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Sample records for binding factor cbf

  1. Ectopic overexpression of SsCBF1, a CRT/DRE-binding factor from the nightshade plant Solanum lycopersicoides, confers freezing and salt tolerance in transgenic Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Lili Zhang

    Full Text Available The C-repeat (CRT/dehydration-responsive element (DRE binding factor (CBF/DREB1 transcription factors play a key role in cold response. However, the detailed roles of many plant CBFs are far from fully understood. A CBF gene (SsCBF1 was isolated from the cold-hardy plant Solanum lycopersicoides. A subcellular localization study using GFP fusion protein indicated that SsCBF1 is localized in the nucleus. We delimited the SsCBF1 transcriptional activation domain to the C-terminal segment comprising amino acid residues 193-228 (SsCBF1(193-228. The expression of SsCBF1 could be dramatically induced by cold, drought and high salinity. Transactivation assays in tobacco leaves revealed that SsCBF1 could specifically bind to the CRT cis-elements in vivo to activate the expression of downstream reporter genes. The ectopic overexpression of SsCBF1 conferred increased freezing and high-salinity tolerance and late flowering phenotype to transgenic Arabidopsis. RNA-sequencing data exhibited that a set of cold and salt stress responsive genes were up-regulated in transgenic Arabidopsis. Our results suggest that SsCBF1 behaves as a typical CBF to contribute to plant freezing tolerance. Increased resistance to high-salinity and late flowering phenotype derived from SsCBF1 OE lines lend more credence to the hypothesis that plant CBFs participate in diverse physiological and biochemical processes related to adverse conditions.

  2. RNA-seq analysis of oil palm under cold stress reveals a different C-repeat binding factor (CBF) mediated gene expression pattern in Elaeis guineensis compared to other species.

    Science.gov (United States)

    Lei, Xintao; Xiao, Yong; Xia, Wei; Mason, Annaliese S; Yang, Yaodong; Ma, Zilong; Peng, Ming

    2014-01-01

    Elaeis guineensis as a tropical oil-crop is particularly sensitive to low temperature. Improvement of cold-tolerance may significantly increase the total cultivation area of this tropical oil-crop worldwide. We sequenced cold-treated and control (untreated) samples of Elaeis guineensis. De novo assembly generated 51,452 unigenes with an average length of 703 bp. Subsequently, these expressed sequences were functionally annotated. In the K category (transcription factors) of COG (Cluster of Orthologous Group) annotation, the largest proportion of genes induced and repressed at least two-fold under cold stress were from the AP2/ERE family, indicating that C-repeat binding factor, (CBFs, members of the AP2/ERE family) may play a central role in cold tolerance in Elaeis guineensis. Subsequently, the CBF-mediated signal transduction pathway was reconstructed based on transcriptome data and the gene expression profile involving the pathway was examined using real-time quantitative RT-PCR (qRT-PCR). CBFs reached maximum transcript level both at medium (4 h) and long period time points (7 days), contrary to the expression pattern of CBFs in Arabidopsis and rice. Moreover, the promoters of downstream Cold Responsive gene (CORs) regulated by CBFs were analyzed. Conservation, mutation and absence of the DRE core motif were detected in the promoters of six CORs. These mutations in DRE motifs suggest that CORs may not be induced via cold stress in Elaeis guineensis. PMID:25479236

  3. RNA-seq analysis of oil palm under cold stress reveals a different C-repeat binding factor (CBF mediated gene expression pattern in Elaeis guineensis compared to other species.

    Directory of Open Access Journals (Sweden)

    Xintao Lei

    Full Text Available Elaeis guineensis as a tropical oil-crop is particularly sensitive to low temperature. Improvement of cold-tolerance may significantly increase the total cultivation area of this tropical oil-crop worldwide. We sequenced cold-treated and control (untreated samples of Elaeis guineensis. De novo assembly generated 51,452 unigenes with an average length of 703 bp. Subsequently, these expressed sequences were functionally annotated. In the K category (transcription factors of COG (Cluster of Orthologous Group annotation, the largest proportion of genes induced and repressed at least two-fold under cold stress were from the AP2/ERE family, indicating that C-repeat binding factor, (CBFs, members of the AP2/ERE family may play a central role in cold tolerance in Elaeis guineensis. Subsequently, the CBF-mediated signal transduction pathway was reconstructed based on transcriptome data and the gene expression profile involving the pathway was examined using real-time quantitative RT-PCR (qRT-PCR. CBFs reached maximum transcript level both at medium (4 h and long period time points (7 days, contrary to the expression pattern of CBFs in Arabidopsis and rice. Moreover, the promoters of downstream Cold Responsive gene (CORs regulated by CBFs were analyzed. Conservation, mutation and absence of the DRE core motif were detected in the promoters of six CORs. These mutations in DRE motifs suggest that CORs may not be induced via cold stress in Elaeis guineensis.

  4. DREB1/CBF transcription factors: their structure, function and role in abiotic stress tolerance in plants

    Indian Academy of Sciences (India)

    M. Akhtar; A. Jaiswal; G. Taj; J. P. Jaiswal; M. I. Qureshi; N. K. Singh

    2012-12-01

    Drought, high salinity and low temperature are major abiotic stresses that influence survival, productivity and geographical distribution of many important crops across the globe. Plants respond to these environmental challenges via physiological, cellular and molecular processes, which results in adjusted metabolic and structural alterations. The dehydration-responsive-element-binding (DREB) protein / C-repeat binding factors (CBFs) belong to APETALA2 (AP2) family transcription factors that bind to DRE/CRT cis-element and regulate the expression of stress-responsive genes. DREB1/CBF genes, therefore, play an important role in increasing stress tolerance in plants and their deployment using transgenic technology seems to be a potential alternative in management of abiotic stresses in crop plants. This review is mainly focussed on the structural characteristics as well as transcriptional regulation of gene expression in response to various abiotic stresses, with particular emphasis on the role of DREB1/CBF regulon in stress-responsive gene expression. The recent progress related to genetic engineering of DREB1/CBF transcription factors in various crops and model plants is also summarized.

  5. The Box H/ACA snoRNP Assembly Factor Shq1p is a Chaperone Protein Homologous to Hsp90 Cochaperones that Binds to the Cbf5p Enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Godin, Katherine S.; Walbott, Helene; Leulliot, Nicolas; van Tilbeurgh, Herman; Varani, Gabriele

    2009-05-06

    Box H/ACA small nucleolar (sno) ribonucleoproteins (RNPs) are responsible for the formation of pseudouridine in a variety of RNAs and are essential for ribosome biogenesis, modification of spliceosomal RNAs, and telomerase stability. A mature snoRNP has been reconstituted in vitro and is composed of a single RNA and four proteins. However, snoRNP biogenesis in vivo requires multiple factors to coordinate a complex and poorly understood assembly and maturation process. Among the factors required for snoRNP biogenesis in yeast is Shq1p, an essential protein necessary for stable expression of box H/ACA snoRNAs. We have found that Shq1p consists of two independent domains that contain casein kinase 1 phosphorylation sites. We also demonstrate that Shq1p binds the pseudourydilating enzyme Cbf5p through the C-terminal domain, in synergy with the N-terminal domain. The NMR solution structure of the N-terminal domain has striking homology to the ‘Chord and Sgt1’ domain of known Hsp90 cochaperones, yet Shq1p does not interact with the yeast Hsp90 homologue in vitro. Surprisingly, Shq1p has stand-alone chaperone activity in vitro. This activity is harbored by the C-terminal domain, but it is increased by the presence of the N-terminal domain. These results provide the first evidence of a specific biochemical activity for Shq1p and a direct link to the H/ACA snoRNP.

  6. 梅花 CBF 转录因子的克隆及表达%Cloning and Expression of CBF Transcription Factor from Prunus mume

    Institute of Scientific and Technical Information of China (English)

    张杰; 张启翔; 杨炜茹

    2012-01-01

    根据GenBank中与梅花同属的桃、甜樱桃等已发表CBFs转录因子序列设计简并引物,采用PCR和RTPCR方法,从梅花基因组DNA和cDNA中克隆CBF转录因子片段.结果表明,两种途径获得的CBF基因序列一致,基因全长821 bp,编码238个氨基酸,其氨基酸序列具有典型的CBF蛋白特征,包含保守的AP2/EREB DNA 结合结构域及CBF家族蛋白特征短多肽序列(PKK/RPAGRxKFxETRHP和DSAWR).氨基酸相似性分析结果表明,该基因与欧洲甜樱桃、矮扁桃等CBF转录因子相似性较高.相对荧光定量PCR结果显示,4℃低温胁迫下,其表达量符合CBF转录因子表达特点,随着胁迫时间的增长表达量呈上升趋势,8h时达峰值,说明该基因在低温胁迫下上调表达.%Primers were designed according to the CBF transcription factors of Peach (Prunus persica), Sweet cherry (Prunus avium) et al from GenBank. Fragments of CBF gene were isolated from Prunus mume by PCR and RT-PCR, The CBF gene was 821 bp long, encoding a putative protein of 238 amino acids;The amino acids sequence owns the characteristics of the CBF protein,which contains an AP2/EREB DNA-binding domain and two special short amino acids sequences;Similarity analysis showed that the nu-cleotide were highly similarity to that of P. avium, P. tenella et al. Relative real-time PCR experiment showed the expression of PmCBFl was coincidence with the expression characteristics of the CBF gene after exposed to 4℃. The expression of PmCBFl increased at beginning and achieved the highest after 8 hour,indicating PmCBFl was induced under low temperature stress.

  7. Transcriptional profiling of Medicago truncatula under salt stress identified a novel CBF transcription factor MtCBF4 that plays an important role in abiotic stress responses

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    Su Zhen

    2011-07-01

    Full Text Available Abstract Background Salt stress hinders the growth of plants and reduces crop production worldwide. However, different plant species might possess different adaptive mechanisms to mitigate salt stress. We conducted a detailed pathway analysis of transcriptional dynamics in the roots of Medicago truncatula seedlings under salt stress and selected a transcription factor gene, MtCBF4, for experimental validation. Results A microarray experiment was conducted using root samples collected 6, 24, and 48 h after application of 180 mM NaCl. Analysis of 11 statistically significant expression profiles revealed different behaviors between primary and secondary metabolism pathways in response to external stress. Secondary metabolism that helps to maintain osmotic balance was induced. One of the highly induced transcription factor genes was successfully cloned, and was named MtCBF4. Phylogenetic analysis revealed that MtCBF4, which belongs to the AP2-EREBP transcription factor family, is a novel member of the CBF transcription factor in M. truncatula. MtCBF4 is shown to be a nuclear-localized protein. Expression of MtCBF4 in M. truncatula was induced by most of the abiotic stresses, including salt, drought, cold, and abscisic acid, suggesting crosstalk between these abiotic stresses. Transgenic Arabidopsis over-expressing MtCBF4 enhanced tolerance to drought and salt stress, and activated expression of downstream genes that contain DRE elements. Over-expression of MtCBF4 in M. truncatula also enhanced salt tolerance and induced expression level of corresponding downstream genes. Conclusion Comprehensive transcriptomic analysis revealed complex mechanisms exist in plants in response to salt stress. The novel transcription factor gene MtCBF4 identified here played an important role in response to abiotic stresses, indicating that it might be a good candidate gene for genetic improvement to produce stress-tolerant plants.

  8. CIR, a corepressor of CBF1, binds to PAP-1 and effects alternative splicing

    International Nuclear Information System (INIS)

    We have reported that PAP-1, a product of a causative gene for autosomal retinitis pigmentosa, plays a role in splicing. In this study, CIR, a protein originally identified as a CBF1-interacting protein and reported to act as a transcriptional corepressor, was identified as a PAP-1 binding protein and its function as a splicing factor was investigated. In addition to a basic lysine and acidic serine-rich (BA) domain and a zinc knuckle-like motif, CIR has an arginine/serine dipeptide repeat (RS) domain in its C terminal region. The RS domain has been reported to be present in the superfamily of SR proteins, which are involved in splicing reactions. We generated CIR mutants with deletions of each BA and RS domain and studied their subcellular localizations and interactions with PAP-1 and other SR proteins, including SC35, SF2/ASF, and U2AF35. CIR was found to interact with U2AF35 through the BA domain, with SC35 and SF2/ASF through the RS domain, and with PAP-1 outside the BA domain in vivo and in vitro. CIR was found to be colocalized with SC35 and PAP-1 in nuclear speckles. Then the effect of CIR on splicing was investigated using the E1a minigene as a reporter in HeLa cells. Ectopic expression of CIR with the E1a minigene changed the ratio of spliced isoforms of E1a that were produced by alternative selection of 5'-splice sites. These results indicate that CIR is a member of the family of SR-related proteins and that CIR plays a role in splicing regulation

  9. Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1).

    OpenAIRE

    Zhang, D E; Fujioka, K.; Hetherington, C J; Shapiro, L H; CHEN, H. M.; Look, A T; Tenen, D. G.

    1994-01-01

    The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E. Zhang, C.J. Hetherington, H.-M. Chen, and D.G. Tenen, Mol. Cell. ...

  10. Factors affecting accuracy of tomographic measurement of rCBF by HEADTOME

    International Nuclear Information System (INIS)

    Factors affecting accuracy of tomographic regional cerebral blood flow (rCBF) were investigated using a Xe-133 inhalation method and a dynamic single photon tomographic system ; HEADTOME. Several improvements were attempted in method and devices based on the investigation. To minimize statistical error, loss of xenon gas and an error in estimating air curve, volume of the spiro-bag was set properly (0.8 - 1.0 l) and a breath-synchronized control of the inhalation system was introduced. To avoid time mismatching between the tomographic head data and the respiratory air curve, the interval of their data acquisition starting was standerdized. For the inhalation of Xe-133, mouthpiece was superior to face-mask because of less effect by scattered radiation from the nasal cavity. Standard deviation of mean rCBF in normal volunteers was improved to 50.0 ± 1.7 ml/100 g/min, from 51.2 ± 7.1 obtained before these improvements. Partial volume effect was also evaluated using simulated rCBF images calculated as a weighted mean value in each 32 x 32 pixels on trans-axial atlas of the normal brain. The rCBF value was noted to change with thickness or form of the cortex, sulci and ventricles. (author)

  11. An apple rootstock overexpressing a peach CBF gene alters growth and flowering in the scion but does not impact cold hardiness or dormancy

    Science.gov (United States)

    The C-repeat Binding Factor (CBF) transcription factor is involved in responses to low temperature and water deficit in many plant species. Overexpression of CBF genes leads to enhanced freezing tolerance and growth inhibition in many species. The overexpression of a peach CBF (PpCBF1) gene in a t...

  12. Potential language and attentional networks revealed through factor analysis of rCBF data measured with SPECT

    DEFF Research Database (Denmark)

    McLaughlin, T; Steinberg, B; Christensen, B;

    1992-01-01

    's area (left hemisphere), when subjects listened to narrative speech, compared to white noise (baseline). No significant rCBF differences were detected with this test during dichotic stimulation vs. white noise. A more sophisticated statistical method (factor analysis) disclosed patterns of functionally...... intercorrelated regions. The factor analysis reduced the highly intercorrelated rCBF measures from 28 regions of interest to a set of three independent factors. These factors accounted for 77% of the total variation in rCBF values. These three factors appeared to represent statistical analogues of independent...

  13. CBF gene expression in peach leaf and bark tissues is gated by a circadian clock

    Science.gov (United States)

    CBF transcription factors are part of the AP2/ERF domain family of DNA-binding proteins that recognize a C-repeat response cis-acting element that regulates a number of cold-responsive genes (CBF-regulon). In peach (Prunus persica), five CBF genes are situated in tandem on scaffold (Linkage Group) ...

  14. The carboxy-terminal domain of Dictyostelium C-module-binding factor is an independent gene regulatory entity.

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    Jörg Lucas

    Full Text Available The C-module-binding factor (CbfA is a multidomain protein that belongs to the family of jumonji-type (JmjC transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD. An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

  15. RNA-Seq Analysis of Oil Palm under Cold Stress Reveals a Different C-Repeat Binding Factor (CBF) Mediated Gene Expression Pattern in Elaeis guineensis Compared to Other Species

    OpenAIRE

    Xintao Lei; Yong Xiao; Wei Xia; Mason, Annaliese S.; Yaodong Yang; Zilong Ma; Ming Peng

    2014-01-01

    Elaeis guineensis as a tropical oil-crop is particularly sensitive to low temperature. Improvement of cold-tolerance may significantly increase the total cultivation area of this tropical oil-crop worldwide. We sequenced cold-treated and control (untreated) samples of Elaeis guineensis. De novo assembly generated 51,452 unigenes with an average length of 703 bp. Subsequently, these expressed sequences were functionally annotated. In the K category (transcription factors) of COG (Cluster of Or...

  16. Sequence analysis of three CBF-like genes from apple (Malus x domestica)

    Science.gov (United States)

    The CBF/DREB family of transcription factors has been demonstrated to have an integral role in the response of plants to low temperatures and water deficit, binding to a cis-acting regulatory element called the C-repeat/dehydration response element (CRT/ DRE) in genes induced by those stresses. CBF...

  17. 不同浓度雌二醇对骨髓基质细胞分化过程中Cbfα1和PPAR-γ2 mRNA表达的影响%Effect of 17 beta-estradiol on the mRNA expression of core-binding factor alpha 1 and proliferator-activated receptor gamma 2 in bone marrow stromal cells

    Institute of Scientific and Technical Information of China (English)

    金小岚; 袁成良; 侯建红; 钱江龙; 黄迎春

    2004-01-01

    /g蛋白(t=29,P<0.01).Ⅰ型胶原含量均随雌二醇浓度增加而降低.结论:雌二醇抑制体外培养的骨髓基质细胞向成骨细胞分化而促进其向脂肪细胞分化.%BACKGROUND: Bone remodeling rate increases precipitously at menopause, which may be explained that loss of sex steroids up-regulates the formation of osteoclasts and osteoblasts in the marrow by up-regulating the production and action of cytokines responsible for osteoclastogenesis and osteoblastogenesis.However, the molecular mechanism of 17β-estradiol(E2)on osteoblastogenesis is not clear yet.OBJECTIVE: To investigate the effects of 17β-estradiol(E2) on the gene expression of core-binding factor alpha 1 (Cbfα1) and peroxisome proliferator-activated receptor gamma 2(PPAR-γ2) in rat bone marrow stromal cells exposured to the differentiation medium and to elucidate the role of E2 on osteoblastogenesis.DESIGN: A nonrandomized controlled experimental study was conducted.SETTING and PARTICIPANTS: All experiments were carried out in the Department of Laboratory, Chengdu Military Command General Hospital and Chengdu Bai' ao Biol-Tech Limited Company. Three-month-old female SD rat weighing (200 ± 20) g used to isolate bone marrow stromal cells was obtained from the Animal Center of Chengdu University of Traditional Chinese Medicine.INTERVENTION: Adherent bone marrow stromal cells were intervened with dexamethasome (DEX) 1 × 10-7 mol/L, 1, 25(OH)2D3 1 × 10-9 mol/L and different concentrations [ (0 - 1) × 10-6 mol/L] of E2.MAIN OUTCOME MEASURES: The effects of E2 on the Cbfα1 and PPAR-γ2 gene expression were assayed by semiquantitative RT-PCR and demonstrated by Northern blot.RESULTS: A dose dependent inhibition of the expression of Cbfα1 mRNA in bone marrow stromal cell by E2 was discovered. When examined under various concentrations of E2 [ (0 - 1) × 10-6 mol/L], the expression of Cbfα1 mRNA decreased from (25.0 ± 3.3)% to (19.8 ± 2.2)% (t =2.62, P <0.05), (14.5±1.3)% (t=5.92, P

  18. Comparison of D2 receptor binding (123I-IBZM) and rCBF (99mTc-HMPAO) in extrapyramidal disorders

    International Nuclear Information System (INIS)

    The aim of this SPECT study was to determine whether there is a correlation between rCBF (99mTc-HMPAO) and D2 receptor binding (123I-IBZM) in disorders of the extrapyramidal system and in which situation the 99MTc-HMPAO scan could predict the outcome of the 123I-IBZM study. 13 patients with Parkinson's syndrome and 13 patients with hyperkinetic extrapyramidal disorders were studied. In all patients the two SPECT studies were performed within 2-7 days. ROIs were placed over the basal ganglia (BG), the frontal cortex (FC) and the cerebellum (CE). The ratios BG/FC and BG/CE were calculated. In both groups the scatter was lower when the frontal cortex was used as reference region. Among the patients with hyperkinetic extrapyramidal hyperkinetic extrapyramidal disorders the two patients with Huntington's chorea had lower rCBF and D2 receptor binding compared to other hyperkinetic extrapyramidal disorders. There was no correlation between D2 receptor binding and rCBF in the basal ganglia. The 99MTc-HMPAO studies did not provide clinically useful information, except in Huntington's chorea. (orig.)

  19. Isolation and Characterization of a C-repeat Binding Transcription Factor from Maize

    Institute of Scientific and Technical Information of China (English)

    Lei Wang; Yanzhong Luo; Lan Zhang; Jun Zhao; Zhiqiu Hu; Yunliu Fan; Chunyi Zhang

    2008-01-01

    C-repeat binding proteins (CBFs) are a group of transcription factors that have been proven to be important for stress tolerance in plants.Many of these transcription factors transactivate the promoters of cold-regulated genes via binding to low temperature- or dehydration-responsive cis-elements,thus conferring plants cold acclimation.In the present study,we Isolated a C-repeat binding transcription factor from maize using the yeast one-hybrid system with the C-repeat motif from the promoter of the Arabidopsis COR15a gene as bait.The isolated transcription factor is highly similar to the Arabidopsis CBF3 in their predicted amino acid sequences,and is therefore designated ZmCBF3.Point mutation analyses of the ZmCBF3-binding cis-element revealed (A/G)(C/T)CGAC as the core binding sequence.Expression analyses showed that ZmCBF3 was upregulated by both abscisic acid and low temperature,and was actively expressed during embryogenesis,suggesting that ZmCBF3 plays a role in stress response in maize.

  20. Comparison of CBF1, CBF2, CBF3 and CBF4 expression in some grapevine cultivars and species under cold stress

    Science.gov (United States)

    Karimi, Maryam; Ebadi, Ali; Mousavi, Seyed Amir; Salami, Seyed Alireza; Zarei, Abdolkarim

    2015-01-01

    Grapevine, an important horticultural crop in the world, is moderately tolerant to cold conditions and is subjected to the cold injuries at different regions. So studies on different aspects of tolerance mechanism to unexpected cold of late spring as well as winter freezing seems necessary about this vine. For this reason, study on genes responsible for acquiring cold tolerance is very important. Transcription factors are among regulatory proteins that are responsible for cold acclimation. In this research work, expression levels of CBF1, CBF2, CBF3, and CBF4 transcription factors were studied on two cvs of Vitis vinifera (“Khalili-Danedar” and “Shahroodi”) as well as one Vitis riparia at different times after treating at 4 °C. Results showed that two vinifera cultivars, “Khalili-Danedar” and “Shahroodi”, had similar trend for each transcription factor. Gene expression increased at the beginning of cold stress and then decreased. Expression of these TF started some minutes (CBF1) after cold treatment and continued for several hours (CBF2), even till the tenth day (CBF4). All together V. riparia which is endemic to the cold regions behaved stronger and showed higher expression for all studied transcription factors. Among two V. vinifera cultivars, “Khalili-Danedar” showed significantly higher expression compared with “Shahroodi”. The comparison of expression levels of these four transcription factors revealed that the least and the greatest expressions were recorded for CBF1 and CBF3 respectively, and two CBF2 and CBF4 had approximately the same expression levels. PMID:26973374

  1. Abortive lytic reactivation of KSHV in CBF1/CSL deficient human B cell lines.

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    Barbara A Scholz

    Full Text Available Since Kaposi's sarcoma associated herpesvirus (KSHV establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade.

  2. Transcriptome Profiling of Pediatric Core Binding Factor AML.

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    Chih-Hao Hsu

    Full Text Available The t(8;21 and Inv(16 translocations disrupt the normal function of core binding factors alpha (CBFA and beta (CBFB, respectively. These translocations represent two of the most common genomic abnormalities in acute myeloid leukemia (AML patients, occurring in approximately 25% pediatric and 15% of adult with this malignancy. Both translocations are associated with favorable clinical outcomes after intensive chemotherapy, and given the perceived mechanistic similarities, patients with these translocations are frequently referred to as having CBF-AML. It remains uncertain as to whether, collectively, these translocations are mechanistically the same or impact different pathways in subtle ways that have both biological and clinical significance. Therefore, we used transcriptome sequencing (RNA-seq to investigate the similarities and differences in genes and pathways between these subtypes of pediatric AMLs. Diagnostic RNA from patients with t(8;21 (N = 17, Inv(16 (N = 14, and normal karyotype (NK, N = 33 were subjected to RNA-seq. Analyses compared the transcriptomes across these three cytogenetic subtypes, using the NK cohort as the control. A total of 1291 genes in t(8;21 and 474 genes in Inv(16 were differentially expressed relative to the NK controls, with 198 genes differentially expressed in both subtypes. The majority of these genes (175/198; binomial test p-value < 10(-30 are consistent in expression changes among the two subtypes suggesting the expression profiles are more similar between the CBF cohorts than in the NK cohort. Our analysis also revealed alternative splicing events (ASEs differentially expressed across subtypes, with 337 t(8;21-specific and 407 Inv(16-specific ASEs detected, the majority of which were acetylated proteins (p = 1.5 x 10(-51 and p = 1.8 x 10(-54 for the two subsets. In addition to known fusions, we identified and verified 16 de novo fusions in 43 patients, including three fusions involving NUP98 in six

  3. The Influence of Light Quality, Circadian Rhythm, and Photoperiod on the CBF-Mediated Freezing Tolerance

    Directory of Open Access Journals (Sweden)

    Chang Ho Kang

    2013-05-01

    Full Text Available Low temperature adversely affects crop yields by restraining plant growth and productivity. Most temperate plants have the potential to increase their freezing tolerance upon exposure to low but nonfreezing temperatures, a process known as cold acclimation. Various physiological, molecular, and metabolic changes occur during cold acclimation, which suggests that the plant cold stress response is a complex, vital phenomenon that involves more than one pathway. The C-Repeat Binding Factor (CBF pathway is the most important and well-studied cold regulatory pathway that imparts freezing tolerance to plants. The regulation of freezing tolerance involves the action of phytochromes, which play an important role in light-mediated signalling to activate cold-induced gene expression through the CBF pathway. Under normal temperature conditions, CBF expression is regulated by the circadian clock through the action of a central oscillator and also day length (photoperiod. The phytochrome and phytochrome interacting factor are involved in the repression of the CBF expression under long day (LD conditions. Apart from the CBF regulon, a novel pathway involving the Z-box element also mediates the cold acclimation response in a light-dependent manner. This review provides insights into the progress of cold acclimation in relation to light quality, circadian regulation, and photoperiodic regulation and also explains the underlying molecular mechanisms of cold acclimation for introducing the engineering of economically important, cold-tolerant plants.

  4. PpCBF3 from Cold-Tolerant Kentucky Bluegrass Involved in Freezing Tolerance Associated with Up-Regulation of Cold-Related Genes in Transgenic Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Lili Zhuang

    Full Text Available Dehydration-Responsive Element Binding proteins (DREB/C-repeat (CRT Binding Factors (CBF have been identified as transcriptional activators during plant responses to cold stress. The objective of this study was to determine the physiological roles of a CBF gene isolated from a cold-tolerant perennial grass species, Kentucky bluegrass (Poa pratensis L., which designated as PpCBF3, in regulating plant tolerance to freezing stress. Transient transformation of Arabidopsis thaliana mesophyll protoplast with PpCBF3-eGFP fused protein showed that PpCBF3 was localized to the nucleus. RT-PCR analysis showed that PpCBF3 was specifically induced by cold stress (4°C but not by drought stress [induced by 20% polyethylene glycol 6000 solution (PEG-6000] or salt stress (150 mM NaCl. Transgenic Arabidopsis overexpressing PpCBF3 showed significant improvement in freezing (-20°C tolerance demonstrated by a lower percentage of chlorotic leaves, lower cellular electrolyte leakage (EL and H2O2 and O2.- content, and higher chlorophyll content and photochemical efficiency compared to the wild type. Relative mRNA expression level analysis by qRT-PCR indicated that the improved freezing tolerance of transgenic Arabidopsis plants overexpressing PpCBF3 was conferred by sustained activation of downstream cold responsive (COR genes. Other interesting phenotypic changes in the PpCBF3-transgenic Arabidopsis plants included late flowering and slow growth or 'dwarfism', both of which are desirable phenotypic traits for perennial turfgrasses. Therefore, PpCBF3 has potential to be used in genetic engineering for improvement of turfgrass freezing tolerance and other desirable traits.

  5. Identification of a nuclear matrix targeting signal in the leukemia and bone-related AML/CBF-α transcription factors

    OpenAIRE

    Zeng, Congmei; van Wijnen, André J.; Stein, Janet L.; Meyers, Shari; Sun, Wuhua; Shopland, Lindsay; Lawrence, Jeanne B.; Penman, Sheldon; Lian, Jane B.; Stein, Gary S.; Hiebert, Scott W.

    1997-01-01

    Transcription factors of the AML (core binding factor-α/polyoma enhancer binding protein 2) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. Alternative splicing of the AML-1 gene results in two major AML variants, AML-1 and AML-1B. We show here that the transcriptionally active AML-1B binds to the nuclear matrix, and the inactive AML-1 does not. The association of AML-1B with the nuclear matrix is independent of DNA binding and requires a nuclear...

  6. Down-regulation of MicroRNAs 222/221 in Acute Myelogenous Leukemia with Deranged Core-Binding Factor Subunits

    Directory of Open Access Journals (Sweden)

    Matteo Brioschi

    2010-11-01

    Full Text Available Core-binding factor leukemia (CBFL is a subgroup of acutemyeloid leukemia (AML characterized by genetic mutations involving the subunits of the core-binding factor (CBF. The leukemogenesis model for CBFL posits that one, or more, gene mutations inducing increased cell proliferation and/or inhibition of apoptosis cooperate with CBF mutations for leukemia development. One of the most commonmutations associated with CBF mutations involves the KIT receptor. A high expression of KIT is a hallmark of a high proportion of CBFL. Previous studies indicate that microRNA (MIR 222/221 targets the 3′ untranslated region of the KIT messenger RNA and our observation that AML1 can bind the MIR-222/221 promoter, we hypothesized that MIR-222/221 represents the link between CBF and KIT. Here, we show that MIR-222/221 expression is upregulated after myeloid differentiation of normal bone marrow AC133+ stem progenitor cells. CBFL blasts with either t(8;21 or inv(16 CBF rearrangements with high expression levels of KIT (CD117 display a significantly lower level of MIR-222/221 expression than non-CBFL blasts. Consistently, we found that the t(8;21 AML1-MTG8 fusion protein binds the MIR-222/221 promoter and induces transcriptional repression of a MIR-222/221-LUC reporter. Because of the highly conserved sequence homology, we demonstrated concomitant MIR-222/221 down-regulation and KIT up-regulation in the 32D/WT1 mouse cell model carrying the AML1-MTG16 fusion protein. This study provides the first hint that CBFL-associated fusion proteins may lead to up-regulation of the KIT receptor by down-regulating MIR-222/221, thus explaining the concomitant occurrence of CBF genetic rearrangements and overexpression of wild type or mutant KIT in AML.

  7. Systemic mastocytosis uncommon in KIT D816V mutation positive core-binding factor acute myeloid leukemia

    DEFF Research Database (Denmark)

    Kristensen, Thomas; Preiss, Birgitte; Broesby-Olsen, Sigurd;

    2012-01-01

    Abstract The KIT D816V mutation is detected in the vast majority of adult cases of systemic mastocytosis (SM). The mutation is also frequently detected in core-binding factor acute myeloid leukemia (CBF-AML) defined by the presence of t(8;21)(q22;q22); RUNX1-RUNX1T1 or inv(16)(p13.1;q22)/t(16;16)(p...

  8. Copy number variation at the HvCBF4-HvCBF2 genomic segment is a major component of frost resistance in barley.

    Science.gov (United States)

    Francia, Enrico; Morcia, Caterina; Pasquariello, Marianna; Mazzamurro, Valentina; Milc, Justyna Anna; Rizza, Fulvia; Terzi, Valeria; Pecchioni, Nicola

    2016-09-01

    A family of CBF transcription factors plays a major role in reconfiguring the plant transcriptome in response to low-freezing temperature in temperate cereals. In barley, more than 13 HvCBF genes map coincident with the major QTL FR-H2 suggesting them as candidates to explain the function of the locus. Variation in copy number (CNV) of specific HvCBFs was assayed in a panel of 41 barley genotypes using RT-qPCR. Taking advantage of an accurate phenotyping that combined Fv/Fm and field survival, resistance-associated variants within FR-H2 were identified. Genotypes with an increased copy number of HvCBF4 and HvCBF2 (at least ten and eight copies, respectively) showed greater frost resistance. A CAPS marker able to distinguish the CBF2A, CBF2B and CBF2A/B forms was developed and showed that all the higher-ranking genotypes in term of resistance harbour only CBF2A, while other resistant winter genotypes harbour also CBF2B, although at a lower CNV. In addition to the major involvement of the HvCBF4-HvCBF2 genomic segment in the proximal cluster of CBF elements, a negative role of HvCBF3 in the distal cluster was identified. Multiple linear regression models taking into account allelic variation at FR-H1/VRN-H1 explained 0.434 and 0.550 (both at p < 0.001) of the phenotypic variation for Fv/Fm and field survival respectively, while no interaction effect between CNV at the HvCBFs and FR-H1/VRN-H1 was found. Altogether our data suggest a major involvement of the CBF genes located in the proximal cluster, with no apparent involvement of the central cluster contrary to what was reported for wheat. PMID:27338258

  9. Melatonin induces the transcripts of CBF/DREB1s and their involvement in both abiotic and biotic stresses in Arabidopsis.

    Science.gov (United States)

    Shi, Haitao; Qian, Yongqiang; Tan, Dun-Xian; Reiter, Russel J; He, Chaozu

    2015-10-01

    Melatonin (N-acetyl-5-methoxytryptamine) is a naturally occurring small molecule that acts as an important secondary messenger in plant stress responses. However, the mechanism underlying the melatonin-mediated signaling pathway in plant stress responses has not been established. C-repeat-binding factors (CBFs)/Drought response element Binding 1 factors (DREB1s) encode transcription factors that play important roles in plant stress responses. This study has determined that endogenous melatonin and transcripts level of CBFs (AtCBF1, AtCBF2, and AtCBF3) in Arabidopsis leaves were significantly induced by salt, drought, and cold stresses and by pathogen Pseudomonas syringe pv. tomato (Pst) DC3000 infection. Moreover, both exogenous melatonin treatment and overexpression of CBFs conferred enhanced resistance to both abiotic and biotic stresses in Arabidopsis. Notably, AtCBFs and exogenous melatonin treatment positively regulated the mRNA expression of several stress-responsive genes (COR15A, RD22, and KIN1) and accumulation of soluble sugars content such as sucrose in Arabidopsis under control and stress conditions. Additionally, exogenous sucrose also conferred improved resistance to both abiotic and biotic stresses in Arabidopsis. Taken together, this study indicates that AtCBFs confer enhanced resistance to both abiotic and biotic stresses, and AtCBF-mediated signaling pathway and sugar accumulation may be involved in melatonin-mediated stress response in Arabidopsis, at least partially. PMID:26182834

  10. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  11. N-termini of fungal CSL transcription factors are disordered, enriched in regulatory motifs and inhibit DNA binding in fission yeast.

    Directory of Open Access Journals (Sweden)

    Martin Převorovský

    Full Text Available BACKGROUND: CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1 transcription factors are the effector components of the Notch receptor signalling pathway, which is critical for metazoan development. The metazoan CSL proteins (class M can also function in a Notch-independent manner. Recently, two novel classes of CSL proteins, designated F1 and F2, have been identified in fungi. The role of the fungal CSL proteins is unclear, because the Notch pathway is not present in fungi. In fission yeast, the Cbf11 and Cbf12 CSL paralogs play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Unusually long N-terminal extensions are typical for fungal and invertebrate CSL family members. In this study, we investigate the functional significance of these extended N-termini of CSL proteins. METHODOLOGY/PRINCIPAL FINDINGS: We identify 15 novel CSL family members from 7 fungal species and conduct bioinformatic analyses of a combined dataset containing 34 fungal and 11 metazoan CSL protein sequences. We show that the long, non-conserved N-terminal tails of fungal CSL proteins are likely disordered and enriched in phosphorylation sites and PEST motifs. In a case study of Cbf12 (class F2, we provide experimental evidence that the protein is proteolytically processed and that the N-terminus inhibits the Cbf12-dependent DNA binding activity in an electrophoretic mobility shift assay. CONCLUSIONS/SIGNIFICANCE: This study provides insight into the characteristics of the long N-terminal tails of fungal CSL proteins that may be crucial for controlling DNA-binding and CSL function. We propose that the regulation of DNA binding by Cbf12 via its N-terminal region represents an important means by which fission yeast strikes a balance between the class F1 and class F2 paralog activities. This mode of regulation might be shared with other CSL-positive fungi, some of which are relevant to human disease and biotechnology.

  12. Identification of CBF14 and NAC2 Genes in Aegilops tauschii Associated with Resistance to Freezing Stress.

    Science.gov (United States)

    Masoomi-Aladizgeh, Farhad; Aalami, Ali; Esfahani, Masoud; Aghaei, Mohamad Jaafar; Mozaffari, Khadijeh

    2015-06-01

    Low temperature as one of the most important environmental factors limits the productivity of plants across the world. Aegilops, as a wild species of Poaceae, contains low temperature-responsive genes. In this study, we analyzed morphological (wilting, chlorosis, and recovery) and physiological (ion leakage) characteristics to identification of a cold-tolerant genotype. In this experiment, we introduced two transcription factors (TFs) in Aegilops species for the first time. Bioinformatics analysis demonstrated that our nucleotide sequences have high similarity with CBF14 (C-repeat-binding factor) and NAC2 (NAM, ATAF, and CUC) in Triticum aestivum. Based on the physiological and morphological data, one genotype (Aladizgeh) was identified as the most resistant genotype which was selected for further gene expression analysis. The real-time PCR results indicated that the CBF14 gene was not expressed 3 h following cold treatment, but the highest expression was observed after 6, 12, and 24 h of cold treatment; however, a sudden decrease was observed in its expression after 30 h. The NAC2 gene also was not expressed 3 h after cold stress, but the highest expression was at 24 h and similar to the CBF14 gene; its expression suddenly decreased after 30 h. Our results indicated that this genotype can tolerate -4 °C for 3 h, but the CBF14 and NAC2 genes were activated when treated for longer durations. Expression of TFs studied in this experiment had decreased after 30 h, in which cell death seems to be the important reason. PMID:25900437

  13. The species-specific RNA polymerase I transcription factor SL-1 binds to upstream binding factor.

    OpenAIRE

    Hempel, W M; Cavanaugh, A H; Hannan, R D; Taylor, L.; Rothblum, L I

    1996-01-01

    Transcription of the 45S rRNA genes is carried out by RNA polymerase I and at least two trans-acting factors, upstream binding factor (UBF) and SL-1. We have examined the hypothesis that SL-1 and UBF interact. Coimmunoprecipitation studies using an antibody to UBF demonstrated that TATA-binding protein, a subunit of SL-1, associates with UBF in the absence of DNA. Inclusion of the detergents sodium dodecyl sulfate and deoxycholate disrupted this interaction. In addition, partially purified UB...

  14. Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum.

    Science.gov (United States)

    Lamed, R; Setter, E; Bayer, E A

    1983-11-01

    The isolation and biochemical characterization of the extracellular form of a cellulose-binding factor (CBF) from Clostridium thermocellum is described. The CBF was isolated from the culture supernatant by a two-step procedure which included affinity chromatography on cellulose and gel filtration on Sepharose 4B. The isolated CBF was homogeneous as determined by immunoelectrophoresis, polyacrylamide gel electrophoresis, gel filtration, and analytical ultracentrifugation analysis. The CBF was found to form a complex which exhibited a molecular weight estimated at 2.1 million. Electron microscopic analysis of negatively stained preparations of the isolated CBF revealed a particulate, multisubunit entity of complicated quaternary structure. The molecule appeared to be about 18 nm in size. Although urea failed to break the complex into its component parts, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolved the CBF complex into 14 polypeptide bands. Immunoprecipitation experiments confirmed that these polypeptides indeed formed part of the same complex. Interestingly, by using the whole-cell immunization procedure described in the accompanying article (Bayer et al., J. Bacteriol., 156:818-827, 1983) only one CBF subunit (Mr = 210,000) was found to be antigenically active. By using a gel-overlay assay technique, at least eight of the remaining CBF-associated polypeptide components were shown to exhibit cellulolytic activity. The results are consistent with the contention that the CBF comprises a discrete, multisubunit complex or group of closely related complexes which exhibit separate antigenic and multiple cellulase activities in addition to the property of cellulose binding. It appears that the CBF is not only responsible for the adherence of the cells to cellulose but also constitutes a major part of the cellulolytic apparatus of this organism. PMID:6195146

  15. An apple rootstock overexpressing a peach CBF gene alters growth and flowering in the scion but does not impact cold hardiness or dormancy

    Science.gov (United States)

    Artlip, Timothy S; Wisniewski, Michael E; Arora, Rajeev; Norelli, John L

    2016-01-01

    The C-repeat binding factor (CBF) transcription factor is involved in responses to low temperature and water deficit in many plant species. Overexpression of CBF genes leads to enhanced freezing tolerance and growth inhibition in many species. The overexpression of a peach CBF (PpCBF1) gene in a transgenic line of own-rooted apple (Malus×domestica) M.26 rootstock (T166) trees was previously reported to have additional effects on the onset of dormancy and time of spring budbreak. In the current study, the commercial apple cultivar ‘Royal Gala’ (RG) was grafted onto either non-transgenic M.26 rootstocks (RG/M.26) or transgenic M.26 (T166) rootstocks (RG/T166) and field grown for 3 years. No PpCBF1 transcript was detected in the phloem or cambium of RG scions grafted on T166 rootstocks indicating that no graft transmission of transgene mRNA had occurred. In contrast to own-rooted T166 trees, no impact of PpCBF1 overexpression in T166 rootstocks was observed on the onset of dormancy, budbreak or non-acclimated leaf-cold hardiness in RG/T166 trees. Growth, however, as measured by stem caliper, current-year shoot extension and overall height, was reduced in RG/T166 trees compared with RG/M.26 trees. Although flowering was evident in both RG/T166 and RG/M.26 trees in the second season, the number of trees in flower, the number of shoots bearing flowers, and the number of flower clusters per shoot was significantly higher in RG/M.26 trees than RG/T166 trees in both the second and third year after planting. Elevated levels of RGL (DELLA) gene expression were observed in RG/T166 trees and T166 trees, which may play a role in the reduced growth observed in these tree types. A model is presented indicating how CBF overexpression in a rootstock might influence juvenility and flower abundance in a grafted scion. PMID:26981253

  16. Nucleotide Polymorphism in the Drought Induced Transcription Factor CBF4 Region of Arabidopsis thaliana and Its Molecular Evolution Analyses%拟南芥抗旱转录因子CBF4基因区域的核苷酸多样性及其分子进化分析

    Institute of Scientific and Technical Information of China (English)

    郝岗平; 吴忠义; 曹鸣庆; Georges Pelletier; Dominique Brunel; 黄丛林; 杨清

    2004-01-01

    以生长于不同气候条件下的17个拟南芥核心生态型为材料,分析了它们的抗旱转录因子CBF4 基因区域的序列多态性.结果表明:拟南芥CBF4基因区域具有高密度的单核苷酸多态性(SNP)和插入缺失(Indel),多态性频率为每35.8 bp一个 SNP,每143 bp一个Indel,基因非编码区的多态性是编码区的4倍;在编码区,SNP的频率为每96.4 bp一个SNP,其中发现25 av、203 av和244 av 3个生态型CBF4基因区域1 034位 (以GenBank登录号AB015478序列第19 696位的核苷酸为1)碱基变化:G(←→)T,引起第205位氨基酸变化:gly(←→)val.核苷酸多样性统计分析显示,该基因内部大范围内存在连锁不平衡(linkage disequilibrium,LD),5′端非编码区有一个重组.与拟南芥等的研究结果类似,选择压力对不同的区域作用不同.3′端非编码区核苷酸多样性程度最高,是平衡性选择的结果,编码区核苷酸变化符合中性突变假说,而5′端非编码区是自然选择作用的靶位点.

  17. Searching for transcription factor binding sites in vector spaces

    OpenAIRE

    Lee Chih; Huang Chun-Hsi

    2012-01-01

    Abstract Background Computational approaches to transcription factor binding site identification have been actively researched in the past decade. Learning from known binding sites, new binding sites of a transcription factor in unannotated sequences can be identified. A number of search methods have been introduced over the years. However, one can rarely find one single method that performs the best on all the transcription factors. Instead, to identify the best method for a particular trans...

  18. Sequential coagulation factor VIIa domain binding to tissue factor

    International Nuclear Information System (INIS)

    Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectory to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the γ-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced

  19. Negative Example Aided Transcription Factor Binding Site Search

    OpenAIRE

    Lee, Chih; Huang, Chun-Hsi

    2011-01-01

    Computational approaches to transcription factor binding site identification have been actively researched for the past decade. Negative examples have long been utilized in de novo motif discovery and have been shown useful in transcription factor binding site search as well. However, understanding of the roles of negative examples in binding site search is still very limited. We propose the 2-centroid and optimal discriminating vector methods, taking into account negative examples. Cross-val...

  20. Effect of Nuclear Binding Energy to K Factor

    Institute of Scientific and Technical Information of China (English)

    HOU Zhao-Yu; GUO Ai-Qiang

    2007-01-01

    We modify the square of virtual photon four-momentum by using nuclear binding energy formula,and calculate the effect of nuclear binding energy to K factor and Compton subprocess and annihilate subprocess in A-A collision Drell-Yan process.The outcome indicates that the effect of nuclear binding energy to K factor is obvious in little x region and it would disappear gradually as x increases.

  1. LASAGNA: A novel algorithm for transcription factor binding site alignment

    OpenAIRE

    Lee, Chih; Huang, Chun-Hsi

    2013-01-01

    Background Scientists routinely scan DNA sequences for transcription factor (TF) binding sites (TFBSs). Most of the available tools rely on position-specific scoring matrices (PSSMs) constructed from aligned binding sites. Because of the resolutions of assays used to obtain TFBSs, databases such as TRANSFAC, ORegAnno and PAZAR store unaligned variable-length DNA segments containing binding sites of a TF. These DNA segments need to be aligned to build a PSSM. While the TRANSFAC database provid...

  2. Alcohol Binding to the Odorant Binding Protein LUSH: Multiple Factors Affecting Binding Affinities

    OpenAIRE

    Ader, Lauren; Jones, David N. M.; Lin, Hai

    2010-01-01

    Density function theory (DFT) calculations have been carried out to investigate the binding of alcohols to the odorant binding protein LUSH from Drosophila melanogaster. LUSH is one of the few proteins known to bind to ethanol at physiologically relevant concentrations and where high-resolution structural information is available for the protein bound to alcohol at these concentrations. The structures of the LUSH–alcohol complexes identify a set of specific hydrogen-bonding interactions as cr...

  3. Factor XII binding to endothelial cells depends on caveolae

    DEFF Research Database (Denmark)

    Schousboe, Inger; Thomsen, Peter; van Deurs, Bo

    2004-01-01

    It is now generally accepted that factor XII (FXII) binds to cellular surfaces in the vascular system. One of the suggested receptors of this binding is the glycosylphosphatidylinositol-anchored urokinase-like plasminogen activator (u-PAR) harbored in caveolae/lipid rafts. However, binding of FXII...... FXII binding to caveolae were performed by sucrose density-gradient centrifugations. This showed that the majority of FXII, chemically cross-linked to HUVEC, could be identified in the same fractions of the gradient as caveolin-1, a marker of caveolae, while the majority of u-PAR was identified in...

  4. Molecular cloning and functional characterization of a DREB1/CBF-like gene (GhDREB1L) from cotton

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The transcription factors DREB1s/CBFs play important roles in the regulation of plant resistance to environmental stresses and are quite useful for generating transgenic plants tolerant to these stresses. In the present work, a cDNA encoding DREB1/CBF-like protein (GhDREB1L) from cotton was isolated, and its sequence features, DNA binding preference, and expression patterns of the transcripts were also characterized. GhDREB1L contained one conserved AP2/ERF domain and its amino acid sequence was similar to the DREB1/CBF group of the DREB family from other plants. The DNA-binding domain of GhDREB1L was successfully expressed as a fusion protein in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Electrophoretic mobility shift assay revealed that the purified GhDREB1L fusion protein had a specific binding activity with the previously characterized DRE ele-ment (core sequence, ACCGAC) and also with the DRE-like sequence (core sequence, GCCGAC) in the promoter of the dehydration-responsive late embryogenesis-abundant gene LEA D113. Semi-quantita- tive RT-PCR showed that GhDREB1L was induced in the cotton cotyledons by low temperature, as well as drought and NaCl treatments. These results suggested that the novel cotton GhDREB1L might play an important role in response to low temperature as well as drought and high salinity through binding to the DRE cis-element.

  5. Molecular cloning and functional characterization of a DREB1/CBF-like gene (GhDREB1L) from cotton

    Institute of Scientific and Technical Information of China (English)

    HUANG Bo; JIN LongGuo; LIU JinYuan

    2007-01-01

    The transcription factors DREB1s/CBFs play important roles in the regulation of plant resistance to environmental stresses and are quite useful for generating transgenic plants tolerant to these stresses. In the present work, a cDNA encoding DREB1/CBF-like protein (GhDREB1L) from cotton was isolated, and its sequence features, DNA binding preference, and expression patterns of the transcripts were also characterized. GhDREB1L contained one conserved AP2/ERF domain and its amino acid sequence was similar to the DREB1/CBF group of the DREB family from other plants. The DNA-binding domain of GhDREB1L was successfully expressed as a fusion protein in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Electrophoretic mobility shift assay revealed that the purified GhDREB1L fusion protein had a specific binding activity with the previously characterized DRE element (core sequence, ACCGAC) and also with the DRE-like sequence (core sequence, GCCGAC) in the promoter of the dehydration-responsive late embryogenesis-abundant gene LEA D113. Semi-quantita- tive RT-PCR showed that GhDREB1L was induced in the cotton cotyledons by low temperature, as well as drought and NaCl treatments. These results suggested that the novel cotton GhDREB1L might play an important role in response to low temperature as well as drought and high salinity through binding to the DRE cis-element.

  6. Oil palm EgCBF3 conferred stress tolerance in transgenic tomato plants through modulation of the ethylene signaling pathway.

    Science.gov (United States)

    Ebrahimi, Mortaza; Abdullah, Siti Nor Akmar; Abdul Aziz, Maheran; Namasivayam, Parameswari

    2016-09-01

    CBF/DREB1 is a group of transcription factors that are mainly involved in abiotic stress tolerance in plants. They belong to the AP2/ERF superfamily of plant-specific transcription factors. A gene encoding a new member of this group was isolated from ripening oil palm fruit and designated as EgCBF3. The oil palm fruit demonstrates the characteristics of a climacteric fruit like tomato, in which ethylene has a major impact on the ripening process. A transgenic approach was used for functional characterization of the EgCBF3, using tomato as the model plant. The effects of ectopic expression of EgCBF3 were analyzed based on expression profiling of the ethylene biosynthesis-related genes, anti-freeze proteins (AFPs), abiotic stress tolerance and plant growth and development. The EgCBF3 tomatoes demonstrated altered phenotypes compared to the wild type tomatoes. Delayed leaf senescence and flowering, increased chlorophyll content and abnormal flowering were the consequences of overexpression of EgCBF3 in the transgenic tomatoes. The EgCBF3 tomatoes demonstrated enhanced abiotic stress tolerance under in vitro conditions. Further, transcript levels of ethylene biosynthesis-related genes, including three SlACSs and two SlACOs, were altered in the transgenic plants' leaves and roots compared to that in the wild type tomato plant. Among the eight AFPs studied in the wounded leaves of the EgCBF3 tomato plants, transcript levels of SlOSM-L, SlNP24, SlPR5L and SlTSRF1 decreased, while expression of the other four, SlCHI3, SlPR1, SlPR-P2 and SlLAP2, were up-regulated. These findings indicate the possible functions of EgCBF3 in plant growth and development as a regulator of ethylene biosynthesis-related and AFP genes, and as a stimulator of abiotic stress tolerance. PMID:27513726

  7. Effects of normal aging on regional CBF and CMRO2 in humans

    International Nuclear Information System (INIS)

    The oxygen-15 continuous inhalation technique and positron emission tomography were used in order to investigate age-related changes of cerebral blood flow (CBF) and oxygen consumption (CMRO2). 27 patients aged 19 to 76 years without evidence of dementia, brain disease or vascular risk factors, were studied. Regional CBF and CMRO2 were calculated in seven different brain structures. In gray matter CBF and CMRO2 decreased with aging (respectively 18% and 17%, p2 was found to be diffuse but to affect principally the frontal and temporo-sylvian cortex. Neuronal loss and/or a diminished metabolic activity of residual neurons are the most likely hypotheses to explain these findings

  8. Alteration of CBF and CMRO/sub 2/ and TRH effects on CBF in spinocerebellar degeneration. The PET study

    Energy Technology Data Exchange (ETDEWEB)

    Harada, Kiyoshi; Fukuyama, Hidenao; Miyoshi, Toshihiko; Namura, Yasuhiro; Kameyama, Masakuni

    1988-05-01

    The purpose of this study is to elucidate the effects of thyrotropin-releasing hormone (TRH) on cerebral blood flow (CBF) in patients with spinocerebellar degeneration (SCD) and to evaluate the cerebral circulation and metabolism in patients with SCD. We performed a positron emission tomography study on each of six SCD patients (mean age 47.7 +- 3.6 : 5 cases; OPCA of Dejerene-Thomas type, 1 case; OPCA of Menzel type) and twelve normal volunteers. In SCD patients there were marked reductions in CBF (p < 0.01) and CMRO2 (p < 0.01) in the cerebellum compared with normal volunteers, while in the cerebral cortices and the thalamus, SCD patients showed normal values. There were no significant changes in regional and global CBF after 2 mg TRH intravenous injection in the SCD patients. But comparing CBF before TRH administration with corrected CBF (CBF after TRH center dot mean global CBF before TRH/mean global CBF after TRH), it is only the CBF of the cerebellum that increased after TRH administration (paired t test, p < 0.02). This elevation of CBF in the cerebellum would be related to some clinical effects of TRH in SCD patients.

  9. Statistic rCBF study of extrapyramidal disorders

    Energy Technology Data Exchange (ETDEWEB)

    Kamei, Hiroshi; Nakajima, Takashi; Fukuhara, Nobuyoshi [National Saigata Hospital, Ogata, Niigata (Japan)

    2002-08-01

    We studied regional cerebral blood flow (rCBF) in 16 patients with Parkinson's disease (PD), 2 patients with dementia with Lewy bodies (DLB), 2 patients with progressive supranuclear palsy (PSP), 2 patients with striatonigral degeneration, and 16 normal volunteers, using Three-dimensional stereotactic surface projections (3D-SSP). Decreased rCBF in PD patients was shown in the posterior parietal and occipital cortex. Decreased rCBF in DLB was shown in the frontal, parietal and occipital cortex with relative sparing of the sensorimotor cortex.. Decreased rCBF in PSP was shown in the frontal cortex. Decreased rCBF in SND was shown in the frontal cortex and cerebellum. Statistic rCBF analysis using 3D-SSP was a useful measure for the early differential diagnosis of extrapyramidal disorders. (author)

  10. Statistic rCBF study of extrapyramidal disorders

    International Nuclear Information System (INIS)

    We studied regional cerebral blood flow (rCBF) in 16 patients with Parkinson's disease (PD), 2 patients with dementia with Lewy bodies (DLB), 2 patients with progressive supranuclear palsy (PSP), 2 patients with striatonigral degeneration, and 16 normal volunteers, using Three-dimensional stereotactic surface projections (3D-SSP). Decreased rCBF in PD patients was shown in the posterior parietal and occipital cortex. Decreased rCBF in DLB was shown in the frontal, parietal and occipital cortex with relative sparing of the sensorimotor cortex.. Decreased rCBF in PSP was shown in the frontal cortex. Decreased rCBF in SND was shown in the frontal cortex and cerebellum. Statistic rCBF analysis using 3D-SSP was a useful measure for the early differential diagnosis of extrapyramidal disorders. (author)

  11. Reliable prediction of transcription factor binding sites by phylogenetic verification

    OpenAIRE

    Li, Xiaoman; Zhong, Sheng; Wong, Wing H.

    2005-01-01

    We present a statistical methodology that largely improves the accuracy in computational predictions of transcription factor (TF) binding sites in eukaryote genomes. This method models the cross-species conservation of binding sites without relying on accurate sequence alignment. It can be coupled with any motif-finding algorithm that searches for overrepresented sequence motifs in individual species and can increase the accuracy of the coupled motif-finding algorithm. Because this method is ...

  12. Quantitative modeling of transcription factor binding specificities using DNA shape.

    Science.gov (United States)

    Zhou, Tianyin; Shen, Ning; Yang, Lin; Abe, Namiko; Horton, John; Mann, Richard S; Bussemaker, Harmen J; Gordân, Raluca; Rohs, Remo

    2015-04-14

    DNA binding specificities of transcription factors (TFs) are a key component of gene regulatory processes. Underlying mechanisms that explain the highly specific binding of TFs to their genomic target sites are poorly understood. A better understanding of TF-DNA binding requires the ability to quantitatively model TF binding to accessible DNA as its basic step, before additional in vivo components can be considered. Traditionally, these models were built based on nucleotide sequence. Here, we integrated 3D DNA shape information derived with a high-throughput approach into the modeling of TF binding specificities. Using support vector regression, we trained quantitative models of TF binding specificity based on protein binding microarray (PBM) data for 68 mammalian TFs. The evaluation of our models included cross-validation on specific PBM array designs, testing across different PBM array designs, and using PBM-trained models to predict relative binding affinities derived from in vitro selection combined with deep sequencing (SELEX-seq). Our results showed that shape-augmented models compared favorably to sequence-based models. Although both k-mer and DNA shape features can encode interdependencies between nucleotide positions of the binding site, using DNA shape features reduced the dimensionality of the feature space. In addition, analyzing the feature weights of DNA shape-augmented models uncovered TF family-specific structural readout mechanisms that were not revealed by the DNA sequence. As such, this work combines knowledge from structural biology and genomics, and suggests a new path toward understanding TF binding and genome function. PMID:25775564

  13. Microbes bind complement inhibitor factor H via a common site.

    Science.gov (United States)

    Meri, T; Amdahl, H; Lehtinen, M J; Hyvärinen, S; McDowell, J V; Bhattacharjee, A; Meri, S; Marconi, R; Goldman, A; Jokiranta, T S

    2013-01-01

    To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19-20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a "superevasion site." PMID:23637600

  14. The Arabidopsis mediator complex subunits MED16, MED14, and MED2 regulate mediator and RNA polymerase II recruitment to CBF-responsive cold-regulated genes.

