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91.
The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines 总被引:5,自引:0,他引:5
Arlen P Tsang KY Marshall JL Chen A Steinberg SM Poole D Hand PH Schlom J Hamilton JM 《Cancer immunology, immunotherapy : CII》2000,49(10):517-529
An enzyme-linked immunosorbent spot (ELISPOT) assay for interferon γ production has been used to analyze specific T cell
responses to a Flu 9-mer peptide, and a 9-mer peptide of carcinoembryonic antigen (CEA). Assays were performed on peripheral
blood mononuclear cells (PBMC) of HLA-A2-positive patients with CEA-expressing carcinomas, both before and after vaccination
with CEA-based vaccines, and from HLA-A2-positive healthy blood donors. The ELISPOT assay utilized aliquots of frozen PBMC,
and assays were performed after 24 h in culture with peptide to rule out any artifacts due to long-term in vitro stimulation
cycles. An internal standard was used for each assay to define reproducibility of the assay, and all samples from a given
patient (pre- and post-vaccination, with both the Flu and CEA peptides) were analyzed simultaneously. The results indicated
a trend towards healthy blood donors having higher levels of Flu-specific T cell precursors than do colon carcinoma patients,
but these results were not statistically significant (P = 0.06). On the other hand, slightly higher CEA-specific T cell responses were observed in cancer patients with CEA-expressing
carcinomas than in healthy blood donors. PBMC from two CEA-based vaccine clinical trials were analyzed for T cell responses
to the same CEA peptide and to the Flu control peptide. The first trial consisted of three monthly vaccinations of CEA peptide
(designated PPP) in adjuvant. The second trial consisted of cohorts receiving three monthly vaccinations of avipox-CEA recombinant
(designated AAA) or cohorts receiving a primary vaccination with recombinant vaccinia-CEA followed by two monthly vaccinations
with avipox-CEA (designated VAA). Few, if any, CEA-specific T cell responses were seen in the PPP vaccinations, while the
majority of patients receiving the poxvirus CEA recombinants demonstrated increases in CEA-specific T cell responses and no
increases in Flu-specific responses. CEA-specific IgG responses were also demonstrated in patients following recombinant CEA
poxvirus vaccinations. Statistical analyses of the T cell responses to the same CEA peptide demonstrated a P value of 0.028 for the recombinant poxvirus vaccines, as compared with the peptide vaccine. There were no differences seen
(P = 0.37) in Flu-specific responses after these two types of CEA vaccination. These results thus provide the first evidence
that poxvirus recombinant-based vaccines are more potent in the initiation of tumor-antigen-specific T cell responses than
vaccines employing peptide in adjuvant, when assays are conducted in an identical manner, and in defining responses to the
same peptide. These results also demonstrate for the first time that an ELISPOT assay, performed over a 24-h period and without
in vitro sensitization, can be successfully used to monitor immune responses to a tumor-associated antigen in cancer patients.
Received: 12 June 2000 / Accepted: 13 July 2000 相似文献
92.
Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells. 下载免费PDF全文
ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract. 相似文献
93.
94.
The effects of the 5-lipoxygenase inhibitors nordihydroguiaretic acid (NDGA), 5, 8, 11, 14-eicosatetraynoic acid (ETYA), 1-phenyl-3-pyrazolidone (phenidone) and BW-755c, on the contractile response to LTC4 or LTD4 were examined on the isolated guinea pig trachea. Responses to either LTC4 or LTD4 were obtained on indomethacin treated tissues, in the presence of either L-serine-borate complex or L-cysteine, respectively, to inhibit metabolic conversion of the leukotrienes. NDGA (30 microM) and ETYA (100 microM) produced a selective competitive antagonism of LTD4-induced contractions, while phenidone antagonized both LTC4- and LTD4-induced responses in a non-competitive manner. In contrast, BW-755c (30 microM) did not significantly antagonize LTC4 or LTD4 concentration-response curves. The results suggest that leukotriene antagonism may be produced by large concentrations of some 5-lipoxygenase inhibitors. 相似文献
95.
