Do the higher oxidation states of the photosynthetic O2-evolving system contain bound H2O?

A modified mass spectrometer was used to determine whether the higher oxidation states of the photosynthetic O2-evolving system contain substrate water that is not freely exchangeable with the external medium. Our data indicated that the higher oxidation states contain no appreciable bound, non-exchangeable H2O. This suggests that H2O oxidation takes place via a rapid, concerted, all-or-none mechanism rather than by a mechanism involving stable, partially oxidized, H2O-derived intermediates. These findings set definite constraints on possible mechanisms of O2 evolution.     

Accounting for Carbon Flux to Mycorrhizal Fungi May Resolve Discrepancies in Forest Carbon Budgets

Ecosystems - Carbon (C) fluxes among different components of plant growth are important to forest ecosystem C cycling and are strongly influenced by species composition and resource availability....     

A Dual Read-Out Assay to Evaluate the Potency of Compounds Active against Mycobacterium tuberculosis

Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors.     

Soil respiration does not acclimatize to warmer temperatures when modeled over seasonal timescales

Soil warming studies have generally demonstrated an ephemeral response of soil respiration to warming suggesting acclimatization to increased temperatures. Many of these studies depict acclimatization as an empirical temperature-respiration model with data collected from late spring through early autumn. We examined the apparent temperature sensitivity of soil respiration in chronically warmed soils over three different timescales: annually, during the growing season, and seasonally during winter, spring, summer, and fall. Temperature sensitivity was evaluated by fitting exponential and flexible temperature functions as mixed effects models. From model coefficients, we estimated annual, growing season, and season-specific Q ₁₀ values, and assessed the ability of model coefficients to predict daily soil respiration rates over a two-year period. We found that respiration in warmed soils can exhibit characteristics of acclimatized temperature sensitivity depending on the timeframe and the function (exponential or flexible) used. Models using growing season data suggested acclimatization while models using data collected in winter or spring indicated enhanced temperature sensitivity with 5 °C of warming. Differences in temperature sensitivity affected predicted daily soil respiration rates, particularly in winter and spring. Models constructed over longer timescales overestimated daily respiration rates by as much 10–40 % whereas season-specific predictions were generally within 2 % of actual values. Failure to use season-specific models to depict changes in temperature dependence may over- or under-estimate carbon losses due to climate warming, especially during the colder months of the year.     

Lysosomal release of cathepsin D precedes relocation of cytochrome c and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential-sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (delta psi(m)) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of delta psi(m), and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.     

Inhibition of cathepsin D prevents free-radical-induced apoptosis in rat cardiomyocytes

Apoptosis was inhibited in rat cardiomyocytes pretreated with the aspartic protease inhibitor pepstatin A and subsequently exposed to naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Cathepsin D was released from lysosomes to the cytosol upon exposure to naphthazarin, and the enzyme activity decreased simultaneously. Later, cathepsin D reappeared in granules of increased size, and enzyme activity was restored. Activation of caspase-3-like proteases was detected, and the number of cells showing apoptotic morphology increased with time. Pepstatin A pretreatment did not prevent release of cathepsin D from lysosomes but did significantly inhibit subsequent naphthazarin-induced caspase activation and apoptotic morphology. This suggests that cathepsin D exerts its apoptosis-stimulating effect upstream of caspase-3-like activation.     

Use of digital webcam images to track spring green-up in a deciduous broadleaf forest

Understanding relationships between canopy structure and the seasonal dynamics of photosynthetic uptake of CO₂ by forest canopies requires improved knowledge of canopy phenology at eddy covariance flux tower sites. We investigated whether digital webcam images could be used to monitor the trajectory of spring green-up in a deciduous northern hardwood forest. A standard, commercially available webcam was mounted at the top of the eddy covariance tower at the Bartlett AmeriFlux site. Images were collected each day around midday. Red, green, and blue color channel brightness data for a 640 × 100-pixel region-of-interest were extracted from each image. We evaluated the green-up signal extracted from webcam images against changes in the fraction of incident photosynthetically active radiation that is absorbed by the canopy (f _APAR), a broadband normalized difference vegetation index (NDVI), and the light-saturated rate of canopy photosynthesis (A _max), inferred from eddy flux measurements. The relative brightness of the green channel (green %) was relatively stable through the winter months. A steady rising trend in green % began around day 120 and continued through day 160, at which point a stable plateau was reached. The relative brightness of the blue channel (blue %) also responded to spring green-up, although there was more day-to-day variation in the signal because blue % was more sensitive to changes in the quality (spectral distribution) of incident radiation. Seasonal changes in blue % were most similar to those in f _APAR and broadband NDVI, whereas changes in green % proceeded more slowly, and were drawn out over a longer period of time. Changes in A _max lagged green-up by at least a week. We conclude that webcams offer an inexpensive means by which phenological changes in the canopy state can be quantified. A network of cameras could offer a novel opportunity to implement a regional or national phenology monitoring program.     

