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111.
目的:探讨HIV-1 Tat蛋白对人外周血B淋巴细胞增殖、凋亡的影响及其机制。方法:采用流式细胞分选术分离HIV阳性患者外周血单核细胞的B淋巴细胞,分别转染pTat或pcDNA3.1各10μg(分别为pTat组与pcDNA3.1组),采用MTT实验检测细胞胞增殖情况,流式细胞术检测凋亡情况,DCHF-DA测定ROS水平,彗星试验检测细胞DNA损伤情况。结果:pTat组转染24h、48h的细胞增殖抑制率、细胞凋亡率及线粒体ROS水平均显著高于pcDNA3.1组(P0.05)。pcDNA3.1组细胞的DNA大部分呈圆形荧光团,无拖尾现象;pTat组的细胞DNA拖尾现象,呈现典型彗星图像。与pcDNA3.1组相比,pTat组细胞DNA尾长、尾部DNA比例均显著增加(P 0.05)。结论:HIV-1 Tat蛋白可能通过增加线粒体ROS产生,诱导DNA损伤,进而抑制人外周血B淋巴细胞增殖并促进其凋亡。 相似文献
112.
113.
Elizabeth P. Harrison Nicola M. Willingham Julie C. Lloyd Christine A. Raines 《Planta》1997,204(1):27-36
Transgenic tobacco (Nicotiana tabacum L. cv. Samsun) plants with reduced levels of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37)
were produced using an antisense construct in which the expression of a tobacco SBPase cDNA clone was driven by the cauliflower
mosaic virus (CaMV) promoter. The reduction in SBPase protein levels observed in the primary transformants correlated with
the presence of the antisense construct and lower levels of the endogenous SBPase mRNA. No changes in the amounts of other
Calvin cycle enzymes were detected using Western blot analysis. The SBPase antisense plants with less than 20% of wild-type
SBPase activity were observed to display a range of phenotypes, including chlorosis and reduced growth rates. Measurements
of photosynthesis, using both light-dosage response and CO2 response curves, of T1 plants revealed a reduction in carbon assimilation rates, which was apparent in plants retaining 57%
of wild-type SBPase activity. Reductions were also observed in the quantum efficiency of photosystem II. This decrease in
photosynthetic capacity was reflected in a reduction in the carbohydrate content of leaves. Analysis of carbohydrate status
in fully expanded source leaves showed a shift in carbon allocation away from starch, whilst sucrose levels were maintained
in all but the most severely affected plants. Plants with less than 15% of wild-type SBPase activity were found to contain
less than 5% of wild-type starch levels. The results of this preliminary analysis indicate that SBPase activity may limit
the rate of carbon assimilation.
Received: 23 February 1997 / Accepted: 2 May 1997 相似文献
114.
Antisense RNA complementary to the Epstein-Barr virus (EBV) Zta gene, an immediate-early gene encoding a transactivator, was applied to inhibit EBV protein synthesis during its lytic cycle. A DNA fragment containing the Zta gene sequence was inserted into an expression vector, pMAMneo, in a sense and antisense direction under a dexamethasone-inducible murine mammary tumor virus LTR promoter, resulting in the construction of plasmids pZ(+) and pZ(–), respectively. Synthesis of Zta protein was reduced in pZ(–)-transfected cells upon dexamethasone induction. Because D-form early antigen and DNA polymerase are essential for viral DNA replication, the contents of these two viral proteins were examined. Amounts of the two lytic proteins were observed to be significantly repressed in pZ(–)-transfected cells. In contrast, both proteins were normally expressed in the sense plasmid pZ(+) or cells transfected with vector alone. Above results demonstrate that Zta antisense RNA can reduce the production of Zta protein and the other lytic proteins, possibly resulting in the inhibition of EBV replication. 相似文献
115.
Eric Vivès Claude Granier Paul Prevot Bernard Lebleu 《International journal of peptide research and therapeutics》1997,4(4-6):429-436
Summary Tat, a 86-amino acid protein involved in the replication of Human Immunodeficiency Virus type 1 (HIV-1), is able to translocate
efficiently through the plasma membrane and to reach the nucleus to transactivate the viral genome. The region 37–72 of the
Tat protein, centered on a cluster of basic amino acids, has been assigned to this translocation activity. Recent data in
our group have attributed this membrane translocating activity to a peptide extending from residues 48 to 60, which contains
a cluster of eight basic amino acids within a linear sequence of nine residues. Internalization of this peptide into cells
occurred within minutes at concentrations as low as 100 nM. In order to define more precisely the involvement of these basic
amino acids in peptide translocation, several analogues carrying deletions or substitutions of one, or several, of the basic
residues were synthesized and tested for their cellular uptake and nuclear translocation. A direct correlation between the
overall charge of the peptide and its cell internalization was found. In addition, the covalent linkage of this short basic
peptide allows the efficient translocation of a non-membrane permeant peptide. 相似文献
116.
The N-terminal portion of HIV-1 Tat covering residues 1-9 is a competitive inhibitor of dipeptidyl peptidase IV (DP IV). We have used 1H NMR techniques, coupled with molecular dynamics methods, to determine the conformation of this peptide in the three diverse media: DMSO-d6, water (pH 2.7) and 40% HFA solution. The results indicate that in both DMSO-d6 and HFA the peptide has a tendency to acquire a type I beta-turn around the segment Asp5-Pro6-Asn7-IIe8. The N-terminal end is seen to be as a random coil. In water, the structure is best described as a left-handed polyproline type II (PPII) helix for the mid segment region Asp2 to Pro6. The structures obtained in this study have been compared with an earlier report on Tat (1-9). 相似文献
117.
The mechanism of fertility inhibition of conjugation by the F plasmid depends on the presence of both the FinO protein and
an antisense RNA, FinP, which together control the expression of the positive regulator of the transfer operon TraJ. FinO
both prevents the degradation of FinP, allowing its intracellular concentration to rise, and promotes duplex formation with
its target, the traJ mRNA. In this study, deletions in finO were constructed and fused to gst, encoded by the pGEX-2T expression vector, to give GST-FinO fusions of varying lengths. These fusions were then tested for
their ability to bind FinP and traJ mRNA, and to promote duplex formation. Our results suggest that the predicted basic N-terminal α-helix is involved in RNA
binding, while the central domain is involved in duplex formation. The presence of the acidic C-terminal domain protects FinP
from ribonucleolase degradation and might enhance binding of the N-terminal α-helical domain in a manner reminiscent of the
Rom protein of ColE1.
Received: 4 May 1998 / Accepted: 15 June 1998 相似文献
118.
Identification of a binding site of the human immunodeficiency virus envelope protein gp120 to neuronal‐specific tubulin
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119.
120.
HIV-1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS-related dementia complex. The HIV-1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat-induced neurotoxicity. Tat increased [Ca2+]i dose-dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium release [8-(diethylamino)octyl-3,4,5-trimethoxybenzoate HCI (TMB-8) and xestospongin], an inhibitor of receptor-Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK-801, (+/-)-2-amino-5-phosphonopentanoic acid, and 6,7-dinitroquinoxaline-2,3-dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB-8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat-induced neurotoxicity. These results suggest that Tat, via pertussis toxin-sensitive phospholipase C activity, induces calcium release from IP3-sensitive intracellular stores, which leads to glutamate receptor-mediated calcium influx, dysregulation of [Ca2+]i, and Tat-induced neurotoxicity. 相似文献