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1.
The efficiency of "LiCl transformation" in Saccharomyces cerevisiae haploid cells by an autonomously replicating pLL12 plasmid carrying yeast LEU2 and LYS2 genes is increased (by an order or more) when the plasmid is linearized by the restriction endonuclease XhoI cleavage of a unique site in LYS2 gene. Transformants were selected on the medium lacking leucine. This phenomenon has been shown to be a result of recombinational repair of double-strand breaks (DSB) of plasmid DNA stimulated by a restriction endonuclease. The kinetic data have shown the process of plasmid DNA DSB repair to consist of two phases. The completion of the first phase occurs during an hour and the second phase occurs in 14-18 hours. DNA double-strand gaps (the deleted sequences of plasmid LYS2 gene in DSB region) with maximal length of 2-2.5 kb are repaired with the same efficiency as DSB. The genetic control of the recombinational repair of plasmid DNA DSB has been studied.  相似文献   
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Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca2+-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca2+-receptor protein with specific subsets of response elements.  相似文献   
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Taxol, a microtubule stabilizing agent, exhibits promise in the treatment of breast and ovarian tumors. Recently, this novel drug has been shown to activate murine macrophages to express TNF-alpha and to down-regulate TNF-alpha receptors, activities shared by bacterial LPS. Our study sought to determine if taxol could regulate gene expression in murine macrophages and to examine further the ability of taxol to generate an LPS-like signal. Toward this end, the ability of taxol to induce TNF-alpha mRNA and five other genes (IL-1 beta, IP-10, D3, D7, and D8) associated with LPS-activation of macrophages was examined by Northern blot analysis. Taxol alone (1-30 microM) induced murine C3H/OuJ macrophages to secrete bioactive TNF-alpha and express increased levels of each of the six genes under investigation. The magnitude and the kinetics of induction of each gene closely resembled that seen with Escherichia coli K235 LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice, however, failed to induce detectably any of the genes in response to taxol, despite being sensitive to the microtubule stabilizing effects of taxol as determined by immunofluorescence microscopy. The gene induction activity of taxol was in marked contrast to an alternative macrophage activator, heat killed Staphylococcus aureus, which induced a distinct gene profile in C3H/OuJ macrophages and which was equally active in C3H/OuJ and C3H/HeJ macrophages. These data are consistent with an ability of taxol to generate an LPS-like signal, possibly through a common signaling intermediate. As a first step toward identifying signal responses shared by taxol and LPS, we have shown that taxol, as shown previously for LPS, rapidly induces the tyrosine phosphorylation of a 41- and 42-kDa protein.  相似文献   
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Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or ‘antispacer’, PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.  相似文献   
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Interleukin (IL)–15 is an inflammatory cytokine that constitutes a validated therapeutic target in some immunopathologies, including rheumatoid arthritis (RA). Previously, we identified an IL‐15 antagonist peptide named [K6T]P8, with potential therapeutic application in RA. In the current work, the metabolic stability of this peptide in synovial fluids from RA patients was studied. Moreover, [K6T]P8 peptide was labeled with 99mTc to investigate its stability in human plasma and its biodistribution pattern in healthy rats. The biological activity of [K6T]P8 peptide and its dimer was evaluated in CTLL‐2 cells, using 3 different additives to improve the solubility of these peptides. The half‐life of [K6T]P8 in human synovial fluid was 5.88 ± 1.73 minutes, and the major chemical modifications included peptide dimerization, cysteinylation, and methionine oxidation. Radiolabeling of [K6T]P8 with 99mTc showed a yield of approximately 99.8%. The 99mTc‐labeled peptide was stable in a 30‐fold molar excess of cysteine and in human plasma, displaying a low affinity to plasma proteins. Preliminary biodistribution studies in healthy Wistar rats suggested a slow elimination of the peptide through the renal and hepatic pathways. Although citric acid, sucrose, and Tween 80 enhanced the solubility of [K6T]P8 peptide and its dimer, only the sucrose did not interfere with the in vitro proliferation assay used to assess their biological activity. The results here presented, reinforce nonclinical characterization of the [K6T]P8 peptide, a potential agent for the treatment of RA and other diseases associated with IL‐15 overexpression.  相似文献   
7.
A two-fold difference in sensitivity to cis-diamminedichloroplatinum(II) (cisplatin), as judged by colony forming assays, has been demonstrated in two human bladder carcinoma continuous cell lines. Approximately twice as many DNA-DNA interstrand cross-links (ISL) and a 2-fold greater inhibition of DNA synthesis occurred in the more sensitive T24 cell line than in the RT112 cell line after exposure to the same concentrations of cisplatin. Equitoxic concentrations of cisplatin resulted in similar extents of ISL and inhibition of DNA synthesis in both cell lines. Although drug uptake was identical, twice as much cisplatin was bound to the DNA of T24 cells than RT112 cells. However after equitoxic concentrations of cisplatin the DNA from both cell lines was platinated to a similar extent. In addition, levels of glutathione (GSH), glutathione reductase (GR) and total glutathione-S-transferases (GST) were higher in the less sensitive RT112 cell line.  相似文献   
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The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.  相似文献   
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