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[3H]Dihydroergocryptine ([3H]DHE) was shown to bind to sites in membranes from neuroblastoma X glioma hybrid cells (NG 108-15) that had the characteristics expected of alpha-adrenergic receptors. The binding was saturable with 0.3 pmol [3H]DHE bound per mg of protein and of high affinity, with an apparent dissociation constant (KD) of 1.8 nM. The specificity of the binding site for various ligands was more similar to that of alpha 2 receptors than to that of alpha 1. No specific binding of [3H]WB-4101 was found in the membranes derived from NG 108 cells. This finding also indicated that the [3H]DHE binding site in the cell is the alpha 2 receptor. GTP lowered the affinity of agonists for the [3H]DHE binding site, although the nucleotide hardly affected the affinity of antagonists including [3H]DHE. 相似文献
3.
The regulatory mechanism of guinea pig lymphokines was investigated in regard to differentiation of myeloid cells to macrophages. The Ml-cell line, established from a myeloid leukemia of an SL-strain mouse, was induced to differentiate in vitro into mature macrophages possessing Fc receptors and the ability to phagocytize latex particles by treatment with crude lymphokines. Both concanavalin A- and antigen-induced lymphokines showed the differentiation-inducing factor (D factor) activity. However, macrophage migration inhibitory factor/ macrophage activation factor (MIF/MAF) purified by an immunoadsorbent column with anti-MIF antibody had no such an activity. The D-factor activity was detected in the lymphokine preparation that was not retained on the immunoadsorbent column. In contrast, colony-stimulating factor (CSF) was adsorbed to the immunoadsorbent column, and could be recovered in the purified MIF/MAF preparation. These findings suggest that the molecular entity of D factor is distinct from MIF/ MAF and CSF. A culture supernatant of guinea pig peritoneal macrophages activated with MIF/ MAF (CSF) exhibited strong D-factor activity. However, the supernatant possessed rather reduced CSF activity as compared to that of the original MIF/MAF (CSF) preparation. Thus, MIF/MAF may play an important role in macrophage differentiation by regulating the production of D factor or CSF from macrophages. 相似文献
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Haruaki Uchiyama Koichi Ohara Kazuko Haga Tatsuya Haga Arata Ichiyama 《Journal of neurochemistry》1990,54(6):1870-1881
Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol. 相似文献
6.
Methenamine-Silver Staining: a Simple and Sensitive Staining Method for Senile Plaques and Neurofibrillary Tangles 总被引:1,自引:0,他引:1
Chie Haga Kenji Ikeda Kiyoshi Iwabuchi Haruhiko Akiyama Hiromi Kondoh Kenji Kosaka 《Biotechnic & histochemistry》1994,69(5):295-300
An improved methenamine-silver impregnation method is presented which exhibits sensitivity for amyloid substances comparable to that of anti-β protein immunostaining. In optimally treated sections, this technique stained both β-amyloid deposits and neurofibrillary tangles, which are known to have a β-pleated structure. This simple procedure allows a large number of sections to be stained for routine examination. 相似文献
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Identification of two novel GTP-binding protein alpha-subunits that lack apparent ADP-ribosylation sites for pertussis toxin 总被引:2,自引:0,他引:2
F Nakamura K Ogata K Shiozaki K Kameyama K Ohara T Haga T Nukada 《The Journal of biological chemistry》1991,266(19):12676-12681
Molecular cloning of cDNAs encoding alpha-subunits of guanine nucleotide-binding regulatory proteins (G-proteins) has revealed the existence of nine species of alpha-subunits. We have identified two additional G-protein alpha-subunits, which we refer to as GL1 alpha and GL2 alpha, by isolating bovine liver cDNA clones that cross-hybridized at reduced stringency with bovine Gi1 alpha-subunit cDNA. The deduced amino acid sequences of GL1 alpha and GL2 alpha share 83% identity with each other and show 45-55% identity with those of other known G-protein alpha-subunits. Both GL1 alpha and GL2 alpha lack a consensus site for ADP-ribosylation by pertussis toxin. Messenger RNA corresponding to GL2 alpha was detected in all tissues examined, but GL1 alpha mRNA was detected only in liver, lung, and kidney. Antiserum prepared against a synthetic pentadecapeptide corresponding to the deduced carboxyl terminus of GL2 alpha specifically reacted with a 40-kDa protein in mouse liver, brain, lung, heart, kidney, and spleen. The amount of the 40-kDa protein was highest in brain and lung. We suggest that GL1 alpha and GL2 alpha are new members of a subfamily of pertussis toxin-insensitive G-proteins. 相似文献
9.
The complete nucleotide sequence of the phosphoprotein (P) gene of the Yamagata-1 strain of a defective subacute sclerosing panencephalitis (SSPE) virus was determined. Comparison with the P gene of the Edmonston strain of measles virus (MV) revealed 44 differences of which 23 nucleotides substitutions were identical with those revealed between other SSPE viruses and MV (Cattaneo et al. (1989) Virology 173, 415-425). The consensus sequence of the G insertion site was completely conserved, whereas mRNAs with one or three non-templated G residue insertions were found in addition to the mRNA of the exact genome copy. As a result of the frameshift downstream of the site of G insertion, the cysteine-rich V protein was predicted from the one G-inserted mRNA besides the P and C proteins predicted from the genome-copied mRNA. 相似文献
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