Specific phospholipid requirement for activity of the purified respiratory chain NADH dehydrogenase of Escherichia coli.

The highly purified respiratory chain NADH dehydrogenase (EC 1.6.99.3) of Escherichia coli is inactive in the absence of detergent or phospholipid. Triton X-100 is the detergent that gives optimal activity, but the Triton X-100-activated enzyme is stimulated an additional 2-fold by E. coli phospholipids. Phosphatidylglycerol and diphosphatidylglycerol are the most effective lipid activators. The activated complex prepared with diphosphatidylglycerol is stable, whereas that with phosphatidylglycerol loses activity rapidly. Maximum activation by phospholipids occurs after preincubation at 0 degrees C and at pH 7. Triton X-100 is required at low concentrations for lipid activation, but high concentrations interfere with the activation. When the enzyme is optimally activated by phospholipids, it may be additionally activated 2-fold by spermidine, but not by magnesium. In contrast, the Triton X-100-activated form of the enzyme is stimulated by several divalent cations, without specificity. Thus, the most stable, active form of the purified NADH dehydrogenase is generated in the presence of diphosphatidylglycerol and spermidine.     

Expression of epidermal growth factor in the kidney and submandibular gland during mouse postnatal development

Earlier work has demonstrated that the salivary glands and kidneys are the major sites of epidermal growth factor (EGF) synthesis in adult mice. The precise timing of the onset of endogenous EGF synthesis in these tissues is not yet clear. In the present study we assessed the ontogenesis of EGF expression in the Swiss-Webster mouse. Paraformaldehyde-fixed frozen sections of neonatal kidneys and salivary glands were probed with proEGF cRNA labelled with 35S for in situ hybridization and with rabbit antisera to mouse EGF for immunocytochemistry. Both EGF mRNA and immunoreactivity were first detected in the developing distal nephron between days 3 and 5 postpartum. Juxtamedullary nephrons underlying the superficial nephrogenic zone were the first to express EGF. During the 2nd week after birth, EGF-expressing tubules became more abundant and distributed to medullary as well as cortical regions, corresponding to the thick ascending limb of Henle and distal convoluted tubule. Initial EGF mRNA and immunoreactivity in the submandibular gland were first detected between days 18 and 20 postpartum and increased notably during the following weeks.     

Enhanced potency of human Sonic hedgehog by hydrophobic modification

Post-translational modifications of the developmental signaling protein Sonic hedgehog (Shh) by a long-chain fatty acid at the N-terminus and cholesterol at the C-terminus greatly activate the protein in a cell-based signaling assay. To investigate the structural determinants of this activation phenomenon, hydrophobic and hydrophilic moieties have been introduced by chemical and mutagenic methods to the soluble N-terminal signaling domain of Shh and tested in both in vitro and in vivo assays. A wide variety of hydrophobic modifications increased the potency of Shh when added at the N-terminus of the protein, ranging from long-chain fatty acids to hydrophobic amino acids, with EC(50) values from 99 nM for the unmodified protein to 0.6 nM for the myristoylated form. The N-myristoylated Shh was as active as the natural form having both N- and C-terminal modifications. The degree of activation appears to correlate with the hydrophobicity of the modification rather than any specific chemical feature of the adduct; moreover, substitution with hydrophilic moieties decreased activity. Hydrophobic modifications at the C-terminus of Shh resulted in only a 2-3-fold increase in activity, and no activation was found with hydrophobic modification at other surface positions. The N-terminal modifications did not appear to alter the binding affinity of the Shh protein for the transfected receptor protein, Patched, and had no apparent effect on structure as measured by circular dichroism, thermal denaturation, and size determination. Activation of Desert Hh through modification of its N-terminus was also observed, suggesting that this is a common feature of Hh proteins.     

Multiphyletic origins of methylotrophy in Alphaproteobacteria,exemplified by comparative genomics of Lake Washington isolates

We sequenced the genomes of 19 methylotrophic isolates from Lake Washington, which belong to nine genera within eight families of the Alphaproteobacteria, two of the families being the newly proposed families. Comparative genomic analysis with a focus on methylotrophy metabolism classifies these strains into heterotrophic and obligately or facultatively autotrophic methylotrophs. The most persistent metabolic modules enabling methylotrophy within this group are the N‐methylglutamate pathway, the two types of methanol dehydrogenase (MxaFI and XoxF), the tetrahydromethanopterin pathway for formaldehyde oxidation, the serine cycle and the ethylmalonyl‐CoA pathway. At the same time, a great potential for metabolic flexibility within this group is uncovered, with different combinations of these modules present. Phylogenetic analysis of key methylotrophy functions reveals that the serine cycle must have evolved independently in at least four lineages of Alphaproteobacteria and that all methylotrophy modules seem to be prone to lateral transfers as well as deletions.     

