Alteration of Cyclic-AMP Response Element Binding Protein in the Postmortem Brain of Subjects with Bipolar Disorder and Schizophrenia

PubMed Central

Ren, Xinguo; Rizavi, Hooriyah S.; Khan, Mansoor A.; Bhaumik, Runa; Dwivedi, Yogesh; Pandey, Ghanshyam N.

2013-01-01

Background Abnormalities of cyclic-AMP (cAMP) response element binding protein (CREB) function has been suggested in bipolar (BP) illness and schizophrenia (SZ), based on both indirect and direct evidence. To further elucidate the role of CREB in these disorders, we studied CREB expression and function in two brain areas implicated in these disorders, i.e., dorsolateral prefrontal cortex (DLPFC) and cingulate gyrus (CG). Methods We determined CREB protein expression using Western blot technique, CRE-DNA binding using gel shift assay, and mRNA expression using real-time RT-polymerase chain reaction (qPCR) in DLPFC and CG of the postmortem brain of BP (n = 19), SZ (n = 20), and normal control (NC, n = 20) subjects. Results We observed that CREB protein and mRNA expression and CRE-DNA binding activity were significantly decreased in the nuclear fraction of DLPFC and CG obtained from BP subjects compared with NC subjects. However, the protein and mRNA expression and CRE-DNA binding in SZ subjects was significantly decreased in CG, but not in DLPFC, compared with NC. Conclusion These studies thus indicate region-specific abnormalities of CREB expression and function in both BP and SZ. They suggest that abnormalities of CREB in CG may be associated with both BP and SZ, but its abnormality in DLPFC is specific to BP illness. PMID:24148789

A Flexible Binding Site Architecture Provides New Insights into CcpA Global Regulation in Gram-Positive Bacteria.

PubMed

Yang, Yunpeng; Zhang, Lu; Huang, He; Yang, Chen; Yang, Sheng; Gu, Yang; Jiang, Weihong

2017-01-24

Catabolite control protein A (CcpA) is the master regulator in Gram-positive bacteria that mediates carbon catabolite repression (CCR) and carbon catabolite activation (CCA), two fundamental regulatory mechanisms that enable competitive advantages in carbon catabolism. It is generally regarded that CcpA exerts its regulatory role by binding to a typical 14- to 16-nucleotide (nt) consensus site that is called a catabolite response element (cre) within the target regions. However, here we report a previously unknown noncanonical flexible architecture of the CcpA-binding site in solventogenic clostridia, providing new mechanistic insights into catabolite regulation. This novel CcpA-binding site, named cre var , has a unique architecture that consists of two inverted repeats and an intervening spacer, all of which are variable in nucleotide composition and length, except for a 6-bp core palindromic sequence (TGTAAA/TTTACA). It was found that the length of the intervening spacer of cre var can affect CcpA binding affinity, and moreover, the core palindromic sequence of cre var is the key structure for regulation. Such a variable architecture of cre var shows potential importance for CcpA's diverse and fine regulation. A total of 103 potential cre var sites were discovered in solventogenic Clostridium acetobutylicum, of which 42 sites were picked out for electrophoretic mobility shift assays (EMSAs), and 30 sites were confirmed to be bound by CcpA. These 30 cre var sites are associated with 27 genes involved in many important pathways. Also of significance, the cre var sites are found to be widespread and function in a great number of taxonomically different Gram-positive bacteria, including pathogens, suggesting their global role in Gram-positive bacteria. In Gram-positive bacteria, the global regulator CcpA controls a large number of important physiological and metabolic processes. Although a typical consensus CcpA-binding site, cre, has been identified, it remains poorly explored for the diversity of CcpA-mediated catabolite regulation. Here, we discovered a novel flexible CcpA-binding site architecture (cre var ) that is highly variable in both length and base composition but follows certain principles, providing new insights into how CcpA can differentially recognize a variety of target genes to form a complicated regulatory network. A comprehensive search further revealed the wide distribution of cre var sites in Gram-positive bacteria, indicating it may have a universal function. This finding is the first to characterize such a highly flexible transcription factor-binding site architecture, which would be valuable for deeper understanding of CcpA-mediated global catabolite regulation in bacteria. Copyright © 2017 Yang et al.

Disruption of SATB2 or its long-range cis-regulation by SOX9 causes a syndromic form of Pierre Robin sequence

PubMed Central

Rainger, Jacqueline K.; Bhatia, Shipra; Bengani, Hemant; Gautier, Philippe; Rainger, Joe; Pearson, Matt; Ansari, Morad; Crow, Jayne; Mehendale, Felicity; Palinkasova, Bozena; Dixon, Michael J.; Thompson, Pamela J.; Matarin, Mar; Sisodiya, Sanjay M.; Kleinjan, Dirk A.; FitzPatrick, David R.

2014-01-01

Heterozygous loss-of-function (LOF) mutations in the gene encoding the DNA-binding protein, SATB2, result in micrognathia and cleft palate in both humans and mice. In three unrelated individuals, we show that translocation breakpoints (BPs) up to 896 kb 3′ of SATB2 polyadenylation site cause a phenotype which is indistinguishable from that caused by SATB2 LOF mutations. This syndrome comprises long nose, small mouth, micrognathia, cleft palate, arachnodactyly and intellectual disability. These BPs map to a gene desert between PLCL1 and SATB2. We identified three putative cis-regulatory elements (CRE1–3) using a comparative genomic approach each of which would be placed in trans relative to SATB2 by all three BPs. CRE1–3 each bind p300 and mono-methylated H3K4 consistent with enhancer function. In silico analysis suggested that CRE1–3 contain one or more conserved SOX9-binding sites, and this binding was confirmed using chromatin immunoprecipitation on cells derived from mouse embryonic pharyngeal arch. Interphase bacterial artificial chromosome fluorescence in situ hybridization measurements in embryonic craniofacial tissues showed that the orthologous region in mice exhibits Satb2 expression-dependent chromatin decondensation consistent with Satb2 being a target gene of CRE1–3. To assess their in vivo function, we made multiple stable reporter transgenic lines for each enhancer in zebrafish. CRE2 was shown to drive SATB2-like expression in the embryonic craniofacial region. This expression could be eliminated by mutating the SOX9-binding site of CRE2. These observations suggest that SATB2 and SOX9 may be acting together via complex cis-regulation to coordinate the growth of the developing jaw. PMID:24363063

Effects of rolipram, a phosphodiesterase 4 inhibitor, in combination with imipramine on depressive behavior, CRE-binding activity and BDNF level in learned helplessness rats.

PubMed

Itoh, Tetsuji; Tokumura, Miwa; Abe, Kohji

2004-09-13

The brain cAMP regulating system and its downstream elements play a pivotal role in the therapeutic effects of antidepressants. We previously reported the increase in activities of phosphodiesterase 4, a major phosphodiesterase isozyme hydrolyzing cAMP, in the frontal cortex and hippocampus of learned helplessness rats, an animal model for depression. The present study was undertaken to examine the combination of effects of rolipram, a phosphodiesterase 4 inhibitor, with imipramine, a typical tricyclic antidepressant, on depressive behavior in learned helplessness rats. Concurrently, cAMP-response element (CRE)-binding activity and brain-derived neurotrophic factor (BDNF) levels related to the therapeutic effects of antidepressants were determined. Repeated administration of imipramine (1.25-10 mg/kg, i.p.) or rolipram (1.25 mg/kg, i.p.) reduced the number of escape failures in learned helplessness rats. Imipramine could not completely ameliorate the escape behavior to a level similar to that of non-stressed rats even at 10 mg/kg. However, repeated coadministration of rolipram with imipramine (1.25 and 2.5 mg/kg, respectively) almost completely eliminated the escape failures in learned helplessness rats. The reduction of CRE-binding activities and BDNF levels in the frontal cortex or hippocampus in learned helplessness rats were ameliorated by treatment with imipramine or rolipram alone. CRE-binding activities and/or BDNF levels of the frontal cortex and hippocampus were significantly increased by treatment with a combination of rolipram and imipramine compared to those in imipramine-treated rats. These results indicated that coadministration of phosphodiesterase type 4 inhibitors with antidepressants may be more effective for depression therapy and suggest that elevation of the cAMP signal transduction pathway is involved in the antidepressive effects.

Differential Transcription Factor Use by the KIR2DL4 Promoter Under Constitutive and IL-2/15-Treated Conditions

PubMed Central

Presnell, Steven R.; Zhang, Lei; Chlebowy, Corrin N.; Al-Attar, Ahmad; Lutz, Charles T.

2012-01-01

KIR2DL4 is unique among human KIR genes in expression, cellular localization, structure, and function, yet the transcription factors required for its expression have not been identified. Using mutagenesis, electrophoretic mobility shift assay, and co-transfection assays, we identified two redundant Runx binding sites in the 2DL4 promoter as essential for constitutive 2DL4 transcription, with contributions by a CRE site and initiator elements. IL-2-and IL-15-stimulated human NK cell lines increased 2DL4 promoter activity, which required functional Runx, CRE, and Ets sites. Chromatin immunoprecipitation experiments show that Runx3 and Ets1 bind the 2DL4 promoter in situ. 2DL4 promoter activity had similar transcription factor requirements in T cells. Runx, CRE, and Ets binding motifs are present in 2DL4 promoters from across primate species, but other postulated transcription factor binding sites are not preserved. Differences between 2DL4 and clonally-restricted KIR promoters suggest a model that explains the unique 2DL4 expression pattern in human NK cells. PMID:22467658

Quantitative interdependence of coeffectors, CcpA and cre in carbon catabolite regulation of Bacillus subtilis.

PubMed

Seidel, Gerald; Diel, Marco; Fuchsbauer, Norbert; Hillen, Wolfgang

2005-05-01

The phosphoproteins HPrSerP and CrhP are the main effectors for CcpA-mediated carbon catabolite regulation (CCR) in Bacillus subtilis. Complexes of CcpA with HPrSerP or CrhP regulate genes by binding to the catabolite responsive elements (cre). We present a quantitative analysis of HPrSerP and CrhP interaction with CcpA by surface plasmon resonance (SPR) revealing small and similar equilibrium constants of 4.8 +/- 0.4 microm for HPrSerP-CcpA and 19.1 +/- 2.5 microm for CrhP-CcpA complex dissociation. Forty millimolar fructose-1,6-bisphosphate (FBP) or glucose-6-phosphate (Glc6-P) increases the affinity of HPrSerP to CcpA at least twofold, but have no effect on CrhP-CcpA binding. Saturation of binding of CcpA to cre as studied by fluorescence and SPR is dependent on 50 microm of HPrSerP or > 200 microm CrhP. The rate constants of HPrSerP-CcpA-cre complex formation are k(a) = 3 +/- 1 x 10(6) m(-1).s(-1) and k(d) = 2.0 +/- 0.4 x 10(-3).s(-1), resulting in a K(D) of 0.6 +/- 0.3 nm. FBP and Glc6-P stimulate CcpA-HPrSerP but not CcpA-CrhP binding to cre. Maximal HPrSerP-CcpA-cre complex formation in the presence of 10 mm FBP requires about 10-fold less HPrSerP. These data suggest a specific role for FBP and Glc6-P in enhancing only HPrSerP-mediated CCR.

Optimization of a cAMP response element signal pathway reporter system.

PubMed

Shan, Qiang; Storm, Daniel R

2010-08-15

A sensitive cAMP response element (CRE) reporter system is essential for studying the cAMP/protein kinase A/cAMP response element binding protein signal pathway. Here we have tested a few CRE promoters and found one with high sensitivity to external stimuli. Using this optimal CRE promoter and the enhanced green fluorescent protein as the reporter, we have established a CRE reporter cell line. This cell line can be used to study the signal pathway by fluorescent microscope, fluorescence-activated cell analysis and luciferase assay. This cell line's sensitivity to forskolin, using the technique of fluorescence-activated cell sorting, was increased to approximately seven times that of its parental HEK 293 cell line, which is currently the most commonly used cell line in the field for the signal pathway study. Therefore, this newly created cell line is potentially useful for studying the signal pathway's modulators, which generally have weaker effect than its mediators. Our research has also established a general procedure for optimizing transcription-based reporter cell lines, which might be useful in performing the same task when studying many other transcription-based signal pathways. (c) 2010 Elsevier B.V. All rights reserved.

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

PubMed

Paca-Uccaralertkun, S; Zhao, L J; Adya, N; Cross, J V; Cullen, B R; Boros, I M; Giam, C Z

1994-01-01

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.

Suppressing cAMP response element-binding protein transcription shortens the duration of status epilepticus and decreases the number of spontaneous seizures in the pilocarpine model of epilepsy.

PubMed

Zhu, Xinjian; Dubey, Deepti; Bermudez, Camilo; Porter, Brenda E

2015-12-01

Current epilepsy therapies directed at altering the function of neurotransmitter receptors or ion channels, or release of synaptic vesicles fail to prevent seizures in approximately 30% of patients. A better understanding of the molecular mechanism underlying epilepsy is needed to provide new therapeutic targets. The activity of cyclic AMP (cAMP) response element-binding protein (CREB), a major transcription factor promoting CRE-mediated transcription, increases following a prolonged seizure called status epilepticus. It is also increased in the seizure focus of patients with medically intractable focal epilepsy. Herein we explored the effect of acute suppression of CREB activity on status epilepticus and spontaneous seizures in a chronic epilepsy model. Pilocarpine chemoconvulsant was used to induce status epilepticus. To suppress CREB activity, a transgenic mouse line expressing an inducible dominant negative mutant of CREB (CREB(IR) ) with a serine to alanine 133 substitution was used. Status epilepticus and spontaneous seizures of transgenic and wild-type mice were analyzed using video-electroencephalography (EEG) to assess the effect of CREB suppression on seizures. Our findings indicate that activation of CREB(IR) shortens the duration of status epilepticus. The frequency of spontaneous seizures decreased in mice with chronic epilepsy during CREB(IR) induction; however, the duration of the spontaneous seizures was unchanged. Of interest, we found significantly reduced levels of phospho-CREB Ser133 upon activation of CREB(IR) , supporting prior work suggesting that binding to the CRE site is important for CREB phosphorylation. Our results suggest that CRE transcription supports seizure activity both during status epilepticus and in spontaneous seizures. Thus, blocking of CRE transcription is a novel target for the treatment of epilepsy. Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.

Abnormal expression and functional characteristics of cyclic adenosine monophosphate response element binding protein in postmortem brain of suicide subjects.

PubMed

Dwivedi, Yogesh; Rao, Jagadeesh Sridhara; Rizavi, Hooriyah S; Kotowski, Jacek; Conley, Robert R; Roberts, Rosalinda C; Tamminga, Carol A; Pandey, Ghanshyam N

2003-03-01

Cyclic adenosine monophosphate response element binding protein (CREB) is a transcription factor that, on phosphorylation by protein kinases, is activated, and in response, regulates the transcription of many neuronally expressed genes. In view of the recent observations that catalytic properties and/or expression of many kinases that mediate their physiological responses through the activation of CREB are altered in the postmortem brain of subjects who commit suicide (hereafter referred to as suicide subjects), we examined the status of CREB in suicidal behavior. These studies were performed in Brodmann area (BA) 9 and hippocampus obtained from 26 suicide subjects and 20 nonpsychiatric healthy control subjects. Messenger RNA levels of CREB and neuron-specific enolase were determined in total RNA by means of quantitative reverse transcriptase-polymerase chain reaction. Protein levels and the functional characteristics of CREB were determined in nuclear fractions by means of Western blot and cyclic adenosine monophosphate response element (CRE)-DNA binding activity, respectively. In the same nuclear fraction, we determined the catalytic activity of cyclic adenosine monophosphate-stimulated protein kinase A by means of enzymatic assay. We observed a significant reduction in messenger RNA and protein levels of CREB, CRE-DNA binding activity, and basal and cyclic adenosine monophosphate-stimulated protein kinase A activity in BA 9 and hippocampus of suicide subjects, without any change in messenger RNA levels of neuron-specific enolase in BA 9. Except for protein kinase A activity, changes in CREB expression and CRE-DNA binding activity were present in all suicide subjects, irrespective of diagnosis. These changes were unrelated to postmortem intervals, age, sex, or antidepressant treatment. Given the significance of CREB in mediating various physiological functions through gene transcription, our results of decreased expression and functional characteristics of CREB in postmortem brain of suicide subjects suggest that CREB may play an important role in suicidal behavior.

Transcriptional regulation of bone sialoprotein gene by Porphyromonas gingivalis lipopolysaccharide.

PubMed

Li, Xinyue; Kato, Naoko; Mezawa, Masaru; Li, Zhengyang; Wang, Zhitao; Yang, Li; Sasaki, Yoko; Kaneko, Takashi; Takai, Hideki; Yoshimura, Atsutoshi; Ogata, Yorimasa

2010-07-01

Lipopolysaccharide (LPS) is a major mediator of inflammatory response. Periodontopathic bacterium Porphyromonas gingivalis LPS has quite different character from Escherichia coli LPS. E. coli LPS is agonist for Toll-like receptor 4 (TLR4), whereas P. gingivalis LPS worked as antagonist for TLR4. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. To investigate the effects of P. gingivalis LPS on BSP transcription, we used rat osteoblast-like ROS17/2.8 cells. BSP mRNA levels were decreased by 0.1 microg/ml and increased by 0.01 microg/ml P. gingivalis LPS at 12 h. Results of luciferase assays showed that 0.1 microg/ml decreased and 0.01 microg/ml P. gingivalis LPS increased BSP transcription in -116 to +60 BSP construct. The effects of P. gingivalis LPS were abrogated by double mutations in cAMP response element (CRE) and FGF2 response element (FRE). Tyrosine kinase inhibitor herbimycin A, ERK1/2 inhibitor and antioxidant N-acetylcystein inhibited effects of P. gingivalis LPS. Protein kinase A inhibitor and PI3-kinase/Akt inhibitor only abolished the effect of 0.01 microg/ml P. gingivalis LPS. Furthermore, 0.1 microg/ml LPS decreased the CRE- and FRE-protein complexes formation, whereas 0.01 microg/ml P. gingivalis LPS increased the nuclear protein binding to CRE and FRE. ChIP assays revealed increased binding of CREB1, JunD, Fra2, Runx2, Dlx5, and Smad1 to a chromatin fragment containing the CRE and FRE by 0.01 microg/ml P. gingivalis LPS. These studies therefore indicated that 0.1 microg/ml suppressed, and 0.01 microg/ml P. gingivalis LPS increased BSP gene transcription mediated through CRE and FRE elements in the rat BSP gene promoter. J. Cell. Biochem. 110: 823-833, 2010. (c) 2010 Wiley-Liss, Inc.

CacyBP/SIP as a regulator of transcriptional responses in brain cells

PubMed Central

Kilanczyk, Ewa; Filipek, Anna; Hetman, Michal

2014-01-01

Summary The Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP) is highly expressed in the brain and was shown to regulate the β-catenin-driven transcription in thymocytes. Therefore, it was investigated whether in brain cells CacyBP/SIP might play a role as a transcriptional regulator. In BDNF- or forskolin-stimulated rat primary cortical neurons, overexpression of CacyBP/SIP enhanced transcriptional activity of the cAMP-response element (CRE). In addition, overexpressed CacyBP/SIP enhanced BDNF-mediated activation of the Nuclear Factor of Activated T-cells (NFAT) but not the Serum Response Element (SRE). These stimulatory effects required an intact C-terminal domain of CacyBP/SIP. Moreover, in C6 rat glioma cells, the overexpressed CacyBP/SIP enhanced activation of CRE- or NFAT- following forskolin- or serum stimulation, respectively. Conversely, knockdown of endogenous CacyBP/SIP reduced activation of CRE- and NFAT but not SRE. Taken together, these results indicate that CacyBP/SIP is a novel regulator of CRE- and NFAT-driven transcription. PMID:25163685

Serine 133 Phosphorylation Is Not Required for Hippocampal CREB-Mediated Transcription and Behavior

ERIC Educational Resources Information Center

Brian, Lisa A.; Lee, Bridgin G.; Lelay, John; Kaestner, Klaus H.; Blendy, Julie A.

2015-01-01

The cAMP response element (CRE)-binding protein, CREB, is a transcription factor whose activity in the brain is critical for long-term memory formation. Phosphorylation of Ser133 in the kinase-inducible domain (KID), that in turn leads to the recruitment of the transcriptional coactivator CREB-binding protein (CBP), is thought to mediate the…

Insulin-like growth factor-II regulates bone sialoprotein gene transcription.

PubMed

Choe, Jin; Sasaki, Yoko; Zhou, Liming; Takai, Hideki; Nakayama, Yohei; Ogata, Yorimasa

2016-09-01

Insulin-like growth factor-I and -II (IGF-I and IGF-II) have been found in bone extracts of several different species, and IGF-II is the most abundant growth factor stored in bone. Bone sialoprotein (BSP) is a noncollagenous extracellular matrix glycoprotein associated with mineralized connective tissues. In this study, we have investigated the regulation of BSP transcription by IGF-II in rat osteoblast-like ROS17/2.8 cells. IGF-II (50 ng/ml) increased BSP mRNA and protein levels after 6-h stimulation, and enhanced luciferase activities of the constructs pLUC3 (-116 to +60), pLUC4 (-425 to +60), pLUC5 (-801 to +60) and pLUC6 (-938 to +60). Effects of IGF-II were inhibited by tyrosine kinase, extracellular signal-regulated kinase1/2 and phosphatidylinositol 3-kinase inhibitors, and abrogated by 2-bp mutations in cAMP response element (CRE), FGF2 response element (FRE) and homeodomain protein-binding site (HOX). The results of gel shift assays showed that nuclear proteins binding to CRE, FRE and HOX sites were increased by IGF-II (50 ng/ml) at 3 and 6 h. CREB1, phospho-CREB1, c-Fos and c-Jun antibodies disrupted the formation of the CRE-protein complexes. Dlx5 and Runx2 antibodies disrupted the FRE- and HOX-protein complex formations. These studies therefore demonstrated that IGF-II increased BSP transcription by targeting CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB1, c-Fos, c-Jun, Dlx5 and Runx2 transcription factors appear to be key regulators of IGF-II effects on BSP transcription.

Endothelin-1 regulates rat bone sialoprotein gene transcription.

PubMed

Li, Xinyue; Wang, Zhitao; Yang, Li; Li, Zhengyang; Ogata, Yorimasa

2010-06-01

Endothelin-1 (ET-1) was originally discovered as a vasoconstrictor protein excreted by vascular endothelial cells. Recently, tumor-produced ET-1 has been considered to stimulate osteoblasts to form new bone, and to be an important mediator of osteoblastic bone metastasis. ET-1 has high affinity for two different membrane receptors, ET(A)R and ET(B)R, which are expressed by many types of cells including osteoblasts. Bone sialoprotein (BSP) is a phosphorylated and sulfated glycoprotein associated with mineralized connective tissues. To investigate the effects of ET-1 on BSP transcription, we used rat osteoblast-like ROS17/2.8 cells. Levels of BSP and osteopontin mRNA were increased at 12 h after treatment with ET-1 (10 ng/ml), and ET-1 at the same concentration induced luciferase activity of a -116 to +60 BSP promoter construct at 6 h. Transcriptional activity of -84BSPLUC, which contains the cAMP response element (CRE), was increased by ET-1. Furthermore, at 6 h, ET-1 (10 ng/ml) increased the binding of nuclear protein to CRE, the FGF2 response element (FRE) and the homeodomain protein-binding site (HOX). Antibodies against CREB1, JunD and Fra2 disrupted the formation of CRE-protein complexes, while antibodies against Runx2 and Dlx5 reduced the formation of FRE- and HOX-protein complexes. These findings indicate that ET-1 increases BSP transcription via the CRE, FRE and HOX sites in the rat BSP gene promoter.

Regulation of human bone sialoprotein gene transcription by platelet-derived growth factor-BB.

PubMed

Mezawa, Masaru; Araki, Shouta; Takai, Hideki; Sasaki, Yoko; Wang, Shuang; Li, Xinyue; Kim, Dong-Soon; Nakayama, Youhei; Ogata, Yorimasa

2009-04-15

Platelet-derived growth factor (PDGF) is produced by mesenchymal cells and released by platelets following aggregation and is synthesized by osteoblasts. In bone, PDGF stimulates proliferation and differentiation of osteoblasts. PDGF also increases bone resorption, most likely by increasing the number of osteoclasts. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone, selectively expressed by differentiated osteoblast. To determine the molecular mechanisms PDGF regulation of human BSP gene transcription, we have analyzed the effects of PDGF-BB on osteoblast-like Saos2 and ROS17/2.8 cells. PDGF-BB (5 ng/ml) increased BSP mRNA and protein levels at 12 h in Saos2 cells, and induced BSP mRNA expression at 3 h, reached maximal at 12 h in ROS17/2.8 cells. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of Saos2 cells with PDGF-BB (5 ng/ml, 12 h) increased luciferase activities of all constructs between -184LUC to -2672LUC including the human BSP gene promoter. Effects of PDGF-BB abrogated in constructs included 2 bp mutations in the two cAMP response elements (CRE1 and CRE2), activator protein 1(3) (AP1(3)) and shear stress response element 1 (SSRE1). Luciferase activities induced by PDGF-BB were blocked by protein kinase A inhibitor H89 and tyrosine kinase inhibitor herbimycin A. Gel mobility shift analyses showed that PDGF-BB increased binding of CRE1, CRE2, AP1(3) and SSRE1 elements. CRE1- and CRE2-protein complexes were supershifted by CREB1 and phospho-CREB1 antibodies. Notably, AP1(3)-protein complexes were supershifted by c-Fos and JunD, and disrupted by CREB1, phospho-CREB1, c-Jun and Fra2 antibodies. These studies, therefore, demonstrate that PDGF-BB stimulates human BSP transcription by targeting the CRE1, CRE2, AP1(3) and SSRE1 elements in the human BSP gene promoter.

