全文获取类型
收费全文 | 40241篇 |
免费 | 4339篇 |
国内免费 | 13篇 |
专业分类
44593篇 |
出版年
2022年 | 300篇 |
2021年 | 520篇 |
2020年 | 343篇 |
2019年 | 423篇 |
2018年 | 543篇 |
2017年 | 511篇 |
2016年 | 895篇 |
2015年 | 1610篇 |
2014年 | 1563篇 |
2013年 | 2079篇 |
2012年 | 2352篇 |
2011年 | 2073篇 |
2010年 | 1441篇 |
2009年 | 1316篇 |
2008年 | 1806篇 |
2007年 | 1783篇 |
2006年 | 1598篇 |
2005年 | 1564篇 |
2004年 | 1469篇 |
2003年 | 1264篇 |
2002年 | 1261篇 |
2001年 | 1226篇 |
2000年 | 1228篇 |
1999年 | 1105篇 |
1998年 | 607篇 |
1997年 | 562篇 |
1996年 | 555篇 |
1995年 | 537篇 |
1994年 | 489篇 |
1993年 | 512篇 |
1992年 | 1003篇 |
1991年 | 748篇 |
1990年 | 770篇 |
1989年 | 751篇 |
1988年 | 652篇 |
1987年 | 595篇 |
1986年 | 609篇 |
1985年 | 707篇 |
1984年 | 530篇 |
1983年 | 412篇 |
1982年 | 340篇 |
1981年 | 312篇 |
1980年 | 253篇 |
1979年 | 378篇 |
1978年 | 343篇 |
1977年 | 241篇 |
1976年 | 225篇 |
1975年 | 196篇 |
1974年 | 289篇 |
1973年 | 250篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
911.
S-layer protein of Lactobacillus acidophilus ATCC 4356: purification, expression in Escherichia coli, and nucleotide sequence of the corresponding gene. 总被引:11,自引:4,他引:7 下载免费PDF全文
The cell surfaces of several Lactobacillus species are covered by a regular layer composed of a single species of protein, the S-protein. The 43-kDa S-protein of the neotype strain Lactobacillus acidophilus ATCC 4356, which originated from the pharynx of a human, was purified. Antibodies generated against purified S-protein were used to screen a lambda library containing chromosomal L. acidophilus ATCC 4356 DNA. Several phages showing expression of this S-protein in Escherichia coli were isolated. A 4.0-kb DNA fragment of one of those phages hybridized to a probe derived from an internal tryptic fragment of the S-protein. The slpA gene, coding for the surface layer protein, was located entirely on the 4.0-kb fragment as shown by deletion analysis. The nucleotide sequence of the slpA gene was determined and appeared to encode a protein of 444 amino acids. The first 24 amino acids resembled a putative secretion signal, giving rise to a mature S-protein of 420 amino acids (44.2 kDa). The predicted isoelectric point of 9.4 is remarkably high for an S-protein but is in agreement with the data obtained during purification. The expression of the entire S-protein or of large, C-terminally truncated S-proteins is unstable in E. coli. 相似文献
912.
Identification of the glmU gene encoding N-acetylglucosamine-1-phosphate uridyltransferase in Escherichia coli. 总被引:5,自引:3,他引:2 下载免费PDF全文
The physiological properties of the EcoURF-1 open reading frame, which precedes the glmS gene at 84 min on the Escherichia coli chromosome (J. E. Walker, N. J. Gay, M. Saraste, and A. N. Eberle, Biochem. J. 224:799-815, 1984), were investigated. A thermosensitive conditional mutant in which the synthesis of the gene product was impaired at 43 degrees C was constructed. The inactivation of the gene in exponentially growing cells rapidly inhibited peptidoglycan synthesis. As a result, various alterations of cell shape were observed, and cell lysis finally occurred when the peptidoglycan content was 37% lower than that of normally growing cells. Analysis of the pools of peptidoglycan precursors revealed a large accumulation of N-acetylglucosamine-1-phosphate and the concomitant depletion of the pools of the seven peptidoglycan nucleotide precursors located downstream in the pathway, a result indicating that the mutational block was in the step leading from N-acetylglucosamine-1-phosphate and UTP to the formation of UDP-N-acetylglucosamine. In vitro assays showed that the overexpression of this gene in E. coli cells, directed by appropriate plasmids, led to a high overproduction (from 25- to 410-fold) of N-acetylglucosamine-1-phosphate uridyltransferase activity. This allowed us to purify this enzyme to homogeneity in only two chromatographic steps. The gene for this enzyme, which is essential for peptidoglycan and lipopolysaccharide biosyntheses, was designated glmU. 相似文献
913.
