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1.
The results described in the accompanying article support the model in
which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the
cytoplasmic face of the ER, and functions as a glucosyl donor for three
Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the
lumenal compartment. In this study, the enzymatic synthesis and structural
characterization by NMR and electrospray-ionization tandem mass
spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing
2-4 isoprene units with either the cis - or trans - stereoconfiguration in
the beta-position are described. The water- soluble analogs were (1) used
to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol
glucosyltransferases (GlcTases) and (2) tested as potential substrates for
a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in
sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated
GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10,
Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c
)Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product
labeled in vitro. A preference was exhibited for C15-20 substrates
containing an internal cis -isoprene unit in the beta-position. In
addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the
lumenal compartment of sealed microsomal vesicles from rat liver and pig
brain via a protein-mediated transport system enriched in the ER. The
properties of the ER transport system have been characterized. Glc-
P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or
bovine erythrocytes. The results of these studies indicate that (1) the
internal cis -isoprene units are important for the utilization of Glc-P-Dol
as a glucosyl donor and (2) the transport of the water- soluble analog may
provide an experimental approach to assay the hypothetical "flippase"
proposed to mediate the transbilayer movement of Glc-P-Dol from the
cytoplasmic face of the ER to the lumenal monolayer.
相似文献
2.
3.
C. Lovatt Evans 《BMJ (Clinical research ed.)》1953,1(4825):1434-1435
4.
CJ von Ruhland 《Biotechnic & histochemistry》2013,88(7):478-484
Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue. 相似文献
5.
The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg?1 diet) or supplemented (220 mg kg?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values. 相似文献
6.
CJ Cooksey 《Biotechnic & histochemistry》2016,91(1):71-76
Rhodamines were first produced in the late 19th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed. 相似文献
7.
CJ Cooksey 《Biotechnic & histochemistry》2016,91(6):438-444
Malachite green was discovered independently by two researchers in Germany in the 19th century and found immediate employment as a dye and a pigment. Subsequently, other uses, such as staining biological specimens, emerged. A much later application was the control of fungal and protozoan infections in fish, for which the dye remains popular, although illegal in many countries owing to a variety of toxicity problems. In solution, malachite green can exist as five different species depending on the pH. The location of the positive charge of the colored cation on a carbon atom or a nitrogen atom is still debated. The original names of this dye, and their origins, are briefly surveyed. 相似文献
8.
Applications of quantitative PCR in the biosafety and genetic stability assessment of biotechnology products 总被引:6,自引:0,他引:6
Lovatt A 《Journal of biotechnology》2002,82(3):279-300
High throughput screening, increased accuracy and the coupling of real-time quantitative PCR (Q-PCR) to robotic set-up systems are beginning to revolutionise biotechnology. Applications of Q-PCR within biotechnology are discussed with particular emphasis on the following areas of biosafety and genetic stability testing: (a) determination of the biodistribution of gene therapy vectors in animals; (b) quantification of the residual DNA in final product therapeutics; (c) detection of viral and bacterial nucleic acid in contaminated cell banks and final products; (d) quantification of the level of virus removal in process validation viral clearance studies; (e) specific detection of retroviral RT activity in vaccines with high sensitivity; and (f) transgene copy number determination for monitoring genetic stability during production. Methods employed for Q-PCR assay validation as required in ICH Topic Q2A Validation of Analytical Methods: Definitions and Terminology (1st June 1995) are also reviewed. 相似文献
9.
Ben C Collins Ludovic CJ Gillet Lorenz C Blum Lin‐Yang Cheng Olga Vitek Jeppe Mouritsen Genevieve Lachance Tim D Spector Emmanouil T Dermitzakis Ruedi Aebersold 《Molecular systems biology》2015,11(2)
The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis‐SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood‐based biomarker studies. 相似文献
10.
Functional defects of SKAP-55-deficient T cells identify a regulatory role for the adaptor in LFA-1 adhesion 总被引:1,自引:0,他引:1
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The ADAP-SKAP-55 module regulates T-cell receptor (TCR)-induced integrin clustering and adhesion in T cells. However, it has been unclear whether ADAP and/or SKAP-55 is an effector of the response. ADAP controls SKAP-55 expression such that ADAP(-/-) T cells are also deficient in SKAP-55 expression. In this study, we report the phenotype of the SKAP-55-deficient mouse. SKAP-55(-/-) T cells retain ADAP expression yet show defects in beta1 and beta2 integrin adhesion, leukocyte function-associated antigen 1 (LFA-1) clustering, production of the cytokines interleukin-2 and gamma interferon, and proliferation. This dependency was also reflected in more-transient conjugation times in response to the superantigen staphylococcal enterotoxin A on dendritic cells and a reduced number of cells with TCR/CD3 microcluster localization at the immunological synapse. SKAP-55(-/-) T cells showed the same general impairment of function as ADAP(-/-) T cells, indicating that SKAP-55 is an effector of the ADAP-SKAP-55 module. At the same time, the requirement for ADAP and SKAP-55 was not absolute, since a subset of peripheral T cells adhered with loss of expression of either adaptor. Further, dependency on SKAP-55 or ADAP differed with the strength of the TCR signal. As with the ADAP(-/-) mouse, SKAP-55-deficient mice showed no major effects on lymphoid development or the appearance of peripheral T cells, B cells, and NK cells. Our findings identify a clear effector role for SKAP-55 in LFA-1 adhesion in peripheral T cells and demonstrate that dependency on SKAP-55 and ADAP differs among T cells and differs with the strength of the TCR signal. 相似文献