NMR studies of membrane proteins

Nuclear magnetic resonance studies of membrane proteins yield valuable insights into their structure and topology. For example, the tilt angle and rotation of the helices in an ion channel can be determined by solid-state NMR spectroscopy in aligned lipid bilayers. Details about the structure of the protein in aligned phospholipids environments are immediately apparent from inspection of the SAMMY spectrum and the data can be further used for the determination of atomic resolution three-dimensional structures. SAR by NMR is a technique that is well suited for the field of membrane transporter proteins. The experiments on protein/phospholipid samples provide a unique insight into the interaction of drugs and the functional proteins.The advances required to transform solid-state NMR from a spectroscopic technique to a generally applicable method for determining molecular structures included multiple-pulse sequences, double-resonance methods, and separated local field spectroscopy. It also required improvements in instrumentation, especially the use of high-field magnets and efficient probes capable of high-power radio-frequency irradiations at high frequencies. The pace of development is accelerating and the local field is being utilized in an increasing number of ways in spectroscopic investigations of molecular structure and dynamics. Applications to many helical membrane proteins are underway and promise to add to our understanding of membrane proteins in health and disease.Chemistr

Expression and isolation of the membrane proteoglycan syndecan-1 from E. coli

Syndecan-1 is a membrane proteoglycan, which is a protein with glycosaminoglycans, or long carbohydrate chains, attached to its extracellular domain. The syndecan family is composed of various proteoglycans that each aid in cell-to-cell communication, and cell signaling pathways. Syndecan-1 plays a role in the initiation and progression of many different types of cancer. The overall goal of our research is to express and isolate the human syndecan-1 protein in bacterial cells, and then proceed with glycosylation of the protein. In order to have effective expression and cleavage of syndecan-1, a His6-TrpΔLE fusion partner was attached to the syndecan-1 protein, and the sequence of syndecan-1 was altered to replace any cysteine and methionine residues with serine residues. The path to achieving this involves a growth of cells with emphasis on the expression of syndecan-1, a chemical cleavage of the TrpΔLE fusion partner from the syndecan-1 protein, purification by size-exclusion chromatography, and each step monitored by SDS-PAGE to show expression and purity. The syndecan-1 protein in its altered form weighs 31.903kDa, and the TrpΔLE weighs approximately 14kDa. We found that we were successful in cleavage and expression via SDS-PAGE, and got separation of protein through size-exclusion chromatography, but have not expressed a large amount of protein from growths, nor have we gotten proof of our protein expression by mass spectrometry. In the future, we plan on making slight adjustments in protocol and doing more growths to create more protein to work with, and find a reason that we are unable to identify the protein by mass spectrometry.Lew Wentz FoundationChemistr

Physio-Mechanical Properties of a New Zinc-Reinforced Glass Ionomer Restorative Material

poster abstractObjective: Zinc-reinforced glass ionomer restorative material (ZRGIC) has been proposed as an improved restorative material. The study compared the mechanical properties of a ZRGIC restorative material (ChemFil Rock, (Dentsply)), with three commercially available glass ionomers (GICs); Fuji IX GP Extra (GC America), Ketac Molar (3M ESPE) and EQUIA Fil (GC America). A resin composite, Premise (Kerr), was included as a control group. Methods: Fracture toughness (KIC) testing was done according to ISO 13586, using single edge notchedbeam specimens (n=10), loaded until failure in a three-point bending test device. Specimens (n=9) for the hardness, roughness and abrasive wear testing were made by mixing and inserting the restorative materials into individual stainless steel molds followed by flattening and polishing. Knoop microhardness (KHN) was performed (25g, 30s),on pre-determined areas of the polished surfaces. For toothbrushing wear resistance and roughness, specimens were brushed in an automated brushing machine (200g) with a suspension of dentifrice and water (1:1w/v) for 20,000 strokes. Specimen surfaces were scanned in an optical profilometer before and after brushing to obtain surface roughness (Ra) and mean height (surface) loss using image subtraction and dedicated software. Data were analyzed using Wilcoxon Rank Sum tests (α=0.05)

