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181.
Li TW Ting JH Yokoyama NN Bernstein A van de Wetering M Waterman ML 《Molecular and cellular biology》2006,26(14):5284-5299
Alternative promoters within the LEF1 locus produce polypeptides of opposing biological activities. Promoter 1 produces full-length LEF-1 protein, which recruits beta-catenin to Wnt target genes. Promoter 2 produces a truncated form that cannot interact with beta-catenin and instead suppresses Wnt regulation of target genes. Here we show that promoter 1 is aberrantly activated in colon cancers because it is a direct target of the Wnt pathway. T-cell factor (TCF)-beta-catenin complexes bind to Wnt response elements in exon 1 and dynamically regulate chromatin acetylation and promoter 1 activity. Promoter 2 is delimited to the intron 2/exon 3 boundary and, like promoter 1, is also directly regulated by TCF-beta-catenin complexes. Promoter 2 is nevertheless silent in colon cancer because an upstream repressor selectively targets the basal promoter leading to destabilized TCF-beta-catenin binding. We conclude that the biological outcome of aberrant LEF1 activation in colon cancer is directed by differential promoter activation and repression. 相似文献
182.
Generation of high-affinity chicken single-chain Fv antibody fragments for measurement of the Pseudonitzschia pungens toxin domoic acid 总被引:3,自引:0,他引:3
Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V(H) and V(L) genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules. 相似文献
183.
Svitel J Tkác J Vostiar I Navrátil M Stefuca V Bucko M Gemeiner P 《Biotechnology letters》2006,28(24):2003-2010
Bacteria belonging to the genus Acetobacter and Gluconobacter, and enzymes isolated from them, have been extensively used for biosensor construction in the last decade. Bacteria used
as a biocatalyst are easy to prepare and use in amperometric biosensors. They contain multiple enzyme activities otherwise
not available commercially. The range of compounds analyzable by Gluconobacter biosensors includes: mono- and poly-alcohols, multiple aldoses and ketoses, several disaccharides, triacylglycerols, and
complex parameters like utilizable saccharides or biological O2 demand. Here, the recent trends in Gluconobacter biosensors and current practical applications are summarized.
An erratum to this article can be found at 相似文献
184.
Cheung MC Brown BG Marino Larsen EK Frutkin AD O'Brien KD Albers JJ 《Biochimica et biophysica acta》2006,1762(1):131-137
Plasma phospholipid lipid transfer protein (PLTP) has several known key functions in lipoprotein metabolism. Recent studies suggest that it also may play a role in the inflammatory response. Inflammatory cell activity contributes to the development of atherosclerosis. To seek further evidence for the association of PLTP with inflammation, we studied the relationship between PLTP activity and five inflammatory markers [C-reactive protein (CRP), serum amyloid A (SAA), interleukin 6 (IL-6), white blood cells (WBC), and fibrinogen] in 93 patients with low HDL and cardiovascular disease (CVD). Plasma PLTP activity had the strongest correlation with CRP (r=0.332, P<0.001) followed by SAA (r=0.239, P=0.021). PLTP, CRP, and SAA were significantly associated with body mass index (BMI), insulin or glucose, apolipoprotein (apo) B, and/or apo E level (r=0.264-0.393, P<0.01). PLTP, SAA, and IL-6 also were associated with the concentration of HDL particles without apo A-II [Lp(A-I)](r=0.373-0.472, P<0.005, n=56), but not particles with apo A-II. Smoking was associated with increased PLTP activity, CRP, and WBC, and hypertension with increased PLTP activity. In linear models, CRP remained significantly associated with PLTP after adjustment of CVD risk factors and insulin resistance. Also, much of the variability of plasma PLTP activity was explained by CRP, BMI, Lp(A-I), smoking, glucose, and blood pressure. These findings show for the first time that plasma PLTP activity is associated positively with CRP in CVD, a state of chronic inflammation. 相似文献
185.
Purified bovine heart cytochrome c oxidase (CcO) has been extracted from aqueous solution into hexane in the presence of phospholipids and calcium ions. In extracts, CcO is in the so-called "slow" form and probably situated in reverse micelles. At low water:phospholipid molar ratios, electron transfer from reduced heme a and Cu(A) to the catalytic center is inhibited and both heme a3 and Cu(B) remain in the oxidized state. The rate of binding of cyanide to heme a3 in this oxidized catalytic center is, however, dependent on the redox state of heme a and Cu(A). When heme a and Cu(A) are reduced, the rate is increased 20-fold compared to the rate when these two centers are oxidized. The enhanced rate of binding of cyanide to heme a3 is explained by the destabilization of an intrinsic ligand, located at the catalytic site, that is triggered by the reduction of heme a and Cu(A). 相似文献
186.
Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding
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Marian Pulido Kenji Saito Shun-Ichi Tanaka Yuichi Koga Masaaki Morikawa Kazufumi Takano Shigenori Kanaya 《Applied microbiology》2006,72(6):4154-4162
Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ~5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a Ki of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier. 相似文献
187.
Gene expression profile of human bone marrow stromal cells: high-throughput expressed sequence tag sequencing analysis 总被引:5,自引:0,他引:5
Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' (97) and 3' (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton. 相似文献
188.
LKB1 is the upstream kinase in the AMP-activated protein kinase cascade 总被引:16,自引:0,他引:16
Woods A Johnstone SR Dickerson K Leiper FC Fryer LG Neumann D Schlattner U Wallimann T Carlson M Carling D 《Current biology : CB》2003,13(22):2004-2008
Inactivating mutations in the protein kinase LKB1 lead to a dominantly inherited cancer in humans termed Peutz-Jeghers syndrome. The role of LKB1 is unclear, and only one target for LKB1 has been identified in vivo [3]. AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that plays a pivotal role in energy homeostasis. AMPK may have a role in protecting the body from metabolic diseases including type 2 diabetes, obesity, and cardiac hypertrophy. We previously reported the identification of three protein kinases (Elm1, Pak1, and Tos3 [9]) that lie upstream of Snf1, the yeast homologue of AMPK. LKB1 shares sequence similarity with Elm1, Pak1, and Tos3, and we demonstrated that LKB1 phosphorylates AMPK on the activation loop threonine (Thr172) within the catalytic subunit and activates AMPK in vitro [9]. Here, we have investigated whether LKB1 corresponds to the major AMPKK activity present in cell extracts. AMPKK purified from rat liver corresponds to LKB1, and blocking LKB1 activity in cells abolishes AMPK activation in response to different stimuli. These results identify a link between two protein kinases, previously thought to lie in unrelated, distinct pathways, that are associated with human diseases. 相似文献
189.
Background:
Concurrent admission of a mother and her newborn to separate intensive care units (herein referred to as co-ICU admission), possibly in different centres, can magnify family discord and stress. We examined the prevalence and predictors of mother–infant separation and mortality associated with co-ICU admissions.Methods:
We completed a population-based study of all 1 023 978 singleton live births in Ontario between Apr. 1, 2002, and Mar. 31, 2010. We included data for maternal–infant pairs that had co-ICU admission (n = 1216), maternal ICU admission only (n = 897), neonatal ICU (NICU) admission only (n = 123 236) or no ICU admission (n = 898 629). The primary outcome measure was mother–infant separation because of interfacility transfer.Results:
The prevalence of co-ICU admissions was 1.2 per 1000 live births and was higher than maternal ICU admissions (0.9 per 1000). Maternal–newborn separation due to interfacility transfer was 30.8 (95% confidence interval [CI] 26.9–35.3) times more common in the co-ICU group than in the no-ICU group and exceeded the prevalence in the maternal ICU group and NICU group. Short-term infant mortality (< 28 days after birth) was higher in the co-ICU group (18.1 per 1000 live births; maternal age–adjusted hazard ratio [HR] 27.8, 95% CI 18.2–42.6) than in the NICU group (7.6 per 1000; age-adjusted HR 11.5, 95% CI 10.4–12.7), relative to 0.7 per 1000 in the no-ICU group. Short-term maternal mortality (< 42 days after delivery) was also higher in the co-ICU group (15.6 per 1000; age-adjusted HR 328.7, 95% CI 191.2–565.2) than in the maternal ICU group (6.7 per 1000; age-adjusted HR 140.0, 95% CI 59.5–329.2) or the NICU group (0.2 per 1000; age-adjusted HR 4.6, 95% CI 2.8–7.4).Interpretation:
Mother–infant pairs in the co-ICU group had the highest prevalence of separation due to interfacility transfer and the highest mortality compared with those in the maternal ICU and NICU groups.Admission of a newborn to a neonatal intensive care unit (NICU) produces a great deal of stress for the parents.1 A new mother who also falls ill may be unable to care for, or bond with, her newborn during the time of her illness.2 Although the father will experience a sense of loss of control when his infant is admitted to the NICU,3,4 this feeling is conceivably worsened when the mother too requires intensive care. In this situation of concurrent ICU admission (herein referred to as “co-ICU”), the mother and newborn may be in different areas of the same hospital or in different facilities. This separation would magnify the degree of mother–infant and family discord and stress and could create competing priorities for family members in terms of decision-making and support. Moreover, the situation would be made more devastating if the mother or newborn died in hospital and the other remained critically ill, or after discharge of one, the other required continuing hospital care or died.Because of the serious, potentially negative consequences of co-ICU admissions, we examined the prevalence and predictors of mother–infant separation and mortality associated with co-ICU admissions. 相似文献190.