Membrane and Integrative Nuclear Fibroblastic Growth Factor Receptor (FGFR) Regulation of FGF-23*

PubMed Central

Han, Xiaobin; Xiao, Zhousheng; Quarles, L. Darryl

2015-01-01

Fibroblastic growth factor receptor 1 (FGFR1) signaling pathways are implicated in the regulation of FGF-23 gene transcription, but the molecular pathways remain poorly defined. We used low molecular weight (LMW, 18 kDa) FGF-2 and high molecular weight (HMW) FGF-2 isoforms, which, respectively, activate cell surface FGF receptors and intranuclear FGFR1, to determine the roles of membrane FGFRs and integrative nuclear FGFR1 signaling (INFS) in the regulation of FGF-23 gene transcription in osteoblasts. We found that LMW-FGF-2 induced NFAT and Ets1 binding to conserved cis-elements in the proximal FGF-23 promoter and stimulated FGF-23 promoter activity through PLCγ/calcineurin/NFAT and MAPK pathways in SaOS-2 and MC3T3-E1 osteoblasts. In contrast, HMW-FGF-2 stimulated FGF-23 promoter activity in osteoblasts through a cAMP-dependent binding of FGFR1 and cAMP-response element-binding protein (CREB) to a conserved cAMP response element (CRE) contiguous with the NFAT binding site in the FGF-23 promoter. Mutagenesis of the NFAT and CRE binding sites, respectively, inhibited the effects of LMW-FGF-2 and HMW-FGF-23 to stimulate FGF-23 promoter activity. FGF-2 activation of both membrane FGFRs and INFS-dependent FGFR1 pathways may provide a means to integrate systemic and local regulation of FGF-23 transcription under diverse physiological and pathological conditions. PMID:25752607

cAMP Response Element-Binding Protein Is Required for Dopamine-Dependent Gene Expression in the Intact But Not the Dopamine-Denervated Striatum

PubMed Central

Andersson, Malin; Konradi, Christine; Cenci, M. Angela

2014-01-01

The cAMP response element-binding protein (CREB) is believed to play a pivotal role in dopamine (DA) receptor-mediated nuclear signaling and neuroplasticity. Here we demonstrate that the significance of CREB for gene expression depends on the experimental paradigm. We compared the role of CREB in two different but related models: L-DOPA administration to unilaterally 6-hydroxydopamine lesioned rats, and cocaine administration to neurologically intact animals. Antisense technology was used to produce a local knockdown of CREB in the lateral caudate–putamen, a region that mediates the dyskinetic or stereotypic manifestations associated with L-DOPA or cocaine treatment, respectively. In intact rats, CREB antisense reduced both basal and cocaine-induced expression of c-Fos, FosB/ΔFosB, and prodynorphin mRNA. In the DA-denervated striatum, CREB was not required for L-DOPA to induce these gene products, nor did CREB contribute considerably to DNA binding activity at cAMP responsive elements (CREs) and CRE-like enhancers. ΔFosB-related proteins and JunD were the main contributors to both CRE and AP-1 DNA–protein complexes in L-DOPA-treated animals. In behavioral studies, intrastriatal CREB knockdown caused enhanced activity scores in intact control animals and exacerbated the dyskinetic effects of acute L-DOPA treatment in 6-OHDA-lesioned animals. These data demonstrate that CREB is not required for the development of L-DOPA-induced dyskinesia in hemiparkinsonian rats. Moreover, our results reveal an unexpected alteration of nuclear signaling mechanisms in the parkinsonian striatum treated with L-DOPA, where AP-1 transcription factors appear to supersede CREB in the activation of CRE-containing genes. PMID:11739600

Fibroblast growth factor 2 regulates bone sialoprotein gene transcription in human breast cancer cells.

PubMed

Li, Zhengyang; Wang, Zhitao; Yang, Li; Li, Xinyue; Sasaki, Yoko; Wang, Shuang; Araki, Shouta; Mezawa, Masaru; Takai, Hideki; Nakayama, Youhei; Ogata, Yorimasa

2010-03-01

Bone sialoprotein (BSP) is a major non-collagenous, extracellular matrix glycoprotein associated with mineralized tissues. Fibroblast growth factor 2 (FGF2) is recognized as a potent mitogen for a variety of mesenchymal cells. FGF2 produced by osteoblasts accumulates in the bone matrix and acts as an autocrine/paracrine regulator of osteoblasts. We previously reported that FGF2 regulates BSP gene transcription through the FGF2 response element (FRE) and activator protein 1 (AP1) binding site overlapping with the glucocorticoid response element in the rat BSP gene promoter. In the present study, FGF2 (10 ng/ml) increased BSP and Runx2 mRNA levels at 6 h in MCF7 human breast cancer cells. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of MCF7 cells with FGF2 (10 ng/ml) increased the luciferase activity of the constructs between -84LUC and -927LUC. Gel mobility shift analyses showed that FGF2 increased the binding of AP1 and CRE2. The CRE2- and AP1-protein complexes were disrupted by antibodies against CREB1, c-Fos, c-Jun, Fra2, p300 and Runx2. These studies demonstrate that FGF2 stimulates BSP transcription in MCF7 human breast cancer cells by targeting the AP1 and CRE2 elements in the human BSP gene promoter.

Androgen receptor stimulates bone sialoprotein (BSP) gene transcription via cAMP response element and activator protein 1/glucocorticoid response elements.

PubMed

Takai, Hideki; Nakayama, Youhei; Kim, Dong-Soon; Arai, Masato; Araki, Shouta; Mezawa, Masaru; Nakajima, Yu; Kato, Naoko; Masunaga, Hiroshi; Ogata, Yorimasa

2007-09-01

Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. Androgens are steroid hormones that are essential for skeletal development. The androgen receptor (AR) is a transcription factor and a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. To determine the molecular mechanism involved in the stimulation of bone formation, we have analyzed the effects of androgens and AR effects on BSP gene transcription. AR protein levels were increased after AR overexpression in ROS17/2.8 cells. BSP mRNA levels were increased by AR overexpression. However, the endogenous and overexpressed BSP mRNA levels were not changed by DHT (10(-8) M, 24 h). Whereas luciferase (LUC) activities in all constructs, including a short construct (nts -116 to +60), were increased by AR overexpression, the basal and LUC activities enhanced by AR overexpression were not induced by DHT (10(-8)M, 24 h). The effect of AR overexpression was abrogated by 2 bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that AR overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE-protein complexes were supershifted by phospho-CREB antibody, and CREB, c-Fos, c-Jun, and AR antibodies disrupted the complexes formation. The AP1/GRE-protein complexes were supershifted by c-Fos antibody and c-Jun, and AR antibodies disrupted the complexes formation. These studies demonstrate that AR stimulates BSP gene transcription by targeting the CRE and AP1/GRE elements in the promoter of the rat BSP gene.

CRE-Mediated Transcription and COX-2 Expression in the Pilocarpine Model of Status Epilepticus

PubMed Central

Lee, Boyoung; Dziema, Heather; Lee, Kyu Hyun; Choi, Yun-Sik; Obrietan, Karl

2007-01-01

Status epilepticus (SE) triggers neuronal death, reactive gliosis and remodeling of synaptic circuitry, thus leading to profound pathological alterations in CNS physiology. These processes are, in part, regulated by the rapid upregulation of both cytotoxic and cytoprotective genes. One pathway that may couple SE to transcriptionally-dependent alterations in CNS physiology is the CREB (cAMP response element-binding protein)/CRE (cAMP response element) cascade. Here, we utilized the pilocarpine model of SE on a mouse strain transgenic for a CRE-reporter construct (β-galactosidase) to begin to characterize how seizure activity regulates the activation state of the CREB/CRE pathway in both glia and neurons of the hippocampus. SE triggered a rapid (4–8 hrs post SE) but transient increase in CRE-mediated gene expression in the neuronal sublayers. In contrast to neurons, SE induced a lasting increase (up to 20 days) in CRE-mediated transcription in both reactive astrocytes and microglia. CRE-mediated gene expression correlated with expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2). To examine the role of CREB in SE-induced COX-2 expression, we generated a transgenic mouse strain that expresses A-CREB, a potent repressor of CREB-dependent transcription. In these animals, the capacity of SE to stimulate COX-2 expression was markedly attenuated, indicating that CREB is a key intermediate in SE-induced COX-2 expression. Collectively these data show that SE triggers two waves of CREB-mediated gene expression, a transient wave in neurons and a long-lasting wave in reactive glial cells, and that CREB couples SE to COX-2 expression. PMID:17029965

Configuration control on the shape memory stiffness of molecularly imprinted polymer for specific uptake of creatinine

NASA Astrophysics Data System (ADS)

Ang, Qian Yee; Zolkeflay, Muhammad Helmi; Low, Siew Chun

2016-04-01

In this study, sol-gel processing was proposed to prepare a creatinine (Cre)-imprinted molecularly imprinted polymer (MIP). The intermolecular interaction constituted by the cross-linkers, i.e., 2-acrylamido-2-methylpropane-sulfonic acid (AMPS) and aluminium ion (Al3+), was studied and compared in order to form a confined matrix that promises the effectiveness of molecular imprinting. In view of the shape recognition, the hydrogen bonded Cre-AMPS did not demonstrate good recognition of Cre, with Cre binding found only at 5.70 ± 0.15 mg g-1 of MIP. Whilst, MIP cross-linked using Al3+ was able to attain an excellent Cre adsorption capacity of 19.48 ± 0.64 mg g-1 of MIP via the stronger ionic interaction of Cre-Al3+. Based on the Scatchard analysis, a higher Cre concentration in testing solution required greater driving force to resolve the binding resistance of Cre molecules, so as to have a precise Cre binding with shape factor. The molecular recognition ability of Cre-MIP in present work was shape-specific for Cre as compared to its structural analogue, 2-pyrrolidinone (2-pyr), by an ideal selectivity coefficient of 6.57 ± 0.10. In overall, this study has come up with a practical approach on the preparation of MIP for the detection of renal dysfunction by point-of-care Cre testing.

FABP4-Cre mediated expression of constitutively active ChREBP protects against obesity

USDA-ARS?s Scientific Manuscript database

Carbohydrate response element binding protein (ChREBP) regulates cellular glucose and lipid homeostasis. Although ChREBP is highly expressed in many key metabolic tissues, the role of ChREBP in most of those tissues and consequent effects on whole-body glucose and lipid metabolism are not well under...

Mechanism of DNA-binding enhancement by the human T-cell leukaemia virus transactivator Tax.

PubMed

Baranger, A M; Palmer, C R; Hamm, M K; Giebler, H A; Brauweiler, A; Nyborg, J K; Schepartz, A

1995-08-17

Tax protein activates transcription of the human T-cell leukaemia virus type I (HTLV-I) genome through three imperfect cyclic AMP-responsive element (CRE) target sites located within the viral promoter. Previous work has shown that Tax interacts with the bZIP element of proteins that bind the CRE target site to promote peptide dimerization, suggesting an association between Tax and bZIP coiled coil. Here we show that the site of interaction with Tax is not the coiled coil, but the basic segment. This interaction increases the stability of the GCN4 bZIP dimer by 1.7 kcal mol-1 and the DNA affinity of the dimer by 1.9 kcal mol-1. The differential effect of Tax on several bZip-DNA complexes that differ in peptide sequence or DNA conformation suggests a model for Tax action based on stabilization of a distinct DNA-bound protein structure. This model may explain how Tax interacts with transcription factors of considerable sequence diversity to alter patterns of gene expression.

Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease

DOE PAGES

Chatterjee, Sumantra; Kapoor, Ashish; Akiyama, Jennifer A.; ...

2016-09-29

Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidencemore » that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.« less

Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chatterjee, Sumantra; Kapoor, Ashish; Akiyama, Jennifer A.

Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidencemore » that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders.« less

Stoichiometry of the Cre recombinase bound to the lox recombining site.

PubMed Central

Mack, A; Sauer, B; Abremski, K; Hoess, R

1992-01-01

The site-specific recombinase Cre from bacteriophage P1 binds and carries out recombination at a 34 bp lox site. The lox site consists of two 13 bp inverted repeats, separated by an 8 bp spacer region. Both the palindromic nature of the site and the results of footprinting and band shift experiments suggest that a minimum of two Cre molecules bind to a lox site. We report here experiments that demonstrate the absolute stoichiometry of the Cre-lox complex to be one molecule of Cre bound per inverted repeat, or two molecules per lox site. Images PMID:1408747

Human osteocalcin and bone sialoprotein mediating osteomimicry of prostate cancer cells: role of cAMP-dependent protein kinase A signaling pathway.

PubMed

Huang, Wen-Chin; Xie, Zhihui; Konaka, Hiroyuki; Sodek, Jaro; Zhau, Haiyen E; Chung, Leland W K

2005-03-15

Osteocalcin and bone sialoprotein are the most abundant noncollagenous bone matrix proteins expressed by osteoblasts. Surprisingly, osteocalcin and bone sialoprotein are also expressed by malignant but not normal prostate epithelial cells. The purpose of this study is to investigate how osteocalcin and bone sialoprotein expression is regulated in prostate cancer cells. Our investigation revealed that (a) human osteocalcin and bone sialoprotein promoter activities in an androgen-independent prostate cancer cell line of LNCaP lineage, C4-2B, were markedly enhanced 7- to 12-fold in a concentration-dependent manner by conditioned medium collected from prostate cancer and bone stromal cells. (b) Deletion analysis of human osteocalcin and bone sialoprotein promoter regions identified cyclic AMP (cAMP)-responsive elements (CRE) as the critical determinants for conditioned medium-mediated osteocalcin and bone sialoprotein gene expression in prostate cancer cells. Consistent with these results, the protein kinase A (PKA) pathway activators forskolin and dibutyryl cAMP and the PKA pathway inhibitor H-89, respectively, increased or repressed human osteocalcin and bone sialoprotein promoter activities. (c) Electrophoretic mobility shift assay showed that conditioned medium-mediated stimulation of human osteocalcin and bone sialoprotein promoter activities occurs through increased interaction between CRE and CRE-binding protein. (d) Conditioned medium was found to induce human osteocalcin and bone sialoprotein promoter activities via increased CRE/CRE-binding protein interaction in a cell background-dependent manner, with marked stimulation in selected prostate cancer but not bone stromal cells. Collectively, these results suggest that osteocalcin and bone sialoprotein expression is coordinated and regulated through cAMP-dependent PKA signaling, which may define the molecular basis of the osteomimicry exhibited by prostate cancer cells.

Kaempferol stimulates bone sialoprotein gene transcription and new bone formation.

PubMed

Yang, Li; Takai, Hideki; Utsunomiya, Tadahiko; Li, Xinyue; Li, Zhengyang; Wang, Zhitao; Wang, Shuang; Sasaki, Yoko; Yamamoto, Hirotsugu; Ogata, Yorimasa

2010-08-15

Kaempferol is a typical flavonol-type flavonoid that is present in a variety of vegetables and fruits, and has a protective effect on postmenopausal bone loss. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone and could be crucial for osteoblast differentiation, bone matrix mineralization and tumor metastasis. In the present study we investigated the regulation of BSP transcription by kaempferol in rat osteoblast-like UMR106 cells, and the effect of kaempferol on new bone formation. Kaempferol (5 microM) increased BSP and Osterix mRNA levels at 12 h and up-regulated Runx2 mRNA expression at 6 h. Kaempferol increased luciferase activity of the construct pLUC3, which including the promoter sequence between nucleotides -116 to +60. Transcriptional stimulation by kaempferol abrogated in constructs included 2 bp mutations in the inverted CCAAT, CRE, and FRE elements. Gel shift analyses showed that kaempferol increased nuclear protein binding to CRE and FRE elements, whereas the CCAAT-protein complex did not change after kaempferol stimulation. Twelve daily injections of 5 microM kaempferol directly into the periosteum of parietal bones of newborn rats increased new bone formation. These data suggest that kaempferol increased BSP gene transcription mediated through inverted CCAAT, CRE, and FRE elements in the rat BSP gene promoter, and could induce osteoblast activities in the early stage of bone formation. (c) 2010 Wiley-Liss, Inc.

Role of cocaine- and amphetamine-regulated transcript in estradiol-mediated neuroprotection

NASA Astrophysics Data System (ADS)

Xu, Yun; Zhang, Wenri; Klaus, Judith; Young, Jennifer; Koerner, Ines; Sheldahl, Laird C.; Hurn, Patricia D.; Martínez-Murillo, Francisco; Alkayed, Nabil J.

2006-09-01

Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine- and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases. ischemia | stroke | estrogen

Binding of FUN14 Domain Containing 1 With Inositol 1,4,5-Trisphosphate Receptor in Mitochondria-Associated Endoplasmic Reticulum Membranes Maintains Mitochondrial Dynamics and Function in Hearts in Vivo.

PubMed

Wu, Shengnan; Lu, Qiulun; Wang, Qilong; Ding, Ye; Ma, Zejun; Mao, Xiaoxiang; Huang, Kai; Xie, Zhonglin; Zou, Ming-Hui

2017-12-05

FUN14 domain containing 1 (FUNDC1) is a highly conserved outer mitochondrial membrane protein. The aim of this study is to examine whether FUNDC1 modulates the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), mitochondrial morphology, and function in cardiomyocytes and intact hearts. The impacts of FUNDC1 on MAMs formation and cardiac functions were studied in mouse neonatal cardiomyocytes, in mice with cardiomyocyte-specific Fundc1 gene knockout ( Fundc1 f/Y /Cre αMyHC+/- ), and in the cardiac tissues of the patients with heart failure. In mouse neonatal cardiomyocytes and intact hearts, FUNDC1 was localized in MAMs by binding to ER-resided inositol 1,4,5-trisphosphate type 2 receptor (IP 3 R2). Fundc1 ablation disrupted MAMs and reduced the levels of IP 3 R2 and Ca 2+ in both mitochondria and cytosol, whereas overexpression of Fundc1 increased the levels of IP 3 R2 and Ca 2+ in both mitochondria and cytosol. Consistently, Fundc1 ablation increased Ca 2+ levels in ER, whereas Fundc1 overexpression lowered ER Ca 2+ levels. Further, Fundc1 ablation in cardiomyocytes elongated mitochondria and compromised mitochondrial functions. Mechanistically, we found that Fundc1 ablation-induced reduction of intracellular Ca 2+ levels suppressed mitochondrial fission 1 protein ( Fis1 ) expression and mitochondrial fission by reducing the binding of the cAMP response element binding protein (CREB) in the Fis1 promoter. Fundc1 f/Y /Cre αMyHC+/- mice but not their littermate control mice ( Fundc1 wt/Y /Cre αMyHC+/- ) exhibited cardiac dysfunction. The ligation of the left ventricle artery of Fundc1 f/Y /Cre αMyHC+/- mice caused more severe cardiac dysfunction than those in sham-treated Fundc1 f/Y /Cre αMyHC+/- mice. Finally, we found that the FUNDC1/MAMs/CREB/Fis1 signaling axis was significantly suppressed in patients with heart failure. We conclude that FUNDC1 binds to IP 3 R2 to modulate ER Ca 2+ release into mitochondria and cytosol. Further, a disruption of the FUNDC1 and IP 3 R2 interaction lowers the levels of Ca 2+ in mitochondria and cytosol, both of which instigate aberrant mitochondrial fission, mitochondrial dysfunction, cardiac dysfunction, and heart failure. © 2017 American Heart Association, Inc.

17beta-estradiol stimulates the growth of human keratinocytes by inducing cyclin D2 expression.

PubMed

Kanda, Naoko; Watanabe, Shinichi

2004-08-01

Estrogen is reported to prevent age-associated epidermal thinning in the skin. We examined if 17beta-estradiol (E2) may enhance the growth of human keratinocytes, focusing on its effects on the expression of cell cycle-regulatory proteins. E2 enhanced proliferation, bromodeoxyuridine incorporation of keratinocytes, and increased the proportion of cells in the S phase. The E2-induced stimulation of proliferation and bromodeoxyuridine incorporation was suppressed by antisense oligonucleotide against cyclin D2, which induces G1 to S phase progression. E2 increased protein and mRNA levels of cyclin D2, and resultantly enhanced assembly and kinase activities of cyclin D2-cyclin-dependent kinases 4 or 6 complexes. E2 enhanced cyclin D2 promoter activity, and the element homologous to cAMP response element (CRE) on the promoter was responsible for the effect. Cyclin D2 expression was enhanced by antiestrogens, ICI 182,780 and 4-hydroxytamoxifen, and membrane-impermeable bovine serum albumin-conjugated E2, indicating the effects via membrane E2-binding sites. E2 increased the enhancer activity of CRE-like element and the amount of phosphorylated cAMP response element binding protein (CREB) binding this element, and the increases were suppressed by H-89, an inhibitor of cAMP-dependent protein kinase A. H-89 also suppressed E2-induced cyclin D2 expression, proliferation, and bromodeoxyuridine incorporation in keratinocytes. Antisense oligonucleotide against G-protein-coupled receptor GPR30 suppressed the E2-induced increases of phosphorylated CREB, cyclin D2 level, proliferation, and bromodeoxyuridine incorporation in keratinocytes. These results suggest that E2 may stimulate the growth of keratinocytes by inducing cyclin D2 expression via CREB phosphorylation by protein kinase A, dependent on cAMP. These effects of E2 may be mediated via cell surface GPR30.

Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts

NASA Technical Reports Server (NTRS)

Thomas, M. J.; Umayahara, Y.; Shu, H.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

1996-01-01

Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal activation of IGF-I gene transcription by cAMP in osteoblasts.

Unliganded estrogen receptor α stimulates bone sialoprotein gene expression.

PubMed

Takai, Hideki; Matsumura, Hiroyoshi; Matsui, Sari; Kim, Kyung Mi; Mezawa, Masaru; Nakayama, Yohei; Ogata, Yorimasa

2014-04-10

Estrogen is one of the steroid hormones essential for skeletal development. The estrogen receptor (ER) is a transcription factor and a member of the steroid receptor superfamily. There are two different forms of the ER, usually referred to as α and β, each encoded by a separate gene. Hormone-activated ERs form dimers, since the two forms are coexpressed in many cell types. Bone sialoprotein (BSP) is a tissue-specific acidic glycoprotein that is expressed by differentiated osteoblasts, odontoblasts and cementoblasts during the initial formation of mineralized tissue. To determine the molecular basis of the tissue-specific expression of BSP and its regulation by estrogen and the ER, we have analyzed the effects of β-estradiol and ERα on BSP gene transcription. ERα protein levels were increased after ERα overexpression in ROS17/2.8 cells. While BSP mRNA levels were increased by ERα overexpression, the endogenous and overexpressed BSP mRNA levels were not changed by β-estradiol (10(-8)M, 24 h). Luciferase activities of different sized BSP promoter constructs (pLUC3~6) were increased by ERα overexpression, whereas basal and induced luciferase activities by ERα overexpression were not influenced by β-estradiol. Effects of ERα overexpression were abrogated by 2 bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that ERα overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE-protein complexes were disrupted by ERα, CREB and phospho-CREB antibodies. The AP1/GRE-protein complexes were supershifted by the c-Fos antibody. These studies demonstrate that ERα stimulates BSP gene transcription in a ligand-independent manner by targeting the CRE and AP1/GRE elements in the rat BSP gene promoter. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

PubMed

Gachon, F; Thebault, S; Peleraux, A; Devaux, C; Mesnard, J M

2000-05-01

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.

Molecular Interactions Involved in the Transactivation of the Human T-Cell Leukemia Virus Type 1 Promoter Mediated by Tax and CREB-2 (ATF-4)

PubMed Central

Gachon, Frederic; Thebault, Sabine; Peleraux, Annick; Devaux, Christian; Mesnard, Jean-Michel

2000-01-01

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation. PMID:10779337

Molecular characterization of thyroid hormone-inhibited atrial L-type calcium channel expression: implication for atrial fibrillation in hyperthyroidism.

PubMed

Chen, Wei-Jan; Yeh, Yung-Hsin; Lin, Kwang-Huei; Chang, Gwo-Jyh; Kuo, Chi-Tai

2011-03-01

Atrial fibrillation (AF) is a common complication in hyperthyroidism. Earlier studies demonstrate that thyroid hormone decreases L-type calcium channel (LCC) current expression with resultant shortening of action potential duration (APD), providing a substrate for AF. The aim of this study was to investigate the potential mechanism underlying the regulatory effect of thyroid hormone on LCC. In a hyperthyroid rat model, thyroid hormone (triiodothyronine [T3]) administration down-regulated atrial LCC expression. In vitro, treatment of murine atrial myocytes (HL-1) with T3 decreased the expression of LCC and its current, resulting in abbreviation of APD. Furthermore, T3 inhibited the activation of cyclic AMP response element (CRE)-binding protein (CREB), including phosphorylation at Ser133 and its nuclear translocation. Transient transfection studies in HL-1 cells indicated that T3 reduced LCC promoter activity. Deletion and mutation analysis of the LCC promoter region along with chromatin immunoprecipitation using anti-CREB antibody showed that CRE was essential for T3-mediated LCC gene expression. Transfection of dominant-negative CREB (mutated Ser133) and mutant thyroid hormone receptor (TR, mutated Cys51) abolished the T3-dependent effects, suggesting an association between both transcriptional factors. Co-immunoprecipitation documented an increased binding of TR with CREB after T3 treatment. The transcriptional cross-talk 3 between TR and CREB bound to CRE mediates T3-inhibited CREB activity and LCC expression. Thyroid hormone-induced TR binding of CREB inhibits CREB activity and LCC current expression, which may contribute to AF. These findings provide an important mechanistic insight into hyperthyroidism-induced AF.

Nicotine suppresses bone sialoprotein gene expression.

PubMed

Nakayama, Y; Mezawa, M; Araki, S; Sasaki, Y; Wang, S; Han, J; Li, X; Takai, H; Ogata, Y

2009-10-01

Tobacco smoking is a risk factor for periodontitis and osteoporosis. Nicotine is a major component of tobacco, and has been reported to inhibit proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. The purpose of this study was to determine the effects of nicotine on bone metabolism. We used rat osteobast-like UMR106 and ROS 17/2.8 cells and rat stromal bone marrow RBMC-D8 cells. To determine the molecular basis of the transcriptional regulation of the BSP gene by nicotine, we conducted Northern hybridization, transient transfection analyses with chimeric constructs of the BSP gene promoter linked to a luciferase reporter gene and gel mobility shift assays. Nicotine (250 microg/mL) decreased the BSP mRNA levels at 12 and 24 h in UMR106 and ROS 17/2.8 cells. From transient transfection assays using various sized BSP promoter-luciferase constructs, nicotine decreased the luciferase activities of the construct, including the promoter sequence nucleotides -116 to +60, in UMR106 and RBMC-D8 cells. Nicotine decreased the nuclear protein binding to the cAMP response element (CRE), fibroblast growth factor 2 response element (FRE) and homeodomain protein-binding site (HOX) at 12 and 24 h. This study indicates that nicotine suppresses BSP transcription mediated through CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene.

Overlapping ETS and CRE Motifs (G/CCGGAAGTGACGTCA) Preferentially Bound by GABPα and CREB Proteins

PubMed Central

Chatterjee, Raghunath; Zhao, Jianfei; He, Ximiao; Shlyakhtenko, Andrey; Mann, Ishminder; Waterfall, Joshua J.; Meltzer, Paul; Sathyanarayana, B. K.; FitzGerald, Peter C.; Vinson, Charles

2012-01-01

Previously, we identified 8-bps long DNA sequences (8-mers) that localize in human proximal promoters and grouped them into known transcription factor binding sites (TFBS). We now examine split 8-mers consisting of two 4-mers separated by 1-bp to 30-bps (X4-N1-30-X4) to identify pairs of TFBS that localize in proximal promoters at a precise distance. These include two overlapping TFBS: the ETS⇔ETS motif (C/GCCGGAAGCGGAA) and the ETS⇔CRE motif (C/GCGGAAGTGACGTCAC). The nucleotides in bold are part of both TFBS. Molecular modeling shows that the ETS⇔CRE motif can be bound simultaneously by both the ETS and the B-ZIP domains without protein-protein clashes. The electrophoretic mobility shift assay (EMSA) shows that the ETS protein GABPα and the B-ZIP protein CREB preferentially bind to the ETS⇔CRE motif only when the two TFBS overlap precisely. In contrast, the ETS domain of ETV5 and CREB interfere with each other for binding the ETS⇔CRE. The 11-mer (CGGAAGTGACG), the conserved part of the ETS⇔CRE motif, occurs 226 times in the human genome and 83% are in known regulatory regions. In vivo GABPα and CREB ChIP-seq peaks identified the ETS⇔CRE as the most enriched motif occurring in promoters of genes involved in mRNA processing, cellular catabolic processes, and stress response, suggesting that a specific class of genes is regulated by this composite motif. PMID:23050235

A Novel Dual-cre Motif Enables Two-Way Autoregulation of CcpA in Clostridium acetobutylicum.

