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51.
A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found. 相似文献
52.
L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three
loci in all vertebrates examined, with the exception of lampreys, which
have a single LDH locus. Biochemical characterizations of LDH proteins have
suggested that a gene duplication early in vertebrate evolution gave rise
to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of
lineages more recently. Although some phylogenetic studies of LDH protein
sequences have supported this pattern of gene duplication, others have
contradicted it. In particular, a number of studies have suggested that
Ldh-C represents the earliest divergence among vertebrate LDHs and that it
may have diverged from the other loci well before the origin of
vertebrates. Such hypotheses make explicit statements about the
relationship of vertebrate and invertebrate LDHs, but to date, no closely
related invertebrate LDH sequences have been available for comparison. We
have attempted to provide further data on the timing of gene duplications
leading to multiple vertebrate LDHs by determining the cDNA sequence of the
LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other
LDH sequences provide strong support for the duplications giving rise to
multiple vertebrate LDHs having occurred after vertebrates diverged from
tunicates. The timing of these LDH duplications is consistent with data
from a number of other gene families suggesting widespread gene duplication
near the origin of vertebrates. With respect to the relationships among
vertebrate LDHs, our data are not consistent with previous claims that
Ldh-C represented the earliest divergence. However, the precise
relationships among some of the main lineages of vertebrate LDHs were not
resolved in our analyses.
相似文献
53.
54.
Maier LM Howlett SK Rainbow KM Clark J Howson JM Todd JA Wicker LS 《Journal of immunology (Baltimore, Md. : 1950)》2008,181(10):7073-7080
NK cells from NOD mice induced with poly(I:C) in vivo exhibit low cytotoxicity against a range of target cells, but the genetic mechanisms controlling this defect are yet to be elucidated. Defects in the expression of NKG2D and its ligands, the RAE-1 molecules, have been hypothesized to contribute to the reduced NK function present in NOD mice. In this study, we show that segregation of the NK-mediated killing phenotype did not correlate with the NOD Raet1 haplotype and that the large alterations in NKG2D expression previously reported on NK cells expanded in vitro were not observed in primary, poly(I:C)-elicited NK cells in vivo. Additional studies indicate a complex genetic control of defective NOD NK cells including genes linked to the MHC and possibly those that are associated with an altered cytokine response to the TLR3-agonist poly(I:C). 相似文献
55.
Ágnes Baross Allen D Delaney H Irene Li Tarun Nayar Stephane Flibotte Hong Qian Susanna Y Chan Jennifer Asano Adrian Ally Manqiu Cao Patricia Birch Mabel Brown-John Nicole Fernandes Anne Go Giulia Kennedy Sylvie Langlois Patrice Eydoux JM Friedman Marco A Marra 《BMC bioinformatics》2007,8(1):1-18
Background
Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.Results
We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.Conclusion
We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity. 相似文献56.
Schilders G Raijmakers R Malmegrim KC Vande Walle L Saelens X Vree Egberts W van Venrooij WJ Vandenabeele P Pruijn GJ 《Arthritis research & therapy》2007,9(1):R12
Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive
systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that
functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly
in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75,
is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75
cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by
caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal
part of the protein at Asp369 (IILD369↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally,
the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed. 相似文献
57.
AB Chang NC Cox J Purcell JM Marchant PJ Lewindon GJ Cleghorn LC Ee GD Withers MK Patrick J Faoagali 《Respiratory research》2005,6(1):1-5
Background and methods
Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.Results
We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 105 viral copies/ml in nasal lavage and 1.88 × 105 viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.Conclusion
HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely. 相似文献58.
59.
Haringman JJ Vinkenoog M Gerlag DM Smeets TJ Zwinderman AH Tak PP 《Arthritis research & therapy》2005,7(4):R862-R867
Analysis of biomarkers in synovial tissue is increasingly used in the evaluation of new targeted therapies for patients with
rheumatoid arthritis (RA). This study determined the intrarater and inter-rater reliability of digital image analysis (DIA)
of synovial biopsies from RA patients participating in clinical trials. Arthroscopic synovial biopsies were obtained before
and after treatment from 19 RA patients participating in a randomized controlled trial with prednisolone. Immunohistochemistry
was used to detect CD3+ T cells, CD38+ plasma cells and CD68+ macrophages. The mean change in positive cells per square millimetre for each marker was determined by different operators
and at different times using DIA. Nonparametric tests were used to determine differences between observers and assessments,
and to determine changes after treatment. The intraclass correlations (ICCs) were calculated to determine the intrarater and
inter-rater reliability. Intrarater ICCs showed good reliability for measuring changes in T lymphocytes (R = 0.87), plasma
cells (R = 0.62) and macrophages (R = 0.73). Analysis by Bland–Altman plots showed no systemic differences between measurements.
The smallest detectable changes were calculated and their discriminatory power revealed good response in the prednisolone
group compared with the placebo group. Similarly, inter-rater ICCs also revealed good reliability for measuring T lymphocytes
(R = 0.68), plasma cells (R = 0.69) and macrophages (R = 0.72). All measurements identified the same cell types as changing
significantly in the treated patients compared with the placebo group. The measurement of change in total positive cell numbers
in synovial tissue can be determined reproducibly for various cell types by DIA in RA clinical trials. 相似文献
60.