Fine Mapping of the 1p36 Deletion Syndrome Identifies Mutation of PRDM16 as a Cause of Cardiomyopathy

Deletion 1p36 syndrome is recognized as the most common terminal deletion syndrome. Here, we describe the loss of a gene within the deletion that is responsible for the cardiomyopathy associated with monosomy 1p36, and we confirm its role in nonsyndromic left ventricular noncompaction cardiomyopathy (LVNC) and dilated cardiomyopathy (DCM). With our own data and publically available data from array comparative genomic hybridization (aCGH), we identified a minimal deletion for the cardiomyopathy associated with 1p36del syndrome that included only the terminal 14 exons of the transcription factor PRDM16 (PR domain containing 16), a gene that had previously been shown to direct brown fat determination and differentiation. Resequencing of PRDM16 in a cohort of 75 nonsyndromic individuals with LVNC detected three mutations, including one truncation mutant, one frameshift null mutation, and a single missense mutant. In addition, in a series of cardiac biopsies from 131 individuals with DCM, we found 5 individuals with 4 previously unreported nonsynonymous variants in the coding region of PRDM16. None of the PRDM16 mutations identified were observed in more than 6,400 controls. PRDM16 has not previously been associated with cardiac disease but is localized in the nuclei of cardiomyocytes throughout murine and human development and in the adult heart. Modeling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish resulted in both contractile dysfunction and partial uncoupling of cardiomyocytes and also revealed evidence of impaired cardiomyocyte proliferative capacity. In conclusion, mutation of PRDM16 causes the cardiomyopathy in 1p36 deletion syndrome as well as a proportion of nonsyndromic LVNC and DCM

Bose-Einstein correlations of same-sign charged pions in the forward region in pp collisions at √s=7 TeV

Bose-Einstein correlations of same-sign charged pions, produced in protonproton collisions at a 7 TeV centre-of-mass energy, are studied using a data sample collected by the LHCb experiment. The signature for Bose-Einstein correlations is observed in the form of an enhancement of pairs of like-sign charged pions with small four-momentum difference squared. The charged-particle multiplicity dependence of the Bose-Einstein correlation parameters describing the correlation strength and the size of the emitting source is investigated, determining both the correlation radius and the chaoticity parameter. The measured correlation radius is found to increase as a function of increasing charged-particle multiplicity, while the chaoticity parameter is seen to decreas

Effect Modification of the Association between Short-term Meteorological Factors and Mortality by Urban Heat Islands in Hong Kong

Background Prior studies from around the world have indicated that very high temperatures tend to increase summertime mortality. However possible effect modification by urban micro heat islands has only been examined by a few studies in North America and Europe. This study examined whether daily mortality in micro heat island areas of Hong Kong was more sensitive to short term changes in meteorological conditions than in other areas. Method An urban heat island index (UHII) was calculated for each of Hong Kong’s 248 geographical tertiary planning units (TPU). Daily counts of all natural deaths among Hong Kong residents were stratified according to whether the place of residence of the decedent was in a TPU with high (above the median) or low UHII. Poisson Generalized Additive Models (GAMs) were used to estimate the association between meteorological variables and mortality while adjusting for trend, seasonality, pollutants and flu epidemics. Analyses were restricted to the hot season (June-September). Results Mean temperatures (lags 0–4) above 29°C and low mean wind speeds (lags 0–4) were significantly associated with higher daily mortality and these associations were stronger in areas with high UHII. A 1°C rise above 29°C was associated with a 4.1% (95% confidence interval (CI): 0.7%, 7.6%) increase in natural mortality in areas with high UHII but only a 0.7% (95% CI: −2.4%, 3.9%) increase in low UHII areas. Lower mean wind speeds (5th percentile vs. 95th percentile) were associated with a 5.7% (95% CI: 2.7, 8.9) mortality increase in high UHII areas vs. a −0.3% (95% CI: −3.2%, 2.6%) change in low UHII areas. Conclusion The results suggest that urban micro heat islands exacerbate the negative health consequences of high temperatures and low wind speeds. Urban planning measures designed to mitigate heat island effects may lessen the health effects of unfavorable summertime meteorological conditions

