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51.
With over 25 monoclonal antibodies (mAbs) currently approved and many more in development, there is considerable interest in gaining improved productivity by increasing cell density and enhancing cell survival of production cell lines. In addition, high costs and growing safety concerns with use of animal products have made the availability of serum-free cell lines more appealing. We elected to transfect the myeloma cell line Sp2/0-Ag14 with Bcl2-EEE, the constitutively active phosphomimetic mutant of Bcl2, for extended cell survival. After adaptation of the initial transfectants to serum-independent growth, a clone with superior growth properties, referred to as SpESF, was isolated and further subjected to iterative rounds of stressful growth over a period of 4 months. The effort resulted in the selection of a promising clone, designated SpESFX-10, which was shown to exhibit robust growth and resist apoptosis induced by sodium butyrate or glutamine deprivation. The advantage of SpESFX-10 as a host for generating mAb-production cell lines was demonstrated by its increased transfection efficiency, culture longevity, and mAb productivity, as well as by the feasibility of accomplishing the entire cell line development process, including transfection, subcloning, and cryopreservation, in the complete absence of serum. 相似文献
52.
Andréia S Lessa Bruno D Paredes Juliana V Dias Adriana B Carvalho Luiz Fernando Quintanilha Christina M Takiya Bernardo R Tura Guilherme FM Rezende Antonio C Campos de Carvalho Célia MC Resende Regina CS Goldenberg 《BMC veterinary research》2010,6(1):1-10
Background
Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.Results
This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.Conclusions
Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage. 相似文献53.
54.
Benoit NE Goldenberg D Deng SX Rosenbaum E Cohen Y Califano JA Shackelford WH Wang XB Sidransky D 《BioTechniques》2005,38(4):635-639
High-throughput genomic mutation screening for primary tumors has characteristically been expensive, labor-intensive, and inadequate to detect low levels of mutation in a background of wild-type signal. We present a new, combined PCR and colorimetric approach that is inexpensive, simple, and can detect the presence of 1% mutation in a background of wild-type. We compared manual dideoxy sequencing of p53 for eight lung cancer samples to a novel assay combining a primer extension step and an enzymatic colorimetric step in a 96-well plate with covalently attached oligonucleotide sequences. For every sample, we were able to detect the presence or absence of the specific mutation with a statistically significant difference between the sample optical density (OD) and the background OD, with a sensitivity and specificity of 100%. This assay is straightforward, accurate, inexpensive, and allows for rapid, high-throughput analysis of samples, making it ideal for genomic mutation or polymorphism screening studies in both clinical and research settings. 相似文献
55.
Development of humanized antibodies as cancer therapeutics 总被引:1,自引:0,他引:1
Qu Z Griffiths GL Wegener WA Chang CH Govindan SV Horak ID Hansen HJ Goldenberg DM 《Methods (San Diego, Calif.)》2005,36(1):84-95
Recent success in the development of monoclonal antibody-based anti-cancer drugs has largely benefitted from the advancements made in recombinant technologies and cell culture production. These reagents, derived from the antibodies of mouse origin, while maintaining the exquisite specificity and affinity to the tumor antigens, have low immunogenicity and toxicity in human. High-level expressing cell clones are generated and used to produce large quantities of the recombinant antibodies in bioreactors in order to meet the clinical demand for therapeutic applications. In this report, the systems and general methodologies developed by us to construct and produce humanized antibodies from the parent mouse antibodies are described. Once the humanized antibodies are available, they can be applied in three principal forms for cancer therapy: (1) naked antibodies, (2) drug- or toxin conjugates, and (3) radioconjugates. Using the humanized anti-CD22 (epratuzumab) and anti-carcinoembryonic antigen (ant-CEA; labetuzumab) antibody prototypes, clinical applications of naked and radiolabeled humanized monoclonal antibodies are described. 相似文献
56.
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58.
Angers S Thorpe CJ Biechele TL Goldenberg SJ Zheng N MacCoss MJ Moon RT 《Nature cell biology》2006,8(4):348-357
Dishevelled is a conserved protein that interprets signals received by Frizzled receptors. Using a tandem-affinity purification strategy and mass spectrometry we have identified proteins associated with Dishevelled, including a Cullin-3 ubiquitin ligase complex containing the Broad Complex, Tramtrack and Bric à Brac (BTB) protein Kelch-like 12 (KLHL12). This E3 ubiquitin ligase complex is recruited to Dishevelled in a Wnt-dependent manner that promotes its poly-ubiquitination and degradation. Functional analyses demonstrate that regulation of Dishevelled by this ubiquitin ligase antagonizes the Wnt-beta-catenin pathway in cultured cells, as well as in Xenopus and zebrafish embryos. Considered with evidence that the distinct Cullin-1 based SCF(beta-TrCP)complex regulates beta-catenin stability, our data on the stability of Dishevelled demonstrates that two distinct ubiquitin ligase complexes regulate the Wnt-beta-catenin pathway. 相似文献
59.