PubMed

Zhang, Lu; Liu, Yanqiang; Yang, Yunpeng; Jiang, Weihong; Gu, Yang

2018-04-15

The master regulator CcpA (catabolite control protein A) manages a large and complex regulatory network that is essential for cellular physiology and metabolism in Gram-positive bacteria. Although CcpA can affect the expression of target genes by binding to a cis -acting catabolite-responsive element ( cre ), whether and how the expression of CcpA is regulated remain poorly explored. Here, we report a novel dual- cre motif that is employed by the CcpA in Clostridium acetobutylicum , a typical solventogenic Clostridium species, for autoregulation. Two cre sites are involved in CcpA autoregulation, and they reside in the promoter and coding regions of CcpA. In this dual- cre motif, cre P , in the promoter region, positively regulates ccpA transcription, whereas cre ORF , in the coding region, negatively regulates this transcription, thus enabling two-way autoregulation of CcpA. Although CcpA bound cre P more strongly than cre ORF in vitro , the in vivo assay showed that cre ORF -based repression dominates CcpA autoregulation during the entire fermentation. Finally, a synonymous mutation of cre ORF was made within the coding region, achieving an increased intracellular CcpA expression and improved cellular performance. This study provides new insights into the regulatory role of CcpA in C. acetobutylicum and, moreover, contributes a new engineering strategy for this industrial strain. IMPORTANCE CcpA is known to be a key transcription factor in Gram-positive bacteria. However, it is still unclear whether and how the intracellular CcpA level is regulated, which may be essential for maintaining normal cell physiology and metabolism. We discovered here that CcpA employs a dual- cre motif to autoregulate, enabling dynamic control of its own expression level during the entire fermentation process. This finding answers the questions above and fills a void in our understanding of the regulatory network of CcpA. Interference in CcpA autoregulation leads to improved cellular performance, providing a new useful strategy in genetic engineering of C. acetobutylicum Since CcpA is widespread in Gram-positive bacteria, including pathogens, this dual- cre -based CcpA autoregulation would be valuable for increasing our understanding of CcpA-based global regulation in bacteria. Copyright © 2018 American Society for Microbiology.

Regulation of Cre recombinase by ligand-induced complementation of inactive fragments.

PubMed

Jullien, Nicolas; Sampieri, François; Enjalbert, Alain; Herman, Jean-Paul

2003-11-01

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05-0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48-72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.

CREB-1 and AP-1 transcription factors JunD and Fra-2 regulate bone sialoprotein gene expression in human breast cancer cells.

PubMed

Detry, C; Lamour, V; Castronovo, V; Bellahcène, A

2008-02-01

Bone sialoprotein (BSP) expression is detected in a variety of human osteotropic cancers. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. In this study, we examined the transcriptional regulation of BSP gene expression in MDA-MB-231 and MCF-7 human breast cancer cells compared with Saos-2 human osteoblast-like cells. BSP human promoter deletion analyses delineated a -56/-84 region, which comprises a cAMP response element (CRE) that was sufficient for maximal promoter activity in breast cancer cell lines. We found that the basic fibroblast growth factor response element (FRE) also located in the proximal promoter was a crucial regulator of human BSP promoter activity in Saos-2 but not in breast cancer cells. Promoter activity experiments in combination with DNA mobility shift assays demonstrated that BSP promoter activity is under the control of the CRE element, through CREB-1, JunD and Fra-2 binding, in MDA-MB-231, MCF-7 and in Saos-2 cells. Forskolin, a protein kinase A pathway activator, failed to enhance BSP transcriptional activity suggesting that CRE site behaves as a constitutive rather than an inducible element in these cell lines. Over-expression of JunD and Fra-2 increased BSP promoter activity and upregulated endogenous BSP protein expression in MCF-7 and Saos-2 cells while siRNA-mediated inhibition of both factors expression significantly reduced BSP protein level in MDA-MB-231. Collectively, these data provide with new transcriptional mechanisms, implicating CREB and AP-1 factors, that control BSP gene expression in breast cancer cells.

Conditional transgenesis using Dimerizable Cre (DiCre).

PubMed

Jullien, Nicolas; Goddard, Isabelle; Selmi-Ruby, Samia; Fina, Jean-Luc; Cremer, Harold; Herman, Jean-Paul

2007-12-26

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCrexR26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters.

o,p'-DDT induces cyclooxygenase-2 gene expression in murine macrophages: Role of AP-1 and CRE promoter elements and PI3-kinase/Akt/MAPK signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Eun Hee; Kim, Ji Young; Kim, Hyung-Kyun

Dichlorodiphenyltrichloroethane (DDT) has been used as an insecticide to prevent the devastation of malaria in tropical zones. However, many reports suggest that DDT may act as an endocrine disruptor and may have possible carcinogenic effects. Cyclooxygenase-2 (COX-2) acts as a link between inflammation and carcinogenesis through its involvement in tumor promotion. In the present study, we examined the effect of o,p'-DDT on COX-2 gene expression and analyzed the molecular mechanism of its activity in murine RAW 264.7 macrophages. Exposure to o,p'-DDT markedly enhanced the production of prostaglandin E{sub 2} (PGE{sub 2}), a major COX-2 metabolite, in murine macrophages. Furthermore, o,p'-DDTmore » dose-dependently increased the levels of COX-2 protein and mRNA. Transfection with human COX-2 promoter construct, electrophoretic mobility shift assays and DNA-affinity protein-binding assay experiments revealed that o,p'-DDT activated the activator protein 1 (AP-1) and cyclic AMP response element (CRE) sites, but not the NF-{kappa}B site. Phosphatidylinositol 3 (PI3)-kinase, its downstream signaling molecule, Akt, and mitogen-activated protein kinases (MAPK) were also significantly activated by the o,p'-DDT-induced AP-1 and CRE activation. These results demonstrate that o,p'-DDT induced COX-2 expression via AP-1 and CRE activation through the PI3-K/Akt/ERK, JNK, and p38 MAP kinase pathways. These findings provide further insight into the signal transduction pathways involved in the carcinogenic effects of o,p'-DDT.« less

Stepwise evolution of pandrug-resistance in Klebsiella pneumoniae

PubMed Central

Zowawi, Hosam M.; Forde, Brian M.; Alfaresi, Mubarak; Alzarouni, Abdulqadir; Farahat, Yasser; Chong, Teik-Min; Yin, Wai-Fong; Chan, Kok-Gan; Li, Jian; Schembri, Mark A.; Beatson, Scott A.; Paterson, David L.

2015-01-01

Carbapenem resistant Enterobacteriaceae (CRE) pose an urgent risk to global human health. CRE that are non-susceptible to all commercially available antibiotics threaten to return us to the pre-antibiotic era. Using Single Molecule Real Time (SMRT) sequencing we determined the complete genome of a pandrug-resistant Klebsiella pneumoniae isolate, representing the first complete genome sequence of CRE resistant to all commercially available antibiotics. The precise location of acquired antibiotic resistance elements, including mobile elements carrying genes for the OXA-181 carbapenemase, were defined. Intriguingly, we identified three chromosomal copies of an ISEcp1-blaOXA-181 mobile element, one of which has disrupted the mgrB regulatory gene, accounting for resistance to colistin. Our findings provide the first description of pandrug-resistant CRE at the genomic level, and reveal the critical role of mobile resistance elements in accelerating the emergence of resistance to other last resort antibiotics. PMID:26478520

ATF3 represses PPARγ expression and inhibits adipocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Min-Kyung; Jung, Myeong Ho, E-mail: jung0603@pusan.ac.kr

Highlights: • ATF3 decrease the expression of PPARγ and its target gene in 3T3-L1 adipocytes. • ATF3 represses the promoter activity of PPARγ2 gene. • ATF/CRE (−1537/−1530) is critical for ATF3-mediated downregulation of PPARγ. • ATF3 binds to the promoter region containing the ATF/CRE. • ER stress inhibits adipocyte differentiation through downregulation of PPARγ by ATF3. - Abstract: Activating transcription factor 3 (ATF3) is a stress-adaptive transcription factor that mediates cellular stress response signaling. We previously reported that ATF3 represses CCAAT/enhancer binding protein α (C/EBPα) expression and inhibits 3T3-L1 adipocyte differentiation. In this study, we explored potential role of ATF3more » in negatively regulating peroxisome proliferator activated receptor-γ (PPARγ). ATF3 decreased the expression of PPARγ and its target gene in 3T3-L1 adipocytes. ATF3 also repressed the activity of −2.6 Kb promoter of mouse PPARγ2. Overexpression of PPARγ significantly prevented the ATF3-mediated inhibition of 3T3-L1 differentiation. Transfection studies with 5′ deleted-reporters showed that ATF3 repressed the activity of −2037 bp promoter, whereas it did not affect the activity of −1458 bp promoter, suggesting that ATF3 responsive element is located between the −2037 and −1458. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds to ATF/CRE site (5′-TGACGTTT-3′) between −1537 and −1530. Mutation of the ATF/CRE site abrogated ATF3-mediated transrepression of the PPARγ2 promoter. Treatment with thapsigargin, endoplasmic reticulum (ER) stress inducer, increased ATF3 expression, whereas it decreased PPARγ expression. ATF3 knockdown significantly blocked the thapsigargin-mediated downregulation of PPARγ expression. Furthermore, overexpression of PPARγ prevented inhibition of 3T3-L1 differentiation by thapsigargin. Collectively, these results suggest that ATF3-mediated inhibition of PPARγ expression may contribute to inhibition of adipocyte differentiation during cellular stress including ER stress.« less

cAMP Response Element-binding Protein (CREB) and Nuclear Factor κB Mediate the Tamoxifen-induced Up-regulation of Glutamate Transporter 1 (GLT-1) in Rat Astrocytes*

PubMed Central

Karki, Pratap; Webb, Anton; Smith, Keisha; Lee, Kyuwon; Son, Deok-Soo; Aschner, Michael; Lee, Eunsook

2013-01-01

Tamoxifen (TX), a selective estrogen receptor modulator, exerts antagonistic effects on breast tissue and is used to treat breast cancer. Recent evidence also suggests that it may act as an agonist in brain tissue. We reported previously that TX enhanced the expression and function of glutamate transporter 1 (GLT-1) in rat astrocytes, an effect that was mediated by TGF-α. To gain further insight into the mechanisms that mediate TX-induced up-regulation of GLT-1 (EAAT2 in humans), we investigated its effect on GLT-1 at the transcriptional level. TX phosphorylated the cAMP response element-binding protein (CREB) and recruited CREB to the GLT-1 promoter consensus site. The effect of TX on astrocytic GLT-1 was attenuated by the inhibition of PKA, the upstream activator of the CREB pathway. In addition, the effect of TX on GLT-1 promoter activity was abolished by the inhibition of the NF-κB pathway. Furthermore, TX recruited the NF-κB subunits p65 and p50 to the NF-κB binding domain of the GLT-1 promoter. Mutation of NF-κB (triple, −583/-282/-251) or CRE (-308) sites on the GLT-1 promoter led to significant repression of the promoter activity, but neither mutant completely abolished the TX-induced GLT-1 promoter activity. Mutation of both the NF-κB (-583/-282/-251) and CRE (-308) sites led to a complete abrogation of the effect of TX on GLT-1 promoter activity. Taken together, our findings establish that TX regulates GLT-1 via the CREB and NF-κB pathways. PMID:23955341

Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter.

PubMed Central

Cousin, E; Medcalf, R L; Bergonzelli, G E; Kruithof, E K

1991-01-01

Gene transcription rates and mRNA levels of plasminogen activator inhibitor type 2 (PAI-2) are markedly induced by the tumor promoting agent phorbol 12-myristate 13-acetate (PMA) in human HT1080 fibrosarcoma cells. To identify promoter elements required for basal-, and phorbol ester-inducible expression, deletion mutants of the PAI-1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, were transiently expressed in HT1080 cells. Constitutive CAT activity was expressed from constructs containing more than 215 bp of promoter sequence, whereas deletion to position -91 bp abolished CAT gene expression. Treatment of transfected cells with PMA resulted in a three- to ten-fold increase in CAT expression from all constructs except from the construct shortened to position -91. DNAse1 protection analysis of the promoter region between -215 and the transcription initiation site revealed numerous protected regions, including two AP1-like binding sites (AP1a and AP1b) and one CRE-like element. Site-directed mutagenesis of the AP1a site or of the CRE-like site resulted in the loss of basal CAT activity and abolished the PMA effect, whereas mutagenesis of AP1b only partially inhibited basal and PMA-mediated expression. Our results suggest that the PAI-2 promoter contains at least two elements required for basal gene transcription and PMA-mediated induction. Images PMID:1650454

Scap is required for sterol synthesis and crypt growth in intestinal mucosa.

PubMed

McFarlane, Matthew R; Cantoria, Mary Jo; Linden, Albert G; January, Brandon A; Liang, Guosheng; Engelking, Luke J

2015-08-01

SREBP cleavage-activating protein (Scap) is an endoplasmic reticulum membrane protein required for cleavage and activation of sterol regulatory element-binding proteins (SREBPs), which activate the transcription of genes in sterol and fatty acid biosynthesis. Liver-specific loss of Scap is well tolerated; hepatic synthesis of sterols and fatty acids is reduced, but mice are otherwise healthy. To determine whether Scap loss is tolerated in the intestine, we generated a mouse model (Vil-Scap(-)) in which tamoxifen-inducible Cre-ER(T2), a fusion protein of Cre recombinase with a mutated ligand binding domain of the human estrogen receptor, ablates Scap in intestinal mucosa. After 4 days of tamoxifen, Vil-Scap(-) mice succumb with a severe enteropathy and near-complete collapse of intestinal mucosa. Organoids grown ex vivo from intestinal crypts of Vil-Scap(-) mice are readily killed when Scap is deleted by 4-hydroxytamoxifen. Death is prevented when culture medium is supplemented with cholesterol and oleate. These data show that, unlike the liver, the intestine requires Scap to sustain tissue integrity by maintaining the high levels of lipid synthesis necessary for proliferation of intestinal crypts. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

An Exposed KID-Like Domain in Human T-Cell Lymphotropic Virus Type 1 Tax Is Responsible for the Recruitment of Coactivators CBP/p300

PubMed Central

Harrod, Robert; Tang, Yong; Nicot, Christophe; Lu, Hsieng S.; Vassilev, Alex; Nakatani, Yoshihiro; Giam, Chou-Zen

1998-01-01

Human T-cell lymphotropic virus type 1 (HTLV-1) transcriptional activation is mediated by the viral transactivator, Tax, and three 21-bp repeats (Tax response element [TxRE]) located in the U3 region of the viral long terminal repeat (LTR). Each TxRE contains a core cyclic AMP response element (CRE) flanked by 5′ G-rich and 3′ C-rich sequences. The TxRE binds CREB (CRE-binding protein) and Tax to form a ternary complex and confers Tax-dependent transactivation. Recent data indicate that Tax functions as a specific link to connect CREB-binding protein (CBP)/p300 in a phosphorylation-independent manner to CREB/ATF-1 assembled on the viral 21-bp repeats. Glutathione S-transferase pull-down performed with Tax deletion mutants and peptide competition have localized the site in Tax critical for binding CBP/p300 to a highly protease-sensitive region around amino acid residues 81 to 95 (81QRTSKTLKVLTPPIT95) which lies between the domains previously proposed to be important for CREB binding and Tax subunit dimerization. Amino acid residues around the trypsin- and chymotrypsin-sensitive sites (88KVL90) of Tax bear resemblance to those in the kinase-inducible domain of CREB (129SRRPSYRKILNE140) surrounding Ser-133, which undergoes signal-induced phosphorylation to recruit CBP/p300. Site-directed mutagenesis of residues in this domain (R82A, K85A, K88A, and V89A) resulted in proteins which failed to transactivate from the HTLV-1 LTR in vivo. These mutants (K85A, K88A, and V89A) bind CREB with similar affinities as wild-type Tax, yet interaction with CBP/p300 is abrogated in various biochemical assays, indicating that the recruitment of CBP/p300 is crucial for Tax transactivation. A Tax mutant, M47, defective in the COOH-terminal transactivation domain, continued to interact with CBP/p300, suggesting that interactions with additional cellular factors are required for proper Tax function. PMID:9710589

Berberine Suppresses Adipocyte Differentiation via Decreasing CREB Transcriptional Activity

PubMed Central

Deng, Ruyuan; Wang, Ning; Zhang, Yuqing; Wang, Yao; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

2015-01-01

Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been demonstrated to lower blood glucose, blood lipid, and body weight in patients with type 2 diabetes mellitus. The anti-obesity effect of berberine has been attributed to its anti-adipogenic activity. However, the underlying molecular mechanism remains largely unknown. In the present study, we found that berberine significantly suppressed the expressions of CCAAT/enhancer-binding protein (C/EBP)α, peroxisome proliferators-activated receptor γ2 (PPARγ2), and other adipogenic genes in the process of adipogenesis. Berberine decreased cAMP-response element-binding protein (CREB) phosphorylation and C/EBPβ expression at the early stage of 3T3-L1 preadipocyte differentiation. In addition, CREB phosphorylation and C/EBPβ expression induced by 3-isobutyl-1-methylxanthine (IBMX) and forskolin were also attenuated by berberine. The binding activities of cAMP responsive element (CRE) stimulated by IBMX and forskolin were inhibited by berberine. The binding of phosphorylated CREB to the promoter of C/EBPβ was abrogated by berberine after the induction of preadipocyte differentiation. These results suggest that berberine blocks adipogenesis mainly via suppressing CREB activity, which leads to a decrease in C/EBPβ-triggered transcriptional cascades. PMID:25928058

Activation of cAMP-dependent signaling pathway induces mouse organic anion transporting polypeptide 2 expression.

PubMed

Chen, Chuan; Cheng, Xingguo; Dieter, Matthew Z; Tanaka, Yuji; Klaassen, Curtis D

2007-04-01

Rodent Oatp2 is a hepatic uptake transporter for such compounds as cardiac glycosides. In the present study, we found that fasting resulted in a 2-fold induction of Oatp2 expression in liver of mice. Because the cAMP-protein kinase A (PKA) signaling pathway is activated during fasting, the role of this pathway in Oatp2 induction during fasting was examined. In Hepa-1c1c7 cells, adenylyl cyclase activator forskolin as well as two cellular membrane-permeable cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, induced Oatp2 mRNA expression in a time- and dose-dependent manner. These three chemicals induced reporter gene activity in cells transfected with a luciferase reporter gene construct containing a 7.6-kilobase (kb) 5'-flanking region of mouse Oatp2. Transient transfection of cells with 5'-deletion constructs derived from the 7.6-kb Oatp2 promoter reporter gene construct, as well as 7.6-kb constructs in which a consensus cAMP response element (CRE) half-site CGTCA (-1808/-1804 bp) was mutated or deleted, confirms that this CRE site was required for the induction of luciferase activity by forskolin. Luciferase activity driven by the Oatp2 promoter containing this CRE site was induced in cells cotransfected with a plasmid encoding the protein kinase A catalytic subunit. Cotransfection of cells with a plasmid encoding the dominant-negative CRE binding protein (CREB) completely abolished the inducibility of the reporter gene activity by forskolin. In conclusion, induction of Oatp2 expression in liver of fasted mice may be caused by activation of the cAMP-dependent signaling pathway, with the CRE site (-1808/-1804) and CREB being the cis- and trans-acting factors mediating the induction, respectively.

Resveratrol stimulates c-Fos gene transcription via activation of ERK1/2 involving multiple genetic elements.

PubMed

Thiel, Gerald; Rössler, Oliver G

2018-06-05

The polyphenol resveratrol is found in many plant and fruits and is a constituent of our diet. Resveratrol has been proposed to have chemopreventive and anti-inflammatory activities. On the cellular level, resveratrol activates stimulus-regulated transcription factors. To identify resveratrol-responsive elements within a natural gene promoter, the molecular pathway leading to c-Fos gene expression by resveratrol was dissected. The c-Fos gene encodes a basic region leucine zipper transcription factor and is a prototype of an immediate-early gene that is regulated by a wide range of signaling molecules. We analyzed chromatin-integrated c-Fos promoter-luciferase reporter genes where transcription factor binding sites were destroyed by point mutations or deletion mutagenesis. The results show that mutation of the binding sites for serum response factor (SRF), activator protein-1 (AP-1) and cAMP response element binding protein (CREB) significantly reduced reporter gene transcription following stimulation of the cells with resveratrol. Inactivation of the binding sites for signal transducer and activator of transcription (STAT) or ternary complex factors did not influence resveratrol-regulated c-Fos promoter activity. Thus, the c-Fos promoter contains three resveratrol-responsive elements, the cAMP response element (CRE), and the binding sites for SRF and AP-1. Moreover, we show that the transcriptional activation potential of the c-Fos protein is increased in resveratrol-stimulated cells, indicating that the biological activity of c-Fos is elevated by resveratrol stimulation. Pharmacological and genetic experiments revealed that the protein kinase ERK1/2 is the signal transducer that connects resveratrol treatment with the c-Fos gene. Copyright © 2018 Elsevier B.V. All rights reserved.

Epigenetic modifications during sex change repress gonadotropin stimulation of cyp19a1a in a teleost ricefield eel (Monopterus albus).

PubMed

Zhang, Yang; Zhang, Shen; Liu, Zhixin; Zhang, Lihong; Zhang, Weimin

2013-08-01

In vertebrates, cytochrome P450 aromatase, encoded by cyp19a1, converts androgens to estrogens and plays important roles in gonadal differentiation and development. The present study examines whether epigenetic mechanisms are involved in cyp19a1a expression and subsequent gonadal development in the hermaphroditic ricefield eel. The expression of the ricefield eel cyp19a1a was stimulated by gonadotropin via the cAMP pathway in the ovary but not the ovotestis or testis. The CpG within the cAMP response element (CRE) of the cyp19a1a promoter was hypermethylated in the ovotestis and testis compared with the ovary. The methylation levels of CpG sites around CRE in the distal region (region II) and around steroidogenic factor 1/adrenal 4 binding protein sites and TATA box in the proximal region (region I) were inversely correlated with cyp19a1a expression during the natural sex change from female to male. In vitro DNA methylation decreased the basal and forskolin-induced activities of cyp19a1a promoter. Chromatin immunoprecipitation assays indicated that histone 3 (Lys9) in both regions I and II of the cyp19a1a promoter were deacetylated and trimethylated in the testis, and in contrast to the ovary, phosphorylated CRE-binding protein failed to bind to these regions. Lastly, the DNA methylation inhibitor 5-aza-2'-deoxycytidine reversed the natural sex change of ricefield eels. These results suggested that epigenetic mechanisms involving DNA methylation and histone deacetylation and methylation may abrogate the stimulation of cyp19a1a by gonadotropins in a male-specific fashion. This may be a mechanism widely used to drive natural sex change in teleosts as well as gonadal differentiation in other vertebrates.

Interaction between C/EBPbeta and Tax down-regulates human T-cell leukemia virus type I transcription.

PubMed

Hivin, P; Gaudray, G; Devaux, C; Mesnard, J-M

2004-01-20

The human T-cell leukemia virus type I (HTLV-I) Tax protein trans-activates viral transcription through three imperfect tandem repeats of a 21-bp sequence called Tax-responsive element (TxRE). Tax regulates transcription via direct interaction with some members of the activating transcription factor/CRE-binding protein (ATF/CREB) family including CREM, CREB, and CREB-2. By interacting with their ZIP domain, Tax stimulates the binding of these cellular factors to the CRE-like sequence present in the TxREs. Recent observations have shown that CCAAT/enhancer binding protein beta (C/EBPbeta) forms stable complexes on the CRE site in the presence of CREB-2. Given that C/EBPbeta has also been found to interact with Tax, we analyzed the effects of C/EBPbeta on viral Tax-dependent transcription. We show here that C/EBPbeta represses viral transcription and that Tax is no more able to form a stable complex with CREB-2 on the TxRE site in the presence of C/EBPbeta. We also analyzed the physical interactions between Tax and C/EBPbeta and found that the central region of C/EBPbeta, excluding its ZIP domain, is required for direct interaction with Tax. It is the first time that Tax is described to interact with a basic leucine-zipper (bZIP) factor without recognizing its ZIP domain. Although unexpected, this result explains why C/EBPbeta would be unable to form a stable complex with Tax on the TxRE site and could then down-regulate viral transcription. Lastly, we found that C/EBPbeta was able to inhibit Tax expression in vivo from an infectious HTLV-I molecular clone. In conclusion, we propose that during cell activation events, which stimulate the Tax synthesis, C/EBPbeta may down-regulate the level of HTLV-I expression to escape the cytotoxic-T-lymphocyte response.

The coactivator CBP stimulates human T-cell lymphotrophic virus type I Tax transactivation in vitro.

PubMed

Kashanchi, F; Duvall, J F; Kwok, R P; Lundblad, J R; Goodman, R H; Brady, J N

1998-12-18

Tax interacts with the cellular cyclic AMP-responsive element binding protein (CREB) and facilitates the binding of the coactivator CREB binding protein (CBP), forming a multimeric complex on the cyclic AMP-responsive element (CRE)-like sites in the human T-cell lymphotrophic virus type I (HTLV-I) promoter. The trimeric complex is believed to recruit additional regulatory proteins to the HTLV-I long terminal repeat, but there has been no direct evidence that CBP is required for Tax-mediated transactivation. We present evidence that Tax and CBP activate transcription from the HTLV-I 21 base pair repeats on naked DNA templates. Transcriptional activation of the HTLV-I sequences required both Tax and CBP and could be mediated by either the N-terminal activation domain of CBP or the full-length protein. Fluorescence polarization binding assays indicated that CBP does not markedly enhance the affinity of Tax for the trimeric complex. Transcription analyses suggest that CBP activates Tax-dependent transcription by promoting transcriptional initiation and reinitiation. The ability of CBP to activate the HTLV-I promoter does not involve the stabilization of Tax binding, but rather depends upon gene activation properties of the co-activator that function in the context of a naked DNA template.

Core binding factor beta (Cbfβ) controls the balance of chondrocyte proliferation and differentiation by upregulating Indian hedgehog (Ihh) expression and inhibiting parathyroid hormone-related protein receptor (PPR) expression in postnatal cartilage and bone formation.

PubMed

Tian, Fei; Wu, Mengrui; Deng, Lianfu; Zhu, Guochun; Ma, Junqing; Gao, Bo; Wang, Lin; Li, Yi-Ping; Chen, Wei

2014-07-01

Core binding factor beta (Cbfβ) is essential for embryonic bone morphogenesis. Yet the mechanisms by which Cbfβ regulates chondrocyte proliferation and differentiation as well as postnatal cartilage and bone formation remain unclear. Hence, using paired-related homeobox transcription factor 1-Cre (Prx1-Cre) mice, mesenchymal stem cell-specific Cbfβ-deficient (Cbfβ(f/f) Prx1-Cre) mice were generated to study the role of Cbfβ in postnatal cartilage and bone development. These mutant mice survived to adulthood but exhibited severe sternum and limb malformations. Sternum ossification was largely delayed in the Cbfβ(f/f) Prx1-Cre mice and the xiphoid process was noncalcified and enlarged. In newborn and 7-day-old Cbfβ(f/f) Prx1-Cre mice, the resting zone was dramatically elongated, the proliferation zone and hypertrophic zone of the growth plates were drastically shortened and disorganized, and trabecular bone formation was reduced. Moreover, in 1-month-old Cbfβ(f/f) Prx1-Cre mice, the growth plates were severely deformed and trabecular bone was almost absent. In addition, Cbfβ deficiency impaired intramembranous bone formation both in vivo and in vitro. Interestingly, although the expression of Indian hedgehog (Ihh) was largely reduced, the expression of parathyroid hormone-related protein (PTHrP) receptor (PPR) was dramatically increased in the Cbfβ(f/f) Prx1-Cre growth plate, indicating that that Cbfβ deficiency disrupted the Ihh-PTHrP negative regulatory loop. Chromatin immunoprecipitation (ChIP) analysis and promoter luciferase assay demonstrated that the Runx/Cbfβ complex binds putative Runx-binding sites of the Ihh promoter regions, and also the Runx/Cbfβ complex directly upregulates Ihh expression at the transcriptional level. Consistently, the expressions of Ihh target genes, including CyclinD1, Ptc, and Pthlh, were downregulated in Cbfβ-deficient chondrocytes. Taken together, our study reveals not only that Cbfβ is essential for chondrocyte proliferation and differentiation for the growth and maintenance of the skeleton in postnatal mice, but also that it functions in upregulating Ihh expression to promoter chondrocyte proliferation and osteoblast differentiation, and inhibiting PPR expression to enhance chondrocyte differentiation. © 2014 American Society for Bone and Mineral Research.