Characterization of IS1001, an insertion sequence element of Bordetella parapertussis. 总被引:4,自引:1,他引:3 下载免费PDF全文
A van der Zee C Agterberg M van Agterveld M Peeters F R Mooi 《Journal of bacteriology》1993,175(1):141-147
By analysis of repetitive DNA in Bordetella parapertussis, an insertion sequence element, designated IS1001, was identified. Sequence analysis revealed that IS1001 comprised 1,306 bp and contained inverted repeats at its termini. Furthermore, several open reading frames that may code for transposition functions were identified. The largest open reading frame coded for a protein comprising 406 amino acid residues and showed homology to TnpA, which is encoded by an insertion sequence element (IS1096) found in Mycobacterium smegmatis. Examination of flanking sequences revealed that insertion of IS1001 occurs preferentially in stretches of T's or A's and results in a duplication of target sequences of 6 to 8 bases. IS1001 was found in about 20 copies in 10 B. parapertussis strains analyzed. No restriction fragment length polymorphism was observed in B. parapertussis when IS1001 was used as a probe. An insertion sequence element similar or identical to IS1001 was found in B. bronchiseptica strains isolated from pigs and a rabbit. In these strains, about five copies of the IS1001-like element were present at different positions in the bacterial chromosome. Neither B. pertussis nor B. bronchiseptica strains isolated from humans and dogs contained an IS1001-like element. Therefore, IS1001 may be used as a specific probe for the detection of B. parapertussis in human clinical samples. 相似文献
914.
Involvement of an activation protein in the methanol:2-mercaptoethanesulfonic acid methyltransferase reaction in Methanosarcina barkeri. 总被引:3,自引:2,他引:1 下载免费PDF全文
P J Daas K A Gerrits J T Keltjens C van der Drift G D Vogels 《Journal of bacteriology》1993,175(5):1278-1283
Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) is the first of two enzymes required for transfer of the methyl group of methanol to 2-mercaptoethanesulfonic acid in Methanosarcina barkeri. MT1 binds the methyl group of methanol to its corrinoid prosthetic group only when the central cobalt atom of the corrinoid is present in the highly reduced Co(I) state. However, upon manipulation of MT1 and even during catalysis, the enzyme becomes inactivated as the result of Co(I) oxidation. Reactivation requires H2, hydrogenase, and ATP. Ferredoxin stimulated the apparent reaction rate of methyl group transfer. Here we report that one more protein fraction was found essential for the overall reaction and, more specifically, for formation of the methylated MT1 intermediate. The more of the protein that was present, the shorter the delay of the start of methyl group transfer. The maximum velocity of methyl transfer was not substantially affected by these varying amounts of protein. This demonstrated that the protein was involved in the activation of MT1. Therefore, it was called methyltransferase activation protein. 相似文献
915.
Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19. 总被引:2,自引:2,他引:0 下载免费PDF全文
L V Bystrykh J Vonck E F van Bruggen J van Beeumen B Samyn N I Govorukhina N Arfman J A Duine L Dijkhuizen 《Journal of bacteriology》1993,175(6):1814-1822
The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 to 50 kDa. Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.7. These cofactors are redox active toward alcohol and aldehyde substrates. Both enzymes contain significant amounts of Zn2+ and Mg2+ ions. The primary amino acid sequences of the A. methanolica and M. gastri MB19 methanol:NDMA oxidoreductases share a high degree of identity, as indicated by N-terminal sequence analysis (63% identity among the first 27 N-terminal amino acids), internal peptide sequence analysis, and overall amino acid composition. The amino acid sequence analysis also revealed significant similarity to a decameric methanol dehydrogenase of Bacillus methanolicus C1. 相似文献
916.
The effect of simulated rainfall frequency on the pathogenicity of Pratylenchus zeae and P. brachyurus was studied in four greenhouse experiments. Corn and grain sorghum were watered at different intervals during predetermined cycles to create a gradient of water-stressed plants. Each experiment included nematode and uninoculated treatments. Growth reaction of plants to different frequencies of watering was significant but was not affected by the presence of nematodes. Pratylenchus zeae numbers differed among watering regimens on corn but not on sorghum. Numbers of P. brachyurus did not differ among watering regimens on corn or sorghum. Both lesion nematode species were harmful to corn, but sorghum increased plant growth in response to P. brachyurus. It is concluded that water stress is not the only environmental factor that influences the pathogenicity of these two species on corn and sorghum. 相似文献
917.
