Enhanced procollagen processing in skeletal muscle after a single bout of eccentric loading in humans.

Increases in procollagen processing within skeletal muscle have previously been reported in small animal models only. While indirect measurements in humans have suggested an increase procollagen processing, no intra-skeletal muscle measurements have confirmed these findings. In this study, eight young healthy male subjects performed a single bout of unaccustomed high intensity eccentric exercise on one leg, with the contralateral leg being the control. A significant increase in the muscle interstitial concentration of the N-terminal propeptide of procollagen type I (PINP) was observed (day 0: 1.96 +/- 0.44 ng ml(-1), day 2: 1.94 +/- 0.32 ng ml(-1), day 4: 3.90 +/- 1.03 ng ml(-1), day 8: 7.23 +/- 2.34 ng ml(-1)*, *P < 0.05 vs. basal and control) with no change being noted in the control leg. By day 2 post-exercise, an increase in the histological immunoreactivity of PINP and the N-terminal propeptide of procollagen type III (PIIINP) was also shown in the exercising leg only. Further, from day 2 post-exercise, immunoreactivity for tenascin C and reactive macrophages (CD68+ cells) was seen within the perimysial and endomysial connective tissue of the exercising leg only, indicating a high mechanical load and inflammation. This study shows that following a single bout of high intensity eccentric exercise there is an increase in procollagen processing within skeletal muscle in humans.     

Leaf litter fungi in a Central Amazonian forest: the influence of rainfall,soil and topography on the distribution of fruiting bodies

Fungi are important components of tropical ecosystems, especially in the recycling of nutrients. However, there is little information on how fungal diversity is structured at scales suitable to plan their conservation. We tested if the distribution of fruiting bodies of litter fungi was random in the landscape (over 25 km²) in a tropical evergreen forest in Central Amazonia. We used linear regressions to evaluate the influence of rainfall, soil characteristics and topography on morphospecies richness and composition. Fungi were collected twice in thirty 0.25 × 250 m plots. Short-term rainfall was represented by the cumulative rainfall in the three days before each plot was surveyed. Plots were classified in two groups based on cumulative rainfall. Clay content in soil and rainfall influenced morphospecies richness, but responses to edaphic factors depended on rainfall. Wetter periods apparently decreased limiting moisture conditions in higher areas, allowing fungal activity and fruiting body production. Morphospecies composition was influenced by clay content, but influence on fungi was probably indirect as clay content was correlated with altitude, plant community and nitrogen availability. Our results suggest that the species of litter fungi are not randomly distributed in the landscape. Furthermore, they indicate that it is viable to conduct mesoscale evaluations of fungal diversity, if the temporal and spatial variation and their interaction are taken into account.     

Simultaneous use of electrochemistry and chemiluminescence to detect reactive oxygen species produced by human neutrophils

A novel approach for the simultaneous optical and electrochemical detection of biologically produced reactive oxygen species has been developed and applied. The set-up consists of a luminol-dependent chemiluminescence assay combined with two amperometric biosensors sensitive to superoxide anion radicals (O(2)(-)) and hydrogen peroxide (H(2)O(2)), respectively. The method permits direct, real-time in vitro determination of both extra- and intracellular O(2)(-) and H(2)O(2) produced by human neutrophil granulocytes. The rate of O(2)(-) production by stimulated neutrophils was calculated to about 10(-17)mol s(-1) per single cell. With inhibited NADPH oxidase, a distinct extracellular release of H(2)O(2) instead of O(2)(-) was obtained from stimulated neutrophils with the rate of about 3 x 10(-18)mol s(-1) per single cell. When the H(2)O(2) release was discontinued, fast H(2)O(2) utilisation was observed. Direct interaction with and possibly attachment of neutrophils to redox protein-modified gold electrodes, resulted in a spontaneous respiratory burst in the population of cells closely associated to the electrode surface. Hence, further stimulation of human neutrophils with a potent receptor agonist (fMLF) did not significantly increase the O(2)(-) sensitive amperometric response. By contrast, the H(2)O(2) sensitive biosensor, based on an HRP-modified graphite electrode, was able to reflect the bulk concentration of H(2)O(2), produced by stimulated neutrophils and would be very useful in modestly equipped biomedical research laboratories. In summary, the system would also be appropriate for assessment of several other metabolites in different cell types, and tissues of varying complexity, with only minor electrode modifications.     

Dissecting sensor functions in cell wall integrity signaling in Kluyveromyces lactis.

KlWSC1, KlWSC2/3 and KlMID2, which encode putative plasma membrane sensors for cell wall integrity signaling in Kluyveromyces lactis, were cloned and characterized. Double and triple deletion mutants show severe cell integrity defects, indicating overlapping functions. The Klwsc1 Klmid2 double deletion phenotype can be suppressed by overexpression of the downstream components KlROM2, KlPKC1 and KlBCK1. KlWsc1 sensor domain analyses showed that an amino-terminal elongation as well as an extension within the cytoplasmic domain are dispensable for function. Heterologous complementation by KlMID2 and KlWSC1 in Saccharomyces cerevisiae is only achieved upon overexpression. In contrast to ScMID2, ScWSC1 complements in K. lactis. Functional studies with chimeric Mid2 constructs indicate that species specificity is mainly conferred by the extracellular domain. Sensor-GFP fusions localize to the plasma membrane, with a cell cycle dependent distribution of KlWsc1-GFP. Both Wsc-type sensors concentrate in discrete spots within the plasma membrane.     

