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781.
NMR analysis of the hydrogen bonding interactions of the RNA-binding domains of the Drosophila sex-lethal protein with target RNA fragments with site-specific [3-15N]uridine substitutions. 总被引:1,自引:0,他引:1 下载免费PDF全文
I Kim Y Muto M Inoue S Watanabe A Kitamura S Yokoyama K Hosono H Takaku A Ono M Kainosho H Sakamoto Y Shimura 《Nucleic acids research》1997,25(8):1565-1569
It has been reported that a 183 residue fragment, consisting of the two RNA-binding domains (RBD1- RBD2) of the Drosophila melanogster Sex-lethal (Sxl) protein, strongly binds an oligonucleotide of the target RNA sequence (5'-GUUUUUUUUC-3') that regulates alternative splicing, and forms four or five hydrogen bonds with the imino groups of the RNA. In the present study, we used site-directed mutagenesis to improve the solubility of the didomain fragment of Sxl, and confirmed that this mutant fragment forms hydrogen bonds with the target RNA in the same manner as that of the wild-type fragment. The mutant fragment was shown to bind the cognate RNA sequences GUUUUUUUUC and AUUUUUUUUC more tightly than UUUUUUUUC. By using a [3-15N]uridine phosphoramidite, we synthesized a series of15N-labeled target RNAs, in which one of the uridine residues was specifically replaced by [3-15N]uridine. By observing the imino1H-15N coupling of the labeled uridine residue, we assigned all four of the hydrogen-bonded imino protons to U1, U2, U5 and U6, respectively, of the target RNA. The imino protons of U2 and U6 exhibited nuclear Overhauser effects with aliphatic protons of the protein. All these results indicate that the A/G, U1, U2, U5 and U6 residues in the target sequence of (G/A)UUUUUUUU are specifically recognized by the two RNA-binding domains of the Sxl protein. 相似文献
782.
Extensive polymorphism of the FUT2 gene in an African (Xhosa) population of South Africa 总被引:2,自引:0,他引:2
Yuhua Liu Yoshiro Koda Mikiko Soejima H. Pang Terry Schlaphoff Ernette D. du Toit H. Kimura 《Human genetics》1998,103(2):204-210
The human secretor type α(1,2)fucosyltrans-ferase gene (FUT2) polymorphism was investigated in Xhosa and Caucasian populations of South Africa by polymerase chain reaction–restriction
fragment length polymorphism and DNA sequencing. Six new base substitutions were found in the coding region of FUT2. A single base (C) deletion at nucleotide 778, which led to a frame shift and produced a stop codon at codon 275, was responsible
for the enzyme inactivation. Three nonsynonymous base substitutions, A40G (Ile14Val), C379T (Arg127Cys), and G481A (Asp161Asn), and two synonymous base substitutions, A375G (Glu125) and C480T (His160), were also identified in functional alleles. As a result, seven new alleles, Se
40
, Se
481
, Se
40,481
, Se
357,480
, Se
357,379,480
, Se
375
, and se
357,480,778
were identified. Population studies revealed that an allele containing a nonsense mutation G428A (Trp143stop) (se
428
) was the common null allele in both Xhosa and Caucasian populations, whereas an allele containing a missense A385T (Ile129Phe) mutation (se
357,385
), which is the common null allele in Orientals, was found to be absent from both populations. The heterozygosity rates of
FUT2 genotypes were as high as 0.75 in the Xhosa population and 0.65 in the Caucasian population. Therefore, the extensive polymorphism
and race specificity of the FUT2 gene make it suitable for application as a new tool in genetic studies of modern human evolutionary history.
Received: 23 March 1998 / Accepted: 9 May 1998 相似文献
783.
