Transverse NMR relaxation as a probe of mesoscopic structure

Transverse NMR relaxation in a macroscopic sample is shown to be extremely sensitive to the structure of mesoscopic magnetic susceptibility variations. Such a sensitivity is proposed as a novel kind of contrast in the NMR measurements. For suspensions of arbitrary shaped paramagnetic objects, the transverse relaxation is found in the case of a small dephasing effect of an individual object. Strong relaxation rate dependence on the objects' shape agrees with experiments on whole blood. Demonstrated structure sensitivity is a generic effect that arises in NMR relaxation in porous media, biological systems, as well as in kinetics of diffusion limited reactions.Comment: 4 pages, 3 figure

Bioactivity in silica/poly(γ-glutamic acid) sol–gel hybrids through calcium chelation

Bioactive glasses and inorganic/organic hybrids have great potential as biomedical implant materials. Sol–gel hybrids with interpenetrating networks of silica and biodegradable polymers can combine the bioactive properties of a glass with the toughness of a polymer. However, traditional calcium sources such as calcium nitrate and calcium chloride are unsuitable for hybrids. In this study calcium was incorporated by chelation to the polymer component. The calcium salt form of poly(γ-glutamic acid) (γCaPGA) was synthesized for use as both a calcium source and as the biodegradable toughening component of the hybrids. Hybrids of 40 wt.% γCaPGA were successfully formed and had fine scale integration of Ca and Si ions, according to secondary ion mass spectrometry imaging, indicating a homogeneous distribution of organic and inorganic components. 29Si magic angle spinning nuclear magnetic resonance data demonstrated that the network connectivity was unaltered with changing polymer molecular weight, as there was no perturbation to the overall Si speciation and silica network formation. Upon immersion in simulated body fluid a hydroxycarbonate apatite surface layer formed on the hybrids within 1 week. The polymer molecular weight (Mw 30–120 kDa) affected the mechanical properties of the resulting hybrids, but all hybrids had large strains to failure, >26%, and compressive strengths, in excess of 300 MPa. The large strain to failure values showed that γCaPGA hybrids exhibited non-brittle behaviour whilst also incorporating calcium. Thus calcium incorporation by chelation to the polymer component is justified as a novel approach in hybrids for biomedical materials

A QUANTITATIVE MAPPING OF ALKALINE PHOSPHATASE IN THE BRAIN OF THE RHESUS MONKEY *

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65477/1/j.1471-4159.1966.tb07513.x.pd

THE IMPACT OF GEL ELECTROPHORESIS UPON OUR UNDERSTANDING OF THE ESTERASES *

The impact of gel electrophoresis upon our understanding of the esterases has been formidable in that it has provided us with new insights and understanding concerning the number and biochemical characteristics of the many esterase-active proteins found in biological material. The relationships between the esterases within a species and among species still remains largely to be determined. With regard to the function of esterases it is to be expected that there will be several. One promising possibility is suggested by the work of Allen and Hunter, which illustrated a dependent relationship between male sex hormone and the esterases in the mouse epididymis. Supporting this work is the observation by Shaw and Koen (1963) demonstrating the presence of an esterase in the mouse kidney, which also was dependent on male sex hormone. The change observed in the serum esterase of the pregnant rabbit reported here and in women by Friedman and Lapman (1961) may also relate to hormone changes associated with pregnancy, although this relationship remains to be demonstrated. A second area where the esterases are likely to be functioning is in relation to protein synthetic activity of the endoplasmic reticulum. The only evidence supporting this suggestion is the abundant presence of esterases found in this location. The seven experiments described and discussed here along with those included in the references may serve as illustrations of the kind of work that can be accomplished by the use of these methods.*Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75632/1/j.1749-6632.1964.tb14224.x.pd

Garlic Accelerates Red Blood Cell Turnover and Splenic Erythropoietic Gene Expression in Mice: Evidence for Erythropoietin-Independent Erythropoiesis

Garlic (Allium sativum) has been valued in many cultures both for its health effects and as a culinary flavor enhancer. Garlic's chemical complexity is widely thought to be the source of its many health benefits, which include, but are not limited to, anti-platelet, procirculatory, anti-inflammatory, anti-apoptotic, neuro-protective, and anti-cancer effects. While a growing body of scientific evidence strongly upholds the herb's broad and potent capacity to influence health, the common mechanisms underlying these diverse effects remain disjointed and relatively poorly understood. We adopted a phenotype-driven approach to investigate the effects of garlic in a mouse model. We examined RBC indices and morphologies, spleen histochemistry, RBC half-lives and gene expression profiles, followed up by qPCR and immunoblot validation. The RBCs of garlic-fed mice register shorter half-lives than the control. But they have normal blood chemistry and RBC indices. Their spleens manifest increased heme oxygenase 1, higher levels of iron and bilirubin, and presumably higher CO, a pleiotropic gasotransmitter. Heat shock genes and those critical for erythropoiesis are elevated in spleens but not in bone marrow. The garlic-fed mice have lower plasma erythropoietin than the controls, however. Chronic exposure to CO of mice on garlic-free diet was sufficient to cause increased RBC indices but again with a lower plasma erythropoietin level than air-treated controls. Furthermore, dietary garlic supplementation and CO treatment showed additive effects on reducing plasma erythropoietin levels in mice. Thus, garlic consumption not only causes increased energy demand from the faster RBC turnover but also increases the production of CO, which in turn stimulates splenic erythropoiesis by an erythropoietin-independent mechanism, thus completing the sequence of feedback regulation for RBC metabolism. Being a pleiotropic gasotransmitter, CO may be a second messenger for garlic's other physiological effects

Analysis of the Promoters Involved in Enterocin AS-48 Expression

The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene) and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2) and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2) promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2) promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.This work was supported in part by the Ministerio de Ciencia e Innovación project BIO2008-01708, the Plan Propio from the University of Granada (Spain) and by the Research Plan Group (BIO 160)

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

Extent: 10p.INTRODUCTION: TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro. METHODS: TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry. RESULTS: TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts. CONCLUSIONS: The expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.Anak A. S. S. K. Dharmapatni, Malcolm D. Smith, Tania N. Crotti, Christopher A. Holding, Cristina Vincent, Helen M. Weedon, Andrew C. W. Zannettino, Timothy S. Zheng, David M. Findlay, Gerald J. Atkins and David R. Hayne

A method for detergent-free isolation of membrane proteins in their local lipid environment.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins