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91.
Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa 总被引:11,自引:0,他引:11
The decline in boar semen quality after cryopreservation may be attributed to changes in intracellular proteins. Thus, the aim of the present study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually mature boars (mean age = 25.5+/-12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15 degrees C, after cooling to 5 degrees C, following thawing after freezing to -100 degrees C, and following thawing after 1 wk of cryopreservation at -196 degrees C. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5x10(6) spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was 90 kDa heat-shock protein (HSP90). Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5 degrees C. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5 degrees C. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100 degrees C. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The length of time between a decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa. 相似文献
92.
Strains of Serratia marcescens (isolated in a hospital during April and August 2000) resistant to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, streptomycin, tetracycline, and gentamicin were characterized. Out of a total of 34 clinical isolates 6 (17.6 %) exhibited the extended spectrum beta-lactamases (ESBL) resistance; they were also resistant to cefotaxime (minimum inhibitory concentration, MIC > or = 128 microg/mL) but susceptible to imipenem (MIC < or = 0.5 microg/mL). This multidrug resistance was shown to be transferred by a conjugative plasmid. Transconjugants revealed similar MIC profiles when compared to the parental strains. Isoelectric focusing revealed one major transferable beta-lactamase (pI 8.4) which was further identified as CTX-M-3 by PCR and gene sequencing. The presence of strains with this type of ESBL showed the evolution of bla genes and their dissemination among at least three species of the family Enterobacteriaceae isolated within a single hospital. The predominance of CTX-M type enzymes found in this area of Taiwan appeared to be similar to that described in Poland. 相似文献
93.
Bergmann DC Lee M Robertson B Tsou MF Rose LS Wood WB 《Development (Cambridge, England)》2003,130(23):5731-5740
The mechanism by which polarity of the left-right (LR) axis is initially established with the correct handedness is not understood for any embryo. C. elegans embryos exhibit LR asymmetry with an invariant handedness that is first apparent at the six-cell stage and persists throughout development. We show here that a strong loss-of-function mutation in a gene originally designated spn-1 affects early spindle orientations and results in near randomization of handedness choice. This mutation interacts genetically with mutations in three par genes that encode localized cortical components. We show that the spn-1 gene encodes the Galpha protein GPA-16, which appears to be required for centrosomal association of a Gbeta protein. We will henceforth refer to this gene as gpa-16. These results demonstrate for the first time involvement of heterotrimeric G proteins in establishment of embryonic LR asymmetry and suggest how they might act. 相似文献
94.
95.
Tsou CH 《American journal of botany》1998,85(11):1531-1547
The early floral development of Camellioideae was studied. Two major evolutionary lineages were recognized for this subfamily. The earlier evolved lineage (Camellia, Polyspora, and Pyrenaria) has normally 11-14 perianth members, which are initiated in a continuous spiral and are differentiated into sepals and petals at late floral development, and numerous stamens initiated individually and centrifugally on the whole androecial region. The later derived lineage (Franklinia, Hartia, Schima, and Stewartia) has five sepals and five petals arranged in two whorls, and numerous individual stamens originating centrifugally from the five petal-opposed zones. Hartia-Stewartia and Franklinia-Schima further diverged as two branches - the former is characterized by having androecial fascicles and axile-basal placentation. The androecial fascicle is considered to be derived within this subfamily. The latter exhibits a higher degree of carpellary congenital fusion and axile-central placentaion, and as a whole, is concluded to be the most advanced group in the Camellioideae. A taxonomic treatment of the Camellioideae at the tribal level is also proposed. 相似文献
96.
Unfolding and refolding of dimeric creatine kinase equilibrium and kinetic studies. 总被引:2,自引:0,他引:2 下载免费PDF全文
Y. X. Fan J. M. Zhou H. Kihara C. L. Tsou 《Protein science : a publication of the Protein Society》1998,7(12):2631-2641
Equilibrium and kinetic studies of the guanidine hydrochloride induced unfolding-refolding of dimeric cytoplasmic creatine kinase have been monitored by intrinsic fluorescence, far ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonate binding. The GuHCl induced equilibrium-unfolding curve shows two transitions, indicating the presence of at least one stable equilibrium intermediate in GuHCl solutions of moderate concentrations. This intermediate is an inactive monomer with all of the thiol groups exposed. The thermodynamic parameters obtained by analysis using a three-state model indicate that this intermediate is similar in energy to the fully unfolded state. There is a burst phase in the refolding kinetics due to formation of an intermediate within the dead time of mixing (15 ms) in the stopped-flow apparatus. Further refolding to the native state after the burst phase follows biphasic kinetics. The properties of the burst phase and equilibrium intermediates were studied and compared. The results indicate that these intermediates are similar in some respects, but different in others. Both are characterized by pronounced secondary structure, compact globularity, exposed hydrophobic surface area, and the absence of rigid side-chain packing, resembling the "molten globule" state. However, the burst phase intermediate shows more secondary structure, more exposed hydrophobic surface area, and more flexible side-chain packing than the equilibrium intermediate. Following the burst phase, there is a fast phase corresponding to folding of the monomer to a compact conformation. This is followed by rapid assembly to form the dimer. Neither of the equilibrium unfolding transitions are protein concentration dependent. The refolding kinetics are also not concentration dependent. This suggests that association of the subunits is not rate limiting for refolding, and that under equilibrium conditions, dissociation occurs in the region between the two unfolding transitions. Based upon the above results, schemes of unfolding and refolding of creatine kinase are proposed. 相似文献
97.
