Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.     

Hypermodified nucleoside carboxyl group as a target site for specific tRNA modification

The free carboxyl group of hypermodified nucleosides N6-methyl-N6-(threoninocarbonyl)adenosine (mt6A37) and 3-(3-amino-3-carboxypropyl)uridine (acp3U20:1) in tRNAmMet (yellow lupine), and N6-(threoninocarbonyl)adenosine (t6A37) in tRNAiMet (yellow lupine) can be converted quantitatively and under very mild conditions into the respective anilides in a reaction with aniline and a water-soluble carbodiimide. The tRNA reactions proceed with rates very similar to that reported previously for t6A nucleoside. Detailed analysis of the products of tRNA modification with [3H]aniline on tRNA (chromatography on BD-DEAE-cellulose), oligonucleotide (polyacrylamide gel electrophoresis) and nucleoside (HPLC on Aminex A6) levels clearly indicates that only the hypermodified nucleoside residues undergo the reaction. The site of modification is confirmed for mono-modified (at mt6A37) and bis-modified (at mt6A37 and acp3U20:1) tRNAmMet, and for mono-modified (at t6A37) tRNAiMet by sequence analysis using 5'end 32P-labeled tRNAs. The modification procedure seems to be universally applicable for all hypermodified nucleosides bearing a free carboxyl group and for different amine reagents designed for the studies on tRNA function.     

Decomposition Patterns of Foliar Litter and Deadwood in Managed and Unmanaged Stands: A 13-Year Experiment in Boreal Mixedwoods

Litter decomposition is a major driver of carbon (C) and nitrogen (N) cycles in forest ecosystems and has major implications for C sequestration and nutrient availability. However, empirical information regarding long-term decomposition rates of foliage and wood remains rare. In this study, we assessed long-term C and N dynamics (12–13 years) during decomposition of foliage and wood for three boreal tree species, under a range of harvesting intensities and slash treatments. We used model selection based on the second-order Akaike’s Information Criterion to determine which decomposition model had the most support. The double-exponential model provided a good fit to C mass loss for foliage of trembling aspen, white spruce, and balsam fir, as well as aspen wood. These litters underwent a rapid initial phase of leaching and mineralisation, followed by a slow decomposition. In contrast, for spruce and fir wood, the single-exponential model had the most support. The long-term average decay rate of wood was faster than that of foliage for aspen, but not of conifers. However, we found no evidence that fir and spruce wood decomposed at slower rates than the recalcitrant fraction of their foliage. The critical C:N ratios, at which net N mineralisation began, were higher for wood than for foliage. Long-term decay rates following clear-cutting were either similar or faster than those observed in control stands, depending on litter material, tree species, and slash treatment. The critical C:N ratios were reached later and decreased for all conifer litters following stem-only clear-cutting, indicating increased N retention in harvested sites with high slash loads. Partial harvesting had weak effects on C and N dynamics of decaying litters. A comprehensive understanding of the long-term patterns and controls of C and N dynamics following forest disturbance would improve our ability to forecast the implications of forest harvesting for C sequestration and nutrient availability.     

Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells

The membrane localization of the plasma membrane Ca²⁺-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.     

TheisfA mutation inhibits mutator activity and processing of UmuD protein inEscherichia coli recA730 strains

Further studies on theisfA mutation responsible for anti-SOS and antimutagenic activities inEscherichia coli are described. We have previously shown that theisfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. TheisfA mutation has now been demonstrated also to suppress mutator activity inE. coli recA730 andrecA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of theisfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in therecA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.     

Plant-growth regulators from common starfish (<Emphasis Type="Italic">Asterias amurensis</Emphasis> Lütken) waste

Starfish waste has been shown to be an effective compost material not only in the promotion of plant growth but also in terms of having insecticidal activity. In the present study, plant growth regulation by chemicals from starfish was examined. The aqueous fraction from a hot water extract of the starfish Asterias amurensis Lütken showed plant-growth activity, while the aqueous fraction from a methanol extract inhibited growth of Brassica campestris. The lipophilic fraction from the methanol extract also exhibited a plant growth-promoting effect. The active components from each extract were identified. Asterubine from the hot water extract promoted plant growth. A ceramide from the lipophilic fraction showed root growth promoting effect, and three glucocerebrosides had promotive effects on the entire plant. Asterosaponins were identified as the main growth inhibitors in the aqueous fraction of the methanol extract. These active compounds from starfish waste could be analyzed as potential plant growth regulators in agricultural applications in the future.     

An Ecteola-cellulose chromatography assay for 3′-phosphoadenosine 5′-phosphosulfate: Phenol sulfotransferase

A new assay procedure for phenol sulfotransferase which employs [35S]-3'-phosphoadenosine-5'-phosphosulfate as a sulfate donor and a variety of phenols as sulfate acceptors was developed. The appearance of the 35S-sulfated products or the disappearance of the [35S]-3'-phosphoadenosine-5'-phosphosulfate are determined simultaneously by chromatography of the assay incubation mixtures on Ecteola-cellulose columns, eluting with an NH4HCO3 step gradient. Various acidic, neutral, and basic phenols can be employed as substrates for phenol sulfotransferase using this procedure.     

Transcriptional profiling of the nucleus pulposus: say yes to notochord

This editorial addresses the debate concerning the origin of adult nucleus pulposus cells in the light of profiling studies by Minogue and colleagues. In their report of several marker genes that distinguish nucleus pulposus cells from other related cell types, the authors provide novel insights into the notochordal nature of the former. Together with recently published work, their work lends support to the view that all cells present within the nucleus pulposus are derived from the notochord. Hence, the choice of an animal model for disc research should be based on considerations other than the cell loss and replacement by non-notochordal cells.