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991.
Keyhole limpet hemocyanin (KLH)-conjugated peptides are routinely used to raise polyclonal antibodies for biochemical or immunolocalization studies. Rats are suitable for producing antisera against plant antigens as they often lack non-specific response towards plant materials. We attempted to obtain rat antisera against peptides derived from several plant proteins. However, most antisera recognized the same background KLH-related plant antigen (KRAP) in Arabidopsis and tobacco. We characterized KRAP with respect to size and cellular localization and examined possible antigen-specific reasons for the failure of most immunizations. We also found no reports of successful use of rat anti-KLH-peptide antibodies in plant studies. We thus believe that the rat-KLH:peptide system is poorly suited for production of antibodies, especially against plant antigens, and should be used with caution, if at all. 相似文献
992.
993.
994.
Sheila M. Levy Flávio Moscardi Reinaldo J. Silva 《Journal of invertebrate pathology》2009,101(1):17-22
In this investigation, the anterior and posterior regions of the midgut of resistant (RL) and non-resistant (SL) Anticarsia gemmatalis larvae were analyzed morphometrically to characterize different regions along their length. Also, this investigation compares the results between SL and RL to improve the understanding of the resistance mechanisms to the virus. Histological sections were analyzed in a computerized system and the data were statistically analyzed by the Kruskal-Wallis test and by multivariate analysis. The midguts are morphometrically different in the two larval populations; we observed higher values in RL. The morphometric analysis of the epithelial cells showed that only columnar and goblet cells were distinct along the midgut, in both larvae, with the higher values found in the anterior region. Comparing the results between the two larval populations, all the epithelial cells presented significant differences, with RL showing the higher morphometric values. We concluded that there are regional differences along the length of midgut in SL and RL that confirm the idea of two morpho-functional distinct regions. The consistently morphometric superior values in RL indicate that this variability can be related with the resistance of A. gemmatalis to its AgMNPV. 相似文献
995.
Fredrik I. Andersson Anders Tryggvesson Michal Sharon Alexander V. Diemand Mirjam Classen Christoph Best Ronny Schmidt Jenny Schelin Tara M. Stanne Bernd Bukau Carol V. Robinson Susanne Witt Axel Mogk Adrian K. Clarke 《The Journal of biological chemistry》2009,284(20):13519-13532
The Clp protease is conserved among eubacteria and most eukaryotes, and
uses ATP to drive protein substrate unfolding and translocation into a chamber
of sequestered proteolytic active sites. The main constitutive Clp protease in
photosynthetic organisms has evolved into a functionally essential and
structurally intricate enzyme. The model Clp protease from the cyanobacterium
Synechococcus consists of the HSP100 molecular chaperone ClpC and a
mixed proteolytic core comprised of two distinct subunits, ClpP3 and ClpR. We
have purified the ClpP3/R complex, the first for a Clp proteolytic core
comprised of heterologous subunits. The ClpP3/R complex has unique functional
and structural features, consisting of twin heptameric rings each with an
identical ClpP33ClpR4 configuration. As predicted by its
lack of an obvious catalytic triad, the ClpR subunit is shown to be
proteolytically inactive. Interestingly, extensive modification to ClpR to
restore proteolytic activity to this subunit showed that its presence in the
core complex is not rate-limiting for the overall proteolytic activity of the
ClpCP3/R protease. Altogether, the ClpP3/R complex shows remarkable
similarities to the 20 S core of the proteasome, revealing a far greater
degree of convergent evolution than previously thought between the development
of the Clp protease in photosynthetic organisms and that of the eukaryotic 26
S proteasome.Proteases perform numerous tasks vital for cellular homeostasis in all
organisms. Much of the selective proteolysis within living cells is performed
by multisubunit chaperone-protease complexes. These proteases all share a
common two-component architecture and mode of action, with one of the best
known examples being the proteasome in archaebacteria, certain eubacteria, and
eukaryotes (1).The 20 S proteasome is a highly conserved cylindrical structure composed of
two distinct types of subunits, α and β. These are organized in
four stacked heptameric rings, with two central β-rings sandwiched
between two outer α-rings. Although the α- and β-protein
sequences are similar, it is only the latter that is proteolytic active, with
a single Thr active site at the N terminus. The barrel-shaped complex is
traversed by a central channel that widens up into three cavities. The
catalytic sites are positioned in the central chamber formed by the
β-rings, adjacent to which are two antechambers conjointly built up by
β- and α-subunits. In general, substrate entry into the core
complex is essentially blocked by the α-rings, and thus relies on the
associating regulatory partner, PAN and 19 S complexes in archaea and
eukaryotes, respectively (1).