Many hazelnut (Corylus avellana L.) cultivars fail to thrive in vitro on standard growth medium and the reasons for poor growth are not well understood. Our initial study of five C. avellana cultivars showed that changes in the mineral nutrients of Driver and Kuniyuki walnut (DKW) medium, including doubling the minor nutrients, produced improved growth and shoot quality. The objectives of this study were to determine the effects of the individual minor mineral nutrients from DKW medium and if added nickel was required for optimal growth. Five factors were tested at 0.5 × to 4× DKW medium concentrations, [H3BO3, CuSO4·5H2O, MnSO4·H2O, Na2MoO4·2H2O and Zn(NO3)2·6H20], in a response surface design with 39 treatment combinations. Ni was not present in the DKW medium formulation so NiSO4·6H2O was varied from 0 to 6 µM. There were many significant interactions among the minor nutrients. Higher concentrations (4×) of B, Mo, and Zn increased overall shoot quality, length, and multiplication. Increased Mo improved some responses for each cultivar, and it interacted significantly with Cu and Zn. The addition of Ni greatly improved the shoot quality and length of ‘Sacajawea.’ Ni interactions were significant for the other cultivars as well, and altered the requirements for the other minor nutrients, but did not necessarily improve the overall shoot response. Improved growth and shoot quality for most cultivars required increased amounts of B, Mo, and Zn and less Mn and Cu. ‘Sacajawea’ required increased B, Cu, Zn, and Ni. All of the cultivars required minor nutrient formulations that differed greatly from DKW medium or other published minor nutrient formulations. 相似文献
96.
Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells. 下载免费PDF全文
ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h. 相似文献
97.
Melanie L Hand Noel OI Cogan Timothy I Sawbridge German C Spangenberg John W Forster 《BMC plant biology》2010,10(1):94
Background
White clover (Trifolium repens L.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumes Lotus japonicus and Medicago truncatula as well as Arabidopsis thaliana were also characterised. 相似文献98.
In spite of the increasing application of DNA fingerprinting to natural
populations and to the genetic identification of humans, explicit methods
for estimation of basic population genetic parameters from DNA
fingerprinting data have not been developed. Contributing to this omission
is the inability to determine, for multilocus fingerprinting probes,
relatively important genetic information, such as the number of loci, the
number of alleles, and the distribution of these alleles into specific
loci. One of the most useful genetic parameters that could be derived from
such data would be the average heterozygosity, which has traditionally been
employed to measure the level of genetic variation within populations and
to compare genetic variation among different loci. We derive here explicit
formulas for both the estimation of average heterozygosity at multiple
hypervariable loci and a maximum value for this estimate. These estimates
are based upon the DNA restriction-pattern matrices that are typical for
fingerprinting studies of humans and natural populations. For several
empirical data sets from our laboratory, estimates of average and maximal
heterozygosity are shown to be relatively close to each other. Furthermore,
variances of these statistics based on simulation studies are relatively
small. These observations, as well as consideration of the effect of
missing alleles and alternate numbers of loci, suggest that the average
heterozygosity can be accurately estimated using phenotypic DNA fingerprint
patterns, because this parameter is relatively insensitive to the lack of
certain genetic information.
相似文献
99.
Localization and enumeration of fimbria-associated adhesins of Bacteroides loescheii. 总被引:7,自引:3,他引:7 下载免费PDF全文
E I Weiss J London P E Kolenbrander A R Hand R Siraganian 《Journal of bacteriology》1988,170(3):1123-1128
Monoclonal antibodies that specifically inhibit coaggregation between Bacteroides loescheii PK1295 and its two gram-positive partners Streptococcus sanguis 34 and Actinomyces israelii PK14 were used to enumerate and localize two distinct types of fimbria-associated adhesins on the surface of B. loescheii. Binding studies with radiolabeled monoclonal antibodies indicated that a maximum (Bmax calculated from Scatchard plots) of approximately 400 adhesin molecules specific for S. sanguis and 310 adhesin molecules specific for A. israelii reside on the surface of the cell. Immunoelectron microscopy revealed that the adhesins were not an integral part of the fimbrial subunit; rather, they were usually found on the distal portion of the structures arranged in a random fashion. 相似文献
100.
Britten CJ; van den Eijnden DH; McDowell W; Kelly VA; Witham SJ; Edbrooke MR; Bird MI; de Vries T; Smithers N 《Glycobiology》1998,8(4):321-327
The alpha3 fucosyltransferase, FucT-VII, is one of the key
glycosyltransferases involved in the biosynthesis of the sialyl Lewis X
(sLex) antigen on human leukocytes. The sialyl Lewis X antigen
(NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential
component of the recruitment of leukocytes to sites of inflammation,
mediating the primary interaction between circulating leukocytes and
activated endothelium. In order to characterize the enzymatic properties of
the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been
expressed in Trichoplusia ni insect cells. The enzyme is capable of
synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from
3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels
of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies
using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors
demonstrate that FucT-VII is able to synthesize both di-fucosylated and
tri-fucosylated structures from mono- fucosylated precursors, but
preferentially fucosylates the distal GlcNAc within a polylactosamine
chain. Furthermore, the rate of fucosylation of the internal GlcNAc
residues is reduced once fucose has been added to the distal GlcNAc. These
results indicate that FucT-VII is capable of generating complex selectin
ligands, in vitro , however the order of fucose addition to the lactosamine
chain affects the rate of selectin ligand synthesis.
相似文献