A Y Tsou S C Ransom J A Gerlt D D Buechter P C Babbitt G L Kenyon 《Biochemistry》1990,29(42):9856-9862
The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells. 相似文献
98.
Comparison of inactivation and conformational changes of D-glyceraldehyde-3-phosphate dehydrogenase during thermal denaturation 总被引:2,自引:0,他引:2
Y Z Lin S J Liang J M Zhou C L Tsou P Q Wu Z K Zhou 《Biochimica et biophysica acta》1990,1038(2):247-252
The inactivation of D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) EC 1.2.1.12) (GAPDH) during thermal denaturation has been compared to its dissociation-aggregation measured by light scattering and changes in secondary structure measured by CD in the far ultraviolet. The inactivation at 38.5 degrees C consists of two stages. The rate of the first stage is too fast to be followed by conventional methods. The extent of this fast stage inactivation increases with increasing temperature and, more markedly, with increasing pH. At this stage, the inactivation is reversible and no appreciable dissociation or change in secondary structure can be detected. The secondary structure of the enzyme is relatively heat stable, showing no appreciable change at 38.5 degrees C. At this temperature, the enzyme first dissociates within several minutes probably into dimers and with prolonged heating, it becomes irreversibly aggregated. The above results are in accord with the earlier suggestion, based on results obtained during denaturation of a number of enzymes by guanidine hydrochloride (GdnHCl) and urea, that for some enzymes the active site is situated in a region more susceptible to perturbation than the molecule as a whole (Tsou, C.-L. (1986) Trends Biochem. Sci. 11, 427). 相似文献
99.
Cynthia S. Ricard Janelle M. Jakubowski John W. Verbsky M. Alejandro Barbieri William M. Lewis G. Esteban Fernandez Marion Vogel Christina Tsou Vinoy Prasad Philip D. Stahl Gabriel Waksman Clarissa M. Cheney 《Genesis (New York, N.Y. : 2000)》2001,31(1):17-29
Summary: Rab GTPases are essential for vesicular transport. Rab GDP dissociation inhibitor (GDI) binds to GDP‐bound rabs, removes rabs from acceptor membranes and delivers rabs to donor membranes. We isolated lethal GDI mutations in Drosophila and analyzed their developmental phenotypes. To learn how these mutations affect GDI structure, the crystal structure of Drosophila GDI was determined by molecular replacement to a resolution of 3.0 Å. Two hypomorphic, missense mutations are located in domain II of GDI at highly conserved positions, but not in previously identified sequence conserved regions. The mutant GDIs were tested for ability to extract rabs from membranes and showed wild‐type levels of rab membrane extraction. The two missense alleles showed intragenic complementation, indicating that domain II of GDI may have two separable functions. This study indicates that GDI function is essential for development of a complex, multicellular organism and that puparium formation and pole cell formation are especially dependent on GDI function. genesis 31:17–29, 2001. © 2001 Wiley‐Liss, Inc. 相似文献
100.
Yang CW Su JY Tsou AP Chau GY Liu HL Chen CH Chien CY Chou CK 《Biochemical and biophysical research communications》2005,330(2):489-497
DNA microarray has been widely used to examine gene expression profile of different human tumors. The information generated from microarray analysis usually represents the overall range of cancer-associated abnormality associated with gene regulation. In order to identify key regulatory genes involved in carcinogenesis of human cancer, hypothesis driven data mining of the microarray data plus experimental validation becomes a critical approach in the post-genome era. Here, we present an integrative genomic analysis of published microarray data and homolog gene database. Over 20,000 genes were examined to reveal 16 genes specific to vertebrates, cell cycle G2/M regulated, and overexpressed in human HCC. Using Affymetrix microarray analysis, we found that all 16 genes were up-regulated in human HCC. Among these 16 genes, we experimentally validated the up-regulation of receptor for hyaluronan-mediated motility (RHAMM) in different cell model systems. We first confirmed elevation of RHAMM in the G2/M phase of synchronized HeLa cells. We also found that RHAMM had an elevated level of expression in all the HCC samples we examined and it was induced during the G2/M phase of regenerating mouse hepatocytes after partial hepatectomy. Thus, the expression of RHAMM appears to be tightly regulated during mammalian cell cycle G2/M progression. The ectopic overexpression of RHAMM in 293T cells resulted in the accumulation of cells at G2/M phase. RHAMM-induced mitotic arrest of cells was predominantly in the prophase. Taken together, using an integrated functional genomic approach, we have uncovered a set of genes that may play specific roles in cell cycle progression and in HCC development. To elucidate the function of these genes in cell cycle regulation may shed light on the control mechanism of human HCC in the future. 相似文献