全文获取类型
收费全文 | 1209篇 |
免费 | 116篇 |
出版年
2022年 | 4篇 |
2021年 | 10篇 |
2020年 | 6篇 |
2019年 | 9篇 |
2018年 | 19篇 |
2017年 | 8篇 |
2016年 | 30篇 |
2015年 | 41篇 |
2014年 | 54篇 |
2013年 | 55篇 |
2012年 | 80篇 |
2011年 | 85篇 |
2010年 | 51篇 |
2009年 | 36篇 |
2008年 | 64篇 |
2007年 | 50篇 |
2006年 | 40篇 |
2005年 | 65篇 |
2004年 | 43篇 |
2003年 | 50篇 |
2002年 | 56篇 |
2001年 | 31篇 |
2000年 | 27篇 |
1999年 | 30篇 |
1998年 | 14篇 |
1997年 | 18篇 |
1996年 | 18篇 |
1995年 | 15篇 |
1994年 | 14篇 |
1993年 | 13篇 |
1992年 | 34篇 |
1991年 | 25篇 |
1990年 | 27篇 |
1989年 | 22篇 |
1988年 | 16篇 |
1987年 | 20篇 |
1986年 | 16篇 |
1985年 | 16篇 |
1984年 | 20篇 |
1983年 | 20篇 |
1982年 | 8篇 |
1981年 | 12篇 |
1980年 | 4篇 |
1979年 | 5篇 |
1978年 | 5篇 |
1975年 | 5篇 |
1972年 | 6篇 |
1971年 | 4篇 |
1969年 | 4篇 |
1966年 | 3篇 |
排序方式: 共有1325条查询结果,搜索用时 31 毫秒
31.
Robin J. Gouka Wim van Hartingsveldt Roel A. L. Bovenberg Cora M. J. van Zeijl Cees A. M. J. J. van den Hondel Robert F. M. van Gorcom 《Applied microbiology and biotechnology》1993,38(4):514-519
A new transformation system for the filamentous fungus Penicillium chrysogenum is described, based on the use of the homologous acetyl-coenzyme A synthetase (facA) gene as a selection marker. Acetate-non-utilizing (Fac–) strains of P. chrysogenum were obtained by positive selection for spontaneous resistance to fluoroacetate. Among these fac mutants putative facA strains were selected for a loss of acetyl-coenzyme A (CoA) synthetase activity. The facA gene, coding for the enzyme acetyl-CoA synthetase, was isolated from a P. chrysogenum genomic library using synthetic oligonucleotides derived from conserved regions from the corresponding genes of Aspergillus nidulans and Neurospora crassa. Vector pPC2-3, comprising a genomic 6.5 kb PstI fragment, was able to complement P. chrysogenum facA strains with frequencies up to 27 transformants·g–1 DNA. Direct selection of transformants was accomplished using acetate and low amounts (0.001%) of glucose as carbon sources. About 50% of the transformants arose by integration of pPC2-3 DNA at the homologous facA locus and 50% by integration elsewhere in the genome. Determination of the nucleotide sequence of part of the cloned fragment showed the presence of an open reading frame of 2007 nucleotides, interrupted by five putative introns. Comparison of the nucleotide and the amino acid sequence of the facA gene of P. chrysogenum with the facA gene of A. nidulans reveals similarities of 80% and 89%, respectively. The putative introns present in the P. chrysogenum facA gene appear at identical positions as those in the A. nidulans facA gene, but show no significant sequence similarity.
Correspondence to: R. F. M.van Gorcom 相似文献
32.
Jacques J. H. Hens Marina De Wit Lodewijk V. Dekker Frans Boomsma A. Beate Oestreicher Frank Margolis† Willem Hendrik Gispen Pierre N. E. De Graan 《Journal of neurochemistry》1993,60(4):1264-1273
Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca2+-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca2+ concentration from 10?8 to 10?5M Ca2+ The Ca2+ sensitivity in the micromolar range is identical for [3H]NA and endogenous NA release, indicating that Ca2+-induced [3H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca2+-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca2+-induced NA release. PKC pseudosubstrate PKC19-36, which inhibited B-50 phosphorylation (IC50 value, 10?5M), failed to inhibit Ca2+-induced NA release, even when added before the Ca2+ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca2+-induced NA release. Concerning the Ca2+ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca2+ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca2+-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca2+- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca2+-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca2+ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca2+ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca2+ influx resulting in exocytosis of NA. 相似文献
33.
The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane. 总被引:14,自引:2,他引:12 下载免费PDF全文
J Blom M Kübrich J Rassow W Voos P J Dekker A C Maarse M Meijer N Pfanner 《Molecular and cellular biology》1993,13(12):7364-7371
The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation. 相似文献
34.
