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1.
Hydrobiologia - Aquatic ecosystems provide vital services, and macrophytes play a critical role in their functioning. Conceptual models indicate that in shallow lakes, plants with different growth...  相似文献   
2.
Han Z  Simpson JT  Fivash MJ  Fisher R  Mori T 《Peptides》2004,25(4):551-561
Cyanovirin-N (CV-N) exerts a potent human immunodeficiency virus (HIV)-inactivating activity against diverse strains of HIV by binding to the viral surface envelope glycoprotein gp120 and blocking its essential interactions with cellular receptors. Based on previous thermodynamic analyses, it has been speculated that discrete protein-protein interactions might play an important ancillary role in the CV-N/gp120 binding event, in addition to the interactions of CV-N with specific oligosaccharides present on gp120. Here, we report the identification and characterization of CV-N-binding peptides, which were isolated by screening of M13 phage-displayed peptide libraries. After performing three rounds of biopanning of the libraries against biotinylated CV-N, a CV-N-binding motif, X3CX6(W/F)(Y/F)CX2(Y/F), was evident. A vector was designed to express CV-N-binding peptides as a fusion with thioredoxin (Trx) containing a penta-His affinity tag. The CV-N-binding peptides fused with His-tagged Trx inhibited binding of the corresponding peptide-bearing phages to CV-N, confirming that the peptides possessed CV-N-binding activity. Optical biosensor binding studies showed that the one of the CV-N-binding peptide, TN10-1, bound to CV-N with a KD value of 1.9 microM. The results of alanine scanning mutagenesis of the peptide showed that aromatic residues at positions 11, 12, and 16, as well as the conformational structure of the peptide secured by a disulfide bond, were important for the binding interactions. A series of competitive binding assays confirmed that gp120 inhibited CV-N binding of the corresponding peptide-bearing phages, and suggested that TN10-1 peptides were mimicking the protein component of gp120 rather than mimicking specific oligosaccharides present on gp120.  相似文献   
3.
The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)(n) oligonucleotides using surface plasmon resonance (SPR) spectroscopy. We found a single NC protein is able to bind to more than one immobilized oligonucleotide, provided that the oligonucleotides are close enough together. As NC is believed to be the nucleic acid binding domain of Gag, we might expect Gag to show the same complex behavior. We wished to analyze the stoichiometry of Gag binding to oligonucleotides without this complication due to tertiary complex formation. We have therefore analyzed Gag binding to extremely low oligonucleotide density on SPR chips. Such low densities of oligonucleotides are difficult to accurately quantitate. We have determined by Fourier transform ion cyclotron (FTICR) mass spectrometry that four molecules of NC bind to d(TG)(10) (a 20-base oligonucleotide). We developed a method of calibrating low-density surfaces using NC calibration injections. Knowing the maximal response and the stoichiometry of binding, we can precisely determine the amount of oligonucleotide immobilized at these very-low-density surfaces (<1 Response Unit). Using this approach, we have measured the binding of Gag to d(TG)(10). Gag binds to a 20-mer with a stoichiometry of greater than 4. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules can bind to this complex.  相似文献   
4.
The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.  相似文献   
5.
The enrichment of HIV-1 macromolecules at the uropod of polarized T cells can significantly promote virus assembly and cell-mediated infection. Using live-cell fluorescence microscopy, we demonstrate that full-length HIV-1 RNA is enriched at the uropod membrane; furthermore, the presence of HIV-1 Gag containing a functional nucleocapsid domain is necessary for this HIV-1 RNA enrichment. The results from these studies provide novel insights into the mechanism of HIV-1 replication in polarized T cells.  相似文献   
6.
Restoration of coastal ecosystem engineers that trap sediment and dampen waves has proven to be difficult, especially in the wave‐exposed and eroding areas where they are needed the most. Environmental stressors, such as hydrodynamic stress and predation, can only be overcome if transplanted organisms are able to establish self‐facilitating feedbacks. We investigate if the artificial lowering of multiple environmental stressors can be used to give transplanted juveniles the opportunity to form a self‐sustainable system and thereby increase their long‐term survival on wave‐exposed and eroding shores. We designed a large field experiment using juvenile mussels (Mytilus edulis) as model species on a wave‐exposed tidal flat in the Oosterschelde estuary (the Netherlands). We tested if the environmental stress caused by a high predation pressure and wave‐driven dislodgement could be reduced by a combination of artificial structures such as fences (to exclude predatory crabs), attachment substrates (such as coir‐net or oyster shells), and breakwaters. Despite a low overall mussel survival (29%), we found that under strong hydrodynamic conditions, experimental fences and attachment substrates increased the retention of transplanted mussel seed. However, modification of local hydrodynamic conditions using breakwaters did not improve mussel coverage preservation. Overall, this study highlights the potential of using techniques that lower multiple environmental stressors to create a window of opportunity for establishment in highly dynamic ecosystems.  相似文献   
7.
Resistance to apoptosis is afforded by inhibitor of apoptosis proteins (IAPs) which bind to and inhibit the caspases responsible for cleavage of substrates leading to apoptotic cell death. Smac (or DIABLO), a proapoptotic protein released from the mitochondrial intermembrane space into the cytosol, promotes apoptosis by binding to IAPs, thus reversing their inhibitory effects on caspases. We have developed a high-throughput fluorescence polarization assay utilizing a fluorescein-labeled peptide similar to the "IAP binding" domain of Smac N terminus complexed with the BIR3 domain of X-linked IAP (XIAP) to identify small-molecule mimics of the action of Smac. The IC(50)s of peptides and a tetrapeptidomimetic homologous to the N terminus of Smac demonstrated the specificity and utility of this assay. We have screened the National Cancer Institute "Training Set" of 230 compounds, with well-defined biological actions, and the "Diversity Set" of 2000 chemically diverse structures for compounds which significantly reduced fluorescence polarization. Highly fluorescing or fluorescence-quenching compounds (false positives) were distinguished from those which interfered with Smac peptide binding to the XIAP-BIR3 in a dose-dependent manner (true positives). This robust assay offers potential for high-throughput screening discovery of novel compounds simulating the action of Smac/DIABLO.  相似文献   
8.
Occupational exposure to nickel has been epidemiologically linked to increased cancer risk in the respiratory tract. Nickel-induced cell transformation is associated with both genotoxic and epigenetic mechanisms that are poorly understood. Prolidase [E.C.3.4.13.9] is a cytosolic Mn(II)-activated metalloproteinase that specifically hydrolyzes imidodipeptides with C-terminal proline or hydroxyproline and plays an important role in the recycling of proline for protein synthesis and cell growth. Prolidase also provides free proline as substrate for proline oxidase, whose gene is activated by p53 during apoptosis. The inhibition of prolidase activity by nickel has not yet been studied. We first showed that Ni(II) chloride specifically inhibited prolidase activity in CHO-K1 cells in situ. This interpretation was possible because CHO-K1 cells are proline auxotrophs requiring added free proline or proline released from added Gly-Pro by prolidase. In a dose-dependent fashion, Ni(II) inhibited growth on Gly-Pro but did not inhibit growth on proline, thereby showing inhibition of prolidase in situ in the absence of nonspecific toxicity. Studies using cell-free extracts showed that Ni(II) inhibited prolidase activity when present during prolidase activation with Mn(II) or during incubation with Gly-Pro. In kinetic studies, we found that Ni(II) inhibition of prolidase varied with respect to Mn(II) concentration. Analysis of these data suggested that increasing concentrations of Mn(II) stabilized the enzyme protein against Ni(II) inhibition. Because prolidase is an important enzyme in collagen metabolism, inhibition of the enzyme activity by nickel could alter the metabolism of collagen and other matrix proteins, and thereby alter cell-matrix and cell-cell interactions involved in gene expression, genomic stability, cellular differentiation, and cell proliferation.  相似文献   
9.
