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1.
Crosslinking of isolated red cell membrane cytoskeletal proteins and hemoglobin mediated by H2O2 was studied. The products of spectrin and hemoglobin interaction were demonstrated electrophoretically to be high-molecular-weight polypeptides crosslinked by nondisulfide covalent bonds. The molecular weight of the protein bands correlated with various combinations of spectrin and hemoglobin chains and the relative amount of the different products was dependent on the molar ratio of the interacting proteins. Free hemin caused spectrin crosslinking as well, but globin in the absence of hemin was inactive. Since the H2O2-mediated reaction resulted in reduction of the spectrin tryptophan fluorescence, the latter was used to monitor the reaction progress under various conditions. Both oxyhemoglobin and methemoglobin were found to be most efficient, whereas cyanmethemoglobin and hemichrome were relatively inactive. Analysis of the data implied that tryptophan oxidation as well as spectrin conformational changes follow an iron-induced crosslinking of the interacting proteins. Actin, the second major protein in the red cell cytoskeleton, behaved similarly to spectrin. The intrinsic fluorescence intensity of both G- and F-actin was decreased upon addition of H2O2 to the mixture of hemoglobin and each of the actin forms. SDS-polyacrylamide gel electrophoresis revealed that G-actin crosslinked one or two hemoglobin chains. F-actin-hemoglobin interaction induced by H2O2 produced very high aggregates that could not penetrate the gel. It is suggested that crosslinking of cytoskeletal proteins in red cells containing membrane-associated hemoglobin provides a rationale for the loss of membrane flexibility. 相似文献
2.
Induction of differentiation in HL-60 and U-937 leukemic cell lines, resulted in 1.5-10-fold increase in 45Ca2+ uptake. The increased 45Ca2+ uptake in the differentiating cells was inhibited by verapamil, cromolyn and amiloride. Elevation in Ca2+ uptake in differentiating cells was also demonstrated using the fluorescent probe, fura-2 acetoxymethyl ester. The increased 45Ca2+ uptake was accompanied by a decrease in ouabain-insensitive and -sensitive 86Rb+ uptake. Furthermore, correlation between the changes in these activities was observed. Modulation of extracellular pH affected differentiation: higher pH increased the extent of differentiation. 相似文献
3.
Spectrin and actin were isolated and their oligomeric state after association with hemin at various conditions was studied. Intact cytoskeletons were prepared by Triton X-100 extraction of red blood cells and incubated with hemin and their stability analyzed by the appearance of dissociated proteins in the supernatant. The cytoskeletons dissociated in a time, temperature and hemin concentration-dependent manner. Following 18 hours incubation in the presence of 0.3 mM hemin there was no dissociation at 4 degrees C, while at the same hemin concentration after 2 hours complete dissociation of the cytoskeletons occurred at 37 degrees C. Microscopy indicated that the cytoskeletons incubated with hemin lost their "cell like" shapes in a time dependent manner. Hemin applied to intact cells also caused dissociation of their cytoskeletons as judged by the failure to separate integer cytoskeletons from red cells treated with hemin. From hemin-induced dissociation profiles of separated actin, spectrin and whole cytoskeletons under various conditions, a mechanism of cytoskeleton breakdown was analyzed, as a release of band 4.1 in the first step which is followed by spectrin dimerization and eventually dissociation of the entire cytoskeletons. 相似文献
4.
Illana Gozes Mati Fridkin Douglas E. Brenneman 《Cellular and molecular neurobiology》1995,15(6):675-687
Summary 1. The 28 amino acid vasoactive intestinal peptide, VIP, was originally isolated from the intestine, following a bioassay measuring vasodilating properties. Immunocytochemistry, receptor binding assays and in situ hybridizations have demonstrated VIP abundance in the nervous system, suggesting multiple bioactivities.2. A pharmacological approach was chosen to dissect VIP activities and a prototype VIP antagonist (Met-Hybrid) consisting of a carboxyl fragment of VIP7–28 and a six amino acid fragment of neurotensin, neurotensin6–11-VIP7–28 was synthesized.3. This hybrid peptide was designed to maintain the binding capacity of one parent molecule (VIP), while loosing the agonistic properties, representing a classical competitive receptor antagonist. Furthermore, the new molecule exhibited increased specificity to central nervous system VIP receptors.4. The Met-Hybrid was originally discovered as a potent inhibitor of VIP functionin vivo. In the adult rodent, acute administration of the antagonist resulted in blockade of VIP-mediated potentiation of sexual behavior and chronic intracerebroventricular application impaired VIP-associated learning abilities. During ontogeny, chronic injections of the molecule resulted in neuronal damage, disruption of the diurnal rhythmicity of motor behavior, and retardation in the acquisition of neonatal reflexes in the rat.5. During gestation, severe microcephaly was induced by acute administration of the Met-Hybrid to pregnant mice. The hybrid antagonist inhibited VIP-stimulated mitosis in whole embryo cultures and in a variety of cancer cell linesin vitro andin vivo, suggesting therapeutical potential. 相似文献
5.
