Escherichia coli Division Inhibitor MinCD Blocks Septation by Preventing Z-Ring Formation

The min system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118-1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410-423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles of ftsZ, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.     

The resident endoplasmic reticulum protein, BAP31, associates with gamma-actin and myosin B heavy chain.

BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two caspase recognition sites that are preferentially cleaved by initiator caspases, such as caspase-8. Recently, we reported that the caspase-resistant BAP31 inhibited Fas-mediated apoptotic membrane fragmentation and the release of cytochrome c from mitochondria in KB epithelial cells (Nguyen M., Breckenridge G., Ducret A & Shore G. (2000) Mol. Cell. Biol.20, 6731-6740). We describe here the characterization by capillary liquid chromatography microelectrospray tandem MS of a BAP31 immunocomplex isolated from a HepG2 cell lysate in the absence of a death signal. We show that BAP31 specifically associates with nonmuscle myosin heavy chain B and nonmuscle gamma-actin, two components of the cytoskeleton actomyosin complex. Collectively, these data confirm that BAP31, in addition to its potential role as a chaperone, may play a fundamental role in the structural organization of the cytoplasm. Here we also show that Fas stimulation of apoptosis releases BAP31 associations with these motor proteins, a step that may contribute to extranuclear events, such as membrane remodelling, during the execution phase of apoptosis.     

Genomic Patterns of Positive Selection at the Origin of Rust Fungi

Understanding the origin and evolution of pathogenicity and biotrophic life-style of rust fungi has remained a conundrum for decades. Research on the molecular mechanisms responsible for rust fungi evolution has been hampered by their biotrophic life-style until the sequencing of some rust fungi genomes. With the availability of multiple whole genomes and EST data for this group, it is now possible to employ genome-wide surveys and investigate how natural selection shaped their evolution. In this work, we employed a phylogenomics approach to search for positive selection and genes undergoing accelerated evolution at the origin of rust fungi on an assembly of single copy genes conserved across a broad range of basidiomycetes. Up to 985 genes were screened for positive selection on the phylogenetic branch leading to rusts, revealing a pervasive signal of positive selection throughout the data set with the proportion of positively selected genes ranging between 19.6–33.3%. Additionally, 30 genes were found to be under accelerated evolution at the origin of rust fungi, probably due to a mixture of positive selection and relaxation of purifying selection. Functional annotation of the positively selected genes revealed an enrichment in genes involved in the biosynthesis of secondary metabolites and several metabolism and transporter classes.     

An IP3R3- and NPY-Expressing Microvillous Cell Mediates Tissue Homeostasis and Regeneration in the Mouse Olfactory Epithelium

Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3^+/−, and IP3R3^−/− mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3^−/− mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3^−/− mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3^−/− mice. The reductions in both NPY release and number of progenitor cells in IP3R3^−/− mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.     

CTCF binding site classes exhibit distinct evolutionary, genomic, epigenomic and transcriptomic features

Background  

CTCF (CCCTC-binding factor) is an evolutionarily conserved zinc finger protein involved in diverse functions ranging from negative regulation of MYC, to chromatin insulation of the beta-globin gene cluster, to imprinting of the Igf2 locus. The 11 zinc fingers of CTCF are known to differentially contribute to the CTCF-DNA interaction at different binding sites. It is possible that the differences in CTCF-DNA conformation at different binding sites underlie CTCF's functional diversity. If so, the CTCF binding sites may belong to distinct classes, each compatible with a specific functional role.     

Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides.

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.     

Biochemical characterization of a strictly specific beta-galactosidase from the digestive juice of the palm weevil Rhynchophorus palmarum larvae