The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization.

PubMed

Romero-López, Cristina; Barroso-delJesus, Alicia; Berzal-Herranz, Alfredo

2017-02-24

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3'UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2'-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome.

The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization

PubMed Central

Romero-López, Cristina; Barroso-delJesus, Alicia; Berzal-Herranz, Alfredo

2017-01-01

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3′UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2′-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome. PMID:28233845

Carbon repression of cellobiose dehydrogenase production in the white rot fungus Trametes versicolor is mediated at the level of gene transcription.

PubMed

Stapleton, P C; Dobson, A D W

2003-04-25

Cellobiose dehydrogenase (CDH) production in Trametes versicolor is induced in the presence of cellulose, but decreases when additional carbon sources such as glucose and maltose are added to the fungal cultures. Using T. versicolor-specific cdh primers in a reverse transcription-polymerase chain reaction-based approach, it appears that this repression in CDH production is being mediated at the level of gene transcription. When a 1.6-kb upstream region of the T. versicolor cdh gene was cloned and sequenced, a number of putative CreA-like binding sites were observed. We propose that these sites may be involved in mediating this repressive effect, based on their similarity to the consensus [5'-SYGGRGG-3'] site for binding of the CreA and Cre1 repressor proteins.

Regulation of anxiety and initiation of sexual behavior by CREB in the nucleus accumbens

PubMed Central

Barrot, Michel; Wallace, Deanna L.; Bolaños, Carlos A.; Graham, Danielle L.; Perrotti, Linda I.; Neve, Rachael L.; Chambliss, Heather; Yin, Jerry C.; Nestler, Eric J.

2005-01-01

Sexual deficits and other behavioral disturbances such as anxiety-like behaviors can be observed in animals that have undergone social isolation, especially in species having important social interactions. Using a model of protracted social isolation in adult rats, we observed increased anxiety-like behavior and deficits in both the latency to initiate sexual behavior and the latency to ejaculate. We show, using transgenic cAMP response element (CRE)-LacZ reporter mice, that protracted social isolation also reduces CRE-dependent transcription within the nucleus accumbens. This decrease in CRE-dependent transcription can be mimicked in nonisolated animals by local viral gene transfer of a dominant negative mutant of CRE-binding protein (CREB). We previously showed that this manipulation increases anxiety-like behavior. We show here that it also impairs initiation of sexual behavior in nonisolated animals, a deficit that can be corrected by anxiolytic drug treatment. This local reduction in CREB activity, however, has no influence on ejaculation parameters. Reciprocally, we used the viral transgenic approach to overexpress CREB in the nucleus accumbens of isolated animals. We show that this local increase in CREB activity completely rescued the anxiety phenotype of the isolated animals, as well as their deficit in initiating sexual behavior, but failed to rescue the deficit in ejaculation. Our data suggest a role for the nucleus accumbens in anxiety responses and in specific aspects of sexual behavior. The results also provide insight into the molecular mechanisms by which social interactions affect brain plasticity and behavior. PMID:15923261

[Divergence of paralogous growth-hormone-encoding genes and their promoters in Salmonidae].

PubMed

Kamenskaya, D N; Pankova, M V; Atopkin, D M; Brykov, V A

2017-01-01

In many fish species, including salmonids, the growth-hormone is encoded by two duplicated paralogous genes, gh1 and gh2. Both genes were already in place at the time of divergence of species in this group. A comparison of the entire sequence of these genes of salmonids has shown that their conserved regions are associated with exons, while their most variable regions correspond to introns. Introns C and D include putative regulatory elements (sites Pit-1, CRE, and ERE), that are also conserved. In chars, the degree of polymorphism of gh2 gene is 2-3 times as large as that in gh1 gene. However, a comparison across all Salmonidae species would not extent this observation to other species. In both these chars' genes, the promoters are conserved mainly because they correspond to putative regulatory sequences (TATA box, binding sites for the pituitary transcription factor Pit-1 (F1-F4), CRE, GRE and RAR/RXR elements). The promoter of gh2 gene has a greater degree of polymorphism compared with gh1 gene promoter in all investigated species of salmonids. The observed differences in the rates of accumulation of changes in growth hormone encoding paralogs could be explained by differences in the intensity of selection.

5′UTR of the Neurogenic bHLH Nex1/MATH-2/NeuroD6 Gene Is Regulated by Two Distinct Promoters Through CRE and C/EBP Binding Sites

PubMed Central

Uittenbogaard, Martine; Martinka, Debra L.; Johnson, Peter F.; Vinson, Charles; Chiaramello, Anne

2009-01-01

Expression of the bHLH transcription factor Nex1/MATH-2/NeuroD6, a member of the NeuroD subfamily, parallels overt neuronal differentiation and synaptogenesis during brain development. Our previous studies have shown that Nex1 is a critical effector of the NGF pathway and promotes neuronal differentiation and survival of PC12 cells in the absence of growth factors. In this study, we investigated the transcriptional regulation of the Nex1 gene during NGF-induced neuronal differentiation. We found that Nex1 expression is under the control of two conserved promoters, Nex1-P1 and Nex1-P2, located in two distinct non-coding exons. Both promoters are TATA-less with multiple transcription start sites, and are activated on NGF or cAMP exposure. Luciferase-reporter assays showed that the Nex1-P2 promoter activity is stronger than the Nex1-P1 promoter activity, which supports the previously reported differential expression levels of Nex1 transcripts throughout brain development. Using a combination of DNaseI footprinting, EMSA assays, and site-directed mutagenesis, we identified the essential regulatory elements within the first 2 kb of the Nex1 5′UTR. The Nex1-P1 promoter is mainly regulated by a conserved CRE element, whereas the Nex1-P2 promoter is under the control of a conserved C/EBP binding site. Overexpression of wild-type C/EBPβ resulted in increased Nex1-P2 promoter activity in NGF-differentiated PC12 cells. The fact that Nex1 is a target gene of C/EBPβ provides new insight into the C/EBP transcriptional cascade known to promote neurogenesis, while repressing gliogenesis. PMID:17075921

Epithelial-mesenchymal transition and nuclear β-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent

PubMed Central

Carter, Emma J.; Barnes, David; Hoppe, Hans-Jürgen; Hughes, Jennifer; Cobbold, Stephen; Harper, James; Morreau, Hans; Surakhy, Mirvat; Hassan, A. Bassim

2016-01-01

Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to β-catenin. Here, we evaluate whether E-cadherin binding can inhibit β-catenin when there is loss of Adenomatous polyposis coli (APC) from the β-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear β-catenin localization, suggesting that E-cadherin inhibits β-catenin. Combination with an intestinal stem cell Cre (Lgr5CreERT2) resulted in ApcΔ/Δ recombination and adenoma, but intact Cdh1fl/fl alleles. Cultured ApcΔ/ΔCdh1fl/fl adenoma cells infected with adenovirus-Cre induced Cdh1fl/fl recombination (Cdh1Δ/Δ), disruption of organoid morphology, nuclear β-catenin localization, and cells with an epithelial-mesenchymal phenotype. Complementation with adenovirus expressing wild-type Cdh1 (Cdh1-WT) rescued adhesion and β-catenin membrane localization, yet an EC1 specific double mutant defective in homophilic adhesion (Cdh1-MutW2A, S78W) did not. These data suggest that E-cadherin inhibits β-catenin in the context of disruption of the APC-destruction complex, and that this function is also EC1 domain dependent. Both binding functions of E-cadherin may be required for its tumour suppressor activity. PMID:27566565

An affinity adsorption media that mimics heparan sulfate proteoglycans for the treatment of drug-resistant bacteremia

NASA Astrophysics Data System (ADS)

McCrea, Keith R.; Ward, Robert S.

2016-06-01

Removal of several drug-resistant bacteria from blood by affinity adsorption onto a heparin-functional media is reported. Heparin is a chemical analogue of heparan sulfate (HS) proteoglycans, found on transmembrane proteins of endothelial cells. Many blood-borne human pathogens, including bacteria, viruses, parasites, and fungi have been reported to target HS as an initial step in their pathogenesis. Here, we demonstrate the binding and removal of Methicillin-resistant Staphylococcus aureus (MRSA), Extended-Spectrum Betalactamase Klebsiella pneumoniae (ESBL), and two Carbapenem-resistant Enterobacteriaceae (both CRE Escherichia coli and CRE K. pneumoniae) using 300 μm polyethylene beads surface modified with end-point-attached heparin. Depending on the specific bacteria, the amount removed ranged between 39% (ESBL) and 99.9% (CRE). The total amount of bacteria adsorbed ranged between 2.8 × 105 and 8.6 × 105 colony forming units (CFU) per gram of adsorption media. Based on a polymicrobial challenge which showed no competitive binding, MRSA and CRE apparently utilize different binding sequences on the immobilized heparin ligand. Since the total circulating bacterial load during bacteremia seldom exceeds 5 × 105 CFUs, it appears possible to significantly reduce bacterial concentration in infected patients by multi-pass recirculation of their blood through a small extracorporeal affinity filter containing the heparin-functional adsorption media. This 'dialysis-like therapy' is expected to improve patient outcomes and reduce the cost of care, particularly when there are no anti-infective drugs available to treat the infection.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Serk In, E-mail: serkin@korea.edu; The BK21 Plus Program for Biomedical Sciences, Korea University College of Medicine, Seoul; Department of Medicine and Center for Bone Biology, Vanderbilt University School of Medicine, Nashville, TN

The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while theremore » was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach. - Highlights: • γ-Irradiation induced CREB phosphorylation and CRE-directed transcription in tumor. • γ-Irradiation-induced transcriptional activation of CREB was via p38 MAPK pathway. • CRE blockade increased radiosensitivity of tumor cells but not of normal cells. • CRE decoy oligonucleotides or p38 MAPK inhibitors can be used as radiosensitizers.« less

Involvement of neuron-derived orphan receptor-1 (NOR-1) in LDL-induced mitogenic stimulus in vascular smooth muscle cells: role of CREB.

PubMed

Rius, Jordi; Martínez-González, José; Crespo, Javier; Badimon, Lina

2004-04-01

Low density lipoproteins (LDLs) modulate the expression of key genes involved in atherogenesis. Recently, we have shown that the transcription factor neuron-derived orphan receptor-1 (NOR-1) is involved in vascular smooth muscle cell (VSMC) proliferation. Our aim was to analyze whether NOR-1 is involved in LDL-induced mitogenic effects in VSMC. LDL induced NOR-1 expression in a time- and dose-dependent manner. Antisense oligonucleotides against NOR-1 inhibit DNA synthesis induced by LDL in VSMCs as efficiently as antisense against the protooncogene c-fos. The upregulation of NOR-1 mRNA levels by LDL involves pertusis-sensitive G protein-coupled receptors, Ca2+ mobilization, protein kinases A (PKA) and C (PKC) activation, and mitogen-activated protein kinase pathways (MAPK) (p44/p42 and p38). LDL promotes cAMP response element binding protein (CREB) activation (phosphorylation in Ser133). In transfection assays a dominant-negative of CREB inhibits NOR-1 promoter activity, while mutation of specific (cAMP response element) CRE sites in the NOR-1 promoter abolishes LDL-induced NOR-1 promoter activity. In VSMCs, LDL-induced mitogenesis involves NOR-1 upregulation through a CREB-dependent mechanism. CREB could play a role in the modulation by LDL of key genes (containing CRE sites) involved in atherogenesis.

Bursting process of large- and small-scale structures in turbulent boundary layer perturbed by a cylinder roughness element

NASA Astrophysics Data System (ADS)

Tang, Zhanqi; Jiang, Nan; Zheng, Xiaobo; Wu, Yanhua

2016-05-01

Hot-wire measurements on a turbulent boundary layer flow perturbed by a wall-mounted cylinder roughness element (CRE) are carried out in this study. The cylindrical element protrudes into the logarithmic layer, which is similar to those employed in turbulent boundary layers by Ryan et al. (AIAA J 49:2210-2220, 2011. doi: 10.2514/1.j051012) and Zheng and Longmire (J Fluid Mech 748:368-398, 2014. doi: 10.1017/jfm.2014.185) and in turbulent channel flow by Pathikonda and Christensen (AIAA J 53:1-10, 2014. doi: 10.2514/1.j053407). The similar effects on both the mean velocity and Reynolds stress are observed downstream of the CRE perturbation. The series of hot-wire data are decomposed into large- and small-scale fluctuations, and the characteristics of large- and small-scale bursting process are observed, by comparing the bursting duration, period and frequency between CRE-perturbed case and unperturbed case. It is indicated that the CRE perturbation performs the significant impact on the large- and small-scale structures, but within the different impact scenario. Moreover, the large-scale bursting process imposes a modulation on the bursting events of small-scale fluctuations and the overall trend of modulation is not essentially sensitive to the present CRE perturbation, even the modulation extent is modified. The conditionally averaging fluctuations are also plotted, which further confirms the robustness of the bursting modulation in the present experiments.

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I.

PubMed

Huang, Y; Ohtani, K; Iwanaga, R; Matsumura, Y; Nakamura, M

2001-03-01

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

Mutations altering the cleavage specificity of a homing endonuclease

PubMed Central

Seligman, Lenny M.; Chisholm, Karen M.; Chevalier, Brett S.; Chadsey, Meggen S.; Edwards, Samuel T.; Savage, Jeremiah H.; Veillet, Adeline L.

2002-01-01

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein–DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences. PMID:12202772

Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase.

PubMed

Lallemand, Y; Luria, V; Haffner-Krausz, R; Lonai, P

1998-03-01

A transgenic mouse strain with early and uniform expression of the Cre site-specific recombinase is described. In this strain, PGK-Crem, Cre is driven by the early acting PGK-1 promoter, but, probably due to cis effects at the integration site, the recombinase is under dominant maternal control. When Cre is transmitted by PGK-Crem females mated to males that carry a reporter transgene flanked by loxP sites, even offspring that do not inherit PGK-Cre delete the target gene. It follows that in the PGK-Crem female Cre activity commences in the diploid phase of oogenesis. In PGK-Crem crosses complete recombination was observed in all organs, including testis and ovary. We prepared a mouse stock that is homozygous for PGK-Crem and at the albino (c) locus. This strain will be useful for the early and uniform induction of ectopic and dominant negative mutations, for the in vivo removal of selective elements from targeted mutations and in connection with the manipulation of targeted loci in 'knock in' and related technologies.

Mutants of Cre recombinase with improved accuracy

PubMed Central

Eroshenko, Nikolai; Church, George M.

2013-01-01

Despite rapid advances in genome engineering technologies, inserting genes into precise locations in the human genome remains an outstanding problem. It has been suggested that site-specific recombinases can be adapted towards use as transgene delivery vectors. The specificity of recombinases can be altered either with directed evolution or via fusions to modular DNA-binding domains. Unfortunately, both wildtype and altered variants often have detectable activities at off-target sites. Here we use bacterial selections to identify mutations in the dimerization surface of Cre recombinase (R32V, R32M, and 303GVSdup) that improve the accuracy of recombination. The mutants are functional in bacteria, in human cells, and in vitro (except for 303GVSdup, which we did not purify), and have improved selectivity against both model off-target sites and the entire E. coli genome. We propose that destabilizing binding cooperativity may be a general strategy for improving the accuracy of dimeric DNA-binding proteins. PMID:24056590

Identification and characterization of an alternative promoter of the human PGC-1{alpha} gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshioka, Toyo; Inagaki, Kenjiro; Noguchi, Tetsuya, E-mail: noguchi@med.kobe-u.ac.jp

2009-04-17

The transcriptional regulator peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) controls mitochondrial biogenesis and energy homeostasis. Although physical exercise induces PGC-1{alpha} expression in muscle, the underlying mechanism of this effect has remained incompletely understood. We recently identified a novel muscle-enriched isoform of PGC-1{alpha} transcript (designated PGC-1{alpha}-b) that is derived from a previously unidentified first exon. We have now cloned and characterized the human PGC-1{alpha}-b promoter. The muscle-specific transcription factors MyoD and MRF4 transactivated this promoter through interaction with a proximal E-box motif. Furthermore, either forced expression of Ca{sup 2+}- and calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A, or the p38 mitogen-activated proteinmore » kinase (p38 MAPK) kinase MKK6 or the intracellular accumulation of cAMP activated the PGC-1{alpha}-b promoter in cultured myoblasts through recruitment of cAMP response element (CRE)-binding protein (CREB) to a putative CRE located downstream of the E-box. Our results thus reveal a potential molecular basis for isoform-specific regulation of PGC-1{alpha} expression in contracting muscle.« less

What Are Space Exposure Histories Telling Us about CM Carbonaceous Chondrites?

NASA Technical Reports Server (NTRS)

Takenouchi, A.; Zolensky, Michael E.; Nishiizumi, K.; Caffee, M.; Velbel, M. A.; Ross, K.; Zolensky, P.; Le, L.; Imae, N.; Yamaguchi, A.;

The rapid spread of carbapenem-resistant Enterobacteriaceae

PubMed Central

Potter, Robert F.; D’Souza, Alaric W.; Dantas, Gautam

2016-01-01

Carbapenems, our one-time silver bullet for multidrug resistant bacterial infections, are now threatened by widespread dissemination of carbapenem-resistant Enterobacteriaceae (CRE). Successful expansion of Enterobacteriaceae clonal groups and frequent horizontal gene transfer of carbapenemase expressing plasmids are causing increasing carbapenem resistance. Recent advances in genetic and phenotypic detection facilitate global surveillance of CRE diversity and prevalence. In particular, whole genome sequencing enabled efficient tracking, annotation, and study of genetic elements colocalized with carbapenemase genes on chromosomes and on plasmids. Improved characterization helps detail the co-occurrence of other antibiotic resistance genes in CRE isolates and helps identify pan-drug resistance mechanisms. The novel β-lactamase inhibitor, avibactam, combined with ceftazidime or aztreonam, is a promising CRE treatment compared to current colistin or tigecycline regimens. To halt increasing CRE-associated morbidity and mortality, we must continue quality, cooperative monitoring and urgently investigate novel treatments. PMID:27912842

Generation of Cre-transgenic mice using Dlx1/Dlx2 enhancers and their characterization in GABAergic interneurons

PubMed Central

Potter, Gregory B.; Petryniak, Magdalena A.; Shevchenko, Eugenia; McKinsey, Gabriel L.; Ekker, Marc; Rubenstein, John L.R.

2009-01-01

DLX1 and DLX2 transcription factors are necessary for forebrain GABAergic neuron differentiation, migration, and survival. We generated transgenic mice that express Cre-recombinase under the control of two ultra-conserved DNA elements near the Dlx1&2 locus termed I12b and URE2. We show that Cre-recombinase is active in a “Dlx-pattern” in the embryonic forebrain of transgenic mice. I12b-Cre is more active than URE2-Cre in the medial ganglionic eminences and its derivatives. Fate-mapping of EGFP+ cells in adult Cre;Z/EG animals demonstrated that GABAergic neurons, but not glia, are labeled. Most NPY+, nNOS+, parvalbumin+, and somatostatin+ cells are marked by I12b-Cre in the cortex and hippocampus, while 25-40% of these interneuron subtypes are labeled by URE2-Cre. Labeling of neurons generated between E12.5 to E15.5 indicated differences in birth-dates of EGFP+ cells that populate the olfactory bulb, hippocampus, and cortex. Finally, we provide the first in vivo evidence that both I12b and URE2 are direct targets of DLX2 and require Dlx1 and Dlx2 expression for proper activity. PMID:19026749

Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities.

PubMed

Nagase, Hiroyuki; Omae, Naoki; Omori, Akiko; Nakagawasai, Osamu; Tadano, Takeshi; Yokosuka, Akihito; Sashida, Yutaka; Mimaki, Yoshihiro; Yamakuni, Tohru; Ohizumi, Yasushi

2005-12-02

cAMP response element (CRE) transcription is dysregulated in neurodegenerative disorders in the central nervous system (CNS), including polyglutamine diseases. As the first step to find natural compounds with protective action against neurodegeneration in the CNS, we here examined whether six citrus flavonoids, namely nobiletin, 5-demethylnobiletin, tangeretin, sinensetin, 6-demethoxytangeretin, and 6-demethoxynobiletin, stimulated CRE-dependent transcription and induced neurite outgrowth in PC12D cells. Among the compounds, nobiletin most potently enhanced CRE-dependent transcription and neurite outgrowth by activating ERK/MAP kinase-dependent signalling to increase CREB phosphorylation. The transcription and neurite outgrowth were stimulated by nobiletin in a concentration-dependent manner, with a strong correlation between them. Furthermore, a 11-day oral administration of nobiletin rescued impaired memory in olfactory-bulbectomized mice documented to be accompanied by a cholinergic neurodegeneration. These results suggest that nobiletin with the activity to improve impaired memory may become a potential leading compound for drug development for neurodegenerative disorders exhibiting the dysregulated CRE-dependent transcription.

Viral Delivery of GFP-Dependent Recombinases to the Mouse Brain.

PubMed

Tang, Jonathan C Y; Rudolph, Stephanie; Cepko, Constance L

2017-01-01

Many genetic tools have been developed that use green fluorescent protein (GFP) and its derivatives for labeling specific cell populations in organisms and in cell culture. To extend the use of GFP beyond labeling purposes, we developed methods and reagents that use GFP as a driver of biological activities. We used nanobodies that bind GFP to engineer CRE-DOG and Flp-DOG, recombinases that can induce Cre/lox and Flp/FRT recombination in a GFP-dependent manner, respectively. Here, we present a protocol to deliver CRE-DOG and Flp-DOG into the mouse brain by recombinant AAV infection. This protocol enables one to manipulate gene expression specifically in GFP-expressing cells, found either in transgenic GFP reporter lines or in cells made to express GFP by other transduction methods.

Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development.

PubMed

Yan, Jianyun; Li, Jun; Hu, Jun; Zhang, Lu; Wei, Chengguo; Sultana, Nishat; Cai, Xiaoqiang; Zhang, Weijia; Cai, Chen-Leng

2018-06-15

Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation remains incompletely understood. In this study, using a Tbx18:Cre ( Tbx18 Cre /+ ) gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 ( Smad4 f/f ) in the limbs of mice. We found that the Smad4 -deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including Col2a1 and Acan , in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy-related markers Col10a1 and Panx3 were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 ( Runx2 ), a critical mediator of chondrocyte hypertrophy, was also down-regulated in Smad4 -deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the Runx2 promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating Runx2 expression during skeletal development. The regulatory mechanism involving Smad4-mediated Runx2 activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia. © 2018 Yan et al.

Conformation of Tax-response elements in the human T-cell leukemia virus type I promoter.

PubMed

Cox, J M; Sloan, L S; Schepartz, A

1995-12-01

HTLV-I Tax is believed to activate viral gene expression by binding bZIP proteins (such as CREB) and increasing their affinities for proviral TRE target sites. Each 21 bp TRE target site contains an imperfect copy of the intrinsically bent CRE target site (the TRE core) surrounded by highly conserved flanking sequences. These flanking sequences are essential for maximal increases in DNA affinity and transactivation, but they are not, apparently, contacted by protein. Here we employ non-denaturing gel electrophoresis to evaluate TRE conformation in the presence and absence of bZIP proteins, and to explore the role of DNA conformation in viral transactivation. Our results show that the TRE-1 flanking sequences modulate the structure and modestly increase the affinity of a CREB bZIP peptide for the TRE-1 core recognition sequence. These flanking sequences are also essential for a maximal increase in stability of the CREB-DNA complex in the presence of Tax. The CRE-like TRE core and the TRE flanking sequences are both essential for formation of stable CREB-TRE-1 and Tax-CREB-TRE-1 complexes. These two DNA segments may have co-evolved into a unique structure capable of recognizing Tax and a bZIP protein.

CcpA and CodY Coordinate Acetate Metabolism in Streptococcus mutans.

PubMed

Kim, Jeong Nam; Burne, Robert A

2017-04-01

In the dental caries pathogen Streptococcus mutans , phosphotransacetylase (Pta) and acetate kinase (Ack) convert pyruvate into acetate with the concomitant generation of ATP. The genes for this pathway are tightly regulated by multiple environmental and intracellular inputs, but the basis for differential expression of the genes for Pta and Ack in S. mutans had not been investigated. Here, we show that inactivation in S. mutans of ccpA or codY reduced the activity of the ackA promoter, whereas a ccpA mutant displayed elevated pta promoter activity. The interactions of CcpA with the promoter regions of both genes were observed using electrophoretic mobility shift and DNase protection assays. CodY bound to the ackA promoter region but only in the presence of branched-chain amino acids (BCAAs). DNase footprinting revealed that the upstream region of both genes contains two catabolite-responsive elements ( cre1 and cre2 ) that can be bound by CcpA. Notably, the cre2 site of ackA overlaps with a CodY-binding site. The CcpA- and CodY-binding sites in the promoter region of both genes were further defined by site-directed mutagenesis. Some differences between the reported consensus CodY binding site and the region protected by S. mutans CodY were noted. Transcription of the pta and ackA genes in the ccpA mutant strain was markedly different at low pH relative to transcription at neutral pH. Thus, CcpA and CodY are direct regulators of transcription of ackA and pta in S. mutans that optimize acetate metabolism in response to carbohydrate, amino acid availability, and environmental pH. IMPORTANCE The human dental caries pathogen Streptococcus mutans is remarkably adept at coping with extended periods of carbohydrate limitation during fasting periods. The phosphotransacetylase-acetate kinase (Pta-Ack) pathway in S. mutans modulates carbohydrate flux and fine-tunes the ability of the organisms to cope with stressors that are commonly encountered in the oral cavity. Here, we show that CcpA controls transcription of the pta and ackA genes via direct interaction with the promoter regions of both genes and that branched-chain amino acids (BCAAs), particularly isoleucine, enhance the ability of CodY to bind to the promoter region of the ackA gene. A working model is proposed to explain how regulation of pta and ackA genes by these allosterically controlled regulatory proteins facilitates proper carbon flow and energy production, which are essential functions during infection and pathogenesis as carbohydrate and amino acid availability continually fluctuate. Copyright © 2017 American Society for Microbiology.