The ability of actin to interact with hemin was studied. It was found that the Soret absorption band of hemin changes in the presence of actin and that hemin is capable of quenching the fluorescence intensity of actin. These findings were indicative of hemin binding to actin. The binding constant for the high affinity site was calculated to be 5.3 X 10(6) M-1. The amounts of native G- and F-actin were estimated by their DNAase I inhibition activity. It was observed that the binding of hemin to G-actin is followed by a slow decrease in the ability of actin to inhibit DNAase I activity and to polymerize upon addition of salts. Binding of hemin to F-actin resulted in a gradual depolymerization of the filaments, to an inactivated form, as expressed by a reduction in the ability of hemin-bound F-actin to inhibit DNAase I activity in the absence as well as in the presence of guanidine-HCl. Electron microscopy studies further corroborated these findings by demonstrating that: (1) hemin-bound G-actin failed to show formation of polymers when salts were added; (2) a marked reduction in the amount of actin polymers was observed in the specimens examined 24 h after mixing with hemin. It is suggested that the elevated amounts of free hemin formed under pathological conditions, might be toxic to cells by interfering with actin polymerization cycles. 相似文献
6.
Yitzhak Stabinsky Philip Gottlieb Mati Fridkin 《Molecular and cellular biochemistry》1980,30(3):165-170
Summary Sixteen new analogs of the phagocytosis-stimulating peptide tuftsin have been synthesized. The biological activities of these synthetic peptides, in which either the C-terminal or both C- and N-terminals are chemically altered, were evaluated by studying their effects on the phagocytosis of heat-killed yeasts and on the reduction of the dye nitroblue tetrazolium by normal human polymorphonuclear leukocytes. The results demonstrate that the integrity of the guanidine side chain of arginine at position four of tuftsin is crucial for maximal activity. Modification, even in side chain length, of the guanidine leads to decreasing activity. Preservation of the positive charge of position four of tuftsin yields analogs possessing considerable activity. Simultaneous alterations of both C- and N-terminal results in diminishing activites. The results of this study are discussed in relation to the structural features of tuftsin. It appears that interaction between the carboxyl of Arg4 and the amino group of Thr1 which would indicate a specific conformation such as a 4 1 -turn are not favored. 相似文献
7.
Nurith Shaklai Hana Abrahami 《Biochemical and biophysical research communications》1980,95(3):1105-1112
The interaction of deoxyhemoglobin with the red cell membrane is characterized by comparing the affinity of deoxyhemoglobin for the membrane with that of oxyhemoglobin. The two techniques used, namely light scattering induced changes and quenching of the fluorescence intensity of a membrane embedded probe, demonstrate that deoxyhemoglobin exhibits a much lower affinity for the membrane than that of oxyhemoglobin. The binding constant of 2×10 M?1 calculated for deoxyhemoglobin at 5 mM phosphate buffer and pH=6.0 is two orders of magnitude lower than the one calculated for oxyhemoglobin. It is estimated that under physiological conditions the only species capable of interacting with the membrane is the oxyhemoglobin. 相似文献
8.
Fluorescence properties of hemocyanin from Levantina hierosolima 总被引:3,自引:0,他引:3
9.
Matić Ivana Z. Ergün Sercan Đorđić Crnogorac Marija Misir Sema Aliyazicioğlu Yüksel Damjanović Ana Džudžević-Čančar Hurija Stanojković Tatjana Konanç Kalbiye Petrović Nina 《Cytotechnology》2021,73(3):373-389
Cytotechnology - Six extracts were obtained from plant species Hypericum perforatum L., collected at Samsun in Turkey. The aim of this study was to examine the mechanisms of the anticancer activity... 相似文献
10.