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43.
IgG-dependent generation of platelet-activating factor by normal and low density human eosinophils 总被引:1,自引:0,他引:1
O Cromwell A J Wardlaw A Champion R Moqbel D Osei A B Kay 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(11):3862-3868
We have compared normal and low density human eosinophils for their ability to generate platelet activating factor (PAF) in response to IgG-dependent and nonimmunologic stimulation. After 45 min incubation with IgG-coated Sepharose beads the concentrations of cell-associated PAF recovered from normal density eosinophils were significantly greater than from low-density eosinophils or neutrophils. Moreover, eosinophils stimulated with calcium ionophore A23187 had a considerably greater capacity to generate PAF than had previously been described. Although the quantities of cell-associated PAF recovered from normal and low density eosinophils and neutrophils after A23187 stimulation were similar, the amounts of extracellular PAF recovered from both eosinophil populations were significantly greater than from neutrophils. The amounts of PAF recovered from the low density eosinophils may not reflect the full synthetic capacity of these cells, because PAF-turnover was found to be more rapid than that observed with normal density eosinophils. When exogenous [3H]PAF was added to the two stimulated eosinophil populations subsequent analysis of the [3H]PAF metabolites by DIOL-HPLC revealed that low density eosinophils incorporated PAF into the phosphatidylcholine (PC) pool more rapidly than did normal density eosinophils or neutrophils. Alkaline hydrolysis of the PC fraction from whole cell extracts followed by treatment with acetic anhydride resulted in all the PC-associated radioactivity being converted to [3H]PAF, confirming PAF incorporation to PC via this pathway. These findings suggest that the contribution of eosinophils to inflammatory processes through the generation of PAF may be greater than previously appreciated, and that Ig-mediated stimulation may be important in initiating generation of the mediator. Low density eosinophils, that are presumed to be similar to tissue eosinophils, may have a role in regulating PAF concentrations in tissues through their enhanced rate of metabolism. 相似文献
44.
Characterization of Opioid Receptors in Cultured Neurons 总被引:1,自引:1,他引:0
Pierre J.-J. Vaysse R. Suzanne Zukin Kay L. Fields John A. Kessler 《Journal of neurochemistry》1990,55(2):624-631
The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors. 相似文献
45.
A Meister S L Weinrich C Nelson W J Rutter 《The Journal of biological chemistry》1989,264(34):20744-20751
46.
J T Griffiths L H Phylip J Konvalinka P Strop A Gustchina A Wlodawer R J Davenport R Briggs B M Dunn J Kay 《Biochemistry》1992,31(22):5193-5200
The sequence requirements for HIV-1 proteinase catalyzed cleavage of oligopeptides containing two distinct types of junctions (-hydrophobic*hydrophobic- or -aromatic*Pro-) has been investigated. For the first type of junction (-hydrophobic*hydrophobic-) the optimal residues in the P2 and P2' positions were found to be Val and Glu, respectively, in accord with recent statistical analysis of natural cleavage sites [Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., & Kézdy, F. J. (1991) J. Biol. Chem. 266, 14554-14561]. For the -aromatic*Pro- type of junction, in the specific sequence context studied here, the value of Glu in the P2' position was again observed. An explanation for the inefficient cleavage observed for peptides with the sequence -Val-Tyr*Pro- has been provided from molecular modeling of the putative enzyme-substrate complex. A significant effect upon cleavage rates due to the amino acid in the P5 position has also been documented. While lysine in the P5 position in one sequence of the -hydrophobic*hydrophobic- type produces a peptide cleaved very efficiently (kcat greater than 15 s-1 for Lys-Ala-Arg-Val-Nle*p-nitrophenylalanine-P2'-Ala-Nle-NH2, for P2' = Glu, Gln, Ile, Val, or Ala), for substrates of the -aromatic*Pro- type, the P5 residue can exert either a positive or negative effect on cleavage rates. These results have again been interpreted in light of molecular modeling. We suggest that interaction of the substrate sequence on the periphery of the active site cleft may influence the match of the enzyme-substrate pair and, hence, control the efficiency of catalysis.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
47.