    Science.gov (United States)

    Hemsley, Piers A; Hurst, Charlotte H; Kaliyadasa, Ewon; Lamb, Rebecca; Knight, Marc R; De Cothi, Elizabeth A; Steele, John F; Knight, Heather

    2014-01-01

    The Mediator16 (MED16; formerly termed SENSITIVE TO FREEZING6 [SFR6]) subunit of the plant Mediator transcriptional coactivator complex regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. Here, we use loss-of-function mutants to show that RNA polymerase II recruitment to CBF-responsive cold-regulated genes requires MED16, MED2, and MED14 subunits. Transcription of genes known to be regulated via CBFs binding to the C-repeat motif/drought-responsive element promoter motif requires all three Mediator subunits, as does cold acclimation-induced freezing tolerance. In addition, these three subunits are required for low temperature-induced expression of some other, but not all, cold-responsive genes, including genes that are not known targets of CBFs. Genes inducible by darkness also required MED16 but required a different combination of Mediator subunits for their expression than the genes induced by cold. Together, our data illustrate that plants control transcription of specific genes through the action of subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced. PMID:24415770

  15. Low-temperature-induced expression of rice ureidoglycolate amidohydrolase is mediated by a C-repeat/dehydration-responsive element that specifically interacts with rice C-repeat-binding factor 3

    Directory of Open Access Journals (Sweden)

    Juan eLi

    2015-11-01

    Full Text Available Nitrogen recycling and redistribution are important for the environmental stress response of plants. In non nitrogen-fixing plants, ureide metabolism is crucial to nitrogen recycling from organic sources. Various studies have suggested that the rate-limiting components of ureide metabolism respond to environmental stresses. However, the underlying regulation mechanism is not well understood. In this report, rice ureidoglycolate amidohydrolase (OsUAH, which is a recently identified enzyme catalyzing the final step of ureide degradation, was identified as low-temperature- (LT but not abscisic acid- (ABA regulated. To elucidate the LT regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region of OsUAH (POsUAH. Series deletions revealed that a minimal region between -522 and -420 relative to the transcriptional start site was sufficient for the cold induction of POsUAH. Detailed analyses of this 103-bp fragment indicated that a C-repeat/dehydration-responsive (CRT/DRE element localized at position -434 was essential for LT-responsive expression. A rice C-repeat-binding factors/DRE-binding proteins 1 (CBFs/DREB1s subfamily member, OsCBF3, was screened to specifically bind to the CRT/DRE element in the minimal region both in yeast one-hybrid assays and in in vitro gel-shift analysis. Moreover, the promoter could be exclusively trans-activated by the interaction between the CRT/DRE element and OsCBF3 in vivo. These findings may help to elucidate the regulation mechanism of stress-responsive ureide metabolism genes and provide an example of the member-specific manipulation of the CBF/DREB1 subfamily.

  16. Architecture and RNA binding of the human negative elongation factor

    Science.gov (United States)

    Vos, Seychelle M; Pöllmann, David; Caizzi, Livia; Hofmann, Katharina B; Rombaut, Pascaline; Zimniak, Tomasz; Herzog, Franz; Cramer, Patrick

    2016-01-01

    Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF. DOI: http://dx.doi.org/10.7554/eLife.14981.001 PMID:27282391

  17. The next generation of transcription factor binding site prediction.

    Directory of Open Access Journals (Sweden)

    Anthony Mathelier

    Full Text Available Finding where transcription factors (TFs bind to the DNA is of key importance to decipher gene regulation at a transcriptional level. Classically, computational prediction of TF binding sites (TFBSs is based on basic position weight matrices (PWMs which quantitatively score binding motifs based on the observed nucleotide patterns in a set of TFBSs for the corresponding TF. Such models make the strong assumption that each nucleotide participates independently in the corresponding DNA-protein interaction and do not account for flexible length motifs. We introduce transcription factor flexible models (TFFMs to represent TF binding properties. Based on hidden Markov models, TFFMs are flexible, and can model both position interdependence within TFBSs and variable length motifs within a single dedicated framework. The availability of thousands of experimentally validated DNA-TF interaction sequences from ChIP-seq allows for the generation of models that perform as well as PWMs for stereotypical TFs and can improve performance for TFs with flexible binding characteristics. We present a new graphical representation of the motifs that convey properties of position interdependence. TFFMs have been assessed on ChIP-seq data sets coming from the ENCODE project, revealing that they can perform better than both PWMs and the dinucleotide weight matrix extension in discriminating ChIP-seq from background sequences. Under the assumption that ChIP-seq signal values are correlated with the affinity of the TF-DNA binding, we find that TFFM scores correlate with ChIP-seq peak signals. Moreover, using available TF-DNA affinity measurements for the Max TF, we demonstrate that TFFMs constructed from ChIP-seq data correlate with published experimentally measured DNA-binding affinities. Finally, TFFMs allow for the straightforward computation of an integrated TF occupancy score across a sequence. These results demonstrate the capacity of TFFMs to accurately model DNA

  18. Structural Fingerprints of Transcription Factor Binding Site Regions

    Directory of Open Access Journals (Sweden)

    Peter Willett

    2009-03-01

    Full Text Available Fourier transforms are a powerful tool in the prediction of DNA sequence properties, such as the presence/absence of codons. We have previously compiled a database of the structural properties of all 32,896 unique DNA octamers. In this work we apply Fourier techniques to the analysis of the structural properties of human chromosomes 21 and 22 and also to three sets of transcription factor binding sites within these chromosomes. We find that, for a given structural property, the structural property power spectra of chromosomes 21 and 22 are strikingly similar. We find common peaks in their power spectra for both Sp1 and p53 transcription factor binding sites. We use the power spectra as a structural fingerprint and perform similarity searching in order to find transcription factor binding site regions. This approach provides a new strategy for searching the genome data for information. Although it is difficult to understand the relationship between specific functional properties and the set of structural parameters in our database, our structural fingerprints nevertheless provide a useful tool for searching for function information in sequence data. The power spectrum fingerprints provide a simple, fast method for comparing a set of functional sequences, in this case transcription factor binding site regions, with the sequences of whole chromosomes. On its own, the power spectrum fingerprint does not find all transcription factor binding sites in a chromosome, but the results presented here show that in combination with other approaches, this technique will improve the chances of identifying functional sequences hidden in genomic data.

  19. Effective antimicrobial activity of Cbf-14, derived from a cathelin-like domain, against penicillin-resistant bacteria.

    Science.gov (United States)

    Ma, Lingman; Wang, Yanrong; Wang, Mengxiao; Tian, Yuwei; Kang, Wei; Liu, Hanhan; Wang, Hui; Dou, Jie; Zhou, Changlin

    2016-05-01

    Cbf-14, a cationic peptide derived from a cathelin-like domain, was designed by inserting the highly α-helical sequence RLLR into an antibacterial sequence and deleting the inactive amino acids in Cbf-K16. Clinical penicillin-resistant isolates as well as NDM-1-carrying Escherichia coli and a correspondingly infected mice model were employed to evaluate Cbf-14 antibacterial activity. The results showed that Cbf-14 possessed potent antimicrobial effects with an MIC of 8-64 μg/ml, and killed almost all bacteria within 240 min. Cbf-14-treated mice achieved an 80% survival rate and approximate 2.5 log unit reduction in CFU in tissues; additionally, this peptide significantly suppressed the production of pro-inflammatory cytokines by the disaggregation of lipopolysaccharide (LPS), suggesting its anti-inflammatory effects. Furthermore, Cbf-14, concentration higher than 2 × MIC value, increased membrane uptake to NPN and PI dye by 96.2% and 63.7%, respectively, neutralised the negative zeta potential of LPS and bacteria surface, and induced 100% leakage of liposome-entrapped calcein and cytoplasmic membrane disruption of E. coli, indicating obvious membrane permeation. Finally, it bound to DNA and respectively evoked 85.0% and 63.3% inhibition of gene replication and protein expression of NDM-1 at sub-MIC concentration in E. coli BL21 (DE3)-NDM-1. These data indicated that Cbf-14 possessed effective antimicrobial activity against penicillin-resistant bacteria in vitro/vivo through membrane disruption, DNA binding, down-regulating NDM-1 expression by plasmid replication inhibition, and anti-inflammatory activity by LPS disaggregation, suggesting a potential anti-infective clinical agent. PMID:26897538

  20. rCBF study with 123I-IMP SPECT of dural arteriovenous fistula

    International Nuclear Information System (INIS)

    It is important for dural arteriovenous fistula (DAVF) to evaluate venous ischemia in the brain related to venous hypertension, but only a few such studies have been performed. In this study, regional cerebral blood flow (rCBF) in DAVF was examined for venous ischemia by 123I-IMP SPECT. The subjects were eighteen patients with DAVF. Of the eighteen patients, nine had DAVF with low perfusion areas and venous ischemia. The factors affecting rCBF in DAVF are: the presence of retrograde leptomeningeal venous drainage, sinus occlusion, and DAVF with high flow. The presence of retrograde leptomeningeal venous drainage was observed in nine patients, sinus occlusion in four patients, high flow in three patients. In two patients, pure leptomeningeal venous drainage was formed by patent sinus, and blood regurgitated from DAVF on the sinus wall to cortical vein. When DAVF was associated with LMVD, most patients had venous hypertension and concomitant venous congestion in the same areas due to reduced venous circulation, resulting in a decrease in rCBF and an increase in regional cerebral blood volume. These hemodynamics suggest venous ischemia in the brain 123I-IMP SPECT was useful for evaluating rCBF and as a parameter of the treatment. (author)

  1. rCBF study with {sup 123}I-IMP SPECT of dural arteriovenous fistula

    Energy Technology Data Exchange (ETDEWEB)

    Kawaguchi, Tsutomu; Kawano, Teruaki; Kaneko, Yoshiro; Ooasa, Takao; Tsutsumi, Masanori; Ogasawara, Sadanobu [Fukuoka Tokushukai Hospital, Kasuga (Japan)

    2000-11-01

    It is important for dural arteriovenous fistula (DAVF) to evaluate venous ischemia in the brain related to venous hypertension, but only a few such studies have been performed. In this study, regional cerebral blood flow (rCBF) in DAVF was examined for venous ischemia by {sup 123}I-IMP SPECT. The subjects were eighteen patients with DAVF. Of the eighteen patients, nine had DAVF with low perfusion areas and venous ischemia. The factors affecting rCBF in DAVF are: the presence of retrograde leptomeningeal venous drainage, sinus occlusion, and DAVF with high flow. The presence of retrograde leptomeningeal venous drainage was observed in nine patients, sinus occlusion in four patients, high flow in three patients. In two patients, pure leptomeningeal venous drainage was formed by patent sinus, and blood regurgitated from DAVF on the sinus wall to cortical vein. When DAVF was associated with LMVD, most patients had venous hypertension and concomitant venous congestion in the same areas due to reduced venous circulation, resulting in a decrease in rCBF and an increase in regional cerebral blood volume. These hemodynamics suggest venous ischemia in the brain {sup 123}I-IMP SPECT was useful for evaluating rCBF and as a parameter of the treatment. (author)

  2. Insulin-like growth factor binding proteins: a structural perspective

    Directory of Open Access Journals (Sweden)

    Briony eForbes

    2012-03-01

    Full Text Available Insulin-like growth factor binding proteins (IGFBP-1 to -6 bind insulin-like growth factors-I and -II (IGF-I and IGF-II with high affinity. These binding proteins maintain IGFs in the circulation and direct them to target tissues, where they promote cell growth, proliferation, differentiation and survival via the type 1 IGF receptor (IGF-1R. IGFBPs also interact with many other molecules, which not only influence their modulation of IGF action but also mediate IGF-independent activities that influence processes such as cell migration and apoptosis by influencing gene transcription.IGFBPs-1 to -6 are structurally similar proteins consisting of three distinct domains, N-terminal, Linker and C-terminal. There have been major advances in our understanding of IGFBP structure in the last decade and a half. While there is still no structure of an intact IGFBP to date, several structures of individual N- and C-domains have been solved. The structure of a complex of N-BP-4:IGF-I:C-BP-4 has also been solved, providing a detailed picture of the structural features of the IGF binding site and the mechanism of binding. Structural studies have also identified features important for interaction with extracellular matrix components and integrins. This review summarises structural studies reported so far and highlights features important for binding not only IGF but also other partners. It also highlights future directions in which structural studies will add to our knowledge of the role played by the IGFBP family in normal growth and development, as well as in disease.

  3. Coagulation Factor Binding Orientation and Dimerization May Influence Infectivity of Adenovirus-Coagulation Factor Complexes

    OpenAIRE

    Irons, Eric E.; Flatt, Justin W.; Doronin, Konstantin; Fox, Tara L.; Acchione, Mauro; Stewart, Phoebe L.; Shayakhmetov, Dmitry M.

    2013-01-01

    Adenoviruses (Ads) are promising vectors for therapeutic interventions in humans. When injected into the bloodstream, Ad vectors can bind several vitamin K-dependent blood coagulation factors, which contributes to virus sequestration in the liver by facilitating transduction of hepatocytes. Although both coagulation factors FVII and FX bind the hexon protein of human Ad serotype 5 (HAdv5) with a very high affinity, only FX appears to play a role in mediating Ad-hepatocyte transduction in vivo...

  4. Microbes Bind Complement Inhibitor Factor H via a Common Site

    OpenAIRE

    Meri, T.; Amdahl, H.; Lehtinen, M. J.; Hyvärinen, S.; McDowell, J.V.; Bhattacharjee, A.; Meri, S.; Marconi, R.; Goldman, A; Jokiranta, T. S.

    2013-01-01

    To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic ...

  5. Cooperative binding of transcription factors promotes bimodal gene expression response.

    Directory of Open Access Journals (Sweden)

    Pablo S Gutierrez

    Full Text Available In the present work we extend and analyze the scope of our recently proposed stochastic model for transcriptional regulation, which considers an arbitrarily complex cis-regulatory system using only elementary reactions. Previously, we determined the role of cooperativity on the intrinsic fluctuations of gene expression for activating transcriptional switches, by means of master equation formalism and computer simulation. This model allowed us to distinguish between two cooperative binding mechanisms and, even though the mean expression levels were not affected differently by the acting mechanism, we showed that the associated fluctuations were different. In the present generalized model we include other regulatory functions in addition to those associated to an activator switch. Namely, we introduce repressive regulatory functions and two theoretical mechanisms that account for the biphasic response that some cis-regulatory systems show to the transcription factor concentration. We have also extended our previous master equation formalism in order to include protein production by stochastic translation of mRNA. Furthermore, we examine the graded/binary scenarios in the context of the interaction energy between transcription factors. In this sense, this is the first report to show that the cooperative binding of transcription factors to DNA promotes the "all-or-none" phenomenon observed in eukaryotic systems. In addition, we confirm that gene expression fluctuation levels associated with one of two cooperative binding mechanism never exceed the fluctuation levels of the other.

  6. Incorporating evolution of transcription factor binding sites into annotated alignments

    Indian Academy of Sciences (India)

    Abha S Bais; Steffen Grossmann; Martin Vingron

    2007-08-01

    Identifying transcription factor binding sites (TFBSs) is essential to elucidate putative regulatory mechanisms. A common strategy is to combine cross-species conservation with single sequence TFBS annotation to yield ``conserved TFBSs”. Most current methods in this field adopt a multi-step approach that segregates the two aspects. Again, it is widely accepted that the evolutionary dynamics of binding sites differ from those of the surrounding sequence. Hence, it is desirable to have an approach that explicitly takes this factor into account. Although a plethora of approaches have been proposed for the prediction of conserved TFBSs, very few explicitly model TFBS evolutionary properties, while additionally being multi-step. Recently, we introduced a novel approach to simultaneously align and annotate conserved TFBSs in a pair of sequences. Building upon the standard Smith-Waterman algorithm for local alignments, SimAnn introduces additional states for profiles to output extended alignments or annotated alignments. That is, alignments with parts annotated as gaplessly aligned TFBSs (pair-profile hits) are generated. Moreover, the pair-profile related parameters are derived in a sound statistical framework. In this article, we extend this approach to explicitly incorporate evolution of binding sites in the SimAnn framework. We demonstrate the extension in the theoretical derivations through two position-specific evolutionary models, previously used for modelling TFBS evolution. In a simulated setting, we provide a proof of concept that the approach works given the underlying assumptions, as compared to the original work. Finally, using a real dataset of experimentally verified binding sites in human-mouse sequence pairs, we compare the new approach (eSimAnn) to an existing multi-step tool that also considers TFBS evolution. Although it is widely accepted that binding sites evolve differently from the surrounding sequences, most comparative TFBS identification

  7. A systems biology approach to transcription factor binding site prediction.

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    Full Text Available BACKGROUND: The elucidation of mammalian transcriptional regulatory networks holds great promise for both basic and translational research and remains one the greatest challenges to systems biology. Recent reverse engineering methods deduce regulatory interactions from large-scale mRNA expression profiles and cross-species conserved regulatory regions in DNA. Technical challenges faced by these methods include distinguishing between direct and indirect interactions, associating transcription regulators with predicted transcription factor binding sites (TFBSs, identifying non-linearly conserved binding sites across species, and providing realistic accuracy estimates. METHODOLOGY/PRINCIPAL FINDINGS: We address these challenges by closely integrating proven methods for regulatory network reverse engineering from mRNA expression data, linearly and non-linearly conserved regulatory region discovery, and TFBS evaluation and discovery. Using an extensive test set of high-likelihood interactions, which we collected in order to provide realistic prediction-accuracy estimates, we show that a careful integration of these methods leads to significant improvements in prediction accuracy. To verify our methods, we biochemically validated TFBS predictions made for both transcription factors (TFs and co-factors; we validated binding site predictions made using a known E2F1 DNA-binding motif on E2F1 predicted promoter targets, known E2F1 and JUND motifs on JUND predicted promoter targets, and a de novo discovered motif for BCL6 on BCL6 predicted promoter targets. Finally, to demonstrate accuracy of prediction using an external dataset, we showed that sites matching predicted motifs for ZNF263 are significantly enriched in recent ZNF263 ChIP-seq data. CONCLUSIONS/SIGNIFICANCE: Using an integrative framework, we were able to address technical challenges faced by state of the art network reverse engineering methods, leading to significant improvement in direct

  8. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  9. Saccharin and Cyclamate Inhibit Binding of Epidermal Growth Factor

    Science.gov (United States)

    Lee, L. S.

    1981-02-01

    The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.

  10. Reliable prediction of transcription factor binding sites by phylogenetic verification.

    Science.gov (United States)

    Li, Xiaoman; Zhong, Sheng; Wong, Wing H

    2005-11-22

    We present a statistical methodology that largely improves the accuracy in computational predictions of transcription factor (TF) binding sites in eukaryote genomes. This method models the cross-species conservation of binding sites without relying on accurate sequence alignment. It can be coupled with any motif-finding algorithm that searches for overrepresented sequence motifs in individual species and can increase the accuracy of the coupled motif-finding algorithm. Because this method is capable of accurately detecting TF binding sites, it also enhances our ability to predict the cis-regulatory modules. We applied this method on the published chromatin immunoprecipitation (ChIP)-chip data in Saccharomyces cerevisiae and found that its sensitivity and specificity are 9% and 14% higher than those of two recent methods. We also recovered almost all of the previously verified TF binding sites and made predictions on the cis-regulatory elements that govern the tight regulation of ribosomal protein genes in 13 eukaryote species (2 plants, 4 yeasts, 2 worms, 2 insects, and 3 mammals). These results give insights to the transcriptional regulation in eukaryotic organisms. PMID:16286651

  11. A study comparing the use of dynamic CT and Xe-CT CBF for ischemic cerebro-vascular disease

    International Nuclear Information System (INIS)

    The simultaneous measurement of dynamic computerized tomography scanning (DCT) with an iodine-contrast enhancement material bolus injection and a simultaneous xenon CT-CBF-study was done on 15 patients (8 cases of unilateral internal carotid occlusion; 3, of unilateral middle cerebral arterial occlusion, and 4, without any major cerebral arterial occlusion or significant arterial stenosis) with ischemic cerebro-vascular diseases at the subacute and/or chronic stage. The value of the width and corrected first moment (cMT1) as well as their functional images, as acquired from DCT data, were compared to the 1-CBF value and the 1-CBF map of the xenon CT-CBF-study. A comparison of the functional images of DCT and 1-CBF showed that there was a good correlation between them in the cases without leptomeningeal anastomosis as a collateral circulation. However, a poor correlation between them was found in the cases with leptomeningeal anastomosis as a collateral circulation. The correlation of 1-CBF and 1/width with 1/cMT1 was significant (r = 0.78, p < 0.01) in the former cases, but it was not significant in the latter cases. The results of our data were thought to be due to the difference in the tracer inflow pattern between the cases without leptomeningeal anastomosis and those with it as a collateral circulation. The factor of cerebral blood volume should be considered in a more detailed study, although our cases did not include any patients with acute cerebral infarction or recanalized cases, which are thought to show various changes in the cerebral blood volume. The 1/width and 1/cMT1 values acquired from DCT well reflected the CBF in the cases without leptomeningeal anastomosis as a collateral circulation. (author)

  12. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    Directory of Open Access Journals (Sweden)

    Ilse Jongerius

    Full Text Available Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH, a negative regulator of the complement system, to its surface via fH binding protein (fHbp, providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.

  13. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    Science.gov (United States)

    Jongerius, Ilse; Lavender, Hayley; Tan, Lionel; Ruivo, Nicola; Exley, Rachel M; Caesar, Joseph J E; Lea, Susan M; Johnson, Steven; Tang, Christoph M

    2013-01-01

    Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups. PMID:23935503

  14. Octamer-binding transcription factors: genomics and functions.

    Science.gov (United States)

    Zhao, Feng-Qi

    2013-01-01

    The Octamer-binding proteins (Oct) are a group of highly conserved transcription factors that specifically bind to the octamer motif (ATGCAAAT) and closely related sequences in promoters and enhancers of a wide variety of genes. Oct factors belong to the larger family of POU domain factors that are characterized by the presence of an amino-terminal specific subdomain (POUS) and a carboxyl-terminal homeo-subdomain (POUH). Eleven Oct proteins have been named (Oct1-11), and currently, eight genes encoding Oct proteins (Oct1, Oct2, Oct3/4, Oct6, Oct7, Oct8, Oct9, and Oct11) have been cloned. Oct1 and Oct2 are widely expressed in adult tissues, while other Oct proteins are much more restricted in their expression patterns. Oct proteins are implicated in crucial and versatile biological events, such as embryogenesis, neurogenesis, immunity, and body glucose and amino acid metabolism. The aberrant expression and null function of Oct proteins have also been linked to various diseases, including deafness, diabetes and cancer. In this review, I will report both the genomic structure and major functions of individual Oct proteins in physiological and pathological processes. PMID:23747866

  15. Protein kinase A binds and activates heat shock factor 1.

    Directory of Open Access Journals (Sweden)

    Ayesha Murshid

    Full Text Available BACKGROUND: Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1, a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. METHODOLOGY/PRINCIPAL FINDINGS: In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA. HSF1 binds avidly to the catalytic subunit of PKA, (PKAcα and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320, both in vitro and in vivo. Intracellular PKAcα levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1 and activate hsp70.1 after stress. Reduction in PKAcα levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, null mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. CONCLUSIONS: These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease.

  16. Minimalistic predictor of protein binding energy: contribution of solvation factor to protein binding.

    Science.gov (United States)

    Choi, Jeong-Mo; Serohijos, Adrian W R; Murphy, Sean; Lucarelli, Dennis; Lofranco, Leo L; Feldman, Andrew; Shakhnovich, Eugene I

    2015-02-17

    It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software. PMID:25692584

  17. Heterogeneity of binding subunits of the human 150K insulin-like growth factor binding protein.

    Science.gov (United States)

    Gelato, M C; Gaynes, L A; Greenstein, L A; Nissley, S P

    1990-04-01

    Models for the structure of the GH-dependent 150K insulin-like growth factor-binding protein (IGF-BP) complex include 1) a binding subunit of 40-60K mol wt associated with a larger nonbinding component, and 2) an oligomeric structure simply made up of six 25-28K monomeric IGF-BP complexes. To evaluate these alternative models we examined the IGF-binding characteristics and behavior on an SP-Sephadex ion exchange column of BP species identified by chemically cross-linking [125I]IGF-I and [125I]IGF-II. In addition, human serum was gel filtered on Sephadex G-200 in 0.05 M NH4HCO3, pH 8.0, and the 150K BP identified by binding of [125I]IGF-II to column fractions. When [125I]IGF-I or [125I]IGF-II was cross-linked to the 150K BP with disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10-15%) and autoradiography, four specifically labeled complexes of 20K, 24K, 33K, and 47K mol wt were identified. We examined the IGF-binding characteristics of these species by cross-linking [125I]IGF-I and [125I]IGF-II after incubation in the presence of increasing concentrations of unlabeled IGF-I or IGF-II. Formation of the 24K complex was inhibited more potently by IGF-II than IGF-I, whereas the relative potency of IGF-I vs. IGF-II for inhibition of the formation of the other complexes depended upon whether [125I]IGF-II or [125I]IGF-I was used. When the 150K BP complex generated from gel filtration on Sephadex G-200 was acid stripped, the only species seen with chemical cross-linking of either [125I]IGF-I or [125I]IGF-II was the 47K complex. By both conventional competitive binding studies and cross-linking [125I]IGF-I and [125I]IGF-II after incubation with increasing concentrations of unlabeled IGF-I or IGF-II, the formation of the 47K complex was usually more potently inhibited by IGF-I than IGF-II. When Cohn fraction IV extract was chromatographed on a SP-Sephadex column (pH 3) and cross-linking performed on the flow-through, the 47K

  18. Effects of cytosine methylation on transcription factor binding sites

    KAUST Repository

    Medvedeva, Yulia A

    2014-03-26

    Background: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important.Results: We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines " traffic lights" We observed a strong selection against CpG " traffic lights" within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions.Conclusions: Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. 2013 Medvedeva et al.; licensee BioMed Central Ltd.

  19. Atrial natriuretic factor binding sites in experimental congestive heart failure

    International Nuclear Information System (INIS)

    A quantitative in vitro autoradiographic study was performed on the aorta, renal glomeruli, and adrenal cortex of cardiomyopathic hamsters in various stages of heart failure and correlated, in some instances, with in vivo autoradiography. The results indicate virtually no correlation between the degree of congestive heart failure and the density of 125I-labeled atrial natriuretic factor [(Ser99, Tyr126)ANF] binding sites (Bmax) in the tissues examined. Whereas the Bmax was increased in the thoracic aorta in moderate and severe heart failure, there were no significant changes in the zona glomerulosa. The renal glomeruli Bmax was lower in mild and moderate heart failure compared with control and severe heart failure. The proportion of ANF B- and C-receptors was also evaluated in sections of the aorta, adrenal, and kidney of control and cardiomyopathic hamsters with severe heart failure. (Arg102, Cys121)ANF [des-(Gln113, Ser114, Gly115, Leu116, Gly117) NH2] (C-ANF) at 10(-6) M displaced approximately 505 of (Ser99, Tyr126)125I-ANF bound in the aorta and renal glomeruli and approximately 20% in the adrenal zona glomerulosa in both series of animals. These results suggest that ANF may exert a buffering effect on the vasoconstriction of heart failure and to a certain extent may inhibit aldosterone secretion. The impairment of renal sodium excretion does not appear to be related to glomerular ANF binding sites at any stage of the disease

  20. The role of octamer binding transcription factors in glioblastoma multiforme.

    Science.gov (United States)

    Rooj, A K; Bronisz, A; Godlewski, J

    2016-06-01

    A group of transcription factors (TF) that are master developmental regulators of the establishment and maintenance of pluripotency during embryogenesis play additional roles to control tissue homeostasis and regeneration in adults. Among these TFs, members of the octamer-binding transcription factor (OCT) gene family are well documented as major regulators controlling the self-renewal and pluripotency of stem cells isolated from different adult organs including the brain. In the last few years a large number of studies show the aberrant expression and dysfunction of OCT in different types of cancers including glioblastoma multiforme (GBM). GBM is the most common malignant primary brain tumor, and contains a subpopulation of undifferentiated stem cells (GSCs), with self-renewal and tumorigenic potential that contribute to tumor initiation, invasion, recurrence, and therapeutic resistance. In this review, we have summarized the current knowledge about OCT family in GBM and their crucial role in the initiation, maintenance and drug resistance properties of GSCs. This article is part of a Special Issue entitled: The Oct Transcription Factor Family, edited by Dr. Dean Tantin. PMID:26968235

  1. MONKEY: Identifying conserved transcription-factor binding sitesin multiple alignments using a binding site-specific evolutionarymodel

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Pollard, Daniel A.; Iyer, VenkyN.; Eisen, Michael B.

    2004-10-28

    We introduce a method (MONKEY) to identify conserved transcription-factor binding sites in multispecies alignments. MONKEY employs probabilistic models of factor specificity and binding site evolution, on which basis we compute the likelihood that putative sites are conserved and assign statistical significance to each hit. Using genomes from the genus Saccharomyces, we illustrate how the significance of real sites increases with evolutionary distance and explore the relationship between conservation and function.

  2. Development of the Chinese Big Five Personality Inventory(CBF-PI) Ⅲ:Psychometric Properties of CBF-PI Brief Version%中国大五人格问卷的初步编制Ⅲ:简式版的制定及信效度检验

    Institute of Scientific and Technical Information of China (English)

    王孟成; 戴晓阳; 姚树桥

    2011-01-01

    Objective: The present study aimed to create a brief inventory (CBF-PI-B) to assess the five dimensions based on Chinese Big Five Personality Inventory (CBF-PI).Methods: A set of good items were extracted from CBF-PI to form a brief scale.A total of 1221 college students were administered the CBF-PI.Other two samples including 54 and 53 college students completed both CBF-PI and NEO-PI-R or BFI, respectively.Results: ①CBF-PI-B included 40 items,which had good psychometric properties.Internal consistency reliabilities (Coefficient Alphas) of the CBF-PI-B ranging from 0.764(A) to 0.814(N); the mean was 0.793.②The test-retest reliabilities 10 weeks after were also excellent, ranging from 0.672 (A) to 0.811 (O); the mean was 0.742.③The CBF-PI brief version had clear factor structure.All items loaded above 0.40 on their intended factor.Five factors explained 43.49% of the total variance, and the variance explained by each factor at the same level, ranging from 8.16% to 9.15%.Equivalence coefficients between the CBF-PI and CBF-PI-B were above 0.85.④The factors of CBF-PI-B correlated strongly with relevant dimensions of NEO-PI-R and BFI, ranging from 0.358(A) to 0.846(C) and 0.584(A) to 0.826(N), respectively.Conclusion: CBF-PI-B has good psychometric properties, which could bo employed to relevant research.%目的:在中国大五人格问卷(CBF-PI)的基础上编制中国大五人格问卷简式版(Chinese Big Five PersonalityInventory brief version,CBF-PI-B ).方法:从CBF-PI中抽取部分条目组成测量大五人格维度的简式量表.结果:①CBF-PI-B含有40个条目,每个维度分别由8个条目测量;②简式版量表各维度具有较好的信度系数,内部一致性系数在0.764(宜人性)~0.814(神经质)之间,平均0.793;间隔10周的重测系数在0.672(宜人性)~0.811(开放性)之间,平均0.742.③简式版五因子结构较为清晰共可解释总方差变异的43.49%,各因子与完整版量表对应因子的相关均在0

  3. Mutations and binding sites of human transcription factors

    KAUST Repository

    Kamanu, Frederick Kinyua

    2012-06-01

    Mutations in any genome may lead to phenotype characteristics that determine ability of an individual to cope with adaptation to environmental challenges. In studies of human biology, among the most interesting ones are phenotype characteristics that determine responses to drug treatments, response to infections, or predisposition to specific inherited diseases. Most of the research in this field has been focused on the studies of mutation effects on the final gene products, peptides, and their alterations. Considerably less attention was given to the mutations that may affect regulatory mechanism(s) of gene expression, although these may also affect the phenotype characteristics. In this study we make a pilot analysis of mutations observed in the regulatory regions of 24,667 human RefSeq genes. Our study reveals that out of eight studied mutation types, insertions are the only one that in a statistically significant manner alters predicted transcription factor binding sites (TFBSs). We also find that 25 families of TFBSs have been altered by mutations in a statistically significant manner in the promoter regions we considered. Moreover, we find that the related transcription factors are, for example, prominent in processes related to intracellular signaling; cell fate; morphogenesis of organs and epithelium; development of urogenital system, epithelium, and tube; neuron fate commitment. Our study highlights the significance of studying mutations within the genes regulatory regions and opens way for further detailed investigations on this topic, particularly on the downstream affected pathways. 2012 Kamanu, Medvedeva, Schaefer, Jankovic, Archer and Bajic.

  4. Binding of epidermal growth factor and insulin-like growth factor I in human myometrium and leiomyomata

    International Nuclear Information System (INIS)

    Samples of uterine myometrium and leiomyoma from 11 women were analyzed for the presence of epidermal growth factor receptors and insulin-like growth factor I receptors. In addition, the content of soluble insulin-like growth factor binding protein (IGF-BP/PP12) was measured in the tissue cytosols. Cell membrane preparations of myoma tissue bound significantly more insulin-like growth factor I than did those of adjacent normal myometrium, whereas myoma tissue bound less epidermal growth factor than did the normal myometrium. The differences in both insulin-like growth factor I and epidermal growth factor binding were due to changes in receptor concentration rather than to alterations in receptor affinity. Neither myoma nor myometrial tissue contained detectable levels of insulin-like growth factor binding protein. The changes in epidermal growth factor and insulin-like growth factor I binding to the myometrium may play a role in the pathogenesis of uterine leiomyomata

  5. A radioisotope dilution assay for unlabelled vitamin B12-intrinsic factor complex employing the binding intrinsic factor antibody: probable evidence for two types of binding antibody

    International Nuclear Information System (INIS)

    A new radioisotope dilution assay for vitamin B12-intrinsic factor complex is described. The method is based on the use of the binding type intrinsic antibody (the binding reagent), which when combined with the intrinsic factor-vitamin B12 complex (labelled ligand), is quantitatively adsorbed onto zirconium phosphate gel pH 6.25. The new assay has been shown to provide a measure of intrinsic factor comparable with other intrinsic factor assays, but it has the important advantage of being able to measure the unlabelled vitamin B12-intrinsic factor complex (unlabelled ligand), and will, therefore, be valuable in the study of physiological events in the gastrointestinal tract. During the study, it was found that there is some evidence for at least two types of binding intrinsic factor antibody: One which combines preferentially with the intrinsic factor-vitamin B12 complex and one which combines equally well with this complex or with free intrinsic factor. (author)

  6. Biological Characteristics of the Leukemia-Associated Transcriptional Factor AML1 Disclosed by Hematopoietic Rescue of AML1-Deficient Embryonic Stem Cells by Using a Knock-in Strategy

    OpenAIRE

    Okuda, Tsukasa; Takeda, Kiyoshi; Fujita, Yasuko; Nishimura, Motohiro; Yagyu, Shigeki; Yoshida, Makie; Akira, Shizuo; Downing, James R.; Abe, Tatsuo

    2000-01-01

    AML1 is one of the most frequently mutated genes associated with human acute leukemia and encodes the DNA-binding subunit of the heterodimering transcriptional factor complex, core-binding factor (CBF) (or polyoma enhancer binding protein 2 [PEBP2]). A null mutation in either AML1 or its dimerizing partner, CBFβ, results in embryonic lethality secondary to a complete block in fetal liver hematopoiesis, indicating an essential role of this transcription complex in the development of definitive...

  7. Probabilistic Inference of Transcription Factor Binding from Multiple Data Sources

    OpenAIRE

    Lähdesmäki, Harri; Rust, Alistair G.; Shmulevich, Ilya

    2008-01-01

    An important problem in molecular biology is to build a complete understanding of transcriptional regulatory processes in the cell. We have developed a flexible, probabilistic framework to predict TF binding from multiple data sources that differs from the standard hypothesis testing (scanning) methods in several ways. Our probabilistic modeling framework estimates the probability of binding and, thus, naturally reflects our degree of belief in binding. Probabilistic modeling also allows for ...

  8. MORPHEUS, a webtool for transcription factor binding analysis using position weight matrices with dependency

    OpenAIRE

    Eugenio Gómez Minguet; Stéphane Segard; Céline Charavay; François Parcy

    2015-01-01

    Transcriptional networks are central to any biological process and changes affecting transcription factors or their binding sites in the genome are a key factor driving evolution. As more organisms are being sequenced, tools are needed to easily predict transcription factor binding sites (TFBS) presence and affinity from mere inspection of genomic sequences. Although many TFBS discovery algorithms exist, tools for using the DNA binding models they generate are relatively scarce and their use ...

  9. JASPAR: an open-access database for eukaryotic transcription factor binding profiles

    OpenAIRE

    Sandelin, Albin; Alkema, Wynand; Engström, Pär; Wasserman, Wyeth W; Lenhard, Boris

    2004-01-01

    The analysis of regulatory regions in genome sequences is strongly based on the detection of potential transcription factor binding sites. The preferred models for representation of transcription factor binding specificity have been termed position-specific scoring matrices. JASPAR is an open-access database of annotated, high-quality, matrix-based transcription factor binding site profiles for multicellular eukaryotes. The profiles were derived exclusively from sets of nucleotide sequences e...

  10. Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

    Directory of Open Access Journals (Sweden)

    B Josh Lane

    2008-03-01

    Full Text Available Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein. TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs, Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K, appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

  11. Dasatinib in high-risk core binding factor acute myeloid leukemia in first complete remission: a French Acute Myeloid Leukemia Intergroup trial

    Science.gov (United States)

    Boissel, Nicolas; Renneville, Aline; Leguay, Thibaut; Lefebvre, Pascale Cornillet; Recher, Christian; Lecerf, Thibaud; Delabesse, Eric; Berthon, Céline; Blanchet, Odile; Prebet, Thomas; Pautas, Cécile; Chevallier, Patrice; Leprêtre, Stéphane; Girault, Stéphane; Bonmati, Caroline; Guièze, Romain; Himberlin, Chantal; Randriamalala, Edouard; Preudhomme, Claude; Jourdan, Eric; Dombret, Hervé; Ifrah, Norbert

    2015-01-01

    Core-binding factor acute myeloid leukemia is a favorable acute myeloid leukemia subset cytogenetically defined by t(8;21) or inv(16)/t(16;16) rearrangements, disrupting RUNX1 (previously CBFA/AML1) or CBFB transcription factor functions. The receptor tyrosine kinase KIT is expressed in the vast majority of these acute myeloid leukemias and frequent activating KIT gene mutations have been associated with a higher risk of relapse. This phase II study aimed to evaluate dasatinib as maintenance therapy in patients with core-binding factor acute myeloid leukemia in first hematologic complete remission, but at higher risk of relapse due to molecular disease persistence or recurrence. A total of 26 patients aged 18–60 years old previously included in the CBF-2006 trial were eligible to receive dasatinib 140 mg daily if they had a poor initial molecular response (n=18) or a molecular recurrence (n=8). The tolerance of dasatinib as maintenance therapy was satisfactory. The 2-year disease-free survival in this high-risk population of patients was 25.7%. All but one patient with molecular recurrence presented subsequent hematologic relapse. Patients with slow initial molecular response had a similar disease-free survival when treated with dasatinib (40.2% at 2 years) or without any maintenance (50.0% at 2 years). The disappearance of KIT gene mutations at relapse suggests that clonal devolution may in part explain the absence of efficacy observed with single-agent dasatinib in these patients (n. EudraCT: 2006-006555-12). PMID:25715404

  12. Effect of positional dependence and alignment strategy on modeling transcription factor binding sites

    OpenAIRE

    Quader Saad; Huang Chun-Hsi

    2012-01-01

    Abstract Background Many consensus-based and Position Weight Matrix-based methods for recognizing transcription factor binding sites (TFBS) are not well suited to the variability in the lengths of binding sites. Besides, many methods discard known binding sites while building the model. Moreover, the impact of Information Content (IC) and the positional dependence of nucleotides within an aligned set of TFBS has not been well researched for modeling variable-length binding sites. In this pape...

  13. A systematic, large-scale comparison of transcription factor binding site models

    OpenAIRE

    Hombach, Daniela; Schwarz, Jana Marie; Peter N. Robinson; Schuelke, Markus; Seelow, Dominik

    2016-01-01

    Background The modelling of gene regulation is a major challenge in biomedical research. This process is dominated by transcription factors (TFs) and mutations in their binding sites (TFBSs) may cause the misregulation of genes, eventually leading to disease. The consequences of DNA variants on TF binding are modelled in silico using binding matrices, but it remains unclear whether these are capable of accurately representing in vivo binding. In this study, we present a systematic comparison ...

  14. Probabilistic inference of transcription factor binding from multiple data sources.

    Science.gov (United States)

    Lähdesmäki, Harri; Rust, Alistair G; Shmulevich, Ilya

    2008-01-01

    An important problem in molecular biology is to build a complete understanding of transcriptional regulatory processes in the cell. We have developed a flexible, probabilistic framework to predict TF binding from multiple data sources that differs from the standard hypothesis testing (scanning) methods in several ways. Our probabilistic modeling framework estimates the probability of binding and, thus, naturally reflects our degree of belief in binding. Probabilistic modeling also allows for easy and systematic integration of our binding predictions into other probabilistic modeling methods, such as expression-based gene network inference. The method answers the question of whether the whole analyzed promoter has a binding site, but can also be extended to estimate the binding probability at each nucleotide position. Further, we introduce an extension to model combinatorial regulation by several TFs. Most importantly, the proposed methods can make principled probabilistic inference from multiple evidence sources, such as, multiple statistical models (motifs) of the TFs, evolutionary conservation, regulatory potential, CpG islands, nucleosome positioning, DNase hypersensitive sites, ChIP-chip binding segments and other (prior) sequence-based biological knowledge. We developed both a likelihood and a Bayesian method, where the latter is implemented with a Markov chain Monte Carlo algorithm. Results on a carefully constructed test set from the mouse genome demonstrate that principled data fusion can significantly improve the performance of TF binding prediction methods. We also applied the probabilistic modeling framework to all promoters in the mouse genome and the results indicate a sparse connectivity between transcriptional regulators and their target promoters. To facilitate analysis of other sequences and additional data, we have developed an on-line web tool, ProbTF, which implements our probabilistic TF binding prediction method using multiple data sources

  15. Factors Affecting the Binding of a Recombinant Heavy Metal-Binding Domain (CXXC motif Protein to Heavy Metals

    Directory of Open Access Journals (Sweden)

    Kamala Boonyodying

    2012-06-01

    Full Text Available A number of heavy metal-binding proteins have been used to study bioremediation. CXXC motif, a metal binding domain containing Cys-X-X-Cys motif, has been identified in various organisms. These proteins are capable of binding various types of heavy metals. In this study, heavy metal binding domain (CXXC motif recombinant protein encoded from mcsA gene of S. aureus were cloned and overexpressed in Escherichia coli. The factors involved in the metal-binding activity were determined in order to analyze the potential of recombinant protein for bioremediation. A recombinant protein can be bound to Cd2+, Co2+, Cu2+ and Zn2+. The thermal stability of a recombinant protein was tested, and the results showed that the metal binding activity to Cu2+ and Zn2+ still exist after treating the protein at 85ºC for 30 min. The temperature and pH that affected the metal binding activity was tested and the results showed that recombinant protein was still bound to Cu2+ at 65ºC, whereas a pH of 3-7 did not affect the metal binding E. coli harboring a pRset with a heavy metal-binding domain CXXC motif increased the resistance of heavy metals against CuCl2 and CdCl2. This study shows that metal binding domain (CXXC motif recombinant protein can be effectively bound to various types of heavy metals and may be used as a potential tool for studying bioremediation.

  16. Detection and properties of A-factor-binding protein from Streptomyces griseus

    International Nuclear Information System (INIS)

    The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The inducing material virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein

  17. Energy-dependent fitness: A quantitative model for the evolution of yeast transcription factor binding sites

    OpenAIRE

    Mustonen, Ville; Kinney, Justin; Callan, Curtis G.; Lässig, Michael

    2008-01-01

    We present a genomewide cross-species analysis of regulation for broad-acting transcription factors in yeast. Our model for binding site evolution is founded on biophysics: the binding energy between transcription factor and site is a quantitative phenotype of regulatory function, and selection is given by a fitness landscape that depends on this phenotype. The model quantifies conservation, as well as loss and gain, of functional binding sites in a coherent way. Its predictions are supported...

  18. Genome-scale study of the importance of binding site context for transcription factor binding and gene regulation

    Directory of Open Access Journals (Sweden)

    Ronne Hans

    2008-11-01

    Full Text Available Abstract Background The rate of mRNA transcription is controlled by transcription factors that bind to specific DNA motifs in promoter regions upstream of protein coding genes. Recent results indicate that not only the presence of a motif but also motif context (for example the orientation of a motif or its location relative to the coding sequence is important for gene regulation. Results In this study we present ContextFinder, a tool that is specifically aimed at identifying cases where motif context is likely to affect gene regulation. We used ContextFinder to examine the role of motif context in S. cerevisiae both for DNA binding by transcription factors and for effects on gene expression. For DNA binding we found significant patterns of motif location bias, whereas motif orientations did not seem to matter. Motif context appears to affect gene expression even more than it affects DNA binding, as biases in both motif location and orientation were more frequent in promoters of co-expressed genes. We validated our results against data on nucleosome positioning, and found a negative correlation between preferred motif locations and nucleosome occupancy. Conclusion We conclude that the requirement for stable binding of transcription factors to DNA and their subsequent function in gene regulation can impose constraints on motif context.

  19. What does rCBF-SPECT offer in schizophrenia?

    International Nuclear Information System (INIS)

    Schizophrenia is a major psychiatric problem common in the younger population. Structural imaging and findings on autopsy have not yet revealed a specific deficit in these patients. Uncertainty in clinical diagnosis based on a set of signs and symptoms is another drawback in the management of this patient population. Regional cerebral blood flow studies (rCBF) using single photon emission computed tomography (SPECT) offers the opportunity to study the underlying phenomenon and to detect the specific functional deficits in schizophrenia. (Author)

  20. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively

    CERN Document Server

    Clifford, Jacob

    2015-01-01

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through Position Weight Matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain a...

  1. A biophysical model for analysis of transcription factor interaction and binding site arrangement from genome-wide binding data.

    Directory of Open Access Journals (Sweden)

    Xin He

    Full Text Available BACKGROUND: How transcription factors (TFs interact with cis-regulatory sequences and interact with each other is a fundamental, but not well understood, aspect of gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: We present a computational method to address this question, relying on the established biophysical principles. This method, STAP (sequence to affinity prediction, takes into account all combinations and configurations of strong and weak binding sites to analyze large scale transcription factor (TF-DNA binding data to discover cooperative interactions among TFs, infer sequence rules of interaction and predict TF target genes in new conditions with no TF-DNA binding data. The distinctions between STAP and other statistical approaches for analyzing cis-regulatory sequences include the utility of physical principles and the treatment of the DNA binding data as quantitative representation of binding strengths. Applying this method to the ChIP-seq data of 12 TFs in mouse embryonic stem (ES cells, we found that the strength of TF-DNA binding could be significantly modulated by cooperative interactions among TFs with adjacent binding sites. However, further analysis on five putatively interacting TF pairs suggests that such interactions may be relatively insensitive to the distance and orientation of binding sites. Testing a set of putative Nanog motifs, STAP showed that a novel Nanog motif could better explain the ChIP-seq data than previously published ones. We then experimentally tested and verified the new Nanog motif. A series of comparisons showed that STAP has more predictive power than several state-of-the-art methods for cis-regulatory sequence analysis. We took advantage of this power to study the evolution of TF-target relationship in Drosophila. By learning the TF-DNA interaction models from the ChIP-chip data of D. melanogaster (Mel and applying them to the genome of D. pseudoobscura (Pse, we found that only about half of the

  2. Melatonin-induced CBF/DREB1s are essential for diurnal change of disease resistance and CCA1 expression in Arabidopsis.

    Science.gov (United States)

    Shi, Haitao; Wei, Yunxie; He, Chaozu

    2016-03-01

    Melatonin (N-acetyl-5-methoxytryptamine) is an important regulator of circadian rhythms and immunity in animals. However, the diurnal changes of endogenous melatonin and melatonin-mediated diurnal change of downstream responses remain unclear in Arabidopsis. Using the publicly available microarray data, we found that the transcript levels of two melatonin synthesis genes (serotonin N-acetyltransferase (SNAT) and caffeate O-methyltransferase (COMT)) and endogenous melatonin level were regulated by diurnal cycles, with different magnitudes of change. Moreover, the transcripts of C-repeat-binding factors (CBFs)/Drought response element Binding 1 factors (DREB1s) were co-regulated by exogenous melatonin and diurnal changes, indicating the possible correlation among clock, endogenous melatonin level and AtCBFs expressions. Interestingly, diurnal change of plant immunity against Pst DC3000 and CIRCADIANCLOCK ASSOCIATED 1 (CCA1) expression were largely lost in AtCBFs knockdown line-amiR-1. Taken together, this study identifies the molecular pathway underlying the diurnal changes of immunity in Arabidopsis. Notably, the diurnal changes of endogenous melatonin may regulate corresponding changes of AtCBF/DREB1s expression and their underlying diurnal cycle of plant immunity and AtCCA1. PMID:26828406

  3. Cytofluorometric identification of plasmin-sensitive factor XIIIa binding to platelets.

    Science.gov (United States)

    Kreager, J A; Devine, D V; Greenberg, C S

    1988-08-30

    We have investigated the binding of blood coagulation factor XIIIa to thrombin-stimulated platelets using cytofluorometric analysis. Washed thrombin-stimulated platelets bound exogenously added factor XIIIa in a calcium-dependent reaction. The expression of endogenous platelet factor XIII was also detected on the surface of thrombin-stimulated platelets. When fluorescence analysis was performed based on particle size, factor XIIIa bound to the surface of greater than 95% of particles which contained more than one platelet, but only 50% of single platelets. The binding of factor XIIIa to thrombin-stimulated platelets was inhibited by plasmin. Plasmin also inhibited thrombin-dependent expression of the factor XIIIa binding site on platelets. Experiments in which thrombin-stimulated platelets were incubated with factor XIIIa in the presence of 125I-dimethylcasein or 3H-putrescine demonstrated that platelets bear both glutamyl and lysyl substrates for factor XIIIa. Thrombin increased the expression of factor XIIIa substrates by platelets. Plasmin inhibited both the expression of factor XIIIa substrates and degraded them. The binding of factor XIIIa to thrombin-stimulated platelets and the availability of factor XIIIa substrates on the platelet surface could provide a mechanism by which factor XIIIa stabilizes the hemostatic plug by promoting crosslinking reactions between platelet membrane proteins and adhesive glycoproteins. In contrast, plasmin inhibition of factor XIIIa binding and crosslinking could disrupt hemostasis. PMID:2903577

  4. Prediction of transcription factor binding to DNA using rule induction methods

    OpenAIRE

    Huss, Mikael; Nordström, Karin

    2005-01-01

    The transcription of DNA into mRNA is initiated and aided by a number of transcription factors (TFs), proteins with DNA-binding regions that attach themselves to binding sites in the DNA (transcription factor binding sites, TFBSs). As it has become apparent that both TFs and TFBSs are highly variable, tools are needed to quantify the strength of the interaction resulting from a certain TF variant binding to a certain TFBS. We used a simple way to predict interactions between protein and DNA: ...

  5. Binding site of MraZ transcription factor in Mollicutes.

    Science.gov (United States)

    Fisunov, G Y; Evsyutina, D V; Semashko, T A; Arzamasov, A A; Manuvera, V A; Letarov, A V; Govorun, V M

    2016-06-01

    Mollicutes (mycoplasmas) feature a significant loss of known regulators of gene expression. Here, we identified the recognition site of the MraZ-family regulator of Mycoplasma gallisepticum, which is conserved in many species of different clades within class Mollicutes. The MraZ binding site is AAAGTG[T/G], in the promoter of mraZ gene it forms a series of direct repeats with a structure (AAAGTG[T/G]N3)k, where k = 3 most frequently. MraZ binds to a single repeat as an octamer complex. MraZ can also bind a single binding site or a series of repeats with different spacer lengths (2-4 nt); thus, it may play a role in the regulation of multiple operons in Mollicutes. In M. gallisepticum, MraZ acts as a transcriptional activator. The overexpression of MraZ leads to moderate filamentation of cells and the formation of aggregates, likely as a result of incomplete cytokinesis. PMID:26945841

  6. A technique for a rapid imaging of regional CBF and partition coefficient using dynamic SPECT and N-isopropyl-p-[123I]iodoamphetamine (123I-IMP)

    International Nuclear Information System (INIS)

    IMP (iodoamphetamine) is a flow tracer due to a large first pass extraction fraction and high affinity in the brain, but significant clearance from the brain causes change of distribution when the beginning time of scan is delayed. The purpose of the present study was to develop a new method to rapidly calculate a quantitative cerebral blood flow (CBF) image by taking clearance effects into account. A dynamic SPECT scan was performed on 5 subjects (4 patients with cerebral infarction and one normal volunteer) following slow intravenous infusion of 123I-IMP. The arterial input function was obtained by frequent blood sampling and by measuring an octanol extraction ratio for each sample. Firstly, non-linear least square fitting (NLS) was performed to investigate the tracer kinetics of 123I-IMP. The 3 compartment model analysis yielded negligibly small k3 (retaining rate constant) (0.0056±0.0128 (ml/ml/min)), and consistent k1 (transport rate constant) with those determined by 2 compartment model (2CM) analysis (r=0.96, p1 was consistent with CBF measured by 15O water PET technique. These observations suggested the validity of using 2CM for describing the IMP tracer kinetics. Secondly, a weighted integration (WI) technique has been implemented to calculate rapidly images of CBF and partition coefficient (Vd). The WI technique yielded values of CBF (k1) and Vd (k1/k2). They were confirmed to be consistent with those determined by NLS technique (CBF; r=0.99, pd; r=0.99, p1 agreed well with PET CBF (r=0.91, pd in infarcted patients. This supports an importance for calculating Vd image. Vd image will provide additional clinical information because 123I-IMP binding mechanism may be related to cell viability. (author)

  7. In vivo detection of prion amyloid plaques using [11C]BF-227 PET

    International Nuclear Information System (INIS)

    In vivo detection of pathological prion protein (PrP) in the brain is potentially useful for the diagnosis of transmissible spongiform encephalopathies (TSEs). However, there are no non-invasive ante-mortem means for detection of pathological PrP deposition in the brain. The purpose of this study is to evaluate the amyloid imaging tracer BF-227 with positron emission tomography (PET) for the non-invasive detection of PrP amyloid in the brain. The binding ability of BF-227 to PrP amyloid was investigated using autoradiography and fluorescence microscopy. Five patients with TSEs, including three patients with Gerstmann-Straeussler-Scheinker disease (GSS) and two patients with sporadic Creutzfeldt-Jakob disease (CJD), underwent [11C]BF-227 PET scans. Results were compared with data from 10 normal controls and 17 patients with Alzheimer's disease (AD). The regional to pons standardized uptake value ratio was calculated as an index of BF-227 retention. Binding of BF-227 to PrP plaques was confirmed using brain samples from autopsy-confirmed GSS cases. In clinical PET study, significantly higher retention of BF-227 was detected in the cerebellum, thalamus and lateral temporal cortex of GSS patients compared to that in the corresponding tissues of normal controls. GSS patients also showed higher retention of BF-227 in the cerebellum, thalamus and medial temporal cortex compared to AD patients. In contrast, the two CJD patients showed no obvious retention of BF-227 in the brain. Although [11C]BF-227 is a non-specific imaging marker of cerebral amyloidosis, it is useful for in vivo detection of PrP plaques in the human brain in GSS, based on the regional distribution of the tracer. PET amyloid imaging might provide a means for both early diagnosis and non-invasive disease monitoring of certain forms of TSEs. (orig.)

  8. In vivo detection of prion amyloid plaques using [{sup 11}C]BF-227 PET

    Energy Technology Data Exchange (ETDEWEB)

    Okamura, Nobuyuki; Yanai, Kazuhiko [Tohoku University School of Medicine, Department of Pharmacology, Sendai (Japan); Shiga, Yusei; Itoyama, Yasuhito [Tohoku University School of Medicine, Department of Neurology, Sendai (Japan); Furumoto, Shozo [Tohoku University School of Medicine, Department of Pharmacology, Sendai (Japan); Tohoku University, Division of Radiopharmaceutical Chemistry, Cyclotron and Radioisotope Center, Sendai (Japan); Tashiro, Manabu [Tohoku University, Division of Cyclotron Nuclear Medicine, Cyclotron and Radioisotope Center, Sendai (Japan); Tsuboi, Yoshio [Fukuoka University School of Medicine, Department of Neurology, Fukuoka (Japan); Furukawa, Katsutoshi; Arai, Hiroyuki [Institute of Development, Aging, and Cancer, Tohoku University, Department of Geriatrics and Gerontology, Division of Brain Sciences, Sendai (Japan); Iwata, Ren [Tohoku University, Division of Radiopharmaceutical Chemistry, Cyclotron and Radioisotope Center, Sendai (Japan); Kudo, Yukitsuka [Tohoku University, Innovation of New Biomedical Engineering Center, Sendai (Japan); Doh-ura, Katsumi [Tohoku University School of Medicine, Department of Prion Research, 2-1 Seiryo-machi, Aoba-ku, Sendai (Japan)

    2010-05-15

    In vivo detection of pathological prion protein (PrP) in the brain is potentially useful for the diagnosis of transmissible spongiform encephalopathies (TSEs). However, there are no non-invasive ante-mortem means for detection of pathological PrP deposition in the brain. The purpose of this study is to evaluate the amyloid imaging tracer BF-227 with positron emission tomography (PET) for the non-invasive detection of PrP amyloid in the brain. The binding ability of BF-227 to PrP amyloid was investigated using autoradiography and fluorescence microscopy. Five patients with TSEs, including three patients with Gerstmann-Straeussler-Scheinker disease (GSS) and two patients with sporadic Creutzfeldt-Jakob disease (CJD), underwent [{sup 11}C]BF-227 PET scans. Results were compared with data from 10 normal controls and 17 patients with Alzheimer's disease (AD). The regional to pons standardized uptake value ratio was calculated as an index of BF-227 retention. Binding of BF-227 to PrP plaques was confirmed using brain samples from autopsy-confirmed GSS cases. In clinical PET study, significantly higher retention of BF-227 was detected in the cerebellum, thalamus and lateral temporal cortex of GSS patients compared to that in the corresponding tissues of normal controls. GSS patients also showed higher retention of BF-227 in the cerebellum, thalamus and medial temporal cortex compared to AD patients. In contrast, the two CJD patients showed no obvious retention of BF-227 in the brain. Although [{sup 11}C]BF-227 is a non-specific imaging marker of cerebral amyloidosis, it is useful for in vivo detection of PrP plaques in the human brain in GSS, based on the regional distribution of the tracer. PET amyloid imaging might provide a means for both early diagnosis and non-invasive disease monitoring of certain forms of TSEs. (orig.)

  9. Detection and properties of A-factor-binding protein from Streptomyces griseus.

    OpenAIRE

    Miyake, K; Horinouchi, S; Yoshida, M; Chiba, N; Mori, K; Nogawa, N; Morikawa, N; Beppu, T

    1989-01-01

    The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel f...