Dörner T Kaufmann J Wegener WA Teoh N Goldenberg DM Burmester GR 《Arthritis research & therapy》2006,8(3):R74-11
B cells play an important role in the pathogenesis of systemic lupus erythematosus (SLE), so the safety and activity of anti-B
cell immunotherapy with the humanized anti-CD22 antibody epratuzumab was evaluated in SLE patients. An open-label, single-center
study of 14 patients with moderately active SLE (total British Isles Lupus Assessment Group (BILAG) score 6 to 12) was conducted.
Patients received 360 mg/m2 epratuzumab intravenously every 2 weeks for 4 doses with analgesic/antihistamine premedication (but no steroids) prior to
each dose. Evaluations at 6, 10, 18 and 32 weeks (6 months post-treatment) follow-up included safety, SLE activity (BILAG
score), blood levels of epratuzumab, B and T cells, immunoglobulins, and human anti-epratuzumab antibody (HAHA) titers. Total
BILAG scores decreased by ≥ 50% in all 14 patients at some point during the study (including 77% with a ≥ 50% decrease at
6 weeks), with 92% having decreases of various amounts continuing to at least 18 weeks (where 38% showed a ≥ 50% decrease).
Almost all patients (93%) experienced improvements in at least one BILAG B- or C-level disease activity at 6, 10 and 18 weeks.
Additionally, 3 patients with multiple BILAG B involvement at baseline had completely resolved all B-level disease activities
by 18 weeks. Epratuzumab was well tolerated, with a median infusion time of 32 minutes. Drug serum levels were measurable
for at least 4 weeks post-treatment and detectable in most samples at 18 weeks. B cell levels decreased by an average of 35%
at 18 weeks and remained depressed at 6 months post-treatment. Changes in routine safety laboratory tests were infrequent
and without any consistent pattern, and there was no evidence of immunogenicity or significant changes in T cells, immunoglobulins,
or autoantibody levels. In patients with mild to moderate active lupus, 360 mg/m2 epratuzumab was well tolerated, with evidence of clinical improvement after the first infusion and durable clinical benefit
across most body systems. As such, multicenter controlled studies are being conducted in broader patient populations. 相似文献
60.
Steinfeld SD Tant L Burmester GR Teoh NK Wegener WA Goldenberg DM Pradier O 《Arthritis research & therapy》2006,8(4):R129
This open-label, phase I/II study investigated the safety and efficacy of epratuzumab, a humanised anti-CD22 monoclonal antibody, in the treatment of patients with active primary Sj?gren's syndrome (pSS). Sixteen Caucasian patients (14 females/2 males, 33-72 years) were to receive 4 infusions of 360 mg/m2 epratuzumab once every 2 weeks, with 6 months of follow-up. A composite endpoint involving the Schirmer-I test, unstimulated whole salivary flow, fatigue, erythrocyte sedimentation rate (ESR), and immunoglobulin G (IgG) was devised to provide a clinically meaningful assessment of response, defined as a > or = 20% improvement in at least two of the aforementioned parameters, with > or = 20% reduction in ESR and/or IgG considered as a single combined criterion. Fourteen patients received all infusions without significant reactions, 1 patient received 3, and another was discontinued due to a mild acute reaction after receiving a partial infusion. Three patients showed moderately elevated levels of Human anti-human (epratuzumab) antibody not associated with clinical manifestations. B-cell levels had mean reductions of 54% and 39% at 6 and 18 weeks, respectively, but T-cell levels, immunoglobulins, and routine safety laboratory tests did not change significantly. Fifty-three percent achieved a clinical response (at > or = 20% improvement level) at 6 weeks, with 53%, 47%, and 67% responding at 10, 18, and 32 weeks, respectively. Approximately 40%-50% responded at the > or = 30% level, while 10%-45% responded at the > or = 50% level for 10-32 weeks. Additionally, statistically significant improvements were observed in fatigue, and patient and physician global assessments. Further, we determined that pSS patients have a CD22 over-expression in their peripheral B cells, which was downregulated by epratuzumab for at least 12 weeks after the therapy. Thus, epratuzumab appears to be a promising therapy in active pSS, suggesting that further studies be conducted. 相似文献