Neurotrophic Effect of Citrus 5-Hydroxy-3,6,7,8,3′,4′-Hexamethoxyflavone: Promotion of Neurite Outgrowth via cAMP/PKA/CREB Pathway in PC12 Cells

PubMed Central

Lai, Hui-Chi; Wu, Ming-Jiuan; Chen, Pei-Yi; Sheu, Ting-Ting; Chiu, Szu-Ping; Lin, Meng-Han; Ho, Chi-Tang; Yen, Jui-Hung

2011-01-01

5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF), a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43). 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB) phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501), a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA) inhibitor, but not MEK1/2, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) or calcium/calmodulin-dependent protein kinase (CaMK) inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action. PMID:22140566

20-hydroxyecdysone enhances the expression of the chitinase 5 via Broad-Complex Zinc-Finger 4 during metamorphosis in silkworm, Bombyx mori.

PubMed

Zhang, X; Zheng, S

2017-04-01

Insect chitinases are hydrolytic enzymes required for the degradation of chitin. They are essential for insect moulting and metamorphosis. In this study, the regulation mechanism of a chitinase gene, Bombyx mori chitinase 5 (BmCHT5), was studied. Quantitative reverse transcription PCR (qRT-PCR) analysis showed that BmCHT5 was up-regulated during the larval-larval and larval-pupa transitions and notably induced by 20-hydroxyecdysone (20E). Analysis of the BmCHT5 promoter revealed the presence of one Bombyx mori Broad-Complex Zinc-Finger Isoform 4 (BR-C Z4), two BR-C Z2 and two ecdysone-induced protein 74A (E74A) cis-regulatory elements (CREs) that are related to 20E. qRT-PCR showed that the expression of both BmBR-C Z4 and BmBR-C Z2 during metamorphosis, and when induced by 20E, was anastomotic with the variations in BmCHT5 mRNA level. In contrast, BmE74A did not follow this trend. An electrophoretic mobility shift assay did not retrieve a binding partner for the two BR-C Z2 CREs in the BmN cell line nuclear extract, whereas BR-C Z4 CRE specifically bound to BmBR-C Z4. Besides, luciferase activity analysis confirmed that BmBR-C Z4 could enhance the activity of the BmCHT5 promoter with BR-C Z4 CRE and could not enhance the promoter activity by mutating BR-C Z4 CRE. Taken together, these data suggest that the transcription factor BmBR-C Z4 enhances the expression of BmCHT5 during metamorphosis. © 2016 The Royal Entomological Society.

Transgenic mouse models in the study of reproduction: insights into GATA protein function.

PubMed

Tevosian, Sergei G

2014-07-01

For the past 2 decades, transgenic technology in mice has allowed for an unprecedented insight into the transcriptional control of reproductive development and function. The key factor among the mouse genetic tools that made this rapid advance possible is a conditional transgenic approach, a particularly versatile method of creating gene deletions and substitutions in the mouse genome. A centerpiece of this strategy is an enzyme, Cre recombinase, which is expressed from defined DNA regulatory elements that are active in the tissue of choice. The regulatory DNA element (either genetically engineered or natural) assures Cre expression only in predetermined cell types, leading to the guided deletion of genetically modified (flanked by loxP or 'floxed' by loxP) gene loci. This review summarizes and compares the studies in which genes encoding GATA family transcription factors were targeted either globally or by Cre recombinases active in the somatic cells of ovaries and testes. The conditional gene loss experiments require detailed knowledge of the spatial and temporal expression of Cre activity, and the challenges in interpreting the outcomes are highlighted. These studies also expose the complexity of GATA-dependent regulation of gonadal gene expression and suggest that gene function is highly context dependent. © 2014 Society for Reproduction and Fertility.

CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

PubMed

Yu, Zhiyuan; Kong, Qun; Kone, Bruce C

2010-03-01

Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

Osteocytes, not Osteoblasts or Lining Cells, are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone

PubMed Central

Xiong, Jinhu; Piemontese, Marilina; Onal, Melda; Campbell, Josh; Goellner, Joseph J.; Dusevich, Vladimir; Bonewald, Lynda; Manolagas, Stavros C.; O’Brien, Charles A.

2015-01-01

The cytokine receptor activator of nuclear factor kappa B ligand (RANKL), encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone. PMID:26393791

Lactoferricin B Inhibits the Phosphorylation of the Two-Component System Response Regulators BasR and CreB*

PubMed Central

Ho, Yu-Hsuan; Sung, Tzu-Cheng; Chen, Chien-Sheng

2012-01-01

Natural antimicrobial peptides provide fundamental protection for multicellular organisms from microbes, such as Lactoferricin B (Lfcin B). Many studies have shown that Lfcin B penetrates the cell membrane and has intracellular activities. To elucidate the intracellular behavior of Lfcin B, we first used Escherichia coli K12 proteome chips to identify the intracellular targets of Lfcin B. The results showed that Lfcin B binds to two response regulators, BasR and CreB, of the two-component system. For further analysis, we conducted several in vitro and in vivo experiments and utilized bioinformatics methods. The electrophoretic mobility shift assays and kinase assays indicate that Lfcin B inhibits the phosphorylation of the response regulators (BasR and CreB) and their cognate sensor kinases (BasS and CreC). Antibacterial assays showed that Lfcin B reduced E. coli's tolerance to environmental stimuli, such as excessive ferric ions and minimal medium conditions. This is the first study to show that an antimicrobial peptide inhibits the growth of bacteria by influencing the phosphorylation of a two-component system directly. PMID:22138548

Lactoferricin B inhibits the phosphorylation of the two-component system response regulators BasR and CreB.

PubMed

Ho, Yu-Hsuan; Sung, Tzu-Cheng; Chen, Chien-Sheng

2012-04-01

Natural antimicrobial peptides provide fundamental protection for multicellular organisms from microbes, such as Lactoferricin B (Lfcin B). Many studies have shown that Lfcin B penetrates the cell membrane and has intracellular activities. To elucidate the intracellular behavior of Lfcin B, we first used Escherichia coli K12 proteome chips to identify the intracellular targets of Lfcin B. The results showed that Lfcin B binds to two response regulators, BasR and CreB, of the two-component system. For further analysis, we conducted several in vitro and in vivo experiments and utilized bioinformatics methods. The electrophoretic mobility shift assays and kinase assays indicate that Lfcin B inhibits the phosphorylation of the response regulators (BasR and CreB) and their cognate sensor kinases (BasS and CreC). Antibacterial assays showed that Lfcin B reduced E. coli's tolerance to environmental stimuli, such as excessive ferric ions and minimal medium conditions. This is the first study to show that an antimicrobial peptide inhibits the growth of bacteria by influencing the phosphorylation of a two-component system directly.

MOLECULAR CHARACTERIZATION OF HTLV-1 TAX INTERACTION WITH THE KIX DOMAIN OF CBP/p300

PubMed Central

Ramírez, Julita A.; Nyborg, Jennifer K.

2007-01-01

Summary The viral oncoprotein Tax mediates transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1). Both Tax and the cellular transcription factor CREB bind to viral cyclic AMP response elements (vCREs) located in the viral promoter. Tax and serine 133 phosphorylated CREB (pCREB) bound to the HTLV-1 promoter facilitate viral transcription via the recruitment of the large cellular coactivators CBP/p300. While the interaction between the phosphorylated kinase inducible domain (pKID) of pCREB and the KIX domain of CBP/p300 has been well-characterized, the molecular interactions between KIX, full-length Tax, and pCREB have not been examined. In this study we biochemically characterized the interaction between Tax and KIX in a physiologically relevant complex containing pCREB and vCRE DNA. Our data show that Tax and pCREB simultaneously and independently bind two distinct surfaces on the KIX domain: Tax binds KIX at the previously-characterized mixed-lineage leukemia (MLL) protein interaction surface while pCREB binds KIX at the pKID-KIX interface. These results provide evidence for a model in which Tax and pCREB bind distinct surfaces of KIX for effective CBP/p300 recruitment to the HTLV-1 promoter. We also show that MLL competes with Tax for KIX binding, suggesting a novel mechanism of Tax oncogenesis in which normal MLL function is disrupted by Tax. PMID:17707401

Production of transgenic pigs using a pGFAP-CreERT2/EGFP LoxP inducible system for central nervous system disease models.

PubMed

Hwang, Seon-Ung; Eun, Kiyoung; Yoon, Junchul David; Kim, Hyunggee; Hyun, Sang-Hwan

2018-05-31

Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein ( pGFAP ) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreER T2 /LCMV-EGFP LoxP and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor ( CreER T2 ) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreER T2 -mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreER T2 /EGFP LoxP recombination system via SCNT.

Characterization of binding preference of polyhydroxyalkanoate biosynthesis-related multifunctional protein PhaM from Ralstonia eutropha.

PubMed

Ushimaru, Kazunori; Tsuge, Takeharu

2016-05-01

The binding preference of a polyhydroxyalkanoate (PHA) biosynthesis-related multifunctional protein from Ralstonia eutropha (PhaMRe) was characterized. In vitro activity assay showed that PHA synthase from R. eutropha (PhaCRe) was activated by the presence of PhaMRe but PHA synthase from Aeromonas caviae (PhaCAc) was not. Additionally, in vitro assays of protein-protein interactions demonstrated that PhaMRe interacted with PhaCRe directly, but did not interact with PhaCAc. These results suggest that the protein-protein interaction is important for the activation of PhaC by PhaMRe. Further analyses indicated that PhaMRe has little or no direct interaction with the PHA polymer chain. Subsequently, PHA biosynthesis genes (phaA Re, phaB Re, and phaC Re/phaC Ac) and the phaM Re gene were introduced into recombinant Escherichia coli and cultivated for PHA accumulation. Contrary to our expectations, the expression of PhaMRe decreased PHA accumulation and changed the morphology of PHA granules to be microscopically obscure shape in PhaCRe-expressing E. coli. No change in the amount of P(3HB) or the morphology of granules by PhaMRe expression was observed in PhaCAc-expressing E. coli. These observations suggest that PhaMRe affects cellular physiology through the PhaM-PhaC interaction.

Antisense protein kinase A RIalpha inhibits 7,12-dimethylbenz(a)anthracene-induction of mammary cancer: blockade at the initial phase of carcinogenesis.

PubMed

Nesterova, Maria V; Cho-Chung, Yoon S

2004-07-01

There are two types of cyclic AMP (cAMP)-dependent protein kinase (PKA), type I (PKA-I) and type II (PKA-II), which share a common catalytic (C) subunit but contain distinct regulatory (R) subunits, RI versus RII, respectively. Evidence suggests that increased expression of PKA-I and its regulatory subunit (RIalpha) correlates with tumorigenesis and tumor growth. We investigated the effect of sequence-specific inhibition of RIalpha gene expression at the initial phase of 7,12-dimethylbenz(alphaa)anthracene (DMBA)-induced mammary carcinogenesis. Antisense RIalpha oligodeoxynucleotide (ODN) targeted against PKA RIalpha was administered (0.1 mg/day/rat, i.p.) 1 day before DMBA intubation and during the first 9 days post-DMBA intubation to determine the anticarcinogenic effects. Antisense RIalpha, in a sequence-specific manner, inhibited the tumor production. At 90 days after DMBA intubation, untreated controls and RIalpha-antisense-treated rats exhibited an average mean number of tumors per rat of 4.2 and 1.8, respectively, and 90% of control and 45% of antisense-treated animals had tumors. The antisense also delayed the first tumor appearance. An increase in RIalpha and PKA-I levels in the mammary gland and liver preceded DMBA-induced tumor production, and antisense down-regulation of RIalpha restored normal levels of PKA-I and PKA-II in these tissues. Antisense RIalpha in the liver induced the phase II enzymes, glutathione S-transferase and quinone oxidoreductase, c-fos protein, and activator protein 1 (AP-1)- and cAMP response element (CRE)-directed transcription. In the mammary glands, antisense RIalpha promoted DNA repair processes. In contrast, the CRE transcription-factor decoy could not mimic these effects of antisense RIalpha. The results demonstrate that RIalpha antisense produces dual anticarcinogenic effects: (a) increasing DMBA detoxification in the liver by increasing phase II enzyme activities, increasing CRE-binding-protein phosphorylation and enhancing CRE- and Ap-1-directed transcription; and (b) activating DNA repair processes in the mammary gland by down-regulating PKA-I.

Characterization of "cis"-regulatory elements ("c"RE) associated with mammary gland function

USDA-ARS?s Scientific Manuscript database

The Bos taurus genome assembly has propelled dairy science into a new era; still, most of the information encoded in the genome has not yet been decoded. The human Encyclopedia of DNA Elements (ENCODE) project has spearheaded the identification and annotation of functional genomic elements in the hu...

Generation and characterization of PDGFRα-GFPCreERT2 knock-In mouse line.

PubMed

Miwa, Hiroyuki; Era, Takumi

2015-05-01

Platelet-derived growth factor (PDGF) and its receptor play an important role in embryogenesis. PDGF receptor α (PDGFRα) is expressed specifically in the embryonic day 7.5 (E7.5) mesoderm and in the E9.5 neural crest among other tissues. PDGFRα-expressing cells and their descendants are involved in the formation of various tissues. To trace PDGFRα-expressing cells in vivo, we generated a knock-in mouse line that expressed a fusion protein of green fluorescent protein (GFP), Cre recombinase (Cre), and mutated estrogen receptor ligand-binding domain (ERT2) under the control of the PDGFRα promoter. In these mice, Cre activity in PDGFRα-expressing cells could be induced by tamoxifen treatment. Taken together, our results suggest that the knock-in mouse line generated here could be useful for studying PDGFRα-expressing cells and their descendants in vivo at various stages of development. © 2015 Wiley Periodicals, Inc.

End-to-end crosstalk within the hepatitis C virus genome mediates the conformational switch of the 3′X-tail region

PubMed Central

Romero-López, Cristina; Barroso-delJesus, Alicia; García-Sacristán, Ana; Briones, Carlos; Berzal-Herranz, Alfredo

2014-01-01

The hepatitis C virus (HCV) RNA genome contains multiple structurally conserved domains that make long-distance RNA–RNA contacts important in the establishment of viral infection. Microarray antisense oligonucelotide assays, improved dimethyl sulfate probing methods and 2′ acylation chemistry (selective 2’-hydroxyl acylation and primer extension, SHAPE) showed the folding of the genomic RNA 3′ end to be regulated by the internal ribosome entry site (IRES) element via direct RNA–RNA interactions. The essential cis-acting replicating element (CRE) and the 3′X-tail region adopted different 3D conformations in the presence and absence of the genomic RNA 5′ terminus. Further, the structural transition in the 3′X-tail from the replication-competent conformer (consisting of three stem-loops) to the dimerizable form (with two stem-loops), was found to depend on the presence of both the IRES and the CRE elements. Complex interplay between the IRES, the CRE and the 3′X-tail region would therefore appear to occur. The preservation of this RNA–RNA interacting network, and the maintenance of the proper balance between different contacts, may play a crucial role in the switch between different steps of the HCV cycle. PMID:24049069

[Analysis of cis-regulatory element distribution in gene promoters of Gossypium raimondii and Arabidopsis thaliana].

PubMed

Sun, Gao-Fei; He, Shou-Pu; Du, Xiong-Ming

2013-10-01

Cotton genomic studies have boomed since the release of Gossypium raimondii draft genome. In this study, cis-regulatory element (CRE) in 1 kb length sequence upstream 5' UTR of annotated genes were selected and scanned in the Arabidopsis thaliana (At) and Gossypium raimondii (Gr) genomes, based on the database of PLACE (Plant cis-acting Regulatory DNA Elements). According to the definition of this study, 44 (12.3%) and 57 (15.5%) CREs presented "peak-like" distribution in the 1 kb selected sequences of both genomes, respectively. Thirty-four of them were peak-like distributed in both genomes, which could be further categorized into 4 types based on their core sequences. The coincidence of TATABOX peak position and their actual position ((-) -30 bp) indicated that the position of a common CRE was conservative in different genes, which suggested that the peak position of these CREs was their possible actual position of transcription factors. The position of a common CRE was also different between the two genomes due to stronger length variation of 5' UTR in Gr than At. Furthermore, most of the peak-like CREs were located in the region of -110 bp-0 bp, which suggested that concentrated distribution might be conductive to the interaction of transcription factors, and then regulate the gene expression in downstream.

Factors predicting emotional cue-responding behaviors of nurses in Taiwan: An observational study.

PubMed

Lin, Mei-Feng; Lee, An-Yu; Chou, Cheng-Chen; Liu, Tien-Yu; Tang, Chia-Chun

2017-10-01

Responding to emotional cues is an essential element of therapeutic communication. The purpose of this study is to examine nurses' competence of responding to emotional cues (CRE) and related factors while interacting with standardized patients with cancer. This is an exploratory and predictive correlational study. A convenience sample of registered nurses who have passed the probationary period in southern Taiwan was recruited to participate in 15-minute videotaped interviews with standardized patients. The Medical Interview Aural Rating Scale was used to describe standardized patients' emotional cues and to measure nurses' CRE. The State-Trait Anxiety Inventory was used to evaluate nurses' anxiety level before the conversation. We used descriptive statistics to describe the data and stepwise regression to examine the predictors of nurses' CRE. A total of 110 nurses participated in the study. Regardless of the emotional cue level, participants predominately responded to cues with inappropriate distancing strategies. Prior formal communication training, practice unit, length of nursing practice, and educational level together explain 36.3% variances of the nurses' CRE. This study is the first to explore factors related to Taiwanese nurses' CRE. Compared to nurses in other countries, Taiwanese nurses tended to respond to patients' emotional cues with more inappropriate strategies. We also identified significant predictors of CRE that show the importance of communication training. Future research and education programs are needed to enhance nurses' CRE and to advocate for emotion-focused communication. Copyright © 2016 John Wiley & Sons, Ltd.

Critical and Independent Role for SOCS3 in Either Myeloid or T Cells in Resistance to Mycobacterium tuberculosis

PubMed Central

Carow, Berit; Reuschl, Ann-Kathrin; Gavier-Widén, Dolores; Jenkins, Brendan J.; Ernst, Matthias; Yoshimura, Akihiko; Chambers, Benedict J.; Rottenberg, Martin E.

2013-01-01

Suppressor of cytokine signalling 3 (SOCS3) negatively regulates STAT3 activation in response to several cytokines such as those in the gp130-containing IL-6 receptor family. Thus, SOCS3 may play a major role in immune responses to pathogens. In the present study, the role of SOCS3 in M. tuberculosis infection was examined. All Socs3fl/fl LysM cre, Socs3fl/fl lck cre (with SOCS3-deficient myeloid and lymphoid cells, respectively) and gp130F/F mice, with a mutation in gp130 that impedes binding to SOCS3, showed increased susceptibility to infection with M. tuberculosis. SOCS3 binding to gp130 in myeloid cells conveyed resistance to M. tuberculosis infection via the regulation of IL-6/STAT3 signalling. SOCS3 was redundant for mycobacterial control by macrophages in vitro. Instead, SOCS3 expression in infected macrophages and DCs prevented the IL-6-mediated inhibition of TNF and IL-12 secretion and contributed to a timely CD4+ cell-dependent IFN-γ expression in vivo. In T cells, SOCS3 expression was essential for a gp130-independent control of infection with M. tuberculosis, but was neither required for the control of infection with attenuated M. bovis BCG nor for M. tuberculosis in BCG-vaccinated mice. Socs3fl/fl lck cre mice showed an increased frequency of γδ+ T cells in different organs and an enhanced secretion of IL-17 by γδ+ T cells in response to infection. Socs3fl/fl lck cre γδ+ T cells impaired the control of infection with M. tuberculosis. Thus, SOCS3 expression in either lymphoid or myeloid cells is essential for resistance against M. tuberculosis via discrete mechanisms. PMID:23853585

Inhibition of autophagy exerts anti-colon cancer effects via apoptosis induced by p53 activation and ER stress.

PubMed

Sakitani, Kosuke; Hirata, Yoshihiro; Hikiba, Yohko; Hayakawa, Yoku; Ihara, Sozaburo; Suzuki, Hirobumi; Suzuki, Nobumi; Serizawa, Takako; Kinoshita, Hiroto; Sakamoto, Kei; Nakagawa, Hayato; Tateishi, Keisuke; Maeda, Shin; Ikenoue, Tsuneo; Kawazu, Shoji; Koike, Kazuhiko

2015-10-24

Although some molecularly targeted drugs for colorectal cancer are used clinically and contribute to a better prognosis, the current median survival of advanced colorectal cancer patients is not sufficient. Autophagy, a basic cell survival mechanism mediated by recycling of cellular amino acids, plays an important role in cancer. Recently, autophagy has been highlighted as a promising new molecular target. The unfolded protein response (UPR) reportedly act in complementary fashion with autophagy in intestinal homeostasis. However, the roles of UPR in colon cancer under autophagic inhibition remain to be elucidated. We aim to clarify the inhibitory effect of autophagy on colon cancer. We crossed K19 (CreERT) and Atg5 (flox/flox) mice to generate Atg5 (flox/flox)/K19 (CreERT) mice. Atg5 (flox/flox)/K19 (CreERT) mice were first treated with azoxymethane/dextran sodium sulfate and then injected with tamoxifen to inhibit autophagy in CK19-positive epithelial cells. To examine the anti-cancer mechanisms of autophagic inhibition, we used colon cancer cell lines harboring different p53 gene statuses, as well as small interfering RNAs (siRNAs) targeting Atg5 and immunoglobulin heavy-chain binding protein (BiP), a chaperone to aid folding of unfolded proteins. Colon tumors in Atg5 (flox/flox)/K19 (CreERT) mice showed loss of autophagic activity and decreased tumor size (the total tumor diameter was 28.1 mm in the control and 20.7 mm in Atg5 (flox/flox)/K19 (CreERT) mice, p = 0.036). We found that p53 and UPR/endoplasmic reticulum (ER) stress-related proteins, such as cleaved caspase 3, and CAAT/enhancer-binding protein homologous protein, are up-regulated in colon tumors of Atg5 (flox/flox)/K19 (CreERT) mice. Although Atg5 and BiP silencing, respectively, increased apoptosis in p53 wild type cells, Atg5 silencing alone did not show the same effect on apoptosis in p53 mutant cells. However, co-transfection of Atg5 and BiP siRNAs led to increased apoptosis in p53 mutant cells. Blocking autophagy has potential in the treatment of colon cancer by inducing apoptosis via p53 and ER stress, and suppressing the UPR pathway is a valid strategy to overcome resistance to autophagic inhibition.

Isolation and Identification of Genes Activating Uas2-Dependent Adh2 Expression in Saccharomyces Cerevisiae

PubMed Central

Donoviel, M. S.; Young, E. T.

1996-01-01

Two cis-acting elements have been identified that act synergistically to regulate expression of the glucose-repressed alcohol dehydrogenase 2 (ADH2) gene. UAS1 is bound by the trans-activator Adr1p. UAS2 is thought to be the binding site for an unidentified regulatory protein. A genetic selection based on a UAS2-dependent ADH2 reporter was devised to isolate genes capable of activating UAS2-dependent transcription. One set of UAS2-dependent genes contained SPT6/CRE2/SSN20. Multicopy SPT6 caused improper expression of chromosomal ADH2. A second set of UAS2-dependent clones contained a previously uncharacterized open reading frame designated MEU1 (Multicopy Enhancer of UAS2). A frame shift mutation in MEU1 abolished its ability to activate UAS2-dependent gene expression. Multicopy MEU1 expression suppressed the constitutive ADH2 expression caused by cre2-1. Disruption of MEU1 reduced endogenous ADH2 expression about twofold but had no effect on cell viability or growth. No homologues of MEU1 were identified by low-stringency Southern hybridization of yeast genomic DNA, and no significant homologues were found in the sequence data bases. A MEU1/β-gal fusion protein was not localized to a particular region of the cell. MEU1 is linked to PPR1 on chromosome XII. PMID:8807288

Extraction of nobiletin from Citrus Unshiu peels by supercritical fluid and its CRE-mediated transcriptional activity.

PubMed

Oba, Chisato; Ota, Masaki; Nomura, Koichiro; Fujiwara, Hironori; Takito, Jiro; Sato, Yoshiyuki; Ohizumi, Yasushi; Inomata, Hiroshi

2017-04-15

Polymethoxyflavone (PMF) is one of bioactive compounds in Citrus Unshiu and included mainly in the peels rather than the fruits, seeds and leaves. Supercritical CO 2 extraction is one candidate for selective extraction of polymethoxyflavone and in this study, supercritical CO 2 extraction with/without ethanol entrainer from Citrus Unshiu peels was examined at a temperature of 333K and a pressure of 30MPa. CRE (cyclic AMP response element)-mediated transcriptional assay was examined by using the extracts from supercritical fluid extraction. The results showed that extracts including nobiletin increased with increasing ethanol concentration in supercritical CO 2 and the elapsed extraction time. Extracts at ethanol concentration of 5 mol% showed high CRE-mediated transcription activity. This can be caused by activity of the extract including nobiletin in addition to the other methoxylated flavonoid species such as tangeretin. Extracts at ethanol concentration of 50% showed the highest CRE-mediated transcription activity, which can be attributed to flavonoid glycoside such as hesperidin. From our investigations, flavonoid glycoside can be one of promoters of CRE-mediated transcription activity. Copyright © 2017 Elsevier GmbH. All rights reserved.

Satb2 Ablation Impairs Hippocampus-Based Long-Term Spatial Memory and Short-Term Working Memory and Immediate Early Genes (IEGs)-Mediated Hippocampal Synaptic Plasticity.

PubMed

Li, Ying; You, Qiang-Long; Zhang, Sheng-Rong; Huang, Wei-Yuan; Zou, Wen-Jun; Jie, Wei; Li, Shu-Ji; Liu, Ji-Hong; Lv, Chuang-Ye; Cong, Jin; Hu, Yu-Ying; Gao, Tian-Ming; Li, Jian-Ming

2017-04-18

Special AT-rich sequence-binding protein 2 (Satb2) is a protein binding to the matrix attachment regions of DNA and important for gene regulation. Patients with SATB2 mutation usually suffer moderate to severe mental retardation. However, the mechanisms for the defects of intellectual activities in patients with SATB2 mutation are largely unclear. Here we established the heterozygous Satb2 mutant mice and Satb2 conditional knockout mice to mimic the patients with SATB2 mutation and figured out the role of Satb2 in mental activities. We found that the spatial memory and working memory were significantly damaged in the heterozygous Satb2 mutant mice, early postnatal Satb2-deficient mice (CaMKIIα-Cre + Satb2 fl/fl mice), and adult Satb2 ablation mice (Satb2 fl/fl mice injected with CaMKIIα-Cre virus). Functionally, late phase long-term potentiation (L-LTP) in these Satb2 mutant mice was greatly impaired. Morphologically, in CA1 neurons of CaMKIIα-Cre + Satb2 fl/fl mice, we found decreased spine density of the basal dendrites and less branches of apical dendrites that extended into lacunar molecular layer. Mechanistically, expression levels of immediate early genes (IEGs) including Fos, FosB, and Egr1 were significantly decreased after Satb2 deletion. And, Satb2 could regulate expression of FosB by binding to the promoter of FosB directly. In general, our study uncovers that Satb2 plays an important role in spatial memory and working memory by regulating IEGs-mediated hippocampal synaptic plasticity.

Measuring Transcription Factor–Binding Site Turnover: A Maximum Likelihood Approach Using Phylogenies

PubMed Central

Otto, Wolfgang; Stadler, Peter F.; López-Giraldéz, Francesc; Townsend, Jeffrey P.; Lynch, Vincent J.

