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71.
Development and Validation of a Real-Time PCR Method To Quantify Rumen Protozoa and Examination of Variability between Entodinium Populations in Sheep Offered a Hay-Based Diet 总被引:2,自引:0,他引:2 下载免费PDF全文
Lucy C. Skillman Andrew F. Toovey Andrew J. Williams Andr-Denis G. Wright 《Applied microbiology》2006,72(1):200-206
PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR () was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R2 = 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (100 and 106 entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required. 相似文献
72.
73.
Kate Bramham Carlos E. Poli-de-Figueiredo Paul T. Seed Annette L. Briley Lucilla Poston Andrew H. Shennan Lucy C. Chappell 《PloS one》2013,8(10)
Objectives
To evaluate occurrence of adverse maternal and perinatal outcomes with different thresholds of proteinuria (300-499mg and ≥500mg/24 hours) in pre-eclamptic women, comparing outcomes against women with chronic and gestational hypertension.Design
Secondary analysis of the Vitamins in Pre-Eclampsia Trial.Setting
25 UK hospitals in ten geographical areas.Population
946 women with pre-existing risk factors for pre-eclampsia.Methods
Women with pre-eclampsia and proteinuria 300-499mg/24h (PE300, referent group, n=60) or proteinuria ≥500 mg/24h (PE500, n=161) were compared with two groups of non-proteinuric women with chronic hypertension (CHT, n=615) or gestational hypertension (GH, n=110).Main Outcome Measures
Maternal: progression to severe hypertension. Perinatal: small for gestational age (SGA) <5th centile, gestation at delivery.Results
Severe hypertension occurred more frequently in PE500 (35%) and PE300 (27%) than CHT (5.9%; P≤0.01) and GH (10%; p≤0.001). Gestation at delivery was earlier in PE500 (33.2w) than PE300 (37.3w; P≤0.001), and later in CHT (38.3w; P≤0.05) and GH (39.1w; P≤0.001). SGA infants were more frequent in PE300 (32%) than in CHT (13.3%; P≤0.001) and GH (16.5%; P≤0.05). Women in PE500 were more likely to have a caesarean section than PE300 (78% vs. 48%; P≤0.001), and to receive magnesium sulphate (17% vs. 1.7%, P≤0.05).Conclusion
Women with PE300 have complication rates above those of women managed as out-patients (GH and CHT), meriting closer surveillance and confirming 300 mg/d as an appropriate threshold for determining in-patient management. Adverse perinatal outcomes are higher still in women with PE500. 相似文献74.
Paul Aia Margaret Kal Evelyn Lavu Lucy N. John Karen Johnson Chris Coulter Julia Ershova Olga Tosas Matteo Zignol Shalala Ahmadova Tauhid Islam 《PloS one》2016,11(3)
Background
Reliable estimates of the burden of multidrug-resistant tuberculosis (MDR-TB) are crucial for effective control and prevention of tuberculosis (TB). Papua New Guinea (PNG) is a high TB burden country with limited information on the magnitude of the MDR-TB problem.Methods
A cross-sectional study was conducted in four PNG provinces: Madang, Morobe, National Capital District and Western Province. Patient sputum samples were tested for rifampicin resistance by the Xpert MTB/RIF assay and those showing the presence of resistance underwent phenotypic susceptibility testing to first- and second-line anti-TB drugs including streptomycin, isoniazid, rifampicin, ethambutol, pyrazinamide, ofloxacin, amikacin, kanamycin and capreomycin.Results
Among 1,182 TB patients enrolled in the study, MDR-TB was detected in 20 new (2.7%; 95% confidence intervals [CI] 1.1–4.3%) and 24 previously treated (19.1%; 95%CI: 8.5–29.8%) TB cases. No case of extensively drug-resistant TB (XDR-TB) was detected. Thirty percent (6/20) of new and 33.3% (8/24) of previously treated cases with MDR-TB were detected in a single cluster in Western Province.Conclusion
In PNG the proportion of MDR-TB in new cases is slightly lower than the regional average of 4.4% (95%CI: 2.6–6.3%). A large proportion of MDR-TB cases were identified from a single hospital in Western Province, suggesting that the prevalence of MDR-TB across the country is heterogeneous. Future surveys should further explore this finding. The survey also helped strengthening the use of smear microscopy and Xpert MTB/RIF testing as diagnostic tools for TB in the country. 相似文献75.
