energy decline as well as compensatory mechanisms that counteract such decline remain unclear. We conducted a discovery proteomic study of skeletal muscle in 57 healthy adults (22 women and 35 men; aged 23–87 years) to identify proteins overrepresented and underrepresented with better muscle oxidative capacity, a robust measure of in vivo mitochondrial function, independent of age, sex, and physical activity. Muscle oxidative capacity was assessed by ³¹P magnetic resonance spectroscopy postexercise phosphocreatine (PCr) recovery time (τ_PCr) in the vastus lateralis muscle, with smaller τ_PCr values reflecting better oxidative capacity. Of the 4,300 proteins quantified by LC‐MS in muscle biopsies, 253 were significantly overrepresented with better muscle oxidative capacity. Enrichment analysis revealed three major protein clusters: (a) proteins involved in key energetic mitochondrial functions especially complex I of the electron transport chain, tricarboxylic acid (TCA) cycle, fatty acid oxidation, and mitochondrial ABC transporters; (b) spliceosome proteins that regulate mRNA alternative splicing machinery, and (c) proteins involved in translation within mitochondria. Our findings suggest that alternative splicing and mechanisms that modulate mitochondrial protein synthesis are central features of the molecular mechanisms aimed at maintaining mitochondrial function in the face of impairment. Whether these mechanisms are compensatory attempt to counteract the effect of aging on mitochondrial function should be further tested in longitudinal studies.     

A correlation between BCL-2 modifying factor,p53 and livin gene expressions in cancer colon patients

Accumulating evidence has revealed that livin gene and BCL-2 modifying factor (BMF) gene are closely associated with the initiation and progression of colon carcinoma by activating or suppressing multiple malignant processes. Those genes that can detect colon - cancer are a promising approach for cancer screening and diagnosis. This study aimed to evaluate correlation between livin, BMF and p53 genes expression in colon cancer tissues of patients included in the study, and their relationship with clinicopathological features and survival outcome in those patients. In this study, 50 pathologically diagnosed early cancer colon patients included and their tissue biopsy with 50 matched adjacent normal tissue, and 50 adenoma tissue specimens were analyzed for livin gene and BMF gene expressions using real time PCR. The relationship of those genes expressions with clinicopathological features, tumor markers, Time to Progression and overall survival for those patients were correlated in cancer colon group. In this study, there was a significant a reciprocal relationship between over expression of livin gene and down regulation of BMF and p53 genes in colon cancer cells. Livin mRNA was significantly higher, while BMF and p53 mRNA were significantly lower in colorectal cancer tissue compared to benign and normal colon tissue specimens (P < 0.001), however, this finding was absent between colon adenomas and normal mucosa. There was a significant association between up regulation of livin and down regulation of BMF and p53 expressions with more aggressive tumor (advanced TNM stage), rapid progression with metastasis and decreased overall survival in cancer colon patients, hence these genes can serve as significant prognostic markers of poor outcome in colon cancer patients. This work highlights the role of livin, BMF and p53 genes in colorectal tumorigenesis and the applicability of using those genes as a diagnostic and prognostic markers in patients with colon carcinoma and as a good target for cancer colon treatment in the future.     

Biocides formulation of essential oils having antimicrobial activity

Essential oils of fennel, peppermint, caraway, eucalyptus, geranium and lemon were tested for their antimicrobial activities against some plant pathogenic micro-organisms (Fusarium oxysporum, Alternaria alternate, Penicilium italicum Penicilium digitatum and Botyritus cinerea). Essential oils of fennel, peppermint, caraway were selected as an active ingredient for the formulation of biocides due to their efficiency in controlling the tested micro-organisms. Successful emulsifiable concentrates (biocides) were prepared from these oils using different emulsifiers (Emulgator B.L.M. Tween20 and Tween80) and different fixed oils (sesame, olive, cotton and soybean oils). Physico-chemical properties of the formulated biocide (spontaneous emulsification, emulsion stability test, cold stability and heat stability tests as well as viscosity, surface tension and pH) were measured. The prepared biocides were ready to be tested for application in a future work as a safe pesticide against different pathogens.     

Metagenomic insights into strategies of carbon conservation and unusual sulfur biogeochemistry in a hypersaline Antarctic lake

Organic Lake is a shallow, marine-derived hypersaline lake in the Vestfold Hills, Antarctica that has the highest reported concentration of dimethylsulfide (DMS) in a natural body of water. To determine the composition and functional potential of the microbial community and learn about the unusual sulfur chemistry in Organic Lake, shotgun metagenomics was performed on size-fractionated samples collected along a depth profile. Eucaryal phytoflagellates were the main photosynthetic organisms. Bacteria were dominated by the globally distributed heterotrophic taxa Marinobacter, Roseovarius and Psychroflexus. The dominance of heterotrophic degradation, coupled with low fixation potential, indicates possible net carbon loss. However, abundant marker genes for aerobic anoxygenic phototrophy, sulfur oxidation, rhodopsins and CO oxidation were also linked to the dominant heterotrophic bacteria, and indicate the use of photo- and lithoheterotrophy as mechanisms for conserving organic carbon. Similarly, a high genetic potential for the recycling of nitrogen compounds likely functions to retain fixed nitrogen in the lake. Dimethylsulfoniopropionate (DMSP) lyase genes were abundant, indicating that DMSP is a significant carbon and energy source. Unlike marine environments, DMSP demethylases were less abundant, indicating that DMSP cleavage is the likely source of high DMS concentration. DMSP cleavage, carbon mixotrophy (photoheterotrophy and lithoheterotrophy) and nitrogen remineralization by dominant Organic Lake bacteria are potentially important adaptations to nutrient constraints. In particular, carbon mixotrophy relieves the extent of carbon oxidation for energy production, allowing more carbon to be used for biosynthetic processes. The study sheds light on how the microbial community has adapted to this unique Antarctic lake environment.     

Positive Feedback Mechanisms among Local Ca Releases,NCX, and ICaL Ignite Pacemaker Action Potentials

Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (I_CaL). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to I_CaL activation threshold. During the ignition phase, I_CaL-mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and I_CaL, as diastolic LCRs begin to occur below and continue within the voltage range of I_CaL activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (～0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.     

Germination of <i>Eryngium regnellii</i>: a major species for ecological restoration of plant-pollinator interactions in the Southern Pampas (Buenos Aires,Argentina)

Eryngium regnellii Malme belongs to the largest genera in the Apiaceae family, with 250 species worldwide and 65 represented in South America. It is a herbaceous species typical of hill plant communities, which, along with remnant grassland patches, are the most relevant natural habitats for the maintenance of diversity in the Southern Pampas. Eryngium regnellii is key to the maintenance of pollination mutualisms, being a generalist (displaying a diverse assemblage of pollinators) and ubiquitous species (present in all studied sierras). However, fragmentation of the Pampean landscape due to agricultural intensification has led to the loss of natural environments. Therefore, the reintroduction of E. regnellii in strategic places would facilitate the occurrence of wild pollinators, while favoring pollination services in the agroecosystem. The germination requirements of E. regnellii were studied because a better knowledge of the reproductive biology of this species would provide information relevant to its reproduction and reintroduction into degraded areas. Germination percentages and mean time to germination were evaluated, using one control and two pre-germination treatments: chemical scarification with sulfuric acid, and mechanical scarification with sand paper. Chemical scarified seeds did not germinate. Mechanically scarified and control seed groups showed no significant differences either in germination percentages (49% and 59% respectively) or in mean germination time (13 and 14 days, respectively). Results indicate that E. regnellii shows no physical dormancy, and does not require specific pre-germination treatments for germination under the studied laboratory conditions. The high germination capacity of E. regnellii, along with its ecological attributes, make it a potential species for restoring plant-pollinator interactions in the fragmented landscapes of the Southern Pampas.     

Nitrate reductase activity,biomass, yield,and quality in cotton in response to nitrogen fertilization

In the production of cotton (Gossypium hirsutum L.), nitrogen fertilization is one of the most costly crop practices, but important to reach high yields. However, high nitrogen (N) content in plants does not always translate into a high fibre production. One way of assessing the efficiency of the N fertilizer is through the enzymatic activity of the nitrate reductase (NR). This is a key enzyme in N assimilation, whose activity is regulated by a number of endogenous and exogenous factors that determine yield. The aim of this study was to assess the effect of N fertilization on yield, fibre quality, biomass, and NR enzymatic activity in vivo in the cotton variety Fiber Max 989. The evaluated application rates were 0, 50, 100, and 150 kg/ha of N, using urea as a source (46% N) in a randomizedblock design with three replicates. At harvest, the maximum yield of seed cotton and the greatest accumulation of total foliar biomass through time was reached after applying 150 kg N/ha. The different N-application rates did not affect the components of cotton-fibre quality. The activity of endogenous NR was greater on plants where 150 kg N/ha were applied. The highest cotton yield and N contents were obtained on these plants. Therefore, the NR activity in vivo could be used as a bioindicator of the N nutritional level in cotton.     

A bioluminescence method for direct measurement of phosphodiesterase activity

We have adapted bioluminescence methods to be able to measure phosphodiesterase (PDE) activity in a one-step technique. The method employs a four-enzyme system (PDE, adenylate kinase (AK) using excess CTP instead of ATP as substrate, pyruvate kinase (PK), and firefly luciferase) to generate ATP, with measurement of the concomitant luciferase-light emission. Since AK, PK, and luciferase reactions are coupled to recur in a cyclic manner, AMP recycling maintains a constant rate of ATP formation, proportional to the steady-state AMP concentration. The cycle can be initiated by the PDE reaction that yields AMP. As long as the PDE reaction is rate limiting, the system is effectively at steady state and the bioluminescence kinetics progresses at a constant rate proportional to the PDE activity. In the absence of cAMP and PDE, low concentrations of AMP trigger the AMP cycling, which allows standardizing the system. The sensitivity of the method enables detection of <1 μU (pmol/min) of PDE activity in cell extracts containing 0.25–10 μg protein. Assays utilizing pure enzyme showed that 0.2 mM IBMX completely inhibited PDE activity. This single-step enzyme- and substrate-coupled cyclic-reaction system yields a simplified, sensitive, reproducible, and accurate method for quantifying PDE activities in small biological samples.