2009-01-01

A major mode of gene expression evolution is based on changes in cis-regulatory elements (CREs) whose function critically depends on the presence of transcription factor–binding sites (TFBS). Because CREs experience extensive TFBS turnover even with conserved function, alignment-based studies of CRE sequence evolution are limited to very closely related species. Here, we propose an alternative approach based on a stochastic model of TFBS turnover. We implemented a maximum likelihood model that permits variable turnover rates in different parts of the species tree. This model can be used to detect changes in turnover rate as a proxy for differences in the selective pressures acting on TFBS in different clades. We applied this method to five TFBS in the fungi methionine biosynthesis pathway and three TFBS in the HoxA clusters of vertebrates. We find that the estimated turnover rate is generally high, with half-life ranging between ∼5 and 150 My and a mode around tens of millions of years. This rate is consistent with the finding that even functionally conserved enhancers can show very low sequence similarity. We also detect statistically significant differences in the equilibrium densities of estrogen- and progesterone-response elements in the HoxA clusters between mammal and nonmammal vertebrates. Even more extreme clade-specific differences were found in the fungal data. We conclude that stochastic models of TFBS turnover enable the detection of shifts in the selective pressures acting on CREs in different organisms. The analysis tool, called CRETO (Cis-Regulatory Element Turn-Over) can be downloaded from http://www.bioinf.uni-leipzig.de/Software/creto/. PMID:20333180

Crosslinking transcription factors to their recognition sequences with PtII complexes

NASA Technical Reports Server (NTRS)

Chu, B. C.; Orgel, L. E.

1992-01-01

We have prepared phosphorothioate-containing cyclic oligodeoxynucleotides that fold into 'dumbbells' containing CRE and TRE sequences, the binding sequences for the CREB and JUN proteins, respectively. Six phosphorothioate residues were introduced into each of the recognition sequences. K2PtCl4 crosslinks CRE to CREB and TRE to JUN. The extent of crosslinking is about eight times greater than that observed with standard oligodeoxynucleotides and amounts to 30-50% of the efficiency of non-covalent association as estimated by gel-shift assays. Crosslinking is reversed by incubation with NaCN. The crosslinking reaction is specific--a dumbbell oligonucleotide with six phosphorothioate groups introduced into the Sp1 recognition sequence could not be crosslinked efficiently to CREB or JUN proteins with K2PtCl4. The binding of TRE to CREB is not strong enough for effective detection by gel-shift assays, but the TRE-CREB complex is crosslinked efficiently by K2PtCl4 and can then readily be detected.

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines

PubMed Central

Vuong, Helen E.; de Sevilla Müller, Luis Pérez; Hardi, Claudia N.; McMahon, Douglas G.; Brecha, Nicholas C.

2015-01-01

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) line with three catecholamine-related Cre recombinase lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ~6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium somal diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines were generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. PMID:26335381

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines.

PubMed

Vuong, H E; Pérez de Sevilla Müller, L; Hardi, C N; McMahon, D G; Brecha, N C

2015-10-29

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. Published by Elsevier Ltd.

Transcriptional machinery of TNF-α-inducible YTH domain containing 2 (YTHDC2) gene.

PubMed

Tanabe, Atsushi; Konno, Junpei; Tanikawa, Kenya; Sahara, Hiroeki

2014-02-01

We previously demonstrated that a cellular factor, cyclosporin A (CsA) associated helicase-like protein (CAHL) that is identical to YTH domain containing 2 (YTHDC2), forms trimer complex with cyclophilin B and NS5B of hepatitis C virus (HCV) and facilitates HCV genome replication. Gene expression of YTHDC2 was shown in tumor cell lines and tumor necrosis factor (TNF)-α-treated hepatocytes, but not in untreated. However, the function of YTHDC2 in the tumor cells and the mechanism by which the YTHDC2 gene is transcribed in these cells is largely unknown. We first evaluated that the role of YTHDC2 in the proliferation of hepatocellular carcinoma (HCC) cell line Huh7 using RNA interference and found that YTHDC2-downregulated Huh7 were significantly decreased cell growth as compared to control. We next demonstrated that the cAMP response element (CRE) site in the promoter region of the YTHDC2 gene is critical for YTHDC2 transcription. To further investigate the transcription factors bound to the CRE site, we performed chromatin immunoprecipitation assays. Our findings demonstrate that c-Jun and ATF-2 bind to the CRE site in Huh7, and that TNF-α induces the biological activity of these transcription factors in hepatocytes as well as Huh7. Moreover, treatment with the HDAC inhibitor, trichostatin A (TSA), reduces YTHDC2 expression in Huh7 and in TNF-α-stimulated hepatocytes. Collectively, these data show that YTHDC2 plays an important role in tumor cells growth and activation/recruitment of c-Jun and ATF-2 to the YTHDC2 promoter is necessary for the transcription of YTHDC2, and that HDAC activity is required for the efficient expression of YTHDC2 in both of hepatocyte and HCC cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of 5-(1-Methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)thiophene-2-Carboxamides as Novel and Selective Monoamine Oxidase B Inhibitors Used to Improve Memory and Cognition.

PubMed

Kaplan, Alan P; Keenan, Terence; Scott, Roderick; Zhou, Xianbo; Bourchouladze, Rusiko; McRiner, Andrew J; Wilson, Mark E; Romashko, Darlene; Miller, Regina; Bletsch, Matthew; Anderson, Gary; Stanley, Jennifer; Zhang, Adia; Lee, Dong; Nikpur, John

2017-12-20

Initial work in Drosophila and mice demonstrated that the transcription factor cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) is a master control gene for memory formation. The relationship between CREB and memory has also been found to be true in other species, including aplysia and rats. It is thus well-established that CREB activation plays a central role in memory enhancement and that CREB is activated during memory formation. On the basis of these findings, a phenotypic high-throughput screening campaign utilizing a CRE-luciferase (CRE-Luci) SK-N-MC cell line was performed to identify compounds that enhance transcriptional activation of the CRE promoter with a suboptimal dose of forskolin. A number of small-molecule hits of unknown mechanisms of action were identified in the screening campaign, including HT-0411. Follow-up studies suggested that the CREB activation by HT-0411 is attributed to its specific and selective inhibition of monoamine oxidase B (MAO-B). Further, HT-0411 was shown to improve 24 h memory in rodents in a contextual fear conditioning model. This report describes the lead optimization of a series of 5-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl) thiophene-2-carboxamides that were identified as novel, potent, and selective inhibitors of MAO-B. Extensive SAR studies and in vivo behavioral evaluations of this and other related analogue series identified a number of potential clinical development candidates; ultimately, compound 8f was identified as a candidate molecule with high selectivity toward MAO-B (29-56 nM) over MAO-A (19% inhibition at a screening concentration of 50 μM), an excellent profile against a panel of other enzymes and receptors, good pharmacokinetic properties in rodents and dogs, and efficacy in multiple rodent memory models.

Corticosteroids inhibit sphingosine 1-phosphate-induced interleukin-6 secretion from human airway smooth muscle via mitogen-activated protein kinase phosphatase 1-mediated repression of mitogen and stress-activated protein kinase 1.

PubMed

Che, Wenchi; Parmentier, Johannes; Seidel, Petra; Manetsch, Melanie; Ramsay, Emma E; Alkhouri, Hatem; Ge, Qi; Armour, Carol L; Ammit, Alaina J

2014-02-01

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays an important proinflammatory role in asthmatic airways. Corticosteroids are first-line antiinflammatories in asthma; however, their repressive effects on S1P-induced cytokine secretion have not been investigated. To address this, our in vitro study reveals the molecular mechanisms by which corticosteroids inhibit S1P-induced IL-6 expression in the pivotal immunomodulatory cell type, airway smooth muscle (ASM). We first uncover the cellular signaling pathways responsible: S1P activates a cyclic adenosine monophosphate/cAMP response-element-binding protein (CREB)/CRE-dependent pathway to induce IL-6 transcription, concomitant with stimulation of the mitogen-activated protein kinase (MAPK) superfamily and downstream mitogen and stress-activated protein kinase 1 (MSK1) and histone H3 phosphorylation. In this way, S1P stimulates parallel signaling pathways to induce IL-6 secretion via CRE-driven transcription of the IL-6 gene promoter in a relaxed chromatin environment achieved through histone H3 phosphorylation. Second, we investigated how corticosteroids mediate their repressive effects. The corticosteroid dexamethasone inhibits S1P-induced IL-6 protein secretion and mRNA expression, but CREB/CRE transrepression, inhibition of IL-6 mRNA stability, or subcellular relocation of MSK1 were not responsible for the repressive effects of dexamethasone. Rather, we show that dexamethasone rapidly induces up-regulation of the MAPK deactivator MAPK phosphatase 1 (MKP-1) and that MKP-1 blocks the MAPK-driven activation of MSK1 and phosphorylation of histone H3. This was confirmed by treatment with triptolide, an inhibitor of MKP-1 up-regulation, where repressive effects of corticosteroids were reversed. Our study reveals the molecular mechanism underlying the antiinflammatory capacity of corticosteroids to repress proinflammatory functions induced by the potent bioactive sphingolipid S1P in the lung.

Fibroblast growth factor receptor-Frs2α signaling is critical for nephron progenitors.

PubMed

Di Giovanni, Valeria; Walker, Kenneth A; Bushnell, Daniel; Schaefer, Caitlin; Sims-Lucas, Sunder; Puri, Pawan; Bates, Carlton M

2015-04-01

Previous studies using transgenic Pax3cre mice have revealed roles for fibroblast growth factor receptors (Fgfrs) and Fgfr substrate 2α (Frs2α) signaling in early metanephric mesenchyme patterning and in ureteric morphogenesis. The role of Fgfr/Frs2α signaling in nephron progenitors is unknown. Thus, we generated mouse models using BAC transgenic Six2EGFPcre (Six2cre) mediated deletion of Fgfrs and/or Frs2α in nephron progenitors. Six2cre mediated deletion of Fgfr1 or Fgfr2 alone led to no obvious kidney defects. Six2creFgfr1(flox/flox)Fgfr2(flox/flox) (Fgfr1/2(NP-/-)) mice generate a discernable kidney; however, they develop nephron progenitor depletion starting at embryonic day 12.5 (E12.5) and later demonstrate severe cystic dysplasia. To determine the role of Frs2α signaling downstream of Fgfr2 in Fgfr1/2(NP-/-) mice, we generated Six2cre(,)Fgfr1(flox/flox)Fgfr2(LR/LR) (Fgfr1(NP-/-)Fgfr2(LR/LR)) mice that have point mutations in the Frs2α binding site of Fgfr2. Like Fgfr1/2(NP-/-) mice, Fgfr1(NP-/-)Fgfr2(LR/LR) develop nephron progenitor depletion, but it does not start until E14.5 and older mice have less severe cystic dysplasia than Fgfr1/2(NP-/-) To determine the role of Frs2α alone in nephron progenitors, we generated Six2creFrs2'A(flox/flox) (Frs2a(NP-/-)) mice. Frs2a(NP-/-)mice also develop nephron progenitor depletion and renal cysts, although these occurred later and were less severe than in the other Six2cre mutant mice. The nephron progenitor loss in all Six2cre mutant lines was associated with decreased Cited1 expression and increased apoptosis versus controls. FAC-sorted nephron progenitors in Six2cre Frs2'A(flox/flox) mice demonstrated evidence of increased Notch activity versus controls, which likely drives the progenitor defects. Thus, Fgfr1 and Fgfr2 have synergistic roles in maintaining nephron progenitors; furthermore, Fgfr signaling in nephron progenitors appears to be mediated predominantly by Frs2α. Copyright © 2015 Elsevier Inc. All rights reserved.

Targeting Signal Transducers and Activators of Transcription-3 (STAT3) as a Novel Strategy in Sensitizing Breast Cancer to EGFR-Targeted Therapy

DTIC Science & Technology

2009-06-01

Osman, F. The human glutathione S-transferase P1 ( GSTP1 ) gene is transactivated by cyclic AMP (cAMP) via a cAMP response element (CRE) proximal to the...transcription start site. Chem-Biol. Interactions 133, 320-321, 2001. 4. Lo, H.-W. and Ali-Osman, F. Cyclic AMP mediated GSTP1 gene activation in...tumor cells involves the interaction of activated CREB-1 with the GSTP1 CRE: a novel mechanism of cellular GSTP1 gene regulation. Journal of Cellular

Redox regulation of stress signals: possible roles of dendritic stellate TRX producer cells (DST cell types).

PubMed

Yodoi, Junji; Nakamura, Hajime; Masutani, Hiroshi

2002-01-01

Thioredoxin (TRX) is a 12 kDa protein with redox-active dithiol (Cys-Gly-Pro-Cys) in the active site. TRX is induced by a variety of stresses including viral infection and inflammation. The promoter sequences of the TRX gene contain a series of stress-responsive elements including ORE, ARE, XRE, CRE and SP-1. TRX promotes DNA binding of transcription factors such as NF-kappaB, AP-1 and p53. TRX interacts with target proteins modulating the activity of those proteins. We have identified TRX binding protein-2 (TBP-2), which was identical to vitamin D3 up-regulated protein 1 (VDUP1). Potential action of TBP-2/VDUP1 as a redox-sensitive tumor suppressor will be discussed. There is accumulating evidence for the involvement of TRX in the protection against infectious and inflammatory disorders. We will discuss the role of TRX-dependent redox regulation of the host defense mechanism, in particular its relation to the emerging concept of constitutive and/or inducible TRX on special cell types with dendritic and stellate morphology in the immune, endocrine and nervous systems, which we provisionally designate as dendritic stellate TRX producer cells (DST cell types).

Computational exploration of cis-regulatory modules in rhythmic expression data using the "Exploration of Distinctive CREs and CRMs" (EDCC) and "CRM Network Generator" (CNG) programs.

PubMed

Bekiaris, Pavlos Stephanos; Tekath, Tobias; Staiger, Dorothee; Danisman, Selahattin

2018-01-01

Understanding the effect of cis-regulatory elements (CRE) and clusters of CREs, which are called cis-regulatory modules (CRM), in eukaryotic gene expression is a challenge of computational biology. We developed two programs that allow simple, fast and reliable analysis of candidate CREs and CRMs that may affect specific gene expression and that determine positional features between individual CREs within a CRM. The first program, "Exploration of Distinctive CREs and CRMs" (EDCC), correlates candidate CREs and CRMs with specific gene expression patterns. For pairs of CREs, EDCC also determines positional preferences of the single CREs in relation to each other and to the transcriptional start site. The second program, "CRM Network Generator" (CNG), prioritizes these positional preferences using a neural network and thus allows unbiased rating of the positional preferences that were determined by EDCC. We tested these programs with data from a microarray study of circadian gene expression in Arabidopsis thaliana. Analyzing more than 1.5 million pairwise CRE combinations, we found 22 candidate combinations, of which several contained known clock promoter elements together with elements that had not been identified as relevant to circadian gene expression before. CNG analysis further identified positional preferences of these CRE pairs, hinting at positional information that may be relevant for circadian gene expression. Future wet lab experiments will have to determine which of these combinations confer daytime specific circadian gene expression.

Computational exploration of cis-regulatory modules in rhythmic expression data using the “Exploration of Distinctive CREs and CRMs” (EDCC) and “CRM Network Generator” (CNG) programs

PubMed Central

Staiger, Dorothee

2018-01-01

Understanding the effect of cis-regulatory elements (CRE) and clusters of CREs, which are called cis-regulatory modules (CRM), in eukaryotic gene expression is a challenge of computational biology. We developed two programs that allow simple, fast and reliable analysis of candidate CREs and CRMs that may affect specific gene expression and that determine positional features between individual CREs within a CRM. The first program, “Exploration of Distinctive CREs and CRMs” (EDCC), correlates candidate CREs and CRMs with specific gene expression patterns. For pairs of CREs, EDCC also determines positional preferences of the single CREs in relation to each other and to the transcriptional start site. The second program, “CRM Network Generator” (CNG), prioritizes these positional preferences using a neural network and thus allows unbiased rating of the positional preferences that were determined by EDCC. We tested these programs with data from a microarray study of circadian gene expression in Arabidopsis thaliana. Analyzing more than 1.5 million pairwise CRE combinations, we found 22 candidate combinations, of which several contained known clock promoter elements together with elements that had not been identified as relevant to circadian gene expression before. CNG analysis further identified positional preferences of these CRE pairs, hinting at positional information that may be relevant for circadian gene expression. Future wet lab experiments will have to determine which of these combinations confer daytime specific circadian gene expression. PMID:29298348

Yes-Associated Protein Promotes Angiogenesis via Signal Transducer and Activator of Transcription 3 in Endothelial Cells.

PubMed

He, Jinlong; Bao, Qiankun; Zhang, Yan; Liu, Mingming; Lv, Huizhen; Liu, Yajin; Yao, Liu; Li, Bochuan; Zhang, Chenghu; He, Shuang; Zhai, Guijin; Zhu, Yan; Liu, Xin; Zhang, Kai; Wang, Xiu-Jie; Zou, Ming-Hui; Zhu, Yi; Ai, Ding

2018-02-16

Angiogenesis is a complex process regulating endothelial cell (EC) functions. Emerging lines of evidence support that YAP (Yes-associated protein) plays an important role in regulating the angiogenic activity of ECs. The objective of this study was to specify the effect of EC YAP on angiogenesis and its underlying mechanisms. In ECs, vascular endothelial growth factor reduced YAP phosphorylation time and dose dependently and increased its nuclear accumulation. Using Tie2Cre-mediated YAP transgenic mice, we found that YAP promoted angiogenesis in the postnatal retina and tumor tissues. Mass spectrometry revealed signal transducer and activator of transcription 3 (STAT3) as a potential binding partner of YAP in ECs. Western blot and immunoprecipitation assays indicated that binding with YAP prolonged interleukin 6-induced STAT3 nuclear accumulation by blocking chromosomal maintenance 1-mediated STAT3 nuclear export without affecting its phosphorylation. Moreover, angiopoietin-2 expression induced by STAT3 was enhanced by YAP overexpression in ECs. Finally, a selective STAT3 inhibitor or angiopoietin-2 blockage partly attenuated retinal angiogenesis in Tie2Cre-mediated YAP transgenic mice. YAP binding sustained STAT3 in the nucleus to enhance the latter's transcriptional activity and promote angiogenesis via regulation of angiopoietin-2. © 2018 American Heart Association, Inc.

Fibroblast growth factor and cyclic AMP (cAMP) synergistically activate gene expression at a cAMP response element.

PubMed Central

Tan, Y; Low, K G; Boccia, C; Grossman, J; Comb, M J

1994-01-01

Growth factors and cyclic AMP (cAMP) are known to activate distinct intracellular signaling pathways. Fibroblast growth factor (FGF) activates ras-dependent kinase cascades, resulting in the activation of MAP kinases, whereas cAMP activates protein kinase A. In this study, we report that growth factors and cAMP act synergistically to stimulate proenkephalin gene expression. Positive synergy between growth factor- and cAMP-activated signaling pathways on gene expression has not been previously reported, and we suggest that these synergistic interactions represent a useful model for analyzing interactions between these pathways. Transfection and mutational studies indicate that both FGF-dependent gene activation and cAMP-dependent gene activation require cAMP response element 2 (CRE-2), a previously characterized cAMP-dependent regulatory element. Furthermore, multiple copies of this element are sufficient to confer FGF regulation upon a minimal promoter, indicating that FGF and cAMP signaling converge upon transcription factors acting at CRE-2. Among many different ATF/AP-1 factors tested, two factors, ATF-3 and c-Jun, stimulate proenkephalin transcription in an FGF- or Ras-dependent fashion. Finally, we show that ATF-3 and c-Jun form heterodimeric complexes in SK-N-MC cells and that the levels of both proteins are increased in response to FGF but not cAMP. Together, these results indicate that growth factor- and cAMP-dependent signaling pathways converge at CRE-2 to synergistically stimulate gene expression and that ATF-3 and c-Jun regulate proenkephalin transcription in response to both growth factor- and cAMP-dependent intracellular signaling pathways. Images PMID:7935470

The Effect of Conditional Inactivation of Beta 1 Integrins using Twist 2 Cre, Osterix Cre and Osteocalcin Cre Lines on Skeletal Phenotype

PubMed Central

Shekaran, Asha; Shoemaker, James T.; Kavanaugh, Taylor E.; Lin, Angela S.; LaPlaca, Michelle C.; Fan, Yuhong; Guldberg, Robert E.; García, Andrés J.

2014-01-01

Skeletal development and growth are complex processes regulated by multiple microenvironmental cues, including integrin-ECM interactions. The β1 sub-family of integrins is the largest integrin sub-family and constitutes the main integrin binding partners of collagen I, the major ECM component of bone. As complete β1 integrin integrin knockout results in embryonic lethality, studies of β1 integrin function in vivo rely on tissue-specific gene deletions. While multiple in vitro studies indicate that β1 integrins are crucial regulators of osteogenesis and mineralization, in vivo osteoblast-specific perturbations of β1 integrins have resulted in mild and sometimes contradictory skeletal phenotypes. To further investigate the role of β1 integrins on skeletal phenotype, we used the Twist2-Cre, Osterix-Cre and Osteocalcin-Cre lines to generate conditional β1 integrin deletions, where cre is expressed primarily in mesenchymal condensation, pre-osteoblast, and mature osteoblast lineage cells respectively within these lines. Mice with Twist2-specific β1 integrin disruption were smaller, had impaired skeletal development, especially in the craniofacial and vertebral tissues at E19.5, and did not survive beyond birth. Osterix-specific β1 integrin deficiency resulted in viable mice which were normal at birth but displayed early defects in calvarial ossification, incisor eruption and growth as well as femoral bone mineral density, structure, and mechanical properties. Although these defects persisted into adulthood, they became milder with age. Finally, a lack of β1 integrins in mature osteoblasts and osteocytes resulted in minor alterations to femur structure but had no effect on mineral density, biomechanics or fracture healing. Taken together, our data indicate that β1 integrin expression in early mesenchymal condensations play an important role in skeletal ossification, while β1 integrin-ECM interactions in pre-osteoblast, odontoblast- and hypertrophic chondryocyte- lineage cells regulate incisor eruption and perinatal bone formation in both intramembranously and endochondrally formed bones in young, rapidly growing mice. In contrast, the Osteocalcin-specific β1 integrin deletion had only minor effects on skeletal phenotype. PMID:25183373

On the Relationship between Cosmic Ray Exposure Ages and Petrography of CM Chondrites

NASA Technical Reports Server (NTRS)

Takenouchi, A.; Zolensky, M. E.; Nishiizumi, K.; Caffee, M.; Velbel, M. A.; Ross, K.; Zolensky, A.; Lee, L.; Imae, N.; Yamaguchi, A.;

Conditional and constitutive expression of a Tbx1-GFP fusion protein in mice

PubMed Central

2013-01-01

Background Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is caused by a 1.5-3 Mb microdeletion of chromosome 22q11.2, frequently referred to as 22q11.2 deletion syndrome (22q11DS). This region includes TBX1, a T-box transcription factor gene that contributes to the etiology of 22q11DS. The requirement for TBX1 in mammalian development is dosage-sensitive, such that loss-of-function (LOF) and gain-of-function (GOF) of TBX1 in both mice and humans results in disease relevant congenital malformations. Results To further gain insight into the role of Tbx1 in development, we have targeted the Rosa26 locus to generate a new GOF mouse model in which a Tbx1-GFP fusion protein is expressed conditionally using the Cre/LoxP system. Tbx1-GFP expression is driven by the endogenous Rosa26 promoter resulting in ectopic and persistent expression. Tbx1 is pivotal for proper ear and heart development; ectopic activation of Tbx1-GFP in the otic vesicle by Pax2-Cre and Foxg1-Cre represses neurogenesis and produces morphological defects of the inner ear. Overexpression of a single copy of Tbx1-GFP using Tbx1Cre/+ was viable, while overexpression of both copies resulted in neonatal lethality with cardiac outflow tract defects. We have partially rescued inner ear and heart anomalies in Tbx1Cre/- null embryos by expression of Tbx1-GFP. Conclusions We have generated a new mouse model to conditionally overexpress a GFP-tagged Tbx1 protein in vivo. This provides a useful tool to investigate in vivo direct downstream targets and protein binding partners of Tbx1. PMID:23971992

Severe Serotonin Depletion after Conditional Deletion of the Vesicular Monoamine Transporter 2 Gene in Serotonin Neurons: Neural and Behavioral Consequences

PubMed Central

Narboux-Nême, Nicolas; Sagné, Corinne; Doly, Stephane; Diaz, Silvina L; Martin, Cédric B P; Angenard, Gaelle; Martres, Marie-Pascale; Giros, Bruno; Hamon, Michel; Lanfumey, Laurence; Gaspar, Patricia; Mongeau, Raymond

2011-01-01

The vesicular monoamine transporter type 2 gene (VMAT2) has a crucial role in the storage and synaptic release of all monoamines, including serotonin (5-HT). To evaluate the specific role of VMAT2 in 5-HT neurons, we produced a conditional ablation of VMAT2 under control of the serotonin transporter (slc6a4) promoter. VMAT2sert−cre mice showed a major (−95%) depletion of 5-HT levels in the brain with no major alterations in other monoamines. Raphe neurons contained no 5-HT immunoreactivity in VMAT2sert−cre mice but developed normal innervations, as assessed by both tryptophan hydroxylase 2 and 5-HT transporter labeling. Increased 5-HT1A autoreceptor coupling to G protein, as assessed with agonist-stimulated [35S]GTP-γ-S binding, was observed in the raphe area, indicating an adaptive change to reduced 5-HT transmission. Behavioral evaluation in adult VMAT2sert−cre mice showed an increase in escape-like reactions in response to tail suspension and anxiolytic-like response in the novelty-suppressed feeding test. In an aversive ultrasound-induced defense paradigm, VMAT2sert−cre mice displayed a major increase in escape-like behaviors. Wild-type-like defense phenotype could be rescued by replenishing intracellular 5-HT stores with chronic pargyline (a monoamine oxidase inhibitor) treatment. Pargyline also allowed some form of 5-HT release, although in reduced amounts, in synaptosomes from VMAT2sert−cre mouse brain. These findings are coherent with the notion that 5-HT has an important role in anxiety, and provide new insights into the role of endogenous 5-HT in defense behaviors. PMID:21814181

Synergistic suppression of early phase of adipogenesis by microsomal PGE synthase-1 (PTGES1)-produced PGE2 and aldo-keto reductase 1B3-produced PGF2α.

PubMed

Fujimori, Ko; Yano, Mutsumi; Ueno, Toshiyuki

2012-01-01

We recently reported that aldo-keto reductase 1B3-produced prostaglandin (PG) F(2α) suppressed the early phase of adipogenesis. PGE(2) is also known to suppress adipogenesis. In this study, we found that microsomal PGE(2) synthase (PGES)-1 (mPGES-1; PTGES1) acted as the PGES in adipocytes and that PGE(2) and PGF(2α) synergistically suppressed the early phase of adipogenesis. PGE(2) production was detected in preadipocytes and transiently enhanced at 3 h after the initiation of adipogenesis of mouse adipocytic 3T3-L1 cells, followed by a quick decrease; and its production profile was similar to the expression of the cyclooxygenase-2 (PTGS2) gene. When 3T3-L1 cells were transfected with siRNAs for any one of the three major PTGESs, i.e., PTGES1, PTGES2 (mPGES-2), and PTGES3 (cytosolic PGES), only PTGES1 siRNA suppressed PGE(2) production and enhanced the expression of adipogenic genes. AE1-329, a PTGER4 (EP4) receptor agonist, increased the expression of the Ptgs2 gene with a peak at 1 h after the initiation of adipogenesis. PGE(2)-mediated enhancement of the PTGS2 expression was suppressed by the co-treatment with L-161982, a PTGER4 receptor antagonist. Moreover, AE1-329 enhanced the expression of the Ptgs2 gene by binding of the cyclic AMP response element (CRE)-binding protein to the CRE of the Ptgs2 promoter; and its binding was suppressed by co-treatment with L-161982, which was demonstrated by promoter luciferase and chromatin immunoprecipitation assays. Furthermore, when 3T3-L1 cells were caused to differentiate into adipocytes in medium containing both PGE(2) and PGF(2α), the expression of the adipogenic genes and the intracellular triglyceride level were decreased to a greater extent than in medium containing either of them, revealing that PGE(2) and PGF(2α) independently suppressed adipogenesis. These results indicate that PGE(2) was synthesized by PTGES1 in adipocytes and synergistically suppressed the early phase of adipogenesis of 3T3-L1 cells in cooperation with PGF(2α) through receptor-mediated activation of PTGS2 expression.

