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231.
Florell SR Schmidt SJ Porter-Gill P Albertine KH Murphy KJ McKinney CB Boucher KM Grossman D Biddle DL Clayton F Layfield LJ Leachman SA 《Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society》2003,16(6):662-669
Confirming melanocytic lineage and purity is important for experiments using cultured human melanocytes. The objective of this study was to develop a simple, reliable method to evaluate and archive cultured melanocytic cells. Melanocytes were isolated from adult skin biopsies or from neonatal foreskins using standard culturing methods. Fibrin cell blocks (FCBs) were prepared from cultured cells at passages two and six. Fibrin blocks were paraffin-embedded and sectioned for immunohistochemical (CD68, Melan-A, and HMB-45) and H & E staining. Flow cytometry was performed (Melan-A) at passage six. A mixing experiment with cultured melanocytes and fibroblasts was performed and cell population purity was determined by manual counts of positively staining cells in the FCBs and by flow cytometry. The FCB method of evaluating population purity was validated experimentally and by correlation with flow cytometry results. Preparation of a FCB followed by immunohistochemical staining is an easy and inexpensive way to confirm melanocytic lineage, estimate population purity, and provide a permanent archive of cultured cells. 相似文献
232.
The Kupffer cell receptor for glycoproteins has been reported to have a role in clearance of galactose- and fucose-terminated glycoproteins from circulation. Although the gene and a cDNA encoding the receptor have been described, there has been little study of the receptor protein. To address some questions about possible ligands and functions for this receptor, fragments representing portions of the extracellular domain have been expressed and characterized. The extracellular domain consists of a trimer stabilized by an extended coiled-coil of alpha-helices. The receptor displays monosaccharide-binding characteristics similar to the hepatic asialoglycoprotein receptor, but with somewhat less selectivity. The two best monosaccharide ligands are GalNAc and galactose. alpha-Methyl fucoside is a particularly poor ligand. Analysis of Kupffer cell receptor binding to glycoproteins and oligosaccharides released from them reveals highest affinity for desialylated, complex N-linked glycans. The best glycoprotein ligands contain multiple highly branched oligosaccharides. A human ortholog of the rat receptor gene does not encode a full-length protein and is not expressed in liver. These characteristics suggest that the receptor may have functions parallel to those of the hepatocyte asialoglycoprotein receptor in some (but not all) mammalian species. 相似文献
233.
Kai Iding Dirk Lütkemeyer Elisabeth Fraune Kurt Gerlach Jürgen Lehmann 《Cytotechnology》2000,34(1-2):141-150
Since 1969 much attention has been devoted to the useof spinfilter systems for retention of mammalian cellsin continuous perfusion cultivations. Previousinvestigations dealt with hydrodynamic conditions,fouling processes and upscaling. But hydrodynamicconditions and fouling processes seem to have asecondary importance in spinfilter performance duringauthentic perfusion cultivations. Obviously,alterations in culture condition are more relevantespecially during long-term processes. Therefore, ourpratical approach focussed on the performance qualityof a commercially available 20 m spinfilterduring a perfusion cultivation of a recombinant CHOcell line in pilot scale regarding the followingissues: 1) retention of viable cells in thebioreactor; 2) removal of dead cells and cell debrisfrom the bioreactor; 3) alterations in culturecondition; and 4) changes in perfusion mode.Furthermore, we tested the performance of 20 mspinfilters in 2 and 100 l pilot scale using solidmodel particles instead of cells. Our investigationsshowed that retention of viable cells in pilot scalewas independent of spinfilter rotation velocity andperfusion rate; the retention increased from 75 to 95%corresponding to operation time, enlarging celldiameter and enhanced formation of aggregates in theculture during the perfusion cultivation. By means ofthe Cell Counter and Analyzer System (CASY) anoperation cut off of 13 m was determined forthis spinfilter. Using solid model particles in 2 lscale, optimal retention was achieved at a tip speedof 0.43 m s-1 (141 rpm) – furtherenhancement of spinfilter rotation velocity up to0.56 m s-1 (185 rpm) decreased the retentionrapidly. In pilot scale best retention performance wasobtained with tip speeds of 0.37 m s-1(35 rpm) and 1.26 m s-1 (120 rpm). Hence,significant retention in pilot scale could already beachieved with low agitation. Therefore, the additionof shear force protectives could be avoided so thatthe purification of the target protein from thesupernatant would be facilitated. 相似文献
234.
Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium 总被引:2,自引:2,他引:0
In this paper it is demonstrated that cross-correlated time modulation of isotropic chemical shifts (`conformational exchange') leads to differential relaxation of double- and zero-quantum coherences, respectively. Quantitative information can be obtained from the time dependence of the interconversion between the two two-spin coherences 2IxSx and 2IySy, induced by the differential relaxation. The effect is illustrated with an application to 13C,15N-labeled quail CRP2(LIM2), by studying 15N-1HN multiple-quantum relaxation. Significant cross-correlated fluctuations of isotropic chemical shifts were observed for residues which are part of a disordered loop region connecting two -strands in CRP2(LIM2). Differential 1HN and 15N exchange contributions to multiple-quantum relaxation observed at these sites illustrate the complex interplay between hydrogen bonding events and conformational reorientations in proteins. 相似文献
235.
This paper describes a [15N,1H]/[13C,1H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide 15N–1H groups, side chain 15N–1H2 groups and aromatic 13C–1H groups in otherwise highly deuterated proteins. The 15N–1H and 13C–1H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the 1H dimension. The side-chain 15N–1H2 groups, which are suppressed in conventional [15N,1H)-TROSY, are observed with high sensitivity in the 15N–1H subspectrum. [15N,1H]/[13C,1H]-TROSY was used as the heteronuclear correlation block in a 3D [1H,1H]-NOESY-[15N,1H]/[13C,1H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain 15N–1H2 groups. 相似文献
236.
Seasonal changes in sediment phosphorus forms in relation to sedimentation and benthic bacterial biomass in Lake Erken 总被引:2,自引:0,他引:2
Surficial sediment and sedimenting material were sampled during spring and summer 1991 in Lake Erken. Sediment was analyzed for redox potential, P concentrations and bacterial biomass. Sedimentation and chlorophyll a concentrations of sedimenting matter were determined. Additionally, different phosphorus forms in surficial sediment were quantified using sequential fractionation. The resulting dataset was used to study the effects of sedimentation events following phytoplankton blooms and benthic bacterial biomass on the size of the various phosphorus pools in the sediment.Sedimentation of spring diatoms caused a rapid increase in the NH4Cl- and NaOH-extractable P (NH4Cl–P and NaOH–rP) in the sediment. During sedimentation, NaOH–rP and NH4Cl–P increased within 3 days from 422 ± 17 g g–1 DW to 537 ± 8.0 g g–1 DW and from 113 ± 13 g g–1 DW to 186 ± 26 g g–1 DW, respectively. The NaOH–nrP (non-reactive P) fraction made up about 17% of Tot-P in sediment samples, whereas NaOH–rP and HCl–P made up 25% each. All P forms showed considerable seasonal variation. Significant relationships were found between bacterial biomass and the NaOH–nrP and NH4Cl–P fractions in the sediment, respectively. Also regressions of NaOH–nrP and NH4Cl–P versus the chlorophyll a concentration of sedimenting matter were highly significant. These regressions lend support to the conjecture that NaOH–nrP is a conservative measure of bacterial poly-P. 相似文献
237.
Benzophenone glycosides from Gnidia involucrata 总被引:2,自引:0,他引:2
Ferrari J Terreaux C Sahpaz S Msonthi JD Wolfender JL Hostettmann K 《Phytochemistry》2000,54(8):883-889
Six compounds have been isolated from the methanol extract of the aerial parts of Gnidia involucrata (Thymelaeaceae). They were identified as 2,3,4',5,6-pentahydroxybenzophenone-4-C-glucoside and 2,4',6-trihydroxy-4-methoxybenzophenone-2-O-glucoside, together with mangiferin, kaempferol-3-O-glucoside, yuankanin and manniflavanone by chemical and spectroscopic means. The structures of three additional C-glycosyl flavones--vitexin, isovitexin and isoorientin--were determined on-line by LC/UV/APCI-MSn analysis of the crude extract. 相似文献
238.
Steffen Dietzel Roland Eils Kurt Sätzler Harald Bornfleth Anna Jauch Christoph Cremer Thomas Cremer 《Experimental cell research》1998,240(2):187
Multicolor fluorescencein situhybridization with a whole chromosome composite probe for the X-chromosome and microdissection probes for the Xp and Xq arms, as well as for the Xp terminal, Xq terminal, and X centromer specific subregional probes, was applied to three-dimensional (3D) preserved human female amniotic fluid cell nuclei. Confocal laser scanning microscopy and three-dimensional image analysis demonstrated distinctly separated Xp arm and Xq arm domains. 3D distance measurements revealed a high variability of intrachromosomal distances between Xpter, Xcen, and Xqter specific probes within both X territories. A 3D distance measurement error of ±70 nm was found in control experiments using quartz glass microspheres labeled with different fluorochromes. Our data argue against the hypothesis of Walkeret al.(1991,Proc. Natl. Acad. Sci. USA88, 6191–6195) that a looped structure of the inactive X territory is formed by tight telomere–telomere associations. 相似文献
239.
240.