RimM and RbfA Are Essential for Efficient Processing of 16S rRNA in <i>Escherichia coli</i>

ABSTRACT The trmD operon is located at 56.7 min on the genetic map of the Escherichia coli chromosome and contains the genes for ribosomal protein (r-protein) S16, a 21-kDa protein (RimM, formerly called 21K), the tRNA (m 1 G37)methyltransferase (TrmD), and r-protein L19, in that order. Previously, we have shown that strains from which the rimM gene has been deleted have a sevenfold-reduced growth rate and a reduced translational efficiency. The slow growth and translational deficiency were found to be partly suppressed by mutations in rpsM , which encodes r-protein S13. Further, the RimM protein was shown to have affinity for free ribosomal 30S subunits but not for 30S subunits in the 70S ribosomes. Here we have isolated several new suppressor mutations, most of which seem to be located close to or within the nusA operon at 68.9 min on the chromosome. For at least one of these mutations, increased expression of the ribosome binding factor RbfA is responsible for the suppression of the slow growth and translational deficiency of a Δ rimM mutant. Further, the RimM and RbfA proteins were found to be essential for efficient processing of 16S rRNA. </jats:p

Abstract 18975: Increased Arterial Blood Pressure and Vascular Fibrosis/remodeling in Mice Lacking Salt-inducible Kinase 1 (sik1)

Arterial fibrosis is one of the main underlying causes of vascular stiffness and is increasingly recognized as an important prognostic factor in the development of high blood pressure (BP), yet its physiophatology is not fully known. Therefore, elucidating the precise mechanism leading to arterial fibrosis will help improving current anti-hypertensive therapies. We previously described in human genetic studies a single nucleotide polymorphism within the salt-inducible kinase 1 (SIK1) gene, affecting the activity of the protein, which is associated with hypertension (lower SIK1 activity is associated with higher BP). Also, lower SIK1 expression induces the expression of transcription factors commonly involved in fibrosis. Therefore, we hypothesized that SIK1 can regulate BP by reducing vascular fibrosis and hence vascular stiffness. To test this, BP and aortic wall structure/composition was evaluated in sik1 -/- and sik1 +/+ mice challenged with a chronic high salt (HS) or normal salt diet (NS). The results showed that the s ik1 -/- mice challenged with HS developed high BP and presented signs of cardiac hypertrophy (increased left ventricle wall thickness and enhanced expression of hypertrophic genes) when compared to their counterparts on NS. On the contrary, BP and cardiac parameters remained unaffected in the sik1 +/+ mice under HS when compared to NS fed mice. Immunohistochemistry analysis revealed increased intima/media thickness and collagen deposition in the aorta of the sik1 -/- vs. sik1 +/+ mice under NS and before becoming hypertensive. Also, qPCR analysis of the aorta of the sik1 -/- mice showed increased expression of other extracellular matrix (ECM) components such as fibronectin and laminin-β1. In addition, the transient knock-down of the SIK1 gene in vascular smooth muscle cell (VSMC) cultures using siRNAs showed a similar increase in the expression of collagen as well as other ECM genes. The present study shows that SIK1 activity is necessary to prevent the development of hypertension, possibly through an anti-fibrotic mechanism regulating vascular wall structure and function. Therefore, SIK1 could become a novel therapeutic target in the treatment of hypertension and other vascular fibrosis-associated diseases

Comparative Analysis of One-Dimensional Protein Fingerprint by Line Shape Enhancement and Two-Dimensional ¹H,¹³C Methyl NMR Methods for Characterization of the Higher Order Structure of IgG1 Monoclonal Antibodies

The use of NMR spectroscopy has emerged as a premier tool to characterize the higher order structure of protein therapeutics and in particular IgG1 monoclonal antibodies (mAbs). Due to their large size, traditional 1H–15N correlation experiments have proven exceedingly difficult to implement on mAbs, and a number of alternative techniques have been proposed, including the one-dimensional (1D) 1H protein fingerprint by line shape enhancement (PROFILE) method and the two-dimensional (2D) 1H–13C methyl correlation-based approach. Both 1D and 2D approaches have relative strengths and weaknesses, related to the inherent sensitivity and resolution of the respective methods. To further increase the utility of NMR to the biopharmaceutical community, harmonized criteria for decision making in employing 1D and 2D approaches for mAb characterization are warranted. To this end, we have conducted an interlaboratory comparative study of the 1D PROFILE and 2D methyl methods on several mAbs samples to determine the degree to which each method is suited to detect spectral difference between the samples and the degree to which results from each correlate with one another. Results from the study demonstrate both methods provide statistical data highly comparable to one another and that each method is capable of complementing the limitations commonly associated with the other, thus providing a better overall picture of higher order structure