  10. Tenascin C promiscuously binds growth factors via its fifth fibronectin type III-like domain.

    Directory of Open Access Journals (Sweden)

    Laura De Laporte

    Full Text Available Tenascin C (TNC is an extracellular matrix protein that is upregulated during development as well as tissue remodeling. TNC is comprised of multiple independent folding domains, including 15 fibronectin type III-like (TNCIII domains. The fifth TNCIII domain (TNCIII5 has previously been shown to bind heparin. Our group has shown that the heparin-binding fibronectin type III domains of fibronectin (FNIII, specifically FNIII12-14, possess affinity towards a large number of growth factors. Here, we show that TNCIII5 binds growth factors promiscuously and with high affinity. We produced recombinant fragments of TNC representing the first five TNCIII repeats (TNCIII1-5, as well as subdomains, including TNCIII5, to study interactions with various growth factors. Multiple growth factors of the platelet-derived growth factor (PDGF family, the fibroblast growth factor (FGF family, the transforming growth factor beta (TGF-β superfamily, the insulin-like growth factor binding proteins (IGF-BPs, and neurotrophins were found to bind with high affinity to this region of TNC, specifically to TNCIII5. Surface plasmon resonance was performed to analyze the kinetics of binding of TNCIII1-5 with TGF-β1, PDGF-BB, NT-3, and FGF-2. The promiscuous yet high affinity of TNC for a wide array of growth factors, mediated mainly by TNCIII5, may play a role in multiple physiological and pathological processes involving TNC.

  11. DNA-binding specificity and molecular functions of NAC transcription factors

    DEFF Research Database (Denmark)

    Olsen, Addie Nina; Ernst, Heidi Asschenfeldt; Lo Leggio, Leila;

    2005-01-01

    The family of NAC (NAM/ATAF1,2/CUC2) transcription factors has been implicated in a wide range of plant processes, but knowledge on the DNA-binding properties of the family is limited. Using a reiterative selection procedure on random oligonucleotides, we have identified consensus binding sites for....... Furthermore, NAC protein binding to the CaMV 35S promoter was shown to depend on sequences similar to the consensus of the selected oligonucleotides. Electrophoretic mobility shift assays demonstrated that NAC proteins bind DNA as homo- or heterodimers and that dimerization is necessary for stable DNA binding....... The ability of NAC proteins to dimerize and to bind DNAwas analysed by structure-based mutagenesis. This identified two salt bridge-forming residues essential for NAC protein dimerization. Alteration of basic residues in a loop region containing several highly conserved residues abolished DNA binding...

  12. Competitive binding of transcription factors drives Mendelian dominance in regulatory genetic pathways

    OpenAIRE

    Porter, Adam H.; Johnson, Norman A.; Tulchinsky, Alexander Y.

    2016-01-01

    We report a new mechanism for allelic dominance in regulatory genetic interactions that we call binding dominance. We investigated a biophysical model of gene regulation, where the fractional occupancy of a transcription factor (TF) on the cis-regulated promoter site it binds to is determined by binding energy (-{\\Delta}G) and TF concentration. Transcription and gene expression proceed when the TF is bound to the promoter. In diploids, individuals may be heterozygous at the cis-site, at the T...

  13. Functional Analyses of Transcription Factor Binding Sites that Differ between Present-Day and Archaic Humans

    Science.gov (United States)

    Weyer, Sven; Pääbo, Svante

    2016-01-01

    We analyze 25 previously identified transcription factor binding sites that carry DNA sequence changes that are present in all or nearly all present-day humans, yet occur in the ancestral state in Neandertals and Denisovans, the closest evolutionary relatives of humans. When the ancestral and derived forms of the transcription factor binding sites are tested using reporter constructs in 3 neuronal cell lines, the activity of 12 of the derived versions of transcription factor binding sites differ from the respective ancestral variants. This suggests that the majority of this class of evolutionary differences between modern humans and Neandertals may affect gene expression in at least some tissue or cell type. PMID:26454764

  14. Insulin-like growth factors, insulin-like growth factor-binding proteins, insulin-like growth factor-binding protein-3 protease, and growth hormone-binding protein in lipodystrophic human immunodeficiency virus-infected patients

    DEFF Research Database (Denmark)

    Haugaard, Steen B; Andersen, Ove; Hansen, Birgitte Rønde;

    2004-01-01

    Human immunodeficiency virus (HIV)-lipodystrophy is associated with impaired growth hormone (GH) secretion. It remains to be elucidated whether insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), IGFBP-3 protease, and GH-binding protein (GHBP) are abnormal in HIV......-lipodystrophy. These parameters were measured in overnight fasting serum samples from 16 Caucasian males with HIV-lipodystrophy (LIPO) and 15 Caucasian HIV-infected males without lipodystrophy (NONLIPO) matched for age, weight, duration of HIV infection, and antiretroviral therapy. In LIPO, abdominal fat mass and insulin...

  15. Complement factor H binding by different Lyme disease and relapsing fever Borrelia in animals and human

    Directory of Open Access Journals (Sweden)

    Gil Horacio

    2009-07-01

    Full Text Available Abstract Background Borreliae employ multiple immune evasive strategies such as binding to complement regulatory proteins [factor H (fH and factor H like-1 (FHL1], differential regulation of surface membrane proteins, antigenic variation, and binding of plasminogen/plasmin and matrix metalloproteinases. As a complement regulatory subunit, fH serves as a cofactor for the factor I-mediated cleavage of C3b. fH binding by Borrelia has been correlated with pathogenesis as well as with host diversity. Here we show the differential binding of borrelial proteins to fH from human and animal sera. Findings Affinity ligand binding experiments, 2-D electrophoresis, and protein identification and peptide de novo sequencing based on mass spectrometry, revealed novel fH putative binding proteins of Lyme- and relapsing fever Borrelia. An OspA serotype-associated differential human and animal fH binding by B. garinii was also observed, which could be related with the ability of some strains from serotypes 4 and 7 to invade non-nervous system tissues. Also, the variable affinity of binding proteins expressed by different Borrelia to animal fH correlated with their host selectivity. Conclusion The novel animal and human putative fH binding proteins (FHBPs in this study underscore the importance of evasion of complement in the pathogenesis of Borrelia infections.

  16. Increased CBF velocity during word fluency in Huntington's disease patients.

    Science.gov (United States)

    Deckel, A W; Cohen, D

    2000-02-01

    1. This study examined the effects of word fluency and reading on cerebral blood flow in Huntington's disease (HD) patients. 2. Changes in cerebral flow velocity in the anterior (ACA) and middle (MCA) cerebral arteries were measured with functional transcranial Doppler ultrasonography (fTCD) in 13 normal controls and 9 gene positive HD patients. To control for motor effects of word fluency, two "control" conditions, including silent word fluency and a reading test, were also administered to all subjects. 3. Cerebral blood flow velocity was increased during the out loud word fluency test in the ACA, but not MCA, in the HD group compared to controls. This increase was due to motor components of the test, as during silent word fluency the HD group had a decrease in cerebral blood flow relative to controls. Significant correlations between blood flow in the ACA and word fluency test scores were found. Cerebral blood flow velocity during testing also was able to predict group assignment (i.e., control vs. mild HD vs. moderate HD). 4. These findings add to a growing body of literature suggesting that CBF velocity in HD is abnormal during cognitive and motor tasks. Although previous work reported that CBF velocity in HD is decreased during hand use on a maze test, the current experiment finds that speech production increases cerebral blood flow velocity in HD patients. Collectively, these results point to a fundamental disturbance in the regulation of CBF in HD. Mechanisms that could account for these findings, including the potential involvement of nitric oxide, are discussed. PMID:10800743

  17. Alterations in transcription factor binding in radioresistant human melanoma cells after ionizing radiation

    International Nuclear Information System (INIS)

    We analyzed alterations in transcription factor binding to specific, known promoter DNA consensus sequences between irradiated and unirradiated radioresistant human melanoma (U1-Mel) cells. The goal of this study was to begin to investigate which transcription factors and DNA-binding sites are responsible for the induction of specific transcripts and proteins after ionizing radiation. Transcription factor binding was observed using DNA band-shift assays and oligonucleotide competition analyses. Confluence-arrested U1-Mel cells were irradiated (4.5 Gy) and harvested at 4 h. Double-stranded oligonucleotides containing known DNA-binding consensus sites for specific transcription factors were used. Increased DNA binding activity after ionizing radiation was noted with oligonucleotides containing the CREB, NF-kB and Sp1 consensus sites. No changes in protein binding to AP-1, AP-2, AP-3, or CTF/NF1, GRE or Oct-1 consensus sequences were noted. X-ray activation of select transcription factors, which bind certain consensus sites in promoters, may cause specific induction or repression of gene transcription. 22 refs., 2 figs

  18. Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

    International Nuclear Information System (INIS)

    Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125I-insulin specific binding was not affected. The decrease in 125I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/105 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125I-epidermal growth factor binding. (au)

  19. Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

    OpenAIRE

    Owczarek, C M; Layton, M. J.; Metcalf, D; Lock, P; Willson, T A; Gough, N M; Nicola, N A

    1993-01-01

    Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF-R), while mouse LIF (mLIF) binds only to mouse LIF-R. Moreover, hLIF binds with higher affinity to the mLIF-R than does mLIF. In order to define the regions of the hLIF molecule responsible for species-specific interaction with the hLIF-R and for the unusual high-affinity binding to the mLIF-R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mappe...

  20. Position specific variation in the rate of evolution intranscription factor binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  1. Evolving Transcription Factor Binding Site Models From Protein Binding Microarray Data

    KAUST Repository

    Wong, Ka-Chun

    2016-02-02

    Protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner. In this paper, we describe the PBM motif model building problem. We apply several evolutionary computation methods and compare their performance with the interior point method, demonstrating their performance advantages. In addition, given the PBM domain knowledge, we propose and describe a novel method called kmerGA which makes domain-specific assumptions to exploit PBM data properties to build more accurate models than the other models built. The effectiveness and robustness of kmerGA is supported by comprehensive performance benchmarking on more than 200 datasets, time complexity analysis, convergence analysis, parameter analysis, and case studies. To demonstrate its utility further, kmerGA is applied to two real world applications: 1) PBM rotation testing and 2) ChIP-Seq peak sequence prediction. The results support the biological relevance of the models learned by kmerGA, and thus its real world applicability.

  2. G = MAT: Linking Transcription Factor Expression and DNA Binding Data

    OpenAIRE

    Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

    2011-01-01

    Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcri...

  3. Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus

    Science.gov (United States)

    Kotova, E. S.; Akopov, S. B.; Didych, D. A.; Petrova, N. V.; Iarovaia, O. V.; Razin, S. V.; Nikolaev, L. G.

    2016-01-01

    A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently – in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected. PMID:27099788

  4. Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus.

    Science.gov (United States)

    Kotova, E S; Akopov, S B; Didych, D A; Petrova, N V; Iarovaia, O V; Razin, S V; Nikolaev, L G

    2016-01-01

    A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected. PMID:27099788

  5. rVISTA for Comparative Sequence-Based Discovery of Functional Transcription Factor Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Loots, Gabriela G.; Ovcharenko, Ivan; Pachter, Lior; Dubchak, Inna; Rubin, Edward M.

    2002-03-08

    Identifying transcriptional regulatory elements represents a significant challenge in annotating the genomes of higher vertebrates. We have developed a computational tool, rVISTA, for high-throughput discovery of cis-regulatory elements that combines transcription factor binding site prediction and the analysis of inter-species sequence conservation. Here, we illustrate the ability of rVISTA to identify true transcription factor binding sites through the analysis of AP-1 and NFAT binding sites in the 1 Mb well-annotated cytokine gene cluster1 (Hs5q31; Mm11). The exploitation of orthologous human-mouse data set resulted in the elimination of 95 percent of the 38,000 binding sites predicted upon analysis of the human sequence alone, while it identified 87 percent of the experimentally verified binding sites in this region.

  6. Comparative evaluation of different methods for calculation of cerebral blood flow (CBF) in nonanesthetized rabbits

    International Nuclear Information System (INIS)

    The measurement of cerebral blood flow (CBF) by the extracranial detection of the radioactivity of 133Xe injected into an internal carotid artery has proved to be of considerable value for the investigation of cerebral circulation in conscious rabbits. Methods are described for calculating CBF from the curves of clearance of 133Xe, and include exponential analysis (two-component model), initial slope, and stochastic method. The different methods of curve analysis were compared in order to evaluate the fitness with the theoretical model. The initial slope and stochastic methods, compared with the biexponential model, underestimate the CBF by 35% and 46% respectively. Furthermore, the validity of recording the clearance curve for 10 min was tested by comparing these CBF values with those obtained from the whole curve. CBF values calculated with the shortened procedure are overestimated by 17%. A correlation exists between the ''10 min'' CBF values and the CBF calculated from the whole curve; in spite of that, the values are not accurate for limited animal populations or for single animals. The extent of the two main compartments into which the CBF is divided was also measured. There is no correlation between CBF values and the extent of the relative compartment. This fact suggests that these two parameters correspond to different biological entities

  7. Monkey and human spect rCBF imaging with 99mTc-ECD

    International Nuclear Information System (INIS)

    The SPECT rCBF studies were performed on 2 monkeys, 4 normal volunteers and 53 patients with brain diseases. The rCBF images of 2 normal volunteers with 99mTc-ECD were similar to 99mTc-HMPAO. The sensitivity of 99mTc-ECD rCBF imaging for detecting ischemic foci of 34 TIA patients was 52.9% coinciding with that of 99mTc-HM-PAO rCBF imaging. 99mTc-ECD seems more ideal for clinical use than 99mTc-HM-PAO due to its stability after labelling, fast excretion and easy production

  8. Regional cerebral blood flow (rCBF) changes in major depression

    International Nuclear Information System (INIS)

    Regional cerebral blood flow (rCBF) in patients with major depression and in normal controls was measured by single photon emission computed tomography (SPECT) using N-isopropyl-p [123I]-iodoamphetamine (IMP). The subjects were 22 patients with major depression and 14 normal controls. The rCBF was calculated by the ratio of activity per pixel in the cortical regions to activity per pixel in the cerebellum. IMP-SPECT was conducted in patients with major depression under the depressive and remitted states. rCBF values in the frontal, parietal, temporal, basal ganglia and the occipital regions, and the mean rCBF values were significantly lower in depressive patients than in the controls. Increased rCBF values were observed, and the mean rCBF became normal in the state of remittence. There was no significant difference in mean rCBF between depressive patients and the controls. Therefore, because the lower rCBF was normalized following improvement in expressive symptoms, the rCBF values could be useful as 'state dependent markers' in patients with major depression. (author)

  9. A role of transcriptional activators as antirepressors for the autoinhibitory activity of TATA box binding of transcription factor IID

    OpenAIRE

    Kotani, Tomohiro; Banno, Ken-ichi; Ikura, Mitsuhiko; Hinnebusch, Alan G.; Nakatani, Yoshihiro; Kawaichi, Masashi; Kokubo, Tetsuro

    2000-01-01

    The TATA box-binding activity of transcription factor IID (TFIID) is autoinhibited by the N-terminal domain of the Drosophila TATA box-binding protein- (TBP) associated factor 230/yeast TBP-associated factor 145 subunit, which binds to the TATA box-binding domain of TBP by mimicking the TATA box structure. Here, we propose a mechanism of transcriptional activation that involves antirepression of this autoinhibitory activity by transcriptional activators. Like the autoinhibitory domain of TFII...

  10. Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3

    OpenAIRE

    TAKAOKA, MUNENORI; Harada, Hideki; Andl, Claudia D; Oyama, Kenji; Naomoto, Yoshio; Dempsey, Kelly L.; Klein-Szanto, Andres J.; El-Deiry, Wafik S; GRIMBERG, ADDA; Nakagawa, Hiroshi

    2004-01-01

    Epidermal growth factor receptor (EGFR) is frequently overexpressed in esophageal carcinoma and its precursor lesions. To gain insights into how EGFR overexpression affects cellular functions in primary human esophageal cells, we performed gene expression profiling and identified insulin-like growth factor-binding protein (IGFBP)-3 as the most up-regulated gene. IGFBP-3 regulates cell proliferation through both insulin-like growth factor-dependent and independent mechanisms. We found that IGF...

  11. Molecular docking studies in factor XIa binding site

    Science.gov (United States)

    Balaji, Govardhan A.; Balaji, Vitukudi N.; Rao, Shashidhar N.

    2016-03-01

    Factor XIa inhibitors have been identified to have potential as anticoagulants with robust efficacy and low bleeding risks. In light of their significance and the availability of their 3-D X-ray data in the PDB, we present molecular docking studies carried out with a view to obtain docking protocols that will successfully reproduce the experimentally observed protein-ligand interactions in the case of various X-ray ligands. In this context, we have specifically investigated the efficacy of various cross-docking protocols in reproducing experimental data. Our studies demonstrate that an ensemble of the three apo proteins is capable of accurately docking a majority of the X-ray ligands accurately without invoking any additional conformational flexibility than that present in their experimental structures. Further, we demonstrate that such an ensemble is successfully able to enrich a collection of known active factor XIa inhibitors embedded in a decoy database of drug-like molecules.

  12. DNA-MATRIX: a tool for constructing transcription factor binding sites Weight matrix

    Directory of Open Access Journals (Sweden)

    Chandra Prakash Singh,

    2009-12-01

    Full Text Available Despite considerable effort to date, DNA transcription factor binding sites prediction in whole genome remains a challenge for the researchers. Currently the genome wide transcription factor binding sites prediction tools required either direct pattern sequence or weight matrix. Although there are known transcription factor binding sites pattern databases and tools for genome level prediction but no tool for weight matrix construction. Considering this, we developed a DNA-MATRIX tool for searching putative transcription factor binding sites in genomic sequences. DNA-MATRIX uses the simple heuristic approach for weight matrix construction, which can be transformed into different formats as per the requirement of researcher’s for further genome wide prediction and therefore provides the possibility to identify the conserved known DNA binding sites in the coregulated genes and also to search for a great variety of different regulatory binding patterns. The user may construct and save specific weight or frequency matrices in different formats derived through user selected set of known motif sequences.

  13. Kit receptor dimerization is driven by bivalent binding of stem cell factor.

    Science.gov (United States)

    Lemmon, M A; Pinchasi, D; Zhou, M; Lax, I; Schlessinger, J

    1997-03-01

    Most growth factors and cytokines activate their receptors by inducing dimerization upon binding. We have studied binding of the dimeric cytokine stem cell factor (SCF) to the extracellular domain of its receptor Kit, which is a receptor tyrosine kinase similar to the receptors for platelet-derived growth factor and colony-stimulating factor-1. Calorimetric studies show that one SCF dimer binds simultaneously to two molecules of the Kit extracellular domain. Gel filtration and other methods show that this results in Kit dimerization. It has been proposed that SCF-induced Kit dimerization proceeds via a conformational change that exposes a key receptor dimerization site in the fourth of the five immunoglobulin (Ig)-like domains in Kit. We show that a form of Kit containing just the first three Ig domains (Kit-123) binds to SCF with precisely the same thermodynamic parameters as does Kit-12345. Analytical ultracentrifugation, light scattering, and gel filtration show that Kit-123 dimerizes upon SCF binding in a manner indistinguishable from that seen with Kit-12345. These data argue that the fourth Ig-like domain of Kit is not required for SCF-induced receptor dimerization and provide additional support for a model in which bivalent binding of the SCF dimer provides the driving force for Kit dimerization. PMID:9045650

  14. Effects of calmodulin on DNA-binding activity of heat shock transcription factor in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The DNA-binding activity of heat shock transcription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific to CaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF for binding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar results were obtained with wheat and tomato. Our observations provide the first example of the involvement of CaM in regulation of the DNA-binding activity of the HSF.

  15. Insulin-like growth factors and insulin-like growth factor binding proteins in mammary gland function

    International Nuclear Information System (INIS)

    Insulin-like growth factor (IGF)-mediated proliferation and survival are essential for normal development in the mammary gland during puberty and pregnancy. IGFs interact with IGF-binding proteins and regulate their function. The present review focuses on the role of IGFs and IGF-binding proteins in the mammary gland and describes how modulation of their actions occurs by association with hormones, other growth factors and the extracellular matrix. The review will also highlight the involvement of the IGF axis in breast cancer

  16. Insulin-like growth factor binding protein 3 in inflammatory bowel disease

    DEFF Research Database (Denmark)

    Kirman, Irena; Whelan, Richard Larry; Jain, Suvinit;

    2005-01-01

    Epithelial cell growth regulation has been reported to be altered in inflammatory bowel disease (IBD) patients. The cell growth regulatory factor, insulin-like growth factor binding protein 3 (IGFBP-3), may be partly responsible for this phenomenon. So far, IGFBP-3 levels have been assessed as...... production or to increased cleavage by proteases other than MMP-9....

  17. Pigment epithelium-derived factor binds to cell-surface F(1)-ATP synthase.

    Science.gov (United States)

    Notari, Luigi; Arakaki, Naokatu; Mueller, David; Meier, Scott; Amaral, Juan; Becerra, S P

    2010-05-01

    Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a approximately 60 kDa PEDF-binding protein in bovine retina with Bos taurus F(1)-ATP synthase beta-subunit, and that F(1)F(o)-ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F(1). Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F(1). Real-time binding as determined by surface plasmon resonance demonstrated that yeast F(1) interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F(1) and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F(1). Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of approximately 60 kDa. Antibodies to F(1)beta-subunit specifically captured PEDF-binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F(1), these results show that PEDF is a ligand for endothelial cell-surface F(1)F(o)-ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity. PMID:20412062

  18. tRNA binding properties of eukaryotic translation initiation factor 2 from Encephalitozoon cuniculi.

    Science.gov (United States)

    Naveau, Marie; Lazennec-Schurdevin, Christine; Panvert, Michel; Mechulam, Yves; Schmitt, Emmanuelle

    2010-10-12

    A critical consequence of the initiation of translation is the setting of the reading frame for mRNA decoding. In eukaryotic and archaeal cells, heterotrimeric initiation factor e/aIF2, in its GTP form, specifically binds Met-tRNA(i)(Met) throughout the translation initiation process. After start codon recognition, the factor, in its GDP-bound form, loses affinity for Met-tRNA(i)(Met) and eventually dissociates from the initiation complex. The role of each aIF2 subunit in tRNA binding has been extensively studied in archaeal systems. The isolated archaeal γ subunit is able to bind tRNA, but the α subunit is required for strong binding. Until now, difficulties during purification have hampered the study of the role of each of the three subunits of eukaryotic eIF2 in specific binding of the initiator tRNA. Here, we have produced the three subunits of eIF2 from Encephalitozoon cuniculi, isolated or assembled into heterodimers or into the full heterotrimer. Using assays following protection of Met-tRNA(i)(Met) against deacylation, we show that the eukaryotic γ subunit is able to bind by itself the initiator tRNA. However, the two peripheral α and β subunits are required for strong binding and contribute equally to tRNA binding affinity. The core domains of α and β probably act indirectly by stabilizing the tRNA binding site on the γ subunit. These results, together with those previously obtained with archaeal aIF2 and yeast eIF2, show species-specific distributions of the roles of the peripheral subunits of e/aIF2 in tRNA binding. PMID:20822097

  19. Binding of the polypyrimidine tract-binding protein-associated splicing factor (PSF) to the hepatitis delta virus RNA

    International Nuclear Information System (INIS)

    The hepatitis delta virus (HDV) has a very limited protein coding capacity and must rely on host proteins for its replication. A ribonucleoprotein complex was detected following UV cross-linking between HeLa nuclear proteins and an RNA corresponding to the right terminal stem-loop domain of HDV genomic RNA. Mass spectrometric analysis of the complex revealed the polypyrimidine tract-binding protein-associated splicing factor (PSF) as a novel HDV RNA-interacting protein. Co-immunoprecipitation demonstrated the interaction between HDV RNA and PSF both in vitro in HeLa nuclear extract and in vivo within HeLa cells containing both polarities of the HDV genome. Analysis of the binding of various HDV-derived RNAs to purified, recombinant PSF further confirmed the specificity of the interaction and revealed that PSF directly binds to the terminal stem-loop domains of both polarities of HDV RNA. Our findings provide evidence of the involvement of a host mRNA processing protein in the HDV life cycle

  20. Effects of Heat Stress on Yeast Heat Shock Factor-Promoter Binding In Vivo

    Institute of Scientific and Technical Information of China (English)

    Ning LI; Le-Min ZHANG; Ke-Qin ZHANG; Jing-Shi DENG; Ralf PR(A)NDL; Fritz SCH(O)FFL

    2006-01-01

    Heat shock factor-DNA interaction is critical for understanding the regulatory mechanisms of stress-induced gene expression in eukaryotes. In this study, we analyzed the in vivo binding of yeast heat shock factor (HSF) to the promoters of target genes ScSSA1, ScSSA4, HSP30 and HSP104, using chromatin immunoprecipitation. Previous work suggested that yeast HSF is constitutively bound to DNA at all temperatures. Expression of HSF target genes is regulated at the post-transcriptional level. However, our results indicated that HSF does not bind to the promoters of ScSSA4 and HSP30 at normal temperature (23 ℃). Binding to these promoters is rapidly induced by heat stress at 39 ℃. HSF binds to ScSSA1 and HSP104 promoters under non-stress conditions, but at a low level. Heat stress rapidly leads to a notable increase in the binding of HSF to these two genes. The kinetics of the level of HSF-promoter binding correlate well with the expression of target genes, suggesting that the expression of HSF target genes is at least partially the result of HSF-promoter binding stability and subsequent transcription stimulation.

  1. INTERNAL REGULATORY INTERACTIONS DETERMINE DNA BINDING SPECIFICITY BY A HOX TRANSCRIPTION FACTOR

    OpenAIRE

    Liu, Ying; Matthews, Kathleen S.; Bondos, Sarah E.

    2009-01-01

    In developing bilaterans, the Hox transcription factor family regulates batteries of downstream genes to diversify serially repeated units. Given Hox homeodomains bind a wider array of DNA binding sites in vitro than are regulated by the full-length protein in vivo, regions outside the homeodomain must aid DNA site selection. Indeed, we find affinity for disparate DNA sequences varies less than 3-fold for the homeodomain isolated from the Drosophila Hox protein Ultrabithorax Ia (UbxHD), where...

  2. Sequence and chromatin determinants of cell-type-specific transcription factor binding.

    Science.gov (United States)

    Arvey, Aaron; Agius, Phaedra; Noble, William Stafford; Leslie, Christina

    2012-09-01

    Gene regulatory programs in distinct cell types are maintained in large part through the cell-type-specific binding of transcription factors (TFs). The determinants of TF binding include direct DNA sequence preferences, DNA sequence preferences of cofactors, and the local cell-dependent chromatin context. To explore the contribution of DNA sequence signal, histone modifications, and DNase accessibility to cell-type-specific binding, we analyzed 286 ChIP-seq experiments performed by the ENCODE Consortium. This analysis included experiments for 67 transcriptional regulators, 15 of which were profiled in both the GM12878 (lymphoblastoid) and K562 (erythroleukemic) human hematopoietic cell lines. To model TF-bound regions, we trained support vector machines (SVMs) that use flexible k-mer patterns to capture DNA sequence signals more accurately than traditional motif approaches. In addition, we trained SVM spatial chromatin signatures to model local histone modifications and DNase accessibility, obtaining significantly more accurate TF occupancy predictions than simpler approaches. Consistent with previous studies, we find that DNase accessibility can explain cell-line-specific binding for many factors. However, we also find that of the 10 factors with prominent cell-type-specific binding patterns, four display distinct cell-type-specific DNA sequence preferences according to our models. Moreover, for two factors we identify cell-specific binding sites that are accessible in both cell types but bound only in one. For these sites, cell-type-specific sequence models, rather than DNase accessibility, are better able to explain differential binding. Our results suggest that using a single motif for each TF and filtering for chromatin accessible loci is not always sufficient to accurately account for cell-type-specific binding profiles. PMID:22955984

  3. Androgen regulation of epidermal growth factor receptor binding activity during fetal rabbit lung development.

    OpenAIRE

    Klein, J M; Nielsen, H C

    1993-01-01

    Fetal lung development progresses in a sex-specific manner with male fetuses exhibiting delayed maturation. Androgens, both exogenous and endogenous, inhibit while epidermal growth factor (EGF) enhances fetal lung development. We hypothesized that one mechanism responsible for the delay in male fetal lung development is an androgen-induced delay in EGF receptor binding activity. We measured EGF binding in sex-specific fetal rabbit lung plasma membranes isolated from control fetuses (days 21, ...

  4. Role of insulin-like growth factor binding protein-3 in glucose and lipid metabolism

    OpenAIRE

    Kim, Ho-Seong

    2013-01-01

    Insulin-like growth factor binding protein (IGFBP)-3 has roles in modulating the effect of IGFs by binding to IGFs and inhibiting cell proliferation in an IGF-independent manner. Although recent studies have been reported that IGFBP-3 has also roles in metabolic regulation, their exact roles in adipose tissue are poorly understood. In this review, we summarized the studies about the biological roles in glucose and lipid metabolism. IGFBP-3 overexpression in transgenic mice suggested that IGFB...

  5. A Novel Alignment-Free Method for Comparing Transcription Factor Binding Site Motifs

    OpenAIRE

    Minli Xu; Zhengchang Su

    2010-01-01

    BACKGROUND: Transcription factor binding site (TFBS) motifs can be accurately represented by position frequency matrices (PFM) or other equivalent forms. We often need to compare TFBS motifs using their PFMs in order to search for similar motifs in a motif database, or cluster motifs according to their binding preference. The majority of current methods for motif comparison involve a similarity metric for column-to-column comparison and a method to find the optimal position alignment between ...

  6. Pigment Epithelium-derived Factor (PEDF) Binds to Cell-surface F1-ATP Synthase

    OpenAIRE

    NOTARI, LUIGI; Arakaki, Naokatu; Mueller, David; Meier, Scott; Amaral, Juan; Becerra, S. Patricia

    2010-01-01

    Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to a yet unknown protein on the surface of endothelial cells. Given that protein fingerprinting suggested a match of a ~60-kDa PEDF-binding protein in bovine retina to Bos taurus F1-ATP synthase β-subunit, and that F1F0-ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding ...

  7. Evidence for serotonin binding in vitro by platelet factor 4 and beta-thromboglobulin

    International Nuclear Information System (INIS)

    Evidence is presented for in vitro high affinity binding of serotonin (5-HT) by beta-thromboglobulin (beta TG) and platelet factor 4 (PF4) from human blood. Results include: 1) identification by radioimmunoassay of PF4 in specifically bound material obtained by 5-HT affinity chromatography of human platelet extracts; 2) binding of 72% and 6% of radiolabelled PF4 on 5-HT and control affinity columns, respectively; and 3) binding of approximately 8 moles of 5-HT per mole of purified beta TG in the presence of ferrous ion and heparin in ultrafiltration studies, with Scatchard analysis indicating a dissociation constant of about 4 X 10(-8) M

  8. Evidence for serotonin binding in vitro by platelet factor 4 and beta-thromboglobulin.

    Science.gov (United States)

    Heemstra, V L

    1983-02-01

    Evidence is presented for in vitro high affinity binding of serotonin (5-HT) by beta-thromboglobulin (beta TG) and platelet factor 4 (PF4) from human blood. Results include: 1) identification by radioimmunoassay of PF4 in specifically bound material obtained by 5-HT affinity chromatography of human platelet extracts; 2) binding of 72% and 6% of radiolabelled PF4 on 5-HT and control affinity columns, respectively; and 3) binding of approximately 8 moles of 5-HT per mole of purified beta TG in the presence of ferrous ion and heparin in ultrafiltration studies, with Scatchard analysis indicating a dissociation constant of about 4 X 10(-8) M. PMID:6189240

  9. Evidence for serotonin binding in vitro by platelet factor 4 and beta-thromboglobulin

    Energy Technology Data Exchange (ETDEWEB)

    Heemstra, V.L.

    1983-02-01

    Evidence is presented for in vitro high affinity binding of serotonin (5-HT) by beta-thromboglobulin (beta TG) and platelet factor 4 (PF4) from human blood. Results include: 1) identification by radioimmunoassay of PF4 in specifically bound material obtained by 5-HT affinity chromatography of human platelet extracts; 2) binding of 72% and 6% of radiolabelled PF4 on 5-HT and control affinity columns, respectively; and 3) binding of approximately 8 moles of 5-HT per mole of purified beta TG in the presence of ferrous ion and heparin in ultrafiltration studies, with Scatchard analysis indicating a dissociation constant of about 4 X 10(-8) M.

  10. JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles

    DEFF Research Database (Denmark)

    Portales-Casamar, Elodie; Thongjuea, Supat; Kwon, Andrew T;

    2009-01-01

    active research community. As binding models are refined by newer data, the JASPAR database now uses versioning of matrices: in this release, 12% of the older models were updated to improved versions. Classification of TF families has been improved by adopting a new DNA-binding domain nomenclature. A......JASPAR (http://jaspar.genereg.net) is the leading open-access database of matrix profiles describing the DNA-binding patterns of transcription factors (TFs) and other proteins interacting with DNA in a sequence-specific manner. Its fourth major release is the largest expansion of the core database...

  11. Competitive binding of viral E2 protein and mammalian core-binding factor to transcriptional control sequences of human papillomavirus type 8 and bovine papillomavirus type 1.

    OpenAIRE

    Schmidt, H. M.; Steger, G; Pfister, H

    1997-01-01

    The promoter P7535 of human papillomavirus type 8 and the promoter P7185 of bovine papillomavirus type 1 are negatively regulated by viral E2 proteins via the promoter proximal binding sites P2 and BS1, respectively. Mutations of these E2 binding sites can reduce basal promoter activity. This suggests binding of a transcription-stimulating factor and may indicate that repression by E2 is due to competitive binding of viral and cellular proteins. A computer search revealed putative binding sit...

  12. [Xenon CT CBF mapping derived from two minutes inhalation].

    Science.gov (United States)

    Toshima, R; Toyohara, K; Ebisawa, T; Ishikawa, K; Karashima, H; Shimojo, S; Miyahara, T

    1988-04-01

    Although xenon enhanced CT method for local cerebral blood flow measurement has been brought into a clinical practice, the technique has inherent limitations including anesthetic effects and expensive cost of xenon by a large consumption. To overcome these problems a modified method with a short-duration inhalation was developed and its validity was attested. Siemens Somatom SF with a resolution of 256 X 256 pixels and a scan time of 10 seconds was used. The subjects inhaled 50% Xe/O2 gas mixture from an apparatus consisted of Douglas bag and an open circuit. Xenon concentration in the expired gas was continuously monitored and estimated for arterial blood concentration by using a hematocrit correction. PaCO2 was monitored throughout the study. At the starting point and the endpoint of the inhalation two scans were performed respectively. Thus obtained four images were processed for CT noise cancellation, summation and subtraction to produce an in vivo autoradiography image. Local CBF was calculated from equations derived from the autoradiographic technique with a fixed partition coefficient of lambda = 1. Computer simulation studies were performed to find the optimal scan point to obtain an autoradiographic image and to estimate the calculation errors of this method. One minute and forty-five seconds was found to be the optimal scan point to gain an autoradiographic image in view of a balance between linearity of CBF/enhancement curve and total amount of tissue enhancement. The theoretical errors due to the assumption for a fixed partition coefficient were calculated to be 8% underestimation for gray matter and 5% overestimation for white matter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3401410

  13. Elevated epidermal growth factor receptor binding in plutonium-induced lung tumors from dogs

    International Nuclear Information System (INIS)

    The objective of this study is to examine and characterize epidermal growth factor receptor (EGF-R) binding in inhaled plutonium-induced canine lung-tumor tissue and to compare it with that in normal canine lung tissue. Crude membrane preparations from normal and lung-tumor tissue from beagle dogs were examined in a radioreceptor assay, using 125I-labeled epidermal growth factor (EGF) as a ligand. Specific EGF receptor binding was determined in the presence of excess unlabeled EGF. We have examined EGF receptor binding in eight lung-tumor samples obtained from six dogs. Epidermal growth factor receptor binding was significantly greater in lung-tumor samples (31.38%) compared with that in normal lung tissue (3.76%). Scatchard plot analysis from the displacement assay revealed that there was no statistical difference in the binding affinity but significantly higher concentration of EGF-R sites in the lung-tumor tissue (619 fmol/mg) than in normal lung tissue (53 fmol/mg). The increase in EGF-R number in plutonium-induced dog lung tumors does not seem to correlate with increase in the initial lung burden exposure to plutonium. Our results demonstrate that there is a significant increase in EGF-R binding in inhaled plutonium-induced dog lung tumors

  14. Binding of human factors X and Xa to HepG2 and J82 human tumor cell lines. Evidence that factor Xa binds to tumor cells independent of factor Va

    International Nuclear Information System (INIS)

    Previous studies demonstrated that several cultured human tumor cell lines potentiate the conversion of prothrombin to thrombin by factor Xa and calcium in the absence of exogenous factor Va. In the present study, the specific binding of radioiodinated preparations of human factor X and factor Xa to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule, and a human bladder carcinoma cell line (J82) that does not synthesize factor V/Va, was examined. Radioiodinated factor Xa bound specifically to J82 and HepG2 cells, whereas no significant specific binding of 125I-factor X to either cell was observed. The binding isotherm of 125I-factor Xa to each tumor cell line exhibited a hyperbolic profile, and Scatchard analysis demonstrated a single class of binding site for factor Xa on each cell surface with Kd values of 1.66 +/- 0.39 and 1.64 +/- 0.52 nM and 566,000 +/- 71,000 and 28,000 +/- 6,000 binding sites/cell for HepG2 and J82 cells, respectively. Thrombin formation by cell-bound factor Xa was hyperbolic and saturable at 5 nM factor Xa on each cell line. Hanes-Woolf plots of the prothrombin activation data indicated that half-maximal rates of thrombin formation occurred at factor Xa concentrations of 1.50 +/- 0.43 nM and 1.42 +/- 0.48 nM on HepG2 and J82 cells, respectively. Pretreatment of J82 cells with polyclonal anti-human factor V IgG had no measurable effect on either the binding of 125I-factor Xa or prothrombin activation. However, pretreatment of HepG2 cells with anti-human factor V IgG inhibited prothrombin activation in a dose-dependent manner, but did not inhibit the binding of factor Xa to this cell. When both cell lines were preincubated with exogenous human factor Va, the binding of factor Xa to either HepG2 or J82 cells was marginally affected

  15. Alpha-contingent EEG feedback reduces SPECT rCBF variability

    DEFF Research Database (Denmark)

    McLaughlin, Thomas; Steinberg, Bruce; Mulholland, Thomas;

    2005-01-01

    EEG feedback methods, which link the occurrence of alpha to the presentation of repeated visual stimuli, reduce the relative variability of subsequent, alpha-blocking event durations. The temporal association between electro-cortical field activation and regional cerebral blood flow (rCBF) led us...... to investigate whether the reduced variability of alpha-blocking durations with feedback is associated with a reduction in rCBF variability. Reduced variability in the rCBF response domain under EEG feedback control might have methodological implications for future brain-imaging studies. Visual stimuli were...... to quantify the variance-reducing effects of ACS across multiple, distributed areas of the brain. Both EEG and rCBF measures demonstrated decreased variability under ACS. This improved control was seen for localized as well as anatomically distributed rCBF measures....

  16. Assessment of cerebral blood flow autoregulation (CBF AR) with rheoencephalography (REG): studies in animals

    International Nuclear Information System (INIS)

    The ability of cerebral vasculature to regulate cerebral blood flow (CBF) in the face of changes in arterial blood pressure (SAP) or intracranial pressure (ICP) is an important guard against secondary ischemia in acute brain injuries, and official guidelines recommend that therapeutic decisions be guided by continuous monitoring of CBF autoregulation (AR). The common method for CBF AR monitoring, which rests on real-time derivation of the correlation coefficient (PRx) between slow oscillations in SAP and ICP is, however, rarely used in clinical practice because it requires invasive ICP measurements. This study investigated whether the correlation coefficient between SAP and the pulsatile component of the non-invasive transcranial bioimpedance signal (rheoencephalography, REG) could be used to assess the state and lower limit of CBF AR. The results from pigs and rhesus macaques affirm the utility of REG; however, additional animal and clinical studies are warranted to assess selectivity of automatic REG-based evaluation of CBF AR.

  17. Assessment of cerebral blood flow autoregulation (CBF AR) with rheoencephalography (REG): studies in animals

    Science.gov (United States)

    Popovic, Djordje; Bodo, Michael; Pearce, Frederick; van Albert, Stephen; Garcia, Alison; Settle, Tim; Armonda, Rocco

    2013-04-01

    The ability of cerebral vasculature to regulate cerebral blood flow (CBF) in the face of changes in arterial blood pressure (SAP) or intracranial pressure (ICP) is an important guard against secondary ischemia in acute brain injuries, and official guidelines recommend that therapeutic decisions be guided by continuous monitoring of CBF autoregulation (AR). The common method for CBF AR monitoring, which rests on real-time derivation of the correlation coefficient (PRx) between slow oscillations in SAP and ICP is, however, rarely used in clinical practice because it requires invasive ICP measurements. This study investigated whether the correlation coefficient between SAP and the pulsatile component of the non-invasive transcranial bioimpedance signal (rheoencephalography, REG) could be used to assess the state and lower limit of CBF AR. The results from pigs and rhesus macaques affirm the utility of REG; however, additional animal and clinical studies are warranted to assess selectivity of automatic REG-based evaluation of CBF AR.

  18. A Human Mitochondrial Transcription Factor Is Related to RNA Adenine Methyltransferases and Binds S-Adenosylmethionine

    OpenAIRE

    McCulloch, Vicki; Seidel-Rogol, Bonnie L.; Shadel, Gerald S.

    2002-01-01

    A critical step toward understanding mitochondrial genetics and its impact on human disease is to identify and characterize the full complement of nucleus-encoded factors required for mitochondrial gene expression and mitochondrial DNA (mtDNA) replication. Two factors required for transcription initiation from a human mitochondrial promoter are h-mtRNA polymerase and the DNA binding transcription factor, h-mtTFA. However, based on studies in model systems, the existence of a second human mito...

  19. Construction of scFv that bind both fibronectin-binding protein A and clumping factor A of Stapylococcus aureus.

    Science.gov (United States)

    Wang, Man; Zhang, Yan; Li, Benqiang; Zhu, Jianguo

    2015-06-01

    Bovine mastitis (BM) causes significant losses to the dairy industry. Vaccines against the causative agent of BM, Staphylococcus aureus, do not confer adequate protection. Because passive immunization with antibodies permits disease prevention, we constructed a recombinant single-chain antibody (scFv) against fibronectin-binding protein A (FnBPA) and clumping factor A (ClfA), two important virulence factors in S. aureus infection. The DNA coding sequences of the variable heavy (VH) and variable light (VL) domains of antibodies produced in the peripheral blood lymphocytes of cows with S. aureus-induced mastitis were obtained using reverse transcription and polymerase chain reaction, and the VH and VL cDNAs were assembled in-tandem using a DNA sequence encoding a (Gly4Ser)3 peptide linker. The scFv cDNAs were cloned into the pOPE101 plasmid for the expression of soluble scFv protein in Escherichia coli. The binding of the scFvs to both FnBPA and ClfA was confirmed using an indirect ELISA and Western blotting. The DNA sequences of the framework regions of the VH and VL domains were highly conserved, and the complementarity-determining regions displayed significant diversity, especially in CDR3 of the VH domain. These novel bovine antibody fragments may be useful as a therapeutic candidate for the prevention and treatment of S. aureus-induced bovine mastitis. PMID:25910693

  20. Heparin-Binding Epidermal Growth Factor-like Growth Factor/Diphtheria Toxin Receptor in Normal and Neoplastic Hematopoiesis

    OpenAIRE

    Fabrizio Vinante; Antonella Rigo

    2013-01-01

    Heparin-binding EGF-like growth factor (HB-EGF) belongs to the EGF family of growth factors. It is biologically active either as a molecule anchored to the membrane or as a soluble form released by proteolytic cleavage of the extracellular domain. HB-EGF is involved in relevant physiological and pathological processes spanning from proliferation and apoptosis to morphogenesis. We outline here the main activities of HB-EGF in connection with normal or neoplastic differentiative or proliferativ...

  1. Eukaryotic initiation factor 4B and the poly(A)-binding protein bind eIF4G competitively.

    Science.gov (United States)

    Cheng, Shijun; Gallie, Daniel R

    2013-01-01

    The eukaryotic translation initiation factor (eIF) 4G functions as a scaffold protein that assembles components of the translation initiation complex required to recruit the 40S ribosomal subunit to an mRNA. Although many eukaryotes express two highly similar eIF4G isoforms, those in plants are highly divergent in size and sequence from one another and are referred to as eIF4G and eIFiso4G. Although the domain organization of eIFiso4G differs substantially from eIF4G orthologs in other species, the domain organization of plant eIF4G is largely unknown despite the fact that it is more similar in size and sequence to eIF4G of other eukaryotes. In this study, we show that eIF4G differs from eIFiso4G in that it contains two distinct interaction domains for the poly(A) binding protein (PABP) and eIF4B but is similar to eIFiso4G in having two eIF4A interaction domains. PABP and eIF4B bind the same N-terminal region of eIF4G as they do to a region C-proximal to the HEAT-1 domain in the middle domain of eIF4G, resulting in competitive binding between eIF4B and PABP to each site. eIF4G also differs from eIFiso4G in that no competitive binding was observed between PABP and eIF4A or between eIF4B and eIF4A to its HEAT-1-containing region. These results demonstrate that despite substantial differences in size, sequence, and domain organization, PABP and eIF4B bind to eIF4G and eIFiso4G competitively. PMID:26824014

  2. Resting state rCBF mapping with single-photon emission tomography and positron emission tomography: magnitude and origin of differences

    International Nuclear Information System (INIS)

    Single-photon emission tomography (SPET), using technetium-99m hexamethylpropylene amine oxime, and positron emission tomography (PET), using oxygen-15 butanol were compared in six healthy male volunteers with regard to the mapping of resting state regional cerebral blood flow (rCBF). A computerized brain atlas was utilized for 3D regional analyses and comparison of 64 selected and normalized volumes of interest (VOIs). The normalized mean rCBF values in SPET, as compared to PET, were higher in most of the Brodmann areas in the frontal and parietal lobes (4.8% and 8.7% respectively). The average differences were small in the temporal (2.3%) and occipital (1.1%) lobes. PET values were clearly higher in small VOIs like the thalamus (12.3%), hippocampus (12.3%) and basal ganglia (9.9%). A resolution phantom study showed that the in-plane SPET/PET system resolution was 11.0/7.5 mm. In conclusion, SPET and PET data demonstrated a fairly good agreement despite the superior spatial resolution of PET. The differences between SPET and PET rCBF are mainly due to physiological and physical factors, the data processing, normalization and co-registration methods. In order to further improve mapping of rCBF with SPET it is imperative not only to improve the spatial resolution but also to apply accurate correction techniques for scatter, attenuation and non-linear extraction. (orig.)

  3. Decreased Transcription Factor Binding Levels Nearby Primate Pseudogenes Suggest Regulatory Degeneration.

    Science.gov (United States)

    Douglas, Gavin M; Wilson, Michael D; Moses, Alan M

    2016-06-01

    Characteristics of pseudogene degeneration at the coding level are well-known, such as a shift toward neutral rates of nonsynonymous substitutions and gain of frameshift mutations. In contrast, degeneration of pseudogene transcriptional regulation is not well understood. Here, we test two predictions of regulatory degeneration along a pseudogenized lineage: 1) Decreased transcription factor (TF) binding and 2) accelerated evolution in putative cis-regulatory regions.We find evidence for decreased TF binding levels nearby two primate pseudogenes compared with functional liver genes. However, the majority of TF-bound sequences nearby pseudogenes do not show evidence for lineage-specific accelerated rates of evolution. We conclude that decreases in TF binding level could be a marker for regulatory degeneration, while sequence degeneration in primate cis-regulatory modules may be obscured by background rates of TF binding site turnover. PMID:26882985

  4. Decreased Transcription Factor Binding Levels Nearby Primate Pseudogenes Suggest Regulatory Degeneration

    Science.gov (United States)

    Douglas, Gavin M.; Wilson, Michael D.; Moses, Alan M.

    2016-01-01

    Characteristics of pseudogene degeneration at the coding level are well-known, such as a shift toward neutral rates of nonsynonymous substitutions and gain of frameshift mutations. In contrast, degeneration of pseudogene transcriptional regulation is not well understood. Here, we test two predictions of regulatory degeneration along a pseudogenized lineage: 1) Decreased transcription factor (TF) binding and 2) accelerated evolution in putative cis-regulatory regions. We find evidence for decreased TF binding levels nearby two primate pseudogenes compared with functional liver genes. However, the majority of TF-bound sequences nearby pseudogenes do not show evidence for lineage-specific accelerated rates of evolution. We conclude that decreases in TF binding level could be a marker for regulatory degeneration, while sequence degeneration in primate cis-regulatory modules may be obscured by background rates of TF binding site turnover. PMID:26882985

  5. Somatomedin-1 binding protein-3: insulin-like growth factor-1 binding protein-3, insulin-like growth factor-1 carrier protein.

    Science.gov (United States)

    2003-01-01

    Somatomedin-1 binding protein-3 [insulin-like growth factor-1 binding protein-3, SomatoKine] is a recombinant complex of insulin-like growth factor-1 (rhIGF-1) and binding protein-3 (IGFBP-3), which is the major circulating somatomedin (insulin-like growth factor) binding protein; binding protein-3 regulates the delivery of somatomedin-1 to target tissues. Somatomedin-1 binding protein-3 has potential as replacement therapy for somatomedin-1 which may become depleted in indications such as major surgery, organ damage/failure and traumatic injury, resulting in catabolism. It also has potential for the treatment of osteoporosis; diseases associated with protein wasting including chronic renal failure, cachexia and severe trauma; and to attenuate cardiac dysfunction in a variety of disease states, including after severe burn trauma. Combined therapy with somatomedin-1 and somatomedin-1 binding protein-3 would prolong the duration of action of somatomedin-1 and would reduce or eliminate some of the undesirable effects associated with somatomedin-1 monotherapy. Somatomedin-1 is usually linked to binding protein-3 in the normal state of the body, and particular proteases clip them apart in response to stresses and release somatomedin-1 as needed. Therefore, somatomedin-1 binding protein-3 is a self-dosing system and SomatoKine would augment the natural supply of these linked compounds. Somatomedin-1 binding protein-3 was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on June 1 2000. Insmed and Avecia, UK, have signed an agreement for the manufacturing of SomatoKine and its components, IGF-1 and binding protein-3. CGMP clinical production of SomatoKine and its components will be done in Avecia's Advanced Biologics Centre, Billingham, UK, which manufactures recombinant-based medicines and vaccines with a capacity of up to 1000 litres. In 2003, manufacturing of SomatoKine is

  6. Elevated epidermal growth factor (EGF) receptor bindings in dog lung tumors

    International Nuclear Information System (INIS)

    The objective of this study is to measure and characterize epidermal growth factor (EGF) receptor binding in dog Pu induced in lung-tumor tissue and to compare then with normal lung tissue in dogs. Epidermal growth factor and its receptors have been shown to play an important role in the proliferation and differentiation of eukaryotic cells. It was therefore hypothesized that inappropriate activation of EGF receptor plays a role in oncogenesis. Microsomal preparations prepared from normal and lung-tumor tissue from beagle dogs were used in a radioreceptor assay, using 125I-EGF as a ligand. Specific EGF receptor bindings were determined in the presence of excess unlabeled EGF. They have examined EGF receptor bindings in 10 lung-tumor samples obtained from seven dogs at necropsy. Epidermal growth factor receptor bindings were significantly higher in eight of the lung-tumor samples compared with both normal lung tissue from control dogs and normal lung tissue from dogs from which tumors were obtained. Their results suggest that the high EGF receptor bindings observed in lung-tumor tissue may play a role in the oncogenesis in dogs

  7. Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated

    Directory of Open Access Journals (Sweden)

    Down Thomas A

    2010-09-01

    Full Text Available Abstract Background DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS" but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq not to be biological transcription factor binding sites ("empirical TFBS". We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. Results Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. Conclusions Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.

  8. The Role of Genome Accessibility in Transcription Factor Binding in Bacteria

    Science.gov (United States)

    Wang, Harris H.

    2016-01-01

    ChIP-seq enables genome-scale identification of regulatory regions that govern gene expression. However, the biological insights generated from ChIP-seq analysis have been limited to predictions of binding sites and cooperative interactions. Furthermore, ChIP-seq data often poorly correlate with in vitro measurements or predicted motifs, highlighting that binding affinity alone is insufficient to explain transcription factor (TF)-binding in vivo. One possibility is that binding sites are not equally accessible across the genome. A more comprehensive biophysical representation of TF-binding is required to improve our ability to understand, predict, and alter gene expression. Here, we show that genome accessibility is a key parameter that impacts TF-binding in bacteria. We developed a thermodynamic model that parameterizes ChIP-seq coverage in terms of genome accessibility and binding affinity. The role of genome accessibility is validated using a large-scale ChIP-seq dataset of the M. tuberculosis regulatory network. We find that accounting for genome accessibility led to a model that explains 63% of the ChIP-seq profile variance, while a model based in motif score alone explains only 35% of the variance. Moreover, our framework enables de novo ChIP-seq peak prediction and is useful for inferring TF-binding peaks in new experimental conditions by reducing the need for additional experiments. We observe that the genome is more accessible in intergenic regions, and that increased accessibility is positively correlated with gene expression and anti-correlated with distance to the origin of replication. Our biophysically motivated model provides a more comprehensive description of TF-binding in vivo from first principles towards a better representation of gene regulation in silico, with promising applications in systems biology. PMID:27104615

  9. The Role of Genome Accessibility in Transcription Factor Binding in Bacteria.

    Science.gov (United States)

    Gomes, Antonio L C; Wang, Harris H

    2016-04-01

    ChIP-seq enables genome-scale identification of regulatory regions that govern gene expression. However, the biological insights generated from ChIP-seq analysis have been limited to predictions of binding sites and cooperative interactions. Furthermore, ChIP-seq data often poorly correlate with in vitro measurements or predicted motifs, highlighting that binding affinity alone is insufficient to explain transcription factor (TF)-binding in vivo. One possibility is that binding sites are not equally accessible across the genome. A more comprehensive biophysical representation of TF-binding is required to improve our ability to understand, predict, and alter gene expression. Here, we show that genome accessibility is a key parameter that impacts TF-binding in bacteria. We developed a thermodynamic model that parameterizes ChIP-seq coverage in terms of genome accessibility and binding affinity. The role of genome accessibility is validated using a large-scale ChIP-seq dataset of the M. tuberculosis regulatory network. We find that accounting for genome accessibility led to a model that explains 63% of the ChIP-seq profile variance, while a model based in motif score alone explains only 35% of the variance. Moreover, our framework enables de novo ChIP-seq peak prediction and is useful for inferring TF-binding peaks in new experimental conditions by reducing the need for additional experiments. We observe that the genome is more accessible in intergenic regions, and that increased accessibility is positively correlated with gene expression and anti-correlated with distance to the origin of replication. Our biophysically motivated model provides a more comprehensive description of TF-binding in vivo from first principles towards a better representation of gene regulation in silico, with promising applications in systems biology. PMID:27104615

  10. Neuroplastin-55 binds to and signals through the fibroblast growth factor receptor

    DEFF Research Database (Denmark)

    Owczarek, Sylwia; Kiryushko, Darya; Hald Larsen, Marianne;

    2010-01-01

    3), whereas Np55 lacks the Ig1 module. Of these two isoforms, only Np65 is involved in homophilic interactions resulting in cell adhesion, whereas the role of Np55 is poorly understood. The present study reports for the first time the crystal structure of the ectodomain of Np55 at 1.95-A resolution...... and demonstrates that Np55 binds to and activates the fibroblast growth factor receptor 1 (FGFR1). Furthermore, we identify a sequence motif in the Ig2 module of Np55 interacting with FGFR1 and show that a synthetic peptide encompassing this motif, termed narpin, binds to and activates FGFR1. We show...... Np55-induced signaling may be involved in synaptic plasticity in vivo. Owczarek, S., Kiryushko, D., Larsen, M. H., Kastrup, J. S., Gajhede, M., Sandi, C., Berezin, V., Bock, E., Soroka, V. Neuroplastin-55 binds to and signals through the fibroblast growth factor receptor....

  11. Low CBF, discontinuous EEG activity, and periventricular brain injury in ill, preterm neonates.

    Science.gov (United States)

    Greisen, G; Pryds, O

    1989-01-01

    Cerebral blood flow was measured by intravenous 133Xe clearance one to four times during the first 48 h of life in 20 infants, born after 27 to 33 weeks of gestation, who were under mechanical ventilation and being monitored by amplitude integrated EEG, resulting in 36 sets of simultaneous CBF and EEG measurements. Mean CBF infinity, a measure of global flow to white as well as grey matter, was 10.0 ml/100 g/min +/- 3.5 SD. Definite EEG activity was seen with CBF infinity values as low as 5 ml/100 g/min. CBF infinity was related to the level of EEG activity, discontinuous EEG activity being associated with low CBF infinity (p = 0.014). It was not possible to determine if this relation indicated the presence of marginal ischaemia or primary inhibition of electrical activity resulting in decreased CBF, two infants developing periventricular leucomalacia, however, and one who developed intraparenchymatous hemorrhage were among the seven with CBF infinity values of less than or equal to 8 ml/100 g/min (p = 0.031). This suggests that ischaemia of periventricular white matter may have been present concurrently with cortical electrical activity. PMID:2751062

  12. Klonierung, Sequenzierung und Charakterisierung der genetischen Determinante des CAMP-Faktors (=cbf-Gen) der Gruppe-B-Streptokokken (Streptococcus agalactiae)

    OpenAIRE

    Blankenstein, Oliver

    2005-01-01

    The CAMP reaction, a synergistic lysis of sheep erythrocytes by Staphylococcus aureus (beta-toxin) and group B streptococci (Streptococcus agalactiae) was first described in 1944 by Christie et al.. This phenomenon is caused by the CAMP-factor, an extra-cellular protein which role pathogenicity and virulence is still unknown. The focus of this work is the molecular isolation and characterization of the CAMP-factor gene of group B streptococci (cbf-Gene). First cloning attempts were made using...

  13. The neural cell adhesion molecule binds to fibroblast growth factor receptor 2

    DEFF Research Database (Denmark)

    Christensen, Claus; Lauridsen, Jes B; Berezin, Vladimir; Bock, Elisabeth; Kiselyov, Vladislav V

    2006-01-01

    The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface...

  14. Identification of candidate transcription factor binding sites in the cattle genome

    Science.gov (United States)

    A resource that provides candidate transcription factor binding sites does not currently exist for cattle. Such data is necessary, as predicted sites may serve as excellent starting locations for future 'omics studies to develop transcriptional regulation hypotheses. In order to generate this resour...

  15. Composite organization of the cobalamin binding and cubilin recognition sites of intrinsic factor

    DEFF Research Database (Denmark)

    Fedosov, Sergey N; Fedosova, Natalya U; Berglund, Lars;

    2005-01-01

    Intrinsic factor (IF(50)) is a cobalamin (Cbl)-transporting protein of 50 kDa, which can be cleaved into two fragments: the 30 kDa N-terminal peptide IF(30) and the 20 kDa C-terminal glycopeptide IF(20). Experiments on binding of Cbl to IF(30), IF(20), and IF(50) revealed comparable association...

  16. Therapy of Core Binding Factor Acute Myeloid Leukemia: Incremental Improvements Toward Better Long-Term Results

    OpenAIRE

    Bhatt, Vijaya Raj; Kantarjian, Hagop; Cortes, Jorge E.; Ravandi, Farhad; Borthakur, Gautam

    2012-01-01

    Despite being considered as good prognostic acute myelogenous leukemia (AML), the long-term survival rate in core binding factor AML leaves room for substantial improvement. We discuss treatments that have improved outcome in this group of patients with AML and ongoing/future strategies that might contribute toward incremental gains.