Broadband Dielectric Spectroscopy on Human Blood

Dielectric spectra of human blood reveal a rich variety of dynamic processes. Achieving a better characterization and understanding of these processes not only is of academic interest but also of high relevance for medical applications as, e.g., the determination of absorption rates of electromagnetic radiation by the human body. The dielectric properties of human blood are studied using broadband dielectric spectroscopy, systematically investigating the dependence on temperature and hematocrit value. By covering a frequency range from 1 Hz to 40 GHz, information on all the typical dispersion regions of biological matter is obtained. We find no evidence for a low-frequency relaxation (alpha-relaxation) caused, e.g., by counterion diffusion effects as reported for some types of biological matter. The analysis of a strong Maxwell-Wagner relaxation arising from the polarization of the cell membranes in the 1-100 MHz region (beta-relaxation) allows for the test of model predictions and the determination of various intrinsic cell properties. In the microwave region beyond 1 GHz, the reorientational motion of water molecules in the blood plasma leads to another relaxation feature (gamma-relaxation). Between beta- and gamma-relaxation, significant dispersion is observed, which, however, can be explained by a superposition of these relaxation processes and is not due to an additional delta-relaxation often found in biological matter. Our measurements provide dielectric data on human blood of so far unsurpassed precision for a broad parameter range. All data are provided in electronic form to serve as basis for the calculation of the absorption rate of electromagnetic radiation and other medical purposes. Moreover, by investigating an exceptionally broad frequency range, valuable new information on the dynamic processes in blood is obtained.Comment: 17 pages, 9 figure

Diversification of importin-α isoforms in cellular trafficking and disease states.

The human genome encodes seven isoforms of importin α which are grouped into three subfamilies known as α1, α2 and α3. All isoforms share a fundamentally conserved architecture that consists of an N-terminal, autoinhibitory, importin-β-binding (IBB) domain and a C-terminal Arm (Armadillo)-core that associates with nuclear localization signal (NLS) cargoes. Despite striking similarity in amino acid sequence and 3D structure, importin-α isoforms display remarkable substrate specificity in vivo. In the present review, we look at key differences among importin-α isoforms and provide a comprehensive inventory of known viral and cellular cargoes that have been shown to associate preferentially with specific isoforms. We illustrate how the diversification of the adaptor importin α into seven isoforms expands the dynamic range and regulatory control of nucleocytoplasmic transport, offering unexpected opportunities for pharmacological intervention. The emerging view of importin α is that of a key signalling molecule, with isoforms that confer preferential nuclear entry and spatiotemporal specificity on viral and cellular cargoes directly linked to human diseases

Dynamical masses of a nova-like variable on the edge of the period gap

We present the first dynamical determination of the binary parameters of an eclipsing SW Sextantis star in the 3–4 h orbital period range during a low state. We obtained time-resolved optical spectroscopy and photometry of HS 0220+0603 during its 2004–2005 low-brightness state, as revealed in the combined Small & Moderate Aperture Research Telescope System, IAC80 and M1 Group long-term optical light curve. The optical spectra taken during primary eclipse reveal a secondary star spectral type of M5.5 ± 0.5 as derived from molecular band-head indices. The spectra also provide the first detection of a DAB white dwarf in a cataclysmic variable. By modelling its optical spectrum we estimate a white dwarf temperature of 30 000 ± 5000 K. By combining the results of modelling the white dwarf eclipse from ULTRACAM light curves with those obtained by simultaneously fitting the emission- and absorption-line radial velocity curves and I-band ellipsoidal light curves, we measure the stellar masses to be M1 = 0.87 ± 0.09 M⊙ and M2 = 0.47 ± 0.05 M⊙ for the white dwarf and the M dwarf, respectively, and an inclination of the orbital plane of i ≈ 79°. A radius of 0.0103 ± 0.0007 R⊙ is obtained for the white dwarf. The secondary star in HS 0220+0603 is likely too cool and undersized for its mass