The kinetics of t-[3H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the γ-aminobutyric acidA (GABAA) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 μM GABA and decelerated by 1 μM bicuculline methochloride, a GABAA antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [3H]TBOB represent multiaffinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation kapp=koff±kon [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B1a (AVM B1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC50, 7.8 nM) followed by displacement at high concentrations (IC50 6.3 μM) of AVM B1a. AVM B1a resulted in similar biphasic effects on t- [35S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [3H]TBOB binding (IC50 125 μM DIDS) leaving the enhancement by AVM B1a unaffected. 相似文献
918.
Characterization of the Scavenger Receptor on Bovine Cerebral Endothelial Cells In Vitro 总被引:1,自引:0,他引:1
H. E. de Vries†J. Kuiper† A. G. de BoerTh. J. C. van Berkel† D. D. Breimer 《Journal of neurochemistry》1993,61(5):1813-1821
Abstract— Primary cultures of bovine brain capillary endo-thelial cells (BCEC), possessing tight junctions and high levels of γ-glutamyl transpeptidase, were used as an in vitro model for the blood-brain barrier. The interaction of acetylated low density lipoprotein (AcLDL) with BCEC was studied to characterize the scavenger receptor on these cells. A saturable high affinity binding site was found with a dissociation constant of AcLDL of 5.4 μg/ml (3.1 n M ) and a maximal binding ranging from 284 to 626 ng of AcLDL/mg of cell protein for eight primary cultures, and independent of the presence of calcium. Cell association was coupled to degradation, and both could be effectively competed for by polyinosinic acid and AcLDL but not by low density lipoprotein or by high density lipoprotein. Prolonged incubation showed an accumulation of the ligand in the cells. The rate of degradation of AcLDL was ∼ 10–20-fold lower in BCEC than that of peripheral endothelial cells. No evidence for lysosomal degradation could be obtained. Binding of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocar-boxyamine perchlorate-labeled AcLDL by BCEC was observed, which could be competed for by an excess of un-labeled AcLDL and polyinosinic acid. We have shown that in vitro BCEC possesses specific binding sites for AcLDL, whereas these cells show a relatively low degradative capacity. 相似文献
919.
Definition of a domain of GLVR1 which is necessary for infection by gibbon ape leukemia virus and which is highly polymorphic between species. 总被引:19,自引:16,他引:3 下载免费PDF全文
Expression of human GLVR1 in mouse cells confers susceptibility to infection by gibbon ape leukemia virus (GALV), while the normally expressed mouse Glvr-1 does not. Since human and murine GLVR1 proteins differ at 64 positions in their sequences, some of the residues differing between the two proteins are critical for infection. To identify these, a series of hybrids and in vitro-constructed mutants were tested for the ability to confer susceptibility to infection. The results indicated that human GLVR1 residues 550 to 551, located in a cluster of seven of the sites that differ between the human and mouse proteins, are the only residues differing between the two which must be in the human protein form to allow infection. Sequencing of a portion of GLVR1 from the rat (which is infectible) confirmed the importance of this cluster in that it contained the only notable differences between the rat and mouse proteins. This region, which also differs substantially between the rat and the human proteins, therefore exhibits a pronounced tendency for polymorphism. 相似文献
920.
Large E1B proteins of adenovirus types 5 and 12 have different effects on p53 and distinct roles in cell transformation. 总被引:8,自引:3,他引:5 下载免费PDF全文
The formation of complexes between oncoproteins of DNA tumor viruses and the cellular protein p53 is thought to result in inactivation of the growth suppressor function of p53. In cells transformed by nononcogenic human adenovirus type 5 (Ad5), the 55-kDa protein encoded by E1B forms a stable complex with p53 and sequesters it in the cytoplasm. However, the homologous 54-kDa protein of highly oncogenic Ad12 does not detectably associate with p53. Yet in Ad12-transformed cells, p53 is metabolically stable, is present at high levels in the nucleus, and contributes to the oncogenicity of the cells. Such properties have previously been described for mutant forms of p53. Here, we show that stable p53 in Ad12-transformed cells is wild type rather than mutant and that stabilization of p53 is a direct consequence of the expression of the Ad12 E1B protein. We also compared the effects of the E1B proteins on transformation of rodent cells by different combinations of oncogenes. A synergistic interaction was observed for the gene encoding the 54-kDa E1B protein of Ad12 with myc plus ras oncogenes, resembling the effect of mutant p53 on myc plus ras. In contrast, the Ad5 55-kDa E1B protein strongly inhibited transformation by myc plus ras but stimulated transformation by E1A plus ras. The data are explained in terms of different interactions of the two E1B proteins with endogenous p53. The results suggest that in cultured rat cells, endogenous wild-type p53 plays an essential role in cell proliferation, even in the presence of myc plus ras. The dependence on p53 is lost, however, when the adenovirus E1A oncogene is present. 相似文献