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951.
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Yuki Ozaki Shingo Suzuki Koichi Kashiwase Atsuko Shigenari Yuko Okudaira Sayaka Ito Anri Masuya Fumihiro Azuma Toshio Yabe Satoko Morishima Shigeki Mitsunaga Masahiro Satake Masao Ota Yasuo Morishima Jerzy K Kulski Katsuyuki Saito Hidetoshi Inoko Takashi Shiina 《BMC genomics》2015,16(1)
Background
HLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1.Results
We developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template.Conclusions
In this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1514-4) contains supplementary material, which is available to authorized users. 相似文献953.
Stołyszewski I Niemcunowicz-Janica A Pepiński W Spólnicka M Zbiec R Janica J 《Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society》2007,45(2):135-136
PURPOSE: The aim of this study was the assessment of vitreous humor as a potential DNA for forensic human postmortem identification. MATERIAL AND METHODS: Vitreous humor samples were collected using two alternative approaches from 25 corpses of either sex during autopsies. DNA was extracted by standard organic method. Recovered DNA was quantitiated fluorometrically. AmpFlSTR SGM Plus kit and ABI 310 Genetic Analyzer (Applera) were used to obtain genetic profiles. RESULTS: Different DNA yields were quantitated in vitreous body depending on cause of death and sampling approach. CONCLUSION: Vitreous humor is a potential DNA for forensic human postmortem identification depending on a sampling method used. 相似文献
954.
Milczarek R Sokolowska E Hallmann A Klimek J 《Molecular and cellular biochemistry》2007,295(1-2):105-111
In pregnant females, placenta is the most important source of lipid hydroperoxides and other reactive oxygen species (ROS).
The increased production of lipid peroxides is often linked to preeclampsia. In our study, we revealed that NADPH- and iron-dependent
lipid peroxidation in human placental microsomes (HPM) occurred. In the presence of Fe2+ ion, HPM produced small amounts of thiobarbituric acid-reactive substances (TBARS) – a final product of lipid peroxidation.
NADPH caused a strong increase of iron stimulated TBARS formation. TBARS formation was inhibited by superoxide dismutase,
butylated hydroxytoluene and α-tocopherol but not by mannitol or catalase. TBARS and superoxide radical production was inhibited
in similar manner by cytochrome P450 inhibitors. The results obtained led us to the following conclusions: (1) microsomal
lipid peroxidation next to mitochondrial lipid peroxidation may by an important source of lipid hydroperoxides in blood during
pregnancy and (2) superoxide radical released by microsomal cytochrome P450 is an important factor in NADPH- and iron-dependent
lipid peroxidation in HPM. 相似文献
955.
The effect of Dithane M-45 (dithiocarbamate fungicide; active substance: mancozeb) was studied on microarthropod fauna inhabiting
dead wood. Although the exposure was almost never 100% lethal for the majority of observed taxa, almost all (Mesostigmata,
Oribatida, some Uropodina, Actinedida, Collembola and Diplopoda) showed very high correlation between concentration of the
fungicide and mortality (r > 0.86). Only Stigmaeidae showed low correlation (r = 0.293). For the majority of taxa LC50 values were close to the concentrations used during agrochemical activities in woods. Only Trachytes aegrota showed full susceptibility to the fungicide within the range of recommended field concentrations used in forestry (characterised
by the low LC95 value). Tolerance of mesostigmatid and oribatid mites was found to differ between juveniles and adults, but not consistently.
Related Uropodina species varied in susceptibility to the fungicide. 相似文献
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Effects of PACAP and VIP on cyclic AMP formation in rat neuronal and astrocyte cultures under normoxic and hypoxic condition 总被引:1,自引:0,他引:1
Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) concentration (0.001-1000 nM)-dependently stimulated cyclic AMP production in rat primary neuronal and glial cell (astrocyte) cultures. The actions of both peptides were much more pronounced in astrocytes than in neuronal cultures. Stimulatory effects of PACAP and VIP on cyclic AMP formation were significantly smaller in cell cultures subjected to 24h lasting hypoxic conditions, induced either chemically (100 microM cobalt chloride) or by low 3% oxygen hypoxia, compared to the normoxic condition (95% air and 5% CO(2)). This picture contrasted with the effects of forskolin that were similar under normoxic and hypoxic conditions. It is suggested that hypoxia leads to changes in PACAP- and VIP-driven cyclic AMP-dependent signaling in the rat brain by influencing molecular processes likely occurring at the level of receptor protein or receptor-Gs protein coupling. 相似文献