Typically, the archaeal core structure is assembled from only one type of
α- and β-subunit, so that the central proteolytic chamber contains
14 catalytic active sites (2).
In contrast, each ring of the eukaryotic 20 S complex has seven distinct
α- and β-subunits. Moreover, only three of the seven
β-subunits in each ring are proteolytically active
(3). Having a strictly
conserved architecture, the main difference between the 20 S proteasomes is
one of complexity. In mammalian cells, the three constitutive active subunits
can even be replaced with related subunits upon induction by
γ-interferon to generate antigenic peptides presented by the class 1
major histocompatibility complex
(4).Two chambered proteases architecturally similar to the proteasome also
exist in eubacteria, HslV and ClpP. HslV is commonly thought to be the
prokaryotic counterpart to the 20 S proteasome mainly because both are Thr
proteases. A single type of HslV protein, however, forms a proteolytic chamber
consisting of twin hexameric rather than heptameric rings
(5). Also displaying structural
similarities to the proteasome is the unrelated ClpP protease. The model Clp
protease from Escherichia coli consists of a proteolytic ClpP core
flanked on one or both sides by the ATP-dependent chaperones ClpA or ClpX
(6). The ClpP proteolytic
chamber is comprised of two opposing homo-heptameric rings with the catalytic
sites harbored within (7). ClpP
alone displays only limited peptidase activity toward short unstructured
peptides (8). Larger native
protein substrates need to be recognized by ClpA or ClpX and then translocated
in an unfolded state into the ClpP proteolytic chamber
(9,
10). Inside, the unfolded
substrate is bound in an extended manner to the catalytic triads (Ser-97,
His-122, and Asp-171) and degraded into small peptide fragments that can
readily diffuse out (11).
Several adaptor proteins broaden the array of substrates degraded by a Clp
protease by binding to the associated HSP100 partner and modifying its protein
substrate specificity (12,
13). One example is the
adaptor ClpS that interacts with ClpA (EcClpA) and targets N-end rule
substrates for degradation by the ClpAP protease
(14).Like the proteasome, the Clp protease is found in a wide variety of
organisms. Besides in all eubacteria, the Clp protease also exist in mammalian
and plant mitochondria, as well as in various plastids of algae and plants. It
also occurs in the unusual plastid in Apicomplexan protozoan
(15), a family of parasites
responsible for many important medical and veterinary diseases such as
malaria. Of all these organisms, photobionts have by far the most diverse
array of Clp proteins. This was first apparent in cyanobacteria, with the
model species Synechococcus elongatus having 10 distinct Clp
proteins, four HSP100 chaperones (ClpB1–2, ClpC, and ClpX), three ClpP
proteins (ClpP1–3), a ClpP-like protein termed ClpR, and two adaptor
proteins (ClpS1–2) (16).
Of particular interest is the ClpR variant, which has protein sequence
similarity to ClpP but appears to lack the catalytic triad of Ser-type
proteases (17). This diversity
of Clp proteins is even more extreme in photosynthetic eukaryotes, with at
least 23 different Clp proteins in the higher plant Arabidopsis
thaliana, most of which are plastid-localized
(18).We have recently shown that two distinct Clp proteases exist in
Synechococcus, both of which contain mixed proteolytic cores. The
first consists of ClpP1 and ClpP2 subunits, and associates with ClpX, whereas
the other has a proteolytic core consisting of ClpP3 and ClpR that binds to
ClpC, as do the two ClpS adaptors
(19). Of these proteases, it
is the more constitutively abundant ClpCP3/R that is essential for cell
viability and growth (20,
21). It is also the ClpP3/R
complex that is homologous to the single type in eukaryotic plastids, all of
which also have ClpC as the chaperone partner
(16). In algae and plants,
however, the complexity of the plastidic Clp proteolytic core has evolved
dramatically. In Arabidopsis, the core complex consists of five ClpP
and four ClpR paralogs, along with two unrelated Clp proteins unique to higher
plants (22). Like ClpP3/R, the
plastid Clp protease in Arabidopsis is essential for normal growth
and development, and appears to function primarily as a housekeeping protease
(23,
24).One of the most striking developments in the Clp protease in photosynthetic
organisms and Apicomplexan parasites is the inclusion of ClpR within the
central proteolytic core. Although this type of Clp protease has evolved into
a vital enzyme, little is known about its activity or the exact role of ClpR
within the core complex. To address these points we have purified the intact
Synechococcus ClpP3/R proteolytic core by co-expression in E.