Binomial mitotic segregation of MYCN-carrying double minutes in neuroblastoma illustrates the role of randomness in oncogene amplification

Background

Amplification of the oncogene MYCN in double minutes (DMs) is a common finding in neuroblastoma (NB). Because DMs lack centromeric sequences it has been unclear how NB cells retain and amplify extrachromosomal MYCN copies during tumour development.

Principal Findings

We show that MYCN-carrying DMs in NB cells translocate from the nuclear interior to the periphery of the condensing chromatin at transition from interphase to prophase and are preferentially located adjacent to the telomere repeat sequences of the chromosomes throughout cell division. However, DM segregation was not affected by disruption of the telosome nucleoprotein complex and DMs readily migrated from human to murine chromatin in human/mouse cell hybrids, indicating that they do not bind to specific positional elements in human chromosomes. Scoring DM copy-numbers in ana/telophase cells revealed that DM segregation could be closely approximated by a binomial random distribution. Colony-forming assay demonstrated a strong growth-advantage for NB cells with high DM (MYCN) copy-numbers, compared to NB cells with lower copy-numbers. In fact, the overall distribution of DMs in growing NB cell populations could be readily reproduced by a mathematical model assuming binomial segregation at cell division combined with a proliferative advantage for cells with high DM copy-numbers.

Conclusion

Binomial segregation at cell division explains the high degree of MYCN copy-number variability in NB. Our findings also provide a proof-of-principle for oncogene amplification through creation of genetic diversity by random events followed by Darwinian selection.     

Screening of novel bacteria for the 2,3-butanediol production

Biotechnologically produced 2,3-butanediol (2,3-BDO) is a potential starting material for industrial bulk chemicals such as butadiene or methyl ethyl ketone which are currently produced from fossil feedstocks. So far, the highest 2,3-BDO concentrations have been obtained with risk group 2 microorganisms. In this study, three risk group 1 microorganisms are presented that are so far unknown for an efficient production of 2,3-BDO. The strains Bacillus atrophaeus NRS-213, Bacillus mojavensis B-14698, and Bacillus vallismortis B-14891 were evaluated regarding their ability to produce high 2,3-BDO concentrations with a broad range of different carbon sources. A maximum 2,3-BDO concentration of 60.4 g/L was reached with the strain B. vallismortis B-14891 with an initial glucose concentration of 200 g/L within 55 h in a batch cultivation. Besides glucose, B. vallismortis B-14891 converts 14 different substrates that can be obtained from residual biomass sources to 2,3-BDO. Therefore B. vallismortis B-14891 is a promising candidate for the large-scale production of 2,3-BDO with low-cost substrates.

     

Estimating predation rates in experimental systems: scale-dependent effects of aggregative behaviour

We studied the effect of arena size on estimates of prey mortality rate in predation experiments. In a laboratory experiment involving two of the dominating benthic species in the northern Baltic Sea, the predacious isopod Saduria entomon , and its main prey, the amphipod Monoporeia affinis , the prey mortality rate increased with container radius. The densities of both predator and prey close to the wall increased with container size. We hypothesised that this scale-dependent coaggregation of predator and prey caused the mortality rates to increase with arena size. We tested the hypothesis with the help of a simple model, by calculating the expected number of prey eaten in containers of different size from experimental data on the distributions of predator and prey within the arenas. A significant relationship between expected and observed numbers eaten supported our hypothesis. As the aggregative response was most pronounced in large arenas, this leads to the counterintuitive conclusion that large containers produced more biased estimates of morality rates than small containers.
To further study the effects of coaggregation we explored a general simulation model where both predator and prey preferred the habitat close to the arena wall. The model predicted a humpshaped relationship between encounter rate and arena size. This suggests that when predators and prey show a scale-dependent tendency to aggregate along arena walls, the most accurate estimates of predation rates may be obtained with very small or very large arenas.     

Is the ArteACC Index a Valid Indicator of Free‐Living Physical Activity in Adolescents?

Objective: The principal aim of this study was to validate a proposed new index of physical activity, the activity‐related time equivalent based on accelerometry (ArteACC), in adolescents. A secondary aim was to develop regression equations for prediction of total energy expenditure (TEE) and activity energy expenditure [AEE = 0.9 × TEE ? resting metabolic rate (RMR)]. Research Methods and Procedures: RMR and energy expenditure (EE) under standardized exercises were measured by indirect calorimetry in 36 adolescents (14 to 19 years old). TEE was measured by the doubly labeled water method, and physical activity was assessed simultaneously with an accelerometer for 14 days. AEE, AEE in relation to body weight (AEE per kilogram), and activity‐related time equivalent based on energy expenditure (ArteEE = AEE/[EE reference activity ? RMR]) were calculated from laboratory and free‐living EE data. ArteACC was calculated as total activity counts/activity counts of reference activity. Results: ArteACC was significantly related to AEE per kilogram (r = 0.57; p < 0.0001) and ArteEE (r = 0.68; p < 0.001). The absolute amount of time (minutes per day) spent in physical activity was significantly lower when calculated from ArteACC than from ArteEE (p < 0.001). TEE was significantly influenced by RMR, sex, and ArteACC (r² = 0.89). AEE was significantly influenced by sex and ArteACC (r² = 0.59). Discussion: Despite an absolute difference between the two indexes, ArteEE and ArteACC, ArteACC seems to be a valid indicator of free‐living physical activity. It contributed significantly, by 3.3% and 12.5%, to the explained variations in TEE and AEE, respectively.