Luis AJ Mur Amanda J Lloyd Simona M Cristescu Frans JM Harren Michael A Hall Aileen R Smith 《Plant signaling & behavior》2009,4(7):610-613
The hypersensitive response (HR) is a cell death phenomenon associated with localized resistance to pathogens. Biphasic patterns in the generation of H2O2, salicylic acid and ethylene have been observed in tobacco during the early stages of the HR. These biphasic models reflect an initial elicitation by pathogen-associated molecular patterns followed by a second phase, induced by pathogen-encoded avirulence gene products. The first phase has been proposed to potentiate the second, to increase the efficacy of plant resistance to disease. This potentiation is comparable to the “priming” of plant defenses which is seen when plants display systemic resistance to disease. The events regulating the generation of the biphasic wave, or priming, remains obscure, however recently we demonstrated a key role for nitric oxide in this process in a HR occurring in tobacco. Here we use laser photoacoustic detection to demonstrate that biphasic ethylene production also occurs during a HR occurring in Arabidopsis. We suggest that ethylene emanation during the HR represents a ready means of visualising biphasic events during the HR and that exploiting the genomic resources offered by this model species will facilitate the development of a mechanistic understanding of potentiating/priming processes.Key words: hypersensitive response, biphasic patterns, potentiation, defense priming, ethylene, ArabidopsisThe Hypersensitive Response (HR) is a cell death process which occurs at the site of attempted pathogen attack and which has been associated with host resistance.1 Much work on the regulation of the HR has indicated the importance of H2O2,2 and NO.3 A feature of H2O2 generation during the HR is its biphasic pattern (Fig. 1A). The first rise reflects elicitation by pathogen-associated molecular patterns (PAMPs)4 and the second reflects the interaction between a pathogen-encoded avirulence (avr) gene product with a plant resistance (R) gene. A key aspect of the first rise is the initiation of salicylic acid (SA) synthesis which potentiates the second rise and hence the potency of plant defense and the HR.5Open in a separate windowFigure 1Patterns of defense signal generation during the Pseudomonas syringae pv. phaseolicola elicited-hypersensitive response in tobacco (Nicotiana tabacum). Generation of (A) H2O2 (●, Mur18); (B) nitric oxide (◇; Mur12 (C) salicylic acid (SA, ■19) and (D) ethylene (○ Mur9) during a HR elicited by Pseudomonas syringae pv. phaseolicola (Psph) in tobacco cv. Samsun NN. In (A) a phase where SA acts to augment the second rise in H2O2—the potentiation phase—is highlighted. The potentiation phase is likely to be similar to defense “priming”.6 Methodological details are contained within the appropriate references. (E) A possible model for biphasic defense signal regulation during the Psph-elicited HR in tobacco. During an initial phase NO and H2O2 act to initiate SA biosynthesis, where SA and NO act to initiate a “H2O2 biphasic switch”. This could initially suppress both SA and the H2O2 generation but subsequently acts to potentiate a second phase of H2O2 generation. This in turn increases SA biosynthesis which could act with NO to initiate the “C2H4 biphasic switch” to potentiate ethylene production. These (and other) signals contribute to initiation of the HR and SAR.This potentiation mechanism appears to be similar to defense priming; when whole plants display systemic resistance to disease as opposed to a localized resistance against pathogens. Priming can be initiated (the “primary stimulus”) following attack with a necrotizing pathogen (leading to “systemic acquired resistance”, SAR) or non-pathogenic rhizosphere bacteria (to confer “induced systemic resistance”, ISR). In the primed state a plant stimulates a range of plant defense genes, produces anti-microbial phytoalexins and deposits cell wall strengthening molecules, but only on imposition of a “secondary stimulus”.6 Such secondary stimuli include SA3 or PAMPs7 and is likely to be mechanistically similar to the potentiation step in the biphasic pattern of H2O2 generation (shaded in Fig. 1A). Accordingly, the two phases in the biphasic wave represent primary and secondary stimuli in priming.Highlighting a similarity between local HR-based events and priming, adds further impetus to efforts aiming to describe the underlying mechanism(s), however both phenomena remain poorly understood. Besides SA, both jasmonates and abscisic acid (ABA) have been shown to prime defenses as have a range of non-plant chemicals, with β-aminobutyric acid (BABA) being perhaps most widely used.6,8 Mutants which fail to exhibit BABA-mediated potentiation were defective in either a cyclin-dependent kinase-like protein, a polyphosphoinositide phosphatase or an ABA biosynthetic enzyme.8We have recently investigated biphasic ethylene production during the HR in tobacco elicited by the nonhost HR-eliciting bacterial pathogen Pseudomonas syringae pv. phaseolicola.9 As with H2O2 generation, this pattern reflected PAMP-and AVR-dependent elicitation events and included a SA-mediated potentiation stage. Crucially, we also showed that NO was a vital component in the SA-potentiation mechanism. When this finding is