Synergistic Suppression of Early Phase of Adipogenesis by Microsomal PGE Synthase-1 (PTGES1)-Produced PGE2 and Aldo-Keto Reductase 1B3-Produced PGF2α

PubMed Central

Fujimori, Ko; Yano, Mutsumi; Ueno, Toshiyuki

2012-01-01

We recently reported that aldo-keto reductase 1B3-produced prostaglandin (PG) F2α suppressed the early phase of adipogenesis. PGE2 is also known to suppress adipogenesis. In this study, we found that microsomal PGE2 synthase (PGES)-1 (mPGES-1; PTGES1) acted as the PGES in adipocytes and that PGE2 and PGF2α synergistically suppressed the early phase of adipogenesis. PGE2 production was detected in preadipocytes and transiently enhanced at 3 h after the initiation of adipogenesis of mouse adipocytic 3T3-L1 cells, followed by a quick decrease; and its production profile was similar to the expression of the cyclooxygenase-2 (PTGS2) gene. When 3T3-L1 cells were transfected with siRNAs for any one of the three major PTGESs, i.e., PTGES1, PTGES2 (mPGES-2), and PTGES3 (cytosolic PGES), only PTGES1 siRNA suppressed PGE2 production and enhanced the expression of adipogenic genes. AE1-329, a PTGER4 (EP4) receptor agonist, increased the expression of the Ptgs2 gene with a peak at 1 h after the initiation of adipogenesis. PGE2-mediated enhancement of the PTGS2 expression was suppressed by the co-treatment with L-161982, a PTGER4 receptor antagonist. Moreover, AE1-329 enhanced the expression of the Ptgs2 gene by binding of the cyclic AMP response element (CRE)-binding protein to the CRE of the Ptgs2 promoter; and its binding was suppressed by co-treatment with L-161982, which was demonstrated by promoter luciferase and chromatin immunoprecipitation assays. Furthermore, when 3T3-L1 cells were caused to differentiate into adipocytes in medium containing both PGE2 and PGF2α, the expression of the adipogenic genes and the intracellular triglyceride level were decreased to a greater extent than in medium containing either of them, revealing that PGE2 and PGF2α independently suppressed adipogenesis. These results indicate that PGE2 was synthesized by PTGES1 in adipocytes and synergistically suppressed the early phase of adipogenesis of 3T3-L1 cells in cooperation with PGF2α through receptor-mediated activation of PTGS2 expression. PMID:22970288

Evolution of UCP1 Transcriptional Regulatory Elements Across the Mammalian Phylogeny

PubMed Central

Gaudry, Michael J.; Campbell, Kevin L.

2017-01-01

Uncoupling protein 1 (UCP1) permits non-shivering thermogenesis (NST) when highly expressed in brown adipose tissue (BAT) mitochondria. Exclusive to placental mammals, BAT has commonly been regarded to be advantageous for thermoregulation in hibernators, small-bodied species, and the neonates of larger species. While numerous regulatory control motifs associated with UCP1 transcription have been proposed for murid rodents, it remains unclear whether these are conserved across the eutherian mammal phylogeny and hence essential for UCP1 expression. To address this shortcoming, we conducted a broad comparative survey of putative UCP1 transcriptional regulatory elements in 139 mammals (135 eutherians). We find no evidence for presence of a UCP1 enhancer in monotremes and marsupials, supporting the hypothesis that this control region evolved in a stem eutherian ancestor. We additionally reveal that several putative promoter elements (e.g., CRE-4, CCAAT) identified in murid rodents are not conserved among BAT-expressing eutherians, and together with the putative regulatory region (PRR) and CpG island do not appear to be crucial for UCP1 expression. The specificity and importance of the upTRE, dnTRE, URE1, CRE-2, RARE-2, NBRE, BRE-1, and BRE-2 enhancer elements first described from rats and mice are moreover uncertain as these motifs differ substantially—but generally remain highly conserved—in other BAT-expressing eutherians. Other UCP1 enhancer motifs (CRE-3, PPRE, and RARE-3) as well as the TATA box are also highly conserved in nearly all eutherian lineages with an intact UCP1. While these transcriptional regulatory motifs are generally also maintained in species where this gene is pseudogenized, the loss or degeneration of key basal promoter (e.g., TATA box) and enhancer elements in other UCP1-lacking lineages make it unlikely that the enhancer region is pleiotropic (i.e., co-regulates additional genes). Importantly, differential losses of (or mutations within) putative regulatory elements among the eutherian lineages with an intact UCP1 suggests that the transcriptional control of gene expression is not highly conserved in this mammalian clade. PMID:28979209

SInCRe—structural interactome computational resource for Mycobacterium tuberculosis

PubMed Central

Metri, Rahul; Hariharaputran, Sridhar; Ramakrishnan, Gayatri; Anand, Praveen; Raghavender, Upadhyayula S.; Ochoa-Montaño, Bernardo; Higueruelo, Alicia P.; Sowdhamini, Ramanathan; Chandra, Nagasuma R.; Blundell, Tom L.; Srinivasan, Narayanaswamy

2015-01-01

We have developed an integrated database for Mycobacterium tuberculosis H37Rv (Mtb) that collates information on protein sequences, domain assignments, functional annotation and 3D structural information along with protein–protein and protein–small molecule interactions. SInCRe (Structural Interactome Computational Resource) is developed out of CamBan (Cambridge and Bangalore) collaboration. The motivation for development of this database is to provide an integrated platform to allow easily access and interpretation of data and results obtained by all the groups in CamBan in the field of Mtb informatics. In-house algorithms and databases developed independently by various academic groups in CamBan are used to generate Mtb-specific datasets and are integrated in this database to provide a structural dimension to studies on tuberculosis. The SInCRe database readily provides information on identification of functional domains, genome-scale modelling of structures of Mtb proteins and characterization of the small-molecule binding sites within Mtb. The resource also provides structure-based function annotation, information on small-molecule binders including FDA (Food and Drug Administration)-approved drugs, protein–protein interactions (PPIs) and natural compounds that bind to pathogen proteins potentially and result in weakening or elimination of host–pathogen protein–protein interactions. Together they provide prerequisites for identification of off-target binding. Database URL: http://proline.biochem.iisc.ernet.in/sincre PMID:26130660

Activation of the Nrf2-ARE Pathway in Hepatocytes Protects Against Steatosis in Nutritionally Induced Non-alcoholic Steatohepatitis in Mice

PubMed Central

Lee, Lung-Yi; Köhler, Ulrike A.; Zhang, Li; Roenneburg, Drew; Werner, Sabine; Johnson, Jeffrey A.; Foley, David P.

2014-01-01

Oxidative stress is implicated in the development of non-alcoholic steatohepatitis (NASH). The Nrf2-antioxidant response element pathway protects cells from oxidative stress. Studies have shown that global Nrf2 deficiency hastens the progression of NASH. The purpose of this study was to determine whether long-term hepatocyte-specific activation of Nrf2 mitigates NASH progression. Transgenic mice expressing a constitutively active Nrf2 construct in hepatocytes (AlbCre+/caNrf2+) and littermate controls were generated. These mice were fed standard or methionine-choline-deficient (MCD) diet, a diet used to induce NASH development in rodents. After 28 days of MCD dietary feeding, mice developed significant increases in steatosis, inflammation, oxidative stress, and HSC activation compared with those mice on standard diet. AlbCre+/caNrf2+ animals had significantly decreased serum transaminases and reduced steatosis when compared with the AlbCre+/caNrf2− animals. This significant reduction in steatosis was associated with increased expression of genes involved in triglyceride export (MTTP) and β-oxidation (CPT2). However, there were no differences in the increased oxidative stress, inflammation, and HSC activation from MCD diet administration between the AlbCre+/caNrf2− and AlbCre+/caNrf2+ animals. We conclude that hepatocyte-specific activation of Nrf2-mediated gene expression decreased hepatocellular damage and steatosis in a dietary model of NASH. However, hepatocyte-specific induction of Nrf2-mediated gene expression alone is insufficient to mitigate inflammation, oxidative stress, and HSC activation in this nutritional NASH model. PMID:25294219

Does a Relationship Between Arctic Low Clouds and Sea Ice Matter?

NASA Technical Reports Server (NTRS)

Taylor, Patrick C.

2016-01-01

Arctic low clouds strongly affect the Arctic surface energy budget. Through this impact Arctic low clouds influence important aspects of the Arctic climate system, namely surface and atmospheric temperature, sea ice extent and thickness, and atmospheric circulation. Arctic clouds are in turn influenced by these elements of the Arctic climate system, and these interactions create the potential for Arctic cloud-climate feedbacks. To further our understanding of potential Arctic cloudclimate feedbacks, the goal of this paper is to quantify the influence of atmospheric state on the surface cloud radiative effect (CRE) and its covariation with sea ice concentration (SIC). We build on previous research using instantaneous, active remote sensing satellite footprint data from the NASA A-Train. First, the results indicate significant differences in the surface CRE when stratified by atmospheric state. Second, there is a weak covariation between CRE and SIC for most atmospheric conditions. Third, the results show statistically significant differences in the average surface CRE under different SIC values in fall indicating a 3-5 W m(exp -2) larger LW CRE in 0% versus 100% SIC footprints. Because systematic changes on the order of 1 W m(exp -2) are sufficient to explain the observed long-term reductions in sea ice extent, our results indicate a potentially significant amplifying sea ice-cloud feedback, under certain meteorological conditions, that could delay the fall freeze-up and influence the variability in sea ice extent and volume. Lastly, a small change in the frequency of occurrence of atmosphere states may yield a larger Arctic cloud feedback than any cloud response to sea ice.

The POU homeodomain transcription factor POUM2 and broad complex isoform 2 transcription factor induced by 20-hydroxyecdysone collaboratively regulate vitellogenin gene expression and egg formation in the silkworm Bombyx mori.

PubMed

Lin, Y; Liu, H; Yang, C; Gu, J; Shen, G; Zhang, H; Chen, E; Han, C; Zhang, Y; Xu, Y; Wu, J; Xia, Q

2017-10-01

Vitellogenin (Vg) is a source of nutrition for embryo development. Our previous study showed that the silkworm (Bombyx mori) transcription factor broad complex isoform 2 (BmBrC-Z2) regulates gene expression of the Vg gene (BmVg) by induction with 20-hydroxyecdysone (20E). However, the mechanism by which 20E regulates BmVg expression was not clarified. In this study, cell transfection experiments showed that the BmVg promoter containing the POU homeodomain transcription factor POUM2 (POUM2) and BrC-Z2 cis-response elements (CREs) showed a more significant response to 20E than that harbouring only the BrC-Z2 or POUM2 CRE. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that BmPOUM2 could bind to the POUM2 CRE of the BmVg promoter. Over-expression of BmPOUM2 and BmBrC-Z2 in B. mori embryo-derived cell line (BmE) could enhance the activity of the BmVg promoter carrying both the POUM2 and BrC-Z2 CREs following 20E induction. Quantitative PCR and immunofluorescence histochemistry showed that the expression pattern and tissue localization of BmPOUM2 correspond to those of BmVg. Glutathione S-transferase pull-down and co-immunoprecipitation assays confirmed that BmPOUM2 interacts only with BmBrC-Z2 to regulate BmVg expression. Down-regulation of BmPOUM2 in female silkworm by RNA interference significantly reduced BmVg expression, leading to abnormal egg formation. In summary, these results indicate that BmPOUM2 binds only to BmBrC-Z2 to collaboratively regulate BmVg expression by 20E induction to control vitellogenesis and egg formation in the silkworm. Moreover, these findings suggest that homeodomain protein POUM2 plays a novel role in regulating insect vitellogenesis. © 2017 The Royal Entomological Society.

Problem-Solving Test: Conditional Gene Targeting Using the Cre/loxP Recombination System

ERIC Educational Resources Information Center

Szeberényi, József

2013-01-01

Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain…

Dwarfism in homozygous Agc1CreERT mice is associated with decreased expression of aggrecan.

PubMed

Rashid, Harunur; Chen, Haiyan; Hassan, Quamarul; Javed, Amjad

2017-10-01

Aggrecan (Acan), a large proteoglycan is abundantly expressed in cartilage tissue. Disruption of Acan gene causes dwarfism and perinatal lethality of homozygous mice. Because of sustained expression of Acan in the growth plate and articular cartilage, Agc Cre model has been developed for the regulated ablation of target gene in chondrocytes. In this model, the IRES-CreERT-Neo-pgk transgene is knocked-in the 3'UTR of the Acan gene. We consistently noticed variable weight and size among the Agc Cre littermates, prompting us to examine the cause of this phenotype. Wild-type, Cre-heterozygous (Agc +/Cre ), and Cre-homozygous (Agc Cre/Cre ) littermates were indistinguishable at birth. However, by 1-month, Agc Cre/Cre mice showed a significant reduction in body weight (18-27%) and body length (19-22%). Low body weight and dwarfism was sustained through adulthood and occurred in both genders. Compared with wild-type and Agc +/Cre littermates, long bones and vertebrae were shorter in Agc Cre/Cre mice. Histological analysis of Agc Cre/Cre mice revealed a significant reduction in the length of the growth plate and the thickness of articular cartilage. The amount of proteoglycan deposited in the cartilage of Agc Cre/Cre mice was nearly half of the WT littermates. Analysis of gene expression indicates impaired differentiation of chondrocyte in hyaline cartilage of Agc Cre/Cre mice. Notably, both Acan mRNA and protein was reduced by 50% in Agc Cre/Cre mice. A strong correlation was noted between the level of Acan mRNA and the body length. Importantly, Agc +/Cre mice showed no overt skeletal phenotype. Thus to avoid misinterpretation of data, only the Agc +/Cre mice should be used for conditional deletion of a target gene in the cartilage tissue. © 2017 Wiley Periodicals, Inc.

Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1.

PubMed

Wong, A O; Le Drean, Y; Liu, D; Hu, Z Z; Du, S J; Hew, C L

1996-05-01

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-dependent pathway and that their promoter activity is dependent on the presence of Pit-1

A short region of the promoter of the breast cancer associated PLU-1 gene can regulate transcription in vitro and in vivo.

PubMed

Catteau, Aurélie; Rosewell, Ian; Solomon, Ellen; Taylor-Papadimitriou, Joyce

2004-07-01

The recently cloned gene PLU-1 shows restricted expression in adult tissues, with high expression being found in testis, and transiently in the pregnant mammary gland. However, both the gene and the protein product are specifically up-regulated in breast cancer. To investigate the control of expression of the PLU-1 gene, we have cloned and functionally characterised the 5' flanking region of the gene, which was found to contain another putative gene. Two transcription start sites of the PLU-1 gene were mapped by 5' RACE. A short proximal 249 bp region was defined using reporter gene assays, which encompasses the major transcription start site and exhibits a strong constitutive promoter activity in all cell lines tested. However, regions upstream of this sequence repress transcription more effectively in a non-malignant breast cell line as compared to breast cancer cell lines. The 249 bp region is GC-rich and includes consensus Sp1 sites, GC boxes, cAMP-responsive element (CRE) and other putative cis-elements. Mutational analysis showed that two intact conserved Sp1 binding sites (shown here to bind Sp1 and/or Sp3) are critical for constitutive promoter activity, while a negative role for a neighbouring GC box is indicated. The sequence of the core promoter is highly conserved in the mouse and Plu-1 expression in the mouse embryo has been documented. Using transgenesis, we therefore examined the ability of the 249 bp fragment to control expression of a reporter gene during embryogenesis. We found that not only is the core promoter sufficient to activate transcription in vivo, but that the expression of the reporter gene coincides both temporally and spatially with regions where endogenous Plu-1 is highly expressed. This suggests that tissue specific controlling elements are found within the short fragment and are functional in the embryonic environment.

Cardiomyocyte A Disintegrin And Metalloproteinase 17 (ADAM17) Is Essential in Post-Myocardial Infarction Repair by Regulating Angiogenesis.

PubMed

Fan, Dong; Takawale, Abhijit; Shen, Mengcheng; Wang, Wang; Wang, Xiuhua; Basu, Ratnadeep; Oudit, Gavin Y; Kassiri, Zamaneh

2015-09-01

A disintegrin and metalloproteinase 17 (ADAM17) is a membrane-bound enzyme that mediates shedding of many membrane-bound molecules, thereby regulating multiple cellular responses. We investigated the role of cardiomyocyte ADAM17 in myocardial infarction (MI). Cardiomyocyte-specific ADAM17 knockdown mice (ADAM17(flox/flox)/α-MHC-Cre; f/f/Cre) and parallel controls (ADAM17(flox/flox); f/f) were subjected to MI by ligation of the left anterior descending artery. Post MI, f/f/Cre mice showed compromised survival, higher rates of cardiac rupture, more severe left ventricular dilation, and suppressed ejection fraction compared with parallel f/f-MI mice. Ex vivo ischemic injury (isolated hearts) resulted in comparable recovery in both genotypes. Myocardial vascular density (fluorescent-labeled lectin perfusion and CD31 immunofluorescence staining) was significantly lower in the infarct areas of f/f/Cre-MI compared with f/f-MI mice. Activation of vascular endothelial growth factor receptor 2 (VEGFR2), its mRNA, and total protein levels were reduced in infarcted myocardium in ADAM17 knockdown mice. Transcriptional regulation of VEGFR2 by ADAM17 was confirmed in cocultured cardiomyocyte-fibroblast as ischemia-induced VEGFR2 expression was blocked by ADAM17-siRNA. Meanwhile, ADAM17-siRNA did not alter VEGFA bioavailability in the conditioned media. ADAM17 knockdown mice (f/f/Cre-MI) exhibited reduced nuclear factor-κB activation (DNA binding) in the infarcted myocardium, which could underlie the suppressed VEGFR2 expression in these hearts. Post MI, inflammatory response was not altered by ADAM17 downregulation. This study highlights the key role of cardiomyocyte ADAM17 in post-MI recovery by regulating VEGFR2 transcription and angiogenesis, thereby limiting left ventricular dilation and dysfunction. Therefore, ADAM17 upregulation, within the physiological range, could provide protective effects in ischemic cardiomyopathy. © 2015 American Heart Association, Inc.

Investigation of the Diversity of Antibiotic Resistance Genes and Mobile Genetic Elements in Salmonella associated with U.S. Food Animals

USDA-ARS?s Scientific Manuscript database

With the emergence of antibiotic resistance (AR), multidrug resistance (MDR), and carbapenem resistant Enterobacteriaceae (CRE), the specter of widespread untreatable bacterial infections threatens human and animal health. The ability of these emerging resistances to transfer between bacteria on mob...

Investigation of the diversity of antibiotic resistance genes and mobile genetic elements in Salmonella associated with U.S. food animals

USDA-ARS?s Scientific Manuscript database

With the emergence of antibiotic resistance (AR), multidrug resistance (MDR), and carbapenem resistant Enterobacteriaceae (CRE), the specter of widespread untreatable bacterial infections threatens human and animal health. The ability of these emerging resistances to transfer between bacteria on mob...

Improved specificity of hippocampal memory trace labeling.

PubMed

Cazzulino, Alejandro S; Martinez, Randy; Tomm, Nicole K; Denny, Christine A

2016-06-01

Recent studies have focused on the identification and manipulation of memory traces in rodent models. The two main mouse models utilized are either a CreER(T2) /loxP tamoxifen (TAM)- or a tetracycline transactivator/tetracycline-response element doxycycline-inducible system. These systems, however, could be improved to label a more specific population of activated neurons corresponding to behavior. Here, we sought to identify an improved selective estrogen receptor (ER) modulator (SERM) in which we could label an individual memory trace in ArcCreER(T2) mice. We found that 4-hydroxytamoxifen (4-OHT) is a selective SERM in the ArcCreER(T2) × Rosa26-CAG-stop(flox) -channelrhodospin (ChR2)-enhanced yellow fluorescent protein (eYFP) mice. The half-life of 4-OHT is shorter than TAM, allowing for more specificity of memory trace labeling. Furthermore, 4-OHT allowed for context-specific labeling in the dentate gyrus and CA3. In summary, we believe that 4-OHT improves the specificity of memory trace labeling and will allow for refined memory trace studies in the future. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

The CreB deubiquitinating enzyme does not directly target the CreA repressor protein in Aspergillus nidulans.

PubMed

Alam, Md Ashiqul; Kamlangdee, Niyom; Kelly, Joan M

2017-08-01

Ubiquitination/deubiquitination pathways are now recognized as key components of gene regulatory mechanisms in eukaryotes. The major transcriptional repressor for carbon catabolite repression in Aspergillus nidulans is CreA, and mutational analysis led to the suggestion that a regulatory ubiquitination/deubiquitination pathway is involved. A key unanswered question is if and how this pathway, comprising CreB (deubiquitinating enzyme) and HulA (ubiquitin ligase) and other proteins, is involved in the regulatory mechanism. Previously, missense alleles of creA and creB were analysed for genetic interactions, and here we extended this to complete loss-of-function alleles of creA and creB, and compared morphological and biochemical phenotypes, which confirmed genetic interaction between the genes. We investigated whether CreA, or a protein in a complex with it, is a direct target of the CreB deubiquitination enzyme, using co-purifications of CreA and CreB, first using strains that overexpress the proteins and then using strains that express the proteins from their native promoters. The Phos-tag system was used to show that CreA is a phosphorylated protein, but no ubiquitination was detected using anti-ubiquitin antibodies and Western analysis. These findings were confirmed using mass spectrometry, which confirmed that CreA was differentially phosphorylated but not ubiquitinated. Thus, CreA is not a direct target of CreB, and nor are proteins that form part of a stable complex with CreA a target of CreB. These results open up new questions regarding the molecular mechanism of CreA repressing activity, and how the ubiquitination pathway involving CreB interacts with this regulatory network.

Amphetamine manipulates monoamine oxidase-A level and behavior using theranostic aptamers of transcription factors AP-1/NF-kB.

PubMed

Liu, Christina H; Ren, Jiaqian; Liu, Philip K

2016-02-03

Monoamine oxidase (MAO) enzymes play a critical role in controlling the catabolism of monoamine neurotransmitters and biogenic trace amines and behavior in humans. However, the mechanisms that regulate MAO are unclear. Several transcription factor proteins are proposed to modulate the transcription of MAO gene, but evidence supporting these hypotheses is controversial. We aimed to investigate the mechanism of gene transcription regulator proteins on amphetamine-induced behavior. We applied aptamers containing a DNA binding sequence, as well as a random sequence (without target) to study the modulation of amphetamine-induced MAO levels and hyperactivity in living mice. We pretreated in adult male C57black6 mice (Taconic Farm, Germantown, NY) (n ≥ 3 litters at a time), 2 to 3 months of age (23 ± 2 gm body weight) with double-stranded (ds) DNA aptamers with sequence specific to activator protein-1 (5ECdsAP1), nuclear factor-kappa beta (5ECdsNF-kB), special protein-1 (5ECdsSP-1) or cyclicAMP responsive element binding (5ECdsCreB) protein binding regions, 5ECdsRan [a random sequence without target], single-stranded AP-1 (5ECssAP-1) (8 nmol DNA per kg) or saline (5 μl, intracerebroventricular [icv] injection) control before amphetamine administration (4 mg/kg, i.p.). We then measured and analyzed locomotor activities and the level of MAO-A and MAO-B activity. In the pathological condition of amphetamine exposure, we showed here that pretreatment with 5ECdsAP1 and 5ECdsNF-kB reversed the decrease of MAO-A activity (p < 0.05, t test), but not activity of the B isomer (MAO-B), in the ventral tegmental area (VTA) and substantia nigra (SN) of C57black6 mice. The change in MAO-A level coincided with a reversed amphetamine-induced restless behavior of mice. Pretreatments with saline, 5ECdsCreB, 5ECdsSP-1, 5ECdsRan or 5ECssAP-1 had no effect. Our data lead us to conclude that elevation of AP-1 or NF-kB indirectly decreases MAO-A protein levels which, in turn, diminishes MAO-A ability in the VTA of the mesolimbic dopaminergic pathway that has been implicated in cells under stress especially in the SN and VTA. This study has implications for design for the treatment of drug exposure and perhaps Parkinson's dementia.

Whole Genome Sequencing and Plasmid Genomics of Antimicrobial Resistance – Salmonella’s mobile genetic elements and the antimicrobial resistance genes they carry

USDA-ARS?s Scientific Manuscript database

With the emergence of antibiotic resistance (AR), multidrug resistance (MDR), and carbapenem resistant Enterobacteriaceae (CRE), the specter of widespread untreatable bacterial infections threatens human and animal health. The ability of these emerging resistances to transfer between bacteria on mob...

Cre/lox-recombinase-mediated cassette exchange for reversible site-specific genomic targeting of the disease vector, Aedes aegypti

USDA-ARS?s Scientific Manuscript database

Site-specific genome modification is an important tool for mosquito functional genomics studies that help to uncover gene functions, identify gene regulatory elements, and perform comparative gene expression studies, all of which contribute to a better understanding of mosquito biology and are thus ...

Catabolite repression in Lactobacillus casei ATCC 393 is mediated by CcpA.

PubMed Central

Monedero, V; Gosalbes, M J; Pérez-Martínez, G

1997-01-01

The chromosomal ccpA gene from Lactobacillus casei ATCC 393 has been cloned and sequenced. It encodes the CcpA protein, a central catabolite regulator belonging to the LacI-GalR family of bacterial repressors, and shows 54% identity with CcpA proteins from Bacillus subtilis and Bacillus megaterium. The L. casei ccpA gene was able to complement a B. subtilis ccpA mutant. An L. casei ccpA mutant showed increased doubling times and a relief of the catabolite repression of some enzymatic activities, such as N-acetylglucosaminidase and phospho-beta-galactosidase. Detailed analysis of CcpA activity was performed by using the promoter region of the L. casei chromosomal lacTEGF operon which is subject to catabolite repression and contains a catabolite responsive element (cre) consensus sequence. Deletion of this cre site or the presence of the ccpA mutation abolished the catabolite repression of a lacp::gusA fusion. These data support the role of CcpA as a common regulatory element mediating catabolite repression in low-GC-content gram-positive bacteria. PMID:9352913

Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2018-02-01

In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Biomechanical investigation into the role of the periodontal ligament in optimising orthodontic force: a finite element case study.

