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1.
Spatial proximity of the glycine-rich loop and the SH2 thiol in myosin subfragment 1 总被引:1,自引:0,他引:1
Subfragment 1 (S1) prepared from rabbit skeletal muscle myosin was digested with trypsin to cleave the 95K heavy chain into three pieces, i.e., the 23K, 50K, and 20K fragments. The trypsin-treated S1 was then cross-linked with p-nitrophenyl iodoacetate. The cross-linker bridged one of the reactive thiols (SH2) in the 20K fragment and a lysine residue in the 23K fragment [Hiratsuka, T. (1987) Biochemistry 26, 3168-3173]. Location of the lysine residue was mapped along the 23K fragment by "end-label fingerprinting", which employed site-directed antibodies against the N-terminus of the 23K fragment and against the C-terminus of the 24K fragment (the 23K fragment plus nine extra residues at its C-terminus). The mapping revealed that Lys-184 or Lys-189 was the residue cross-linked with SH2. Since the cross-linker used here spans only several angstroms, the result indicates that Lys-184 or Lys-189 is very close to SH2 in the three-dimensional structure of myosin head. Examination of the primary structure of the 23K fragment has revealed that these lysine residues are in and very close to the so-called "glycine-rich loop", whose sequence is highly homologous to those of nucleotide-binding sites of various nucleotide-binding proteins. 相似文献
2.
T Watabe A Hiratsuka K Ogura 《Biochemical and biophysical research communications》1986,134(1):100-105
Potent mutagenicity of 7,12-dihydroxymethylbenz[a]anthracene (DHBA) toward Salmonella typhimurium TA 98 in the presence of rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate (PAPS) was completely retarded by the addition of glutathione (GSH). The reactive and intrinsically mutagenic metabolite, DHBA 7-sulfate, formed by hepatic cytosolic sulfotransferase disappeared from the incubation mixture by the addition of GSH. Non-mutagenic S-(12-hydroxymethylbenz[a]anthracen-7-yl)methylglutathione was isolated from the incubation mixture consisting of the hepatic cytosol, DHBA, PAPS, and GSH and proved to be formed by GSH S-transferase directly from DHBA 7-sulfate as an obligatory intermediate. 相似文献
3.
T Watabe A Hiratsuka K Ogura K Endoh 《Biochemical and biophysical research communications》1985,131(2):694-699
The carcinogen, 7,12-dihydroxymethylbenz[alpha]anthracene (DHBA), was regioselectively conjugated in the presence of 3'-phosphoadenosine 5'-phosphosulfate by male rat liver cytosolic sulfotransferase to DHBA 7-sulfate. The sulfate ester was highly reactive and showed a potent, intrinsic mutagenicity toward Salmonella typhimurium TA 98. 相似文献
4.
I Miki T Shimizu Y Seyama S Kitamura K Yamaguchi H Sano H Ueno A Hiratsuka T Watabe 《The Journal of biological chemistry》1989,264(10):5799-5805
(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro. 相似文献
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7.
S Itonori M Hiratsuka N Sonku H Tsuji O Itasaka T Hori M Sugita 《Biochimica et biophysica acta》1992,1123(3):263-268
Antiserum against Man beta 1-4Glc beta 1-1Ceramide (MIOse2Cer), a mannolipid isolated from spermatozoa of the fresh-water bivalve, Hyriopsis schlegellii, has been elicited in rabbits by repeated injection of a mixture of hapten-bovine serum albumin (1:1, mg/ml) with Freund's adjuvant. The specificity of the affinity-purified antibody (immunoglobulin G type) obtained from the serum was examined, using other glycosphingolipids and glyco-proteins structurally related to MIOse2Cer, by means of ELISA and TLC-immunostaining. The purified antibody was highly specific to MIOse2Cer and lacked reactivity with other glycolipids and glycoproteins including glucosylceramide, lactosylceramide, dimannosylglucosylceramide (MIOse3Cer), glucosaminylmannosylglucosylceramide (ArOse3Cer), thyroglobulin and alpha 1-acid glycoprotein. The antibody was found to bind, although less efficiently, to certain other compounds containing the group Man beta 1-4Glc and/or Man beta 1-4GlcNAc at their termini, such as MIOse2-sphingosine and Man beta 1-4GlcNAc beta 1-p-aminobenzoic acid ethylester derivatives. The present antibody was applied to the detection of the natural hapten in crustacean glycolipids. The purified antibody reacted with a neutral glycosphingolipid present in the two kinds of crustacean, Euphausia superba (antarctic krill) and Macrobrachium nipponense (fresh-water shrimp) as shown by TLC-immunostaining. The crustacean glycolipid antigen was isolated and characterized to be the Man beta 1-4Glc-Cer. This is the first report on the presence of a mannose-containing glycosphingolipid in the crustacean. 相似文献
8.
