Molecular and proteomic analyses highlight the importance of ubiquitination for the stress resistance,metabolic adaptation,morphogenetic regulation and virulence of Candida albicans

How cells co‐ordinate size with growth and development is a major, unresolved question in cell biology. In previous work we identified the glucosyltransferase UgtP as a division inhibitor responsible for increasing the size of Bacillus subtilis cells under nutrient‐rich conditions. In nutrient‐rich medium, UgtP is distributed more or less uniformly throughout the cytoplasm and concentrated at the cell poles and/or the cytokinetic ring. Under these conditions, UgtP interacts directly with FtsZ to inhibit division and increase cell size. Conversely, under nutrient‐poor conditions, UgtP is sequestered away from FtsZ in punctate foci, and division proceeds unimpeded resulting in a reduction in average cell size. Here we report that nutrient‐dependent changes in UgtP's oligomerization potential serve as a molecular rheostat to precisely co‐ordinate B. subtilis cell size with nutrient availability. Our data indicate UgtP interacts with itself and the essential cell division protein FtsZ in a high‐affinity manner influenced in part by UDP glucose, an intracellular proxy for nutrient availability. These findings support a model in which UDP‐glc‐dependent changes in UgtP's oligomerization potential shift the equilibrium between UgtP?UgtP and UgtP?FtsZ, fine‐tuning the amount of FtsZ available for assembly into the cytokinetic ring and with it cell size.     

Iron overload,measured as serum ferritin,increases brain damage induced by focal ischemia and early reperfusion

High levels of iron, measured as serum ferritin, are associated to a worse outcome after stroke. However, it is not known whether ischemic damage might increase ferritin levels as an acute phase protein or whether iron overload affects stroke outcome. The objectives are to study the effect of stroke on serum ferritin and the contribution of iron overload to ischemic damage.     

Continuous biosynthesis of abscisic acid (ABA) may be required for maintaining dormancy of isolated embryos and intact seeds of Euonymus alatus

Euonymus alatus (Thunb.) Sieb. is a popular landscape plant in the United States due to its brilliant red fall foliage. It is also an important ornamental plant in many other areas of the world such as China, Japan and Europe. However, E. alatus is considered as a highly invasive plant species in the US. Mutation breeding can be used to create sterile, non-invasive cultivars. Seeds are the most commonly used explants for mutagen treatments, but E. alatus mature seeds possess prolonged dormancy and only a low percentage of them germinate even after 18?months of cold stratification. Here we report an immature embryo culture method for E. alatus ??Compactus?? to circumvent the seed dormancy problem. Also, we have found that activated charcoal, gibberellic acid (GA₃) and 6-benzyladenine (BA) can reduce the dormancy of isolated embryos, which suggests that abscisic acid (ABA) might play a role in controlling seed dormancy. We have further demonstrated that exogenous ABA enhances dormancy of isolated E. alatus embryos while fluridone, an inhibitor for ABA biosynthesis, can effectively break their dormancy. These results, particularly the effect of fluridone, suggest that continuous ABA biosynthesis plays an important role in controlling the dormancy of E. alatus seeds.     

Overexpression of manganese superoxide dismutase ameliorates high-fat diet-induced insulin resistance in rat skeletal muscle

Elevated mitochondrial reactive oxygen species have been suggested to play a causative role in some forms of muscle insulin resistance. However, the extent of their involvement in the development of diet-induced insulin resistance remains unclear. To investigate, manganese superoxide dismutase (MnSOD), a key mitochondrial-specific enzyme with antioxidant modality, was overexpressed, and the effect on in vivo muscle insulin resistance induced by a high-fat (HF) diet in rats was evaluated. Male Wistar rats were maintained on chow or HF diet. After 3 wk, in vivo electroporation (IVE) of MnSOD expression and empty vectors was undertaken in right and left tibialis cranialis (TC) muscles, respectively. After one more week, insulin action was evaluated using hyperinsulinemic euglycemic clamp, and tissues were subsequently analyzed for antioxidant enzyme capacity and markers of oxidative stress. MnSOD mRNA was overexpressed 4.5-fold, and protein levels were increased by 70%, with protein detected primarily in the mitochondrial fraction of muscle fibers. This was associated with elevated MnSOD and glutathione peroxidase activity, indicating that the overexpressed MnSOD was functionally active. The HF diet significantly reduced whole body and TC muscle insulin action, whereas overexpression of MnSOD in HF diet animals ameliorated this reduction in TC muscle glucose uptake by 50% (P < 0.05). Decreased protein carbonylation was seen in MnSOD overexpressing TC muscle in HF-treated animals (20% vs. contralateral control leg, P < 0.05), suggesting that this effect was mediated through an altered redox state. Thus interventions causing elevation of mitochondrial antioxidant activity may offer protection against diet-induced insulin resistance in skeletal muscle.     