Interactions of Ni(II) with the base moieties of 2'-deoxynucleosides and 2'-deoxynucleotides were studied by means of UV difference spectroscopy in order to elucidate the mechanisms of site-specific enhancement by Ni(II) of DNA base oxidation with active oxygen species, observed previously (Kasprzak et al., Cancer Res., 49 (1989) 5964; Carcinogenesis, 11 (1990) 647). The interactions were generally weak and could be quantitated only at pH 7.2-7.9. The resulting coordination binding of Ni(II) was stronger with the purine derivatives, especially these of guanine, than with pyrimidine derivatives. Also, Ni(II) interacted more strongly with the bases of 2'-deoxynucleotides than with the bases of 2'-deoxynucleosides. The apparent stability constants for the interactions calculated with the use of a non-linear regression method, equalled 102 +/- 14, 159 +/- 30 and 290 +/- 70 M-1 for Ni(II) coordinated by 5'dAMP, 5'dADP and 5'dATP, respectively, and 305 +/- 73, 191 +/- 54, and 270 +/- 28 M-1 for 5'dGMP, 5'dGDP and 5'dGTP, respectively. Stability constant for the dG Ni(II) interaction was 39 +/- 7 M-1. Interactions of Ni(II) with the bases of dA, dC, dT and the dC- and dT- mono-, di- and tri-phosphates were too weak for meaningful quantitation. The strongest relative Ni(II) interaction with dG may explain high sensitivity of the dG site at the DNA molecule to Ni(II)-mediated oxidation observed in vitro and in vivo. The present results contrast with Ni(II)-directed site specific cleavage of DNA with H2O2 that occurs preferentially at the pyrimidine bases (Kawanishi et al., Carcinogenesis, 10 (1989) 2231).  相似文献   
10.
We have analyzed the binding of recombinant human immunodeficiency virus type 1 nucleocapsid protein (NC) to very short oligonucleotides by using surface plasmon resonance (SPR) technology. Our experiments, which were conducted at a moderate salt concentration (0.15 M NaCl), showed that NC binds more stably to runs of d(G) than to other DNA homopolymers. However, it exhibits far more stable binding with the alternating base sequence d(TG)n than with any homopolymeric oligodeoxyribonucleotide; thus, it shows a strong sequence preference under our experimental conditions. We found that the minimum length of an alternating d(TG) sequence required for stable binding was five nucleotides. Stable binding to the tetranucleotide d(TG)2 was observed only under conditions where two tetranucleotide molecules were held in close spatial proximity. The stable, sequence-specific binding to d(TG)n required that both zinc fingers be present, each in its proper position in the NC protein, and was quite salt resistant, indicating a large hydrophobic contribution to the binding. Limited tests with RNA oligonucleotides indicated that the preferential sequence-specific binding observed with DNA also occurs with RNA. Evidence was also obtained that NC can bind to nucleic acid molecules in at least two distinct modes. The biological significance of the specific binding we have detected is not known; it may reflect the specificity with which the parent Gag polyprotein packages genomic RNA or may relate to the functions of NC after cleavage of the polyprotein, including its role as a nucleic acid chaperone.A single protein species, the Gag polyprotein, is sufficient for assembly of retrovirus particles. Since this process includes the selective encapsidation of viral RNA, this protein is evidently capable of specific interactions with nucleic acids. The nature of these interactions is not well understood as yet. After the virion is released from the cell, the polyprotein is cleaved by the virus-encoded protease; one of the cleavage products, termed the nucleocapsid protein (NC), then binds to the genomic RNA, forming the ribonucleoprotein core of the mature particle (21, 35, 41).The interaction between Gag and the genomic RNA is known to involve the NC domain of the polyprotein, since mutants within this domain of Gag are defective in RNA packaging (e.g., references 2, 16, 17, 2427, 31, 36, 37, and 39) and since the specificity of encapsidation tends to be determined by the NC domain in chimeric Gag molecules (9, 18, 49). However, NC is a basic protein and has frequently been described as binding to single-stranded DNA or RNA in a sequence-independent manner. Indeed, it is probably capable of binding to any single-stranded nucleic acid under appropriate conditions. This binding activity appears to be crucial at several stages of virus replication (13, 19, 28, 46).In the experiments described here, we have analyzed the binding of recombinant human immunodeficiency virus type 1 (HIV-1) NC to short oligonucleotides. These studies were performed at moderate ionic strengths, at which the nonspecific electrostatic interaction between NC and nucleic acids is minimized. We find that under these conditions, the protein exhibits profound sequence preferences. This sequence-specific binding is dependent upon the zinc fingers of the protein and has a strong hydrophobic component. The biological significance of this sequence specificity is not clear at present, but the results suggest that studies with very short oligonucleotides may provide important insights into NC function and perhaps functions of Gag as well.  相似文献   
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