Characterizing the impact of pressure on virus filtration processes and establishing design spaces to ensure effective parvovirus removal 下载免费PDF全文
Daniel Strauss Joshua Goldstein Tomoko Hongo‐Hirasaki Yoshiro Yokoyama Naokatsu Hirotomi Tomoyuki Miyabayashi Dominick Vacante 《Biotechnology progress》2017,33(5):1294-1302
Virus filtration provides robust removal of potential viral contaminants and is a critical step during the manufacture of biotherapeutic products. However, recent studies have shown that small virus removal can be impacted by low operating pressure and depressurization. To better understand the impact of these conditions and to define robust virus filtration design spaces, we conducted multivariate analyses to evaluate parvovirus removal over wide ranges of operating pressure, solution pH, and conductivity for three mAb products on Planova? BioEX and 20N filters. Pressure ranges from 0.69 to 3.43 bar (10.0–49.7 psi) for Planova BioEX filters and from 0.50 to 1.10 bar (7.3 to 16.0 psi) for Planova 20N filters were identified as ranges over which effective removal of parvovirus is achieved for different products over wide ranges of pH and conductivity. Viral clearance at operating pressure below the robust pressure range suggests that effective parvovirus removal can be achieved at low pressure but that Minute virus of mice (MVM) logarithmic reduction value (LRV) results may be impacted by product and solution conditions. These results establish robust design spaces for Planova BioEX and 20N filters where high parvovirus clearance can be expected for most antibody products and provide further understanding of viral clearance mechanisms. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1294–1302, 2017 相似文献
784.
Involvement of KC, MIP-2, and MCP-1 in leukocyte infiltration following injection of necrotic cells into the peritoneal cavity 总被引:1,自引:0,他引:1
Tanimoto N Terasawa M Nakamura M Kegai D Aoshima N Kobayashi Y Nagata K 《Biochemical and biophysical research communications》2007,361(2):533-536
Although necrotic cells are known to induce inflammation in vivo, the underlying mechanism remains largely unexplored. In order to examine the mechanism, we used an inflammation model induced by injection of necrotic leukemic P388 cells into the peritoneal cavity in this study. The injection of necrotic cells induced the infiltration of neutrophils and subsequently that of monocytes/macrophages. In agreement with this, the injection also induced the production of KC and MIP-2, and subsequently that of MCP-1. Although the level of KC was higher than that of MIP-2, both anti-KC Ab and anti-MIP-2 Ab significantly inhibited the infiltration of neutrophils. Antibodies against CXCR2, a sole receptor for KC and MIP-2, almost completely inhibited the infiltration of neutrophils and monocytes/macrophages. Anti-MCP-1 Ab, on the other hand, inhibited the infiltration of monocytes/macrophages but not neutrophils. These results indicate that KC and MIP-2 play important roles in the infiltration of neutrophils into the site of injection of necrotic cells and that neutrophils may regulate monocyte/macrophage infiltration in our model. 相似文献
785.
Niki E Fukuhara A Omata Y Saito Y Yoshida Y 《Bioorganic & medicinal chemistry letters》2008,18(7):2464-2466
The role of radical-scavenging antioxidant against oxidative stress has received much attention. The antioxidant capacity has been assessed by various methods. Above all, oxygen radical absorbance capacity (ORAC) has been frequently employed [Prior et.al., J. Agric. Food Chem.2005, 53, 4290]. In the present study, the antioxidant capacity of 2,3-dihydro-5-hydroxy-4,6-di-tert-butyl-2,2-dipentylbenzofuran (BO-653) and uric acid was assessed by ORAC method using pyranine as a reference probe and compared with that against lipid peroxidation of human plasma. It was found that BO-653 was assessed to be much less potent than uric acid by ORAC method, whereas BO-653 exerted much higher antioxidant activity than uric acid against plasma lipid peroxidation. The reason for such discrepancy is discussed. The results suggest that ORAC method is suitable for the assessment of free radical scavenging capacity, but not for the assessment of antioxidant capacity against lipid peroxidation in plasma. 相似文献
786.
Minagawa M Watanabe H Miyaji C Tomiyama K Shimura H Ito A Ito M Domen J Weissman IL Kawai K 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(8):4153-4160
IL-2/IL-15Rbeta-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rbeta-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rbeta-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rbeta-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development. 相似文献
787.
