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1.
Proalbumin is processed to serum albumin in COS-1 cells transfected with cDNA for rat albumin 总被引:2,自引:0,他引:2
K Oda N Takami T Fujiwara Y Misumi Y Ikehara 《Biochemical and biophysical research communications》1989,163(1):194-200
Synthesis and processing of rat albumin were investigated in COS-1 cells transiently expressing rat albumin. Analysis using isoelectric focusing revealed that serum-type albumin, which is indistinguishable from the counterpart isolated from rat hepatocyte cuture medium, was secreted from the transfected COS-1 cells, indicating that proalbumin is effectively converted into serum albumin in the COS-1 cells, if not completely. Furthermore methylamine was found to cause the diminution of serum albumin released from the cells, substantiating that the proteolytical conversion of proalbumin occurs in the Golgi complex before discharge from the COS-1 cells. 相似文献
2.
Primary structure of rat liver alkaline phosphatase deduced from its cDNA. 总被引:5,自引:1,他引:4 下载免费PDF全文
Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver. 相似文献
3.
Disparate effects of monensin and colchicine on intracellular processing of secretory proteins in cultured rat hepatocytes 总被引:8,自引:0,他引:8
We have studied the biosynthesis and intracellular processing of three major secretory proteins, albumin, alpha 1-protease inhibitor and alpha 2u-globulin, in cultured rat hepatocytes. The effect of secretion-blocking agents, monensin, a monovalent ionophore, and the microtubule-affecting agents colchicine and taxol was determined. In the control cells, alpha 1-protease inhibitor, a glycoprotein, was first synthesized as an endoglycosidase-H-sensitive form with Mr 51 000, and then processed to two endoglycosidase-H-resistant forms having Mr 51 000 and 56 000, the latter of which was secreted into the medium. Initially synthesized proalbumin was converted with chase to serum-type albumin, while no pro-type precursor was identified for alpha 2u-globulin. In the cells treated with colchicine or taxol, in which secretion was greatly inhibited, the fully processed alpha 1-protease inhibitor and albumin accumulated and were finally secreted into the medium. In the monensin-treated cells, however, most of the newly synthesized alpha 1-protease inhibitor and albumin were not processed to the final mature forms, resulting in accumulation of two 51 000-Mr forms and proalbumin, respectively. Moreover in treated cells, proalbumin and the endoglycosidase-H-resistant alpha 1-protease inhibitor were finally secreted into the medium. Such an effect was not caused by NH4Cl which also inhibited the secretion and is known to exert the similar effect as monensin on the receptor-mediated endocytosis pathway. Based on these results, the use of monensin may prove valuable for more detailed analysis of intracellular processing of various proteins. 相似文献
4.
K Oda Y Misumi Y Ikehara S O Brennan K Hatsuzawa K Nakayama 《Biochemical and biophysical research communications》1992,189(3):1353-1361
We have recently purified and characterized a truncated soluble form of furin from which the predicted transmembrane domain and cytoplasmic tail were deleted (Hatsuzawa, K., Nagahama, M., Takahashi, S., Takada, K., Murakami, K., and Nakayama, K. (1992) J. Biol. Chem. 267, 16094-16099). Our results showed that furin resembles the yeast Kex2 protease with respect to both its enzymic properties and substrate specificity. Here we demonstrate that the soluble form of furin is capable of converting the precursors of albumin and the third component of complement (proalbumin and pro-C3, respectively) in vitro to mature proteins. Thus furin mimics the Ca(2+)-dependent proalbumin and pro-C3 convertases found in the Golgi membranes (Brennan, S. O., and Peach, R. J. (1988) FEBS Lett. 229, 167-170; Oda, K. (1992) J. Biol. Chem. 267, 17465-17471). Furthermore we show that the variant alpha 1-antitrypsin Pittsburgh, which is a specific inhibitor of the Golgi proalbumin convertase, inhibits not only the Golgi pro-C3 convertase, but also the soluble furin. These results suggest a role for furin in the cleavage of proproteins transported via the constitutive pathway. 相似文献
5.
Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock 总被引:3,自引:0,他引:3
Sun Nyunt Wai Tetsuhiro Moriya Katsuhiko Kondo Hiroyasu Misumi Kazunobu Amako 《FEMS microbiology letters》1996,136(2):187-191
Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH4 Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation. 相似文献
6.
Occurrence of a factor(s) which can selectively inhibit ribosomal RNA synthesis in isolated neurula cells of Xenopus laevis was examined in oocytes, unfertilized eggs, and embryos of Xenopus laevis. It was found that acid-soluble materials from full-sized oocytes, white-banded mature oocytes, unfertilized eggs, and pregastrular embryos were all active in significantly reducing the relative ratio of the [3H]uridine incorporation into 18S and 28S ribosomal RNA to that into 4S RNA from the control value. These results suggest that the inhibitor appears in the terminal step of oogenesis and, hence, may be assumed as a maternal regulator. 相似文献
7.
T Muta T Miyata Y Misumi F Tokunaga T Nakamura Y Toh Y Ikehara S Iwanaga 《The Journal of biological chemistry》1991,266(10):6554-6561
8.
5'-nucleotidase from the electric ray electric lobe. Primary structure and relation to mammalian and procaryotic enzymes. 总被引:1,自引:0,他引:1
W Volknandt M Vogel J Pevsner Y Misumi Y Ikehara H Zimmermann 《European journal of biochemistry》1991,202(3):855-861
A cDNA encoding a 5'-nucleotidase was identified by screening a lambda gt10 cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63,833 Da. The N-terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C-terminus contains a stretch of hydrophobic amino acids, considered to be cleaved on post-translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N-linked glycosylation sites. Electric ray 5'-nucleotidase shares 61% amino acid identity with the enzymes from rat liver and human placenta, and about 23% with bacterial proteins possessing 5'-nucleotidase activity and also additional enzyme activities like UDP-glucose hydrolase. Polyclonal antibodies raised against 5'-nucleotidase from mammalian sources or the electric ray electric organ reveal mutual cross-reactivity. Interestingly, there are 5-7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5'-nucleotidase, 3'-nucleotidase or phosphodiesterase activity. 5'-nucleotidase isolated from Torpedo electric organ hydrolyzes UDP-glucose at 8% of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5'-nucleotidase from multifunctional nucleotide hydrolases is discussed. 相似文献
9.
K Oda Y Misumi M Sohda N Takami Y Sakaki Y Ikehara 《Biochemical and biophysical research communications》1991,175(2):690-696
Rat proalbumin is cleaved at the dibasic pair Arg-Arg and converted into a mature form with Glu at the NH2 terminus. In the present study site-directed mutagenesis of the albumin cDNA was designed to generate proalbumin variants in which Glu1 was substituted with various amino acid residues. The expression plasmids constructed were transfected into COS-1 cells, and the intracellular processing of proalbumins expressed was examined by labeling experiments. Substitution of Glu1----Ser allowed the expressed proalbumin to be processed as observed for the wild-type precursor. However, replacement of Glu1 with a hydrophobic residue (Val, Leu or Ile) resulted in no processing of proalbumin, despite retaining the same cleavage signal Arg-Arg as above. The results indicate that the residue at position 1 adjacent to the dibasic pair is also important for recognition by the proalbumin-processing enzyme. 相似文献
10.
K Shiokawa Y Misumi Y Yasuda Y Nishio S Kurata M Sameshima K Yamana 《Developmental biology》1979,68(2):503-514
Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs. 相似文献