  17. Asap: a framework for over-representation statistics for transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Troels T Marstrand

    Full Text Available BACKGROUND: In studies of gene regulation the efficient computational detection of over-represented transcription factor binding sites is an increasingly important aspect. Several published methods can be used for testing whether a set of hypothesised co-regulated genes share a common regulatory regime based on the occurrence of the modelled transcription factor binding sites. However there is little or no information available for guiding the end users choice of method. Furthermore it would be necessary to obtain several different software programs from various sources to make a well-founded choice. METHODOLOGY: We introduce a software package, Asap, for fast searching with position weight matrices that include several standard methods for assessing over-representation. We have compared the ability of these methods to detect over-represented transcription factor binding sites in artificial promoter sequences. Controlling all aspects of our input data we are able to identify the optimal statistics across multiple threshold values and for sequence sets containing different distributions of transcription factor binding sites. CONCLUSIONS: We show that our implementation is significantly faster than more naïve scanning algorithms when searching with many weight matrices in large sequence sets. When comparing the various statistics, we show that those based on binomial over-representation and Fisher's exact test performs almost equally good and better than the others. An online server is available at http://servers.binf.ku.dk/asap/.

  18. Functional and phylogenetic analyses of chromosome 21 promoters and hominid-specific transcription factor binding sites

    OpenAIRE

    Querfurth, Robert

    2011-01-01

    The focus of this work addresses functional studies of human and primate promoters, and the genome-wide localization and validation of human-specific transcription factor binding sites of the essential transcription factor GABPa. In this context, the development of an improved PCR protocol, including the careful adjustment of PCR additives to compose an efficient enhancer mix, was central to the amplification of large GC-rich promoter fragments used as source for the functional studies. Based...

  19. SPIC: A novel similarity metric for comparing transcription factor binding site motifs based on information contents

    OpenAIRE

    Zhang, Shaoqiang; Zhou, Xiguo; Du, Chuanbin; Su, Zhengchang

    2013-01-01

    Background Discovering transcription factor binding sites (TFBS) is one of primary challenges to decipher complex gene regulatory networks encrypted in a genome. A set of short DNA sequences identified by a transcription factor (TF) is known as a motif, which can be expressed accurately in matrix form such as a position-specific scoring matrix (PSSM) and a position frequency matrix. Very frequently, we need to query a motif in a database of motifs by seeking its similar motifs, merge similar ...

  20. Purification and characterization of native human insulin-like growth factor binding protein-6

    OpenAIRE

    Taferner, Andrea; Micutkova, Lucia; Hermann, Martin; Jansen-Dürr, Pidder; Pircher, Haymo

    2011-01-01

    Insulin-like growth factor binding proteins (IGFBPs) are key regulators of insulin-like growth factor (IGF) mediated signal transduction and thereby can profoundly influence cellular phenotypes and cell fate. Whereas IGFBPs are extracellular proteins, intracellular activities were described for several IGFBP family members, such as IGFBP-3, which can be reinternalized by endocytosis and reaches the nucleus through routes that remain to be fully established. Within the family of IGFBPs, IGFBP-...

  1. FF Domains of CA150 Bind Transcription and Splicing Factors through Multiple Weak Interactions

    OpenAIRE

    Smith, Matthew J.; Kulkarni, Sarang; Pawson, Tony

    2004-01-01

    The human transcription factor CA150 modulates human immunodeficiency virus type 1 gene transcription and contains numerous signaling elements, including six FF domains. Repeated FF domains are present in several transcription and splicing factors and can recognize phosphoserine motifs in the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Using mass spectrometry, we identify a number of nuclear binding partners for the CA150 FF domains and demonstrate a direct interaction between CA15...

  2. Decrease of CBF measured by SPECT correlates to neurological features in Wilson's disease

    International Nuclear Information System (INIS)

    Objective: To investigate changes of cerebral blood flow and its association with neurological features in the patients with Wilson's disease (WD). Methods: In 13 WD patients and 12 age-matched control subjects, the authors quantitatively determined CBF and rCBF with SPECT using the non-blood-withdraw method. Five neurological signs including dysarthria, dystonia, bradykinesia/rigidity, tremor and gait ataxia were assessed semi-quantitatively. The severity of each sign was scored from 0 (absent) to 3 (severe). The individual scores collected from a subject were then summed up, resulting in a 'total clinical score' (TCS). The authors studied the correlations between the CBF or rCBF and semiquantitative clinical scores. Results: CBF in case group was (40.94 +- 2.33) mL·100 g-1·min-1 and was significantly lower than that in control group [(43.82 +- 1.58) mL·100 g-1·min-1] (P<0.01). Statistical comparison found more significant rCBF reduction in bilateral basal ganglia, thalamus, front lobe (P<0.01) and left temporal lobe (P<0.05). The reduction in CBF was correlated with the TCS (r = -0.620, P<0.05). The reduction in rCBF of bilateral basal ganglia correlated with the severity of dysarthria (r = -0.770, P<0.01 and -0.576, P<0.05). The reduction in rCBF of right parietal lobe and bilateral thalamus correlated with the severity of bradykinesia/rigidity (r = -0.757, P<0.01 and -0.635, P<0.05). Conclusions: These results suggest Wilson's disease patients have CBF or rCBF reduction to different degrees, and lower rCBF was found in bilateral basal ganglia, thalamus, front lobe and left temporal lobe. The neurological deficits may have relationship with reduction of cerebral blood flow in Wilson's disease. SPECT is useful for evaluation of cerebral function in Wilson's disease patients

  3. Novel Bioluminescent Binding Assays for Ligand–Receptor Interaction Studies of the Fibroblast Growth Factor Family

    Science.gov (United States)

    Song, Ge; Shao, Xiao-Xia; Wu, Qing-Ping; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand–receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand–receptor interaction studies. PMID:27414797

  4. Conversion of MyoD to a Neurogenic Factor: Binding Site Specificity Determines Lineage

    Directory of Open Access Journals (Sweden)

    Abraham P. Fong

    2015-03-01

    Full Text Available MyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG and a “private” sequence (CAGGTG or CAGATG, respectively. To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

  5. Alteration in transcription factor binding in murine thymocytes after low dose radiation

    International Nuclear Information System (INIS)

    Objective: To study the effect of low dose radiation on gene transcription regulation of murine thymocytes. Methods: Alteration in transcription factor binding in murine thymocytes 4 h after whole body irradiation (WBI) with 75 mGy X-rays was investigated with gel mobility shift assay. Results: Increased binding to CREB, NF-kB and APl consensus sequences was found with nuclear extracts prepared from thymocytes of irradiated versus sham-irradiated mice. Binding to the CREB, NF-kB and APl consensus sequences by nuclear extracts derived from irradiated mice was 6-fold, 4,3-fold and 2-fold higher than that from sham-irradiated respectively. The present report demonstrates that WBI with 75 mGy X-rays is capable of increasing expression of CREB, NF-kB and APl in murine thymocytes. competition with the cold oligonucleotide containing the consensus sequences for CREB and NF-kB resulted in loss of the shifted band, indicating specific binding. Conclusions: X-ray activation of select transcription factors, which bind certain consensus sites in promoters, may cause specific induction of gene transcription

  6. Effects of class I heparin binding growth factor and fibronectin on platelet adhesion and aggregation

    International Nuclear Information System (INIS)

    Fibronectin and heparin binding growth factor-type 1 have been affixed to vascular graft surfaces to enhance the attachment and the proliferation of transplanted endothelial cells, respectively. The current study examines the effect of fibronectin and heparin binding growth factor-type 1 on platelet adhesion and activation in vivo and on platelet aggregation in vitro. Expanded polytetrafluoroethylene prostheses (5 cm x 4 mm internal diameter) were treated either with fibronectin (n = 9), fibronectin/heparin/heparin binding growth factor-type 1/heparin (n = 12), or neither (n = 13) and were interposed into canine aortoiliac systems bilaterally. Autogenous radiolabeled (Indium 111 oxine, 650 microCi) platelets were injected intravenously before reestablishment of circulation. Perfusion was maintained for 30 minutes, and prostheses were removed with segments of native aorta and distal iliac arteries bilaterally. Specimens were examined for thrombus-free surface area, by gamma well counting for adherent radiolabeled platelets, and by light microscopy and transmission and scanning electron microscopic techniques. Results showed that both the fibronectin and fibronectin/heparin/heparin binding growth factor-type 1/heparin pretreated prostheses contained significantly greater numbers of platelets and adherent radioactivity than did control graft segments when normalized to their ipsilateral iliac arteries. Fibronectin/heparin/heparin binding growth factor-type 1/heparin pretreated prostheses contained 27 +/- 16 times more radioactivity per square millimeter than ipsilateral iliac arteries, fibronectin pretreated prostheses had 13 +/- 8 times more radioactivity per square millimeter than ipsilateral iliac arteries, and untreated expanded polytetrafluoroethylene had 4 +/- 3 times more radioactivity per square millimeter than ipsilateral iliac arteries

  7. Inhibition of 125I-epidermal growth factor binding to cultured keratinocytes by antiproliferative molecules gamma interferon, cyclosporin A, and transforming growth factor-beta

    International Nuclear Information System (INIS)

    The growth of cultured human keratinocytes (KC) is inhibited by gamma interferon (IFN-gamma), cyclosporin A and transforming growth factor-beta, but not by tumor necrosis factor. When these antiproliferative molecules were added to KC they induced a concentration and time-dependent inhibition of 125I-epidermal growth factor (I-EGF) binding. These anti-proliferative molecules primarily reduced the number of binding sites by approximately 25%-50% without affecting the binding affinity. Tumor necrosis factor did not influence the ligand binding by I-EGF. In parallel with the ability of the antiproliferative molecules to inhibit I-EGF binding, there was an increase in transforming growth factor-alpha production. These results suggest that several different antiproliferative molecules may share a common mechanism to inhibit cell growth by reducing I-EGF binding to KC

  8. Estimating binding free energy of a putative growth factors EGF-VEGF complex - a computational bioanalytical study.

    Science.gov (United States)

    Lin, Meng-Han; Chang, C Allen; Fischer, Wolfgang B

    2016-08-01

    Epidermal growth factor (EGF) and homodimeric vascular endothelial growth factor (VEGF) bind to cell surface receptors. They are responsible for cell growth and angiogenesis, respectively. Docking of the individual proteins as monomeric units using ZDOCK 2.3.2 reveals a partial blocking of the receptor binding site of VEGF by EGF. The receptor binding site of EGF is not affected by VEGF. The calculated binding energy is found to be intermediate between the binding energies calculated for Alzheimer's Aß42 and the barnase/barstar complex. PMID:26338536

  9. Liver-type fatty acid binding protein interacts with hepatocyte nuclear factor

    OpenAIRE

    McIntosh, Avery L.; Petrescu, Anca D.; Heather A. Hostetler; Kier, Ann B.; Schroeder, Friedhelm

    2013-01-01

    Hepatocyte nuclear factor 4α (HNF4α) regulates liver type fatty acid binding protein (L-FABP) gene expression. Conversely as shown herein, L-FABP structurally and functionally also interacts with HNF4α. Fluorescence resonance energy transfer (FRET) between Cy3-HNF4α (donor) and Cy5-L-FABP (acceptor) as well as FRET microscopy detected L-FABP in close proximity (~80 Å) to HNF4α, binding with high affinity Kd ~250–300 nM. Circular dichroism (CD) determined that the HNF4α/L-FABP interaction alte...

  10. JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles

    OpenAIRE

    Portales-Casamar, E; Thongjuea, S.; Kwon, A. T.; Arenillas, D.; Zhao, X; Valen, E.; Yusuf, D; Lenhard, B.; Wasserman, W. W.; Sandelin, A

    2010-01-01

    JASPAR (http://jaspar.genereg.net) is the leading open-access database of matrix profiles describing the DNA-binding patterns of transcription factors (TFs) and other proteins interacting with DNA in a sequence-specific manner. Its fourth major release is the largest expansion of the core database to date: the database now holds 457 non-redundant, curated profiles. The new entries include the first batch of profiles derived from ChIP-seq and ChIP-chip whole-genome binding experiments, and 177...

  11. Free Energy Simulations of a GTPase: GTP and GDP Binding to Archaeal Initiation Factor 2

    OpenAIRE

    Satpati, Priyadarshi; Clavaguéra, Carine; Ohanessian, Gilles; Simonson, Thomas

    2011-01-01

    Archaeal initiation factor 2 (aIF2) is a protein involved in the initiation of protein biosynthesis. In its GTP-bound, “ON” conformation, aIF2 binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, “OFF” conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and its dependence on the ON or OFF conformational state of aIF2, molecular dynamics free energy simulations (MDFE) are a tool of choice. However, the validity of the computed free ene...

  12. Insulin-like Growth Factor Binding Protein 7 Mediates Glioma Cell Growth and Migration1

    OpenAIRE

    Jiang, Wei; Xiang, Cunli; Cazacu, Simona; Brodie, Chaya; Mikkelsen, Tom

    2008-01-01

    Insulin-like growth factor binding protein 7 (IGFBP-7) is the only member of the IGFBP superfamily that binds strongly to insulin, suggesting that IGFBP-7 may have different functions from other IGFBPs. Unlike other IGFBPs, the expression and functions of IGFBP-7 in glioma tumors have not been reported. Using cDNA microarray analysis, we found that expression of IGFBP-7 correlated with the grade of glioma tumors and the overall patient survival. This finding was further validated by real-time...

  13. Insulin-like Growth Factor Binding Protein 7 Mediates Glioma Cell Growth and Migration

    OpenAIRE

    Wei Jiang; Cunli Xiang; Simona Cazacu; Chaya Brodie; Tom Mikkelsen

    2008-01-01

    Insulin-like growth factor binding protein 7 (IGFBP-7) is the only member of the IGFBP superfamily that binds strongly to insulin, suggesting that IGFBP-7 may have different functions from other IGFBPs. Unlike other IGFBPs, the expression and functions of IGFBP-7 in glioma tumors have not been reported. Using cDNA microarray analysis, we found that expression of IGFBP-7 correlated with the grade of glioma tumors and the overall patient survival. This finding was further validated by real-time...

  14. G =  MAT: linking transcription factor expression and DNA binding data.

    Science.gov (United States)

    Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

    2011-01-01

    Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/. PMID:21297945

  15. G =  MAT: linking transcription factor expression and DNA binding data.

    Directory of Open Access Journals (Sweden)

    Konstantin Tretyakov

    Full Text Available Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/.

  16. DNA binding by the plant-specific NAC transcription factors in crystal and solution

    DEFF Research Database (Denmark)

    Welner, Ditte Hededam; Lindemose, Søren; Grossmann, J. Günter;

    2012-01-01

    angle X-ray scattering on complexes with oligonucleotides, mutagenesis and (DNase I and uranyl photo-) footprinting, is combined to form a structural view of DNA-binding, and for the first time provide experimental evidence for the speculated relationship between plant-specific NAC proteins, WRKY...... transcription factors and the mammalian GCM (Glial cell missing) transcription factors, which all use a ß-strand motif for DNA-binding. The structure shows that the NAC domain inserts the edge of its core ß-sheet into the major groove, while leaving the DNA largely undistorted. The structure of the NAC......NAC (NAM/ATAF/CUC) plant transcription factors regulate essential processes in development, stress responses and nutrient distribution in important crop and model plants (rice, Populus, Arabidopsis), which makes them highly relevant in the context of crop optimization and bioenergy production. The...

  17. Differential Regulatory Mechanisms of CBF Regulon Between Nipponbare (Japonica) and 93-11 (Indica) During Cold Acclimation

    Institute of Scientific and Technical Information of China (English)

    PAN Xiao-wu; LI Yong-chao; LI Xiao-xiang; LIU Wen-qiang; MING Jun; LU Ting-ting; TAN Jiang

    2013-01-01

    Nine CBF/DREB1 homologous genes in rice were obtained by BLAST search in the NCBI database,which share conserved amino acid sequences with DREB1 protein in Arabidopsis.Three CBF genes organized in tandem,named OsCBF1,OsCBF2 and OsCBF3,showed a transient induction in the process of cold acclimation,much stronger in indica rice 93-11 compared with japonica rice Nipponbare.The candidate downstream genes OsLIP5 and OsLIP9 were induced in 93-11 but not in Nipponbare.The differential expression of CBF regulon might be caused by polymorphisms within promoter sequences between these two rice varieties.These results could be useful for utilization of CBF/DREB1 genes and illustration of differences in chilling tolerance between indica and japonica rice varieties.

  18. Quantitative models of the mechanisms that control genome-wide patterns of transcription factor binding during early Drosophila development.

    Directory of Open Access Journals (Sweden)

    Tommy Kaplan

    Full Text Available Transcription factors that drive complex patterns of gene expression during animal development bind to thousands of genomic regions, with quantitative differences in binding across bound regions mediating their activity. While we now have tools to characterize the DNA affinities of these proteins and to precisely measure their genome-wide distribution in vivo, our understanding of the forces that determine where, when, and to what extent they bind remains primitive. Here we use a thermodynamic model of transcription factor binding to evaluate the contribution of different biophysical forces to the binding of five regulators of early embryonic anterior-posterior patterning in Drosophila melanogaster. Predictions based on DNA sequence and in vitro protein-DNA affinities alone achieve a correlation of ∼0.4 with experimental measurements of in vivo binding. Incorporating cooperativity and competition among the five factors, and accounting for spatial patterning by modeling binding in every nucleus independently, had little effect on prediction accuracy. A major source of error was the prediction of binding events that do not occur in vivo, which we hypothesized reflected reduced accessibility of chromatin. To test this, we incorporated experimental measurements of genome-wide DNA accessibility into our model, effectively restricting predicted binding to regions of open chromatin. This dramatically improved our predictions to a correlation of 0.6-0.9 for various factors across known target genes. Finally, we used our model to quantify the roles of DNA sequence, accessibility, and binding competition and cooperativity. Our results show that, in regions of open chromatin, binding can be predicted almost exclusively by the sequence specificity of individual factors, with a minimal role for protein interactions. We suggest that a combination of experimentally determined chromatin accessibility data and simple computational models of transcription

  19. Wnts grasp the WIF domain of Wnt Inhibitory Factor 1 at two distinct binding sites.

    Science.gov (United States)

    Kerekes, Krisztina; Bányai, László; Patthy, László

    2015-10-01

    Wnts have a structure resembling a hand with "thumb" and "index" fingers that grasp the cysteine rich domains of Frizzled receptors at two distinct binding sites. In the present work we show that the WIF domain of Wnt Inhibitory Factor 1 is also bound by Wnts at two sites. Using C-terminal domains of Wnt5a and Wnt7a and arginine-scanning mutagenesis of the WIF domain we demonstrate that, whereas the N-terminal, lipid-modified "thumb" of Wnts interacts with the alkyl-binding site of the WIF domain, the C-terminal domain of Wnts (Wnt-CTD) binds to a surface on the opposite side of the WIF domain. PMID:26342861

  20. A Common Structural Motif in the Binding of Virulence Factors to Bacterial Secretion Chaperones

    International Nuclear Information System (INIS)

    Salmonella invasion protein A (SipA) is translocated into host cells by a type III secretion system (T3SS) and comprises two regions: one domain binds its cognate type III secretion chaperone, InvB, in the bacterium to facilitate translocation, while a second domain functions in the host cell, contributing to bacterial uptake by polymerizing actin. We present here the crystal structures of the SipA chaperone binding domain (CBD) alone and in complex with InvB. The SipA CBD is found to consist of a nonglobular polypeptide as well as a large globular domain, both of which are necessary for binding to InvB. We also identify a structural motif that may direct virulence factors to their cognate chaperones in a diverse range of pathogenic bacteria. Disruption of this structural motif leads to a destabilization of several chaperone-substrate complexes from different species, as well as an impairment of secretion in Salmonella

  1. Identification and Structural Basis of Binding to Host Lung Glycogen by Streptococcal Virulence Factors

    Energy Technology Data Exchange (ETDEWEB)

    Lammerts van Bueren,A.; Higgins, M.; Wang, D.; Burke, R.; Boraston, A.

    2007-01-01

    The ability of pathogenic bacteria to recognize host glycans is often essential to their virulence. Here we report structure-function studies of previously uncharacterized glycogen-binding modules in the surface-anchored pullulanases from Streptococcus pneumoniae (SpuA) and Streptococcus pyogenes (PulA). Multivalent binding to glycogen leads to a strong interaction with alveolar type II cells in mouse lung tissue. X-ray crystal structures of the binding modules reveal a novel fusion of tandem modules into single, bivalent functional domains. In addition to indicating a structural basis for multivalent attachment, the structure of the SpuA modules in complex with carbohydrate provides insight into the molecular basis for glycogen specificity. This report provides the first evidence that intracellular lung glycogen may be a novel target of pathogenic streptococci and thus provides a rationale for the identification of the streptococcal {alpha}-glucan-metabolizing machinery as virulence factors.

  2. Molecular determinants of epidermal growth factor binding: a molecular dynamics study.

    Directory of Open Access Journals (Sweden)

    Jeffrey M Sanders

    Full Text Available The epidermal growth factor receptor (EGFR is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of

  3. Molecular determinants of epidermal growth factor binding: a molecular dynamics study.

    Science.gov (United States)

    Sanders, Jeffrey M; Wampole, Matthew E; Thakur, Mathew L; Wickstrom, Eric

    2013-01-01

    The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF

  4. Measurement of immunotargeted plasmonic nanoparticles' cellular binding: a key factor in optimizing diagnostic efficacy

    International Nuclear Information System (INIS)

    In this study, we use polarized light scattering to study immunotargeted plasmonic nanoparticles which bind to live SK-BR-3 human breast carcinoma cells. Gold nanoparticles can be conjugated to various biomolecules in order to target specific molecular signatures of disease. This specific targeting provides enhanced contrast in scattering-based optical imaging techniques. While there are papers which report the number of antibodies that bind per nanoparticle, there are almost no reports of the key factor which influences diagnostic or therapeutic efficacy using nanoparticles: the number of targeted nanoparticles that bind per cell. To achieve this goal, we have developed a 'negative' method of determining the binding concentration of those antibody/nanoparticle bioconjugates which are targeted specifically to breast cancer cells. Unlike previously reported methods, we collected unbound nanoparticle bioconjugates and measured the light scattering from dilute solutions of these particles so that quantitative binding information can be obtained. By following this process, the interaction effects of adjacent bound nanoparticles on the cell membrane can be avoided simply by measuring the light scattering from the unbound nanoparticles. Specifically, using nanoshells of two different sizes, we compared the binding concentrations of anti-HER2/nanoshell and anti-IgG/nanoshell bioconjugates targeted to HER2-positive SK-BR-3 breast cancer cells. The results indicate that, for anti-HER2/nanoshell bioconjugates, there are approximately 800-1600 nanoshells bound per cell; for anti-IgG/nanoshell bioconjugates, the binding concentration is significantly lower at nearly 100 nanoshells bound per cell. These results are also supported by dark-field microscopy images of the cells labeled with anti-HER2/nanoshell and anti-IgG/nanoshell bioconjugates

  5. Promoter binding factors regulating cyclin B transcription in the sea urchin embryo.

    Science.gov (United States)

    Thatcher, J D; McBride, B; Katula, K S

    1995-10-01

    Cyclin B is a key regulatory protein of the cell cycle, central to the control of the G2/M transition. In the developing sea urchin embryo, the cyclin B gene is transcriptionally regulated in concert with changing patterns of cell division. In an effort to understand the mechanism controlling cyclin B expression during development, we have conducted an analysis of the Strongylocentrotus purpuratus cyclin B gene promoter. DNase I foot-printing of the cyclin B upstream region revealed eight binding regions within 435 bp of the start of transcription; seven of these sites were within 215 bp. Found within these regions were consensus sequences for two CCAAT boxes, TATA, and E-boxes and sequences with some similarity to E2F and octamer binding motifs. Upstream sequences were functionally defined by generating cyclin B-CAT fusion genes, containing deletions and base specific mutations, and testing for relative levels of expression by gene transfer. Both CCAAT boxes were found to be essential for maximal levels of expression. A third binding site (PR7) with no recognizable consensus sequence was also found to act as a positive element. Our results suggest that protein binding to the E2F-like sequences may act to reduce expression. Protein binding was further characterized by gel mobility-shift and methylation interference. The CCAAT boxes were found to bind similar, if not identical, proteins. Sequence comparisons and methylation interference data indicate that the likely protein binding these CCAAT sequences is the characterized CCAAT-binding protein CP1. A probe containing site PR7 formed multiple gel shift complexes that, by methylation interference, appeared to be interrelated. One major complex was formed with an oligonucleotide containing the two E2F-like sequences. Protein binding to this probe was specific and required bases within the E2F-like sequences. Our results indicate that cyclin B is subject to positive and negative regulation, involving multiple factors

  6. JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles.

    OpenAIRE

    Mathelier, Anthony; Fornes, Oriol; Arenillas, David J.; Chen, Chih-Yu; Denay, Grégoire; Lee, Jessica; Shi, Wenqiang; Shyr, Casper; Tan, Ge; Worsley-Hunt, Rebecca; Zhang, Allen W.; Parcy, François; Lenhard, Boris; Sandelin, Albin; Wasserman, Wyeth W

    2016-01-01

    JASPAR (http://jaspar.genereg.net) is an open-access database storing curated, non-redundant transcription factor (TF) binding profiles representing transcription factor binding preferences as position frequency matrices for multiple species in six taxonomic groups. For this 2016 release, we expanded the JASPAR CORE collection with 494 new TF binding profiles (315 in vertebrates, 11 in nematodes, 3 in insects, 1 in fungi and 164 in plants) and updated 59 profiles (58 in vertebrates and 1 in f...

  7. The absolute CBF response to activation is preserved during elevated perfusion: Implications for neurovascular coupling measures

    OpenAIRE

    Whittaker, Joseph R.; Driver, Ian D.; Bright, Molly G.; Murphy, Kevin

    2016-01-01

    Functional magnetic resonance imaging (fMRI) techniques in which the blood oxygenation level dependent (BOLD) and cerebral blood flow (CBF) response to a neural stimulus are measured, can be used to estimate the fractional increase in the cerebral metabolic rate of oxygen consumption (CMRO2) that accompanies evoked neural activity. A measure of neurovascular coupling is obtained from the ratio of fractional CBF and CMRO2 responses, defined as n, with the implicit assumption that relative rath...

  8. Prognostic value of rCBF measurements and CT in focal cerebral ischemia

    International Nuclear Information System (INIS)

    CT is the most effective examination technique for studying the evolution of ischemic attacks, but if performed within the first 3 weeks it does not allow prognosis of possible evolution towards necrosis. CBF measurement and vasoreactivity tests under Althesin do allow this prognosis. Thirty patients whose evolution was checked clinically and by repeated CT examinations (89 in all) underwent CBF measurements (intra-arterial xenon 133) and vasoreactivity had completely disappeared (inverse steal), the infarct evolved towards necrosis. (orig.)

  9. A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA-DNA Complex Structure and Binding Specificities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Xun; Guanga, Gerald P; Wan, Cheng; Rose, Robert B [Z; (W Elec.); (NCSU)

    2012-11-13

    MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA–DNA complex. MafA forms base-specific hydrogen bonds with the flanking G–5C–4 and central C0/G0 bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse–chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases.

  10. Binding of complement regulators factor H and C4b binding protein to group A streptococcal strains isolated from tonsillar tissue and blood.

    Science.gov (United States)

    Suvilehto, Jari; Jarva, Hanna; Seppänen, Mikko; Siljander, Tuula; Vuopio-Varkila, Jaana; Meri, Seppo

    2008-06-01

    Group A streptococcus (GAS) is the most common pathogen causing bacterial pharyngitis. We isolated streptococcal strains from tonsils removed from patients with tonsillar disease (n=202) and studied their ability to bind the complement regulators factor H (FH) and C4b binding protein (C4BP) using 125 I-labeled proteins. Blood isolates of GAS (n=10) were obtained from patients with bacteraemia. Streptococci were isolated from 21% of the tonsillitis patients. The emm and T types of the GAS strains were determined. Of the 26 GAS strains studied, only six could bind FH and/or C4BP above the threshold levels. The fraction of the offered radioactive protein bound ranged between 6-12% for FH and 19-56% for C4BP. The clinical course of the tonsillar disease was not related to the binding of FH or C4BP by GAS. The binding strains were mostly of the T4M4 or T28M28 type. From the invasive strains (n=10), three bound FH (binding level: 8-11%) and two C4BP (36-39%). The binding correlated only partially to M-protein (emm) type suggesting that the binding was not exclusively due to M-protein. The results indicate that complement regulator binding by GAS is only partially related to pathogenicity and not a universal property of all group A streptococci. PMID:18538613

  11. Nonspecific transcription factor binding can reduce noise in the expression of downstream proteins

    Science.gov (United States)

    Soltani, M.; Bokes, P.; Fox, Z.; Singh, A.

    2015-10-01

    Transcription factors (TFs) interact with a multitude of binding sites on DNA and partner proteins inside cells. We investigate how nonspecific binding/unbinding to such decoy binding sites affects the magnitude and time-scale of random fluctuations in TF copy numbers arising from stochastic gene expression. A stochastic model of TF gene expression, together with decoy site interactions is formulated. Distributions for the total (bound and unbound) and free (unbound) TF levels are derived by analytically solving the chemical master equation under physiologically relevant assumptions. Our results show that increasing the number of decoy binding sides considerably reduces stochasticity in free TF copy numbers. The TF autocorrelation function reveals that decoy sites can either enhance or shorten the time-scale of TF fluctuations depending on model parameters. To understand how noise in TF abundances propagates downstream, a TF target gene is included in the model. Intriguingly, we find that noise in the expression of the target gene decreases with increasing decoy sites for linear TF-target protein dose-responses, even in regimes where decoy sites enhance TF autocorrelation times. Moreover, counterintuitive noise transmissions arise for nonlinear dose-responses. In summary, our study highlights the critical role of molecular sequestration by decoy binding sites in regulating the stochastic dynamics of TFs and target proteins at the single-cell level.

  12. Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification

    Science.gov (United States)

    Jiang, Chao; Wilkinson, Mark C.

    2016-01-01

    The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein–polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans. PMID:27030175

  13. Polymerase (Pol) III TATA Box-Binding Protein (TBP)-Associated Factor Brf Binds to a Surface on TBP Also Required for Activated Pol II Transcription

    OpenAIRE

    Shen, Yuhong; Kassavetis, George A.; Bryant, Gene O.; Berk, Arnold J.

    1998-01-01

    The TATA box-binding protein (TBP) plays an essential role in transcription by all three eukaryotic nuclear RNA polymerases, polymerases (Pol) I, II, and III. In each case, TBP interacts with class-specific TBP-associated factors (TAFs) to form class-specific transcription initiation factors. For yeast Pol III transcription, TBP associates with Brf (from TFIIB-related factor) and B", two Pol III TAFs, to form Pol III transcription factor TFIIIB. Here, we identify TBP surface residues that are...

  14. MORPHEUS, a Webtool for Transcription Factor Binding Analysis Using Position Weight Matrices with Dependency.

    Science.gov (United States)

    Minguet, Eugenio Gómez; Segard, Stéphane; Charavay, Céline; Parcy, François

    2015-01-01

    Transcriptional networks are central to any biological process and changes affecting transcription factors or their binding sites in the genome are a key factor driving evolution. As more organisms are being sequenced, tools are needed to easily predict transcription factor binding sites (TFBS) presence and affinity from mere inspection of genomic sequences. Although many TFBS discovery algorithms exist, tools for using the DNA binding models they generate are relatively scarce and their use is limited among the biologist community by the lack of flexible and user-friendly tools. We have developed a suite of web tools (called Morpheus) based on the proven Position Weight Matrices (PWM) formalism that can be used without any programing skills and incorporates some unique features such as the presence of dependencies between nucleotides positions or the possibility to compute the predicted occupancy of a large regulatory region using a biophysical model. To illustrate the possibilities and simplicity of Morpheus tools in functional and evolutionary analysis, we have analysed the regulatory link between LEAFY, a key plant transcription factor involved in flower development, and its direct target gene APETALA1 during the divergence of Brassicales clade. PMID:26285209

  15. MORPHEUS, a Webtool for Transcription Factor Binding Analysis Using Position Weight Matrices with Dependency.

    Directory of Open Access Journals (Sweden)

    Eugenio Gómez Minguet

    Full Text Available Transcriptional networks are central to any biological process and changes affecting transcription factors or their binding sites in the genome are a key factor driving evolution. As more organisms are being sequenced, tools are needed to easily predict transcription factor binding sites (TFBS presence and affinity from mere inspection of genomic sequences. Although many TFBS discovery algorithms exist, tools for using the DNA binding models they generate are relatively scarce and their use is limited among the biologist community by the lack of flexible and user-friendly tools. We have developed a suite of web tools (called Morpheus based on the proven Position Weight Matrices (PWM formalism that can be used without any programing skills and incorporates some unique features such as the presence of dependencies between nucleotides positions or the possibility to compute the predicted occupancy of a large regulatory region using a biophysical model. To illustrate the possibilities and simplicity of Morpheus tools in functional and evolutionary analysis, we have analysed the regulatory link between LEAFY, a key plant transcription factor involved in flower development, and its direct target gene APETALA1 during the divergence of Brassicales clade.

  16. Altered [125I]epidermal growth factor binding and receptor distribution in psoriasis

    International Nuclear Information System (INIS)

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [125I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers

  17. A constitutive heat shock element-binding factor is immunologically identical to the Ku autoantigen.

    Science.gov (United States)

    Kim, D; Ouyang, H; Yang, S H; Nussenzweig, A; Burgman, P; Li, G C

    1995-06-23

    Analysis of the heat shock element (HSE)-binding proteins in extracts of rodent cells, during heat shock and their post-heat shock recovery, indicates that the regulation of heat shock response involves a constitutive HSE-binding factor (CHBF), in addition to the heat-inducible heat shock factor HSF1. We purified the CHBF to apparent homogeneity from HeLa cells using column chromatographic techniques including an HSE oligonucleotide affinity column. The purified CHBF consists of two polypeptides with apparent molecular masses of 70 and 86 kDa. Immunoblot and gel mobility shift analysis verify that CHBF is identical or closely related to the Ku autoantigen. The DNA binding characteristics of CHBF to double-stranded or single-stranded DNA are similar to that of Ku autoantigen. In gel mobility shift analysis using purified CHBF and recombinant human HSF1, CHBF competes with HSF1 for the binding of DNA sequences containing HSEs in vitro. Furthermore, when Rat-1 cells were co-transfected with human Ku expression vectors and the hsp70-promoter-driven luciferase reporter gene, thermal induction of luciferase is significantly suppressed relative to cells transfected with only the hsp70-luciferase construct. These data suggest a role of CHBF (or Ku protein) in the regulation of heat response in vivo. PMID:7797514

  18. Detection of growth factor binding to gelatin and heparin using a photonic crystal optical biosensor

    International Nuclear Information System (INIS)

    Drug-carrier interactions are important to protein controlled release systems to protect the protein from denaturation and ensure properly timed release. A novel photonic crystal biosensor was used to investigate a gelatin-protein controlled release system to determine the amount of protein bound to the carrier at physiological conditions. The Biomolecular Interaction Detection (BIND) system reflects a narrow band of wavelengths when white light is shone incident to the grating. As mass is deposited onto the surface, the peak wavelength value is shifted due to changes in the optical density of the biosensor. The BIND system was used to detect the binding of growth factors onto acidic gelatin, basic gelatin, and heparin on the sensor surface. Through a series of experiments, including functionalizing the sensor, adjusting the ionic strength of the solution, adjusting the substrate concentration, and minimizing non-specific signal, the adsorption of the gelatins and heparin on the sensor was enhanced. The binding interaction of recombinant human transforming growth factor (rhTGF)-β1 and bone morphogenetic protein (rhBMP)-2 with the two types of gelatin and heparin were investigated. The strength of the interaction between rhTGF-β1 and the substrates is in the following order: heparin > acidic gelatin > basic gelatin. RhBMP-2 bound to the substrates but with less intensity than TGF-β1: heparin > basic gelatin > acidic gelatin. This work provides support for the controlled release mechanism through degradation of the gelatin carrier.

  19. The effect of education on rCBF changes in Alzheimer's disease: a longitudinal SPECT study

    International Nuclear Information System (INIS)

    To determine the relationship of differing levels of education on regional cerebral blood flow (rCBF) in patients with Alzheimer's disease (AD). Fifty-three patients with AD followed-up for an average of 36 months were divided into the high-educated group (HE, ≥12 years of schooling) and low-educated group (LE, 123I]-iodoamphetamine and the SPECT data were analyzed by 3D-stereotactic surface projections. At initial evaluation, the HE group had greater rCBF deficits in the parietotemporal regions than did the LE group, even though both groups had comparable MMSE and FAST scores. When compared with initial SPECT, follow-up SPECT showed a significant rCBF reduction in widespread regions, including the frontal, parietal, temporal, and limbic lobes of the HE group, while it a significant rCBF reduction in scattered and small regions of the parietotemporal, cingulate, and occipital areas of the LE group, as the HE group had faster cognitive and functional decline than the LE group. The HE group showed lower rCBF at initial SPECT than the LE group, suggesting more advanced AD pathology. As a result, the HE group demonstrated a more extensive and severe reduction of rCBF on follow-up SPECT in association with faster cognitive and functional decline than the LE group. Our SPECT study provides stronger support for the cognitive reserve effects of education in AD. (orig.)

  20. Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry

    Directory of Open Access Journals (Sweden)

    Gisa Gerold

    2015-08-01

    Full Text Available Hepatitis C virus (HCV enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1, which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion.

  1. Asap: a framework for over-representation statistics for transcription factor binding sites

    DEFF Research Database (Denmark)

    Marstrand, Troels T; Frellsen, Jes; Moltke, Ida;

    2008-01-01

    promoter sequences. Controlling all aspects of our input data we are able to identify the optimal statistics across multiple threshold values and for sequence sets containing different distributions of transcription factor binding sites. CONCLUSIONS: We show that our implementation is significantly faster...... regime based on the occurrence of the modelled transcription factor binding sites. However there is little or no information available for guiding the end users choice of method. Furthermore it would be necessary to obtain several different software programs from various sources to make a well...... than more naïve scanning algorithms when searching with many weight matrices in large sequence sets. When comparing the various statistics, we show that those based on binomial over-representation and Fisher's exact test performs almost equally good and better than the others. An online server is...

  2. Modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of RNA polymerase I transcription.

    Science.gov (United States)

    Banerjee, Rajeev; Weidman, Mary K; Navarro, Sonia; Comai, Lucio; Dasgupta, Asim

    2005-08-01

    Soon after infection, poliovirus (PV) shuts off host-cell transcription, which is catalysed by all three cellular RNA polymerases. rRNA constitutes more than 50 % of all cellular RNA and is transcribed from rDNA by RNA polymerase I (pol I). Here, evidence has been provided suggesting that both pol I transcription factors, SL-1 (selectivity factor) and UBF (upstream binding factor), are modified and inactivated in PV-infected cells. The viral protease 3C(pro) appeared to cleave the TATA-binding protein-associated factor 110 (TAF(110)), a subunit of the SL-1 complex, into four fragments in vitro. In vitro protease-cleavage assays using various mutants of TAF(110) and purified 3C(pro) indicated that the Q(265)G(266) and Q(805)G(806) sites were cleaved by 3C(pro). Both SL-1 and UBF were depleted in PV-infected cells and their disappearance correlated with pol I transcription inhibition. rRNA synthesis from a template containing a human pol I promoter demonstrated that both SL-1 and UBF were necessary to restore pol I transcription fully in PV-infected cell extracts. These results suggested that both SL-1 and UBF are transcriptionally inactivated in PV-infected HeLa cells. PMID:16033979

  3. Insulin-like growth factor binding protein-3 in preterm infants with retinopathy of prematurity

    OpenAIRE

    Manizheh Mostafa Gharehbaghi; Ali Peirovifar; Karim Sadeghi; Haleh Mostafidi

    2012-01-01

    Background: Retinopathy of prematurity (ROP) is the main cause of visual impairment in preterm newborn infants. Objective: This study was conducted to determine whether insulin-like growth factor binding protein -3 (IGFBP-3) is associated with proliferative ROP and has a role in pathogenesis of the disease in premature infants. Materials and Methods: A total of 71 preterm infants born at or before 32 weeks of gestation participated in this study. Studied patients consisted of 41 neonates with...

  4. JASPAR 2014: an extensively expanded and updated open-access database of transcription factor binding profiles

    OpenAIRE

    Mathelier, Anthony; Zhao, Xiaobei; Zhang, Allen W.; Parcy, François; Worsley-Hunt, Rebecca; Arenillas, David J.; Buchman, Sorana; Chen, Chih-Yu; Chou, Alice; Ienasescu, Hans; Lim, Jonathan; Shyr, Casper; Tan, Ge; Zhou, Michelle; Lenhard, Boris

    2013-01-01

    JASPAR (http://jaspar.genereg.net) is the largest open-access database of matrix-based nucleotide profiles describing the binding preference of transcription factors from multiple species. The fifth major release greatly expands the heart of JASPAR—the JASPAR CORE subcollection, which contains curated, non-redundant profiles—with 135 new curated profiles (74 in vertebrates, 8 in Drosophila melanogaster, 10 in Caenorhabditis elegans and 43 in Arabidopsis thaliana; a 30% increase in total) and ...

  5. Insulin-like growth factor binding protein 2 promotes ovarian cancer cell invasion

    OpenAIRE

    Liu Jinsong; Wang Huamin; Shmulevich Ilya; Mircean Cristian; Lee Eun-Ju; Niemistö Antti; Kavanagh John J; Lee Je-Ho; Zhang Wei

    2005-01-01

    Abstract Background Insulin-like growth factor binding protein 2 (IGFBP2) is overexpressed in ovarian malignant tissues and in the serum and cystic fluid of ovarian cancer patients, suggesting an important role of IGFBP2 in the biology of ovarian cancer. The purpose of this study was to assess the role of increased IGFBP2 in ovarian cancer cells. Results Using western blotting and tissue microarray analyses, we showed that IGFBP2 was frequently overexpressed in ovarian carcinomas compared wit...

  6. Binding energy of finite nuclei in Jastrow-Factor-Cluster method with applications in helium

    International Nuclear Information System (INIS)

    A general expression for the binding energy of finite nuclei in the Jastrow-Factor-Cluster method is developed upto the three-body cluster term with centre of mass correction. It is applied in mass A = 3 and 4 with the standard Chakkalakal function and the average Pauli condition. Compared to the previous work of Afnan (1969) better saturation properties are obtained for two- and three-body truncation. Convergence is also better. (orig.)

  7. Orthogonal matrix factorization enables integrative analysis of multiple RNA binding proteins

    OpenAIRE

    Stražar, Martin; Žitnik, Marinka; Zupan, Blaž; Ule, Jernej; Curk, Tomaž

    2016-01-01

    Motivation: RNA binding proteins (RBPs) play important roles in post-transcriptional control of gene expression, including splicing, transport, polyadenylation and RNA stability. To model protein–RNA interactions by considering all available sources of information, it is necessary to integrate the rapidly growing RBP experimental data with the latest genome annotation, gene function, RNA sequence and structure. Such integration is possible by matrix factorization, where current approaches hav...

  8. Perfect Hiding and Perfect Binding Universally Composable Commitment Schemes with Constant Expansion Factor

    DEFF Research Database (Denmark)

    Damgård, Ivan Bjerre; Nielsen, Jesper Buus

    Canetti and Fischlin have recently proposed the security notion universal composability for commitment schemes and provided two examples. This new notion is very strong. It guarantees that security is maintained even when an unbounded number of copies of the scheme are running concurrently, also ...... versions. These are the rst schemes to show that constant expansion factor, perfect hiding, and perfect binding can be obtained for universally composable commitments....

  9. Hypoxia-inducible factor 2alpha binds to cobalt in vitro.

    Science.gov (United States)

    Yuan, Y; Beitner-Johnson, D; Millhorn, D E

    2001-11-01

    The hypoxia-inducible factor (HIF) activates the expression of genes that contain a hypoxia response element (HRE). The alpha subunit of the HIF transcription factors is degraded by proteasome pathways during normoxia, but stabilized under hypoxic conditions. It has previously been established that cobalt causes accumulation of HIF-2alpha and HIF-1alpha. However, little is known about the mechanism by which cobalt mimics hypoxia and stabilizes these transcription factors. We show here that cobalt binds directly to HIF-2alpha in vitro with a high affinity and in an oxygen-dependent manner. We found that HIF-2alpha, which had been stabilized with a proteasome inhibitor, could bind to cobalt, whereas hypoxia-stabilized HIF-2alpha could not. Mutations within the oxygen-dependent degradation domain of HIF-2alpha prevented cobalt binding and led to accumulation of HIF-2alpha during normoxia. This suggests that transition metal such as iron may play a role in regulation of HIF-2alpha in vivo. PMID:11688986

  10. A DNA-binding-site landscape and regulatory network analysis for NAC transcription factors in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Lindemose, Søren; Jensen, Michael Krogh; de Velde, Jan Van; O'Shea, Charlotte; Heyndrickx, Ken S.; Workman, Christopher; Vandepoele, Klaas; Skriver, Karen; De Masi, Federico

    2014-01-01

    regulatory networks of 12 NAC transcription factors. Our data offer specific single-base resolution fingerprints for most TFs studied and indicate that NAC DNA-binding specificities might be predicted from their DNA-binding domain's sequence. The developed methodology, including the application of...... the DNA-binding preferences of individual members. Here, we present a TF-target gene identification workflow based on the integration of novel protein binding microarray data with gene expression and multi-species promoter sequence conservation to identify the DNA-binding specificities and the gene...

  11. Antibody Responses in Patients with Staphylococcal Septicemia against Two Staphylococcus aureus Fibrinogen Binding Proteins: Clumping Factor and an Extracellular Fibrinogen Binding Protein

    OpenAIRE

    Colque-Navarro, Patricia; Palma, Marco; Söderquist, Bo; Flock, Jan-Ingmar; Möllby, Roland

    2000-01-01

    We analyzed the serum antibody responses against two Staphylococcus aureus fibrinogen binding proteins, the cell-bound clumping factor (Clf) and an extracellular fibrinogen binding protein (Efb). The material consisted of 105 consecutive serum samples from 41 patients suffering from S. aureus septicemia and 72 serum samples from healthy individuals. An enzyme-linked immunosorbent assay (ELISA) was developed. Healthy individuals showed variable levels of antibodies against the studied antigens...

  12. The relationship between transcription initiation RNAs and CCCTC-binding factor (CTCF localization

    Directory of Open Access Journals (Sweden)

    Taft Ryan J

    2011-08-01

    Full Text Available Abstract Background Transcription initiation RNAs (tiRNAs are nuclear localized 18 nucleotide RNAs derived from sequences immediately downstream of RNA polymerase II (RNAPII transcription start sites. Previous reports have shown that tiRNAs are intimately correlated with gene expression, RNA polymerase II binding and behaviors, and epigenetic marks associated with transcription initiation, but not elongation. Results In the present work, we show that tiRNAs are commonly found at genomic CCCTC-binding factor (CTCF binding sites in human and mouse, and that CTCF sites that colocalize with RNAPII are highly enriched for tiRNAs. To directly investigate the relationship between tiRNAs and CTCF we examined tiRNAs originating near the intronic CTCF binding site in the human tumor suppressor gene, p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A. Inhibition of CTCF-proximal tiRNAs resulted in increased CTCF localization and increased p21 expression, while overexpression of CTCF-proximal tiRNA mimics decreased CTCF localization and p21 expression. We also found that tiRNA-regulated CTCF binding influences the levels of trimethylated H3K27 at the alternate upstream p21 promoter, and affects the levels of alternate p21 (p21alt transcripts. Extending these studies to another randomly selected locus with conserved CTCF binding we found that depletion of tiRNA alters nucleosome density proximal to sites of tiRNA biogenesis. Conclusions Taken together, these data suggest that tiRNAs modulate local epigenetic structure, which in turn regulates CTCF localization.

  13. Characterization of transcription factors binding to-120 GATA motif of rat βbminy-globin promoter

    OpenAIRE

    Pavlović Sonja T.; Mitrović Tatjana; Karan-Đurašević Teodora; Nikčević Gordana T.

    2005-01-01

    The aim of this study was to elucidate the regulation of rat adult βbminy-globin gene transcription. We used DNasel foot printing, gel mobility shift and super shift assays to characterize transcription factors involved in this regulation. In this study we analyzed GATA motif at-120 bp in the distal promoter of βbminy-globin gene. Footprint analysis revealed the binding of nuclear factors from MEL cells to the GATA motif. By using gel mobility shift assay two protein complexes were detected. ...

  14. Functional modulation of insulin-like growth factor binding protein-3 expression in melanoma

    OpenAIRE

    Dar, Altaf A.; Majid, Shahana; Nosrati, Mehdi; deSemir, David; Federman, Scot; Kashani-Sabet, Mohammed

    2010-01-01

    Insulin-like growth factor binding protein-3 (IGFBP3) is a member of the IGFBP family, which regulates mitogenic and anti-apoptotic effects of insulin-like growth factors. In this report we evaluated the role of IGFBP3 in melanoma. Quantitative real-time PCR (qRT-PCR), western and ELISA analysis indicated a significant downregulation of IGFBP3 expression in melanoma cell lines as compared to a normal melanocyte cell line. Melanoma cell lines treated with the demethylating agent 5-AZA-2′ deoxy...

  15. Insulin-like Growth Factor Binding Proteins in the Plasma of Growing Horses

    OpenAIRE

    Burk, John Robert

    2002-01-01

    Insulin-like growth factor binding proteins (IGFBP) are modulators of insulin-like growth factor I (IGF-I), which functions as a regulator of cartilage and bone development. Rapid growth and high starch diets have been associated with increased circulating concentrations of IGF-I, which lead to developmental orthopedic disorders in foals. The objective of this study was to assess the effects of age, diet, growth and season on plasma IGFBP and IGF-I concentrations from birth to 16 mo of age in...

  16. Altered relationships between rCBF in different brain regions of never-treated schizophrenics

    Energy Technology Data Exchange (ETDEWEB)

    Sabri, O.; Schreckenberger, M.; Cremerius, U.; Dickmann, C.; Schulz, G.; Zimny, M.; Buell, U. [Technische Hochschule Aachen (Germany). Dept. of Nuclear Medicine; Erkwoh, R.; Owega, A.; Sass, H. [Technische Hochschule Aachen (Germany). Dept. of Psychiatry

    1997-09-01

    Aim of this study was to investigate the relations between regional cerebral blood flow (rCBF) of different brain regions in acute schizophrenia and following neuroleptic treatment. Methods: Twenty-two never-treated, acute schizophrenic patients were examined with HMPAO brain SPECT and assessed psychopathologically, and reexamined following neuroleptic treatment (over 96.8 days) and psychopathological remission. rCBF was determined by region/cerebellar count quotients obtained from 98 irregular regions of interest (ROIs), summed up to 11 ROIs on each hemisphere. In acute schizophrenics, interregional rCBF correlations of each ROI to every other ROI were compared to the interregional correlations following neuroleptic treatment and to those of controls. Results: All significant correlations of rCBF ratios of different brain regions were exclusively positive in controls and patients. In controls, all ROIs of one hemisphere except the mesial temporal ROI correlated significantly to its contralateral ROI. Each hemisphere showed significant frontal-temporal correlations, as well as cortical-subcortical and some cortico-limbic. In contrast, in acute schizophrenics nearly every ROI correlated significantly with every other ROI, without a grouping or relation of the rCBF of certain ROIs as in controls. After neuroleptic treatment and clinical improvement, this diffuse pattern of correlations remained. Conclusions: These results indicate differences in the neuronal interplay between regions in schizophrenic and healthy subjects. In nevertreated schizophrenics, diffuse interregional rCBF correlations can be seen as a sign of change and dysfunction of the systems regulating specificity and diversity of the neuronal functions. Neuroleptic therapy and psychopathologic remission showed no normalizing effect on interregional correlations. (orig.) [Deutsch] Ziel der vorliegenden Studie war es, die Beziehungen zwischen den rCBF-Werten von verschiedenen Hirnregionen bei noch nie

  17. Hepatic binding and uptake kinetics of epidermal growth factor: studies with isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Hepatocytes are known to bind and internalize a variety of small molecular weight proteins by a process known as receptor-mediated endocytosis (RME). The purpose of this investigation was to characterize the binding and uptake kinetics of a small protein known to be taken up by the liver by RME, epidermal growth factor (EGF), using suspensions of freshly isolated rat hepatocytes. Rat hepatocytes accumulated 125I-EGF (90pM) in a temperature-dependent fashion. Isolated hepatocytes incubated at 370C with 125I-EGF began to release a TCA-soluble radiolabeled material into the incubation medium with a lag period of 20 min. EGF uptake by isolated hepatocytes was linear for only 60 seconds and displayed saturation kinetics. Hepatocytes incubated at 40C bound, but did not internalize, EGF. Under these conditions, EGF binding was saturable at concentrations above 8 nM. A Scatchard analysis revealed that the average number of receptors per hepatocyte was 7.7 x 104 with a dissociation constant of 2.6 nM. These data demonstrate that freshly isolated hepatocytes are capable of binding, internalizing and metabolizing EGF and thus are a good model to study RME of small molecular weight proteins. 15 references, 5 figures

  18. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

    Science.gov (United States)

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-01-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency. PMID:27432161

  19. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting.

    Science.gov (United States)

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-01-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency. PMID:27432161

  20. Cytosine methylation does not affect binding of transcription factor Sp1

    International Nuclear Information System (INIS)

    DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. The authors used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Sp1 to its target sequence (a G+C-rich sequence known as a GC box). Concatemers of double-stranded 14-mers containing a GC box successfully competed with the human metallothionein IIA promoter for binding to Sp1 in DNase I protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Sp1 binding. The result was confirmed using double-stranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Sp1-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Sp1-DNA interactions

  1. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    Directory of Open Access Journals (Sweden)

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  2. 结缕草CBF基因的同源克隆及其转基因拟南芥的抗寒性验证%Cloning of Zoysiagrass CBF Gene and Validation of Cold Tolerance in Trans-genic Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    冯勋伟; 才宏伟

    2014-01-01

    结缕草是优良的暖季型草坪草之一,主要用于亚热带和热带地区的草坪种植。抗冷性是结缕草栽培范围的限制因子。本研究以日本最北部原产的结缕草品系为材料,根据其他植物的已知的抗寒基因 CBF 序列,通过同源克隆的方法获得结缕草中相对应的同源基因ZjCBF;根据和其他已报告的CBF序列的比对结果,确定ZjCBF基因属于CBF转录因子家族基因中CBF1型基因。利用半定量PCR和实时定量PCR分析该基因在寒冷条件下的表达情况,发现ZjCBF基因受冷胁迫的诱导,在4℃处理6 h时表达量最高。在此基础上,本研究构建了该基因的过表达载体,并将其转化到拟南芥中,通过低温冷处理实验发现,不论是否经过冷驯化,转 ZjCBF 基因植株由于 ZjCBF 的过量表达均比野生型植株抗寒性强。%Zoysiagrass is recognized as an excellent warm-season turfgrass and mainly used in subtropical and tropical regions. Cold stress is a major constraint factor for the cultivation of zoysiagrass. In this study, according to the sequences of cold tolerance gene CBF had been reported in other plant species, we cloned the corresponding homologous of the ZjCBF gene by homology cloning method in Zoysia japonica using a material originated from the most northern area of Japan. Based on the alignment re-sults compared with other reported CBF genes, we found the ZjCBF gene belongs to the CBF1 familiy. By semi-quantitative PCR and real-time quantitative PCR, we analyzed the expression level of the ZjCBF gene in the cold condition and found that ZjCBF was induced by cold stress, and the ZjCBF expression reached peak at six hours post 4°C treatment. In addition, we also con-structed ZjCBF over expression vector and generated transgenic Arabidopsis plants, with better cold tolerance than the wild-type, whether through cold acclimation or not.

  3. Memory-provoked rCBF-SPECT as a diagnostic tool in Alzheimer's disease?

    International Nuclear Information System (INIS)

    Alzheimer's disease (AD) is a primary degenerative disease that progressively affects all brain functions, with devastating consequences for the patient, the patient's family and society. Rest regional cerebral blood flow (rCBF) could have a strategic role in differentiating between AD patients and normal controls, but its use for this purpose has a low discriminatory capacity. The purpose of this study was to evaluate whether the diagnostic sensitivity of rCBF single-photon emission computed tomography (SPECT) could be increased by using an episodic memory task provocation, i.e. memory-provoked rCBF-SPECT (MP-SPECT). Eighteen persons (73.2±4.8 years) with mild AD and 18 healthy elderly (69.4±3.9 years) were included in the study. The subjects were injected with99mTc-hexamethylpropylene amine oxime (HMPAO) during memory provocation with faces and names, followed by an rCBF-SPECT study. The rCBF99mTc-HMPAO SPECT images were analysed using statistical parametric mapping (SPM2). Peaks with a false discovery rate corrected value of 0.05 were considered significant. On MP-SPECT, the AD group showed a significant rCBF reduction in the left parietal cortex in comparison with healthy elderly. At rest, no significant group differences were seen. Memory provocation increased the sensitivity of rCBF-SPECT for the detection of AD-related blood flow changes in the brain at the group level. Further studies are needed to evaluate MP-SPECT as a diagnostic tool at the individual level. If a higher sensitivity for AD at the individual level is verified in future studies, a single MP-SPECT study might be sufficient in the clinical setting. (orig.)

  4. How well do lipophilicity parameters, MEEKC microemulsion capacity factor, and plasma protein binding predict CNS tissue binding?

    Science.gov (United States)

    Zamek-Gliszczynski, Maciej J; Sprague, Karen E; Espada, Alfonso; Raub, Thomas J; Morton, Stuart M; Manro, Jason R; Molina-Martin, Manuel

    2012-05-01

    Brain fraction unbound (Fu) is critical to understanding the pharmacokinetics/dynamics of central nervous system (CNS) drugs, thus several surrogate predictors have been proposed. At present, correlations between brain Fu, microemulsion electrokinetic chromatography capacity factor (MEEKC k'), plasma Fu, octanol-water partition coefficient (clogP), and LogP at pH 7.4 (clogD(7.4) ) were compared for 94 diverse molecules, and additionally for 587 compounds. MEEKC k' was a better predictor of brain Fu (r(2) = 0.74) than calculated lipophilicity parameters (clogP r(2) = 0.51-0.54, clogD(7.4) r(2) = 0.41-0.44), but it was not superior to plasma Fu (r(2) = 0.74-0.85) as a predictor of brain Fu. MEEKC k' did not predict plasma Fu(r(2) = 0.58) as well as brain Fu, and the extent of improvement over clogP or clogD(7.4) (r(2) = 0.41-0.49) was less pronounced. Although log-log-correlation analysis supported seemingly strong prediction of brain Fu both by MEEKC k' and by plasma Fu (r(2) ≥ 0.74), analysis of prediction error estimated a 10-fold and 6.9-8.6-fold prediction interval for brain Fu estimated using MEEKC k' and plasma Fu, respectively. Therefore, MEEKC k' and plasma Fu can predict the log order of CNS tissue binding, but they cannot provide truly quantitative brain Fu predictions necessary to support in-vitro-to-in-vivo extrapolations and pharmacokinetic/dynamic data interpretation. PMID:22344827

  5. A structural-based strategy for recognition of transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Beisi Xu

    Full Text Available Scanning through genomes for potential transcription factor binding sites (TFBSs is becoming increasingly important in this post-genomic era. The position weight matrix (PWM is the standard representation of TFBSs utilized when scanning through sequences for potential binding sites. However, many transcription factor (TF motifs are short and highly degenerate, and methods utilizing PWMs to scan for sites are plagued by false positives. Furthermore, many important TFs do not have well-characterized PWMs, making identification of potential binding sites even more difficult. One approach to the identification of sites for these TFs has been to use the 3D structure of the TF to predict the DNA structure around the TF and then to generate a PWM from the predicted 3D complex structure. However, this approach is dependent on the similarity of the predicted structure to the native structure. We introduce here a novel approach to identify TFBSs utilizing structure information that can be applied to TFs without characterized PWMs, as long as a 3D complex structure (TF/DNA exists. This approach utilizes an energy function that is uniquely trained on each structure. Our approach leads to increased prediction accuracy and robustness compared with those using a more general energy function. The software is freely available upon request.