coli. The recombinant ClpP3/R forms a double heptameric ring complex,
with each ring having a specific ClpP3/R stoichiometry and arrangement.
Together with ClpC, the ClpP3/R complex degrades several polypeptide
substrates, but at a rate considerably slower than that by the E.
coli ClpAP protease. Interestingly, although ClpR is shown to be
proteolytically inactive, its inclusion in the core complex is not
rate-limiting to the overall activity of the ClpCP3/R protease. In general,
the results reveal remarkable similarities between the evolutionary
development of the Clp protease in photosynthetic organisms and the eukaryotic
proteasome relative to their simpler prokaryotic counterparts. 相似文献
996.
Dabros M Dennewald D Currie DJ Lee MH Todd RW Marison IW von Stockar U 《Bioprocess and biosystems engineering》2009,32(2):161-173
This work evaluates three techniques of calibrating capacitance (dielectric) spectrometers used for on-line monitoring of
biomass: modeling of cell properties using the theoretical Cole–Cole equation, linear regression of dual-frequency capacitance
measurements on biomass concentration, and multivariate (PLS) modeling of scanning dielectric spectra. The performance and
robustness of each technique is assessed during a sequence of validation batches in two experimental settings of differing
signal noise. In more noisy conditions, the Cole–Cole model had significantly higher biomass concentration prediction errors
than the linear and multivariate models. The PLS model was the most robust in handling signal noise. In less noisy conditions,
the three models performed similarly. Estimates of the mean cell size were done additionally using the Cole–Cole and PLS models,
the latter technique giving more satisfactory results. 相似文献
997.
Olivier Harismendy Pauline C Ng Robert L Strausberg Xiaoyun Wang Timothy B Stockwell Karen Y Beeson Nicholas J Schork Sarah S Murray Eric J Topol Samuel Levy Kelly A Frazer 《Genome biology》2009,10(3):R32-13
Background
Next generation sequencing (NGS) platforms are currently being utilized for targeted sequencing of candidate genes or genomic intervals to perform sequence-based association studies. To evaluate these platforms for this application, we analyzed human sequence generated by the Roche 454, Illumina GA, and the ABI SOLiD technologies for the same 260 kb in four individuals.Results
Local sequence characteristics contribute to systematic variability in sequence coverage (>100-fold difference in per-base coverage), resulting in patterns for each NGS technology that are highly correlated between samples. A comparison of the base calls to 88 kb of overlapping ABI 3730xL Sanger sequence generated for the same samples showed that the NGS platforms all have high sensitivity, identifying >95% of variant sites. At high coverage, depth base calling errors are systematic, resulting from local sequence contexts; as the coverage is lowered additional 'random sampling' errors in base calling occur.Conclusions
Our study provides important insights into systematic biases and data variability that need to be considered when utilizing NGS platforms for population targeted sequencing studies. 相似文献998.
Michal Amir Shabtai Romano Shlomit Goldman Eliezer Shalev 《Reproductive biology and endocrinology : RB&E》2009,7(1):152-8
Background
To study the expression of Plexin-B1, Glycodelin, and MMP7 during the menstrual cycle in the endometrium and in the fallopian tube. 相似文献999.
NA-Seq: A Discovery Tool for the Analysis of Chromatin Structure and Dynamics during Differentiation
Gaetano Gargiulo Samuel Levy Gabriele Bucci Mauro Romanenghi Lorenzo Fornasari Karen Y. Beeson Susanne M. Goldberg Matteo Cesaroni Marco Ballarini Fabio Santoro Natalie Bezman Gianmaria Frigè Philip D. Gregory Michael C. Holmes Robert L. Strausberg Pier Giuseppe Pelicci Fyodor D. Urnov Saverio Minucci 《Developmental cell》2009,16(3):466-481
1000.