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31.
32.
Cho YS Dobos KM Prenni J Yang H Hess A Rosenkrands I Andersen P Ryoo SW Bai GH Brennan MJ Izzo A Bielefeldt-Ohmann H Belisle JT 《Proteomics》2012,12(7):979-991
Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations. 相似文献
33.
D. James Morr V. Schirrmacher Peter Robinson Kristine Hess Werner W. Franke 《Experimental cell research》1979,119(2):265-275
Purified fractions of plasma membrane, Golgi apparatus, rough endoplasmic reticulum vesicles, nuclear envelope, and mitochondria were isolated from mouse liver and the distribution of H-2 histocompatibility antigens determined by indirect radioimmunoassay before and after membrane disruptive treatments. Fractions enriched in plasma membrane (surface membrane) revealed H-2 antigens in highest concentration; disruptive treatments were not necessary to reveal H-2 antigens with surface membranes. In contrast, internal membranes did not possess H-2 antigens which were accessible to antibody. Golgi apparatus fractions or some component of these fractions (e.g. secretory vesicles) possessed the antigens but in a latent form where accessibility was provided by simple rupture of the membrane vesicles. With endoplasmic reticulum, detergent solubilization of the membranes was required before H-2 antigen could be detected. Nuclear envelope preparations contained little or no demonstrable H-2 activity. These results were confirmed by several techniques including immunoprecipitation of labelled solubilized membrane components with anti-H-2 serum and subsequent analysis in SDS-PAGE. 相似文献
34.
F Z Stanczyk D L Hess P C Namkung J W Senner P H Petra M J Novy 《Biology of reproduction》1986,35(1):126-132
Temporal relationships between concentrations of sex steroid-binding protein (SBP), corticosteroid-binding globulin (CBG), total and free estradiol, total and free testosterone, cortisol, and progesterone were studied in plasma obtained at 1- to 3-day intervals throughout gestation in six rhesus macaques. Concentrations of SBP and CBG were measured by diethylaminoethyl cellulose filter assays. Total and free steroids were estimated by radioimmunoassay and ultrafiltration dialysis, respectively. We found that SBP was elevated between days 30 and 50 and CBG between days 60 and 140; both then declined until term (167 days). Estradiol increased gradually throughout gestation. Testosterone was elevated between days 10 and 40, then declined, and rose slightly in late gestation until approximately 15 days before delivery, when it increased markedly. Free estradiol and testosterone increased dramatically before parturition. Progesterone was elevated between days 25 and 45 and declined to relatively constant levels thereafter. Cortisol was essentially unchanged throughout gestation. Our data show that in the pregnant rhesus, levels of SBP and CBG vary independently of one another, but both decline before term; concentrations of both total and free estradiol and testosterone increase markedly before parturition; in late gestation, elevated estrogen is not associated with increased levels of SBP or CBG (as it is in human females). 相似文献
35.
Purification, characterization, and immunological properties for two isoforms of glutathione reductase from eastern white pine needles 总被引:1,自引:3,他引:1 下载免费PDF全文
Glutathione reductase (EC 1.6.4.2) was purified from Eastern white pine (Pinus strobus L.) needles. The purification steps included affinity chromatography using 2′, 5′-ADP-Sepharose, FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLC-gel filtration. Separation of proteins by FPLC-anion-exchange resulted in the recovery of two distinct isoforms of glutathione reductase (GRA and GRB). Purified GRA had a specific activity of 1.81 microkatals per milligram of protein and GRB had a specific activity of 6.08 microkatals per milligram of protein. GRA accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GRB accounted for 83%. The native molecular mass for GRA was 103 to 104 kilodaltons and for GRB was 88 to 95 kilodaltons. Both isoforms of glutathione reductase were dimers composed of identical subunit molecular masses which were 53 to 54 kilodaltons for GRA and 57 kilodaltons for GRB. The pH optimum for GRA was 7.25 to 7.75 and for GRB was 7.25. At 25°C the Km for GSSG was 15.3 and 39.8 micromolar for GRA and GRB, respectively. For NADPH, the Km was 3.7 and 8.8 micromolar for GRA and GRB, respectively. Antibody produced from purified GRB was reactive with both native and denatured GRB, but was cross-reactive with only native GRA. 相似文献
36.