  6. A Novel Alignment-Free Method for Comparing Transcription Factor Binding Site Motifs

    Science.gov (United States)

    Xu, Minli; Su, Zhengchang

    2010-01-01

    Background Transcription factor binding site (TFBS) motifs can be accurately represented by position frequency matrices (PFM) or other equivalent forms. We often need to compare TFBS motifs using their PFMs in order to search for similar motifs in a motif database, or cluster motifs according to their binding preference. The majority of current methods for motif comparison involve a similarity metric for column-to-column comparison and a method to find the optimal position alignment between the two compared motifs. In some applications, alignment-free methods might be preferred; however, few such methods with high accuracy have been described. Methodology/Principal Findings Here we describe a novel alignment-free method for quantifying the similarity of motifs using their PFMs by converting PFMs into k-mer vectors. The motifs could then be compared by measuring the similarity among their corresponding k-mer vectors. Conclusions/Significance We demonstrate that our method in general achieves similar performance or outperforms the existing methods for clustering motifs according to their binding preference and identifying similar motifs of transcription factors of the same family. PMID:20098703

  7. A novel alignment-free method for comparing transcription factor binding site motifs.

    Directory of Open Access Journals (Sweden)

    Minli Xu

    Full Text Available BACKGROUND: Transcription factor binding site (TFBS motifs can be accurately represented by position frequency matrices (PFM or other equivalent forms. We often need to compare TFBS motifs using their PFMs in order to search for similar motifs in a motif database, or cluster motifs according to their binding preference. The majority of current methods for motif comparison involve a similarity metric for column-to-column comparison and a method to find the optimal position alignment between the two compared motifs. In some applications, alignment-free methods might be preferred; however, few such methods with high accuracy have been described. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a novel alignment-free method for quantifying the similarity of motifs using their PFMs by converting PFMs into k-mer vectors. The motifs could then be compared by measuring the similarity among their corresponding k-mer vectors. CONCLUSIONS/SIGNIFICANCE: We demonstrate that our method in general achieves similar performance or outperforms the existing methods for clustering motifs according to their binding preference and identifying similar motifs of transcription factors of the same family.

  8. Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α.

    Directory of Open Access Journals (Sweden)

    Hyeong-Jun Han

    Full Text Available Peptidyl prolyl isomerase (PIN1 regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF-1α in human colon cancer (HCT116 cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target.

  9. Identification of potential nuclear reprogramming and differentiation factors by a novel selection method for cloning chromatin-binding proteins

    Institute of Scientific and Technical Information of China (English)

    LiuWang; AihuaZheng; LingYi; ChongrenXu; MingxiaoDing; HongkuiDeng

    2005-01-01

    Nuclear reprogramming is critical for animal cloning and stem cell creation through nuclear transfer, which requires extensive remodeling of chromosomal architecture involving dramatic changes in chromatin-binding proteins. To understand the mechanism of nuclear reprogramming, it is critical to identify chromatin-binding factors specify the reprogramming process. In this report, we have developed a high-throughput selection method, based on T7 phage display and chromatin immunoprecipitation, to isolate chromatin-binding factors expressed in mouse embryonic stem cells using primary mouse embryonic fibroblast chromatin. Seven chromatin-binding proteins have been isolated by this method. We have also isolated several chromatin-binding proteins involved in hepatocyte differentiation. Our method provides a powerful tool to rapidly and selectively identify chromatin-binding proteins. The method can be used to study epigenetic modification of chromatin during nuclear reprogramming, cell differentiation, and transdifferentiation.

  10. Roles for GTP-binding proteins in Vigna unguiculata responding to Nod factors or chitin elicitors

    International Nuclear Information System (INIS)

    μNod factors are lipo-chito-oligosaccharides secreted by Rhizobium to initiate deformation of legume root hairs and other changes such as increases in intracellular calcium in responsive root hairs. We studied the effects of Nod factors and G-protein modulators on root hair deformation and found significant deformation of root hairs after 30 min exposure to the compounds. Since G-proteins have been implicated in the root hair response to Nod factors in vivo, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of the legume Vigna unguiculata. GTP competitively binds to microsomal membrane fractions labelled with [35S]GTPγS with a high affinity, yielding a two-site displacement curve with displacement constants (Ki) of 0.58 μM and 0.16 μM. Competition with either ATP or GDP revealed a one-site displacement curve with Ki of 4.14 and 11.7 μM respectively. To test if exposure to Nod factors affect the GTP-binding profile, we isolated microsomal membrane fractions from roots pretreated with either NodNGR[S] (from Rhizobium sp. NGR234) or the four-sugar, tetracetylchitotetraose (TACT) backbone of Nod factors. Pretreatment with NodNGR[S] results in an increased affinity for GTP of several hundred-fold. Roots pretreated with TACT also showed a similar but slightly smaller increase in affinity for GTP. To begin identification of possible candidates microsomal proteins were separated by SDS-PAGE and GTP-binding proteins were probed with [35S]GTPγS. Microsomal membrane factions isolated from roots pretreated with NodNGR[S] revealed two proteins (27 kDa and 25 kDa) with a higher affinity for GTPγS. Western blotting of the microsomal membrane preparation with anti-Rac antibodies also showed changes in Rac associated signal in microsomal membranes prepared from either NodNGR[S] or TACT pretreated roots. These results provide further support for a role for small, monomeric G-proteins in the Nod factor signal

  11. Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1.

    Science.gov (United States)

    Guja, Kip E; Venkataraman, Krithika; Yakubovskaya, Elena; Shi, Hui; Mejia, Edison; Hambardjieva, Elena; Karzai, A Wali; Garcia-Diaz, Miguel

    2013-09-01

    Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a role analogous to KsgA/Dim1 proteins. This modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and diabetes. Here, we present the first structural characterization of the mammalian TFB1 factor. We have solved two X-ray crystallographic structures of TFB1M with (2.1 Å) and without (2.0 Å) its cofactor S-adenosyl-L-methionine. These structures reveal that TFB1M shares a conserved methyltransferase core with other KsgA/Dim1 methyltransferases and shed light on the structural basis of S-adenosyl-L-methionine binding and methyltransferase activity. Together with mutagenesis studies, these data suggest a model for substrate binding and provide insight into the mechanism of methyl transfer, clarifying the role of this factor in an essential process for mitochondrial function. PMID:23804760

  12. Binding and aggregation of pro-atrial natriuretic factor by calcium.

    Science.gov (United States)

    Thibault, G; Doubell, A F

    1992-04-01

    Analysis of atrial secretory granule content by sodium dodecyl sulfate-gel electrophoresis followed by a 45Ca2+ overlay assay indicates that a 17,000 protein binds 45Ca2+. This protein, which can be immunostained by atrial natriuretic factor (ANF) antiserum, corresponds to proANF. Ca2+ binding is proportional to the amount of proANF and pH dependent. Generation of ANF-(1-98) by thrombin digestion of proANF does not affect Ca2+ binding. Blocking the carboxyl groups of proANF and the use of NH2-terminal fragments bearing those carboxyl groups demonstrated that the Ca(2+)-interaction site is probably located within the highly acidic portion (11-30) of the propeptide. Ca2+ binding to proANF induces its aggregation that can be verified by sedimentation. ProANF aggregation is Ca2+ dependent, being optimal at 10 mM, partially pH dependent, and greatly increased by high concentrations of proANF. However, because of its relatively low-binding affinity, Ca2+ can be substituted by other divalent cations such as Sr2+, Ba2+, or Mg2+. The high level of Ca2+ in atrial secretory granules and the aggregation of proANF in the presence of Ca2+ suggest a possible involvement of these physicochemical properties in the condensed state of the matrix of secretory granules. Indeed, detergent solubilization of the membrane of the secretory granules in presence of Ca2+ resulted only in a partial dissolution of the dense core matrix. We therefore postulate that, in the Golgi complex, proANF and Ca2+ associate to form a condensed aggregate that helps package secretory material into secretory vesicles. PMID:1533094

  13. Insulin-like growth factor binding protein-6 delays replicative senescence of human fibroblasts

    DEFF Research Database (Denmark)

    Micutkova, Lucia; Diener, Thomas; Li, Chen;

    2011-01-01

    Cellular senescence can be induced by a variety of mechanisms, and recent data suggest a key role for cytokine networks to maintain the senescent state. Here, we have used a proteomic LC-MS/MS approach to identify new extracellular regulators of senescence in human fibroblasts. We identified 26...... extracellular proteins with significantly different abundance in conditioned media from young and senescent fibroblasts. Among these was insulin-like growth factor binding protein-6 (IGFBP-6), which was chosen for further analysis. When IGFBP-6 gene expression was downregulated, cell proliferation was inhibited...... and apoptotic cell death was increased. Furthermore, downregulation of IGFBP-6 led to premature entry into cellular senescence. Since IGFBP-6 overexpression increased cellular lifespan, the data suggest that IGFBP-6, in contrast to other IGF binding proteins, is a negative regulator of cellular...

  14. Oligonucleotide that binds nuclear factor NF-kappa-B acts as a lymphoid-specific and inducible enhancer element

    OpenAIRE

    Pierce, J. W.; Lenardo, M; Baltimore, D

    1988-01-01

    The immunoglobulin kappa light chain gene contains a lymphoid-specific enhancer that includes several short protein-binding sequences. The sequence that binds the nuclear factor NF-kappa B was tested for its ability to act independently as an enhancer element by inserting it into test plasmids containing the chloramphenicol acetyltransferase gene. When analyzed for activity by transient transfection into lymphoid and nonlymphoid cells, a single copy of the NF-kappa B binding site could act as...

  15. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

    Directory of Open Access Journals (Sweden)

    Janet L Smith

    2015-05-01

    Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

  16. Complement factor H binding by different Lyme disease and relapsing fever Borrelia in animals and human

    OpenAIRE

    Gil Horacio; Camafeita Emilio; Escudero Raquel; Bhide Mangesh R; Jado Isabel; Anda Pedro

    2009-01-01

    Abstract Background Borreliae employ multiple immune evasive strategies such as binding to complement regulatory proteins [factor H (fH) and factor H like-1 (FHL1)], differential regulation of surface membrane proteins, antigenic variation, and binding of plasminogen/plasmin and matrix metalloproteinases. As a complement regulatory subunit, fH serves as a cofactor for the factor I-mediated cleavage of C3b. fH binding by Borrelia has been correlated with pathogenesis as well as with host diver...

  17. Heparan sulfate proteoglycans mediate factor XIIa binding to the cell surface.

    Science.gov (United States)

    Wujak, Lukasz; Didiasova, Miroslava; Zakrzewicz, Dariusz; Frey, Helena; Schaefer, Liliana; Wygrecka, Malgorzata

    2015-03-13

    Hageman factor (FXIIa) initiates the intrinsic coagulation pathway and triggers the kallikrein-kinin and the complement systems. In addition, it functions as a growth factor by expressing promitogenic activities toward several cell types. FXIIa binds to the cell surface via a number of structurally unrelated surface receptors; however, the underlying mechanisms are not yet fully understood. Here, we demonstrate that FXIIa utilizes cell membrane-bound glycosaminoglycans to interact with the cell surface of human lung fibroblasts (HLF). The combination of enzymatic, inhibitory, and overexpression approaches identified a heparan sulfate (HS) component of proteoglycans as an important determinant of the FXIIa binding capacity of HLF. Moreover, cell-free assays and competition experiments revealed preferential binding of FXIIa to HS and heparin over dextran sulfate, dermatan sulfate, and chondroitin sulfate A and C. Finally, we demonstrate that fibroblasts isolated from the lungs of the patients suffering from idiopathic pulmonary fibrosis (IPF) exhibit enhanced FXIIa binding capacity. Increased sulfation of HS resulting from elevated HS 6-O-sulfotransferase-1 expression in IPF HLF accounted, in part, for this phenomenon. Application of RNA interference technology and inhibitors of intracellular sulfation revealed the cooperative action of cell surface-associated HS and urokinase-type plasminogen activator receptor in the accumulation of FXIIa on the cell surface of IPF HLF. Moreover, FXIIa stimulated IPF HLF migration, which was abrogated by pretreatment of cells with heparinase I. Collectively, our study uncovers a novel role of HS-type glycosaminoglycans in a local accumulation of FXIIa on the cell membrane. The enhanced association of FXIIa with IPF HLF suggests its contribution to fibrogenesis. PMID:25589788

  18. Transcription Factors Bind Thousands of Active and InactiveRegions in the Drosophila Blastoderm

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiao-Yong; MacArthur, Stewart; Bourgon, Richard; Nix, David; Pollard, Daniel A.; Iyer, Venky N.; Hechmer, Aaron; Simirenko, Lisa; Stapleton, Mark; Luengo Hendriks, Cris L.; Chu, Hou Cheng; Ogawa, Nobuo; Inwood, William; Sementchenko, Victor; Beaton, Amy; Weiszmann, Richard; Celniker, Susan E.; Knowles, David W.; Gingeras, Tom; Speed, Terence P.; Eisen, Michael B.; Biggin, Mark D.

    2008-01-10

    Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. Here, we use whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior-posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over forty well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal-ventral patterning genes, whose expression we show to be quantitatively modulated by anterior-posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly-bound regions are not involved in early

  19. 椰子CBF基因的克隆研究%Study on Cloning of CBF Gene from Coconut (Cocos nucifera)

    Institute of Scientific and Technical Information of China (English)

    肖勇; 杨耀东; 曹红星; 雷新涛; 范海阔; 赵松林; 马子龙

    2012-01-01

    In order to elucidate the molecular mechanism of coconuts cold resistance, this study designed primers according to the CBF gene sequence of oil palm, and then amplified the genomic DNA of Cocos nucifera by using PCR method. The PCR products were retrieved, linked, transformed and sequenced; finally, a DNA sequence of Cocos nucifera was obtained. This sequence had the highest identity (99%) with CBF gene of oil palm, and it was confirmed to be CBF gene. Through the clustering analysis of the cloned CBF gene of coconut and CBF genes of graminaceous crops, the high homology was found between the cloned CBF gene and HvCBF1, Os-DREB1E, HvCBF11, TaCBF11.%为了从分子水平上解析椰子抗寒的机理,以油棕的CBF基因序列设计引物,对椰子的基因组DNA进行PCR扩增,并对PCR产物进行回收、连接、转化以及测序,最后获得了1条DNA序列.这条序列与油棕的CBF基因的同源性高达99%,被证实为CBF基因.将克隆的椰子的CBF基因与禾本科作物的CBF基因进行聚类分析,结果显示:所克隆的CBF基因与HvCBF1、OsDREB1E、HvCB F11和TaCBF11具有较高的同源性.

  20. Divergence of Pumilio/fem-3 mRNA Binding Factor (PUF) Protein Specificity through Variations in an RNA-binding Pocket*

    Science.gov (United States)

    Qiu, Chen; Kershner, Aaron; Wang, Yeming; Holley, Cynthia P.; Wilinski, Daniel; Keles, Sunduz; Kimble, Judith; Wickens, Marvin; Hall, Traci M. Tanaka

    2012-01-01

    mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species. PMID:22205700

  1. Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket.

    Science.gov (United States)

    Qiu, Chen; Kershner, Aaron; Wang, Yeming; Holley, Cynthia P; Wilinski, Daniel; Keles, Sunduz; Kimble, Judith; Wickens, Marvin; Hall, Traci M Tanaka

    2012-02-24

    mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species. PMID:22205700

  2. Inhibition of human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site

    NARCIS (Netherlands)

    S.R. Eberhardy; J. Goncalves; S. Coelho; D.J. Segal; B. Berkhout; C.F. Barbas

    2006-01-01

    The human immunodeficiency virus type 1 (HIV-1) primer-binding site (PBS) is a highly conserved region in the HIV genome and represents an attractive target for the development of new anti-HIV therapies. In this study, we designed four artificial zinc finger transcription factors to bind at or adjac

  3. Casein kinase II phosphorylation increases the rate of serum response factor-binding site exchange.

    OpenAIRE

    Marais, R M; Hsuan, J J; McGuigan, C.; Wynne, J; Treisman, R

    1992-01-01

    Recombinant baculoviruses were used to express wild-type serum response factor (SRF) and a mutant, SRF.CKIIA, which lacks all four serine residues in the major casein kinase II (CKII) site at residues 77-90. Purified recombinant SRF binds DNA with an affinity and specificity indistinguishable from that of HeLa cell SRF, and activates transcription in vitro. Comparative phosphopeptide analysis of the wild-type and mutant proteins demonstrated that the wild-type protein is phosphorylated at the...

  4. Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A

    OpenAIRE

    Gangelhoff, Todd A.; Mungalachetty, Purnima S.; Nix, Jay C.; Mair E A Churchill

    2009-01-01

    The mitochondrial transcription factor A (mtTFA) is central to assembly and initiation of the mitochondrial transcription complex. Human mtTFA (h-mtTFA) is a dual high mobility group box (HMGB) protein that binds site-specifically to the mitochondrial genome and demarcates the promoters for recruitment of h-mtTFB1, h-mtTFB2 and the mitochondrial RNA polymerase. The stoichiometry of h-mtTFA was found to be a monomer in the absence of DNA, whereas it formed a dimer in the complex with the light...

  5. Heparin Binds Endothelial Cell Growth Factor, the Principal Endothelial Cell Mitogen in Bovine Brain

    Science.gov (United States)

    Maciag, Thomas; Mehlman, Tevie; Friesel, Robert; Schreiber, Alain B.

    1984-08-01

    Endothelial cell growth factor (ECGF), an anionic polypeptide mitogen, binds to immobilized heparin. The interaction between the acidic polypeptide and the anionic carbohydrate suggests a mechanism that is independent of ion exchange. Monoclonal antibodies to purified bovine ECGF inhibited the biological activity of ECGF in crude preparations of bovine brain. These data indicate that ECGF is the principal mitogen for endothelial cells from bovine brain, that heparin affinity chromatography may be used to purify and concentrate ECGF, and that the affinity of ECGF for heparin may have structural and perhaps biological significance.

  6. Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

    OpenAIRE

    Friesel, R; Burgess, W H; Maciag, T

    1989-01-01

    Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130...

  7. Detection of insulin-like growth factor binding proteins (IGFBPs) in porcine serum

    OpenAIRE

    Masnikosa Romana; Baričević Ivona; Lagundžin Dragana; Nedić Olgica

    2010-01-01

    Multiple insulin-like growth factor binding proteins (IGFBPs) are found in sera of various species. This study was conducted to investigate whether IGFBPs in porcine serum could be detected using antibodies and antisera raised against human IGFBP-1, -2, -3 and -4. Western ligand blot procedure was used for the identification of c in porcine serum. The results presented in this work showed that the heterologous antibodies can be used to identify IGFBP-1, IGFBP-2 and IGFBP-3, but not IGFBP-4 in...

  8. Molecular and physiological changes in response to salt stress in Citrus macrophylla W plants overexpressing Arabidopsis CBF3/DREB1A.

    Science.gov (United States)

    Alvarez-Gerding, Ximena; Espinoza, Carmen; Inostroza-Blancheteau, Claudio; Arce-Johnson, Patricio

    2015-07-01

    Plant stress induced by high salinity has leading to an important reduction in crop yields. Due to their tropical origin, citrus fruits are highly sensitive to salts. Rootstocks are the root system of fruit trees, regulating ion uptake and transport to the canopy. Therefore, increasing their salt tolerance could improve the salt tolerance of the fruit tree. For this, we genetically-transformed an important rootstock for lemon, Citrus macrophylla W, to constitutively express the CBF3/DREB1A gene from Arabidopsis, a well-studied salinity tolerance transcription factor. Transgenic lines showed normal size, with no dwarfism. Under salt stress, some transgenic lines showed greater growth, similar accumulation of chloride and sodium in the leaves and better stomatal conductance, in comparison to wild-type plants. Quantitative real-time analyses showed a similar expression of several CBF3/DREB1A target genes, such as COR15A, LEA 4/5, INV, SIP1, P5CS, GOLS, ADC2 and LKR/SDH, in transgenic lines and wild type plants, with the exception of INV that shows increased expression in line 4C15. Under salt stress, all measured transcript increased in both wild type and transgenics lines, with the exception of INV. Altogether, these results suggest a higher salt tolerance of transgenic C. macrophylla plants induced by the overexpression of AtCBF3/DREB1A. PMID:25914135

  9. LASAGNA-Search: an integrated web tool for transcription factor binding site search and visualization.

    Science.gov (United States)

    Lee, Chic; Huang, Chun-Hsi

    2013-03-01

    The release of ChIP-seq data from the ENCyclopedia Of DNA Elements (ENCODE) and Model Organism ENCyclopedia Of DNA Elements (modENCODE) projects has significantly increased the amount of transcription factor (TF) binding affinity information available to researchers. However, scientists still routinely use TF binding site (TFBS) search tools to scan unannotated sequences for TFBSs, particularly when searching for lesser-known TFs or TFs in organisms for which ChIP-seq data are unavailable. The sequence analysis often involves multiple steps such as TF model collection, promoter sequence retrieval, and visualization; thus, several different tools are required. We have developed a novel integrated web tool named LASAGNA-Search that allows users to perform TFBS searches without leaving the web site. LASAGNA-Search uses the LASAGNA (Length-Aware Site Alignment Guided by Nucleotide Association) algorithm for TFBS alignment. Important features of LASAGNA-Search include (i) acceptance of unaligned variable-length TFBSs, (ii) a collection of 1726 TF models, (iii) automatic promoter sequence retrieval, (iv) visualization in the UCSC Genome Browser, and (v) gene regulatory network inference and visualization based on binding specificities. LASAGNA-Search is freely available at http://biogrid.engr.uconn.edu/lasagna_search/. PMID:23599922

  10. Mitochondrial transcription termination factor 2 binds to entire mitochondrial DNA and negatively regulates mitochondrial gene expression

    Institute of Scientific and Technical Information of China (English)

    Weiwei Huang; Min Yu; Yang Jiao; Jie Ma; Mingxing Ma; Zehua Wang; Hong Wu; Deyong Tan

    2011-01-01

    Mitochondrial transcription termination factor 2 (mTERF2) is a mitochondriai matrix protein that binds to the mitochondriai DNA.Previous studies have shown that overexpression of mTERF2 can inhibit cell proliferation, but the mechanism has not been well defined so far.This study aimed to present the binding pattern of mTERF2 to the mitochondrial DNA (mtDNA) in vivo, and investigated the biological function of mTERF2 on the replication of mtDNA, mRNA transcription, and protein translation.The mTERF2 binding to entire mtDNA was identified via the chromatin immunoprecipitation analysis.The mtDNA replication efficiency and expression levels of mitochondria genes were significantly inhibited when the mTERF2 was overexpressed in HeLa cells.The inhibition level of mtDNA content was the same with the decreased levels of mRNA and mitochondrial protein expression.Overall, the mTERF2 might be a cell growth inhibitor based on its negative effect on mtDNA replication, which eventually own-regulated all of the oxidative phosphorylation components in the mitochondria that were essential for the cell's energy metabolism.

  11. The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

    Science.gov (United States)

    Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

    2014-12-01

    The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

  12. Complement Factor H and Simian Virus 40 bind the GM1 ganglioside in distinct conformations.

    Science.gov (United States)

    Blaum, Bärbel S; Frank, Martin; Walker, Ross C; Neu, Ursula; Stehle, Thilo

    2016-05-01

    Mammalian cell surfaces are decorated with a variety of glycan chains that orchestrate development and defense and are exploited by pathogens for cellular attachment and entry. While glycosidic linkages are, in principle, flexible, the conformational space that a given glycan can sample is subject to spatial and electrostatic restrictions imposed by its overall chemical structure. Here, we show how the glycan moiety of the GM1 ganglioside, a branched, monosialylated pentasaccharide that serves as a ligand for various proteins, undergoes differential conformational selection in its interactions with different lectins. Using STD NMR and X-ray crystallography, we found that the innate immune regulator complement Factor H (FH) binds a previously not reported GM1 conformation that is not compatible with the GM1-binding sites of other structurally characterized GM1-binding lectins such as the Simian Virus 40 (SV40) capsid. Molecular dynamics simulations of the free glycan in explicit solvent on the 10 μs timescale reveal that the FH-bound conformation nevertheless corresponds to a minimum in the Gibbs free energy plot. In contrast to the GM1 conformation recognized by SV40, the FH-bound GM1 conformation is associated with poor NOE restraints, explaining how it escaped(1)H-(1)H NOE-restrained modeling in the past and highlighting the necessity for ensemble representations of glycan structures. PMID:26715202

  13. HOCOMOCO: A comprehensive collection of human transcription factor binding sites models

    KAUST Repository

    Kulakovskiy, Ivan V.

    2012-11-21

    Transcription factor (TF) binding site (TFBS) models are crucial for computational reconstruction of transcription regulatory networks. In existing repositories, a TF often has several models (also called binding profiles or motifs), obtained from different experimental data. Having a single TFBS model for a TF is more pragmatic for practical applications. We show that integration of TFBS data from various types of experiments into a single model typically results in the improved model quality probably due to partial correction of source specific technique bias. We present the Homo sapiens comprehensive model collection (HOCOMOCO, http://autosome.ru/HOCOMOCO/, http://cbrc.kaust.edu.sa/ hocomoco/) containing carefully hand-curated TFBS models constructed by integration of binding sequences obtained by both low- and high-throughput methods. To construct position weight matrices to represent these TFBS models, we used ChIPMunk software in four computational modes, including newly developed periodic positional prior mode associated with DNA helix pitch. We selected only one TFBS model per TF, unless there was a clear experimental evidence for two rather distinct TFBS models. We assigned a quality rating to each model. HOCOMOCO contains 426 systematically curated TFBS models for 401 human TFs, where 172 models are based on more than one data source. The Author(s) 2012.

  14. Cerebral embolism: local CBF and edema measured by CT scanning and Xe inhalation

    International Nuclear Information System (INIS)

    Serial CT scans were made in baboons after cerebral embolization during stable Xe inhalation for measuring local values for CBF and lambda (brain-blood partition or solubility coefficients), followed by iodine infusion for detecting blood-brain barrier (BBB) damage. Supplementary 133Xe CBF measurements were made at corresponding intervals. Persistent zones of zero flow surrounded by reduced flow were measured predominantly in subcortical regions, which showed gross and microscopic evidence of infarction at necropsy. Overylng cortex was relatively spared. Reduced lambda values attributed to edema appeared with in 3 to 5 minutes and progressed up to 60 minutes. Damage to BBB with visible transvascular seepage of iodine began to appear 1-11/2 hours after embolism. In chronic animals, lambda values were persistently reduced in areas showing histologic infarction. Contralateral hemispheric CBF increased for the first 15 minutes after embolism, followed by progressive reduction after 30 minutes

  15. Free energy simulations of a GTPase: GTP and GDP binding to archaeal initiation factor 2.

    Science.gov (United States)

    Satpati, Priyadarshi; Clavaguéra, Carine; Ohanessian, Gilles; Simonson, Thomas

    2011-05-26

    Archaeal initiation factor 2 (aIF2) is a protein involved in the initiation of protein biosynthesis. In its GTP-bound, "ON" conformation, aIF2 binds an initiator tRNA and carries it to the ribosome. In its GDP-bound, "OFF" conformation, it dissociates from tRNA. To understand the specific binding of GTP and GDP and its dependence on the ON or OFF conformational state of aIF2, molecular dynamics free energy simulations (MDFE) are a tool of choice. However, the validity of the computed free energies depends on the simulation model, including the force field and the boundary conditions, and on the extent of conformational sampling in the simulations. aIF2 and other GTPases present specific difficulties; in particular, the nucleotide ligand coordinates a divalent Mg(2+) ion, which can polarize the electronic distribution of its environment. Thus, a force field with an explicit treatment of electronic polarizability could be necessary, rather than a simpler, fixed charge force field. Here, we begin by comparing a fixed charge force field to quantum chemical calculations and experiment for Mg(2+):phosphate binding in solution, with the force field giving large errors. Next, we consider GTP and GDP bound to aIF2 and we compare two fixed charge force fields to the recent, polarizable, AMOEBA force field, extended here in a simple, approximate manner to include GTP. We focus on a quantity that approximates the free energy to change GTP into GDP. Despite the errors seen for Mg(2+):phosphate binding in solution, we observe a substantial cancellation of errors when we compare the free energy change in the protein to that in solution, or when we compare the protein ON and OFF states. Finally, we have used the fixed charge force field to perform MDFE simulations and alchemically transform GTP into GDP in the protein and in solution. With a total of about 200 ns of molecular dynamics, we obtain good convergence and a reasonable statistical uncertainty, comparable to the force

  16. Engineered proteins with Pumilio/fem-3 mRNA binding factor scaffold to manipulate RNA metabolism.

    Science.gov (United States)

    Wang, Yang; Wang, Zefeng; Tanaka Hall, Traci M

    2013-08-01

    Pumilio/fem-3 mRNA binding factor proteins are characterized by a sequence-specific RNA-binding domain. This unique single-stranded RNA recognition module, whose sequence specificity can be reprogrammed, has been fused with functional modules to engineer protein factors with various functions. We summarize the advances made with respect to developing RNA regulatory tools, as well as opportunities for the future. PMID:23731364

  17. Nuclear factor I revealed as family of promoter binding transcription activators

    Directory of Open Access Journals (Sweden)

    Plasari Genta

    2011-04-01

    Full Text Available Abstract Background Multiplex experimental assays coupled to computational predictions are being increasingly employed for the simultaneous analysis of many specimens at the genome scale, which quickly generates very large amounts of data. However, inferring valuable biological information from the comparisons of very large genomic datasets still represents an enormous challenge. Results As a study model, we chose the NFI/CTF family of mammalian transcription factors and we compared the results obtained from a genome-wide study of its binding sites with chromatin structure assays, gene expression microarray data, and in silico binding site predictions. We found that NFI/CTF family members preferentially bind their DNA target sites when they are located around transcription start sites when compared to control datasets generated from the random subsampling of the complete set of NFI binding sites. NFI proteins preferably associate with the upstream regions of genes that are highly expressed and that are enriched in active chromatin modifications such as H3K4me3 and H3K36me3. We postulate that this is a causal association and that NFI proteins mainly act as activators of transcription. This was documented for one member of the family (NFI-C, which revealed as a more potent gene activator than repressor in global gene expression analysis. Interestingly, we also discovered the association of NFI with the tri-methylation of lysine 9 of histone H3, a chromatin marker previously associated with the protection against silencing of telomeric genes by NFI. Conclusion Taken together, we illustrate approaches that can be taken to analyze large genomic data, and provide evidence that NFI family members may act in conjunction with specific chromatin modifications to activate gene expression.

  18. Altered relationships between rCBF in different brain regions of never-treated schizophrenics

    International Nuclear Information System (INIS)

    Aim of this study was to investigate the relations between regional cerebral blood flow (rCBF) of different brain regions in acute schizophrenia and following neuroleptic treatment. Methods: Twenty-two never-treated, acute schizophrenic patients were examined with HMPAO brain SPECT and assessed psychopathologically, and reexamined following neuroleptic treatment (over 96.8 days) and psychopathological remission. rCBF was determined by region/cerebellar count quotients obtained from 98 irregular regions of interest (ROIs), summed up to 11 ROIs on each hemisphere. In acute schizophrenics, interregional rCBF correlations of each ROI to every other ROI were compared to the interregional correlations following neuroleptic treatment and to those of controls. Results: All significant correlations of rCBF ratios of different brain regions were exclusively positive in controls and patients. In controls, all ROIs of one hemisphere except the mesial temporal ROI correlated significantly to its contralateral ROI. Each hemisphere showed significant frontal-temporal correlations, as well as cortical-subcortical and some cortico-limbic. In contrast, in acute schizophrenics nearly every ROI correlated significantly with every other ROI, without a grouping or relation of the rCBF of certain ROIs as in controls. After neuroleptic treatment and clinical improvement, this diffuse pattern of correlations remained. Conclusions: These results indicate differences in the neuronal interplay between regions in schizophrenic and healthy subjects. In nevertreated schizophrenics, diffuse interregional rCBF correlations can be seen as a sign of change and dysfunction of the systems regulating specificity and diversity of the neuronal functions. Neuroleptic therapy and psychopathologic remission showed no normalizing effect on interregional correlations. (orig.)

  19. Proliferative responses and binding properties of hematopoietic cells transfected with low-affinity receptors for leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor.

    OpenAIRE

    Gearing, D P; Ziegler, S F; Comeau, M R; Friend, D; Thoma, B; Cosman, D; Park, L.; Mosley, B

    1994-01-01

    Specific low-affinity receptors for leukemia inhibitory factor (LIF), oncostatin M (OSM; gp130), and ciliary neurotrophic factor (CNTF; receptor alpha, CNTFR alpha) may be utilized in various combinations to generate high-affinity binding sites and signal transduction. We have tested the ability of combinations of these receptors to transduce a proliferative signal in BAF-B03 cells. Coexpression of the LIF receptor and gp130 in these cells conferred high-affinity LIF and OSM binding and respo...

  20. An erythrocyte-specific DNA-binding factor recognizes a regulatory sequence common to all chicken globin genes

    International Nuclear Information System (INIS)

    The authors have identified a protein present only in erythroid cells that binds to two adjacent sites within an enhancer region of the chicken β-globin locus. Mutation of the sites, so that binding by the factor can no longer be detected in vitro, leads to a loss of enhancing ability, assayed by transient expression in primary erythrocytes. Binding sites for the erythroid-specific factor (Eryf1) are found within regulatory regions for all chicken globin genes. A strong Eryf1 binding site is also present within the enhancer of at least one human globin gene, and proteins from human erythroid cells (but not HeLa cells) bind to both the chicken and the human sites

  1. The sensitivity of CT and rCBF-studies for the pathology of strokes

    International Nuclear Information System (INIS)

    A localized pathology of the cerebral circulation or cerebral metabolism and the morphology of the brain tissue can be visualized by the use of new techniques in radiology and nuclear medicine: measurements of rCBF by radioactive diffusible isotopes, of the densitiy of the brain tissue, by CT, and of the cerebral metabolism regionally by positron-emission tomography. Having studied 475 stroke patients par rCBF measurements and 335 by CT we think it useful to compare the value of these two methods in revealing information on the pathology underlying focal disorders of brain functions due to stroke. (orig./VJ)

  2. A general pairwise interaction model provides an accurate description of in vivo transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Marc Santolini

    Full Text Available The identification of transcription factor binding sites (TFBSs on genomic DNA is of crucial importance for understanding and predicting regulatory elements in gene networks. TFBS motifs are commonly described by Position Weight Matrices (PWMs, in which each DNA base pair contributes independently to the transcription factor (TF binding. However, this description ignores correlations between nucleotides at different positions, and is generally inaccurate: analysing fly and mouse in vivo ChIPseq data, we show that in most cases the PWM model fails to reproduce the observed statistics of TFBSs. To overcome this issue, we introduce the pairwise interaction model (PIM, a generalization of the PWM model. The model is based on the principle of maximum entropy and explicitly describes pairwise correlations between nucleotides at different positions, while being otherwise as unconstrained as possible. It is mathematically equivalent to considering a TF-DNA binding energy that depends additively on each nucleotide identity at all positions in the TFBS, like the PWM model, but also additively on pairs of nucleotides. We find that the PIM significantly improves over the PWM model, and even provides an optimal description of TFBS statistics within statistical noise. The PIM generalizes previous approaches to interdependent positions: it accounts for co-variation of two or more base pairs, and predicts secondary motifs, while outperforming multiple-motif models consisting of mixtures of PWMs. We analyse the structure of pairwise interactions between nucleotides, and find that they are sparse and dominantly located between consecutive base pairs in the flanking region of TFBS. Nonetheless, interactions between pairs of non-consecutive nucleotides are found to play a significant role in the obtained accurate description of TFBS statistics. The PIM is computationally tractable, and provides a general framework that should be useful for describing and predicting

  3. Isolation of bovine corneal keratan sulfate and its growth factor and morphogen binding.

    Science.gov (United States)

    Weyers, Amanda; Yang, Bo; Solakyildirim, Kemal; Yee, Vienna; Li, Lingyun; Zhang, Fuming; Linhardt, Robert J

    2013-05-01

    Keratan sulfate (KS) is an important glycosaminoglycan that is found in cartilage, reproductive tissues, and neural tissues. Corneal KS glycosaminoglycan is found N-linked to lumican, keratocan and mimecan proteoglycans, and has been widely studied by investigators interested in corneal development and diseases. Recently, the availability of corneal KS has become severely limited, owing to restrictions on the shipment of bovine central nervous system byproducts across international borders in an effort to prevent additional cases of mad cow disease. We report a simple method for the purification of multi-milligram quantities of bovine corneal KS, and characterize its structural properties. We also examined its protein-binding properties, and discovered that corneal KS bound with high affinity to fibroblast growth factor-2 and sonic hedgehog, a growth factor and a morphogen involved in corneal development and healing. PMID:23402351

  4. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites

    DEFF Research Database (Denmark)

    Tornøe, Jens; Kusk, P.; Johansen, T.E.;

    2002-01-01

    fluorescent protein and secreted alkaline phosphatase reporter assays. By replacing sequences separating the transcription factor binding sites with randomized sequences of the same length, sets of new promoters with different strengths, spanning a 10-fold range of transcriptional activity in cell culture......The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C...... promoters. The JeT promoter was made by separating the included consensus boxes by the same distances in base pairs as found in the wild-type promoters, thus preserving transcription factor interaction. The resulting promoter was shown to drive reporter expression to high levels in enhanced green...

  5. Pluripotency factor binding and Tsix expression act synergistically to repress Xist in undifferentiated embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Nesterova Tatyana B

    2011-10-01

    Full Text Available Abstract Background Expression of Xist, the master regulator of X chromosome inactivation, is extinguished in pluripotent cells, a process that has been linked to programmed X chromosome reactivation. The key pluripotency transcription factors Nanog, Oct4 and Sox2 are implicated in Xist gene extinction, at least in part through binding to an element located in Xist intron 1. Other pathways, notably repression by the antisense RNA Tsix, may also be involved. Results Here we employ a transgene strategy to test the role of the intron 1 element and Tsix in repressing Xist in ES cells. We find that deletion of the intron 1 element causes a small increase in Xist expression and that simultaneous deletion of the antisense regulator Tsix enhances this effect. Conclusion We conclude that Tsix and pluripotency factors act synergistically to repress Xist in undifferentiated embryonic stem cells. Double mutants do not exhibit maximal levels of Xist expression, indicating that other pathways also play a role.

  6. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites

    DEFF Research Database (Denmark)

    Tornøe, Jens; Kusk, P.; Johansen, T.E.; Jensen, Peter Ruhdal

    The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C...... promoters. The JeT promoter was made by separating the included consensus boxes by the same distances in base pairs as found in the wild-type promoters, thus preserving transcription factor interaction. The resulting promoter was shown to drive reporter expression to high levels in enhanced green...... fluorescent protein and secreted alkaline phosphatase reporter assays. By replacing sequences separating the transcription factor binding sites with randomized sequences of the same length, sets of new promoters with different strengths, spanning a 10-fold range of transcriptional activity in cell culture...

  7. HOCOMOCO: expansion and enhancement of the collection of transcription factor binding sites models.

    Science.gov (United States)

    Kulakovskiy, Ivan V; Vorontsov, Ilya E; Yevshin, Ivan S; Soboleva, Anastasiia V; Kasianov, Artem S; Ashoor, Haitham; Ba-Alawi, Wail; Bajic, Vladimir B; Medvedeva, Yulia A; Kolpakov, Fedor A; Makeev, Vsevolod J

    2016-01-01

    Models of transcription factor (TF) binding sites provide a basis for a wide spectrum of studies in regulatory genomics, from reconstruction of regulatory networks to functional annotation of transcripts and sequence variants. While TFs may recognize different sequence patterns in different conditions, it is pragmatic to have a single generic model for each particular TF as a baseline for practical applications. Here we present the expanded and enhanced version of HOCOMOCO (http://hocomoco.autosome.ru and http://www.cbrc.kaust.edu.sa/hocomoco10), the collection of models of DNA patterns, recognized by transcription factors. HOCOMOCO now provides position weight matrix (PWM) models for binding sites of 601 human TFs and, in addition, PWMs for 396 mouse TFs. Furthermore, we introduce the largest up to date collection of dinucleotide PWM models for 86 (52) human (mouse) TFs. The update is based on the analysis of massive ChIP-Seq and HT-SELEX datasets, with the validation of the resulting models on in vivo data. To facilitate a practical application, all HOCOMOCO models are linked to gene and protein databases (Entrez Gene, HGNC, UniProt) and accompanied by precomputed score thresholds. Finally, we provide command-line tools for PWM and diPWM threshold estimation and motif finding in nucleotide sequences. PMID:26586801

  8. Insulin-like Growth Factor Binding Protein 7 Mediates Glioma Cell Growth and Migration

    Directory of Open Access Journals (Sweden)

    Wei Jiang

    2008-12-01

    Full Text Available Insulin-like growth factor binding protein 7 (IGFBP-7 is the only member of the IGFBP superfamily that binds strongly to insulin, suggesting that IGFBP-7 may have different functions from other IGFBPs. Unlike other IGFBPs, the expression and functions of IGFBP-7 in glioma tumors have not been reported. Using cDNA microarray analysis, we found that expression of IGFBP-7 correlated with the grade of glioma tumors and the overall patient survival. This finding was further validated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. We used RNAi to examine the role of IGFBP-7 in glioma cells, inhibiting IGFBP-7 expression by short interfering RNA transfection. Cell proliferation was suppressed after IGFBP-7 expression was inhibited for 5 days, and glioma cell growth was stimulated consistently by the addition of recombinant IGFBP-7 protein. Moreover, glioma cell migration was attenuated by IGFBP-7 depletion but enhanced by IGFBP-7 overexpression and addition. Overexpression of AKT1 in IGFBP-7-overxpressed cells attenuated the IGFBP-7-promoted migration and further enhanced inhibition of IGFBP-7 depletion on the migration. Phosphorylation of AKT and Erk1/2 was also inversely regulated by IGFBP-7 expression. These two factors together suggest that IGFBP-7 can regulate glioma cell migration through the AKT-ERK pathway, thereby playing an important role in glioma growth and migration.

  9. HOCOMOCO: expansion and enhancement of the collection of transcription factor binding sites models

    KAUST Repository

    Kulakovskiy, Ivan V.

    2015-11-19

    Models of transcription factor (TF) binding sites provide a basis for a wide spectrum of studies in regulatory genomics, from reconstruction of regulatory networks to functional annotation of transcripts and sequence variants. While TFs may recognize different sequence patterns in different conditions, it is pragmatic to have a single generic model for each particular TF as a baseline for practical applications. Here we present the expanded and enhanced version of HOCOMOCO (http://hocomoco.autosome.ru and http://www.cbrc.kaust.edu.sa/hocomoco10), the collection of models of DNA patterns, recognized by transcription factors. HOCOMOCO now provides position weight matrix (PWM) models for binding sites of 601 human TFs and, in addition, PWMs for 396 mouse TFs. Furthermore, we introduce the largest up to date collection of dinucleotide PWM models for 86 (52) human (mouse) TFs. The update is based on the analysis of massive ChIP-Seq and HT-SELEX datasets, with the validation of the resulting models on in vivo data. To facilitate a practical application, all HOCOMOCO models are linked to gene and protein databases (Entrez Gene, HGNC, UniProt) and accompanied by precomputed score thresholds. Finally, we provide command-line tools for PWM and diPWM threshold estimation and motif finding in nucleotide sequences.

  10. Nanopore current transduction analysis of protein binding to non-terminal and terminal DNA regions: analysis of transcription factor binding, retroviral DNA terminus dynamics, and retroviral integrase-DNA binding

    OpenAIRE

    Amin Iftekhar; Davis Amanda; Winters-Hilt Stephen; Morales Eric

    2007-01-01

    Abstract Background Synthetic transcription factors (STFs) promise to offer a powerful new therapeutic against Cancer, AIDS, and genetic disease. Currently, 10% of drugs are of this type, including salicylate and tamoxifen. STFs that can appropriately target (and release) their transcription factor binding sites on native genomic DNA provide a means to directly influence cellular mRNA production. An effective mechanism for screening amongst transcription factor (TF) candidates would itself be...

  11. Two cell surface proteins bind the sponge Microciona prolifera aggregation factor.

    Science.gov (United States)

    Varner, J A; Burger, M M; Kaufman, J F

    1988-06-15

    Two extracellular matrix cell surface proteins which bind the proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) and which may function as physiological receptors for MAF were identified and characterized for the first time. By probing nitrocellulose blots of nonreducing sodium dodecyl sulfate gels containing whole sponge cell protein with iodinated MAF, a 210- and a 68-kDa protein, which have native molecular masses of approximately 200-400 and 70 kDa, were identified. MAF binding to blots is species-specific. It is also sensitive to reduction and is completely abolished by pretreatment of live cells with proteases, as was cellular aggregation, indicating that the 210- and 68-kDa proteins may be located on the cell surface. The additional observations that the 68 kDa is an endoglycosidase F-sensitive glycoprotein and that antisera against whole sponge cells or membranes can immunoprecipitate the 210 kDa when prebound to intact cells are consistent with a cell surface location. Both proteins can be isolated from sponge cell membranes and from the sponge skeleton (insoluble extracellular matrix), but the 210-kDa MAF-binding protein can also be found in the soluble extracellular matrix (buffer washes of cells and skeleton) as well. A third MAF-binding protein of molecular mass 95 kDa was also found in the sponge extracellular matrix but rarely on cells. Both of the cell-associated 210- and 68-kDa proteins are nonintegral membrane proteins, based on Triton X-114 phase separation, flotation of liposomes containing sponge membrane lysates, and their extraction from membranes by buffer washes. Both proteins bind MAF affinity resins, indicating that they each exhibit a moderate affinity for MAF under native conditions. They can also be separated from each other and from the bulk of the protein in an octylpolyoxyethylene extract of membranes by fast protein liquid chromatography Mono Q anion exchange chromatography, as assessed by native

  12. Analysis of Specific Binding and Subcellular Localization of Wheat ERF Transcription Factor W17

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yun-xiang; LIU Pei; XU Zhao-shi; CHEN Ming; LI Lian-cheng; CHEN Yao-feng; XIONG Xiang-jin; MA You-zhi

    2008-01-01

    The study aims to detect the subcellular localization of ERF (ethylene-responsive element binding factor) transcription factor W17 protein, the interaction between W17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24h at 22℃ in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST (glutathione-S-transferase) in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 (DE3). IPTG (0.5mmol L-1) was added to induce the expression of recombinant GST/W17 for 3h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32P-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses (e. g., salt-) induced signaling pathway.

  13. Vitamin B12 Phosphate Conjugation and Its Effect on Binding to the Human B12 -Binding Proteins Intrinsic Factor and Haptocorrin.

    Science.gov (United States)

    Ó Proinsias, Keith; Ociepa, Michał; Pluta, Katarzyna; Chromiński, Mikołaj; Nexo, Ebba; Gryko, Dorota

    2016-06-01

    The binding of vitamin B12 derivatives to human B12 transporter proteins is strongly influenced by the type and site of modification of the cobalamin original structure. We have prepared the first cobalamin derivative modified at the phosphate moiety. The reaction conditions were fully optimized and its limitations examined. The resulting derivatives, particularly those bearing terminal alkyne and azide groups, were isolated and used in copper-catalyzed alkyne-azide cycloaddition reactions (CuAAC). Their sensitivity towards light revealed their potential as photocleavable molecules. The binding abilities of selected derivatives were examined and compared with cyanocobalamin. The interaction of the alkylated derivatives with haptocorrin was less affected than the interaction with intrinsic factor. Furthermore, the configuration of the phosphate moiety was irrelevant to the binding process. PMID:27120016

  14. CBF and CMRo2 during craniotomy for small supratentorial cerebral tumours in enflurane anaesthesia. A dose-response study

    International Nuclear Information System (INIS)

    In 14 patients with supratentorial cerebral tumours with midline shift ≤ 10 mm, cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRo2) were measured twice on the contralateral side of the craniotomy, using a modification of the Kety and Schmidt method. For induction of anaesthesia, thiopental, fentanyl and pancuronium were used. The anaesthesia was maintained with enflurane 1% in nitrous oxide 67%. Moderate hypocapnia to a level averaging 4.3 kPa was achieved. The patients were divided into two groups. In group 1 (n=7), 1% enflurane was used throughout the anaesthesia, and CBF and CMRo2 measured about 70 min after induction averaged 30.1 ml 100 g-1 min-1 and 1.98 ml O2 100 g-1 min-1, respectively. During the second CBF study 1 h later, CBF and CMRo2 were unchanged (P>0.05). In group 2 (n=7), the inspiratory enflurane concentration was increased from 1 to 2% after the first CBF measurement. In this group a significant decrease in CMRo2 was observed, while CBF was unchanged. In six patients EEG was recorded simultaneously with the CBF measurements. In patients subjected to increasing enflurane concentration (Group 2), a suppression in the EEG activity was observed without spike waves. It is concluded that enflurane induces a dose-related decrease in CMRo2 and suppression in the EEG activity, whereas CBF was unchanged (author)

  15. Lyso-Sulfatide Binds Factor Xa and Inhibits Thrombin Generation by the Prothrombinase Complex.

    Directory of Open Access Journals (Sweden)

    Subramanian Yegneswaran

    Full Text Available Blood coagulation reactions are strongly influenced by phospholipids, but little is known about the influence of sphingolipids on coagulation mechanisms. Lysosulfatide (lyso-SF (sulfogalactosyl sphingosine prolonged factor Xa (fXa 1-stage plasma clotting assays, showing it had robust anticoagulant activity. In studies using purified clotting factors, lyso-SF inhibited >90% of prothrombin (II activation for reaction mixtures containing fXa/factor Va (fVa/II, and also inhibited II activation generation by fXa/ phospholipids and by Gla-domainless-fXa/fVa/phospholipids. When lyso-SF analogs were tested, results showed that N-acetyl-sulfatide was not anticoagulant, implying that the free amine group was essential for the anticoagulant effects of lyso-SF. Lyso-SF did not inhibit fXa enzymatic hydrolysis of small peptide substrates, showing it did not directly inhibit the fXa activity. In surface plasmon resonance studies, lyso-SF bound to immobilized inactivated fXa as well as inactivated Gla-domainless-fXa. Confirming this lyso-SF:fXa interaction, fluorescence studies showed that fluorescently-labeled-fXa in solution bound to lyso-SF. Thus, lyso-SF is an anticoagulant lipid that inhibits fXa when this enzyme is bound to either phospholipids or to fVa. Mechanisms for inhibition of procoagulant activity are likely to involve lyso-SF binding to fXa domain(s that are distinct from the fXa Gla domain. This suggests that certain sphingolipids, including lyso-SF and some of its analogs, may down-regulate fXa activity without inhibiting the enzyme's active site or binding to the fXa Gla domain.

  16. A functional YY1 binding site is necessary and sufficient to activate Surf-1 promoter activity in response to serum growth factors.

    OpenAIRE

    Cole, E G; Gaston, K

    1997-01-01

    The human Surf-1 and Surf-2 housekeeping genes are divergently transcribed and share a bi-directional, TATA-less promoter. Housekeeping promoters typically contain complex arrays of transcription factor binding sites and several studies have suggested that many of these sites might be functionally redundant. The Surf-1/Surf-2 promoter region contains four factor binding sites; members of the ETS family of transcription factors bind to two of these sites whilst YY1 binds to a third site immedi...

  17. Structure of the active core of human stem cell factor and analysis of binding to its receptor Kit

    OpenAIRE

    Jiang, Xuliang; Gurel, Ogan; Mendiaz, Elizabeth A.; Stearns, George W.; Clogston, Christi L.; Lu, Hsieng S; Osslund, Timothy D.; Syed, Rashid S.; Langley, Keith E.; Hendrickson, Wayne A

    2000-01-01

    Stem cell factor (SCF) is an early-acting hematopoietic cytokine that elicits multiple biological effects. SCF is dimeric and occurs in soluble and membrane-bound forms. It transduces signals by ligand- mediated dimerization of its receptor, Kit, which is a receptor tyrosine kinase related to the receptors for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF). All of these have extracellular ligand-binding p...

  18. Monitoring CBF in clinical routine by dynamic single photon emission tomography (SPECT) of inhaled xenon-133

    DEFF Research Database (Denmark)

    Sugiyama, H; Christensen, J; Skyhøj Olsen, T;

    1986-01-01

    A very simple and low-cost brain dedicated, rapidly rotating Single Photon Emission Tomograph SPECT is described. Its use in following patients with ischemic stroke is illustrated by two middle cerebral artery occlusion cases, one with persistent occlusion and low CBF in MCA territory, and one with...

  19. CDNA cloning and mRNA expression of the six mouse insulin-like growth factor binding proteins

    NARCIS (Netherlands)

    A.G.P. Schuller (Alwin); C. Groffen (Cora); J.W. van Neck (Han); E.C. Zwarthoff (Ellen); S.L.S. Drop (Stenvert)

    1994-01-01

    textabstractThe insulin-like growth factor binding proteins (IGFBPs) comprise a family of six distinct proteins which modulate insulin-like growth factor action. We have isolated cDNAs encoding the six mouse IGFBPs (mIGFBPs). In addition, we studied the mRNA expression of the six mIGFBPs during deve

  20. Gene regulation knowledge commons: community action takes care of DNA binding transcription factors.

    Science.gov (United States)

    Tripathi, Sushil; Vercruysse, Steven; Chawla, Konika; Christie, Karen R; Blake, Judith A; Huntley, Rachael P; Orchard, Sandra; Hermjakob, Henning; Thommesen, Liv; Lægreid, Astrid; Kuiper, Martin

    2016-01-01

    A large gap remains between the amount of knowledge in scientific literature and the fraction that gets curated into standardized databases, despite many curation initiatives. Yet the availability of comprehensive knowledge in databases is crucial for exploiting existing background knowledge, both for designing follow-up experiments and for interpreting new experimental data. Structured resources also underpin the computational integration and modeling of regulatory pathways, which further aids our understanding of regulatory dynamics. We argue how cooperation between the scientific community and professional curators can increase the capacity of capturing precise knowledge from literature. We demonstrate this with a project in which we mobilize biological domain experts who curate large amounts of DNA binding transcription factors, and show that they, although new to the field of curation, can make valuable contributions by harvesting reported knowledge from scientific papers. Such community curation can enhance the scientific epistemic process.Database URL: http://www.tfcheckpoint.org. PMID:27270715

  1. ECRbase: Database of Evolutionary Conserved Regions, Promoters, and Transcription Factor Binding Sites in Vertebrate Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Loots, G; Ovcharenko, I

    2006-08-08

    Evolutionary conservation of DNA sequences provides a tool for the identification of functional elements in genomes. We have created a database of evolutionary conserved regions (ECRs) in vertebrate genomes entitled ECRbase that is constructed from a collection of pairwise vertebrate genome alignments produced by the ECR Browser database. ECRbase features a database of syntenic blocks that recapitulate the evolution of rearrangements in vertebrates and a collection of promoters in all vertebrate genomes presented in the database. The database also contains a collection of annotated transcription factor binding sites (TFBS) in all ECRs and promoter elements. ECRbase currently includes human, rhesus macaque, dog, opossum, rat, mouse, chicken, frog, zebrafish, and two pufferfish genomes. It is freely accessible at http://ECRbase.dcode.org.

  2. [Expression and Clinical Significance of Lymphoid Enhancer-Binding Factor 1 in Acute Leukemias].

    Science.gov (United States)

    Huo, Wan-Ying; Gao, Ju

    2015-06-01

    Lymphoid enhancer-binding factor 1 (LEF1), a key downstream effector of Wnt/β-catenin signal transduction pathway, plays a crucial role in the maintenance, proliferation and differentiation of normal hematopoietic stem/progenitor cells through regulating the transcription of its target genes. Aberrant LEF1 expression has been documented in a variety of leukemias, and implicated in the prediction of prognosis. Nevertheless, discrepancies exist regarding the expression level and clinical implication of LEF1 in different types of leukemias, suggesting LEF1 might exert distinct roles in different types of leukemia. In the present article, recent research advances of the relationship of LEF1 and regulation of hematopoiesis and leukemogenesis are reviewed. PMID:26117056

  3. A Nonradioactive Method for Detecting DNA-binding Activity of Nuclear Transcription Factors

    Institute of Scientific and Technical Information of China (English)

    张宁; 徐永健; 张珍祥; 熊维宁

    2003-01-01

    To determine the feasibility of a nonradioactive electrophoresis mobility shift assay fordetecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus tar-get sequence of NF-κB were labled with DIG by terminal transferase. After nuclear protein stimula-ted with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDT C)electrophoresed on 8 % nondenaturing poliacrylamide gel together with oligeonucleotide probe, theywere electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemilu-minescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded spe-cifically to the NF-κB consensus sequence in the EMSA by chemiluminescent technique method andthe activity of NF-κB in PMA group was more than that in PMA+PDTC group. It is suggestedthat detection of NF-κB by EMSA with chemiluminescent technique is feasible and simple, whichcan be performed in ordinary laboratories.

  4. Acute effects of electroconvulsive therapy on regional cerebral blood flow (rCBF) in psychiatric disorders

    International Nuclear Information System (INIS)

    Electroconvulsive therapy (ECT) is frequently used in the treatment of major depression and other psychiatric disorders; its mechanism of action is not established, but previous evidence suggests that it is associated with postictal metabolic suppression. The authors have used measurements of rCBF as an index of cortical metabolic activity to study the acute effects of ECT. Measurements of rCBF were made in 32 cortical regions in 10 patients (pts) following one minute breathing of Xe-133 (5mCi/L); the measurements were performed 30min before and 50min after ECT. Bilateral ECT was administered to six pts (five diagnosed as major depressives and one schizophrenic) and unilateral ECT to four (all diagnosed as unipolar or bipolar affective disorder). The total rCBF material consists of 52 measurements in these pts, made before and after 16 bilateral and 10 unilateral treatments. ECT was found to cause significant reduction of rCBF. Mean hemispheric flows (using the Initial Slope Index to measure grey-matter flow) were reduced by about 5% in both hemispheres following bilateral treatment. Unilateral treatment caused a 9% reduction of flow in the treated hemisphere, but only 2% contralaterally. Regional patterns of flow decreases also differed between the two treatment modes: bilateral frontal reductions were found after bilateral treatment, whereas unilateral ECT caused a widespread flow reduction in the treated hemisphere, and almost no effect contralaterally. These results suggest that rCBF studies are useful for assessing ECT, and indicate that the acute cerebral effects of ECT vary with the mode of treatment

  5. Expression and Critical Role of Interleukin Enhancer Binding Factor 2 in Hepatocellular Carcinoma

    Science.gov (United States)

    Cheng, Shaobing; Jiang, Xu; Ding, Chaofeng; Du, Chengli; Owusu-Ansah, Kwabena Gyabaah; Weng, Xiaoyu; Hu, Wendi; Peng, Chuanhui; Lv, Zhen; Tong, Rongliang; Xiao, Heng; Xie, Haiyang; Zhou, Lin; Wu, Jian; Zheng, Shusen

    2016-01-01

    Interleukin enhancer binding factor 2 (ILF2), a transcription factor, regulates cell growth by inhibiting the stabilization of mRNA. Currently, its role has gained recognition as a factor in the tumorigenic process. However, until now, little has been known about the detailed role ILF2 plays in hepatocellular carcinoma (HCC). In this study, we investigated the expression levels of ILF2 in HCC tissue with Western blot and immunohistochemical assays. To examine the effect of ILF2 on liver cancer cell growth and apoptosis, small interfering RNAs (siRNAs) targeting ILF2 were recombined to create lentiviral overexpression vectors. Our results showed higher expression levels of ILF2 mRNA and ILF2 protein in HCC tissue compared with matched peritumoral tissue. Expression of ILF2 may regulate cell growth and apoptosis in liver cancer cells via regulation of B-cell lymphoma 2 (Bcl-2), Bcl-2 related ovarian killer (Bok), Bcl-2-associated X protein (BAX), and cellular inhibitor of apoptosis 1 (cIAP1). Moreover, we inoculated nude mice with liver cancer cells to investigate the effect of ILF2 on tumorigenesis in vivo. As expected, a rapid growth was observed in cancer cells inoculated with a lentiviral vector coding Flag-ILF2 (Lenti-ILF2) compared with the control cells. Hence, these results promote a better understanding of ILF2’s potential role as a therapeutic target in HCC. PMID:27556459

  6. Insulin-like growth factor II: complexity of biosynthesis and receptor binding

    DEFF Research Database (Denmark)

    Gammeltoft, S; Christiansen, Jan; Nielsen, F C;

    1991-01-01

    Insulin-like growth factor II (IGF-II) belongs to the insulin family of peptides and acts as a growth factor in many fetal tissues and tumors. The gene expression of IGF-II is initiated at three different promoters which gives rise to multiple transcripts. In a human rhabdomyosarcoma cell line IN...... the 4.8-kb mRNA is translated to IGF-II. The cell line secretes two forms of immunoreactive and bioactive IGF-II to the medium of molecular size 10 kd and 7.5 kd which may be involved in autocrine control of cell growth. IGF-II binds to two receptors on the surface of many cell types: the IGF...... types, however, Man-6-P induces cellular responses. We have studied rat brain neuronal precursor cells where Man-6-P acted as a mitogen suggesting that phosphomannosylated proteins may act as growth factors via the Man-6-P/IGF-II receptor. In conclusion, the gene expression and mechanism of action of...