Osamu Kaneko Lucy Gong Jingli Zhang Johanna K. Hansen Raffit Hassan Byungkook Lee Mitchell Ho 《The Journal of biological chemistry》2009,284(6):3739-3749
Ovarian cancer and malignant mesothelioma frequently express both
mesothelin and CA125 (also known as MUC16) at high levels on the cell surface.
The interaction between mesothelin and CA125 may facilitate the implantation
and peritoneal spread of tumors by cell adhesion, whereas the detailed nature
of this interaction is still unknown. Here, we used truncated mutagenesis and
alanine replacement techniques to identify a binding site on mesothelin for
CA125. We examined the molecular interaction by Western blot overlay assays
and further quantitatively analyzed by enzyme-linked immunosorbent assay. We
also evaluated the binding on cancer cells by flow cytometry. We identified
the region (296–359) consisting of 64 amino acids at the N-terminal of
cell surface mesothelin as the minimum fragment for complete binding activity
to CA125. We found that substitution of tyrosine 318 with an alanine abolished
CA125 binding. Replacement of tryptophan 321 and glutamic acid 324 with
alanine could partially decrease binding to CA125, whereas mutation of
histidine 354 had no effect. These results indicate that a
conformation-sensitive structure of the region (296–359) is required and
sufficient for the binding of mesothelin to CA125. In addition, we have shown
that a single chain monoclonal antibody (SS1) recognizes this CA125-binding
domain and blocks the mesothelin-CA125 interaction on cancer cells. The
identified CA125-binding domain significantly inhibits cancer cell adhesion
and merits evaluation as a new therapeutic agent for preventing or treating
peritoneal malignant tumors.Ovarian cancer largely is confined to the peritoneal cavity for much of its
natural history (1). Peritoneal
mesothelioma is a highly invasive tumor originating from the mesothelial
linings of the peritoneum (2).
The development of effective drug regimens against ovarian cancer and
mesothelioma has proven extremely difficult.Mesothelin was first identified in 1992 by the monoclonal antibody
(mAb)2 K1 that was
generated by the immunization of mice with human ovarian carcinoma (OVCAR-3)
cells (3). The mesothelin gene
encodes a 71-kDa precursor protein that is processed to a 40-kDa protein
termed mesothelin, which is a glycosylphosphatidylinositol (GPI)-anchored
glycoprotein present on the cell surface
(4). Mesothelin is a
differentiation antigen that is present on a restricted set of normal adult
tissues such as the mesothelium. In contrast, it is overexpressed in a variety
of cancers including mesothelioma, ovarian cancer, and pancreatic cancer
(5). In addition, mesothelin is
also expressed on the surface of non-small cell lung cancer cells
(6,
7), especially most lung
adenocarcinomas (8).We and others have shown that mesothelin is shed from tumor cells
(9,
10), and antibodies specific
for mesothelin are elevated in the sera of patients with mesothelioma and
ovarian cancer (11). Shed
serum mesothelin has been approved by the United States Food and Drug
Administration (FDA) as a new diagnostic biomarker in mesothelioma. In a Phase
I clinical study of an intrapleural interferon-β gene transfer using an
adenoviral vector in patients with mesotheliomas, we found that antitumor
immune responses targeting mesothelin were elicited in several patients
(12). A recent study indicated
that anti-mesothelin antibodies and circulating mesothelin relate to the
clinical state in ovarian cancer patients
(13). Pastan and colleagues
(14) developed an immunotoxin
(SS1P) with a Fv for mesothelin. Two Phase I clinical trials were completed at
the National Cancer Institute (National Institutes of Health, Bethesda, MD)
and there was sufficient antitumor activity of SS1P to justify a Phase II
trial. A chimeric antibody containing the mouse SS1 Fv for mesothelin was also
developed and is currently examined in a Phase I clinical trial for ovarian
cancer, mesothelioma, pancreatic cancer, and non-small cell lung cancer
(15).Mucins are heavily glycosylated proteins found in the mucus layer or at the
cell surface of many epitheliums
(16). There are two
structurally distinct families of mucins, secreted and membrane-bound forms.