Glucoamylase overexpression inAspergillus niger: Molecular genetic analysis of strains containing multiple copies of theglaA gene 总被引:1,自引:0,他引:1
35.
Marjon J. H. van Haandel I. M. C. M. Rietjens Ans E. M. F. Soffers Cees Veeger Jacques Vervoort Sandeep Modi Madhu S. Mondal Prasanta K. Patel Digambar V. Behere 《Journal of biological inorganic chemistry》1996,1(5):460-467
The second-order rate constants for the oxidation of a series of phenol derivatives by horseradish peroxidase compound II
were compared to computer-calculated chemical parameters characteristic for this reaction step. The phenol derivatives studied
were phenol, 4-chlorophenol, 3-hydroxyphenol, 3-methylphenol, 4-methylphenol, 4-hydroxybenzoate, 4-methoxyphenol and 4-hydroxybenzaldehyde.
Assuming a reaction of the phenolic substrates in their non-dissociated, uncharged forms, clear correlations (r = 0.977 and r = 0.905) were obtained between the natural logarithm of the second-order rate constants (ln k
app and ln k
2 respectively) for their oxidation by compound II and their calculated ionisation potential, i.e. minus the energy of their
highest occupied molecular orbital [E(HOMO)]. In addition to this first approach in which the quantitative structure-activity
relationship (QSAR) was based on a calculated frontier orbital parameter of the substrate, in a second and third approach
the relative heat of formation (ΔΔHF) calculated for the process of one-electron abstraction and H• abstraction from the phenol derivatives was used as a parameter. Plots of the natural logarithms of the second-order rate
constants (k
app and k
2) for the reaction and the calculated ΔΔHF values for the process of one-electron abstraction also provide clear QSARs with
correlation coefficients of –0.968 and –0.926 respectively. Plots of the natural logarithms of the second-order rate constants
(k
app and k
2) for the reaction and the calculated ΔΔHF values for the process of H• abstraction provide QSARs with correlation coefficients of –0.989 and –0.922 respectively. Since both mechanisms considered,
i.e. initial electron abstraction versus initial H• abstraction, provided clear QSARs, the results could not be used to discriminate between these two possible mechanisms for
phenol oxidation by horseradish peroxidase compound II. The computer calculation-based QSARs thus obtained for the oxidation
of the various phenol derivatives by compound II from horseradish peroxidase indicate the validity of the approaches investigated,
i.e. both the frontier orbital approach and the approach in which the process is described by calculated relative heats of
formation. The results also indicate that outcomes from computer calculations on relatively unrelated phenol derivatives can
be reliably compared to one another. Furthermore, as the actual oxidation of peroxidase substrates by compound II is known
to be the rate-limiting step in the overall catalysis by horseradish peroxidase, the QSARs of the present study may have implications
for the differences in the overall rate of substrate oxidation of the phenol derivatives by horseradish peroxidase.
Received: 29 March 1996 / Accepted: 17 July 1996 相似文献
36.
Woodward's reagent K inactivation of Escherichia coli L-threonine dehydrogenase: increased absorbance at 340-350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups. 下载免费PDF全文
A. R. Johnson E. E. Dekker 《Protein science : a publication of the Protein Society》1996,5(2):382-390
L-Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L-threonine (60 mM) is approximately 50% as effective. Tryptic digestion of WRK-modified TDH followed by HPLC fractionation (pH 6.2) yields four 340-nm-absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35-43), whereas peptide II is TAICGTDVHIY (residues 35-45). Peptides not protected are TMLDTMNHGGR (III, residues 248-258) and NCRGGRTHLCR (IV, residues 98-108). Absorbance spectra of these WRK-peptides were compared with WRK adducts of imidazole, 2-hydroxyethanethiolate, and acetate. Peptides III and IV have pH-dependent lambda max values (340-350 nm), consistent with histidine modification. Peptide I has pH-independent lambda max (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys-38 in the active site of TDH; modification of His-105 and His-255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340-350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports. 相似文献
37.