  7. In silico cloning and characterization of the TGA (TGACG MOTIF-BINDING FACTOR) transcription factors subfamily in Carica papaya.

    Science.gov (United States)

    Idrovo Espín, Fabio Marcelo; Peraza-Echeverria, Santy; Fuentes, Gabriela; Santamaría, Jorge M

    2012-05-01

    The TGA transcription factors belong to the subfamily of bZIP group D that play a major role in disease resistance and development. Most of the TGA identified in Arabidopsis interact with the master regulator of SAR, NPR1 that controls the expression of PR genes. As a first approach to determine the possible involvement of these transcription factors in papaya defense, we characterized Arabidopsis TGA orthologs from the genome of Carica papaya cv. SunUp. Six orthologs CpTGA1 to CpTGA6, were identified. The predicted CpTGA proteins were highly similar to AtTGA sequences and probably share the same DNA binding properties and transcriptional regulation features. The protein sequences alignment evidenced the presence of conserved domains, characteristic of this group of transcription factors. The phylogeny showed that CpTGA evolved into three different subclades associated with defense and floral development. This is the first report of basal expression patterns assessed by RT-PCR, from the whole subfamily of CpTGA members in different tissues from papaya cv. Maradol mature plants. Overall, CpTGA1, CpTGA3 CpTGA6 and CpTGA4 showed a basal expression in all tissues tested; CpTGA2 expressed strongly in all tissues except in petioles while CpTGA5 expressed only in petals and to a lower extent in petioles. Although more detailed studies in anthers and other floral structures are required, we suggest that CpTGA5 might be tissue-specific, and it might be involved in papaya floral development. On the other hand, we report here for the first time, the expression of the whole family of CpTGA in response to salicylic acid (SA). The expression of CpTGA3, CpTGA4 and CpTGA6 increased in response to SA, what would suggest its involvement in the SAR response in papaya. PMID:22410205

  8. Nuclear actions of insulin-like growth factor binding protein-3.

    Science.gov (United States)

    Baxter, Robert C

    2015-09-10

    In addition to its actions outside the cell, cellular uptake and nuclear import of insulin-like growth factor binding protein-3 (IGFBP-3) has been recognized for almost two decades, but knowledge of its nuclear actions has been slow to emerge. IGFBP-3 has a functional nuclear localization signal and interacts with the nuclear transport protein importin-β. Within the nucleus IGFBP-3 appears to have a role in transcriptional regulation. It can bind to the nuclear receptor, retinoid X receptor-α and several of its dimerization partners, including retinoic acid receptor, vitamin D receptor (VDR), and peroxisome proliferator-activated receptor-γ (PPARγ). These interactions modulate the functions of these receptors, for example inhibiting VDR-dependent transcription in osteoblasts and PPARγ-dependent transcription in adipocytes. Nuclear IGFBP-3 can be detected by immunohistochemistry in cancer and other tissues, and its presence in the nucleus has been shown in many cell culture studies to be necessary for its pro-apoptotic effect, which may also involve interaction with the nuclear receptor Nur77, and export from the nucleus. IGFBP-3 is p53-inducible and in response to DNA damage, forms a complex with the epidermal growth factor receptor (EGFR), translocating to the nucleus to interact with DNA-dependent protein kinase. Inhibition of EGFR kinase activity or downregulation of IGFBP-3 can inhibit DNA double strand-break repair by nonhomologous end joining. IGFBP-3 thus has the ability to influence many cell functions through its interactions with intranuclear pathways, but the importance of these interactions in vivo, and their potential to be targeted for therapeutic benefit, require further investigation. PMID:26074086

  9. Emerging role of insulin-like growth factor-binding protein 7 in hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Akiel M

    2014-03-01

    Full Text Available Maaged Akiel, Devaraja Rajasekaran, Rachel Gredler, Ayesha Siddiq, Jyoti Srivastava, Chadia Robertson, Nidhi Himanshu Jariwala, Paul B Fisher, Devanand SarkarDepartment of Human and Molecular Genetics, Massey Cancer Center, VCU Institute of Molecular Medicine, Virginia Commonwealth University, Richmond, Virginia, USAAbstract: Hepatocellular carcinoma (HCC is a vicious and highly vascular cancer with a dismal prognosis. It is a life-threatening illness worldwide that ranks fifth in terms of cancer prevalence and third in cancer deaths. Most patients are diagnosed at an advanced stage by which time conventional therapies are no longer effective. Targeted molecular therapies, such as the multikinase inhibitor sorafenib, provide a modest increase in survival for advanced HCC patients and display significant toxicity. Thus, there is an immense need to identify novel regulators of HCC that might be targeted effectively. The insulin-like growth factor (IGF axis is commonly abnormal in HCC. Upon activation, the IGF axis controls metabolism, tissue homeostasis, and survival. Insulin-like growth factor-binding protein 7 (IGFBP7 is a secreted protein of a family of low-affinity IGF-binding proteins termed “IGFBP-related proteins” that have been identified as a potential tumor suppressor in HCC. IGFBP7 has been implicated in regulating cellular proliferation, senescence, and angiogenesis. In this review, we provide a comprehensive discussion of the role of IGFBP7 in HCC and the potential use of IGFBP7 as a novel biomarker for drug resistance and as an effective therapeutic strategy.Keywords: HCC, IGFBP7, gene therapy, angiogenesis, senescence, apoptosis

  10. Brain binding sites for atrial natriuretic factor (ANF): alterations in prehypertensive Dahl salt-sensitive (S/JR) rats

    International Nuclear Information System (INIS)

    The binding of radioiodinated atrial natriuretic factor (125I-ANF-28) to discrete areas of brain in 7 week old, inbred Dahl salt-sensitive (S/JR) and salt-resistant (R/JR) rats was studied utilizing quantitative film autoradiography. At this age, S/JR rats exhibit systolic blood pressures that are prehypertensive and tend to be slightly higher than systolic blood pressures of age-matched R/JR rats. Scatchard analysis of 125I-ANF-28 binding in forebrain revealed that S/JR rats have a significantly increased number of binding sites for 125I-ANF-28 in the subfornical organ as compared to R/JR controls. In contrast, values for 125I-ANF-28 binding capacity in the choroid plexus and area postrema were similar for both strains, and binding affinity constants for 125I-ANF-28 binding revealed no strain differences in any brain area examined. The elevation in the number of binding sites for atrial natriuretic factor may serve as a compensatory mechanism acting in part to lower fluid volume and sodium levels prior to the precipitous increase in blood pressure which occurs in S/JR rats by 10 weeks of age

  11. Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor.

    OpenAIRE

    Kiefer, M C; Stephans, J C; Crawford, K; Okino, K.; Barr, P.J.

    1990-01-01

    Expression cloning of cDNAs encoding a basic fibroblast growth factor (FGF) binding protein confirms previous hypotheses that this molecule is a cell-surface heparan sulfate proteoglycan. A cDNA library constructed from a hamster kidney cell line rich in FGF receptor activity was transfected into a human lymphoblastoid cell line. Clones expressing functional basic FGF binding proteins at their surfaces were enriched by panning on plastic dishes coated with human basic FGF. The amino acid sequ...

  12. The HMG-box mitochondrial transcription factor xl-mtTFA binds DNA as a tetramer to activate bidirectional transcription.

    OpenAIRE

    Antoshechkin, I; Bogenhagen, D F; Mastrangelo, I A

    1997-01-01

    The mitochondrial HMG-box transcription factor xl-mtTFA activates bidirectional transcription by binding to a site separating two core promoters in Xenopus laevis mitochondrial DNA (mtDNA). Three independent approaches were used to study the higher order structure of xl-mtTFA binding to this site. First, co-immunoprecipitation of differentially tagged recombinant mtTFA derivatives established that the protein exists as a multimer. Second, in vitro chemical cross-linking experiments provided e...

  13. The bidirectional upstream element of the adenovirus-2 major late promoter binds a single monomeric molecule of the upstream factor.

    OpenAIRE

    Lennard, A C; Egly, J M

    1987-01-01

    The adenovirus-2 major late promoter (Ad2MLP) upstream element (Ad2MLP-UE) contains a sequence of interrupted dyad symmetry. By inverting this element we have found that it functions in a bidirectional manner both in vivo and in vitro. Footprinting and binding kinetics studies have demonstrated that both orientations of the upstream element bind the sequence-specific upstream factor (UEF) in a similar fashion. These data strongly suggest that the dyad symmetric sequence is sufficient for full...

  14. The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity

    OpenAIRE

    Cicero, Marco P.; T. Hubl, Susan; Harrison, Celia J.; Littlefield, Otis; Hardy, Jeanne A.; Nelson, Hillary C. M.

    2001-01-01

    The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins. HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively. Unlike other winged helix–turn–helix proteins, HSF’s wing does not appear to contact DNA, as based on a previously solved crystal structure. Instead, the structure implies that the wing is involved in protein–protein interactions, possibly within a trimer or between adjacent trimers. To unders...

  15. Nuclear Factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA

    OpenAIRE

    Jayachandran, Uma; Grey, Heather; Cook, Atlanta

    2016-01-01

    Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the posttranscriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3' untranslated regions of specific mRNAs and several noncoding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tande...

  16. Aggravation of post-ischemic liver injury by overexpression of insulin-like growth factor binding protein 3

    OpenAIRE

    Lu Zhou; Hyoung-Won Koh; Ui-Jin Bae; Byung-Hyun Park

    2015-01-01

    Insulin-like growth factor-1 (IGF-1) is known to inhibit reperfusion-induced apoptosis. IGF-binding protein-3 (IGFBP-3) is the major circulating carrier protein for IGF-1 and induces apoptosis. In this study, we determined if IGFBP-3 was important in the hepatic response to I/R. To deliver IGFBP-3, we used an adenovirus containing IGFBP-3 cDNA (AdIGFBP-3) or an IGFBP-3 mutant devoid of IGF binding affinity but retaining IGFBP-3 receptor binding ability (AdIGFBP-3GGG). Mice subjected to I/R in...

  17. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA

    OpenAIRE

    Smith, Janet L.; Grossman, Alan D.

    2015-01-01

    DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep ...

  18. Nuclear factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA

    OpenAIRE

    Jayachandran, Uma; Grey, Heather; Cook, Atlanta

    2015-01-01

    Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3′ untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tan...

  19. A general integrative genomic feature transcription factor binding site prediction method applied to analysis of USF1 binding in cardiovascular disease

    Directory of Open Access Journals (Sweden)

    Wang Tianyuan

    2009-04-01

    Full Text Available Abstract Transcription factors are key mediators of human complex disease processes. Identifying the target genes of transcription factors will increase our understanding of the biological network leading to disease risk. The prediction of transcription factor binding sites (TFBSs is one method to identify these target genes; however, current prediction methods need improvement. We chose the transcription factor upstream stimulatory factor l (USF1 to evaluate the performance of our novel TFBS prediction method because of its known genetic association with coronary artery disease (CAD and the recent availability of USF1 chromatin immunoprecipitation microarray (ChIP-chip results. The specific goals of our study were to develop a novel and accurate genome-scale method for predicting USF1 binding sites and associated target genes to aid in the study of CAD. Previously published USF1 ChIP-chip data for 1 per cent of the genome were used to develop and evaluate several kernel logistic regression prediction models. A combination of genomic features (phylogenetic conservation, regulatory potential, presence of a CpG island and DNaseI hypersensitivity, as well as position weight matrix (PWM scores, were used as variables for these models. Our most accurate predictor achieved an area under the receiver operator characteristic curve of 0.827 during cross-validation experiments, significantly outperforming standard PWM-based prediction methods. When applied to the whole human genome, we predicted 24,010 USF1 binding sites within 5 kilobases upstream of the transcription start site of 9,721 genes. These predictions included 16 of 20 genes with strong evidence of USF1 regulation. Finally, in the spirit of genomic convergence, we integrated independent experimental CAD data with these USF1 binding site prediction results to develop a prioritised set of candidate genes for future CAD studies. We have shown that our novel prediction method, which employs

  20. The isolation of transcription factors from lambda gt11 cDNA expression libraries: human steroid 5 alpha-reductase 1 has sequence-specific DNA binding activity.

    OpenAIRE

    Gaston, K; Fried, M

    1992-01-01

    The Surf-1/Surf-2 bi-directional promoter contains binding sites for at least three transcription factors (Su1, Su2, and Su3). By screening a lambda gt11 HeLa cell cDNA expression library with a concatenated Su2 factor binding site, we isolated a cDNA which encodes a protein with sequence-specific DNA binding activity. Gel retardation assays showed that the cloned factor binds specifically to the Su2 factor binding site present in the human Surf-1/Surf-2 promoter but not to an Su2 site contai...

  1. Oxidative stress effect on progesterone-induced blocking factor (PIBF) binding to PIBF-receptor in lymphocytes.

    Science.gov (United States)

    de la Haba, Carlos; Palacio, José R; Palkovics, Tamas; Szekeres-Barthó, Júlia; Morros, Antoni; Martínez, Paz

    2014-01-01

    Receptor-ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor-ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor-ligand binding and reduces lipid raft clustering. PMID:23954806

  2. JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles.

    Science.gov (United States)

    Mathelier, Anthony; Fornes, Oriol; Arenillas, David J; Chen, Chih-Yu; Denay, Grégoire; Lee, Jessica; Shi, Wenqiang; Shyr, Casper; Tan, Ge; Worsley-Hunt, Rebecca; Zhang, Allen W; Parcy, François; Lenhard, Boris; Sandelin, Albin; Wasserman, Wyeth W

    2016-01-01

    JASPAR (http://jaspar.genereg.net) is an open-access database storing curated, non-redundant transcription factor (TF) binding profiles representing transcription factor binding preferences as position frequency matrices for multiple species in six taxonomic groups. For this 2016 release, we expanded the JASPAR CORE collection with 494 new TF binding profiles (315 in vertebrates, 11 in nematodes, 3 in insects, 1 in fungi and 164 in plants) and updated 59 profiles (58 in vertebrates and 1 in fungi). The introduced profiles represent an 83% expansion and 10% update when compared to the previous release. We updated the structural annotation of the TF DNA binding domains (DBDs) following a published hierarchical structural classification. In addition, we introduced 130 transcription factor flexible models trained on ChIP-seq data for vertebrates, which capture dinucleotide dependencies within TF binding sites. This new JASPAR release is accompanied by a new web tool to infer JASPAR TF binding profiles recognized by a given TF protein sequence. Moreover, we provide the users with a Ruby module complementing the JASPAR API to ease programmatic access and use of the JASPAR collection of profiles. Finally, we provide the JASPAR2016 R/Bioconductor data package with the data of this release. PMID:26531826

  3. Upstream binding factor stabilizes Rib 1, the TATA-binding-protein-containing Xenopus laevis RNA polymerase I transcription factor, by multiple protein interactions in a DNA-independent manner.

    OpenAIRE

    Bodeker, M; Cairns, C; McStay, B

    1996-01-01

    Initiation of RNA polymerase I transcription in Xenopus laevis requires Rib 1 and upstream binding factor (UBF). UBF and Rib 1 combine to form a stable transcription complex on the Xenopus ribosomal gene promoter. Here we show that Rib 1 comprises TATA-binding protein (TBP) and TBP-associated factor components. Thus, Rib 1 is the Xenopus equivalent of mammalian SL 1. In contrast to SL 1, Rib 1 is an unstable complex that readily dissociates into TBP and associated components. We identify a no...

  4. Characterization of the DNA-binding properties of the Mohawk homeobox transcription factor.

    Science.gov (United States)

    Anderson, Douglas M; George, Rajani; Noyes, Marcus B; Rowton, Megan; Liu, Wenjin; Jiang, Rulang; Wolfe, Scot A; Wilson-Rawls, Jeanne; Rawls, Alan

    2012-10-12

    The homeobox transcription factor Mohawk (Mkx) is a potent transcriptional repressor expressed in the embryonic precursors of skeletal muscle, cartilage, and bone. MKX has recently been shown to be a critical regulator of musculoskeletal tissue differentiation and gene expression; however, the genetic pathways through which MKX functions and its DNA-binding properties are currently unknown. Using a modified bacterial one-hybrid site selection assay, we determined the core DNA-recognition motif of the mouse monomeric Mkx homeodomain to be A-C-A. Using cell-based assays, we have identified a minimal Mkx-responsive element (MRE) located within the Mkx promoter, which is composed of a highly conserved inverted repeat of the core Mkx recognition motif. Using the minimal MRE sequence, we have further identified conserved MREs within the locus of Sox6, a transcription factor that represses slow fiber gene expression during skeletal muscle differentiation. Real-time PCR and immunostaining of in vitro differentiated muscle satellite cells isolated from Mkx-null mice revealed an increase in the expression of Sox6 and down-regulation of slow fiber structural genes. Together, these data identify the unique DNA-recognition properties of MKX and reveal a novel role for Mkx in promoting slow fiber type specification during skeletal muscle differentiation. PMID:22923612

  5. Pipeline for Efficient Mapping of Transcription Factor Binding Sites and Comparison of Their Models

    KAUST Repository

    Ba alawi, Wail

    2011-06-01

    The control of genes in every living organism is based on activities of transcription factor (TF) proteins. These TFs interact with DNA by binding to the TF binding sites (TFBSs) and in that way create conditions for the genes to activate. Of the approximately 1500 TFs in human, TFBSs are experimentally derived only for less than 300 TFs and only in generally limited portions of the genome. To be able to associate TF to genes they control we need to know if TFs will have a potential to interact with the control region of the gene. For this we need to have models of TFBS families. The existing models are not sufficiently accurate or they are too complex for use by ordinary biologists. To remove some of the deficiencies of these models, in this study we developed a pipeline through which we achieved the following: 1. Through a comparison analysis of the performance we identified the best models with optimized thresholds among the four different types of models of TFBS families. 2. Using the best models we mapped TFBSs to the human genome in an efficient way. The study shows that a new scoring function used with TFBS models based on the position weight matrix of dinucleotides with remote dependency results in better accuracy than the other three types of the TFBS models. The speed of mapping has been improved by developing a parallelized code and shows a significant speed up of 4x when going from 1 CPU to 8 CPUs. To verify if the predicted TFBSs are more accurate than what can be expected with the conventional models, we identified the most frequent pairs of TFBSs (for TFs E4F1 and ATF6) that appeared close to each other (within the distance of 200 nucleotides) over the human genome. We show unexpectedly that the genes that are most close to the multiple pairs of E4F1/ATF6 binding sites have a co-expression of over 90%. This indirectly supports our hypothesis that the TFBS models we use are more accurate and also suggests that the E4F1/ATF6 pair is exerting the

  6. Usefulness of cerebral blood flow (CBF) measurements to predict the functional outcome for rehabilitation in patients with cerebrovascular disease (CVD)

    International Nuclear Information System (INIS)

    The objectives of this study were to elucidate the relationship between the mean CBF in the whole brain (Av.mCBF) before rehabilitation of CVD patients and the BI score before and after rehabilitation, determine whether the efficacy of rehabilitation can be predicted by measurement of the Av.mCBF, and investigate what part of the brain was most important to improving the BI score. The Av.mCBFs in 160 patients with CVD were calculated by Patlak plots with 99mTc-HMPAO before rehabilitation, and we determined the BI score before and after rehabilitation. Based on the BI scores before and after rehabilitation, patients were divided into four groups: Group A, BI=100; Group B, 80≤BI≤99; Group C, 60≤BI≤79; Group D, 0≤BI≤59. We evaluated the relationship between the Av.mCBF and BI score before and after rehabilitation. The Av.mCBF before rehabilitation showed a tendency to be more correlated with the BI score after rehabilitation (r=0.414, p<0.0001) than before rehabilitation (r=0.272, p=0.0006). After rehabilitation, there was a tendency for the Av.mCBF value to increase in direct proportion to the BI score of the group: it was highest in Group A and lowest in Group D. The strongest correlation was found between the frontal lobe regional CBF before rehabilitation and the BI score after rehabilitation (r=0.343, p<0.0001). Measurement of the Av.mCBF before rehabilitation of CVD patients will permit prediction of the efficacy of rehabilitation. Also the regional CBF of the frontal lobe is most important for improving the BI score. (author)

  7. Memory functions and rCBF 99mTc-HMPAO SPET: developing diagnostics in Alzheimer's disease

    International Nuclear Information System (INIS)

    Alzheimer's disease (AD) is a primary degenerative disease of the brain. The prevalence increases with age, with devastating consequences for the individual and society. The aim of this study was to evaluate whether patients with early AD show an altered regional cerebral blood flow (rCBF) compared with control persons. Furthermore, we aimed to investigate the correlation between rCBF in sublobar volumes of the brain and performance on memory tests. Memory tests were chosen to evaluate episodic and semantic memory. Fourteen patients (aged 75.2±8.8 years) with early AD and 15 control persons (aged 71.4±3.2 years) were included. rCBF measurements with single-photon emission tomography (SPET) using technetium-99m hexamethylpropylene amine oxime (HMPAO) were performed. The rCBF 99mTc-HMPAO SPET images were spatially transformed to fit a brain atlas and normalised for differences in rCBF (Computerised Brain Atlas software). Cortical and subcortical volumes of interest (VOIs) were analysed and compared. Compared with the controls, AD patients showed a significantly lower rCBF ratio in temporoparietal regions, including the left hippocampus. The diagnostic sensitivity and specificity for AD were high in temporoparietal regions. AD patients had significantly reduced performance on semantic and, in particular, episodic memory tests compared with age-matched normative data, and their performance on several episodic tests correlated with rCBF ratios in parietal and temporal regions, including the left hippocampus. The correlation between rCBF ratio and level of episodic memory performance suggests that abnormalities in rCBF pattern underlie impaired episodic memory functioning in AD. (orig.)

  8. Memory functions and rCBF-99mTc-HMPAO-SPECT: Developing diagnostics in Alzheimer's Disease

    International Nuclear Information System (INIS)

    Alzheimer's disease (AD) is a primary degenerative disease of the brain. The prevalence increases with age with devastating consequences for the individual and for the society. The aim of this study was to evaluate if patients with early AD show an altered regional cerebral blood flow (rCBF) compared to control persons. The aim was furthermore to investigate the correlation between rCBF in sub-lobar volumes of the brain and performance on memory tests. Memory tests were chosen to evaluate episodic and semantic memory. Fourteen patients (75.2±8.8 yrs) with early AD, and 15 control persons (71.4±3.2 yrs) were included. rCBF measurements with single photon emission computerized tomography (SPECT) using 99mTc-hexamethyl propylenamine oxime (HMPAO) were performed. The rCBF-99mTc-HMPAO-SPECT images were spatially transformed to fit a brain atlas and normalized for differences in rCBF (Computerized Brain Atlas software). Cortical and sub-cortical volumes of interests (VOI) were analyzed and compared. Compared to the controls, AD-patients showed a significantly lower rCBF ratio in temporoparietal regions including left hippocampus. The diagnostic sensitivity and specificity for AD were high in temporoparietal regions. AD-patients had significantly reduced performance on semantic and, in particular, episodic memory tests compared to age matched normative data, and their performance on several episodic tests correlated with rCBF ratios in parietal and temporal regions including left hippocampus. The correlation between rCBF ratio and level of episodic memory performance suggests that abnormalities in rCBF pattern underlie impaired episodic memory functioning in AD

  9. Usefulness of cerebral blood flow (CBF) measurements to predict the functional outcome for rehabilitation in patients with cerebrovascular disease (CVD)

    Energy Technology Data Exchange (ETDEWEB)

    Tamamoto, Fumihiko; Nakanishi, Atsushi; Okayasu, Katsuhiko [Tokyo Metropolitan Ohtsuka Hospital (Japan); Sumi, Yukiharu; Maehara, Tadayuki; Katayama, Hitoshi

    2000-02-01

    The objectives of this study were to elucidate the relationship between the mean CBF in the whole brain (Av.mCBF) before rehabilitation of CVD patients and the BI score before and after rehabilitation, determine whether the efficacy of rehabilitation can be predicted by measurement of the Av.mCBF, and investigate what part of the brain was most important to improving the BI score. The Av.mCBFs in 160 patients with CVD were calculated by Patlak plots with {sup 99m}Tc-HMPAO before rehabilitation, and we determined the BI score before and after rehabilitation. Based on the BI scores before and after rehabilitation, patients were divided into four groups: Group A, BI=100; Group B, 80{<=}BI{<=}99; Group C, 60{<=}BI{<=}79; Group D, 0{<=}BI{<=}59. We evaluated the relationship between the Av.mCBF and BI score before and after rehabilitation. The Av.mCBF before rehabilitation showed a tendency to be more correlated with the BI score after rehabilitation (r=0.414, p<0.0001) than before rehabilitation (r=0.272, p=0.0006). After rehabilitation, there was a tendency for the Av.mCBF value to increase in direct proportion to the BI score of the group: it was highest in Group A and lowest in Group D. The strongest correlation was found between the frontal lobe regional CBF before rehabilitation and the BI score after rehabilitation (r=0.343, p<0.0001). Measurement of the Av.mCBF before rehabilitation of CVD patients will permit prediction of the efficacy of rehabilitation. Also the regional CBF of the frontal lobe is most important for improving the BI score. (author)

  10. Structure of the fMet-tRNAfMet-binding domain of B.stearothermophilus initiation factor IF2

    Science.gov (United States)

    Meunier, Sylvie; Spurio, Roberto; Czisch, Michael; Wechselberger, Rainer; Guenneugues, Marc; Gualerzi, Claudio O.; Boelens, Rolf

    2000-01-01

    The three-dimensional structure of the fMet-tRNAfMet -binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel β-strands, connected via loops, and forms a closed β-barrel similar to domain II of elongation factors EF-Tu and EF-G, despite low sequence homology. Two structures of the ternary complexes of the EF-Tu⋅aminoacyl-tRNA⋅ GDP analogue have been reported and were used to propose and discuss the possible fMet-tRNAfMet-binding site of IF2. PMID:10775275

  11. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development

    DEFF Research Database (Denmark)

    Nielsen, J; Christiansen, J; Lykke-Andersen, J;

    1999-01-01

    day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5' UTR-binding proteins control IGF-II biosynthesis during late mammalian development.......Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5' untranslated regions (5' UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins...

  12. Insulin-like growth factor binding protein-5 modulates muscle differentiation through an insulin-like growth factor-dependent mechanism

    OpenAIRE

    1996-01-01

    The insulin-like growth factor binding proteins (IGFBPs) are a family of six secreted proteins which bind to and modulate the actions of insulin-like growth factors-I and -II (IGF-I and -II). IGFBP-5 is more conserved than other IGFBPs characterized to date, and is expressed in adult rodent muscle and in the developing myotome. We have shown previously that C2 myoblasts secrete IGFBP-5 as their sole IGFBP. Here we use these cells to study the function of IGFBP-5 during myogenesis, a process s...

  13. Recruitment of TATA-Binding Protein–TAFI Complex SL1 to the Human Ribosomal DNA Promoter Is Mediated by the Carboxy-Terminal Activation Domain of Upstream Binding Factor (UBF) and Is Regulated by UBF Phosphorylation

    OpenAIRE

    Tuan, JoAnn C.; Zhai, Weiguo; Comai, Lucio

    1999-01-01

    Human rRNA synthesis by RNA polymerase I requires at least two auxiliary factors, upstream binding factor (UBF) and SL1. UBF is a DNA binding protein with multiple HMG domains that binds directly to the CORE and UCE elements of the ribosomal DNA promoter. The carboxy-terminal region of UBF is necessary for transcription activation and has been shown to be extensively phosphorylated. SL1, which consists of TATA-binding protein (TBP) and three associated factors (TAFIs), does not have any seque...

  14. Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors

    Institute of Scientific and Technical Information of China (English)

    Paul C. SEHNKE; Beth J. LAUGHNER; Carla R. LYERLY LINEBARGER; William B. GURLEY; Robert J.FERL

    2005-01-01

    Environmental control of the alcohol dehydrogenase (Adh) and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors (GBFs).The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood.In an effort to identify potential GBF binding and control partners, maize GBF1 was used as bait in a yeast two-hybrid screen of an A. thaliana cDNA library. GBF Interacting Protein 1 (GIP1) arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs. Northern analysis of A.thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript, predominantly in roots. Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus. In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A. thaliana GBF3 or maize GBF1, showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration, suggesting a transient association between GIP1 and GBF. Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP. These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar, and potentially regulates the multimeric state of GBFs, thereby contributing to bZIP-mediated gene regulation.

  15. Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

    International Nuclear Information System (INIS)

    The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purified 34 K IGF-BP specifically bound [125I]iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of [125I] iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of [125I] iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with [125I]iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-[3H]aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells

  16. Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Ritvos, O.; Ranta, T.; Jalkanen, J.; Suikkari, A.M.; Voutilainen, R.; Bohn, H.; Rutanen, E.M.

    1988-05-01

    The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purified 34 K IGF-BP specifically bound (125I)iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of (125I) iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of (125I) iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with (125I)iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-(3H)aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells.

  17. The Association between Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Protein-3 Levels and Clinical Prognosis in Patients with Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Hasan Yaşar

    2015-04-01

    Full Text Available OBJECTIVE: Recent studies report that the insulin-like growth factor system may be involved in stroke pathogenesis, and is reported to increase myelination, maturation, cell proliferation and neuronal sprouting of the central nervous system. The aim of the present study is to demonstrate the role of insulin-like growth factor system in ischemic stroke pathogenesis and its association with the prognosis by investigating insulin-like growth factor-1 and insulin-like growth factor binding protein-3 levels in patients diagnosed with acute ischemic stroke. METHODS: : Sixty-eight patients and 20 healthy individuals were included to this study. Clinical evaluation of the patients was performed according to National Institute of Health Stroke Scale and functional outcomes were graded according to Modified Rankin Scale. Bamford classification was used for the clinical classification of ischemic strokes, and the TOAST system for etiological classification. Each patient's levels of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 were measured on the first, fifth and thirtieth day of ischemic stroke. RESULTS: Only the levels of insulin-like growth factor binding protein-3 on the day of 5 were significantly decreased compared to the control group. The decrease in IGF-1 values was associated with an increased risk of death and was accompanied by clinical worsening and decreased functionality. CONCLUSION: It has been concluded that the levels of investigating insulin-like growth factor-1 and insulin-like growth factor binding protein-3 may affect mortality risk, clinical condition and functionality outcomes in patients presenting with ischemic stroke, and further studies are needed for the investigation of different effects of insulin-like growth factor-1 in future.

  18. Insulin-like growth factor binding protein-3 in preterm infants with retinopathy of prematurity

    Directory of Open Access Journals (Sweden)

    Manizheh Mostafa Gharehbaghi

    2012-01-01

    Full Text Available Background: Retinopathy of prematurity (ROP is the main cause of visual impairment in preterm newborn infants. Objective: This study was conducted to determine whether insulin-like growth factor binding protein -3 (IGFBP-3 is associated with proliferative ROP and has a role in pathogenesis of the disease in premature infants. Materials and Methods: A total of 71 preterm infants born at or before 32 weeks of gestation participated in this study. Studied patients consisted of 41 neonates without vaso-proliferative findings of ROP as the control group and 30 preterm infants with evidence of severe ROP in follow up eye examination as the case group. Blood samples obtained from these infants 6-8 weeks after birth and blood levels of IGFBP-3 were measured using enzyme-linked immunosorbent assay (ELISA. Results: The mean gestation age and birth weight of the studied patients were 28.2±1.6 weeks and 1120.7±197 gram in the case group and 28.4±1.6 weeks and 1189.4±454 gram in the control group (P=0.25 and P=0.44 respectively. The infants in the case group had significantly lower Apgar score at first and 5 min after birth. Insulin-like growth factor binding protein -3 (IGFBP-3 was significantly lower in the patients with proliferative ROP than the patients without ROP [592.5±472.9 vs. 995.5±422.2 ng/ml (P=0.009]. Using a cut-off point 770.45 ng/ml for the plasma IGFBP-3, we obtained a sensitivity of 65.9% and a specificity of 66.7% in the preterm infants with vasoproliferative ROP. Conclusion: Our data demonstrated that the blood levels IGFBP-3 was significantly lower in the patients with ROP and it is suspected that IGFBP-3 deficiency in the premature infants may have a pathogenetic role in proliferative ROP.

  19. Insulin-like growth factor binding protein-2, 28 kDa an 24 kDa insulin-like growth factor binding protein levels are decreased in fluid of dominant follicles, obtained from normal and polycystic ovaries

    NARCIS (Netherlands)

    A.G.P. Schuller (Alwin); D.J. Lindenbergh-Kortleve (Dicky); T.D. Pache; E.C. Zwarthoff (Ellen); B.C.J.M. Fauser (Bart); S.L.S. Drop (Stenvert)

    1993-01-01

    textabstractIn order to investigate potential changes in insulin-like growth factor binding proteins (IGFBPs) during human follicle maturation, we examined the IGFBP profiles in follicular fluid from follicles in different stages of maturation. Samples were obtained from ovaries of women with regula

  20. Characterization of Diverse Subvariants of the Meningococcal Factor H (fH) Binding Protein for Their Ability To Bind fH, To Mediate Serum Resistance, and To Induce Bactericidal Antibodies

    NARCIS (Netherlands)

    K.L. Seib; B. Brunelli; B. Brogioni; E. Palumbo; S. Bambini; A. Muzzi; F. Dimarcello; S. Marchi; A. van den Ende; B. Aricó; S. Savino; M. Scarselli; M. Comanducci; R. Rappuoli; M.M. Giuliani; M. Pizza

    2011-01-01

    Neisseria meningitidis is a commensal of the human nasopharynx but is also a major cause of septicemia and meningitis. The meningococcal factor H binding protein (fHbp) binds human factor H (fH), enabling down-regulation of complement activation on the bacterial surface. fHbp is a component of two s

  1. Cerebral blood flow in asymptomatic individuals; Relationship with cerebrovascular risk factors and magnetic resonance imaging signal abnormalities

    Energy Technology Data Exchange (ETDEWEB)

    Isaka, Yoshinari; Iiji, Osamu; Ashida, Keiichi; Imaizumi, Masatoshi (National Osaka Hospital (Japan))

    1993-04-01

    We studied the relationship between cortical grey matter flow (CBF) and age, cerebrovascular risk factors and the severity of subcortical hypersignals (HS, hyperintensity score in MRI) in 47 asymptomatic subjects with cerebrovascular risk factors. Multiple regression analysis revealed that HS was most strongly related to CBF, and that hematocrit, age and evidence of ischemic change detected in the electrocardiogram also appeared to be independent determinants of CBF. Both the severity and location of hypersignals were correlated with CBF. The most significant negative correlation observed was that between CBF and HS in the basal ganglia-thalamic region, where the degree of signal abnormality was modest. Decreased CBF in asymptomatic subjects with cerebrovascular risk factors may be related to microcirculatory disturbance associated with elevated hematocrit and an increase in the number of risk factors, and functional suppression of cerebral cortex due to the neuronal disconnection associated with subcortical lesions. In addition, impaired cerebral circulation may be related to MRI signal abnormalities. (author).

  2. Focal hyperemia followed by spreading oligemia and impaired activation of rCBF in classic migraine

    DEFF Research Database (Denmark)

    Olesen, J; Larsen, B; Lauritzen, M

    1981-01-01

    migraine was similarly followed during his only classic attack. The attacks were initiated by focal hyperemia in 3 patients. During prodromes all patients displayed occipitoparietal rCBF reduction (oligemia), but in only 1 case did the reduction approach critical values. Oligemia gradually spread...... anteriorly in the course of 15 to 45 minutes. In 4 patients a global oligemia was observed. In 4 patients severe headache was present concomitantly with oligemia and with no sign of hyperemia or nonhomogeneous brain perfusion. The normal rCBF increase during cortical activity (hand movement, speech, and...... similar activities) was impaired in 6 patients. The results indicate that the vasospastic model of the migraine attack is too simplistic....

  3. Cultured fibroblast monolayers secrete a protein that alters the cellular binding of somatomedin-C/insulinlike growth factor I

    International Nuclear Information System (INIS)

    We studied somatomedin-C/insulinlike growth factor (Sm-C/IGF-I) binding to human fibroblasts in both adherent monolayers and in suspension cultures. The addition of Sm-C/IGF-I in concentrations between 0.5 and 10 ng/ml to monolayers cultures resulted in a paradoxical increase in 125I-Sm-C/IGF-I binding and concentrations between 25 and 300 ng/ml were required to displace the labeled peptide. The addition of unlabeled insulin resulted in no displacement of labeled Sm-C/IGF-I from the adherent cells. When fibroblast suspensions were used Sm-C/IGF-I concentrations between 1 and 10 ng/ml caused displacement, the paradoxical increase in 125I-Sm-C/IGF-I binding was not detected, and insulin displaced 60% of the labeled peptide. Affinity cross-linking to fibroblast monolayers revealed a 43,000-mol wt 125I-Sm-C-binding-protein complex that was not detected after cross-linking to suspended cells. The 43,000-mol wt complex was not detected after cross-linking to smooth muscle cell monolayers, and binding studies showed that 125I-Sm-C/IGF-I was displaced greater than 90% by Sm-C/IGF-I using concentrations between 0.5 and 10 ng/ml. Because fibroblast-conditioned medium contains the 43,000-mol wt complex, smooth muscle cells were incubated with conditioned medium for 24 h prior to initiation of the binding studies. 125I-Sm-C/IGF-I-binding increased 1.6-fold compared to control cultures and after cross-linking the 43,000-mol wt complex could be detected on the smooth muscle cell surface. Human fibroblast monolayers secrete a protein that binds 125I-Sm-C/IGF-I which can be transferred to the smooth muscle cell surface and alters 125I-Sm-C/IGF-I binding

  4. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    International Nuclear Information System (INIS)

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

  5. Changes of rCBF 99mTc-HMPAO SPECT in a selected disorders

    International Nuclear Information System (INIS)

    With single photon emission computer tomography (SPECT) and HMPAO a noninvasive examination of regional cerebral blood flow can be performed (rCBF). The purpose of the SPECT examinations was to define the location and magnitude of blood flow disorder in selected diseases and the assessment of the results as complementary to morphological tests CT and MRI or functional tests, such as EEG. The examinations were carried out in 455 patients: 91 - patients with depression, 29 - congenital hypothyroidism, 66 - migraine, 34 - epilepsy, 6 - Landau-Kleffner syndrome, 20 - Alzheimer disease, 55 - with suspected Alzheimer disease, 105 - cerebral stroke, and 48 - transient cerebral ischemia. The control group comprised of 26 subjects. The SPECT method was used to perform examinations. The images were evaluated based on semiquantitative method. The asymmetry of activity and activity referred to the referential region were measured using symmetrical ROIs localized in hypoperfusion foci. The differences in perfusion in symmetric locations exceeding 10% were considered abnormal. During the depression stage, in patients with depression, a decrease of rCBF occurred. Regression of depression results in an increase of rCBF almost in the entire cerebrum. In patients with depression, SPECT examination is a useful method to monitor course of a disease and to objectively verify the results of treatment. 2. Disorders of rCBF, a decrease and/or asymmetry, occurred in patients with congenital hypothyroidism, migraine, and epilepsy. 3. There is a relation between patterns of cerebral perfusion in stroke, visible in SPECT image, and an extent and intensity of cerebral ischemia. (author)

  6. Creating PWMs of transcription factors using 3D structure-based computation of protein-DNA free binding energies

    Directory of Open Access Journals (Sweden)

    Stegmaier Philip

    2010-05-01

    Full Text Available Abstract Background Knowledge of transcription factor-DNA binding patterns is crucial for understanding gene transcription. Numerous DNA-binding proteins are annotated as transcription factors in the literature, however, for many of them the corresponding DNA-binding motifs remain uncharacterized. Results The position weight matrices (PWMs of transcription factors from different structural classes have been determined using a knowledge-based statistical potential. The scoring function calibrated against crystallographic data on protein-DNA contacts recovered PWMs of various members of widely studied transcription factor families such as p53 and NF-κB. Where it was possible, extensive comparison to experimental binding affinity data and other physical models was made. Although the p50p50, p50RelB, and p50p65 dimers belong to the same family, particular differences in their PWMs were detected, thereby suggesting possibly different in vivo binding modes. The PWMs of p63 and p73 were computed on the basis of homology modeling and their performance was studied using upstream sequences of 85 p53/p73-regulated human genes. Interestingly, about half of the p63 and p73 hits reported by the Match algorithm in the altogether 126 promoters lay more than 2 kb upstream of the corresponding transcription start sites, which deviates from the common assumption that most regulatory sites are located more proximal to the TSS. The fact that in most of the cases the binding sites of p63 and p73 did not overlap with the p53 sites suggests that p63 and p73 could influence the p53 transcriptional activity cooperatively. The newly computed p50p50 PWM recovered 5 more experimental binding sites than the corresponding TRANSFAC matrix, while both PWMs showed comparable receiver operator characteristics. Conclusions A novel algorithm was developed to calculate position weight matrices from protein-DNA complex structures. The proposed algorithm was extensively validated

  7. rVISTA 2.0: Evolutionary Analysis of Transcription Factor Binding Sites

    Energy Technology Data Exchange (ETDEWEB)

    Loots, G G; Ovcharenko, I

    2004-01-28

    Identifying and characterizing the patterns of DNA cis-regulatory modules represents a challenge that has the potential to reveal the regulatory language the genome uses to dictate transcriptional dynamics. Several studies have demonstrated that regulatory modules are under positive selection and therefore are often conserved between related species. Using this evolutionary principle we have created a comparative tool, rVISTA, for analyzing the regulatory potential of noncoding sequences. The rVISTA tool combines transcription factor binding site (TFBS) predictions, sequence comparisons and cluster analysis to identify noncoding DNA regions that are highly conserved and present in a specific configuration within an alignment. Here we present the newly developed version 2.0 of the rVISTA tool that can process alignments generated by both zPicture and PipMaker alignment programs or use pre-computed pairwise alignments of seven vertebrate genomes available from the ECR Browser. The rVISTA web server is closely interconnected with the TRANSFAC database, allowing users to either search for matrices present in the TRANSFAC library collection or search for user-defined consensus sequences. rVISTA tool is publicly available at http://rvista.dcode.org/.

  8. Gains and Losses of Transcription Factor Binding Sites in Saccharomyces cerevisiae and Saccharomyces paradoxus.

    Science.gov (United States)

    Schaefke, Bernhard; Wang, Tzi-Yuan; Wang, Chuen-Yi; Li, Wen-Hsiung

    2015-08-01

    Gene expression evolution occurs through changes in cis- or trans-regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus) and within species (comparing two closely related S. cerevisiae strains). PMID:26220934

  9. Model-based Comparative Prediction of Transcription-Factor Binding Motifs in Anabolic Responses in Bone

    Institute of Scientific and Technical Information of China (English)

    Andy B. Chen; Kazunori Hamamura; Guohua Wang; Weirong Xing; Subburaman Mohan; Hiroki Yokota; Yunlong Liu

    2007-01-01

    Understanding the regulatory mechanism that controls the alteration of global gene expression patterns continues to be a challenging task in computational biology. We previously developed an ant algorithm, a biologically-inspired computational technique for microarray data, and predicted putative transcription-factor binding motifs (TFBMs) through mimicking interactive behaviors of natural ants. Here we extended the algorithm into a set of web-based software, Ant Modeler, and applied it to investigate the transcriptional mechanism underlying bone formation. Mechanical loading and administration of bone morphogenic proteins (BMPs) are two known treatments to strengthen bone. We addressed a question: Is there any TFBM that stimulates both "anabolic responses of mechanical loading" and "BMP-mediated osteogenic signaling"? Although there is no significant overlap among genes in the two responses, a comparative model-based analysis suggests that the two independent osteogenic processes employ common TFBMs, such as a stress responsive element and a motif for peroxisome proliferator-activated recep- tor (PPAR). The post-modeling in vitro analysis using mouse osteoblast cells sup- ported involvements of the predicted TFBMs such as PPAR, Ikaros 3, and LMO2 in response to mechanical loading. Taken together, the results would be useful to derive a set of testable hypotheses and examine the role of specific regulators in complex transcriptional control of bone formation.

  10. Predicting transcription factor binding sites using local over-representation and comparative genomics

    Directory of Open Access Journals (Sweden)

    Touzet Hélène

    2006-08-01

    Full Text Available Abstract Background Identifying cis-regulatory elements is crucial to understanding gene expression, which highlights the importance of the computational detection of overrepresented transcription factor binding sites (TFBSs in coexpressed or coregulated genes. However, this is a challenging problem, especially when considering higher eukaryotic organisms. Results We have developed a method, named TFM-Explorer, that searches for locally overrepresented TFBSs in a set of coregulated genes, which are modeled by profiles provided by a database of position weight matrices. The novelty of the method is that it takes advantage of spatial conservation in the sequence and supports multiple species. The efficiency of the underlying algorithm and its robustness to noise allow weak regulatory signals to be detected in large heterogeneous data sets. Conclusion TFM-Explorer provides an efficient way to predict TFBS overrepresentation in related sequences. Promising results were obtained in a variety of examples in human, mouse, and rat genomes. The software is publicly available at http://bioinfo.lifl.fr/TFM-Explorer.

  11. Comparative Analysis of Regulatory Motif Discovery Tools for Transcription Factor Binding Sites

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In the post-genomic era, identification of specific regulatory motifs or transcription factor binding sites (TFBSs) in non-coding DNA sequences, which is essential to elucidate transcriptional regulatory networks, has emerged as an obstacle that frustrates many researchers. Consequently, numerous motif discovery tools and correlated databases have been applied to solving this problem. However, these existing methods, based on different computational algorithms, show diverse motif prediction efficiency in non-coding DNA sequences. Therefore, understanding the similarities and differences of computational algorithms and enriching the motif discovery literatures are important for users to choose the most appropriate one among the online available tools. Moreover, there still lacks credible criterion to assess motif discovery tools and instructions for researchers to choose the best according to their own projects. Thus integration of the related resources might be a good approach to improve accuracy of the application. Recent studies integrate regulatory motif discovery tools with experimental methods to offer a complementary approach for researchers, and also provide a much-needed model for current researches on transcriptional regulatory networks. Here we present a comparative analysis of regulatory motif discovery tools for TFBSs.

  12. Myocyte-specific enhancer binding factor 2A expression is downregulated during temporal lobe epilepsy.

    Science.gov (United States)

    Huang, Yunyi; Wu, Xuling; Guo, Jing; Yuan, Jinxian

    2016-09-01

    Myocyte-specific enhancer binding factor 2A (MEF2A) is a multifunctional nuclear protein that regulates synaptogenesis, dendritic morphogenesis, and neuronal survival. This study aimed to investigate the expression pattern of MEF2A in epileptogenic processes. MEF2A expression was detected in 20 temporal neocortex tissue samples from patients with temporal lobe epilepsy (TLE) and 20 samples from trauma patients without epilepsy by real-time quantitative polymerase chain reaction, immunohistochemistry, double-label immunofluorescent staining, and western blot analysis. In addition, the expression patterns of MEF2A in the hippocampus and adjacent cortex of a lithium-pilocarpine-induced TLE rat model and control rats were examined. MEF2A was found to be expressed in the nuclei of neurons but not in the dendrites of neurons and astrocytes. MEF2A expression was significantly downregulated in temporal neocortex of humans and rats with TLE compared to the control groups. In addition, in the lithium-pilocarpine-induced TLE model, MEF2A expression dynamically decreased within 2 months. Taken together, these data suggest that MEF2A is involved in the pathogenesis of TLE. PMID:26439092

  13. Bridging macroscopic and microscopic methods for the measurements of cerebral blood flow: Toward finding the determinants in maintaining the CBF homeostasis.

    Science.gov (United States)

    Kanno, I; Masamoto, K

    2016-01-01

    Methods exist to evaluate the cerebral blood flow (CBF) at both the macroscopic and microscopic spatial scales. These methods provide complementary information for understanding the mechanism in maintaining an adequate blood supply in response to neural demand. The macroscopic CBF assesses perfusion flow, which is usually measured using radioactive tracers, such as diffusible, nondiffusible, or microsphere. Each of them determines CBF based on indicator dilution principle or particle fraction principle under the assumption that CBF is steady state during the measurement. Macroscopic CBF therefore represents averaged CBF over a certain space and time domains. On the other hand, the microscopic CBF assesses bulk flow, usually measures using real-time microscopy. The method assesses hemodynamics of microvessels, ie, vascular dimensions and flow velocities of fluorescently labeled or nonlabeled RBC and plasma markers. The microscopic CBF continuously fluctuates in time and space. Smoothing out this heterogeneity may lead to underestimation in the macroscopic CBF. To link the two measurements, it is needed to introduce a common parameter which is measurable for the both methods, such as mean transit time. Additionally, applying the defined physiological and/or pharmacological perturbation may provide a good exercise to determine how the specific perturbations interfere the quantitative relationships between the macroscopic and microscopic CBF. Finally, bridging these two-scale methods potentially gives a further indication how the absolute CBF is regulated with respect to a specific type of the cerebrovascular tones or capillary flow velocities in the brain. PMID:27130412

  14. Focal hyperemia followed by spreading oligemia and impaired activation of rCBF in classic migraine

    International Nuclear Information System (INIS)

    Regional cerebral blood flow (rCBF) was measured in 254 areas of a hemisphere with the xenon 133 intraarterial injection method. Six cases of classic migraine were followed from the normal state into the prodromal phase, and in 3 cases further into the headache phase. One patient with common migraine was similarly followed during his only classic attack. The attacks were initiated by focal hyperemia in 3 patients. During prodromes all patients displayed occipitoparietal rCBF reduction (oligemia), but in only 1 case did the reduction approach critical values. Oligemia gradually spread anteriorly in the course of 15 to 45 minutes. In 4 patients a global oligemia was observed. In 4 patients severe headache was present concomitantly with oligemia and with no sign of hyperemia or nonhomogeneous brain perfusion. The normal rCBF increase during cortical activity (hand movement, speech, and similar activities) was impaired in 6 patients. The results indicate that the vasospastic model of the migraine attack is too simplistic

  15. rCBF change in the brain of patients with major depressive disorder

    International Nuclear Information System (INIS)

    Purpose Major depressive disorder is a frequent emotional mood disorder. To evaluate the changes of brain blood flow in patients with depressive disorder and the correlation between rCBF and clinical feature is very important to diagnosis and treatment of this decease. Methods: Regional cerebral perfusion was investigated using SPECT in 75 patients with depressive disorders. The mean ages of the patients were 41.9 (17-74) Years old. The course of disease was different from several days to over 20 years. Results: 97.3 per cent of patients (73/75) had relative hypoperfusions in some cerebral regions. The patients had a significant decrease of rCBF in the frontal lobesbilaterally, and temporal lobes, basal ganglia, thalamus and parietal lobe. The course of disease and age of the patients had a negative correlation with the changes of rCBF. Conclusion: According to the results of our study, patients with depressive disorders had profound dysfunction of the frontal lobes bilaterally. The temporal cortices and basal ganglia were involved in most patients too. It is coincident with the results of other studies. The function of frontal lobes and temporal lobes is close relation close with affective action, attention, memory, thinking, abstraction, and other brain cognitive function. The clinical symptom of depressive disorder may be relevant with hypoperfusions of frontal lobes and temporal lobes. (authors)

  16. Usefulness of rCBF SPECT in patients with encephalitis. Comparison study with MRI

    Energy Technology Data Exchange (ETDEWEB)

    Nagamachi, Shigeki; Jinnouchi, Seishi; Flores, L.G. II; Kodama, Takao; Itokazu, Naoya; Nakahara, Hiroshi; Ohnishi, Takashi; Futami, Shigemi; Watanabe, Katsushi [Miyazaki Medical Coll., Kiyotake (Japan)

    1997-01-01

    Twenty-nine rCBF SPECT study was done in 17 patients with encephalitis. Five of 6 patients (83.3%) showed regional high uptake in acute phase within a week after onset and 16 of 23 studies (69.6%) showed regional low uptake in subacute and chronic phase. Six of 19 lesions (31.6%) with regional high uptake changed to low uptake and 11 lesions (57.9%) improved to normal uptake on follow up studies. Seventeen of 51 lesions with low uptake (33.3%) improved to normal uptake. On the comparative study with MRI, 8 of 18 (44.4%) high uptake area showed cortical thickness or high intensity on T{sub 2} weighted images. Thirty-six of 74 low uptake area (48.6%) showed cortical thickness, brain atrophy or high intensity on T{sub 2} weighted images. Forty-eight of 212 regions (22.6%) with normal MRI findings showed abnormal accumulation of cerebral tracer on rCBF SPECT studies. rCBF SPECT was useful tool for diagnosis and follow up management in patients with encephalitis. (author)

  17. Transcription factor CCAAT/enhancer binding protein alpha up-regulates microRNA let-7a-1 in lung cancer cells by direct binding

    OpenAIRE

    Lin, Yani; Zhao, Jian; Hu, Xiaoyan; Wang, Lina; Liang, Liming; Chen, Weiwen

    2016-01-01

    Aims The transcription factor CCAAT/enhancer binding protein α (C/EBPα) and microRNA (miRNA) let-7a-1 act as tumor suppressors in many types of cancers including lung cancer. In the present study, we aim to investigate whether let-7a-1 is a novel important target of C/EBPα in lung cancer cells. Methods The DNA sequence of the 2.1 kb let-7a-1 promoter was analyzed with MatInspector 4.1 (http://www.genomatix.de). Human lung cancer cell lines A549 and H1299, and human cervical cancer cell line H...

  18. Physical capacity influences the response of insulin-like growth factor and its binding proteins to training

    DEFF Research Database (Denmark)

    Rosendal, Lars; Langberg, Henning; Flyvbjerg, Allan;

    2002-01-01

    The influence of initial training status on the response of circulating insulin-like growth factor (IGF) and its binding proteins (IGFBP) to prolonged physical training was studied in young men. It was hypothesized that highly standardized training would result in more extensive changes in the...

  19. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F

    DEFF Research Database (Denmark)

    Helin, K; Lees, J A; Vidal, M;

    1992-01-01

    The retinoblastoma protein (pRB) plays an important role in the control of cell proliferation, apparently by binding to and regulating cellular transcription factors such as E2F. Here we describe the characterization of a cDNA clone that encodes a protein with properties of E2F. This clone, RBP3...

  20. JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update

    DEFF Research Database (Denmark)

    Bryne, J.C.; Valen, E.; Tang, M.H.E.; Marstrand, T.; Winther, Ole; da Piedade, I.; Krogh, A.; Lenhard, B.; Sandelin, A.

    2008-01-01

    JASPAR is a popular open-access database for matrix models describing DNA-binding preferences for transcription factors and other DNA patterns. With its third major release, JASPAR has been expanded and equipped with additional functions aimed at both casual and power users. The heart of the JASPAR...

  1. Identification and characterization of a pituitary corticotropin-releasing factor binding protein by chemical cross-linking

    DEFF Research Database (Denmark)

    Nishimura, E; Billestrup, Nils; Perrin, M;

    1987-01-01

    A corticotropin-releasing factor (CRF) binding protein has been identified based on the chemical cross-linking of ovine [Nle21,m-125I-Tyr32]CRF (125I-oCRF) to bovine anterior pituitary membranes using disuccinimidyl suberate (DSS). The apparent molecular weight of the cross-linked complex...

  2. Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein

    OpenAIRE

    Lee, Chang Hoon; Melchers, Mark; Wang, Hongsheng; Torrey, Ted A.; Slota, Rebecca; Qi, Chen-Feng; Kim, Ji Young; Lugar, Patricia; Kong, Hee Jeong; Farrington, Lila; van der Zouwen, Boris; ZHOU, JEFF X.; Lougaris, Vassilios; Lipsky, Peter E.; Grammer, Amrie C.

    2006-01-01

    Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphoma...

  3. Modulation of enteroviral proteinase cleavage of poly(A)-binding protein (PABP) by conformation and PABP-associated factors

    OpenAIRE

    Rivera, Carlos I.; Lloyd, Richard E.

    2008-01-01

    Poliovirus (PV) causes a drastic inhibition of cellular cap-dependant protein synthesis due to the cleavage of translation factors eukaryotic initiation factor 4G (eIF4G) and poly (A) binding protein (PABP). Only about half of cellular PABP is cleaved by viral 2A and 3C proteinases during infection. We have investigated PABP cleavage determinants that regulate this partial cleavage. PABP cleavage kinetics analyses indicate that PABP exists in multiple conformations, some of which are resistan...

  4. Reassessing the Role of Staphylococcus aureus Clumping Factor and Fibronectin-Binding Protein by Expression in Lactococcus lactis

    OpenAIRE

    Que, Yok-Ai; François, Patrice; Haefliger, Jacques-Antoine; Entenza, José-Manuel; Vaudaux, Pierre; Moreillon, Philippe

    2001-01-01

    Since Staphylococcus aureus expresses multiple pathogenic factors, studying their individual roles in single-gene-knockout mutants is difficult. To circumvent this problem, S. aureus clumping factor A (clfA) and fibronectin-binding protein A (fnbA) genes were constitutively expressed in poorly pathogenic Lactococcus lactis using the recently described pOri23 vector. The recombinant organisms were tested in vitro for their adherence to immobilized fibrinogen and fibronectin and in vivo for the...

  5. Andrographolide interferes with binding of nuclear factor-κB to DNA in HL-60-derived neutrophilic cells

    OpenAIRE

    María A. Hidalgo; Romero, Alex; Figueroa, Jaime; Cortés, Patricia; Concha, Ilona I; Hancke, Juan L.; Burgos, Rafael A

    2005-01-01

    Andrographolide, the major active component from Andrographis paniculata, has shown to possess anti-inflammatory activity. Andrographolide inhibits the expression of several proinflammatory proteins that exhibit a nuclear factor kappa B (NF-κB) binding site in their gene.In the present study, we analyzed the effect of andrographolide on the activation of NF-κB induced by platelet-activating factor (PAF) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) in HL-60 cells differentiated to neutro...