PubMed

Liao, Zhipeng; Chen, Junning; Li, Wei; Darendeliler, M Ali; Swain, Michael; Li, Qing

2016-06-01

This paper aimed to precisely locate centres of resistance (CRe) of maxillary teeth and investigate optimal orthodontic force by identifying the effective zones of orthodontic tooth movement (OTM) from hydrostatic stress thresholds in the periodontal ligament (PDL). We applied distally-directed tipping and bodily forces ranging from 0.075 N to 3 N (7.5 g to 300 g) onto human maxillary teeth. The hydrostatic stress was quantified from nonlinear finite element analysis (FEA) and compared with normal capillary and systolic blood pressure for driving the tissue remodelling. Two biomechanical stimuli featuring localised and volume-averaged hydrostatic stresses were introduced to describe OTM. Locations of CRe were determined through iterative FEA simulation. Accurate locations of CRes of teeth and ranges of optimal orthodontic forces were obtained. By comparing with clinical results in literature, the volume average of hydrostatic stress in PDL was proved to describe the process of OTM more indicatively. The optimal orthodontic forces obtained from the in-silico modelling study echoed with the clinical results in vivo. A universal moment to force (M/F) ratio is not recommended due to the variation in patients and loading points. Accurate computational determination of CRe location can be applied in practice to facilitate orthodontic treatment. Global measurement of hydrostatic pressure in the PDL better characterised OTM, implying that OTM occurs only when the majority of PDL volume is critically stressed. The FEA results provide new insights into relevant orthodontic biomechanics and help establish optimal orthodontic force for a specific patient. Copyright © 2016 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, G.; Thom, R.; Whiting, A.

Habitat restoration in the Columbia River estuary (CRE) is an important off-site mitigation action in the 2000 Biological Opinion (BiOp), an operation of the Federal Columbia River Power System. The CRE, defined as the tidally influenced stretch of river from the mouth to Bonneville Dam 146 miles upstream, is part of the migration pathway for anadromous fish in the Columbia Basin, including salmon listed under the Endangered Species Act (ESA). Salmon in various stages of life, from fry to adults, use tidal channels and wetlands in the CRE to feed, find refuge from predators, and transition physiologically from freshwater tomore » saltwater. Over the last 100 years, however, the area of some wetland habitats has decreased by as much as 70% because of dike and levee building, flow regulation, and other activities. In response to the decline in available habitat, the BiOp's Reasonable and Prudent Alternative (RPA) included mandates to 'develop a plan addressing the habitat needs of juvenile salmon and steelhead in the estuary' (RPA Action 159) and 'develop and implement an estuary restoration program with a goal of protecting and enhancing 10,000 acres of tidal wetlands and other key habitats' (RPA Action 160). To meet Action 159 and support Action 160, this document develops a science-based approach designed to improve ecosystem functions through habitat restoration activities in the CRE. The CRE habitat restoration program's goal and principles focus on habitat restoration projects in an ecosystem context. Since restoration of an entire ecosystem is not generally practical, individual habitat restoration projects have the greatest likelihood of success when they are implemented with an ecosystem perspective. The program's goal is: Implementation of well-coordinated, scientifically sound projects designed to enhance, protect, conserve, restore, and create 10,000 acres of tidal wetlands and other key habitats to aid rebuilding of ESA-listed salmon populations and native species using the CRE. The program's underlying principles are: (1) projects are founded on the best available ecological restoration science, implemented in an ecosystem context, and developed with the intent to restore relevant ecological processes; (2) projects incorporate adaptive management practices with testable hypotheses to track ecological responses to a given restoration effort; and (3) projects are implemented in a coordinated, open process and scientific results from monitoring and evaluation are communicated widely and readily accessible. With this goal and these principles in mind, we developed an approach for CRE habitat restoration. The intent of this document is to provide a scientific basis and implementation guidelines for a habitat restoration program designed to improve ecosystem functions and enhance juvenile salmonid survival in the CRE. The stepwise approach to CRE habitat restoration outlined is somewhat general and broad because the available scientific information is incomplete, e.g., juvenile salmon usage of various CRE wetland habitats. As new data become available, a more specific, detailed plan than was possible here can be produced as an outgrowth of this document. In conclusion, this document provides a scientific basis and implementation guidelines for a habitat restoration program designed to improve ecosystem functions and enhance juvenile salmonid survival in the CRE. As more experience is gained with CRE habitat restoration and scientific uncertainties are resolved, this document should be used as a basis for a detailed habitat restoration plan that specifically addresses (1) which habitat types offer the greatest ecological benefit to salmon, (2) the location of potential sites that if restored would likely provide these habitat types, and (3) how and when the restoration work should be done. This document supports the use of adaptive management so that all elements of salmonid habitat restoration actions in the CRE are under continual evaluation and revision at both the project and program levels. Lessons learned from current and proposed habitat restoration projects need to be applied to all future work, such as the Estuary Partnership's habitat restoration program and the U.S. Army Corps of Engineers General Investigation Study for the CRE, to ensure the most effective use of resources and the best possible long term environment for salmonid growth and survival in the CRE.« less

Post-surgical mediastinitis due to carbapenem-resistant Enterobacteriaceae: Clinical, epidemiological and survival characteristics.

PubMed

Abboud, C S; Monteiro, J; Stryjewski, M E; Zandonadi, E C; Barbosa, V; Dantas, D; Sousa, E E; Fonseca, M J; Jacobs, D M; Pignatari, A C; Kiffer, C; Rao, G G

2016-05-01

Invasive infections due to carbapenem-resistant Enterobacteriaceae (CRE), including polymyxin-resistant (PR-CRE) strains, are being increasingly reported. However, there is a lack of clinical data for several life-threatening infections. Here we describe a cohort of patients with post-surgical mediastinitis due to CRE, including PR-CRE. This study was a retrospective cohort design at a single cardiology centre. Patients with mediastinitis due to CRE were identified and were investigated for clinically relevant variables. Infecting isolates were studied using molecular techniques. Patients infected with polymyxin-susceptible CRE (PS-CRE) strains were compared with those infected with PR-CRE strains. In total, 33 patients with CRE mediastinitis were studied, including 15 patients (45%) with PR-CRE. The majority (61%) were previously colonised. All infecting isolates carried blaKPC genes. Baseline characteristics of patients with PR-CRE mediastinitis were comparable with those with PS-CRE mediastinitis. Of the patients studied, 70% received at least one agent considered active in vitro and most patients received at least three concomitant antibiotics. Carbapenem plus polymyxin B was the most common antibiotic combination (73%). Over 90% of patients underwent surgical debridement. Overall, in-hospital mortality was 33% and tended to be higher in patients infected with PR-CRE (17% vs. 53%; P=0.06). In conclusion, mediastinitis due to CRE, including PR-CRE, can become a significant challenge in centres with CRE and a high cardiac surgery volume. Despite complex antibiotic treatments and aggressive surgical procedures, these patients have a high mortality, particularly those infected with PR-CRE. Copyright © 2016. Published by Elsevier B.V.

A Study of Plazomicin Compared With Colistin in Patients With Infection Due to Carbapenem-Resistant Enterobacteriaceae (CRE)

ClinicalTrials.gov

2016-10-03

Bloodstream Infections (BSI) Due to CRE; Hospital-Acquired Bacterial Pneumonia (HABP) Due to CRE; Ventilator-Associated Bacterial Pneumonia (VABP) Due to CRE; Complicated Urinary Tract Infection (cUTI) Due to CRE; Acute Pyelonephritis (AP) Due to CRE

Characteristics of sensory neuronal groups in CGRP-cre-ER reporter mice: Comparison to Nav1.8-cre, TRPV1-cre and TRPV1-GFP mouse lines.

PubMed

Patil, Mayur J; Hovhannisyan, Anahit H; Akopian, Armen N

2018-01-01

Peptidergic sensory neurons play a critical role in nociceptive pathways. To precisely define the function and plasticity of sensory neurons in detail, new tools such as transgenic mouse models are needed. We employed electrophysiology and immunohistochemistry to characterize in detail dorsal root ganglion (DRG) neurons expressing an inducible CGRPcre-ER (CGRP-cre+); and compared them to DRG neurons expressing Nav1.8cre (Nav1.8-cre+), TRPV1cre (TRPV1-cre+) and TRPV1-GFP (V1-GFP+). Tamoxifen effectively induced CGRPcre-ER production in DRG. ≈87% of CGRPcre-ER-expressing neurons were co-labeled CGRP antibody. Three small and two medium-large-sized (5HT3a+/NPY2R- and NPY2R+) neuronal groups with unique electrophysiological profiles were CGRP-cre+. Nav1.8-cre+ neurons were detected in all CGRP-cre+ groups, as well as in 5 additional neuronal groups: MrgprD+/TRPA1-, MrgprD+/TRPA1+, TRPV1+/CGRP-, vGLUT3+ and ≈30% of trkC+ neurons. Differences between TRPV1cre and Nav1.8cre reporters were that unlike TRPV1-cre+, Nav1.8-cre+ expression was detected in non-nociceptive vGLUT3+ and trkC+ populations. Many TRPV1-cre+ neurons did not respond to capsaicin. In contrast, V1-GFP+ neurons were in 4 groups, each of which was capsaicin-sensitive. Finally, none of the analyzed reporter lines showed cre-recombination in trkB+, calbindin+, 70% of trkC+ or parvalbumin+ neurons, which together encompassed ≈20% of Nav1.8-cre- DRG neurons. The data presented here increases our knowledge of peptidergic sensory neuron characteristics, while showing the efficiency and specificity manipulation of peptidergic neurons by the CGRPcre-ER reporter. We also demonstrate that manipulation of all C- and A-nociceptors is better achieved with TRPV1-cre reporter. Finally, the described approach for detailed characterization of sensory neuronal groups can be applied to a variety of reporter mice.

Infection control implications of heterogeneous resistance mechanisms in carbapenem-resistant Enterobacteriaceae (CRE).

PubMed

Goodman, K E; Simner, P J; Tamma, P D; Milstone, A M

2016-01-01

The Centers for Disease Control and Prevention (CDC) defines carbapenem-resistant Enterobacteriaceae (CRE) based upon a phenotypic demonstration of carbapenem resistance. However, considerable heterogeneity exists within this definitional umbrella. CRE may mechanistically differ by whether they do or do not produce carbapenemases. Moreover, patients can acquire CRE through multiple pathways: endogenously through antibiotic selective pressure on intestinal microbiota, exogenously through horizontal transmission or through a combination of these factors. Some evidence suggests that non-carbapenemase-producing CRE may be more frequently acquired by antibiotic exposure and carbapenemase-producing CRE via horizontal transmission, but definitive data are lacking. This review examines types of CRE resistance mechanisms, antibiotic exposure and horizontal transmission pathways of CRE acquisition, and the implications of these heterogeneities to the development of evidence-based CRE healthcare epidemiology policies. In our Expert Commentary & Five-Year View, we outline specific nosocomial CRE knowledge gaps and potential methodological approaches for their resolution.

Cerebellar associative sensory learning defects in five mouse autism models

PubMed Central

Kloth, Alexander D; Badura, Aleksandra; Li, Amy; Cherskov, Adriana; Connolly, Sara G; Giovannucci, Andrea; Bangash, M Ali; Grasselli, Giorgio; Peñagarikano, Olga; Piochon, Claire; Tsai, Peter T; Geschwind, Daniel H; Hansel, Christian; Sahin, Mustafa; Takumi, Toru; Worley, Paul F; Wang, Samuel S-H

2015-01-01

Sensory integration difficulties have been reported in autism, but their underlying brain-circuit mechanisms are underexplored. Using five autism-related mouse models, Shank3+/ΔC, Mecp2R308/Y, Cntnap2−/−, L7-Tsc1 (L7/Pcp2Cre::Tsc1flox/+), and patDp(15q11-13)/+, we report specific perturbations in delay eyeblink conditioning, a form of associative sensory learning requiring cerebellar plasticity. By distinguishing perturbations in the probability and characteristics of learned responses, we found that probability was reduced in Cntnap2−/−, patDp(15q11-13)/+, and L7/Pcp2Cre::Tsc1flox/+, which are associated with Purkinje-cell/deep-nuclear gene expression, along with Shank3+/ΔC. Amplitudes were smaller in L7/Pcp2Cre::Tsc1flox/+ as well as Shank3+/ΔC and Mecp2R308/Y, which are associated with granule cell pathway expression. Shank3+/ΔC and Mecp2R308/Y also showed aberrant response timing and reduced Purkinje-cell dendritic spine density. Overall, our observations are potentially accounted for by defects in instructed learning in the olivocerebellar loop and response representation in the granule cell pathway. Our findings indicate that defects in associative temporal binding of sensory events are widespread in autism mouse models. DOI: http://dx.doi.org/10.7554/eLife.06085.001 PMID:26158416

Characterization of the human UDP-galactose:ceramide galactosyltransferase gene promoter.

PubMed

Tencomnao, T; Yu, R K; Kapitonov, D

2001-02-16

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthesis of galactocerebroside, the most abundant glycosphingolipid in the myelin sheath. An 8 kb fragment upstream from the transcription initiation site of CGT gene was isolated from a human genomic DNA library. Primer extension analysis revealed a single transcription initiation site 329 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start site; however, this region contains a high GC content and multiple putative regulatory elements. To investigate the transcriptional regulation of CGT, a series of 5' deletion constructs of the 5'-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in the human oligodendroglioma (HOG) and human neuroblastoma (LAN-5) cell lines, we found that the CGT promoter functions in a cell type-specific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at -292/-256 which contains the potential binding sites for known transcription factors (TFs) such as Ets and SP1 (GC box), a distal region at -747/-688 comprising a number of binding sites such as the ERE half-site, NF1-like, TGGCA-BP, and CRE, and a third positive cis-acting region distally localized at -1325/-1083 consisting of binding sites for TFs such as nitrogen regulatory, TCF-1, TGGCA-BP, NF-IL6, CF1, bHLH, NF1-like, GATA, and gamma-IRE. A negative cis-acting domain localized in a far distal region at -1594/-1326 was also identified. Our results suggest the presence of both positive and negative cis-regulatory regions essential for the cell-specific expression in the TATA-less promoter of the human CGT gene.

Impaired Embryonic Development in Mice Overexpressing the RNA-Binding Protein TIAR

PubMed Central

Kharraz, Yacine; Salmand, Pierre-Adrien; Camus, Anne; Auriol, Jacques; Gueydan, Cyril; Kruys, Véronique; Morello, Dominique

2010-01-01

Background TIA-1-related (TIAR) protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs). Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. Methodology/Principal Findings To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR) allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2α that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. Conclusions/Significance This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming. PMID:20596534

Strain activation of bovine aortic smooth muscle cell proliferation and alignment: study of strain dependency and the role of protein kinase A and C signaling pathways

NASA Technical Reports Server (NTRS)

Mills, I.; Cohen, C. R.; Kamal, K.; Li, G.; Shin, T.; Du, W.; Sumpio, B. E.

1997-01-01

Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0-7% center; 7-24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7-24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0-7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 microM Rp-cAMP) or PKC (with 5-20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses.

Heterotrimeric G Stimulatory Protein α Subunit Is Required for Intestinal Smooth Muscle Contraction in Mice.

PubMed

Qin, Xiaoteng; Liu, Shangming; Lu, Qiulun; Zhang, Meng; Jiang, Xiuxin; Hu, Sanyuan; Li, Jingxin; Zhang, Cheng; Gao, Jiangang; Zhu, Min-Sheng; Feil, Robert; Li, Huashun; Chen, Min; Weinstein, Lee S; Zhang, Yun; Zhang, Wencheng

2017-04-01

The α subunit of the heterotrimeric G stimulatory protein (Gsa), encoded by the guanine nucleotide binding protein, α-stimulating gene (Gnas, in mice), is expressed ubiquitously and mediates receptor-stimulated production of cyclic adenosine monophosphate and activation of the protein kinase A signaling pathway. We investigated the roles of Gsa in vivo in smooth muscle cells of mice. We performed studies of mice with Cre recombinase-mediated disruption of Gnas in smooth muscle cells (Gsa SMKO and SM22-CreER T2 , induced in adult mice by tamoxifen). Intestinal tissues were collected for histologic, biochemical, molecular, cell biology, and physiology analyses. Intestinal function was assessed in mice using the whole-gut transit time test. We compared gene expression patterns of intestinal smooth muscle from mice with vs without disruption of Gnas. Biopsy specimens from ileum of patients with chronic intestinal pseudo-obstruction and age-matched control biopsies were analyzed by immunohistochemistry. Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene (Foxf1) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo-obstruction, compared with tissues from controls. Gsa is required for intestinal smooth muscle contraction in mice, and its levels are reduced in ileum biopsies of patients with chronic intestinal pseudo-obstruction. Mice with disruption of Gnas might be used to study human chronic intestinal pseudo-obstruction. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Comment on "Cosmic-ray-driven reaction and greenhouse effect of halogenated molecules: Culprits for atmospheric ozone depletion and global climate change"

NASA Astrophysics Data System (ADS)

Müller, Rolf; Grooß, Jens-Uwe

2014-04-01

Lu's "cosmic-ray-driven electron-induced reaction (CRE) theory" is based on the assumption that the CRE reaction of halogenated molecules (e.g., chlorofluorocarbons (CFCs), HCl, ClONO2) adsorbed or trapped in polar stratospheric clouds in the winter polar stratosphere is the key step in forming photoactive halogen species that are the cause of the springtime ozone hole. This theory has been extended to a warming theory of halogenated molecules for climate change. In this comment, we discuss the chemical and physical foundations of these theories and the conclusions derived from the theories. First, it is unclear whether the loss rates of halogenated molecules induced by dissociative electron attachment (DEA) observed in the laboratory can also be interpreted as atmospheric loss rates, but even if this were the case, the impact of DEA-induced reactions on polar chlorine activation and ozone loss in the stratosphere is limited. Second, we falsify several conclusions that are reported on the basis of the CRE theory: There is no polar ozone loss in darkness, there is no apparent 11-year periodicity in polar total ozone measurements, the age of air in the polar lower stratosphere is much older than 1-2 years, and the reported detection of a pronounced recovery (by about 20-25%) in Antarctic total ozone measurements by the year 2010 is in error. There are also conclusions about the future development of sea ice and global sea level which are fundamentally flawed because Archimedes' principle is neglected. Many elements of the CRE theory are based solely on correlations between certain datasets which are no substitute for providing physical and chemical mechanisms causing a particular behavior noticeable in observations. In summary, the CRE theory cannot be considered as an independent, alternative mechanism for polar stratospheric ozone loss and the conclusions on recent and future surface temperature and global sea level change do not have a physical basis.

Carbapenem-resistant Enterobacteriaceae colonization (CRE) and subsequent risk of infection and 90-day mortality in critically ill patients, an observational study.

PubMed

McConville, Thomas Howe; Sullivan, Sean Berger; Gomez-Simmonds, Angela; Whittier, Susan; Uhlemann, Anne-Catrin

2017-01-01

Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as an urgent public health threat. Intestinal colonization with CRE has been identified as a risk factor for the development of systemic CRE infection, but has not been compared to colonization with third and/or fourth generation cephalosporin-resistant (Ceph-R) Enterobacteriaceae. Moreover, the risk conferred by colonization on adverse outcomes is less clear, particularly in critically ill patients admitted to the intensive care unit (ICU). We carried out a cohort study of consecutive adult patients screened for rectal colonization with CRE or Ceph-R upon ICU entry between April and July 2013. We identified clinical variables and assessed the relationship between CRE or Ceph-R colonization and subsequent systemic CRE infection within 30 days (primary outcome) and all-cause mortality within 90 days (secondary outcome). Among 338 ICU patients, 94 (28%) were colonized with either Ceph-R or CRE. 26 patients developed CRE infection within 30 days of swab collection; 47% (N = 17/36) of CRE-colonized and 3% (N = 2/58) of Ceph-R colonized patients. 36% (N = 13/36) of CRE-colonized patients died within 90 days compared to 31% (N = 18/58) of Ceph-R-colonized and 15% (N = 37/244) of non-colonized patients. In a multivariable analysis, CRE colonization independently predicted development of a systemic CRE infection at 30 days (aOR 10.8, 95% CI2.8-41.9, p = 0.0006); Ceph-R colonization did not (aOR 0.5, 95% CI0.1-3.3, p = 0.5). CRE colonization was associated with increased 90-day mortality in a univariable analysis (p-value 0.001), in a multivariable model, previous hospitalization and medical ICU admission were independent predictors of 90-day mortality whereas CRE colonization approached significance (aOR 2.3, 95% CI1.0-5.3, p = 0.056). Our study highlights the increased risk of CRE infection and mortality in patients with CRE colonization at the time of ICU admission. Future studies are needed to assess how CRE colonization can guide empiric antibiotic choices and to develop novel decolonization strategies.

A critical developmental role for tgfbr2 in myogenic cell lineages is revealed in mice expressing SM22-Cre, not SMMHC-Cre.

PubMed

Frutkin, Andrew D; Shi, Haikun; Otsuka, Goro; Levéen, Per; Karlsson, Stefan; Dichek, David A

2006-10-01

Smooth muscle cell (SMC)-specific deletion of transforming growth factor beta (TGF-beta) signaling would help elucidate the mechanisms through which TGF-beta signaling contributes to vascular development and disease. We attempted to generate mice with SMC-specific deletion of TGF-beta signaling by mating mice with a conditional ("floxed") allele for the type II TGF-beta receptor (tgfbr2flox) to mice with SMC-targeted expression of Cre recombinase. We bred male mice transgenic for smooth muscle myosin heavy chain (SMMHC)-Cre with females carrying tgfbr2flox. Surprisingly, SMMHC-Cre mice recombined tgfbr2flox at low levels in SMC and at high levels in the testis. Recombination of tgfbr2flox in testis correlated with high-level expression of SMMHC-Cre in testis and germline transmission of tgfbr2null. In contrast, mice expressing Cre from a SM22alpha promoter (SM22-Cre) efficiently recombined tgfbr2flox in vascular and visceral SMC and the heart, but not in testis. Use of the R26R reporter allele confirmed that Cre-mediated recombination in vascular SMC was inefficient for SMMHC-Cre mice and highly efficient for SM22-Cre mice. Breedings that introduced the SM22-Cre allele into tgfbr2flox/flox zygotes in order to generate adult mice that are hemizygous for SM22-Cre and homozygous for tgfbr2flox- and would have conversion of tgfbr2flox/flox to tgfbr2null/null in SMC-produced no live SM22-Cre : tgfbr2flox/flox pups (P<0.001). We conclude: (1) "SMC-targeted" Cre lines vary significantly in specificity and efficiency of Cre expression; (2) TGF-beta signaling in the subset of cells that express SM22alpha is required for normal development; (3) generation of adult mice with absent TGF-beta signaling in SMC remains a challenge.

Flp and Cre expressed from Flp–2A–Cre and Flp–IRES–Cre transcription units mediate the highest level of dual recombinase-mediated cassette exchange

PubMed Central

Anderson, Rachelle P.; Voziyanova, Eugenia; Voziyanov, Yuri

2012-01-01

Recombinase-mediated cassette exchange (RMCE) is a powerful tool for unidirectional integration of DNA fragments of interest into a pre-determined genome locale. In this report, we examined how the efficiency of dual RMCE catalyzed by Flp and Cre depends on the nature of transcription units that express the recombinases. The following recombinase transcription units were analyzed: (i) Flp and Cre genes expressed as individual transcription units located on different vectors, (ii) Flp and Cre genes expressed as individual transcription units located on the same vector, (iii) Flp and Cre genes expressed from a single promoter and separated by internal ribosome entry sequence and (iv) Flp and Cre coding sequences separated by the 2A peptide and expressed as a single gene. We found that the highest level of dual RMCE (35–45% of the transfected cells) can be achieved when Flp and Cre recombinases are expressed as Flp–2A–Cre and Flp–IRES–Cre transcription units. In contrast, the lowest level of dual RMCE (∼1% of the transfected cells) is achieved when Flp and Cre are expressed as individual transcription units. The analysis shows that it is the relative Flp–to–Cre ratio that critically affects the efficiency of dual RMCE. Our results will be helpful for maximizing the efficiency of dual RMCE aimed to engineer and re-engineer genomes. PMID:22270085

Urinary coagulation-fibrinolysis, kallirein-kinin systems and kininase in cases of preclampsia.

PubMed

Mutoh, S; Kobayashi, M; Hirata, J; Itoh, N; Maki, M; Komatsu, Y; Yoshida, A; Sasa, H; Kuroda, K; Kikuchi, Y

1992-01-01

Urinary kallikrein and kallikrein activity significantly decreased in cases of preeclampsia (u-kall./CRE.index 42.39 +/- 9.66 ng/mg, u-kall. act./CRE.index 0.26 +/- 0.06 ng/min/mg), and urinary kininase II and kininase activity significantly increased (u-kininase/CRE.index 10.91 +/- 1.26 x 10(-3) IU/min/mg, u-kininase act./CRE.index 506.37 +/- 178.45 pg/min/mg) when compared with those of normal gravidas from 28 weeks to 42 weeks of gestation (u-kall./CRE.index 189.31 +/- 14.17 ng/mg, u-kall. act./CRE index 1.08 +/- 0.10 ng/min/mg, u-kininase/CRE.index 6.24 +/- 0.31 x 10(-3) IU/min/mg, u-kininase act./CRE.index 15.64 +/- 0.10 pg/min/mg). Urinary FPA, B beta 5-42, alpha 2-PI, and alpha 2PI-plasmin-complex (PIC) significantly increased in preeclampsia (u-FPA/CRE.index 23.59 +/- 8.47 ng/mg, u-B beta/CRE.index 105.26 +/- 29.30 ng/mg, u-alpha 2PI/CRE.index 121.53 +/- 43.57 ng/mg, u-PIC/CRE index 278.39 +/- 60.50 ng/mg) when compared with those of normal control group (u-FPA/CRE.index 0.92 +/- 0.04 ng/mg, u-B beta/CRE.index 12.15 +/- 0.44 ng/mg, u-alpha 2PI/CRE.index 4.18 +/- 0.33 ng/mg, u-PIC/CRE.index 5.98 +/- 1.15 ng/mg). Urinary urokinase markedly increased and urinary D-dimer was detected in severe cases of preeclampsia (u-UK/CRE.index 58.20 +/- 43.69 ng/mg, u-D-dimer 54.76 +/- 9.89 ng/ml) when compared with those of normal control group. These findings suggest that deficiency in urinary kinin excretion may induce hypertension in addition to the changes of urinary coagulation-fibrinolysis system that represents the occurrence of either the endothelial cell injury in the glomerulus or the renal tulbular damage in mild cases of preeclampsia, eventually resulting in the intra-renal vascular coagulation.

Isolation of Novel CreERT2-Driver Lines in Zebrafish Using an Unbiased Gene Trap Approach

PubMed Central

Jungke, Peggy; Hammer, Juliane; Hans, Stefan; Brand, Michael

2015-01-01

Gene manipulation using the Cre/loxP-recombinase system has been successfully employed in zebrafish to study gene functions and lineage relationships. Recently, gene trapping approaches have been applied to produce large collections of transgenic fish expressing conditional alleles in various tissues. However, the limited number of available cell- and tissue-specific Cre/CreERT2-driver lines still constrains widespread application in this model organism. To enlarge the pool of existing CreERT2-driver lines, we performed a genome-wide gene trap screen using a Tol2-based mCherry-T2a-CreERT2 (mCT2aC) gene trap vector. This cassette consists of a splice acceptor and a mCherry-tagged variant of CreERT2 which enables simultaneous labeling of the trapping event, as well as CreERT2 expression from the endogenous promoter. Using this strategy, we generated 27 novel functional CreERT2-driver lines expressing in a cell- and tissue-specific manner during development and adulthood. This study summarizes the analysis of the generated CreERT2-driver lines with respect to functionality, expression, integration, as well as associated phenotypes. