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41.
Swelling and Ca2+-activated Anion Conductances in C127 Epithelial Cells Expressing WT and ΔF508-CFTR
CFTR is a chloride channel that is required for fluid secretion and salt absorption in many exocrine epithelia. Mutations
in CFTR cause cystic fibrosis. CFTR expression influences some ion channels, but the range of channels influenced, the mechanism
of the interaction and the significance for cystic fibrosis are not known. Possible interactions between CFTR and other ion
channels were studied in C127 mouse mammary epithelial cell lines stably transfected with CFTR, ΔF508-CFTR, or vector. Cell
lines were compared quantitatively using an 125I efflux assay and qualitatively using whole-cell patch-clamp recording. As expected, 125I efflux was significantly increased by forskolin only in the CFTR line, and forskolin-stimulated whole-cell currents were
time- and voltage independent. All three lines responded to hypotonic challenge with large 125I efflux responses of equivalent magnitude, and whole-cell currents were outwardly rectified and inactivated at positive voltages.
Unexpectedly, basal 125I efflux was significantly smaller in the ΔF508-CFTR cell line than in either the CFTR or control cell lines (P < 0.0001), and the magnitude of the efflux response to ionomycin was largest in the vector cell line and smallest in the
cell line expressing ΔF508-CFTR (P < 0.01). Whole-cell responses to ionomycin had a linear instantaneous I-V relation and activated at depolarizing voltages. Forskolin responses showed simple summation with responses to ionomycin
or hypotonic challenge. Thus, we found no evidence for interactions between CFTR and the channels responsible for swelling-mediated
responses. Differences were found in basal and ionomycin-stimulated efflux, but these may arise from variations in the clonally
selected cell lines that are unrelated to CFTR expression.
Received: 15 November 1995/Revised: 16 February 1996 相似文献
42.
Role of protein kinase A and the serine-rich region of herpes simplex virus type 1 ICP4 in viral replication. 总被引:8,自引:6,他引:2 下载免费PDF全文
Efficient expression of herpes simplex virus genes requires the synthesis of functional ICP4, a nuclear phosphoprotein that contains a prominent serine-rich region between amino acids 142 and 210. Residues in this region not only are potential sites for phosphorylation but also are involved in the functions of ICP4. By comparing the growth of a virus in which this region is deleted (d8-10) with wild-type virus (KOS) in PC12 cells or PC12 cells that are deficient in cyclic AMP-dependent protein kinase (PKA), two observations were made: (i) the growth of wild-type virus was impaired by 1 to 2 orders of magnitude in the PKA-deficient cells, indicating the involvement of PKA in the growth cycle of herpes simplex virus type 1, and (ii) while the growth of d8-10 was impaired by almost 2 orders of magnitude in wild-type cells, it was not further impaired (as was that of wild-type virus) in PKA-deficient cells, implicating the region deleted in d8-10 as a possible target for cellular PKA. In trigeminal'ganglia of mice, the d8-10 mutant virus grew poorly; however, it established latency in nearly 90% of ganglia tested. Studies of the phosphorylation of wild-type and d8-10 ICP4 proteins revealed that the serine-rich region is a major determinant for phosphorylation of ICP4 in vivo and that the phosphorylation state could change as a function of the PKA activity. Consistent with this observation, the serine-rich region of ICP4 was shown to be a target for PKA in vitro. While intact ICP4 was readily phosphorylated by ICP4 in vitro, the d8-10 mutant ICP4 was not. Moreover, a synthethic peptide representing a sequence in the serine tract that is predicted to be a substrate for PKA was phosphorylated by PKA in vitro, having a Km within the physiological range. These data suggest that PKA plays a role in viral growth through phosphorylation of one or more sites on the ICP4 molecule. 相似文献
43.
44.
中国野豌豆属的分类研究 总被引:8,自引:0,他引:8
本文报道了国产野豌豆属43种,4变种及6变型,其中包括4个新种(多叶野豌豆,三尖野豌豆,武山野豌豆,长齿野豌豆);一个新变种(三叶歪头菜)及一个新等级(千山野豌豆)。 相似文献
45.
Stanislav D. Zakharov Xia Li Taya P. Red'ko Richard A. Dilley 《Journal of bioenergetics and biomembranes》1996,28(6):483-494
The 8-kDa subunit c of theE. coli F0 ATP-synthase proton channel was tested for Ca++ binding activity using a45Ca++ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds45Ca++ strongly with Ca++ binding properties very similar to those of the 8-kDa CF0 subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca++ binding capacity, shown by loss of Ca++ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca++ binding, shown by Ca++ binding being retained in twoE. coli mutants, Asp61Asn and Asp61Gly. The Ca++ binding is pH dependent in both theE. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca++ binding affinity to its F0 binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca++ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca++ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80–100 nM was obtained using an EGTA-Ca++ buffer system to control free Ca++ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca++ bound to the periplasimic side of theE. coli F0 proton channel can block H+ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca++ binding site is on the lumen side of the thylakoid, where Ca++ binding can modulate acid-base jump ATP formation. The Ca++ binding to the F0 and CF0 complexes is consistent with a pH-dependent gating mechanism for control of H+ ion flux across the opening of the H+ channel.This work was supported in part by grants from the Department of Energy and the U.S. Department of Agriculture.On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Science, Pushchino, Russia. 相似文献
46.