  6. Prognostic Significance of Lymphoid Enhancer-Binding Factor-1 Expression in Egyptian Adult B-Acute Lymphocytic Leukemia Patients

    OpenAIRE

    Aly, Rabab M.; Ansaf B. Yousef

    2015-01-01

    Objective: Lymphoid enhancer-binding factor-1 (LEF-1) is a key transcription factor of wingless-type (Wnt) signaling in various tumors and it is associated with a number of malignant diseases such as leukemia. We explored the expression profile of LEF-1 in acute lymphoblastic leukemia (ALL) and determined its specific prognostic significance in this disease. Materials and Methods: We studied LEF-1 expression in 56 newly diagnosed B-acute ALL adult patients using real-time quantitative polymer...

  7. Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells

    DEFF Research Database (Denmark)

    Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani;

    2015-01-01

    Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on...... procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2....

  8. Serum levels of insulin-like growth factor-1 and insulin-like growth factor binding protein-3 in relapsing and primary progressive multiple sclerosis

    NARCIS (Netherlands)

    Wilczak, N; Ramsaransing, GSM; Mostert, J; Chesik, D; De Keyser, J

    2005-01-01

    Using radioimmunoassay we measured serum levels of insulin- like growth factor ( IGF)- 1 and IGF binding protein ( IGFBP)- 3 in patients with relapsing multiple sclerosis ( MS) and a benign course ( Expanded Disability Status Scale ( EDSS) less than or equal to 3 despite > 10 years disease duration)

  9. Promoter recognition in archaea is mediated by transcription factors: identification of transcription factor aTFB from Methanococcus thermolithotrophicus as archaeal TATA-binding protein.

    OpenAIRE

    Gohl, H P; Gröndahl, B; Thomm, M

    1995-01-01

    At least two transcription factors, aTFB and aTFA, are required for accurate and faithful in vitro transcription of homologous templates in cell-free extracts from the methanogenic Archaeon Methanococcus thermolithotrophicus. We have recently shown that the function of aTFB can be replaced by eucaryal TATA-binding proteins. Here we demonstrate using template commitment experiments that promoter recognition in an Archaeon is mediated by transcription factors. The archaeal TATA box was identifi...

  10. Cell surface syndecan-1 contributes to binding and function of macrophage migration inhibitory factor (MIF) on epithelial tumor cells.

    Science.gov (United States)

    Pasqualon, Tobias; Lue, Hongqi; Groening, Sabine; Pruessmeyer, Jessica; Jahr, Holger; Denecke, Bernd; Bernhagen, Jürgen; Ludwig, Andreas

    2016-04-01

    Surface expressed proteoglycans mediate the binding of cytokines and chemokines to the cell surface and promote migration of various tumor cell types including epithelial tumor cells. We here demonstrate that binding of the chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) to epithelial lung and breast tumor cell lines A549 and MDA-MB231 is sensitive to enzymatic digestion of heparan sulphate chains and competitive inhibition with heparin. Moreover, MIF interaction with heparin was confirmed by chromatography and a structural comparison indicated a possible heparin binding site. These results suggested that proteoglycans carrying heparan sulphate chains are involved in MIF binding. Using shRNA-mediated gene silencing, we identified syndecan-1 as the predominant proteoglycan required for the interaction with MIF. MIF binding was decreased by induction of proteolytic shedding of syndecan-1, which could be prevented by inhibition of the metalloproteinases involved in this process. Finally, MIF induced the chemotactic migration of A549 cells, wound closure and invasion into matrigel without affecting cell proliferation. These MIF-induced responses were abrogated by heparin or by silencing of syndecan-1. Thus, our study indicates that syndecan-1 on epithelial tumor cells promotes MIF binding and MIF-mediated cell migration. This may represent a relevant mechanism through which MIF enhances tumor cell motility and metastasis. PMID:26852939

  11. Localization, characterization, and quantification of insulin-like growth factor-I-binding sites in the ewe ovary

    Energy Technology Data Exchange (ETDEWEB)

    Monget, P.; Monniaux, D.; Durand, P. (INRA, Station de Physiologie de la Reproduction, Nouzilly (France))

    1989-11-01

    To assess a potential role of insulin-like growth factor-I (IGF-I) in the ewe ovary, the presence of IGF-I receptors and IGF-I-binding proteins was studied by binding assays performed on granulosa cell suspensions, in follicular fluid, and on ovarian sections. On the ovarian sections, labeling was quantified after autoradiography by microphotometry. Competition studies with IGF-I and insulin allowed us to estimate the relative proportions of binding proteins and type I receptors in the different compartments of the ewe ovary. Our results clearly show that saturable, specific, and high affinity IGF-I receptors are present on the ovine granulosa cells. At equilibrium for both granulosa cell suspensions and frozen sections, the Kd value was close to 2 nM. IGF-I binding proteins were also present in follicular fluid and stroma, thecal, and granulosa cells. At equilibrium for follicular fluid, the Kd value was 0.91 +/- 0.27 nM (mean +/- SE). Moreover, on frozen sections, it was shown that atresia of small follicles (less than 2 mm) was accompanied by a decrease in the number of IGF-I receptors and an increase in the number of IGF-I-binding proteins on granulosa cells. By contrast, this phenomenon was not observed in large follicles. These data indicate that granulosa cells of ewe ovary possess type I receptors, and IGF-I-binding proteins may modulate IGF-I action in the process of follicular growth and atresia.

  12. Localization, characterization, and quantification of insulin-like growth factor-I-binding sites in the ewe ovary

    International Nuclear Information System (INIS)

    To assess a potential role of insulin-like growth factor-I (IGF-I) in the ewe ovary, the presence of IGF-I receptors and IGF-I-binding proteins was studied by binding assays performed on granulosa cell suspensions, in follicular fluid, and on ovarian sections. On the ovarian sections, labeling was quantified after autoradiography by microphotometry. Competition studies with IGF-I and insulin allowed us to estimate the relative proportions of binding proteins and type I receptors in the different compartments of the ewe ovary. Our results clearly show that saturable, specific, and high affinity IGF-I receptors are present on the ovine granulosa cells. At equilibrium for both granulosa cell suspensions and frozen sections, the Kd value was close to 2 nM. IGF-I binding proteins were also present in follicular fluid and stroma, thecal, and granulosa cells. At equilibrium for follicular fluid, the Kd value was 0.91 +/- 0.27 nM (mean +/- SE). Moreover, on frozen sections, it was shown that atresia of small follicles (less than 2 mm) was accompanied by a decrease in the number of IGF-I receptors and an increase in the number of IGF-I-binding proteins on granulosa cells. By contrast, this phenomenon was not observed in large follicles. These data indicate that granulosa cells of ewe ovary possess type I receptors, and IGF-I-binding proteins may modulate IGF-I action in the process of follicular growth and atresia

  13. 125I-human epidermal growth factor specific binding to placentas and fetal membranes from varoius pregnancy states

    International Nuclear Information System (INIS)

    Specific binding of 125I-human epidermal growth factor (hEGF) to homogenates of term human placentas and fetal membranes from normal and appropriate for gestational age (N = 20), intrauterine growth retarded (N = 9), twin (N = 11), White class A/B diabetic (N = 12), and large for gestational age (N = 13) pregnancies was measured. In all pregnancy states, placentas bound approximately four times more 125I-hEGF than did fetal membranes (P125I-hEGF binding to fetal membranes from the various pregnancy states (P125I-hEGF specific binding to placentas from intrauterine growth retarded or twin pregnancies was significantly greater compared with placentas from normal and appropriate for gestational age pregnancies (P125I-hEGF specific binding did not differ between placentas from intrauterine growth retarded or twin pregnancies (P125I-hEGF binding did not vary with fetal sex, maternal race, placental weight, or gestational age between 37 to 42 weeks (P125I-hEGF binding increased with increasing infant weight when appropriate for gestational age and large for gestational age infants were included (P<0.05, r = 0.38, N = 32) but not for intrauterine growth retarded, appropriate for gestational age, or large for gestational age infants alone. (author)

  14. A deeper look into transcription regulatory code by preferred pair distance templates for transcription factor binding sites

    KAUST Repository

    Kulakovskiy, Ivan V.

    2011-08-18

    Motivation: Modern experimental methods provide substantial information on protein-DNA recognition. Studying arrangements of transcription factor binding sites (TFBSs) of interacting transcription factors (TFs) advances understanding of the transcription regulatory code. Results: We constructed binding motifs for TFs forming a complex with HIF-1α at the erythropoietin 3\\'-enhancer. Corresponding TFBSs were predicted in the segments around transcription start sites (TSSs) of all human genes. Using the genome-wide set of regulatory regions, we observed several strongly preferred distances between hypoxia-responsive element (HRE) and binding sites of a particular cofactor protein. The set of preferred distances was called as a preferred pair distance template (PPDT). PPDT dramatically depended on the TF and orientation of its binding sites relative to HRE. PPDT evaluated from the genome-wide set of regulatory sequences was used to detect significant PPDT-consistent binding site pairs in regulatory regions of hypoxia-responsive genes. We believe PPDT can help to reveal the layout of eukaryotic regulatory segments. © The Author 2011. Published by Oxford University Press. All rights reserved.

  15. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

    Science.gov (United States)

    Du, Juan; Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  16. Comparison of rCBF between patients with medial temporal lobe epilepsy and normal controls using H215O PET

    International Nuclear Information System (INIS)

    The aim of this study was to identify the brain areas whose regional cerebral blood flow (rCBF) was changed in medial temporal lobe epilepsy (mTLE) using H215O-PET. 12 patients with mTLE (6 left, 6 right mTLE) and 6 normal controls were scanned during a fixation baseline period and a sensory-motor condition where subjects pressed a button to an upward arrow. A voxel-based analysis using SPM99 software was performed to compare the patient groups with the normal controls for the rCBF during fixation baseline period and for relative changes of rCBF during the sensory-motor task relative to fixation. Duirng the fixation baseline, a significant reduction of rCBF was found posterior insula bilaterally and right frontopolar regions in right mTLE patients compared to the normal controls. In left mTLE patients, the reduction was found in left frontopolar and temporal regions. During the sensory-motor task, rCBF increase over the fixation period, was reduced in left frontal and superior temporal regions in the right mTLE patients whereas in various areas of right hemisphere in left mTLE patients, relative to normal controls. However, the increased rCBF was also found in the left inferior parietal and anterior thalamic/fornix regions in both right and left mTLE patients compared to normal controls. Epilepsy induced changes were found not only in relative increase/ decrease of rCBF during a simple sensory-motor control condition relative to a fixation rest condition but also in the relative rCBF distribution during the rest period

  17. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha.

    Science.gov (United States)

    Yuan, Yong; Hilliard, George; Ferguson, Tsuneo; Millhorn, David E

    2003-05-01

    The hypoxia-inducible factor (HIF) activates the expression of genes that contain a hypoxia response element. The alpha-subunits of the HIF transcription factors are degraded by proteasomal pathways during normoxia but are stabilized under hypoxic conditions. The von Hippel-Lindau protein (pVHL) mediates the ubiquitination and rapid degradation of HIF-alpha (including HIF-1alpha and HIF-2alpha). Post-translational hydroxylation of a proline residue in the oxygen-dependent degradation (ODD) domain of HIF-alpha is required for the interaction between HIF and VHL. It has previously been established that cobalt mimics hypoxia and causes accumulation of HIF-1alpha and HIF-2alpha. However, little is known about the mechanism by which this occurs. In an earlier study, we demonstrated that cobalt binds directly to the ODD domain of HIF-2alpha. Here we provide the first evidence that cobalt inhibits pVHL binding to HIF-alpha even when HIF-alpha is hydroxylated. Deletion of 17 amino acids within the ODD domain of HIF-2alpha that are required for pVHL binding prevented the binding of cobalt and stabilized HIF-2alpha during normoxia. These findings show that cobalt mimics hypoxia, at least in part, by occupying the VHL-binding domain of HIF-alpha and thereby preventing the degradation of HIF-alpha. PMID:12606543

  18. DNA binding during expanded bed adsorption and factors affecting adsorbent aggregation

    DEFF Research Database (Denmark)

    Arpanaei, Ayyoob; Mathiasen, N.; Hobley, Timothy John

    2008-01-01

    tolerance of anion exchangers when binding DNA. However, more importantly. with the adsorbents examined here. attempts to reduce bed aggregation by feedstock conditioning with added salt may increase DNA binding leading to a reduction in expanded bed adsorption performance compromising protein capture...... ligand densities to be examined. Very high dynamic binding capacities at 10% breakthrough were found in the absence of added salt. However, the highest binding capacities (similar to 10 and similar to 19mg DNA ml(-1) gel) were found in buffers containing added salt at concentrations of either 0.25 or 0......) even though the dynamic binding capacity was reduced as DNA concentration was increased. The extent of bed contraction during DNA loading was found to be a function of added salt concentration and ligand density of the adsorbent. The results imply that ligand density significantly affects the salt...

  19. Usefulness of CBF measurement in the hyperacute phase of ischemic stroke

    International Nuclear Information System (INIS)

    Thrombolytic therapy has emerged as a new treatment option in the hyperacute stage of ischemic stroke. Although a 15% increase in meaningful recovery has been observed at three months, it can also trigger devastating hemorrhagic transformations. Therefore, it is important to select suitable patients to undergo this treatment. The purpose of the present study was to evaluate the feasibility of cerebral blood flow (CBF) measurement by xenon-enhanced CT (Xe-CT) in patients with acute ischemic stroke as well as its potential in identifying the occluded artery and the stroke subtype. In effect, this study examines the potential for Xe-CT to select the appropriate candidates for thrombolytic therapy. In 36 sequential patients (average age: 64.1±13.1) with sudden-onset of ischemic stroke (except for lacunar stroke) who had presented to our hospital within two hours after the onset, we performed Xe-CT and MRI diffusion-weighted imaging (DWI). A selective cerebral angiography was also done if further evaluation was warranted. We examined the sensitivity of Xe-CT in demonstrating the ischemic area compared with that of DWI, its potential to identify the stroke subtype, its ability to determine infarction and hemorrhage based on CBF thresholds, and its contribution to selection of thrombolysis candidates. Xe-CT was completed safely in 31 out of 34 patients (91%). Within three hours after symptom onset, Xe-CT detected the ischemic area in most of the patients (94%), whereas DWI failed to do so in 24%. Of 14 patients who underwent both Xe-CT and angiography, the diagnosis of the stroke subtype determined by Xe-CT was confirmed to be correct by angiography in 11 patients (79%). The CBF threshold of non-hemorrhagic infarction in the gray matter became constant (19 ml/100 g/min) at 3-5 hours after the onset. In hemorrhagic infarction, however, the threshold was initially found to be lower (9 ml/100 g/min) at 3-5 hours and reaching comparable levels with non-hemorrhagic subtypes

  20. The study of correlation between neurological function rehabilitation and dynamic change of rCBF in patients with aphasia

    International Nuclear Information System (INIS)

    Objective: To evaluate the result of SPECT and CT in the patients with acute cerebral infarction and further more, to study the correlation between aphasia and dynamic change of regional cerebral blood flow (rCBF) in patients. Methods: Thirty cases with cerebral infarction of left basal ganglia were divided into two groups according to the presence or absence of aphasia; the vision and semi-ration analysis were used in photograph reading and region of interest (ROI) technology, respectively. Results: 1) Group A: there was a low rCBF in left basal ganglia, the dimension was larger than that in CT. There was also a low rCBF in frontal lobe and temporal lobe. Group B: there was only a low rCBF in left basal ganglia. 2) There were 6 cases with crossed cerebellar diaschisis (CCD) in the patients with aphasia. 3) The comparison about aphasia: the rCBF was higher in language center in the patients with improved language function than that in the patients without language function improvement and the difference between them was significant. Conclusions: The neurological function can be indirectly reflected through the study of the rCBF. At the same time, it may conduce to the locating of the damage in the central nervous system and to the differentiation diagnosis. It may also conduce to the programming of the therapeutic course and prognostication. (authors)

  1. Molecular dissection of the intrinsic factor-vitamin B12 receptor, cubilin, discloses regions important for membrane association and ligand binding

    DEFF Research Database (Denmark)

    Kristiansen, M; Kozyraki, R; Jacobsen, Christian; Nexø, E; Verroust, P J; Moestrup, S K

    1999-01-01

    addition to binding the vitamin B12-carrier complex, cubilin also binds receptor-associated protein. To delineate the structures for membrane association and ligand binding we established a panel of stable transfected Chinese hamster ovary cells expressing overlapping segments of rat cubilin. Analysis of......Cubilin, the receptor for intrinsic factor-vitamin B12, is a novel type of high molecular weight receptor consisting of a 27 CUB (complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domain cluster preceded by 8 epidermal growth factor repeats and a short N-terminal sequence. In...... and surface plasmon resonance analysis of the secreted cubilin fragments showed ligand binding in the CUB domain region. Further dissection of binding-active fragments localized the binding site for intrinsic factor-vitamin B12 to CUB domains 5-8 and a receptor-associated protein-binding site to CUB...

  2. Non-p53 p53RE binding protein, a human transcription factor functionally analogous to P53

    OpenAIRE

    Zeng, Xiaoya; Levine, Arnold J.; Lu, Hua

    1998-01-01

    The transactivation activity of the p53 tumor suppressor protein is critical for regulating cell growth and apoptosis. We describe the identification of a transcription factor that is functionally similar to p53 and contains the same DNA binding and transcription activities specific for the p53 responsive DNA element (p53RE). This protein was highly purified through chromatography from HeLa cell extracts. The purified protein was able to bind specifically to the p53RE derived from a p21waf1 p...

  3. Crp of Streptomyces Coelicolor Is the Third Transcription Factor of the Large Crp-Fnr Superfamily Able to Bind Camp

    OpenAIRE

    Derouaux, Adeline; Dehareng, Dominique; Lecocq, Elke; Halici, Serkan; Nothaft, Harald; Giannotta, Fabrizio; Moutzourelis, Georgios; Dusart, Jean; Devreese, Bart; Titgemeyer, Fritz; Van Beeumen, Jozef; Rigali, Sébastien

    2004-01-01

    The chromosomal inactivation of the unique transcription factor of Streptomyces coelicolor that displays a cyclic-nucleotide-binding domain, Crp(Sco), led to a germination-defective phenotype similar to the mutant of the adenylate cyclase gene (cya) unable to produce cAMP. By means of cAMP affinity chromatography we demonstrate the specific cAMP-binding ability of Crp(Sco), which definitely demonstrate that a Cya/cAMP/Crp system is used to trigger germination in S. coelicolor. However, electr...

  4. Proteolysis of insulin-like growth factor-binding protein-1, -2 and -4 : function of fragments

    OpenAIRE

    Brandt, Katrin

    2012-01-01

    The insulin-like growth factors (IGF-I and IGF-II) stimulate cell growth, survival and differentiation, and have insulin-like activity. A family of six IGF-binding proteins (IGFBP-1 through -6) bind to IGFs with high affinity and modulate their activity in the circulation and locally at cell-surface receptors. IGFBPs can potentiate or inhibit IGF-activity. Proteolysis is recognised as the predominant mechanism for IGF release from IGFBPs. IGFBPs have also IGF-independent effects, through the ...

  5. mRNA decay factor AUF1 binds the internal ribosomal entry site of enterovirus 71 and inhibits virus replication.

    Directory of Open Access Journals (Sweden)

    Jing-Yi Lin

    Full Text Available AU-rich element binding factor 1 (AUF1 has a role in the replication cycles of different viruses. Here we demonstrate that AUF1 binds the internal ribosome entry site (IRES of enterovirus 71 (EV71 and negatively regulates IRES-dependent translation. During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells. AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins. Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.

  6. Discrimination between thermodynamic models of cis-regulation using transcription factor occupancy data.

    Science.gov (United States)

    Zeigler, Robert D; Cohen, Barak A

    2014-02-01

    Many studies have identified binding preferences for transcription factors (TFs), but few have yielded predictive models of how combinations of transcription factor binding sites generate specific levels of gene expression. Synthetic promoters have emerged as powerful tools for generating quantitative data to parameterize models of combinatorial cis-regulation. We sought to improve the accuracy of such models by quantifying the occupancy of TFs on synthetic promoters in vivo and incorporating these data into statistical thermodynamic models of cis-regulation. Using chromatin immunoprecipitation-seq, we measured the occupancy of Gcn4 and Cbf1 in synthetic promoter libraries composed of binding sites for Gcn4, Cbf1, Met31/Met32 and Nrg1. We measured the occupancy of these two TFs and the expression levels of all promoters in two growth conditions. Models parameterized using only expression data predicted expression but failed to identify several interactions between TFs. In contrast, models parameterized with occupancy and expression data predicted expression data, and also revealed Gcn4 self-cooperativity and a negative interaction between Gcn4 and Nrg1. Occupancy data also allowed us to distinguish between competing regulatory mechanisms for the factor Gcn4. Our framework for combining occupancy and expression data produces predictive models that better reflect the mechanisms underlying combinatorial cis-regulation of gene expression. PMID:24288374

  7. DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

    International Nuclear Information System (INIS)

    Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor

  8. Insulin-like growth factor binding protein production and regulation in fetal rat lung cells.

    Science.gov (United States)

    Price, W A; Moats-Staats, B M; D'Ercole, A J; Stiles, A D

    1993-04-01

    Insulin-like growth factor binding proteins (IGFBPs) are expressed in lung from early in gestation and may modulate IGF-stimulated fetal lung cell proliferation and/or differentiation. To begin to define IGFBP production and regulation in lung cells during development, we prepared primary cultures of 19 day gestation fetal rat lung fibroblasts and epithelial cells and identified IGFBPs secreted into medium. Ligand blot analysis of conditioned media (CM) from both cell types demonstrated IGFBP bands of approximately 39,000-45,000, 32,000, 24,000, and 22,000 M(r). These migration characteristics allowed the identification of the 39,000-45,000 M(r) bands as IGFBP-3 and the 24,000 M(r) band as IGFBP-4, while Western immunoblot analyses localized IGFBP-2 to the 32,000 M(r) band and IGFBP-5 to the 22,000 M(r) band. Polymerase chain reaction amplification of cDNAs generated by reverse transcription of fibroblast and epithelial cell RNA using specific oligodeoxynucleotide primers for IGFBPs 1 through 6, demonstrated the presence of amplified products for IGFBP-2, -3, -4, -5, and -6. In both cell types, IGFBP-2 and -3 production was sustained during 48 h of incubation in serum-free medium, whereas IGFBP-4 abundance increased only during the first 6 to 12 h of incubation. CM from fibroblasts and epithelial cells plated at low densities contained a high abundance of IGFBP-2 per microgram cellular DNA compared with cells at higher densities. In contrast, IGFBP-3 and -4 abundance normalized to cell DNA did not change with differing cell densities.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7682822

  9. Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor.

    Science.gov (United States)

    Kozma, Noemi; Halasz, Melinda; Polgar, Beata; Poehlmann, Tobias G; Markert, Udo R; Palkovics, Tamas; Keszei, Marton; Par, Gabriella; Kiss, Katalin; Szeberenyi, Jozsef; Grama, Laszlo; Szekeres-Bartho, Julia

    2006-01-15

    Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor. PMID:16393965

  10. Insulin-like growth factor binding protein-5 influences pancreatic cancer cell growth

    Institute of Scientific and Technical Information of China (English)

    Sarah K Johnson; Randy S Haun

    2009-01-01

    AIM: To investigate the functional significance of insulin-like growth factor binding protein-5 (IGFBP-5) overexpression in pancreatic cancer (PaC).METHODS: The effects of IGFBP-5 on cell growth were assessed by stable transfection of BxPC-3 and PANC-1 cell lines and measuring cell number and DNA synthesis. Alterations in the cell cycle were assessed by flow cytometry and immunoblot analyses.Changes in cell survival and signal transduction were evaluated after mitogen activated protein kinase and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.RESULTS: After serum depr ivat ion, IGFBP-5 expression increased both cell number and DNA synthesis in BxPC-3 cells, but reduced cell number in PANC-1 cells. Consistent with this observation, cell cycle analysis of IGFBP-5-expressing cells revealed accelerated cell cycle progression in BxPC-3 and G2/M arrest of PANC-1 cells. Signal transduction analysis revealed that Akt activation was increased in BxPC-3, but reduced in PANC-1 cells that express IGFBP-5. Inhibition of PI3K with LY294002 suppressed extracellular signal-regulated kinase-1 and -2 (ERK1/2) activation in BxPC-3, but enhanced ERK1/2 activation in PANC-1 cells that express IGFBP-5. When MEK1/2 was blocked, Akt activation remained elevated in IGFBP-5 expressing PaC cells; however, inhibition of PI3K or MEK1/2 abrogated IGFBP-5-mediated cell survival.CONCLUSION: These results indicate that IGFBP-5 expression affects the cell cycle and survival signal pathways and thus it may be an important mediator of PaC cell growth.

  11. Chromatin remodeling occurs independent of transcription factor binding during 5-azacytidine reactivation of the human HPRT gene

    Energy Technology Data Exchange (ETDEWEB)

    Hornstra, L.K.; Litt, M.D.; Yang, T.P. [Univ. of Florida College of Medicine, Gainesville, FL (United States)] [and others

    1994-09-01

    A novel system of differential gene expression in mammals is established during normal female embryogenesis by X chromosome inactivation. Studies of 5-aza-2{prime}-deoxycytidine (5aCdr)-induced reactivation of genes on the inactive human X chromosome strongly implicate DNA methylation in maintaining the transcriptional repression of discrete loci on the inactive X. During the process of 5aCdr-induced reactivation of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the inactive X chromosome, changes in nuclease sensitivity of chromatin in the 5{prime} region of the HPRT gene and HPRT mRNA levels have been analyzed from 0-72 hrs. after 5aCdr exposure. Increased nuclease sensitivity is first detectable at 6 hrs. and reaches a maximum at 24 hrs. after initial exposure to 5aCdr, while the appearance of HPRT mRNA levels is first detectable by RT-PCR at 24 hrs. and reaches a maximum of 48 hrs. after 5aCdr exposure. Thus, the change in chromatin structure of the 5{prime} region as a result of 5aCdr treatment appears to occur prior to active transcription of the gene. However, it is unclear if the remodeling of chromatin requires the binding of transcription factors to the 5{prime} region, or if the binding of transcription factors is only required for transcription of the HPRT gene. We now have assayed the binding of transcription factors to the 5{prime} region of the HPRT gene on the inactive X chromosome during 5aCdr reactivation. We find that the change in chromatin structure as a result of 5aCdr treatment occurs independent of transcription factor binding, and that the binding of factors is correlated with active transcription of the gene rather than remodeling of chromatin structure. These data suggest that the differential binding of transcriptional activators (and differential expression of the HPRT gene) to the active and inactive HPRT genes is modulated by the accessibility of their binding sites due to chromatin structure.

  12. Klebsiella 'modifying factor': binding studies with HLA-B27+ and B27- lymphocytes.

    OpenAIRE

    Trapani, J A; McKenzie, I. F.

    1985-01-01

    On the basis that extracts of some klebsiella organisms bind selectively to the lymphocytes of HLA-B27+ individuals and induce the appearance of new antigens, attempts were made to detect the binding of klebsiella products to HLA-B27+ and B27- lymphocytes by a number of different techniques. Firstly, blocking of the binding of two different HLA-B27 specific monoclonal antibodies to HLA-B27+ lymphocytes has been examined following exposure of the lymphocytes to a cell-free culture filtrate fro...

  13. Coupling of folding and DNA-binding in the bZIP domains of Jun-Fos heterodimeric transcription factor.

    Science.gov (United States)

    Seldeen, Kenneth L; McDonald, Caleb B; Deegan, Brian J; Farooq, Amjad

    2008-05-01

    In response to mitogenic stimuli, the heterodimeric transcription factor Jun-Fos binds to the promoters of a diverse array of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryogenic development and cancer. In so doing, Jun-Fos heterodimer regulates gene expression central to physiology and pathology of the cell in a specific and timely manner. Here, using the technique of isothermal titration calorimetry (ITC), we report detailed thermodynamics of the bZIP domains of Jun-Fos heterodimer to synthetic dsDNA oligos containing the TRE and CRE consensus promoter elements. Our data suggest that binding of the bZIP domains to both TRE and CRE is under enthalpic control and accompanied by entropic penalty at physiological temperatures. Although the bZIP domains bind to both TRE and CRE with very similar affinities, the enthalpic contributions to the free energy of binding to CRE are more favorable than TRE, while the entropic penalty to the free energy of binding to TRE is smaller than CRE. Despite such differences in their thermodynamic signatures, enthalpy and entropy of binding of the bZIP domains to both TRE and CRE are highly temperature-dependent and largely compensate each other resulting in negligible effect of temperature on the free energy of binding. From the plot of enthalpy change versus temperature, the magnitude of heat capacity change determined is much larger than that expected from the direct association of bZIP domains with DNA. This observation is interpreted to suggest that the basic regions in the bZIP domains are largely unstructured in the absence of DNA and only become structured upon interaction with DNA in a coupled folding and binding manner. Our new findings are rationalized in the context of 3D structural models of bZIP domains of Jun-Fos heterodimer in complex with dsDNA oligos containing the TRE and CRE consensus sequences. Taken together, our study demonstrates that enthalpy is

  14. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

    International Nuclear Information System (INIS)

    Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map the distribution on frozen skin sections of an extracellular epitope on the EGF receptor. The [125I]EGF binding experiments showed accessible, unoccupied EGF receptors to be present on the epidermal basal cells (with reduced binding to spinous cells), the basal cells of the hair shaft and sebaceous gland, the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing presumptive cortex cells, excluding the medulla, lying around and above the upper dermal papilla of anagen hair follicles, epithelial cells around the lower dermal papilla region, and in some tissue samples the cell margins of the viable differentiating layers of the epidermis. In a control study, to clarify whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar, indicating that, at least for SVK14 cells, EGF-R1 binding provides a reliable marker for EGF binding. Explanations for the discrepancies between these two methods for determining EGF receptor distribution in human skin are discussed

  15. /sup 125/I-human epidermal growth factor specific binding to placentas and fetal membranes from varoius pregnancy states

    Energy Technology Data Exchange (ETDEWEB)

    Hofmann, G.E.; Siddiqi, T.A.; Rao, Ch. V.; Carman, F.R.

    1988-01-01

    Specific binding of /sup 125/I-human epidermal growth factor (hEGF) to homogenates of term human placentas and fetal membranes from normal and appropriate for gestational age (N = 20), intrauterine growth retarded (N = 9), twin (N = 11), White class AB diabetic (N = 12), and large for gestational age (N = 13) pregnancies was measured. In all pregnancy states, placentas bound approximately four times more /sup 125/I-hEGF than did fetal membranes (P<0.0001). There was no significant differnce in /sup 125/I-hEGF binding to fetal membranes from the various pregnancy states (P<0.05). /sup 125/I-hEGF specific binding to placentas from intrauterine growth retarded or twin pregnancies was significantly greater compared with placentas from normal and appropriate for gestational age pregnancies (P<0.05). The binding to placentas from pregnancies complicated by White class AB diabetes or large for gestational age infants, on the other hand, was not significantly different from that to placentas from normal and appropriate for gestational age pregnancies. /sup 125/I-hEGF specific binding did not differ between placentas from intrauterine growth retarded or twin pregnancies (P<0.05). Placental and fetal membrane /sup 125/I-hEGF binding did not vary with fetal sex, maternal race, placental weight, or gestational age between 37 to 42 weeks (P<0.05). Placental but not fetal membrane /sup 125/I-hEGF binding increased with increasing infant weight when appropriate for gestational age and large for gestational age infants were included (P<0.05, r = 0.38, N = 32) but not for intrauterine growth retarded, appropriate for gestational age, or large for gestational age infants alone.

  16. Binding of Streptococcus mutans SR protein to human monocytes: production of tumor necrosis factor, interleukin 1, and interleukin 6.

    Science.gov (United States)

    Soell, M; Holveck, F; Schöller, M; Wachsmann, R D; Klein, J P

    1994-05-01

    To examine the possible implication of protein SR, an I/II-related antigen from Streptococcus mutans OMZ 175 (serotype f), in inflammatory reactions, we tested the immunomodulatory effects of protein SR on human monocytes. Using biotinylated protein, we provide evidence that protein SR binds to human monocytes in dose-, time-, and calcium-dependent manners through specific interactions. These results were confirmed by competition experiments using either soluble human monocyte extract or anti-SR immunoglobulin G. Binding occurred through lectin-like interactions between SR and carbohydrate portions of monocyte membrane glycoproteins, since binding could be inhibited by several sugars, especially fucose and N-acetylneuraminic acid (NANA), which were confirmed by ligand blotting to be the primer ligands recognized by SR on human monocyte extracts. The ability of protein SR to stimulate the production of cytokines by human circulating monocytes was then examined. The release of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta, and interleukin 6 is time and dose dependent and not affected by the addition of polymyxin B. Activation of monocytes resulted from specific binding of SR to NANA and fucose present on cell surface glycoproteins since TNF-alpha release could be inhibited by sialidase and pronase treatment of monocytes and by NANA and fucose. These results confirm that sialic acid and fucose present on cell surface macromolecules and especially glycoproteins are needed for the binding of SR to monocytes and for the release of TNF-alpha. PMID:8168943

  17. Selectivity in glycosaminoglycan binding dictates the distribution and diffusion of fibroblast growth factors in the pericellular matrix.

    Science.gov (United States)

    Sun, Changye; Marcello, Marco; Li, Yong; Mason, David; Lévy, Raphaël; Fernig, David G

    2016-03-01

    The range of biological outcomes generated by many signalling proteins in development and homeostasis is increased by their interactions with glycosaminoglycans, particularly heparan sulfate (HS). This interaction controls the localization and movement of these signalling proteins, but whether such control depends on the specificity of the interactions is not known. We used five fibroblast growth factors with an N-terminal HaloTag (Halo-FGFs) for fluorescent labelling, with well-characterized and distinct HS-binding properties, and measured their binding and diffusion in pericellular matrix of fixed rat mammary 27 fibroblasts. Halo-FGF1, Halo-FGF2 and Halo-FGF6 bound to HS, whereas Halo-FGF10 also interacted with chondroitin sulfate/dermatan sulfate, and FGF20 did not bind detectably. The distribution of bound FGFs in the pericellular matrix was not homogeneous, and for FGF10 exhibited striking clusters. Fluorescence recovery after photobleaching showed that FGF2 and FGF6 diffused faster, whereas FGF1 diffused more slowly, and FGF10 was immobile. The results demonstrate that the specificity of the interactions of proteins with glycosaminoglycans controls their binding and diffusion. Moreover, cells regulate the spatial distribution of different protein-binding sites in glycosaminoglycans independently of each other, implying that the extracellular matrix has long-range structure. PMID:27009190

  18. Cellular receptor for 125I-labeled tumor necrosis factor: specific binding, affinity labeling, and relationship to sensitivity

    International Nuclear Information System (INIS)

    Tumor necrosis factor (TNF) is a proteinaceous toxin shed by stimulated myeloid cells. Murine TNF was radioiodinated to a specific activity of 1 mCi/nmol (1 Ci = 37 GBq) of monomer. 125I-labeled TNF (125-TNF) retained complete cytotoxic activity and it was immunochemically identical to the native toxin in a quantitative immunoprecipitation assay. It could be shown by competition binding that 125I-TNF bound to intact L929 cells with a specificity equal to that of native toxin. The conditions of time, temperature, and concentration involved in equilibrium specific binding to intact cells were studied in detail. J774.1 cells, the source of the toxin, demonstrated similar binding but were not sensitive to 125I-TNF cytotoxicity. Normal lymphoid organ cell suspensions and two human tumorigenic cell lines were not sensitive and failed to demonstrate specific binding. 125I-TNF, covalently cross-linked to its receptor on sensitive L-M cells with disuccinimidyl suberate, was isolated and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography. Two specific bands were identified. The presence of the binding site appears to be necessary but not sufficient to explain the sensitivity of cells to the cytotoxic action of TNF

  19. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    International Nuclear Information System (INIS)

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1, Val1, Asn2, Gln3, His4, Ser8, His9, Glu12, Tyr15, Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3, Ala4] IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15, Leu16] IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, [Gln3, Ala4, Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I

  20. Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.

    1996-01-01

    Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination of th...

  1. The influence of experimental conditions on the association constant for binding of vitamin B12 by human intrinsic factor coupled to Sepharose 4B

    International Nuclear Information System (INIS)

    The association constant for binding of vitamin B12 (cyanocobalamin) by human intrinsic factor coupled to Sepharose 4B and the concentration of binding sites of the coupled intrinsic factor have been measured as a function of pH, temperature, ionic strength and the presence of various (ionic and non-ionic) components in the solution. (author)

  2. Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

    Science.gov (United States)

    Sorrentino, Luca; Calogero, Alessandra Maria; Pandini, Vittorio; Vanoni, Maria Antonietta; Sevrioukova, Irina F; Aliverti, Alessandro

    2015-12-01

    Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide. PMID:26535916

  3. Isolation and characterization of a factor from rat liver which inhibits 3H-nicotine binding to brain nicotine receptors

    International Nuclear Information System (INIS)

    In studies of 3H-nicotine binding sites in rat brain membranes, it was observed that crude rat liver homogenates are capable of inhibiting this binding. The purpose of this work was to isolate from the liver homogenate the factor (or factors) responsible for this nicotinelike activity and to identify it. Isolation procedures, including heat denaturation, ultrafiltration, and reverse phase high pressure liquid chromatography, resulted in an incompletely purified substance whose chemical properties were compatible with those of dimethylaminoethyl (DMAE) carbamate, as reflected in infrared, ultraviolet, NMR, and mass spectra; in HPLC elution characteristics into mobile phases; and in IC50's with respect to 3H-nicotine binding to neural membranes, although additional purification and characterization will be necessary to confirm or reject this identification. This compound has not previously been described in mammalian tissue, although its two component functional moieties, dimethylaminoethanol and carbamic acid, are present in the liver. The binding properties of DMAE carbamate are compared with those of structurally and pharmacological related compounds to assess its potential as an endogenous cholinergic ligand

  4. Structure of the fMet-tRNA(fMet)-binding domain of B. stearothermophilus initiation factor IF2.

    Science.gov (United States)

    Meunier, S; Spurio, R; Czisch, M; Wechselberger, R; Guenneugues, M; Gualerzi, C O; Boelens, R

    2000-04-17

    The three-dimensional structure of the fMet-tRNA(fMet) -binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel beta-strands, connected via loops, and forms a closed beta-barrel similar to domain II of elongation factors EF-Tu and EF-G, despite low sequence homology. Two structures of the ternary complexes of the EF-Tu small middle dotaminoacyl-tRNA small middle dot GDP analogue have been reported and were used to propose and discuss the possible fMet-tRNA(fMet)-binding site of IF2. PMID:10775275

  5. Accuracy of PET rCBF measurements: Effect of transit time delay

    International Nuclear Information System (INIS)

    Analytic expressions were derived for estimating the error in PET rCBF measurements associated with the time lag between brain and blood radioactivity (1) following 0-15 water injection and (2) during non-steady-state 0-15 CO/sub 2/ inhalation. This lag time reflects the physiological difference in arrival times of 0-15 activity at brain and radial arterial sampling site as well as the experimentally introduced resistance to flow offered by the arterial catheter/stopcock assembly. Multiple measurements of transit time delay were made in 2 patients using Rb-82. The arrival of radioactivity in the brain was detected by a pair of PET detectors operating in coincidence. The arrival of radioactivity at the radial arterial catheter was estimated from consecutive 5-sec blood samples (catheter flow rate 7-10 ml/min). Transit time delays varied between 1 and 8 sec. For non-steady-state 0-15 CO/sub 2//PET measurements, estimated errors in rCBF ranged from 0.02 to 30% for delays of 2-8 sec and scan lengths of 30-180 sec. In the range 20-100 ml/min/100 g, variations in rCBF only marginally affected these errors. Errors increased with scan length and with longer delays but decreased sharply with scan duration > 60 sec. For 30-180 sec scans, even larger errors are associated with the 0-15 water injection technique (peak blood activity at 10 sec): 1-60% for delays of 2-8 sec. A ''slow'' bolus peaking at 20 sec decreased the error by 40%. For the 0-15 water method it is essential to determine the transit time delay to within 2 sec if accurate flow measurements (error < 5%) are to be obtained from 40-60 sec scans

  6. Insulin-like growth factor-binding protein-3 inhibition of prostate cancer growth involves suppression of angiogenesis.

    Science.gov (United States)

    Liu, B; Lee, K-W; Anzo, M; Zhang, B; Zi, X; Tao, Y; Shiry, L; Pollak, M; Lin, S; Cohen, P

    2007-03-15

    Insulin-like growth factor-binding protein-3 (IGFBP-3) is a multifunctional protein that induces apoptosis utilizing both insulin-like growth factor receptor (IGF)-dependent and -independent mechanisms. We investigated the effects of IGFBP-3 on tumor growth and angiogenesis utilizing a human CaP xenograft model in severe-combined immunodeficiency mice. A 16-day course of IGFBP-3 injections reduced tumor size and increased apoptosis and also led to a reduction in the number of vessels stained with CD31. In vitro, IGFBP-3 inhibited both vascular endothelial growth factor- and IGF-stimulated human umbilical vein endothelial cells vascular network formation in a matrigel assay. This action is primarily IGF independent as shown by studies utilizing the non-IGFBP-binding IGF-1 analog Long-R3. Additionally, we used a fibroblast growth factor-enriched matrigel-plug assay and chick allantoic membrane assays to show that IGFBP-3 has potent antiangiogenic actions in vivo. Finally, overexpression of IGFBP-3 or the non-IGF-binding GGG-IGFBP-3 mutant in Zebrafish embryos confirmed that both IGFBP-3 and the non-IGF-binding mutant inhibited vessel formation in vivo, indicating that the antiangiogenic effect of IGFBP-3 is an IGF-independent phenomenon. Together, these studies provide the first evidence that IGFBP-3 has direct, IGF-independent inhibitory effects on angiogenesis providing an additional mechanism by which it exerts its tumor suppressive effects and further supporting its development for clinical use in the therapy of patients with prostate cancer. PMID:16983336

  7. Effects of Vitamin D-binding Protein-derived Macrophage-activating Factor on Human Breast Cancer Cells

    OpenAIRE

    S. Pacini; T.Punzi; G.Morucci; Gulisano, M; Ruggiero, M

    2012-01-01

    BACKGROUND: Searching for additional therapeutic tools to fight breast cancer, we investigated the effects of vitamin D-binding protein-derived macrophage activating factor (DBP-MAF, also known as GcMAF) on a human breast cancer cell line (MCF-7). MATERIALS AND METHODS: The effects of DBP-MAF on proliferation, morphology, vimentin expression and angiogenesis were studied by cell proliferation assay, phase-contrast microscopy, immunohistochemistry and western blotting, and chorioallantoic m...

  8. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein α-deficient mice

    OpenAIRE

    Zhang, Dong-Er; Zhang, Pu; Wang, Nai-dy; Hetherington, Christopher J.; Darlington, Gretchen J.; Tenen, Daniel G.

    1997-01-01

    Transcription factors are master regulatory switches of differentiation, including the development of specific hematopoietic lineages from stem cells. Here we show that mice with targeted disruption of the CCAAT enhancer binding protein α gene (C/EBPα) demonstrate a selective block in differentiation of neutrophils. Mature neutrophils and eosinophils are not observed in the blood or fetal liver of mutant animals, while other hematopoietic lineages, including monocytes, are not affected. Inste...

  9. JASPAR, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update

    OpenAIRE

    Bryne, Jan Christian; Valen, Eivind; Tang, Man-Hung Eric; Marstrand, Troels; Winther, Ole; da Piedade, Isabelle; Krogh, Anders; Lenhard, Boris; Sandelin, Albin

    2007-01-01

    JASPAR is a popular open-access database for matrix models describing DNA-binding preferences for transcription factors and other DNA patterns. With its third major release, JASPAR has been expanded and equipped with additional functions aimed at both casual and power users. The heart of the JASPAR database—the JASPAR CORE sub-database—has increased by 12% in size, and three new specialized sub-databases have been added. New functions include clustering of matrix models by similarity, generat...

  10. oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes

    OpenAIRE

    Ho Sui, Shannan J; Mortimer, James R.; Arenillas, David J.; Brumm, Jochen; Walsh, Christopher J.; Kennedy, Brian P; Wasserman, Wyeth W.

    2005-01-01

    Targeted transcript profiling studies can identify sets of co-expressed genes; however, identification of the underlying functional mechanism(s) is a significant challenge. Established methods for the analysis of gene annotations, particularly those based on the Gene Ontology, can identify functional linkages between genes. Similar methods for the identification of over-represented transcription factor binding sites (TFBSs) have been successful in yeast, but extension to human genomics has la...

  11. IGF binding protein 2 is a cell-autonomous factor supporting survival and migration of acute leukemia cells

    OpenAIRE

    Chen, Xiaoli; Zheng, Junke; Zou, Yizhou; Song, Chun; Hu, Xuemei; Zhang, Cheng Cheng

    2013-01-01

    Background The role of IGF binding protein 2 (IGFBP2) in cancer development is intriguing. Previously we identified IGFBP2 as an extrinsic factor that supports the activity of hematopoietic stem cells (HSCs). Methods and results Here we investigated the role of IGFBP2 in in human leukemia cells and in the retroviral AML1-ETO9a transplantation acute myeloid leukemia (AML) mouse model. Results IGFBP2 is highly expressed in certain human AML and acute lymphoblastic leukemia (ALL) cells. Inhibiti...

  12. Insulin-like growth factor-binding protein 5 (Igfbp5) compromises survival, growth, muscle development, and fertility in mice

    OpenAIRE

    Salih, Dervis A. M.; Tripathi, Gyanendra; Holding, Cathy; Szestak, Tadge A. M.; Gonzalez, M. Ivelisse; Carter, Emma J.; Cobb, Laura J.; Eisemann, Joan E.; Pell, Jennifer M.

    2004-01-01

    The insulin-like growth factors (IGFs) are essential for development; bioavailable IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). Igfbp5 is the most conserved and is developmentally up-regulated in key lineages and pathologies; in vitro studies suggest that IGFBP-5 functions independently of IGF interaction. Genetic ablation of individual Igfbps has yielded limited phenotypes because of substantial compensation by remaining family members. Therefore, to reveal Igfbp5 a...

  13. Interaction of Insulin-like Growth Factor-binding Protein-3 and BAX in Mitochondria Promotes Male Germ Cell Apoptosis

    OpenAIRE

    Jia, Yue; Lee, Kuk-Wha; Swerdloff, Ronald; Hwang, David; Cobb, Laura J.; Sinha Hikim, Amiya; Lue, Yan He; Cohen, Pinchas; Wang, Christina

    2009-01-01

    Germ cell apoptosis is crucial for spermatogenesis and can be triggered by various stimuli, including intratesticular hormone deprivation. This study proposes a role for insulin-like growth factor binding protein-3 (IGFBP-3) in male germ cell apoptosis. Groups of adult Sprague-Dawley male rats received one of the following treatments for 5 days: (i) daily intratesticular (IT) injections with saline (control); (ii) a single subcutaneous injection of the gonadotropin-releasing hormone antagonis...

  14. Phylogenetic Analysis of the Insulin-like Growth Factor Binding Protein (IGFBP) and IGFBP-related Protein Gene Families

    OpenAIRE

    Rodgers, Buel D.; Roalson, Eric H.; Thompson, Cullen

    2007-01-01

    Insulin-like growth factor (IGF) activity is regulated by six high affinity binding proteins (IGFBPs) and possibly by some of the nine IGFBP-related proteins (IGFBP-rPs). To determine the phylogenetic relationship of this proposed gene superfamily, we conducted maximum likelihood (ML) and Bayesian inference analyses on a matrix of amino acid sequences from a diversity of vertebrate species. A single most likely phylogram, ML bootstrap, and Bayesian consensus tree of 10,000,000 generations rev...

  15. A selective, slow binding inhibitor of factor VIIa binds to a nonstandard active site conformation and attenuates thrombus formation in vivo.

    Science.gov (United States)

    Olivero, Alan G; Eigenbrot, Charles; Goldsmith, Richard; Robarge, Kirk; Artis, Dean R; Flygare, John; Rawson, Thomas; Sutherlin, Daniel P; Kadkhodayan, Saloumeh; Beresini, Maureen; Elliott, Linda O; DeGuzman, Geralyn G; Banner, David W; Ultsch, Mark; Marzec, Ulla; Hanson, Stephen R; Refino, Canio; Bunting, Stuart; Kirchhofer, Daniel

    2005-03-11

    The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo. PMID:15632123

  16. Nuclear factor I acts as a transcription factor on the MMTV promoter but competes with steroid hormone receptors for DNA binding.

    OpenAIRE

    Brüggemeier, U; Rogge, L.; Winnacker, E L; Beato, M

    1990-01-01

    Several steroid hormones induce transcription of the mouse mammary tumor virus (MMTV) promoter, through an interaction of their respective receptors with the hormone responsive elements (HREs) in the long terminal repeat (LTR) region. The molecular mechanism underlying transcriptional activation is not known, but binding of nuclear factor I (NFI) to a site adjacent to the HRE appears to be required for efficient transcription of the MMTV promoter. In JEG-3 choriocarcinoma cells the MMTV promo...

  17. Serum insulin-like growth factor-binding protein-2 levels and metabolic and cardiovascular risk factors in young adults and children born small for gestational age

    OpenAIRE

    Kort, Sandra; Doorn, Jaap van; Sande, Ad; Leunissen, Ralph; Hokken-Koelega, Anita

    2010-01-01

    textabstractBackground: IGF binding protein (IGFBP)-2 might protect against cardiovascular disease. Small for gestational age (SGA) birth could be associated with a higher risk for type 2 diabetes mellitus and cardiovascular disease in later life. No data are available on the relationship between serum IGFBP-2 levels and cardiovascular risk factors in young adults and children born SGA. Objective: The aim of the study was to determine circulating IGFBP-2 levels in subjects born SGA and to inv...

  18. Thrombin binds to murine bone marrow-derived macrophages and enhances colony-stimulating factor-1-driven mitogenesis

    International Nuclear Information System (INIS)

    The binding and mitogenic properties of thrombin have been established in various transformed cell lines. In such systems, thrombin induces cell division in the absence of exogenous growth factors, and the enzyme is considered to act directly as a mitogen. This study explores thrombin's interaction with nontransformed, growth factor-dependent cells. Binding of 125I-alpha-thrombin to colony-stimulating factor (CSF)-1-dependent bone marrow-derived macrophages is saturable, time-dependent, and displaceable by both unlabeled alpha-thrombin, and esterolytically inactive thrombin. Both dissociation studies of pre-bound radio-labeled thrombin and Scatchard analysis assisted by the program Ligand suggest adherence of thrombin-binding data to a multi-site model. There are an estimated 2 x 10(4) high affinity sites (Kd = 7 x 10(-9)M) and 2 x 10(6) low affinity sites (Kd = 9 x 10(-7)M) per cell. Quiescent bone marrow-derived macrophages were cultured with either 10(-8)M thrombin, 1000 units of CSF-1/ml, or both and [3H]thymidine incorporation was determined. Thrombin alone did not induce mitogenesis. CSF-1 induced mitogenesis with peak [3H] thymidine incorporation occurring 24 h after addition of the mitogen. This CSF-1-dependent mitogenic influence was enhanced greater than 2-fold by treatment with thrombin

  19. Integration host factor and LuxR synergistically bind DNA to coactivate quorum-sensing genes in Vibrio harveyi.

    Science.gov (United States)

    Chaparian, Ryan R; Olney, Stephen G; Hustmyer, Christine M; Rowe-Magnus, Dean A; van Kessel, Julia C

    2016-09-01

    The cell-cell signaling process called quorum sensing allows bacteria to control behaviors in response to changes in population density. In Vibrio harveyi, the master quorum-sensing transcription factor LuxR is a member of the TetR family of transcription factors that both activates and represses genes to coordinate group behaviors, including bioluminescence. Here, we show that integration host factor (IHF) is a key coactivator of the luxCDABE bioluminescence genes that is required together with LuxR for precise timing and expression levels of bioluminescence during quorum sensing. IHF binds to multiple sites in the luxCDABE promoter and bends the DNA in vitro. IHF and LuxR synergistically bind luxCDABE promoter DNA at overlapping, essential binding sites that are required for maximal gene expression in vivo. RNA-seq analysis demonstrated that IHF regulates 300 genes in V. harveyi, and among these are a core set of 19 genes that are also directly bound and regulated by LuxR. We validated these global analyses by demonstrating that both IHF and LuxR are required for transcriptional activation of the osmotic stress response genes betIBA-proXWV. These data suggest that IHF plays an integral role in one mechanism of transcriptional activation by the LuxR-type family of quorum-sensing regulators in vibrios. PMID:27191515

  20. Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs

    Science.gov (United States)

    Ohmura, Sakie; Mizuno, Seiya; Oishi, Hisashi; Ku, Chia-Jui; Hermann, Mary; Hosoya, Tomonori; Takahashi, Satoru; Engel, James Douglas

    2016-01-01

    The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell–specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte–specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte–specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell–regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell–specific transcriptional activity. PMID:26808502

  1. Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs.

    Science.gov (United States)

    Ohmura, Sakie; Mizuno, Seiya; Oishi, Hisashi; Ku, Chia-Jui; Hermann, Mary; Hosoya, Tomonori; Takahashi, Satoru; Engel, James Douglas

    2016-03-01

    The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell-specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte-specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte-specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell-regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell-specific transcriptional activity. PMID:26808502

  2. Effect of antemortem and postmortem factors on [3H]MK-801 binding in the human brain: Transient elevation during early childhood

    International Nuclear Information System (INIS)

    The effect of a number of antemortem and postmortem factors on [3H]MK-801 binding was investigated under equilibrium conditions in the frontal cortex of human brains of 38 controls. Binding values transiently increased during the early postnatal period reaching a maximum at the age of about 2 years. After age 10 years [3H]MK-801 binding sites disappeared at 5.7% per decade. The storage time of brain tissue had a reducing effect on these binding sites. There was no effect of gender, brain weight or postmortem time interval and the binding sites were bilaterally symmetrically distributed in the frontal cortex

  3. The presence of a pregnenolone-binding factor in the copulatory organ of the migratory locust, Locusta migratoria migratorioides R. & F.

    Science.gov (United States)

    Paesen, G; Novak, F; Swevers, L; De Clerck, D; De Loof, A

    1988-12-01

    The presence of binding sites for nonecdysteroid steroids was investigated in the cytosol of several tissues of the migratory locust, Locusta migratoria migratorioides. Binding of androgens was not observed. Most tissues, however, showed nonsaturable binding of estrogens and in some tissues saturable progestin binding could be demonstrated. A pregnenolone binder, that was found to be present in the male copulatory organ, was further studied. It showed a dissociation constant of 4.4 (+/- 1.6) X 10(-8) M. This is the first report of a nonecdysteroid steroid-binding factor in an insect tissue. PMID:3240850

  4. Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2).

    OpenAIRE

    Binkert, C; Landwehr, J; Mary, J L; J. Schwander; Heinrich, G

    1989-01-01

    Insulin-like growth factors bind with high affinity to specific binding proteins in extracellular fluids. To identify structural characteristics of IGF-binding proteins that might define their physiological roles, we determined the complete primary structure of a novel human IGF-binding protein (IGFBP-2) from a cloned cDNA. The cDNA encodes a 328 amino acid IGF-binding protein precursor which contains a 39-residue signal peptide. The mature 289 amino acid IGFBP-2 has a predicted Mr of 31,325....

  5. Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor

    International Nuclear Information System (INIS)

    Epidermal growth factor receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization

  6. DIFFERENT REQUIREMENTS OF THE KINASE AND UHM DOMAINS OF KIS FOR ITS NUCLEAR LOCALIZATION AND BINDING TO SPLICING FACTORS

    OpenAIRE

    Manceau, Valérie; Kielkopf, Clara L.; Sobel, André; Maucuer, Alexandre

    2008-01-01

    The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF Homology Motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA splicing factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF65. KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF65 and the 3′ splice site, an event known to take place at ...

  7. Microphysiometry Studies of Rapid Binding of Insulin-Like Growth Factor I by Parental and Transfected Mammary Epithelial Cell Lines

    OpenAIRE

    Robinson, Rose Marie

    1998-01-01

    Breast cancer is a leading cause of cancer death of women in the U.S. today. Members of the family of insulin-like growth factors (IGFs) are proposed to play a major role in the development and subsequent uncontrolled proliferation of breast cancer cells. Insulin-like growth factor-I (IGF-I) is known to be a potent mitogen for mammary epithelial cells. IGF-I acts by binding to cell surface receptors, thereby stimulating a cascade of events leading to cell division. In the interest of inte...

  8. Structure of the fMet-tRNAfMet-binding domain of B.stearothermophilus initiation factor IF2

    OpenAIRE

    Meunier, Sylvie; Spurio, Roberto; Czisch, Michael; Wechselberger, Rainer; Guenneugues, Marc; GUALERZI, CLAUDIO O.; Boelens, Rolf

    2000-01-01

    The three-dimensional structure of the fMet-tRNAfMet -binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel β-strands, connected via loops, and forms a closed β-barrel similar to domain II of elongation factors EF-Tu and EF-G, despite low sequence homology. Two structures of the ternary complexes of the EF-Tu⋅aminoacyl-tRNA⋅ GDP analogue have been reported and were ...

  9. Transcription Factors KLF1 and KLF2 Positively Regulate Embryonic and Fetal β-Globin Genes through Direct Promoter Binding*

    OpenAIRE

    Yousef N Alhashem; Vinjamur, Divya S.; Basu, Mohua; Klingmüller, Ursula; Gaensler, Karin M. L.; Lloyd, Joyce A.

    2011-01-01

    Krüppel-like factors (KLFs) control cell differentiation and embryonic development. KLF1 (erythroid Krüppel-like factor) plays essential roles in embryonic and adult erythropoiesis. KLF2 is a positive regulator of the mouse and human embryonic β-globin genes. KLF1 and KLF2 have highly homologous zinc finger DNA-binding domains. They have overlapping roles in embryonic erythropoiesis, as demonstrated using single and double KO mouse models. Ablation of the KLF1 or KLF2 gene causes embryonic le...

  10. The Role of Insulin-like Growth Factor-I and IGF-binding Proteins in Mammary Gland Development

    OpenAIRE

    Weber, Miriam S

    1998-01-01

    Development of the mammary gland is likely mediated by locally produced growth factors acting in concert with circulating mitogens. To investigate the importance of mammary synthesis of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBP), the initial objective was to evaluate the physiological effects of recombinant IGF-I synthesis in the mouse mammary gland. Expression of recombinant IGF-I was targeted by the mouse mammary tumor virus - long terminal repeat (MMTV-LTR) to ...

  11. Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I

    OpenAIRE

    1993-01-01

    Insulin-like growth factor binding proteins (IGFBPs) have been shown to serve as carrier proteins for the insulin-like growth factors (IGFs) and to modulate their biologic effects. Since extracellular matrix (ECM) has been shown to be a reservoir for IGF-I and IGF-II, we examined the ECM of cultured human fetal fibroblasts and found that IGFBP-5 was incorporated intact into ECM, while mostly inert proteolytic fragments were found in the medium. In contrast, two other forms of IGFBP that are s...

  12. Regulation of insulin-like growth factor binding protein-1 (IGFBP-1) and implications in catabolic conditions

    OpenAIRE

    Lindgren, Björn

    1997-01-01

    This thesis has studied the regulation of IGFBP-1 (insulin-like growth factor binding protein 1), which is one factor regulating the bioavailability of IGF-I with special interest how IGFBP-1 is regulated in vitro and in humans, especially in diabetes and catabolic conditions. The IGFBP-1 cDNA was cloned and used for studies in human hepatoma cells, HepG2, which showed that both insulin and IGF-I could decrease IGFBP-1 in the cell conditioned medium. IGF-I inhibited also IGF...