Subunits of forming pollen exine and Ubisch bodies as seen in freeze substitutedLedebouria socialis Roth (Hyacinthaceae) 总被引:1,自引:0,他引:1
Summary High-pressure freezing/freeze substitution/TEM was employed to investigate anthers of the monocotyledonous angiospermLedebouria socialis Roth (Hyacinthaceae) during early tetrad stage. The initials of the outer sporopollenous pollen wall stratum (=sexine) and of the homologous tapetal products (=Ubisch bodies) are composed of highly regular subunits: clustered globules with a constant diameter of approximately 28 nm. The clusters develop within diffuse accumulations of electron-dense material. This process, interpreted as sporopollenin polymerization, does not necessarily depend on the presence of membrane-bound enzymes. Immunogold labeling with JIM 5 and JIM 7 antibodies revealed that the primexine as well as the dissolving tapetal cell walls, the sites of sexine and Ubisch body formation, respectively, contain un-esterified and methyl-esterified pectins.Abbreviations E-PTA
ethanolic phosphotungstic acid
- PA
periodic acid
- UA/Pb
uranyl acetate/lead 相似文献
37.
The activation of voltage-gated ion channels is controlled by the S4 helix, with arginines every third residue. The x-ray structures are believed to reflect an open-inactivated state, and models propose combinations of translation, rotation, and tilt to reach the resting state. Recently, experiments and simulations have independently observed occurrence of 310-helix in S4. This suggests S4 might make a transition from α- to 310-helix in the gating process. Here, we show 310-helix structure between Q1 and R3 in the S4 segment of a voltage sensor appears to facilitate the early stage of the motion toward a down state. We use multiple microsecond-steered molecular simulations to calculate the work required for translating S4 both as α-helix and transformed to 310-helix. The barrier appears to be caused by salt-bridge reformation simultaneous to R4 passing the F233 hydrophobic lock, and it is almost a factor-two lower with 310-helix. The latter facilitates translation because R2/R3 line up to face E183/E226, which reduces the requirement to rotate S4. This is also reflected in a lower root mean-square deviation distortion of the rest of the voltage sensor. This supports the 310 hypothesis, and could explain some of the differences between the open-inactivated- versus activated-states. 相似文献
38.
Gunewardene MS Subach FV Gould TJ Penoncello GP Gudheti MV Verkhusha VV Hess ST 《Biophysical journal》2011,(6):1019-1528
Localization-based superresolution optical imaging is rapidly gaining popularity, yet limited availability of genetically encoded photoactivatable fluorescent probes with distinct emission spectra impedes simultaneous visualization of multiple molecular species in living cells. We introduce PAmKate, a monomeric photoactivatable far-red fluorescent protein, which facilitates simultaneous imaging of three photoactivatable proteins in mammalian cells using fluorescence photoactivation localization microscopy (FPALM). Successful probe identification was achieved by measuring the fluorescence emission intensity in two distinct spectral channels spanning only ∼100 nm of the visible spectrum. Raft-, non-raft-, and cytoskeleton-associated proteins were simultaneously imaged in both live and fixed fibroblasts coexpressing Dendra2-hemagglutinin, PAmKate-transferrin receptor, and PAmCherry1-β-actin fusion constructs, revealing correlations between the membrane proteins and membrane-associated actin structures. 相似文献
39.
RP Tucker K Drabikowski JF Hess J Ferralli R Chiquet-Ehrismann JC Adams 《BMC evolutionary biology》2006,6(1):60-17
Background
Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes. 相似文献40.
Site-directed spin labeling and electron paramagnetic resonance were used to probe residues 281-304 of human vimentin, a region that has been predicted to be a non-alpha-helical linker and the beginning of coiled-coil domain 2B. Though no direct test of linker structure has ever been made, this region has been hypothesized to be flexible with the polypeptide chains looping away from one another. EPR analysis of spin-labeled mutants indicates that (a) several residues reside in close proximity, suggesting that adjacent linker regions in a dimer run in parallel, and that (b) the polypeptide backbone is relatively rigid and inflexible in this region. However, this region does not show the characteristics of a coiled-coil as has been identified elsewhere in the molecule. Within this region, spectra from positions 283 and 291 are unique from all others thus far examined. These positions, predicted to be in a noncoiled-coil structure, display a significantly stronger interaction than the a-d contact positions of coiled-coil regions. Analysis of the early stages of assembly by dialysis from 8 M urea and progressive thermal denaturation shows the close apposition and structural rigidity at residues 283 and 291 occurs very early in assembly and with a relatively sudden onset, well before coiled-coil formation in other parts of the molecule. These features are inconsistent with hypotheses that envision the linkers as flexible regions, or as looping away from one another, and raise the possibility that the linker may be the site at which dimer alignment and/or formation is initiated. Spin labels placed further downstream yield spectra suggesting that the first regular heptad of rod domain 2 begins at position 302. In conjunction with our previous characterization of region 305-336 and the solved structure of rod 2B from 328-405, the full extent of coiled-coil domain in rod 2B is now known, spanning from vimentin positions 302-405. 相似文献