Effects of inhibitors of the arachidonic acid cascade on primary muscle culture from a Duchenne muscular dystrophy patient

The aim of this study was to elucidate the mechanisms of action for potential targets of therapeutic intervention related to the arachidonic acid cascade in muscular dystrophy. Primary cultures from a Duchenne patient were used to study the expression of dystrophin-1, utrophin, desmin, neonatal myosin heavy chain (MHCn) and Bcl-2 during inhibition of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX). Hypo-osmotic treatment was applied in order to trigger Ca2+ influx and PLA2 activity. Inhibition of PLA2 and LOX with prednisolone and nordihydroguaiaretic acid (NDGA) caused a semi-quantitative increase of utrophin and Bcl-2-, and a dose-dependent, quantitative increase of desmin expression, an effect that was augmented by hypo-osmotic treatment. Our results indicate that LOX inhibitors, similarly to corticosteroids, can be beneficial in the treatment of muscular dystrophies.     

An unusual presentation of a malignant jejunal tumor and a different management strategy

BACKGROUND: Malignant small bowel tumors are very rare and leiomyosarcoma accounts for less than 15% of the cases. Management of these tumors is challenging in view of nonspecific symptoms, unusual presentation and high incidence of metastasis. In this case report, an unusual presentation of jejunal sarcoma and management of liver metastasis with radiofrequency ablation (RFA) is discussed. CASE PRESENTATION: A 45-year-old male presented with anemia and features of small bowel obstruction. Operative findings revealed a mass lesion in jejunum with intussusception of proximal loop. Resection of bowel mass was performed. Histopathological findings were suggestive of leiomyosarcoma. After 3-years of follow-up, the patient developed recurrence in infracolic omentum and a liver metastasis. The omental mass was resected and liver lesion was managed with radiofrequency ablation. CONCLUSION: Jejunal leiomyosarcoma is a rare variety of malignant small bowel tumor and a clinical presentation with intussusception is unusual. We suggest that an aggressive management approach using a combination of surgery and a newer technique like RFA can be attempted in patients with limited metastatic spread to liver to prolong the long-term survival in a subset of patients.     

Inhibition of the sole type I signal peptidase of Mycobacterium tuberculosis is bactericidal under replicating and nonreplicating conditions

Proteins secreted by bacteria perform functions vital for cell survival and play a role in virulence in Mycobacterium tuberculosis. M. tuberculosis lepB (Rv2903c) encodes the sole homolog of the type I signal peptidase (SPase). The lepB gene is essential in M. tuberculosis, since we could delete the chromosomal copy only when a second functional copy was provided elsewhere. By placing expression under the control of an anhydrotetracycline-inducible promoter, we confirmed that reduced lepB expression was detrimental to growth. Furthermore, we demonstrated that a serine-lysine catalytic dyad, characteristic for SPase function, is required for LepB function. We confirmed the involvement of LepB in the secretion of a reporter protein fused to an M. tuberculosis signal peptide. An inhibitor of LepB (MD3; a beta-aminoketone) was active against M. tuberculosis, exhibiting growth inhibition and bactericidal activity. Overexpression of lepB reduced the susceptibility of M. tuberculosis to MD3, and downregulation resulted in increased susceptibility, suggesting that LepB is the true target of MD3. MD3 lead to a rapid loss of viability and cell lysis. Interestingly, the compound had increased potency in nonreplicating cells, causing a reduction in viable cell numbers below the detection limit after 24 h. These data suggest that protein secretion is required to maintain viability under starvation conditions and that secreted proteins play a critical role in generating and surviving the persistent state. We conclude that LepB is a promising novel target for drug discovery in M. tuberculosis, since its inhibition results in rapid killing of persistent and replicating organisms.