CA125 (also known as MUC16) was first identified in 1981 by OC125, a mAb that
had been developed from mice immunized with human ovarian cancer cells
(17). The first cDNA clones
were reported in 2001 (18,
19). CA125 is a very large
membrane-bound cell surface mucin, with an average molecular mass between 2.5
and 5 million daltons. It is also heavily glycosylated with both
O-linked and N-linked oligosaccharides
(20). The peptide backbone of
CA125 is composed of the N-terminal region, extensive Ser/Thr/Pro-rich tandem
repeats (TR) with 156 amino acids each with both N- and
O-glycosylations, a SEA domain with high levels of
O-glycosylation and a C-terminal region with a short cytoplasmic tail
(19). The SEA domain was first
identified as a module commonly found in sea urchin sperm protein,
enterokinase and agrin (21,
22). The significance of the
SEA domain in CA125 is not clear.CA125 was originally used as a biomarker in ovarian cancer due to its high
expression in ovarian carcinomas and that it is shed into the serum
(23). A majority (88%) of
mesotheliomas are also CA125 positive on the cell membrane
(24). It was shown that 25% of
peritoneal mesotheliomas have high CA125 expression
(25). The intensity of CA125
membranous expression is indistinguishable between ovarian carcinomas and
peritoneal mesotheliomas. Gene expression analysis using the SAGE tag data
base has shown that mesothelioma has the second highest co-expression of CA125
and mesothelin after ovarian cancer
(26). Rump and colleagues
(26) have shown that
mesothelin binds to CA125 and that this interaction may mediate cell adhesion.
Scholler et al. (27)
recently showed that CA125/mesothelin-dependent cell attachment could be
blocked with anti-CA125 antibodies. Because mesothelin is present on
peritoneal mesothelium, there may be an important role for the
mesothelin-CA125 interaction in the tumorigenesis of ovarian cancer and
mesothelioma in the peritoneal cavity. The mesothelin binding site on CA125
may lie within the 156-amino acid TR units, indicating multimeric binding of
mesothelin to CA125. It has been found that the extraordinarily abundant
N-glycans on CA125, presumably in the TR region, are required for
binding to both glycosylated and non-glycosylated mesothelin
(28).Here, we identified the binding site of CA125 on mesothelin by use of
truncated mutagenesis and alanine replacement approaches. We measured binding
qualitatively by Western blot overlay assays and quantitatively by
enzyme-linked immunosorbent assay (ELISA). We also evaluated the interaction
of CA125 and mesothelin on cancer cells by flow cytometry. Furthermore, we
have shown that a single chain mAb (SS1) recognized the CA125-binding domain
and blocked the mesothelin-CA125 interaction on cancer cells. The identified
CA125-binding domain-Fc fusion protein also significantly inhibited cancer
cell adhesion. Our results suggest that conformation-sensitive structures of
the region (296–359) are required and sufficient for specific binding of
mesothelin to CA125. The domain proteins or the antibodies that block the
mesothelin-CA125 interaction merit evaluation as new therapeutic agents in
treating peritoneal malignant tumors. 相似文献
76.
Sally E. Thomas Elke Malzer Adriana Ordó?ez Lucy E. Dalton Emily F. A. van ′t Wout Elizabeth Liniker Damian C. Crowther David A. Lomas Stefan J. Marciniak 《The Journal of biological chemistry》2013,288(11):7606-7617
Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G1 phase, although recent studies have identified a novel ER stress G2 checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G2 delay involving CHK1 and a later induction of G1 arrest associated both with the induction of p53 target genes and loss of cyclin D1. We show that substitution of p53/47 for p53 impairs the ER stress G1 checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G2 progression prior to ultimate G1 arrest. 相似文献
77.
