Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori.

Half the world's population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease produces NH(3) and CO(2), neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast <it>Pichia pastoris</it>.</p> <p>Results</p> <p>By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.</p> <p>Conclusions</p> <p>We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant <it>P. pastoris </it>clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of <it>P. pastoris </it>clones with high expression levels of aquaporins and other classes of membrane proteins.</p

HYPERFINE INTERACTIONS IN IRON BORATE FeBO3

For the FeBO3 single crystal, the values of isomer shift, quadrupole constant and magnetic hyperfine field were determined for over a wide temperature range.Исследования выполнены при финансовой поддержке РФФИ, проект № 19-29-12016-мк, в части мёссбауэровских исследований и теоретического анализа. Рентгеновские измерения проведены при поддержке Министерства науки и высшего образования РФ в рамках выполнения работ по Государственному заданию ФНИЦ «Кристаллография и фотоника» с использованием оборудования ЦКП (проект RFMEFI62119X0035)

EFFECT OF THE GAS MEDIA AND TEMPERATURE REGIMES OF ANNEALING ON THE PHASE COMPOSITION OF IRON BORATE FeBO3

Iron borate FeBO3 crystals are annealed in neutral, oxidizing, and reducing gas media on a specially designed setup. The effect of the chemical medium and annealing modes on the surface morphology and phase composition of the samples is established.Исследования выполнены при финансовой поддержке РФФИ, проект № 19-29-12016-мк, в части разработки экспериментальной установки и получения кристаллических образцов. Рентгеновские и электронно-микроскопические измерения проведены при под-держке Министерства науки и высшего образования РФ в рамках выполнения работ по Государственному заданию ФНИЦ «Кристаллография и фотоника» с использованием оборудования ЦКП (проект RFMEFI62119X0035)

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Iron-Doped Gallium Borate Crystals: Synthesis and ESR Study of Local Disorder

High quality FexGa1-xBO3 single crystals with 0 ≤ x ≤ 1 were synthesized and studied by electron magnetic resonance in the temperature range from 4 to 290 K. At low x, only the electron spin resonance (ESR) of diluted Fe3+ ions is present. Fitting to the experimental ESR spectra, assuming distributions of Fe3+ ligand coordinates, has shown the existence of local disorder in the crystals

Anisotropic energy gap of low-frequency AFMR mode in Fe x Ga 1-x BO 3 single crystals

International audienc

Iron Borate Based Monocrystals for Research in Magneto-Ordered State Physics

Elemental composition and crystal structure of synthesized monocrystal series FexGa1-xBO3, which is model object for fundamental research of magneto-ordered state physics, was investigated. It was established that the crystal lattice parameters weakly grow with magnetic ion concentration increase

Mutual orientation of electric intracrystalline and magnetic fields in iron borate single crystals

X-ray structural analysis, Mssbauer spectroscopy, and ab-initio calculations were used to study the structural properties and refine the orientation of intracrystalline fields in FeBO3 crystals in the region of the magnetic phase transition. It was found that in the temperature range of 293 - 403 K, the trigonal lattice parameters increase monotonically. Analysis of the electron density distribution maps did not show visible local disordering over the entire investigated temperature range. It was found that iron borate has an axially symmetric electric field gradient (EFG), whose main axis is directed along [001]. This orientation is maintained above and below the Neel point. In magnetically ordered state of the crystal, the main axis of the EFG is orthogonal to the direction of the hyperfine magnetic field at iron nuclei. The results obtained will be used to develop a theoretical model of the formation of hyperfine structure in iron borate, which is important for applications of such crystals in the synchrotron technologies of a new generation