Takahashi M Takahashi K Hiratsuka Y Uchida K Yamagishi A Uyeda TQ Yazawa M 《The Journal of biological chemistry》2001,276(2):1034-1040
The alternatively spliced isoform of nonmuscle myosin II heavy chain B (MHC-IIB) with an insert of 21 amino acids in the actin-binding surface loop (loop 2), MHC-IIB(B2), is expressed specifically in the central nervous system of vertebrates. To examine the role of the B2 insert in the motor activity of the myosin II molecule, we expressed chimeric myosin heavy chain molecules using the Dictyostelium myosin II heavy chain as the backbone. We replaced the Dictyostelium native loop 2 with either the noninserted form of loop 2 from human MHC-IIB or the B2-inserted form of loop 2 from human MHC-IIB(B2). The transformant Dictyostelium cells expressing only the B2-inserted chimeric myosin formed unusual fruiting bodies. We then assessed the function of chimeric proteins, using an in vitro motility assay and by measuring ATPase activities and binding to F-actin. We demonstrate that the insertion of the B2 sequence reduces the motor activity of Dictyostelium myosin II, with reduction of the maximal actin-activated ATPase activity and a decrease in the affinity for actin. In addition, we demonstrate that the native loop 2 sequence of Dictyostelium myosin II is required for the regulation of the actin-activated ATPase activity by phosphorylation of the regulatory light chain. 相似文献
9.
Evaluation of the electroosmotic medium pump system for preparative disk gel electrophoresis 总被引:1,自引:0,他引:1
Hayakawa M Hosogi Y Takiguchi H Saito S Shiroza T Shibata Y Hiratsuka K Kiyama-Kishikawa M Abiko Y 《Analytical biochemistry》2001,288(2):168-175
This paper describes an improved electroosmotic elution system for preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on the epochal idea of H. V. Tan et al. (Nucleic Acids Res. 1988, 16, 1921-1930). In this elution system, a semipermeable membrane, mounted under the gel terminal end, works as the elution pump as well as the partition of the elution chamber. We refer to this system as the "electroosmotic medium pump system." Operation of the constructed apparatus (3.6 cm i.d. disk gel column) and resolution of the protein bands were examined by separation of the model protein mixture (bovine serum albumin (BSA), ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor) and purification of the membrane protein, dipeptidyl peptidase IV (DPP IV). The Spectra/Por 7 dialysis membrane provided a better flow profile for the elution buffer. The four model proteins of the protein mixture were able to be completely separated from each other and recovered without dilution. The maximum protein concentration of eluate achieved was 93 mg/ml, when applying a single component, BSA fraction V, as a sample. Furthermore, the multifunctional ectoenzyme, DPP IV, was purified in a single step. 相似文献
10.
Tsukamoto H Hishinuma T Suzuki N Tayama R Hiratsuka M Yoshihisa T Mizugaki M Goto J 《Prostaglandins & other lipid mediators》2004,73(3-4):191-213
Thiazolidinedione, peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, has been used as an anti-diabetic drug and as an useful tool to elucidate multiple PPARgamma functions by in vitro and in vivo studies. We investigated the effects of thiazolidinediones on prostanoid production in lipopolysaccharide-stimulated cells. The high concentrations (>10 microM) of rosiglitazone and pioglitazone significantly increased lipopolysaccharide-stimulated prostanoid production such as thromboxane A2 and prostaglandin E2. However, PPARgamma antagonist could not inhibit them. In PPARgamma-deficient cells, thiazolidinediones increased prostaglandin E2 production. Thiazolidinediones increased arachidonic acid (AA) release from the cell membrane by not stimulating AA releasing process involving several phospholipase A2s but inhibiting AA reuptaking process. The expression of cyclooxygenase-1 and cyclooxygenase-2 were not affected by thiazolidinediones. In this study, we demonstrated that high concentrations of TZDs increased AA release by the inhibition of AA reuptaking process, leading to subsequent increase in the prostanoid production in a PPARgamma-independent manner. This mechanism provides useful information for the elucidation of multiple PPARgamma functions and diabetic drug therapy. 相似文献