A beta‐galactosidase from the digestive juice of the palm weevil Rhynchophorus palmarum L. larvae was purified by chromatography on ion exchange, gel filtration, and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gel. Beta‐galactosidase was a monomeric protein with a molecular weight of 62 kDa based on its mobility in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 60 kDa based on gel filtration. Maximal enzyme activity occurred at 55°C and pH 5.0. The purified beta‐galactosidase was stable at 37°C and its pH stability was in the range of 4.6–6.0. Beta‐galactosidase was highly specific for the beta‐d ‐galactosyl residue and beta‐(1‐4) linkage. The catalytic efficiency (V_max/K_m) values for p‐nitrophenyl‐beta‐d ‐galactopyranoside, beta‐d ‐galactosyl(1‐4)‐d ‐glucose (lactose), beta‐d ‐galactosyl(1‐4)‐d ‐galactose and beta‐d ‐galactosyl(1‐4)‐beta‐d ‐galactosyl(1‐4)‐d ‐glucose were, respectively, 72.95, 10.97, 20.74 and 12.73. 5,5‐Dithio‐bis(2‐nitrobenzoate) and sodium dodecyl sulfate inhibited completely the beta‐galactosidase activity. The enzyme was capable of catalyzing transgalactosylation reactions. The yield of galactosylation of 2‐phenylethanol (43%), catalyzed by the beta‐galactosidase in the presence of lactose as galactosyl donor, is higher than those reported previously with conventional sources of beta‐galactosidases. In addition, the optimum pH is different for the hydrolysis (pH 5.0) and transgalactosylation reactions (pH 6.0).     

Sex‐specific allelic transmission bias suggests sexual conflict at MC1R

Sexual conflict arises when selection in one sex causes the displacement of the other sex from its phenotypic optimum, leading to an inevitable tension within the genome – called intralocus sexual conflict. Although the autosomal melanocortin‐1‐receptor gene (MC1R) can generate colour variation in sexually dichromatic species, most previous studies have not considered the possibility that MC1R may be subject to sexual conflict. In the barn owl (Tyto alba), the allele MC1R_WHITE is associated with whitish plumage coloration, typical of males, and the allele MC1R_RUFOUS is associated with dark rufous coloration, typical of females, although each sex can express any phenotype. Because each colour variant is adapted to specific environmental conditions, the allele MC1R_WHITE may be more strongly selected in males and the allele MC1R_RUFOUS in females. We therefore investigated whether MC1R genotypes are in excess or deficit in male and female fledglings compared with the expected Hardy–Weinberg proportions. Our results show an overall deficit of 7.5% in the proportion of heterozygotes in males and of 12.9% in females. In males, interannual variation in assortative pairing with respect to MC1R explained the year‐specific deviations from Hardy–Weinberg proportions, whereas in females, the deficit was better explained by the interannual variation in the probability of inheriting the MC1R_WHITE or MC1R_RUFOUS allele. Additionally, we observed that sons inherit the MC1R_RUFOUS allele from their fathers on average slightly less often than expected under the first Mendelian law. Transmission ratio distortion may be adaptive in this sexually dichromatic species if males and females are, respectively, selected to display white and rufous plumages.     

Ecoinvent 3: assessing water use in LCA and facilitating water footprinting

Purpose

Water footprinting and the assessment of water use in life cycle assessment have become of major interest in sustainability assessments. Various initiatives for combining water resource issues with consumption of products and services have been initiated in the last decade. However, comprehensive databases fulfilling the requirements for addressing these issues have been lacking and are necessary to facilitate efficient and consistent assessments of products and services. To this purpose, ecoinvent focused on integrating appropriate water use data into version 3, since previously water use data has been inconsistently reported and some essential flows were missing. This paper describes the structure of the water use data in ecoinvent, how the data has been compiled and the way it can be used for water footprinting.

Methods

The main changes required for proper assessment of water use are the addition of environmental and product flows in order to allow a water balance over each process. This is in accordance with the strict paradigm in ecoinvent 3 to focus on mass balances, which requires the inclusion of water contents of all products (also for e.g. waste water flows), as well as emissions of water to soil, air and various water bodies. Water inputs from air (e.g. rainwater harvesting) is introduced but is not yet used by any activity.

Results and discussion

Ecoinvent version 3.1 consistently includes the relevant flows to address water use in life cycle assessment (LCA) and calculate water footprints on the product level for most processes including uncertainty information. Although some problems regarding data quality and spatial resolution remain, this is an important step forward and can limit efforts for detailed data collection to the most sensitive processes in the product system. With the combination of data on water use and emissions to water for each process, concentration and corresponding water classes can also be calculated and assessed with existing impact assessment methods.

Conclusions

This comprehensive collection of water use data on the process level facilitates the proper assessment of water use within an LCA and water footprints beyond agricultural production. Especially in LCA, but also in tools for eco-design and specific water footprint, this data is essential and leads to a cost-efficient way of assessing consumption choices and product design decisions with full transparency. It enhances the effectiveness of investing in data collection by performing sensitivity analyses using ecoinvent data to identify the most relevant flows and processes.