L K Nicholson L E Kay D M Baldisseri J Arango P E Young A Bax D A Torchia 《Biochemistry》1992,31(23):5253-5263
This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having Mr = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-13C2]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the 13C T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau f (5-80 ps) and tau s (less than 2 ns). The fast motion is identified with the rapid rotation of the methyl group about the C gamma-C delta bond axis, while the slow motion is associated with reorientation of the C gamma-C delta bond axis itself. The mean squared order parameters associated with the latter motion, Ss2, lie in the range 0.34-0.92. The values of Ss2 correlate reasonably well with the temperature factors of the leucine methyl carbons obtained from the crystal structures, but some are smaller than anticipated on the basis of the fact that nearly all leucine methyl carbons are buried and have temperature factors no larger than that of the leucine backbone atoms. Five leucine residues in liganded SNase and eight in unliganded SNase have values of Ss2 less than 0.71. These order parameters correspond to large amplitude motions (angular excursions of 27-67 degrees) of the C gamma-C delta bond axis. These results indicate that, in solution, the internal motions of the leucine side chains of SNase are significantly larger than suggested by the X-ray structures or by qualitative analysis of NOESY spectra. Comparison of Ss2 values obtained from liganded and unliganded SNase reveals a strong correlation between delta Ss2 and distance between the leucine methyl carbon and the ligands.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
48.
A soluble ATP-dependent enzyme which phosphorylates myo-inositol has been characterized in Dictyostelium. The myo-inositol kinase activity was partially purified from amoebae by chromatography on DEAE-Sepharose and phenyl-Sepharose columns. The product of both the partially purified activity and of a crude cytosolic fraction was myo-inositol 3-phosphate. The partially purified preparations of myo-inositol kinase (a) possessed a Km for myo-inositol of 120 microM (in the presence of 5 mM-ATP) and for ATP of 125 microM (in the presence of 1 microM-myo-inositol), (b) did not recognize allo-, epi-, muco-, neo-, scyllo-, 1 D-chiro or 1 L-chiro-inositol as substrates, (c) were competitively inhibited by three naturally occurring analogues of myo-inositol: 1 L-chiro-inositol (Ki 49.5 +/- 0.7 microM: the structural equivalent of myo-inositol, except that the D-3 hydroxy moiety is axial), D-3-deoxy-myo-inositol [Ki 103 +/- 1 microM: (-)-viburnitol], and sequoyitol (Ki 271 +/- 7 microM; unlike 1 L-chiro-inositol and D-3-deoxy-myo-inositol, this was a substrate for the kinase), and finally (d) were apparently non-competitively inhibited by myo-inositol 3-phosphate. The product of myo-inositol kinase could be detected in intact amoebae and was a substrate for the first in a series of inositol polyphosphate kinases present in Dictyostelium which ultimately yield myo-inositol hexakisphosphate. The activity of myo-inositol D-3-hydroxykinase in Dictyostelium lysates showed evidence of developmental regulation. 相似文献
49.
Kay Robinson-Beers Th. D. Sharkey R. F. Evert 《Plant biology (Stuttgart, Germany)》1990,103(4):424-429
Sink-to-source transition was studied in developing sugarcane (Saccharum interspecific variety L62–96) leaves. Fully-expanded, mature sugarcane leaves were fed 14CO2 for 20 minutes, incorporating about 617 Bq. After five hours the leaves of each plant were cut into 1-cm-length segments that were weighed and then placed in scintillation cocktail for counting. All leaves younger than the leaf fed 14CO2 imported labeled photoassimilate. Three to four leaves had both importing and non-importing regions within the blade and a distinct transition region between them. A transition region was observed in leaves which had expanded to between 30 and 90 % of final blade length. Radioactivity per gram fresh weight was calculated as a measure of sink strength. Sink strength was greatest in the youngest leaf and declined with leaf age. The results of this study indicate that 1) import of photosynthate by developing sugarcane leaves occurs over a longer span of developmental ages than in dicotyledonous leaves and 2) the actual tissue region undergoing transition within such a leaf can be resolved as narrow zone between the importing and non-importing regions. 相似文献
50.
The prion protein gene is dispensable for the development of spongiform myeloencephalopathy induced by the neurovirulent Cas-Br-E murine leukemia virus. 下载免费PDF全文
The Cas-Br-E murine leukemia virus (MuLV) induces paralysis in susceptible mice that is accompanied by a severe spongiform myeloencephalopathy. These neurodegenerative lesions are very similar to those observed in prion diseases. To determine whether the prion protein gene (Prn-p) product was a downstream effector of this neurovirulent MuLV, we inoculated Prn-p(-/-) knockout homozygote and control heterozygote or wild-type mice with this retrovirus. All groups developed typical paralysis and spongiform encephalopathy, and no differences in clinical or histological phenotypes were observed between these groups. These results indicate that the Cas-Br-E MuLV does not require the prion protein to induce lesions. Thus, MuLV and prion proteins may induce a very similar disease through distinct pathways, or the viral Env protein, which harbors the primary determinant of pathogenicity, may act in a common pathway but downstream of the prion protein. 相似文献