Syncytium-inducing (SI) phenotype suppression at seroconversion after intramuscular inoculation of a non-syncytium-inducing/SI phenotypically mixed human immunodeficiency virus population. 总被引:14,自引:10,他引:4 下载免费PDF全文
M Cornelissen G Mulder-Kampinga J Veenstra F Zorgdrager C Kuiken S Hartman J Dekker L van der Hoek C Sol R Coutinho et al. 《Journal of virology》1995,69(3):1810-1818
Two distinct biological phenotypes of human immunodeficiency virus (HIV) have been described: the non-syncytium-inducing (NSI) phenotype, best characterized by the inability to infect MT-2 cells, and the syncytium-inducing (SI) phenotype, with the ability to infect MT-2 cells. The earliest virus population observed following HIV transmission is generally of the NSI phenotype, even after exposure to inocula of mixed NSI/SI phenotype. In this study, the issue of intrapatient selection of virus phenotype following transmission was addressed by studying two cases of accidental transmission. A comparison of the sequences of the V1-V2 and the V3 coding regions of the envelope gene and the p17 region of the gag gene showed that the donor-recipient pairs were tightly clustered in all gene segments, but away from local and published transmission controls. The intrasample variation of the p17 sequence was greater in the recipients and smaller in the donors than that of the V3 region sequence, indicating selection of V3 at transmission. In these transmission cases, the effects of an intravenous inoculation of a small quantity of blood containing predominantly SI V3 sequences (6 of 8 clonal sequences) were compared with those of an intramuscular inoculation of a large quantity of blood containing predominantly NSI viruses (14 of 16 clonal sequences). Both SI and NSI V3 regions were demonstrated to be phenotypic expressions of genetically related viral strains. The inoculation of the predominantly SI virus population resulted in the persistence of an SI virus population in the recipient and a rapid CD4+ T-cell decline. The inoculation of the predominantly NSI population resulted in a selective amplification of SI viruses before seroconversion, followed by a suppression of SI viruses at seroconversion and a rapid decline of CD4+ T-cell numbers. These data suggest that the suppression of SI viruses can be accomplished following the development of HIV-specific immunity and that the ability to suppress SI viruses does not prevent the development of immunodeficiency. 相似文献
38.
Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: A problem of ancient DNA and molecular phylogenies 总被引:10,自引:0,他引:10
Antoinette C. van der Kuyl Carla L. Kuiken John T. Dekker Wladimir R. K. Perizonius Jaap Goudsmit 《Journal of molecular evolution》1995,40(6):652-657
Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.Correspondence to: A.C. van der Kuyl 相似文献
39.
Complete nucleotide sequence of simian endogenous type D retrovirus with intact genome organization: evidence for ancestry to simian retrovirus and baboon endogenous virus. 总被引:3,自引:3,他引:0 下载免费PDF全文
A complete endogenous type D viral genome has been isolated from a baboon genomic library. The provirus, simian endogenous retrovirus (SERV), is 8,393 nucleotides long and contains two long terminal repeats and complete genes for gag, pro, pol, and env. The primer binding site is complementary to tRNA(Lys)3, like in lentiviruses. The env GP70 protein is highly homologous to that of baboon endogenous virus (BaEV). PCR analysis of primate DNA showed that related proviral sequences are present in Old World monkeys of the subfamily Cercopithecinae but not in apes and humans. Analysis of virus and host sequences indicated that the proviral genomes were inherited from a common ancestor. Comparison of the evolution of BaEV, exogenous simian retrovirus types 1 to 3 (SRV1 to SRV3), and SERV suggests that SERV is ancestral to both BaEV and the SRVs. 相似文献
40.
Setaria viridis is an important self-pollinating, cosmopolitan weed of temperate regions worldwide. Allozyme markers were used to investigate genetic diversity and structure in 168 accessions (including four S. italica) collected mainly from North America and Eurasia. Genetic diversity in green foxtail, and its population genetic structure, provided important clues about this weed's evolutionary history. Genetic diversity was low, with marked population differentiation: the percentage of polymorphic loci was 25% (0.95 criterion); mean number of alleles per locus was 1.86; mean panmictic heterozygosity was 0.07; and the coefficient of population genetic differentiation was 0.65. A common genotype occurred in 25 accessions distributed in six countries from both the Old World and New World, in a wide variety of ecological situations. Relatively little genetic divergence occurred between Eurasia and North America, with Nei's unbiased genetic identity between the two regions equaling 1.0. Populations from these two continents also had equivalent genetic diversity. Within North America, regional differentiation was indicated by northern and southern groups separated at 43.5° N latitude. No geographic pattern in genetic diversity was found within Eurasia. The size of the geographic range from which populations were sampled was not an accurate indicator of the extent of genetic diversity found among populations from that region. These results suggest that present patterning among green foxtail populations in North America is the consequence of multiple introductions into the New World followed by local adaptation and regional differentiation. Finally, S. italica and several green foxtail varieties did not differ isozymatically from typical forms of green foxtail. This supports the view that S. italica and S. viridis are conspecific, that the former (foxtail millet) is a domesticated form of the latter, and also questions the taxonomic validity of formally recognizing morphological varieties within green foxtail. 相似文献