The occurrence of the transposable elementpogo inDrosophila melanogaster

We examined the genomic occurrence of the transposable elementpogo in over 120 strains ofDrosophila melanogaster, from around the world and from different eras. All had multiple copies of a 2.1 kilobase (kb)pogo element, and multiple copies of several size classes between 1.0 and 1.8 kb. There were differences between strains in intensities or presences of deletion-derivative size classes, suggesting current or recent mobility in the species. We were unable to find anypogo-hybridization in eight other species in the genus, in three subgenera, or in the relatedScaptomyza pallida. Thepogo element may be a ‘middle-aged’ element in the genome ofD. melanogaster, having entered the species since its divergence from its sibling species, but long before theP andhobo elements.     

Rejoinder for discussion on “Horseshoe-based Bayesian nonparametric estimation of effective population size trajectories”

Phylodynamics is an area of population genetics that uses genetic sequence data to estimate past population dynamics. Modern state-of-the-art Bayesian nonparametric methods for recovering population size trajectories of unknown form use either change-point models or Gaussian process priors. Change-point models suffer from computational issues when the number of change-points is unknown and needs to be estimated. Gaussian process-based methods lack local adaptivity and cannot accurately recover trajectories that exhibit features such as abrupt changes in trend or varying levels of smoothness. We propose a novel, locally adaptive approach to Bayesian nonparametric phylodynamic inference that has the flexibility to accommodate a large class of functional behaviors. Local adaptivity results from modeling the log-transformed effective population size a priori as a horseshoe Markov random field, a recently proposed statistical model that blends together the best properties of the change-point and Gaussian process modeling paradigms. We use simulated data to assess model performance, and find that our proposed method results in reduced bias and increased precision when compared to contemporary methods. We also use our models to reconstruct past changes in genetic diversity of human hepatitis C virus in Egypt and to estimate population size changes of ancient and modern steppe bison. These analyses show that our new method captures features of the population size trajectories that were missed by the state-of-the-art methods.     

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

Background

Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture.

Results

Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins.

Conclusions

The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.

     

Involvement of Reactive Oxygen Species in a Feed-forward Mechanism of Na/K-ATPase-mediated Signaling Transduction

Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-l-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial ²²Na⁺ transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, ouabain stimulated direct carbonylation of two amino acid residues in the actuator domain of the Na/K-ATPase α1 subunit. Taken together, the data indicate that carbonylation modification of the Na/K-ATPase α1 subunit is involved in a feed-forward mechanism of regulation of ouabain-mediated renal proximal tubule Na/K-ATPase signal transduction and subsequent sodium transport.     

Hydrologic and Vegetative Removal of Cryptosporidium parvum,Giardia lamblia,and Toxoplasma gondii Surrogate Microspheres in Coastal Wetlands

Constructed wetland systems are used to reduce pollutants and pathogens in wastewater effluent, but comparatively little is known about pathogen transport through natural wetland habitats. Fecal protozoans, including Cryptosporidium parvum, Giardia lamblia, and Toxoplasma gondii, are waterborne pathogens of humans and animals, which are carried by surface waters from land-based sources into coastal waters. This study evaluated key factors of coastal wetlands for the reduction of protozoal parasites in surface waters using settling column and recirculating mesocosm tank experiments. Settling column experiments evaluated the effects of salinity, temperature, and water type (“pure” versus “environmental”) on the vertical settling velocities of C. parvum, G. lamblia, and T. gondii surrogates, with salinity and water type found to significantly affect settling of the parasites. The mesocosm tank experiments evaluated the effects of salinity, flow rate, and vegetation parameters on parasite and surrogate counts, with increased salinity and the presence of vegetation found to be significant factors for removal of parasites in a unidirectional transport wetland system. Overall, this study highlights the importance of water type, salinity, and vegetation parameters for pathogen transport within wetland systems, with implications for wetland management, restoration efforts, and coastal water quality.