The rat mutant allele as is located on chromosome 12. Homozygous (as/as) males show arrested spermatogenesis, mainly at the pachytene spermatocyte stage. It is not clear whether this defective spermatogenesis is caused by a failure in a somatic cell component that supports spermatogenesis or in the germ cell itself. Spermatogonial transplantation was performed to identify the genetically defective site in the as/as testis. In experiment 1, germ cells collected from as/as testes were transplanted into the testes of immunodeficient mice and normal rats. In experiment 2, normal rat germ cells were transplanted into as/as testes. The results of experiment 1 showed arrest of spermatogenesis at the pachytene spermatocyte stage, accompanied by a characteristic morphological feature, i.e., the formation of inclusion-like bodies in the cytoplasm, in both rat and mouse recipients. These results revealed the intrinsic effect of the mutant gene(s) on germ cells. In experiment 2, no restoration of spermatogenesis was detected in the recipient testes despite thorough histological examination. These results suggest that defects in a somatic cell component in as/as testes prevent the donor germ cells from colonizing and regaining their spermatogenetic ability. When the seminiferous epithelium of the as/as testis was examined by electron microscopy, no morphological abnormalities, including the formation of ectoplasmic specializations between adjacent Sertoli cells, were observed in the somatic cell components. However, when cytochrome c was applied as a tracer material, it penetrated the tight junctions between the Sertoli cells, indicating dysfunction of the blood-testis barrier in the as/as testis. The lack of restoration of spermatogenesis in the as/as testis after transplantation of normal germ cells may have been caused by the unfavorable environment in the seminiferous epithelium resulting from the incomplete barrier system between adjoining Sertoli cells. The gene(s) at the as locus may have a role in both germ cell differentiation and the establishment of the blood-testis barrier. 相似文献
788.
ATF3 stimulated promoter activity of EphA1 by 3.4-fold in ATF3-dependent angiogenesis in vitro. Although tyrosine kinase activation of EphA1 was dispensable, binding of EphA1 to fibronectin through its type I repeat played an essential role in the angiogenesis. Recombinant proteins containing fibronectin 10th to 12th type I repeat (I 10-12) but not I 12 could inhibit the angiogenesis in vitro by competitively targeting EphA1 with the full-length fibronectin. However, I 12 acquired a higher affinity toward EphA2 with K(d) 18 nm and inhibited vascular endothelial growth factor-dependent angiogenic invasion in a Matrigel plug assay. 相似文献
789.
Welsh GI Hale LJ Eremina V Jeansson M Maezawa Y Lennon R Pons DA Owen RJ Satchell SC Miles MJ Caunt CJ McArdle CA Pavenstädt H Tavaré JM Herzenberg AM Kahn CR Mathieson PW Quaggin SE Saleem MA Coward RJ 《Cell metabolism》2010,12(4):329-340
Diabetic nephropathy (DN) is the leading cause of renal failure in the world. It is characterized by albuminuria and abnormal glomerular function and is considered a hyperglycemic "microvascular" complication of diabetes, implying a primary defect in the endothelium. However, we have previously shown that human podocytes have robust responses to insulin. To determine whether insulin signaling in podocytes affects glomerular function in vivo, we generated mice with specific deletion of the insulin receptor from their podocytes. These animals develop significant albuminuria together with histological features that recapitulate DN, but in a normoglycemic environment. Examination of "normal" insulin-responsive podocytes in vivo and in vitro demonstrates that insulin signals through the MAPK and PI3K pathways via the insulin receptor and directly remodels the actin cytoskeleton of this cell. Collectively, this work reveals the critical importance of podocyte insulin sensitivity for kidney function. 相似文献
790.
Suzuki Masahiro; Sugatani Junko; Ino Mitsuhiro; Shimura Masahiko; Akiyama Masaki; Yamazaki Ryuta; Suzuki Yasuo; Miwa Masao 《American journal of physiology. Cell physiology》1998,274(1):C47
Human and rabbit platelets fully aggregated byplatelet-activating factor (PAF) underwent slow disaggregation but wererapidly disaggregated by the PAF receptor antagonists WEB-2086,Y-24180, SM-12502, and CV-3988. Whereas the1-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine([3H]acetyl-PAF)specifically bound to platelet receptors underwent slow and spontaneousdissociation, it dissociated promptly from its receptor when WEB-2086was added, in parallel with platelet disaggregation and disappearanceof P-selectin on the cell surface. Extracellular[3H]acetyl-PAF wasrapidly deacetylated by normal rabbit platelets; some of the[3H]acetyl-PAF wasbound to the cells and a very small amount of [3H]acetate wasdetected in the cells. In contrast, when1-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholinewas added to the platelets, the radioactivity was rapidly incorporatedinto the 1-alkyl-2-acyl-sn-glycero-3-phosphocholinefraction. These results indicate that1) continuous binding of PAF to itsreceptor is necessary for prolonged platelet aggregation, which may bemediated through an unknown signaling system for a long-term cellresponse rather than a transient signaling system, and2) most of the[3H]acetyl-PAF boundto platelets is metabolized extracellularly by ecto-type PAFacetylhydrolase, with the lyso-PAF generated being incorporated rapidlyinto the cells and converted to1-alkyl-2-acyl-sn-glycero-3-phosphocholine. 相似文献