  13. Regional differences in distribution volume of I-123 IMP in the human brain. Effect on CBF calculated by ARG method

    International Nuclear Information System (INIS)

    Two methods of quantitating cerebral blood flow (CBF) with iodine-123-labeled N-isopropyl-p-iodoamphetamine (I-123 IMP) and a two-compartment model had been proposed; one is the table look-up (TLU) method and the other is the autoradiographic (ARG) method. The TLU method provides values of the cerebral blood flow (CBF) values and distribution volume of I-123 IMP (Vd) independently. In the ARG method, a fixed Vd is applied for the entire brain to calculate CBF. Our purpose was to evaluate regional differences in Vd in the human brain, or possible effects of regional differences in Vd on CBF calculated by the ARG method. In the present study, two SPECT scans were acquired from each of eight normal subjects (aged 44.0±16.7) at 40 min and 180 min of mid-scan-time after intravenous 1 min infusion of 111 MBq IMP. A single arterial blood sampling was performed 10 min after the IMP infusion. All images were anatomically normalized and analyzed with SPM99 and Matlab. We generated CBF and Vd images for each subject by the TLU method and evaluated differences in Vd among brain structures. We subsequently generated another set of CBF images by the ARG method and examined differences between CBF calculated by the TLU method and that by the ARG method. Significant main effects of subject and brain structure in Vd were observed (two-way ANOVA). Vd values were higher in the deep gray matter than in the cerebral cortical regions. Among the cerebral cortical regions, no significant difference in Vd was observed. In spite of the significant differences in Vd among the brain structures, the voxel-by-voxel analyses as well as the ROI analyses revealed no statistically significant difference between CBF calculated by the TLU method and that by the ARG method. Although regional differences in Vd were observed, the present results support the assumption that a fixed Vd does not cause significant error in the calculation of CBF by the ARG method. (author)

  14. Potentiation of Growth Factor Signaling by Insulin-like Growth Factor-binding Protein-3 in Breast Epithelial Cells Requires Sphingosine Kinase Activity*

    OpenAIRE

    Martin, Janet L; Mike Z. Lin; Eileen M. McGowan; Baxter, Robert C.

    2009-01-01

    We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1...

  15. Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies.

    OpenAIRE

    Conover, C A; Kiefer, M C; Zapf, J

    1993-01-01

    Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85% decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This p...

  16. TATA-binding protein-associated factor(s) in TFIID function through the initiator to direct basal transcription from a TATA-less class II promoter.

    OpenAIRE

    Martinez, E; Chiang, C M; Ge, H.; Roeder, R. G.

    1994-01-01

    The RNA polymerase II (Pol II) basal transcription factor TFIID is composed of the TATA box-binding protein (TBP) and several TBP-associated factors (TAFs). TBP is required for Pol II transcription from TATA-containing and TATA-less promoters. TATA-less promoters of mRNA-encoding genes often contain an initiator element at the transcription start site that is sufficient to direct accurate Pol II transcription. Here we address the mechanisms of functional TBP recruitment to the TATA-less initi...

  17. IFN-gamma reduces specific binding of tumor necrosis factor on murine macrophages

    International Nuclear Information System (INIS)

    Because IFN-gamma is the main cytokine activating macrophages and TNF cooperates in this activation, we assessed TNF binding capacity during the course of murine macrophage activation by IFN-gamma. TNF binding to elicited macrophages increased with time, was maximal by 8 h of culture, and required de novo protein synthesis. 125I-TNF bound to about 40,000 sites/cell with a Kd of 1 x 10(-9) M. Cross-linking experiments performed with a bifunctional cross-linking agent revealed a specific band with a m.w. of 94,000. Preincubation of macrophages with IFN-gamma prevented the binding of TNF to receptors. This effect was dose-dependent and maximal at 100 U/ml. IFN-gamma also reduced specific TNF binding to preexisting receptors (50% inhibition in 3 h), but IFN-gamma did not change the internalization rate of TNF. These studies showed that the number of TNF receptors increased on macrophages vs maturation in culture and was negatively controlled by IFN-gamma

  18. The retinoblastoma protein binds to a family of E2F transcription factors

    DEFF Research Database (Denmark)

    Lees, J A; Saito, M; Vidal, M;

    1993-01-01

    retinoblastoma protein in vivo. Finally, E2F-2 and E2F-3 were able to activate transcription of E2F-responsive genes in a manner that was dependent upon the presence of at least one functional E2F binding site. These observations suggest that the E2F activities described previously result from the combined...

  19. Heterogeneous dynamics in DNA site discrimination by the structurally homologous DNA-binding domains of ETS-family transcription factors.

    Science.gov (United States)

    He, Gaofei; Tolic, Ana; Bashkin, James K; Poon, Gregory M K

    2015-04-30

    The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only for DNA complexes of PU.1. Free solution measurements by dynamic light scattering showed PU.1 to be more dynamic than Ets-1; moreover, dynamic changes are strongly coupled to site discrimination by PU.1, but not Ets-1. Interrogation of the protein/DNA interface by DNA footprinting showed similar accessibility to dimethyl sulfate for PU.1/DNA and Ets-1/DNA complexes, indicating that the dynamics of PU.1/DNA complexes reside primarily outside that interface. An information-based analysis of the two homologs' binding motifs suggests a role for dynamic coupling in PU.1's ability to enforce a more stringent sequence preference than Ets-1 and its proximal sequence homologs. PMID:25824951

  20. Theory on the mechanism of rapid binding of transcription factor proteins at specific-sites on DNA

    CERN Document Server

    Murugan, Rajamanickam

    2014-01-01

    We develop revised theoretical ideas on the mechanism by which the transcription factor proteins locate their specific binding sites on DNA faster than the three-dimensional (3D) diffusion controlled rate limit. We demonstrate that the 3D-diffusion controlled rate limit can be enhanced when the protein molecule reads several possible binding stretches of the template DNA via one-dimensional (1D) diffusion upon each 3D-diffusion mediated collision or nonspecific binding event. The overall enhancement of site-specific association rate is directly proportional to the maximum possible sliding length (LA, square root of (6Do/kr) where Do is the 1D-diffusion coefficient and kr is the dissociation rate constant associated with the nonspecific DNA-protein complex) associated with the 1D-diffusion of protein molecule along DNA. Upon considering several possible mechanisms we find that the DNA binding proteins can efficiently locate their cognate sites on DNA by switching across fast-moving, slow-moving and reading sta...

  1. Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

    International Nuclear Information System (INIS)

    The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity

  2. The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding

    Science.gov (United States)

    Velazquez, Gilberto; Sousa, Rui; Brieba, Luis G

    2015-01-01

    Single subunit RNA polymerases have evolved 2 mechanisms to synthesize long transcripts without falling off a DNA template: binding of nascent RNA and interactions with an RNA:DNA hybrid. Mitochondrial RNA polymerases share a common ancestor with T-odd bacteriophage single subunit RNA polymerases. Herein we characterized the role of the thumb subdomain of the yeast mtRNA polymerase gene (RPO41) in complex stability, processivity, and fidelity. We found that deletion and point mutants of the thumb subdomain of yeast mtRNA polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. Mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (Mtf1). The latter suggests that the thumb subdomain is part of an extended binding surface area involved in binding Mtf1. PMID:25654332

  3. Transforming growth factor beta stimulation of biglycan gene expression is potentially mediated by sp1 binding factors

    DEFF Research Database (Denmark)

    Heegaard, Anne-Marie; Xie, Zhongjian; Young, Marian Frances;

    2004-01-01

    construct was co-transfected with Sp1 and Sp3 expression vectors in Sp1-deficient Drosophila Schneider-2 cells, Sp1 induced the transcriptional activity of biglycan. Addition of Sp3 augmented the effect of Sp1 on biglycan gene expression. Induction of biglycan mRNA expression in response to TGF-beta in MG......-63 cells was abrogated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. A mutation in the Sp1 site at -216 to -208 within the -218 biglycan promoter construct substantially diminished the transcriptional up-regulation by TGF-beta(1). Taken together this data shows for the first...

  4. Carotid artery disease in cerebrovasculas asymptomatic volunteerscorrelations with risk factors, CBF and CT findings

    International Nuclear Information System (INIS)

    In an effort to determine the prevalence of carotid artery disease in cerebrovascular asymptomatic volunteers, 125 randomly chosen active and retired employees of the state of Styria, Austria (82 men, 43 women, age 24 - 75, mean 49.6 +/- 10.5 years) were examined. High resolution Duplex-scanning reveales minimal to mild atherosclerotic plaques (<50% diameter stenosis), predominantly in the carotid bifurcation in 37/125 cases (29.6%). When controlling for age and sex, the incidences of hypertension, diabeter mallitus, cardiac disorders, peripheral vascular disease, cigarette smoking and elevated serum cholesterol were not significally different in the sonographycally affected and not affected group. Besides age (p=0.0002) to be significant predictors of the presence of asymptomatic atherosclerotic carotid artery disease. (author). 7 refs.; 2 tabs

  5. A study of cerebral hemodynamics in various cerebrovascular disorders by means of rCBF measurement with single photon emission computed tomography

    International Nuclear Information System (INIS)

    Using single photon emission computed tomography (SPECT) with Xe-133 inhalation method, regional cerebral blood flow (rCBF) was measured for the purpose of analyzing the pathophysiology of various cerebrovascular disorders. Included in this series were 38 normal volunteers (N), 72 patients with ischemic cerebrovascular disease (ICD), 16 with subarachnoid hemorrhage (SAH), 9 with arteriovenous malformation (AVM), 6 with Moyamoya disease (MD), and 4 with hypertensive intracerebral hematoma (HIH). In the N group, rCBF was independent of sex and laterality. Increased rCBF was observed in the frontal region, as compared with other regions. A significantly increased rCBF was observed in the thirties decade of life; the difference in rCBF was, however, not statistically significant above the age of 30 years. In the ICD group, rCBF decreased in association with severer disorder. In cases of severe disorder, a significantly decreased rCBF was observed in the whole area, as compared with the control group. SPECT allowed early detection of decreased rCBF due to vaso-spasm in the SAH group. The groups of AVM, MD, and HIH showed decreased rCBF in the surrounding areas of the lesions. (Namekawa, K.)

  6. Statistical parametric mapping in the detection of rCBF changes in mild Alzheimer's disease

    International Nuclear Information System (INIS)

    Full text: Reduction in temporoparietal regional cerebral blood flow (rCBF) is proportional to the degree of cognitive deficit in patients with Alzheimer's Disease (AD). The characteristic pattern is readily apparent in advanced disease but is often subtle in early stage AD, reducing the clinical value of SPECT in the management of this condition. We have previously reported that Statistical Parametric Mapping (SPM95) revealed significant temporoparietal hypoperfusion when 10 patients with mild AD (classified by the Clinical Dementia Rating Scale) were compared to 10 age matched normals. We have now begun to evaluate the sensitivity and specificity of SPM95 in individuals with mild AD by comparison to our bank of 39 normals (30 female, 9 male, age range 26 to 74, mean age 52). Preliminary results reveal low sensitivity (<40%) when the standard reference region for normalization (i.e. global brain counts) is used. Better results are expected from normalizing to the cerebellum or basal ganglia and this is under investigation. An objective method to improve the accuracy of rCBF imaging for the diagnosis of early AD would be very useful in clinical practice. This study will demonstrate whether SPM can fulfill this role

  7. Functional roles and clinical values of insulin-like growth factor-binding protein-5 in different types of cancers

    Institute of Scientific and Technical Information of China (English)

    G(o)k(c)e Güllü; Sevgi Karabulut; Mustafa Akkiprik

    2012-01-01

    Insulin-like growth factor-binding proteins (IGFBPs) are critical regulators of the mitogenic activity of insulin-like growth factors (IGFs).IGFBP5,one of these IGFBPs,has special structural features,including a nuclear transport domain,heparin-binding motif,and IGF/extracellular matrix/acid-labile subunit-binding sites.Furthermore,IGFBP5 has several functional effects on carcinogenesis and even normal cell processes,such as cell growth,death,motility,and tissue remodeling.These biological effects are sometimes related with IGF (IGF-dependent effects) and sometimes not (IGF-independent effects).The functional role of IGFBP5 is most likely determined in a cell-type and tissue-type specific manner but also depends on cell context,especially in terms of the diversity of interacting proteins and the potential for nuclear localization.Clinical findings show that IGFBP5 has the potential to be a useful clinical biomarker for predicting response to therapy and clinical outcome of cancer patients.In this review,we summarize the functional diversity and clinical importance of IGFBP5 in different types of cancers.

  8. Influence of geometrical factor on binding energy of Cooper pairs in YBa2Cu3O7-δ compound

    Science.gov (United States)

    Ibrahim, Saeed O.; Mustafa, Bassam M.

    2016-03-01

    In this research the influence of geometrical factors on binding energy between Cooper pairs are investigated for the high temperature superconducting (YBa2Cu3O7-δ) compound. This is done by using a model considering that the formation of Cooper pairs happens during tunneling mechanism of holes between the two CuO2 layers in the YBCO crystal which are considered as forming adjacent ridged potential wells. According to this model binding energy is: Eb i n=4/√{2 }m3/2 π2ħ3p ×∫U-0(U -0-E) exp [-2/(d -Rc) ħ √{2 m (U -E ) } ] √{E } d E 0 is a work function, ħ is the Plank's constant, m is the electron mass, and U is the height of the potential barrier, E is the energy, Rc is the electron cloud radius in tunneling direction and d is the distance between the two CuO2 planes. In the above model two effects of geometrical factors were ignored, considering that ridges in the two CuO2 layers as having rectangular shape, but in reality the ridges in the potential well are of triangular shape. Also the model considers the distance between layers as constant but really the distant d is variable due to the triangular geometry. In this work we consider both effects that affect the density of state in the potential well and the tunneling probability. Then binding energy between the Cooper pairs, is calculated.

  9. Poly(A) binding protein abundance regulates eukaryotic translation initiation factor 4F assembly in human cytomegalovirus-infected cells.

    Science.gov (United States)

    McKinney, Caleb; Perez, Cesar; Mohr, Ian

    2012-04-10

    By commandeering cellular translation initiation factors, or destroying those dispensable for viral mRNA translation, viruses often suppress host protein synthesis. In contrast, cellular protein synthesis proceeds in human cytomegalovirus (HCMV)-infected cells, forcing viral and cellular mRNAs to compete for limiting translation initiation factors. Curiously, inactivating the host translational repressor 4E-BP1 in HCMV-infected cells stimulates synthesis of the cellular poly(A) binding protein (PABP), significantly increasing PABP abundance. Here, we establish that new PABP synthesis is translationally controlled by the HCMV-encoded UL38 mammalian target of rapamycin complex 1-activator. The 5' UTR within the mRNA encoding PABP contains a terminal oligopyrimidine (TOP) element found in mRNAs, the translation of which is stimulated in response to mitogenic, growth, and nutritional stimuli, and proteins encoded by TOP-containing mRNAs accumulated in HCMV-infected cells. Furthermore, UL38 expression was necessary and sufficient to regulate expression of a PABP TOP-containing reporter. Remarkably, preventing the rise in PABP abundance by RNAi impaired eIF4E binding to eIF4G, thereby reducing assembly of the multisubunit initiation factor eIF4F, viral protein production, and replication. This finding demonstrates that viruses can increase host translation initiation factor concentration to foster their replication and defines a unique mechanism whereby control of PABP abundance regulates eIF4F assembly. PMID:22431630

  10. Cyclosporine A, FK506, and NIM811 ameliorate prolonged CBF reduction and impaired neurovascular coupling after cortical spreading depression

    DEFF Research Database (Denmark)

    Hansen, Henning Piilgaard; Witgen, Brent Marvin; Rasmussen, Peter;

    2011-01-01

    Cortical spreading depression (CSD) is associated with mitochondrial depolarization, increasing intracellular Ca(2+), and the release of free fatty acids, which favor opening of the mitochondrial permeability transition pore (mPTP) and activation of calcineurin (CaN). Here, we test the hypothesis...... that cyclosporine A (CsA), which blocks both mPTP and CaN, ameliorates the persistent reduction of cerebral blood flow (CBF), impaired vascular reactivity, and a persistent rise in the cerebral metabolic rate of oxygen (CMRO(2)) following CSD. In addition to CsA, we used the specific mPTP blocker NIM......, CMRO(2), and neurovascular and neurometabolic coupling were unaffected by all three drugs under control conditions. NIM811 augmented the rise in CBF observed during CSD. Cyclosporine A and FK506 ameliorated the persistent decrease in CBF after CSD. All three drugs prevented disruption of neurovascular...

  11. Cyclosporine A, FK506, and NIM811 ameliorate prolonged CBF reduction and impaired neurovascular coupling after cortical spreading depression

    DEFF Research Database (Denmark)

    Hansen, Henning Piilgaard; Witgen, Brent Marvin; Rasmussen, Peter;

    2011-01-01

    Cortical spreading depression (CSD) is associated with mitochondrial depolarization, increasing intracellular Ca(2+), and the release of free fatty acids, which favor opening of the mitochondrial permeability transition pore (mPTP) and activation of calcineurin (CaN). Here, we test the hypothesis......811 and the specific CaN blocker FK506. Cortical spreading depression was induced in rat frontal cortex. Electrocortical activity was recorded by glass microelectrodes, CBF by laser Doppler flowmetry, and tissue oxygen tension with polarographic microelectrodes. Electrocortical activity, basal CBF...... coupling after CSD; the rise in CMRO(2) was unchanged. Our data suggest that blockade of mPTP formation and CaN activation may prevent persistent CBF reduction and vascular dysfunction after CSD....

  12. Clinical application of brain SPECT imaging and rCBF measurement in alzeimers diseases and multi-intarcted dementia

    International Nuclear Information System (INIS)

    Brain SPECT imaging and rCBF measurement was performed in 10 normal controls and 25 patients including 17 Alzeimers Diseases (AD) and 8 Multi-intarcted Dementia (MID). The results showed that in SPECT imaging all of the patients visualized regional cerebral blood supply insufficiency, and also there was the presence of its own characteristic changes among AD and MID. Thereby SPECT imaging has important significance for the early and differential diagnosis for both diseases. rCBF can quantify the brain blood flow, it can be used not only for diagnostic purpose but also for the monitoring therapeutic effect during treatment. In combination with SPECT imaging, it was proved that the diagnostic effect can be improved. In conclusion, it was proved that the diagnostic effect can be improved. In conclusion, it is considered that in comparison with X-CT showing only anatomical abnormalities, SPECT and rCBF has better sensitivity and specificity

  13. Determination of relative CMRO2 from CBF and BOLD changes: significant increase of oxygen consumption rate during visual stimulation

    DEFF Research Database (Denmark)

    Kim, S.G.; Rostrup, Egill; Larsson, H.B.; Ogawa, S; Paulson, Olaf B.

    1999-01-01

    The blood oxygenation level-dependent (BOLD) effect in functional magnetic resonance imaging depends on at least partial uncoupling between cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO2) changes. By measuring CBF and BOLD simultaneously, the relative change in CMRO2 can be......(-1), which corresponds to BOLD signal change of 2.4 +/- 0.7% with a gradient echo time of 50 msec. During black/white visual stimulation reversing at 8 Hz, regional CBF increase in the visual cortex was 43.6 +/- 9.4% (n = 18), and deltaR2* was -0.114 +/- 0.086 sec(-1), corresponding to a BOLD signal...

  14. Dynamic Factors Affecting Gaseous Ligand Binding in an Artificial Oxygen Transport Protein‡

    OpenAIRE

    Zhang, Lei; Andersen, Eskil M.E.; Khajo, Abdelahad; Magliozzo, Richard S.; Koder, Ronald L.

    2013-01-01

    We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7 this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities and oxyferrous state l...

  15. Isolation of complexes formed between insulin-like growth factor-binding protein-3 and transferrin from the human serum

    Directory of Open Access Journals (Sweden)

    Miljuš Goran

    2012-01-01

    Full Text Available Insulin-like growth factors (IGFs play an important role in the regulation of cell growth, differentiation and metabolism. The amount of free, biologically active IGFs is regulated by the IGF-binding proteins (IGFBPs. IGFBP-3 is the most abundant binding protein and it is known to interact with other circulating proteins, including transferrin (Tf. In order to elucidate the possible role of IGF/IGFBP-3 in the iron metabolism, it is necessary to isolate IGFBP-3/Tf complexes. Several affinity-based techniques were employed. Results have shown that only double immunoprecipitation method with anti-Tf and anti-IGFBP-3 antibodies selectively separated complexes from other molecular forms, such as monomers, oligomers or fragments of IGFBP-3 and Tf. Isolated complexes can now be used to investigate the relationship between IGF/IGFBP-3 and iron, both in structural and metabolic tеrms.

  16. ORF13 in the Type III secretion system gene cluster of Edwardsiella tarda binds to the mammalian factor Cugbp2.

    Science.gov (United States)

    Okuda, Jun; Takeuchi, Yusuke; Yasuda, Masashi; Nakai, Toshihiro

    2016-05-01

    The Type III secretion system (TTSS) is essential for the intracellular replication of Edwardsiella tarda in phagocytes of fish and mammals, and a hypothetical gene (orf13) located in the TTSS gene cluster is required for intracellular replication and virulence of E. tarda. Here, we show that under TTSS-inducing conditions, the protein ORF13 was secreted into culture supernatant. Then, using a yeast 2-hybrid screen, we show that the mammalian factor Cugbp2, which regulates apoptosis in breast cancer cells, directly interacts with ORF13. A pull-down assay revealed that ORF13 binds to the C-terminal region of Cugbp2. Our results suggest that ORF13 may facilitate E. tarda replication in phagocytes by binding to Cugbp2. PMID:27137075

  17. Crystal Structure of the Bovine lactadherin C2 Domain, a Membrane Binding Motif, Shows Similarity of the C2 Domains of Factor V and Factor VIII

    Energy Technology Data Exchange (ETDEWEB)

    Lin,L.; Huai, Q.; Huang, M.; Furie, B.; Furie, B.

    2007-01-01

    Lactadherin, a glycoprotein secreted by a variety of cell types, contains two EGF domains and two C domains with sequence homology to the C domains of blood coagulation proteins factor V and factor VIII. Like these proteins, lactadherin binds to phosphatidylserine (PS)-containing membranes with high affinity. We determined the crystal structure of the bovine lactadherin C2 domain (residues 1 to 158) at 2.4 Angstroms. The lactadherin C2 structure is similar to the C2 domains of factors V and VIII (rmsd of C? atoms of 0.9 Angstroms and 1.2 Angstroms, and sequence identities of 43% and 38%, respectively). The lactadherin C2 domain has a discoidin-like fold containing two ?-sheets of five and three antiparallel ?-strands packed against one another. The N and C termini are linked by a disulfide bridge between Cys1 and Cys158. One ?-turn and two loops containing solvent-exposed hydrophobic residues extend from the C2 domain ?-sandwich core. In analogy with the C2 domains of factors V and VIII, some or all of these solvent-exposed hydrophobic residues, Trp26, Leu28, Phe31, and Phe81, likely participate in membrane binding. The C2 domain of lactadherin may serve as a marker of cell surface phosphatidylserine exposure and may have potential as a unique anti-thrombotic agent.

  18. Crystal Structure of the Bovine lactadherin C2 Domain, a Membrane Binding Motif, Shows Similarity to the C2 Domains of Factor V and Factor VIII

    Energy Technology Data Exchange (ETDEWEB)

    Lin,L.

    2007-01-01

    Lactadherin, a glycoprotein secreted by a variety of cell types, contains two EGF domains and two C domains with sequence homology to the C domains of blood coagulation proteins factor V and factor VIII. Like these proteins, lactadherin binds to phosphatidylserine (PS)-containing membranes with high affinity. We determined the crystal structure of the bovine lactadherin C2 domain (residues 1 to 158) at 2.4 {angstrom}. The lactadherin C2 structure is similar to the C2 domains of factors V and VIII (rmsd of C{sub {alpha}} atoms of 0.9 {angstrom} and 1.2 {angstrom}, and sequence identities of 43% and 38%, respectively). The lactadherin C2 domain has a discoidin-like fold containing two {beta}-sheets of five and three antiparallel {beta}-strands packed against one another. The N and C termini are linked by a disulfide bridge between Cys1 and Cys158. One {beta}-turn and two loops containing solvent-exposed hydrophobic residues extend from the C2 domain {beta}-sandwich core. In analogy with the C2 domains of factors V and VIII, some or all of these solvent-exposed hydrophobic residues, Trp26, Leu28, Phe31, and Phe81, likely participate in membrane binding. The C2 domain of lactadherin may serve as a marker of cell surface phosphatidylserine exposure and may have potential as a unique anti-thrombotic agent.

  19. A major binding protein for leukemia inhibitory factor in normal mouse serum: identification as a soluble form of the cellular receptor.

    OpenAIRE

    Layton, M. J.; Cross, B. A.; Metcalf, D; Ward, L. D.; Simpson, R. J.; Nicola, N A

    1992-01-01

    A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-a...

  20. Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

    International Nuclear Information System (INIS)

    The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of [125I]EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of [125I]EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of [125I]EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland

  1. Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

    Energy Technology Data Exchange (ETDEWEB)

    Chabot, J.G.; Walker, P.; Pelletier, G.

    1986-01-01

    The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of (/sup 125/I)EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of (/sup 125/I)EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of (/sup 125/I)EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland.

  2. Membrane-associated insulin-like growth factor (IGF binding structures in placental cells

    Directory of Open Access Journals (Sweden)

    ROMANA MASNIKOSA

    2003-11-01

    Full Text Available The biological activities of IGF-I and –II are mediated mainly by the type 1 IGF receptor (IGF 1R and controlled by their interaction with soluble proteins, the IGF binding proteins (IGFBPs. Although there is a growing body of evidence that some IGFBPs may be cell surface-bound, published data concerning cell association of IGFBP-1 are scarce and none of them concern placental cells. The cell membranes used in this study were isolated from term human placentae. Detergent-solubilized membranes were shown to contain two types of IGF binding structures that were separated by gel filtration on a Sephadex G-100 column. Proteins in the first peak were eluted at V0 (Mr > 100 kD and they bound IGF-I with greater specificity and affinity than IGF-II and insulin. Most likely, they represented the IGF 1R. Small proteins (Mr ~ 45 kD were eluted with the membrane proteins in the second maximum. They were able to bind IGF-I and IGF-II, but not insulin. The identity of these proteins was shown to be IGFBP-1 on the basis of their reaction with specific anti-IGFBP-1 antibodies. To the best of our knowledge, the existence of IGFBP-1 associated with human placental cell membranes has not been reported in the literature before. Colocalisation of IGFBP-1 with IGF 1R in cell membranes could provide efficient modulation of IGF 1R receptor-ligand interactions.

  3. Binding characteristics of brain-derived neurotrophic factor to its receptors on neurons from the chick embryo

    International Nuclear Information System (INIS)

    Brain-derived neurotrophic factor (BDNF), a protein known to support the survival of embryonic sensory neurons and retinal ganglion cells, was derivatized with 125I-Bolton-Hunter reagent and obtained in a biologically active, radioactive form (125I-BDNF). Using dorsal root ganglion neurons from chick embryos at 9 d of development, the basic physicochemical parameters of the binding of 125I-BDNF with its receptors were established. Two different classes of receptors were found, with dissociation constants of 1.7 x 10(-11) M (high-affinity receptors) and 1.3 x 10(-9) M (low-affinity receptors). Unlabeled BDNF competed with 125I-BDNF for binding to the high-affinity receptors with an inhibition constant essentially identical to the dissociation constant of the labeled protein: 1.2 x 10(-11) M. The association and dissociation rates from both types of receptors were also determined, and the dissociation constants calculated from these kinetic experiments were found to correspond to the results obtained from steady-state binding. The number of high-affinity receptors (a few hundred per cell soma) was 15 times lower than that of low-affinity receptors. No high-affinity receptors were found on sympathetic neurons, known not to respond to BDNF, although specific binding of 125I-BDNF to these cells was detected at a high concentration of the radioligand. These results are discussed and compared with those obtained with nerve growth factor on the same neuronal populations

  4. Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.

    OpenAIRE

    Di Marco, A; Gloaguen, I; Graziani, R; Paonessa, G; Saggio, I; Hudson, K R; Laufer, R

    1996-01-01

    Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by ...

  5. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bayne, M.L.; Applebaum, J.; Chicchi, G.G.; Hayes, N.S.; Green, B.G.; Cascieri, M.A.

    1988-05-05

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. (Phe/sup -1/, Val/sup 1/, Asn/sup 2/, Gln/sup 3/, His/sup 4/, Ser/sup 8/, His/sup 9/, Glu/sup 12/, Tyr/sup 15/, Leu/sup 16/)IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. (Gln/sup 3/, Ala/sup 4/) IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. (Tyr/sup 15/, Leu/sup 16/) IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, (Gln/sup 3/, Ala/sup 4/, Tyr/sup 15/,Leu/sup 16/)IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.

  6. Brain regions involved in voluntary movements as revealed by radioisotopic mapping of CBF or CMR-glucose changes

    DEFF Research Database (Denmark)

    Lassen, N A; Ingvar, D H

    1990-01-01

    Mapping of cortical and subcortical grey matter active during voluntary movements by means of measurements of local increases of CBF or CMR-Glucose is reviewed. Most of the studies concern observations in man during hand movements using the intracarotid Xenon-133 injection technique, an approach...... motor area SMA on both sides increase in CBF/CMR-glucose and even internally ("mentally") going through the trained movements, causes such changes; complex purposeful movements also activate the premotor cortex, a response that is bilateral with greatest response contralaterally. Studies in patients...

  7. Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

    Science.gov (United States)

    Oliva, M L V; Andrade, S A; Juliano, M A; Sallai, R C; Torquato, R J; Sampaio, M U; Pott, V J; Sampaio, C A M

    2003-07-01

    The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays. PMID:12678803

  8. Second Intron of Mouse Nestin Gene Directs its Expression in Pluripotent Embryonic Carcinoma Cells through POU Factor Binding Site

    Institute of Scientific and Technical Information of China (English)

    Zhi-Gang JIN; Li LIU; Hua ZHONG; Ke-Jing ZHANG; Yong-Feng CHEN; Wei BIAN; Le-Ping CHENG; Nai-He JING

    2006-01-01

    Nestin, an intermediate filament protein, is expressed in the neural stem cells of the developing central nervous system. This tissue-specific expression is driven by the neural stem cell-specific enhancer in the second intron of the nestin gene. In this study, we showed that the mouse nestin gene was expressed in pluripotent embryonic carcinoma (EC) P19 and F9 cells, not in the differentiated cell types. This cell typespecific expression was conferred by the enhancer in the second intron. Mutation of the conserved POU factor-binding site in the enhancer abolished the reporter gene expression in EC cells. Oct4, a Class V POU factor, was found to be coexpressed with nestin in EC cells. Electrophoretic mobility-shift assays and supershift assays showed that a unique protein-DNA complex was formed specifically with nuclear extracts of EC cells, and Oct4 protein was included. Together, these results suggest the functional relevance between the conserved POU factor-binding site and the expression of the nestin gene in pluripotent EC cells.

  9. Berberine inhibits cyclin D1 expression via suppressed binding of AP-1 transcription factors to CCND1 AP-1 motif

    Institute of Scientific and Technical Information of China (English)

    Ye LUO; Yu HAO; Tai-ping SHI; Wei-wei DENG; Na LI

    2008-01-01

    Aim: To verify the suppressive effect of berberine on the proliferation of the human pulmonary giant cell carcinoma cell line PG and to demonstrate the mecha-nisms behind the antitumoral effects of berberine. Methods: The proliferative effects of PG cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetry. The cell cycle was examined by flow cytometry. The expression level of cyclin D1 was detected by RT-PCR. The activities of the activating protein-1 (AP-1) and NF-κB signaling pathways related to cyclin D1 were examined by luciferase assay. The cytoplasmic level of c-Jun was detected by Western blot analysis. An electrophoretic mobility shift assay was used to examiae the binding of transcription factors to the cyclin D1 gene (CCNDl) AP-1 motif. Results: The results showed that the proliferation of PG cells treated with different concentrations (10, 20, and 40 μg/mL) of berberine for 24 and 48 h was suppressed significantly compared to the control group. After treatment with berberine, the proportion of PG cells at the G0/G1 phase increased, while cells at the S and G2/M phases decreased. Berberine could inhibit the expression of cyclin D1 in PG cells. Berberine inhibited the activity of the AP-1 signaling pathway, but had no significant effect on the NF-κB signaling pathway. Berberine suppressed the expression of c-Jun and decreased the binding of tran-scription factors to the CCND1 AP-1 motif. Conclusion: Berberine suppresses the activity of the AP-1 signaling pathway and decreases the binding of transcrip-tion factors to the CCND1 AP-1 motif. This is one of the important mechanisms behind the antitumoral effects of berberine as a regulator of cyclin D1.

  10. Evidence that IGF-binding protein-5 functions as a growth factor

    OpenAIRE

    Miyakoshi, Naohisa; Richman, Charmaine; Kasukawa, Yuji; Linkhart, Thomas A.; David J. Baylink; Mohan, Subburaman

    2001-01-01

    Recent studies support the concept that IGF-binding protein-5 (IGFBP-5) stimulates bone formation, at least in part, via IGF-independent mechanisms. To evaluate this hypothesis further, we evaluated in vitro and in vivo effects of IGFBP-5 on bone formation parameters using the IGF-I knockout (KO) mouse. Treatment of serum-free cultures of osteoblast clones derived from IGF-I KO mice with recombinant human IGFBP-5 increased both proliferation and alkaline phosphatase (ALP) activity in a dose-d...

  11. Purification and characterization of porcine growth hormone-dependent insulin-like growth factor binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Walton, P.E.

    1988-01-01

    The objectives of this thesis were (1) to evaluate molecular weight (M{sub r}) profiles of IGF-I and {sup 125}I-IGF-I; binding activity in pig serum; (2) to determine whether exogenous IGF-I partitions to IGFBPs in the circulation in pigs; (3) to purify and characterize the GH-dependent IGFBP from pig serum; (4) to assess the effects of the IGFBP on IGF-I action in vitro; (5) to establish a radioimmunoassay for the IGFBP; and (6) to measure IGFBP concentrations in serum from GH- and IGF-I-treated pigs.

  12. Complement factor H binds malondialdehyde epitopes and protects from oxidative stress

    DEFF Research Database (Denmark)

    Weismann, David; Hartvigsen, Karsten; Lauer, Nadine;

    2011-01-01

    Oxidative stress and enhanced lipid peroxidation are linked to many chronic inflammatory diseases, including age-related macular degeneration (AMD). AMD is the leading cause of blindness in Western societies, but its aetiology remains largely unknown. Malondialdehyde (MDA) is a common lipid...... polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy...

  13. Effect of X-irradiation on vitamin B12 binding capacity to intrinsic factor

    International Nuclear Information System (INIS)

    The effects of whole-body X-irradiation on vitamin bB12-protein complex formation in gastric juice after oral administration of [57Co]-B12 have been studied. Two proteins with B-12-binding activity have been isolated by gel filtration from gastric juice. 57Co-activity, recovered from B12-protein complex in gastric juice, is found to be about 30 percent less in the X-irradiated rat. In serum, vitamin B12 is mainly associated with alpha1-globulin. Radioactivity distribution in serum globulins after intraperitoneal injection of [57Co]-B12 was similar in control and X-irradiated rats. (author)

  14. Collagen and Stretch Modulate Autocrine Secretion of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Proteins from Differentiated Skeletal Muscle Cells

    Science.gov (United States)

    Perrone, Carmen E.; Fenwick-Smith, Daniela; Vandenburgh, Herman H.

    1995-01-01

    Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micro-g of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.

  15. Characterization of a Wnt-binding site of the WIF-domain of Wnt inhibitory factor-1.

    Science.gov (United States)

    Bányai, László; Kerekes, Krisztina; Patthy, László

    2012-09-21

    A Wnt-binding site of the WIF-domain of Wnt inhibitory factor-1 was localized by structure-guided arginine-scanning mutagenesis in combination with surface plasmon resonance assays. Our observation that substitution of some residues of WIF resulted in an increased affinity for Wnt5a, but decreased affinity for Wnt3a, suggests that these residues may define the specificity spectrum of WIF for Wnts. These results hold promise for a more specific targeting of Wnt family members with WIF variants in various forms of cancer. PMID:22986341

  16. Epigenetic analyses of the insulin-like growth factor binding protein 1 gene in type 1 diabetes and diabetic nephropathy

    OpenAIRE

    Gu, Tianwei; Falhammar, Henrik; Gu, Harvest F.; Brismar, Kerstin

    2014-01-01

    Background Clinical observations have demonstrated that high levels of circulating insulin-like growth factor binding protein-1 (IGFBP-1) are associated with type 1 diabetes (T1D), whereas low serum IGFBP-1 levels are associated with the risk of type 2 diabetes (T2D). Recently, we reported that increased DNA methylation levels in the IGFBP1 gene were associated with T2D. In the present study, we evaluated the epigenetic changes of IGFBP1 in T1D and diabetic nephropathy (DN). Results In total,...

  17. Salmon serum 22 kDa insulin-like growth factor-binding protein (IGFBP) is IGFBP-1

    OpenAIRE

    Shimizu, M; Dickey, J T; Fukada, H; Dickhoff, W W

    2005-01-01

    Western ligand blotting of salmon serum typically reveals three insulin-like growth factor binding proteins (IGFBPs) at 22, 28 and 41 kDa. Physiological regulation of the 22-kDa IGFBP is similar to that of mammalian IGFBP-1; it is increased under catabolic states such as fasting and stress. On the other hand, its molecular weight on Western ligand blotting is closest to mammalian IGFBP-4. The conflict between physiology and molecular weight makes it difficult to conclude the identity of the 2...

  18. The acid-labile subunit of human ternary insulin-like growth factor binding protein complex in serum

    DEFF Research Database (Denmark)

    Juul, A; Møller, S; Mosfeldt-Laursen, E; Rasmussen, M H; Scheike, Thomas Harder; Pedersen, S A; Kastrup, K W; Yu, Hao; Mistry, J; Rasmussen, S; Müller, J; Henriksen, J; Skakkebaek, N E

    1998-01-01

    not measurable by this approach or, alternatively, that the liver is not the primary source of circulating ALS, IGF-I, or IGFBP-3 in humans. In conclusion, we have provided extensive normal data for a novel ALS assay and found that circulating ALS levels exhibit minor diurnal variation. We suggest......Circulating insulin-like growth factor-I (IGF-I) is predominantly bound in the trimeric complex comprised of IGF binding protein-3 (IGFBP-3) and acid-labile subunit (ALS). Circulating concentrations of IGF-I, IGFBP-3 and ALS are believed to reflect the GH secretory status, but the clinical use of...

  19. High-affinity insulin binding to an atypical insulin-like growth factor-I receptor in human breast cancer cells.

    OpenAIRE

    Milazzo, G.; Yip, C. C.; Maddux, B A; Vigneri, R; Goldfine, I D

    1992-01-01

    We studied the nature of insulin receptor binding in MCF-7 breast cancer cells. In both intact cells and solubilized receptor preparations, high-affinity insulin binding was seen. However, unlabeled insulin-like growth factor-I (IGF-I) was five-fold more potent in inhibiting 125I-insulin binding than insulin itself. With monoclonal antibodies to the insulin receptor, 30% of 125I-insulin binding was inhibited. In contrast when alpha-IR3, a monoclonal antibody that recognizes typical IGF-I rece...

  20. Studying the evolution of transcription factor binding events using multi-species ChIP-Seq data.

    Science.gov (United States)

    Zheng, Wei; Zhao, Hongyu

    2013-03-01

    Recent technology advances make it possible to collect whole-genome transcription factor binding (TFB) profiles from multiple species through the ChIP-Seq data. This provides rich information to understand TFB evolution. However, few rigorous statistical models are available to infer TFB evolution from these data. We have developed a phylogenetic tree based method to model the on/off rates of TFB events. There are two unique features of our method compared to existing models. First, we mask nucleotide substitutions and focus on INDEL disruption of TFB events, which are rarer evolution events and more appropriate for divergent species and non-coding regulatory regions. Second, we correct for ascertainment bias in ChIP-Seq data by maximizing likelihood conditional on the observed (incomplete) data. Simulations show that our method works well in model selection and parameter estimation when there are sufficient aligned TFB events. When this method is applied to a ChIP-Seq data set with five vertebrates, we find that the instantaneous transition rates to INDELs are higher in TFB regions than in homologous non-binding regions. This is driven by an excess of alignment columns showing binding in one species but gaps in all other species. When we compare the inferred transition rates between the conserved and non-conserved regions, as expected, the conserved regions are estimated to have lower transition rates. The R package TFBphylo that implements the described model can be downloaded from http://bioinformatics.med.yale.edu/. PMID:23446869

  1. Modulation of the binding of basic fibroblast growth factor and heparanase activity by purified λ-carrageenan oligosaccharides.

    Science.gov (United States)

    Niu, Ting-Ting; Zhang, Dong-Sheng; Chen, Hai-Min; Yan, Xiao-Jun

    2015-07-10

    Inhibitors of angiogenesis and tumor metastasis are increasingly emerging as promising agents for cancer therapy. Here, we report λ-carrageenan oligosaccharides (λ-COs), highly-sulfated oligosaccharides acting as a basic fibroblast growth factor (bFGF) antagonist and heparanase inhibitor. λ-COs with degree of polymerization (DP) from 2 to 8 degraded by λ-carrageenase were separated and purified. The structures were identified by mass spectrometry. The activities of λ-COs are closely related with DP. λ-COs showed no cytotoxicity, but inactivated bFGF-induced cell proliferation; among them, λ-carraheptaose showed highest capability. Only λ-carraheptaose can effectively bind to bFGF. Binding kinetics showed that λ-carraheptaose and suramin had different binding modes, i.e., suramin displayed a fast association and fast dissociation, but λ-carraheptaose exhibited a slow association and slow dissociation. In addition, λ-COs showed the highest heparanase inhibitory ability and abolished the endothelial cell invasion. Thus, λ-COs may provide a tool to develop of new carbohydrate-based therapeutics against cancer and angiogenesis. PMID:25857962

  2. Changes in the level of growth hormone, insulin like growth factor-1 and insulin like growth factor binding proteine-3 in young males 24 hours after submaximaltraining

    OpenAIRE

    ÇOKNAZ, Hakkı

    2011-01-01

    The study was accomplished as a control study, under the question whether 6-weeks endurance training affects the growth hormone (GH), insulin like growth factor-1 (IGF-1) and the IGF bindings protein-3 (IGFBP-3) levels. Sixty male subjects participated in the study. The subjects were separated into 2 groups as control (n=30; mean age=21,13±1,16 years) and study (n=30; mean age=21,53±1,61 years) randomly, prior to the runtest. Blood samples were drawn before breakfast and analyzed in the labor...

  3. Mannose-binding Lectin (MBL) as a susceptible host factor influencing Indian Visceral Leishmaniasis.

    Science.gov (United States)

    Mishra, Anshuman; Antony, Justin S; Gai, Prabhanjan; Sundaravadivel, Pandarisamy; Van, Tong Hoang; Jha, Aditya Nath; Singh, Lalji; Velavan, Thirumalaisamy P; Thangaraj, Kumarasamy

    2015-12-01

    Visceral Leishmaniasis (VL), caused by Leishmania donovani is endemic in the Indian sub-continent. Mannose-binding Lectin (MBL) is a complement lectin protein that binds to the surface of Leishmania promastigotes and results in activation of the complement lectin cascade. We utilized samples of 218 VL patients and 215 healthy controls from an Indian population. MBL2 functional variants were genotyped and the circulating MBL serum levels were measured. MBL serum levels were elevated in patients compared to the healthy controls (adjusted P=0.007). The MBL2 promoter variants -78C/T and +4P/Q were significantly associated with relative protection to VL (-78C/T, OR=0.7, 95% CI=0.5-0.96, adjusted P=0.026 and +4P/Q, OR=0.66, 95% CI=0.48-0.9, adjusted P=0.012). MBL2*LYQA haplotypes occurred frequently among controls (OR=0.69, 95% CI=0.5-0.97, adjusted P=0.034). MBL recognizes Leishmania and plays a relative role in establishing L. donovani infection and subsequent disease progression. In conclusion, MBL2 functional variants were associated with VL. PMID:26297290

  4. TATA binding protein associated factor 3 (TAF3 interacts with p53 and inhibits its function

    Directory of Open Access Journals (Sweden)

    Tora Laszlo

    2008-06-01

    Full Text Available Abstract Background The tumour suppressor protein p53 is a sequence specific DNA-binding transcription regulator, which exerts its versatile roles in genome protection and apoptosis by affecting the expression of a large number of genes. In an attempt to obtain a better understanding of the mechanisms by which p53 transcription function is regulated, we studied p53 interactions. Results We identified BIP2 (Bric-à-brac interacting protein 2, the fly homolog of TAF3, a histone fold and a plant homeodomain containing subunit of TFIID, as an interacting partner of Drosophila melanogaster p53 (Dmp53. We detected physical interaction between the C terminus of Dmp53 and the central region of TAF3 both in yeast two hybrid assays and in vitro. Interestingly, DmTAF3 can also interact with human p53, and mammalian TAF3 can bind to both Dmp53 and human p53. This evolutionarily conserved interaction is functionally significant, since elevated TAF3 expression severely and selectively inhibits transcription activation by p53 in human cell lines, and it decreases the level of the p53 protein as well. Conclusion We identified TAF3 as an evolutionarily conserved negative regulator of p53 transcription activation function.

  5. Interruption of intrachromosomal looping by CCCTC binding factor decoy proteins abrogates genomic imprinting of human insulin-like growth factor II

    Science.gov (United States)

    Zhang, He; Niu, Beibei; Ge, Shengfang; Wang, Haibo; Li, Tao; Ling, Jianqun; Steelman, Brandon N.; Qian, Guanxiang

    2011-01-01

    Monoallelic expression of IGF2 is regulated by CCCTC binding factor (CTCF) binding to the imprinting control region (ICR) on the maternal allele, with subsequent formation of an intrachromosomal loop to the promoter region. The N-terminal domain of CTCF interacts with SUZ12, part of the polycomb repressive complex-2 (PRC2), to silence the maternal allele. We synthesized decoy CTCF proteins, fusing the CTCF deoxyribonucleic acid–binding zinc finger domain to CpG methyltransferase Sss1 or to enhanced green fluorescent protein. In normal human fibroblasts and breast cancer MCF7 cell lines, the CTCF decoy proteins bound to the unmethylated ICR and to the IGF2 promoter region but did not interact with SUZ12. EZH2, another part of PRC2, was unable to methylate histone H3-K27 in the IGF2 promoter region, resulting in reactivation of the imprinted allele. The intrachromosomal loop between the maternal ICR and the IGF2 promoters was not observed when IGF2 imprinting was lost. CTCF epigenetically governs allelic gene expression of IGF2 by orchestrating chromatin loop structures involving PRC2. PMID:21536749

  6. In vivo and in vitro evaluation of Cy5.5 conjugated epidermal growth factor receptor binding peptide

    International Nuclear Information System (INIS)

    The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor and plays an important role in carcinogenesis. In this study, the epidermal growth factor receptor binding peptide (EGBP) was identified using a phage display method and evaluated in U87MG cells in order to investigate the possibility to target the EGFR using an optical imaging system. Cyanine dye 5.5 (Cy5.5) was conjugated with EGBP-GGG-SC, EGBP-AOC-SC, and EGBP-AM2BA-SC. Cellular binding study of EGBP-Linker-Cy5.5 conjugates or 125I-EGBP-Linker compounds was performed in U87MG cells. Optical imaging studies were performed in U87MG bearing mice. Three of seven clones from the 12-mer peptide library showed a specific binding affinity to rhEGFR, and they encoded the same 12 amino acid peptide sequence, FPMFNHWEQWPP. Confocal images show that the fluorescent signal of EGBP-Linker-Cy5.5 conjugates was decreased in the order: EGBP-AOC-Cy5.5≫EGBP-AM2BA-Cy5.5>EGBP-GGG-Cy5.5. EGBP-AOC-Cy5.5 appeared in cell cytoplasm and surface, and it was inhibited by free EGBP apparently. The cellular binding of EGBP-AOC-Cy5.5 exhibited a higher average radiance value than EGBP-GGG-Cy5.5 and EGBP-AM2BA-Cy5.5. Among various 125I-EGBP-Linker compounds, EGBP-GGG showed a higher binding than other compounds. However, uptake of 125I-EGBP-AOC was clearly inhibited by free EGBP in inhibition study. In an in vivo study, the fluorescent signal of EGBP-AOC-Cy5.5 treated mouse was mainly detected in the tumor and kidney. Among the three derivatives, EGBP-AOC-Cy5.5 was the optimized optical imaging agent for U87MG EGFR positive tumors in the animal model. This study demonstrated the EGBP-Linker-Cy5.5 conjugates may be useful as a potential EGFR target optical probe.

  7. The usefulness of CBF brain SPECT in forensic medicine. A description of four cases

    International Nuclear Information System (INIS)

    The aim of this study was to show the applications of cerebral blood flow SPECT scanning in forensic medicine using four cases: two suspects and two victims of crime. Cerebral blood flow studies were performed with the use of 99mTc-ECD and a triple head gammacamera. Qualitative and quantitative analysis was performed, utilising an asymmetry index for unilateral perfusion deficits and a comparison to cerebellar perfusion for assessing the regional cerebral perfusion. For assessing the normal values, a control group of 30 patients was studied. In these cases CBF SPECT scanning proved its usefulness in medico-legal argument and played an important role in formulating the final forensic expertTMs opinion. Radionuclide cerebral blood flow studies may play a role in forensic medicine, where this method it is mostly under-utilised at present. (author)

  8. The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococci.

    Science.gov (United States)

    Kolberg, Jan; Hammerschmidt, Sven; Frank, Ronald; Jonák, Jirí; Sanderová, Hana; Aase, Audun

    2008-07-01

    Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and polyclonal rabbit antibodies against pneumococcal EF-Tu. These antibodies were used to investigate the amount of surface-exposed EF-Tu on viable bacteria using a flow cytometric analysis. The control antibodies recognizing the pneumococcal surface protein A and phosphorylcholine showed a significant binding to viable pneumococci. In contrast, anti-EF-Tu antibodies did not recognize pneumococcal EF-Tu. However, heat killing of pneumococci lacking capsular polysaccharides resulted in specific antibody binding to EF-Tu and, moreover, increased the exposure of recognized phosphorylcholine epitopes. Similarly, our EF-Tu-specific antibodies did not recognize EF-Tu of viable Neisseria meningitidis. However, pretreatment of meningococci with ethanol resulted in specific antibody binding to EF-Tu on outer membranes. Importantly, these treatments did not destroy the membrane integrity as analysed with control mAbs directed against cytoplasmic proteins. In conclusion, our flow cytrometric assays emphasize the importance of using viable bacteria and not heat-killed or ethanol-treated bacteria for surface-localization experiments of proteins, because these treatments modulate the cytoplasmic and outer membranes of bacteria and the binding results may not reflect the situation under physiological conditions. PMID:18462389

  9. Draft Genome Sequence of Pseudomonas putida CBF10-2, a Soil Isolate with Bioremediation Potential in Agricultural and Industrial Environmental Settings.

    Science.gov (United States)

    Iyer, Rupa; Damania, Ashish

    2016-01-01

    Pseudomonas putida CBF10-2 is a microorganism isolated from farmland soil in Fairchild, TX, found to degrade high-impact xenobiotics, including organophosphate insecticides, petroleum hydrocarbons, and both monocyclic and polycyclic aromatics. The versatility of CBF10-2 makes it useful for multipurpose bioremediation of contaminated sites in agricultural and industrial environments. PMID:27417844

  10. Identification of a functional hepatocyte nuclear factor 4 binding site in the neutral ceramidase promoter

    DEFF Research Database (Denmark)

    Maltesen, Henrik R; Troelsen, Jesper T; Olsen, Jørgen

    2010-01-01

    The brush border membrane of the differentiated small intestinal epithelial cell is studded with membrane bound hydrolytic ectoenzymes involved in digestion. Previous studies of the regulation of genes encoding brush border enzymes have especially implicated the transcription factors hepatocyte...... nuclear factor HNF-1 and Cdx2. Recent genome-wide studies have, however, also identified HNF-4a as a transcription factor with a high number of target genes in the differentiated small intestinal epithelial cell. The Asah2 gene encodes neutral ceramidase, which is a hydrolytic brush border enzyme involved...

  11. Coagulation factor Va binds to human umbilical vein endothelial cells and accelerates protein C activation.

    OpenAIRE

    Maruyama, I.; Salem, H H; Majerus, P W

    1984-01-01

    In vitro the rate of protein C activation by thrombin is significantly accelerated by two distinct cofactors (a) the endothelial cell surface protein, thrombomodulin, and (b) human coagulation Factor Va. We have recently reported that the activity of Factor Va is contained in the 78,000-D light chain. In this study we have investigated the effects of Factor Va and its light chain on the activation of protein C in the presence of cultured endothelial cells. Thrombin-catalyzed protein C activat...

  12. Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

    Institute of Scientific and Technical Information of China (English)

    齐眉; 黄美娟; 陈凡

    2002-01-01

    Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hy- brid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.

  13. Binding mode of the breakthrough inhibitor AZD9291 to epidermal growth factor receptor revealed.

    Science.gov (United States)

    Yosaatmadja, Yuliana; Silva, Shevan; Dickson, James M; Patterson, Adam V; Smaill, Jeff B; Flanagan, Jack U; McKeage, Mark J; Squire, Christopher J

    2015-12-01

    The discovery of genetic drivers of lung cancer in patient sub-groups has led to their use as predictive biomarkers and as targets for selective drug therapy. Some of the most important lung cancer drivers are mutations in the EGFR gene, for example, the exon 19 deletions and the L858R variant that confer sensitivity to the front line drugs erlotinib and gefitinib; the acquired T790M variants confer drug resistance and a poor prognosis. A challenge then in targeting EGFR is to produce drugs that inhibit both sensitising variants and resistance variants, leaving wild type protein in healthy cells unaffected. One such agent is AstraZeneca's "breakthrough" AZD9291 molecule that shows a 200-fold selectivity for T790M/L858R over wild type EGFR. Our X-ray crystal structure reveals the binding mode of AZD9291 to the kinase domain of wild type EGFR. PMID:26522274

  14. Release of IgD-binding factor by T cells under the influence of interleukin 2, interleukin 4, or cross-linked IgD.

    OpenAIRE

    Amin, A R; Coico, R F; Finkelman, F; Siskind, G W; Thorbecke, G. J.

    1988-01-01

    Helper T cells with receptors specific for IgD have immunoaugmenting properties. We have now detected soluble IgD-binding factor in cell supernatants immobilized on nitrocellulose paper by their ability to bind 125I-labeled IgD. IgD-binding factor is released by normal splenic T cells stimulated with recombinant interleukin 2, recombinant interleukin 4, or crosslinked IgD in amounts paralleling the induction of IgD receptors on the cells. IgD receptors are constitutively produced by antigen-s...

  15. Reconfiguring the connectivity of a multiprotein complex: Fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB

    OpenAIRE

    Kassavetis, George A.; Soragni, Elisabetta; Driscoll, Robert; Geiduschek, E.Peter

    2005-01-01

    Transcription factor (TF) IIIB, the central transcription initiation factor of RNA polymerase III (pol III), is composed of three subunits, Bdp1, Brf1 and TATA-binding protein (TBP), all essential for normal function in vivo and in vitro. Brf1 is a modular protein: Its N-proximal half is related to TFIIB and binds similarly to the C-terminal stirrup of TBP; its C-proximal one-third provides most of the affinity for TBP by binding along the entire length of the convex surface and N-terminal la...

  16. Serum free and total insulin-like growth factor-1, insulin-like growth factor binding protein-1 and insulin-like growth factor binding protein-3 levels in healthy elderly individuals - Relation to self-reported duality of health and disability

    NARCIS (Netherlands)

    Janssen, JAMJL; Stolk, RP; Pols, HAP; Grobbee, DE; Lamberts, SWJ

    1998-01-01

    Background: Little is known about the influence of the free insulin-like growth factor-I/insulin-like growth factor binding protein (IGF-I/IGFBP) system on the quality of health and on disability in the elderly population. Design: In a cross-sectional population based study of 218 healthy elderly su

  17. Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1

    OpenAIRE

    Guja, Kip E.; Venkataraman, Krithika; Yakubovskaya, Elena; Hui SHI; Mejia, Edison; Hambardjieva, Elena; Karzai, A. Wali; Garcia-Diaz, Miguel

    2013-01-01

    Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a role analogous to KsgA/Dim1 proteins. This modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and di...

  18. Defining transcriptional networks through integrative modeling of mRNA expression and transcription factor binding data

    Directory of Open Access Journals (Sweden)

    Bussemaker Harmen J

    2004-03-01

    Full Text Available Abstract Background Functional genomics studies are yielding information about regulatory processes in the cell at an unprecedented scale. In the yeast S. cerevisiae, DNA microarrays have not only been used to measure the mRNA abundance for all genes under a variety of conditions but also to determine the occupancy of all promoter regions by a large number of transcription factors. The challenge is to extract useful information about the global regulatory network from these data. Results We present MA-Networker, an algorithm that combines microarray data for mRNA expression and transcription factor occupancy to define the regulatory network of the cell. Multivariate regression analysis is used to infer the activity of each transcription factor, and the correlation across different conditions between this activity and the mRNA expression of a gene is interpreted as regulatory coupling strength. Applying our method to S. cerevisiae, we find that, on average, 58% of the genes whose promoter region is bound by a transcription factor are true regulatory targets. These results are validated by an analysis of enrichment for functional annotation, response for transcription factor deletion, and over-representation of cis-regulatory motifs. We are able to assign directionality to transcription factors that control divergently transcribed genes sharing the same promoter region. Finally, we identify an intrinsic limitation of transcription factor deletion experiments related to the combinatorial nature of transcriptional control, to which our approach provides an alternative. Conclusion Our reliable classification of ChIP positives into functional and non-functional TF targets based on their expression pattern across a wide range of conditions provides a starting point for identifying the unknown sequence features in non-coding DNA that directly or indirectly determine the context dependence of transcription factor action. Complete analysis results are

  19. Dynamic Nucleocytoplasmic Shuttling of an Arabidopsis SR Splicing Factor: Role of the RNA-Binding Domains

    OpenAIRE

    Rausin, Glwadys; Tillemans, Vinciane; Stankovic, Nancy; Hanikenne, Marc; Motte, Patrick

    2010-01-01

    Serine/arginine-rich (SR) proteins are essential nuclear-localized splicing factors. We have investigated the dynamic subcellular distribution of the Arabidopsis (Arabidopsis thaliana) RSZp22 protein, a homolog of the human 9G8 SR factor. Little is known about the determinants underlying the control of plant SR protein dynamics, and so far most studies relied on ectopic transient overexpression. Here, we provide a detailed analysis of the RSZp22 expression profile and describe its nucleocytop...

  20. Mapping Escherichia coli elongation factor Tu residues involved in binding of aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Wiborg, Ove; Andersen, C; Knudsen, Charlotte Rohde;

    1996-01-01

    were characterized with respect to thermal and chemical stability, GTPase activity, tRNA affinity, and activity in an in vitro translation assay. Most conspicuously tRNA affinities were reduced for all mutants. The results verify our structural analysis of elongation factor Tu in complex with aminoacyl....... Their functional roles are discussed in relation to the structure of elongation factor Tu in complex with aminoacyl-tRNA. Udgivelsesdato: 1996-Aug-23...