懒猴属的核糖体DNA变异及其种间分化关系 总被引:5,自引:2,他引:3
用15种限制性内切酶和人28S、18SrDNA探针构建了懒猴属各物种核糖体DNA重复单位的限制性内切酶图谱。在进化速率较高的非转录间隔区,在大、中、小懒猴中分别定位了23、24、24个酶切位点。大懒猴与中懒猴有12个位点不同,与小懒猴有14个位点不同,而中、小懒猴间则只有一个位点的差异。经过计算,大懒猴与中懒猴的遗传距离值为12.65%,与小懒猴的差异为14.24%,中、小懒猴间的差异则仅为0.7 相似文献
47.
云南姬鼠的蛋白多态性及其遗传分化关系 总被引:3,自引:0,他引:3
本文采用蛋白电泳技术对来源于云南省若干地区的姬鼠属(Apodemus)的3种姬鼠──高山姬鼠(A.chevrieri)8只,中华姬鼠(A.draco)3只和大耳姬鼠(A.latronum)1只,以及作为外群的同科的绒鼠属的大绒鼠(Hapalomysdelalori)3只进行了分析。共检测遗传座位27个,发现21个座位存在多态性。根据蛋白多态的数据对研究对象进行遗传分化关系的探讨,用系统分析软件PHYLIP计算它们之间的分化关系,得到了一棵无根系统树。结果表明,作为外群的大绒鼠明显不同于其它3种姬鼠而聚在最外面。8只高山姬鼠个体汇聚成独立的一支,中华姬鼠的3个个体也聚成一支,但大耳姬鼠却聚在中华姬鼠一支中,因此我们认为大耳姬鼠同中华姬鼠的分化时间可能比较晚近。 相似文献
48.
48例原发性闭经患者的细胞遗传学分析 总被引:9,自引:1,他引:8
郑克勤 李永全 潘超仁 周汝滨 廖霞 陈小萍ZHENG Ke-Qin LI Yong-Quan PAN Chao-Ren ZHOU Ru-Bin LIAO Xia CHEN Xiao-Ping 《遗传》1996,18(1):33-35
本文报告对48例原发闭经患者的临床和细胞遣传学分析,共发现染色体异常17例,占35.4%,其中包括45,X,7例;45,X/46,XX,2例;X染色体结构异常5例;核型中有Y染色体3例。讨论了原发闭经的细胞遗传学病因及异常核型与表型的关系。 相似文献
49.
Gene transfer is a major factor in bacterial evolution 总被引:17,自引:3,他引:14
Lateral gene transfer in four strains of Salmonella enterica has been
assessed using genomic subtraction. Strain LT2 (subspecies I serovar
Typhimurium) chromosomal DNA was used as target and subtracted by three
subspecies I strains of serovars Typhimurium (S21), Muenchen (S71), Typhi
(M229), and a subspecies V strain (M321). Data from probing random cosmids
of LT2 DNA with preparations of the residual LT2 DNA after subtraction were
used to estimate the amounts of LT2 DNA not able to hybridize to strains
S21, S71, M229, and M321 to be in the range of 84-106, 191-355, 305-629,
and 778-1,286 kb, respectively. Several lines of evidence indicate that
most of this DNA is from genes not present in strain M321 and not from
genes that have diverged in sequence. The amounts correlate with the
divergence of the four strains as revealed by multilocus enzyme
electrophoresis and sequence variation of housekeeping genes. Sequence of
39 of the fragments from the M321 subtracted residual LT2 DNA revealed only
six inserts of known gene function with evidence of both gain and loss of
genes during the development of S. enterica clones. Sixteen of the 39
segments have 45% or lower G+C content, below the species average, but over
half are within the normal range for the species. We conclude that even
within a species, clones may differ by up to 20% of chromosomal DNA,
indicating a major role for lateral transfer, and that on the basis of G+C
content, a significant proportion of the DNA is from distantly related
species.
相似文献
50.
将菠菜叶片匀浆后.用差速离心和梯度率心分离叶绿体、过氧物酶体、微粒体等细胞器和100000×g上清法部分。用酶活测定法测定各部分甜菜碱醛脱氢酶(BADH)的活性;用免疫扩散法鉴定各组分的BADH。除叶绿体外,过氧物酶体、微粒体.以及100000×g上清液中也存在BADH。 相似文献