HIV-1 trafficking to the dendritic cell-T-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse 总被引:3,自引:0,他引:3
Garcia E Pion M Pelchen-Matthews A Collinson L Arrighi JF Blot G Leuba F Escola JM Demaurex N Marsh M Piguet V 《Traffic (Copenhagen, Denmark)》2005,6(6):488-501
Dendritic cells (DCs) are essential components of the early events of HIV infection. Here, we characterized the trafficking pathways that HIV-1 follows during its capture by DCs and its subsequent presentation to CD4(+) T cells via an infectious synapse. Immunofluorescence microscopy indicates that the virus-containing compartment in mature DCs (mDCs) co-labels for the tetraspanins CD81, CD82, and CD9 but contains little CD63 or LAMP-1. Using ratio imaging of pH-reporting fluorescent virions in live DCs, we show that HIV-1 is internalized in an intracellular endocytic compartment with a pH of 6.2. Significantly, we demonstrate that the infectivity of cell-free virus is more stable at mildly acidic pH than at neutral pH. Using electron microscopy, we confirm that HIV-1 accumulates in intracellular vacuoles that contain CD81 positive internal membranes but overlaps only partially with CD63. When allowed to contact T cells, HIV-1-loaded DCs redistribute CD81, and CD9, as well as internalized HIV-1, but not the immunological synapse markers MHC-II and T-cell receptor to the infectious synapse. Together, our results indicate that HIV-1 is internalized into a non-conventional, non-lysosomal, endocytic compartment in mDCs and further suggest that HIV-1 is able to selectively subvert components of the intracellular trafficking machinery required for formation of the DC-T-cell immunological synapse to facilitate its own cell-to-cell transfer and propagation. 相似文献
78.
Two TIR:NB:LRR genes are required to specify resistance to Peronospora parasitica isolate Cala2 in Arabidopsis 总被引:6,自引:0,他引:6
Sinapidou E Williams K Nott L Bahkt S Tör M Crute I Bittner-Eddy P Beynon J 《The Plant journal : for cell and molecular biology》2004,38(6):898-909
Resistance responses that plants deploy in defence against pathogens are often triggered following a recognition event mediated by resistance (R) genes. The encoded R proteins usually contain a nucleotide-binding site (NB) and a leucine-rich repeat (LRR) domain. They are further classified into those that contain an N-terminal coiled coil (CC) motif or a Toll interleukin receptor (TIR) domain. Such R genes, when transferred into a susceptible plant of the same or closely related species, usually impart full resistance capability. We have used map-based cloning and mutation analysis to study the recognition of Peronospora parasitica (RPP)2 (At) locus in Arabidopsis accession Columbia (Col-0), which is a determinant of specific recognition of P. parasitica (At) isolate Cala2. Genetic mapping located RPP2 to a 200-kb interval on chromosome 4, which contained four adjacent TIR:NB:LRR genes. Mutational analysis revealed three classes of genes involved in specifying resistance to Cala2. One class, which resulted in pleiotropic effects on resistance to other P. parasitica (At) isolates, was unlinked to the RPP2 locus; this class included AtSGT1b. The other two classes were mapped within the interval and were specific to Cala2 resistance. Representatives of each of these classes were sequenced, and mutations were found in one or the other of two (RPP2A and RPP2B) of the four TIR:NB:LRR genes. RPP2A and RPP2B complemented their specific mutations, but failed to impart resistance when present alone, and it is concluded that both genes are essential determinants for isolate-specific recognition of Cala2. RPP2A has an unusual structure with a short LRR domain at the C-terminus, preceded by two potential but incomplete TIR:NB domains. In addition, the RPP2A LRR domain lacks conserved motifs found in all but three other TIR:NB:LRR class proteins. In contrast, RPP2B has a complete TIR:NB:LRR structure. It is concluded that RPP2A and RPP2B cooperate to specify Cala2 resistance by providing recognition or signalling functions lacked by either partner protein. 相似文献
79.
Biolog system was evaluated for the identification of strains of Paenibacillus azotofixans as no data concerning this species were in the list of Bacillus currently identified using Biolog data base. The P. azotofixans type strain P3L5 was first tested with the results recorded manually or using the automatic plate reader. In both cases, P3L5 utilized 22 carbon sources and when the results obtained were compared to data of Biolog software (Release 3.50), P3L5 was identified as B. azotoformans with a similarity coefficient of 0.913 (data recorded manually) and of 0.791 (data recorded automatically). Metabolic profiles of P3L5 were also compared after readings of 4 and 24 h using the computer-driven automatic plate reader. No significant difference was observed in both cases and P3L5 was identified again as B. azotoformans with indices of similarity considered only for excellent identification. Besides P3L5, other 15 P. azotofixans strains were tested with the Biolog system and all were identified as B. azotoformans with similarity coefficients varying from 0.511 to 0.927. Phenotypic and genetic characteristics of B. azotoformans were compared to those described for P. azotofixans to explain the misidentification of the latter species. We could conclude that these two species are quite different and that data of Biolog software are from P. azotofixans and not from B. azotoformans. 相似文献