Hectare-scale demonstration of high rate algal ponds for enhanced wastewater treatment and biofuel production

High rate algal ponds (HRAPs) are shallow, paddlewheel-mixed open raceway ponds that are an efficient and cost-effective upgrade for the conventional wastewater treatment ponds used by communities and farms the world over. HRAPs provide improved natural disinfection and nutrient removal and can be further enhanced by carbon dioxide (CO₂) addition to promote algal growth which is often carbon limited. This paper discusses the construction and operation of a 5-ha demonstration HRAP system treating primary settled wastewater at the Christchurch wastewater treatment plant, New Zealand. The system consisted of four 1.25-ha HRAPs that were constructed from an existing conventional pond. Algae were harvested from the HRAP effluent in specially designed settlers, which concentrated the algal/bacterial biomass to 1–2% organic solids for conversion to bio-crude oil following dewatering. Performance data from the first 15?months of HRAP operation (without CO₂ addition) are presented. The four demonstration HRAPs had reasonable replication of both treatment performance and algal/bacterial productivity with similar annual average wastewater treatment efficiency (~50% removal of BOD₅, ~87% removal of fBOD₅, ~65% removal of ammoniacal-N, ~19% removal of dissolved reactive phosphorus and ~2 log removal of Escherichia coli), algal species composition and algal/bacterial biomass production (~8?g?m^?2?day ?1 volatile suspended solids). These results were in good agreement with the results for pilot-scale HRAP without CO₂ addition in New Zealand. This study provides further indication of the potential for energy efficient and effective wastewater treatment using HRAP, while biofuel conversion of the harvested algal bacterial biomass could provide a valuable niche distributed energy source for local communities.     

A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs

Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack.     

Vascular endothelial growth factor enhances macrophage clearance of apoptotic cells

Efficient clearance of apoptotic cells from the lung by alveolar macrophages is important for the maintenance of tissue structure and function. Lung tissue from humans with emphysema contains increased numbers of apoptotic cells and decreased levels of vascular endothelial growth factor (VEGF). Mice treated with VEGF receptor inhibitors have increased numbers of apoptotic cells and develop emphysema. We hypothesized that VEGF regulates apoptotic cell clearance by alveolar macrophages (AM) via its interaction with VEGF receptor 1 (VEGF R1). Our data show that the uptake of apoptotic cells by murine AMs and human monocyte-derived macrophages is inhibited by depletion of VEGF and that VEGF activates Rac1. Antibody blockade or pharmacological inhibition of VEGF R1 activity also decreased apoptotic cell uptake ex vivo. Conversely, overexpression of VEGF significantly enhanced apoptotic cell uptake by AMs in vivo. These results indicate that VEGF serves a positive regulatory role via its interaction with VEGF R1 to activate Rac1 and enhance AM apoptotic cell clearance.     

Membrane association, mechanism of action, and structure of Arabidopsis embryonic factor 1 (FAC1)

Embryonic factor 1 (FAC1) is one of the earliest expressed plant genes and encodes an AMP deaminase (AMPD), which is also an identified herbicide target. This report identifies an N-terminal transmembrane domain in Arabidopsis FAC1, explores subcellular fractionation, and presents a 3.3-A globular catalytic domain x-ray crystal structure with a bound herbicide-based transition state inhibitor that provides the first glimpse of a complete AMPD active site. FAC1 contains an (alpha/beta)(8)-barrel characterized by loops in place of strands 5 and 6 that places it in a small subset of the amidohydrolase superfamily with imperfect folds. Unlike tetrameric animal orthologs, FAC1 is a dimer and each subunit contains an exposed Walker A motif that may be involved in the dramatic combined K(m) (25-80-fold lower) and V(max) (5-6-fold higher) activation by ATP. Normal mode analysis predicts a hinge motion that flattens basic surfaces on each monomer that flank the dimer interface, which suggests a reversible association between the FAC1 globular catalytic domain and intracellular membranes, with N-terminal transmembrane and disordered linker regions serving as the anchor and attachment to the globular catalytic domain, respectively.     

Extracellular calpains increase tubular epithelial cell mobility. Implications for kidney repair after ischemia

Calpains are intracellular Ca2+-dependent cysteine proteases that are released in the extracellular milieu by tubular epithelial cells following renal ischemia. Here we show that externalized calpains increase epithelial cell mobility and thus are critical for tubule repair. In vitro, exposure of human tubular epithelial cells (HK-2 cells) to mu-calpain limited their adhesion to extracellular matrix and increased their mobility. Calpains acted primarily by promoting the cleavage of fibronectin, thus preventing fibronectin binding to the integrin alphavbeta3. Analyzing downstream integrin effects, we found that the cyclic AMP-dependent protein kinase A pathway was activated in response to alphavbeta3 disengagement and was essential for calpain-mediated increase in HK-2 cell mobility. In a murine model of ischemic acute renal failure, injection of a fragment of calpastatin, which specifically blocked calpain activity in extracellular milieu, markedly delayed tubule repair, increasing functional and histological lesions after 24 and 48 h of reperfusion. These findings suggest that externalized calpains are critical for tubule repair process in acute renal failure.