Gene Cluster of Rhodothermus marinus High-Potential Iron-Sulfur Protein:Oxygen Oxidoreductase, a caa(3)-Type Oxidase Belonging to the Superfamily of Heme-Copper Oxidases

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named rcoxA to rcoxD) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream of rcoxD. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the R. marinus enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E(278), Paracoccus denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the Cu(A) domain, that is characteristic of caa(3) oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this caa(3) oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the caa(3) class of heme-copper cytochrome c oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases

Intricate Role of Water in Proton Transport through Cytochrome c

Cytochrome c oxidase (CytcO), the final electron acceptor in the respiratory chain, catalyzes the reduction of O(2) to H(2)O while simultaneously pumping protons across the inner mitochondrial or bacterial membrane to maintain a transmembrane electrochemical gradient that drives, for example, ATP synthesis. In this work mutations that were predicted to alter proton translocation and enzyme activity in preliminary computational studies are characterized with extensive experimental and computational analysis. The mutations were introduced in the D pathway, one of two proton-uptake pathways, in CytcO from R. sphaeroides. Serine residues 200 and 201, which are hydrogen-bonded to crystallographically resolved water molecules half way up the D pathway, were replaced by more bulky hydrophobic residues (Ser200Ile, Ser200Val/Ser201Val and Ser200Val/Ser201Tyr) in order to query the effects of changing the local structure on enzyme activity as well as proton uptake, release and intermediate transitions. In addition, the effects of these mutations on internal proton transfer were investigated by blocking proton uptake at the pathway entrance (Asp132Asn replacement in addition to the above-mentioned mutations). Even though the overall activities of all mutant CytcOs were lowered, both the Ser200Ile and Ser200Val/Ser201Val variants maintained the ability to pump protons. The lowered activities were shown to be due to slowed oxidation kinetics during the P(R) → F and F → O transitions. Furthermore, the P(R) → F transition is shown to be essentially pH independent up to pH 12 (i.e. the apparent pK(a) of Glu286 is increased from 9.4. by at least 3 pK(a) units) in the S200V/S201V mutant. Explicit simulations of proton transport in the mutated enzymes revealed that the solvation dynamics can cause intriguing energetic consequences and hence provide mechanistic insights that would never be detected in static structures or simulations of the system with fixed protonation states (i.e., lacking explicit proton transport). The results are discussed in terms of the proton-pumping mechanism of CytcO

Study on stomach content of fish to update databases and analyse possible changes in diet or food web interactions

Fish stomach content is a compelling data source, as stomachs can provide information about diet of predators, distribution of prey fish, predator-prey preference or changes in diet over time. For instance, stomach samples have historically been used to inform the natural mortality of stock assessments in the Baltic and North Seas. Since stomach samples tend to vary substantially over time, due to differences in biomass of predator and prey, time series must be constructed to provide critical information on food web interactions in time and space. In this project, we aimed (1) to analyse new stomach samples, from the North and Baltic Seas; (2) to continue historical time series; (3) to update and create an online ICES database for the broader community to explore new and old data in conjunction; and (4) to perform preliminary analysis on the newly added data. In total, 10 087 new stomachs will be provided from the two case study areas. We analysed and uploaded 5 512 new stomach samples so far. Additionally, 27 744 historical Baltic cod stomach data have been uploaded. The analysis provided here show that the predation on saduria and sprat by cod in the Baltic Sea has changed over the last 30 years. In the North Sea, the new samples consisted primarily of unidentified matter, benthic food and crustaceans, whereas the historical samples contain a larger number of commercial fish

Macromolecular Crowding Fails To Fold a Globular Protein in Cells

Proteins perform their function in cells where macromolecular solutes reach concentrations of >300 g/L and occupy >30% of the volume. The volume excluded by these macromolecules will stabilize globular proteins because the native state occupies less space than the denatured state. Theory predicts that crowding can increase the ratio of folded to unfolded protein by a factor of 100, amounting to 3 kcal/mol of stabilization at room temperature. We tested the idea that volume exclusion dominates the crowding effect in cells with a variant of protein L, a 7-kDa globular protein with seven lysine residues replaced by glutamic acids. Eighty-four percent of the variant molecules populate the denatured state in dilute buffer at room temperature, compared to 0.1% for the wild-type protein. We then used in-cell nuclear magnetic resonance spectroscopy to show that the cytoplasm of Escherichia coli does not overcome even this modest (~1 kcal/mol) free energy deficit. The data are consistent with the idea that non-specific interactions between cytoplasmic components can overcome the excluded volume effect. Evidence for these interactions is provided by the observation that adding simple salts folds the variant in dilute solution, but increasing the salt concentration inside E. coli does not fold the protein. Our data are consistent with other studies of protein stability in cells, and suggest that stabilizing excluded volume effects, which must be present under crowded conditions, can be ameliorated by non-specific interactions between cytoplasmic components

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of −18%. There were no significant betweengroup differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo.</p