integrated with our other measurements of defense signal generation in the same host-pathogen system the complexity in the signaling network is revealed (Fig. 1). NO generation (Fig. 1B) appeared to be coincident with the first rise in H2O2 (Fig. 1A) which initiated SA biosynthesis10,11 and together would contribute to the first small, but transient, rise in that hormone (Fig. 1C). In line with established models5 this momentary rise in SA coincides with the potentiation phase (shaded in Fig. 1A) required to augment the second rise in ROS. However, ethylene production seems to be correlated poorly with the patterns of NO, H2O2 and SA (Fig. 1D). Nevertheless, biphasic ethylene production was found to reflect PAMP and AVR-dependent recognition and included a SA-mediated potentiation step.9 Hence, ethylene production could be used as a post-hoc indicator of the potentiation mechanism. Therefore, our discovery that the second wave of ethylene production—a “biphasic switch”—is influenced by NO acting with SA could also be relevant to the H2O2 generation. Significantly, the second phases in both H2O2 and ethylene production occur exactly where SA and NO production coincides; in the case of H2O2 generation 2–4 h post challenge and with ethylene 6 h onwards (Fig. 1E).Thus, ethylene production represents a readily assayable marker to indicate perturbations in the underlying biphasic and possible priming mechanisms. As we have demonstrated, laser photoacoustic detection (LAPD) is a powerful on-line approach to determine in planta ethylene production in tobacco9,12 but any mechanistic investigations would be greatly facilitated if the genetic resources offered by the model species Arabidopsis could be exploited.To address this, Arabidopsis Col-0 rosettes were vacuum infiltrated with either Pseudomonas syringae pv. tomato (Pst) avrRpm1 (HR-eliciting), the virulent Pst strain and the non-HR eliciting and non-virulent Pst hrpA strain. Ethylene production was monitored by LAPD (Fig. 2A). Significantly, Pst avrRpm1 initiated a biphasic pattern of ethylene production whose kinetics were very similar to that seen in tobacco (compare Figs. 2A with with1D).1D). Inoculations with Pst and Pst hrpA only displayed the first PAMP-dependent rise in ethylene production. Thus, these data establish that Arabidopsis can be used to investigate biphasic switch mechanism(s) in ethylene production during the HR and possibly defense priming. When considering such mechanisms, it is relevant to highlight the work of Foschi et al.13 who observed that biphasic activation of a monomeric G protein to cause phase-specific activation of different kinase cascades. Interestingly, ethylene has been noted to initiate biphasic activation of G proteins and kinases in Arabidopsis, although differing in kinetics to the phases seen during the HR.14 Further, plant defense priming has been associated with the increased accumulation of MAP kinase protein.6Open in a separate windowFigure 2Ethylene in the Pseudomonas syringae pv. tomato elicited-hypersensitive response in Arabidopsis thaliana. (A) Ethylene production from 5 week old short day (8 h light 100 µmol.m2.sec−1) grown Arabidopsis rosette leaves which were vacuum infiltrated with bacterial suspensions (2 × 106 colony forming units.ml−1) of Pseudomonas syringae pv. tomato (Pst) strains detected using laser photoacoustic detection (LAPD). Experimental details of the ethylene detection by LAPD are detailed in Mur et al.9 The intercellular spaces in leaves were infiltrated with the HR-eliciting strain Pst avrRpm1, (■), the virulent strain Pst (△) or the non-virulent and non-HR eliciting derivative, Pst hrpA (◇). (B) The appearance of Arabidopsis Col-0 and etr1-1 leaves at various h following injection with 2 × 106 c.f.u.mL−1 with of Pst avrRpm1. (C) Explants (1 cm diameter discs) from Arabidopsis leaf areas infiltrated with suspensions of Pst avrRpm1 were placed in a 1.5 cm diameter well, bathed in 1 mL de-ionized H2O. Changes in the conductivity of the bathing solution, as an indicator of electrolyte leakage from either wild type Col-0 (◆), mutants which were compromised in ethylene signaling; etr1-1 (□), ein2-2 (▲) or which overproduced ethylene; eto2-1 (●) were measured using a conductivity meter. Methodological details are set out in Mur et al.9A further point requires consideration; the role of ethylene as a direct contributor to plant defense.15 The contribution of ethylene to the HR has been disputed,16 but in tobacco we have observed that altered ethylene production influenced the formation of a P. syringae pv. phaseolicola elicited HR.9 In Arabidopsis, cell death in the ethylene receptor mutant etr1-1 following inoculation with Pst avrRpm1 is delayed compared to wild type (Fig. 2B). When electrolyte leakage was used to quantify Pst avrRpm1 cell death, both etr1-1 and the ethylene insensitive signaling mutant ein2-1 exhibited slower death than wild-type but in the ethylene overproducing mutant eto2, cell death was augmented (Fig. 2C). These data indicate that ethylene influences the kinetics of the HR.Taking these data together we suggest that the complexity of signal interaction during the HR or in SAR/ISR could be further dissected by combining the genetic resources of Arabidopsis with measurements of ethylene production using such sensitive approaches as LAPD. 相似文献