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101.
Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5′-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg2+-ATPase, (Na+ + K+)-ATPase, γ-glutamyl transpeptidase, phosphodiesterase I, akaline phosphatase and xanthine oxidase were also high (12–18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide compositions in both light and heavy membranes were similar upon SDS-polyacrylamide gel electrophoresis. 相似文献
102.
Kiyoshi Takahashi Katsuya Miyatani Hiroyuki Yanai Ho Jong Jeon Kotaro Fujiwara Tadashi Yoshino Kazuhiko Hayashi Tadaatsu Akagi Ken Tsutsui Koichi Mizobuchi 《Virchows Archiv. B, Cell pathology including molecular pathology》1992,62(1):105-113
Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating
various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary
phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features.
Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed
for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became
intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly
down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme
and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell
function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned
medium significantly down-regulated the expression of CD14 antigen.
Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human
cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic
lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is
necessary for the differentiation and maturation of IDC. 相似文献
103.
The levels of free amino acids in the cerebral cortex of acute and chronic uremic rats were examined. Amino acids significantly elevated were aspartate, glutamine, glycine, histidine, ornithine, phenylalanine, phosphoethanolamine and taurine, whereas 1-methyl histidine and 3-methyl histidine were specifically detected in uremic rats. Glutamate, arginine and carnosine disclosed a significant reduction. There was no change in the concentrations of γ-aminobutyrate and alanine. The above findings were essentially identical in both acute and chronic uremia. It was conjectured that these changes of amino acid levels in the brain might participate in the progress of uremic encephalopathy. 相似文献
104.
Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H2O2. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD. 相似文献
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107.
Takashi Kobayashi Hiroko Habuchi Ken Nogami Satoko Ashikari‐Hada Koji Tamura Hiroyuki Ide Koji Kimata 《Development, growth & differentiation》2010,52(2):146-156
Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O‐sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O‐sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS‐6‐O‐sulfotransferases (HS6ST‐1 and HS6ST‐2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf‐8 and Fgf‐10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST‐2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf‐8 and Fgf‐10 expressions than that achieved with HS6ST‐1 RNAi. HS6ST‐1 RNAi and HS6ST‐2 RNAi caused a significant but distinct reduction in the levels of different 6‐O‐sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6‐O‐sulfation of HS play essential roles in chick limb bud development. 相似文献
108.
Circadian rhythm is a self-sustaining oscillation whose period length coincides with the 24-hour day-night cycle. A powerful tool for circadian clock research is the real-time automated bioluminescence monitoring system in which a promoter region of a clock-controlled gene is fused to a luciferase reporter gene and rhythmic regulation of the promoter activity is monitored as bioluminescence. In the present study, we greatly improved the bioluminescence reporter system in the cyanobacterium Synechocystis sp. strain PCC 6803. We fused an 805-bp promoter region of the dnaK gene seamlessly to the luxA coding sequence and integrated the P(dnaK)::luxAB fusion gene into a specific intergenic region of the Synechocystis genome (targeting site 1). The resulting new reporter strain, PdnaK::luxAB(-), showed 12 times the bioluminescence intensity of the standard reporter strain, CFC2. Furthermore, we generated strain PdnaK::luxAB(+), in which the P(dnaK)::luxAB fusion gene and the selection-marker spectinomycin resistance gene are transcribed in opposite directions. The PdnaK::luxAB(+) strain showed 19 times the bioluminescence intensity of strain CFC2. The procedures used to increase the bioluminescence intensity are especially useful for bioluminescence monitoring of genes with low promoter activity. In addition, these reporter constructs facilitate bioluminescence monitoring of any gene because the promoter fragments they contain can easily be replaced by digestion with unique restriction enzymes. They would therefore contribute to a genome-wide analysis of gene expression in Synechocystis. 相似文献
109.
Ito S Taguchi H Hamada S Kawauchi S Ito H Senoura T Watanabe J Nishimukai M Ito S Matsui H 《Applied microbiology and biotechnology》2008,79(3):433-441
The gene for cellobiose 2-epimerase (CE) from Ruminococcus albus NE1 was overexpressed in Escherichia coli cells. The recombinant CE was purified to homogeneity by a simple purification procedure with a high yield of 88%, and the molecular mass was 43.1 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and 44.0 kDa on gel chromatography. It exhibited optimal activity around at 30 degrees C and pH 7.5, and the enzyme activity was inhibited by Al3+, Fe3+, Co2+, Cu2+, Zn2+, Pb2+, Ag+, N-bromosuccinimide, iodoacetate, and 4-chloromercuribenzoate. In addition to cello-oligosaccharides, the enzyme was found to effectively 2-epimerize lactose to yield 4-O-beta-D-galactopyranosyl-D-mannose (epilactose), which occurs in cow milk as a rare oligosaccharide. The Km and kcat/Km values toward lactose were 33 mM and 1.6 s(-1) mM(-1), and those toward cellobiose were 13.8 mM and 4.6 s(-1) mM(-1), respectively. N-Acetyl-D-glucosamine, uridine 5'-diphosphate-glucose, D-glucose 6-phosphate, maltose, sophorose, laminaribiose, and gentiobiose were inert as substrates for the recombinant CE. We demonstrated that epilactose was resistant to rat intestinal enzymes, utilized by human adult bifidobacteria, and stimulated the tight junction permeability in Caco-2 cells. These results strongly suggest that this rare disaccharide is promising for use as a prebiotic. 相似文献
110.
Sakiyama H Wynn RM Lee WR Fukasawa M Mizuguchi H Gardner KH Repa JJ Uyeda K 《The Journal of biological chemistry》2008,283(36):24899-24908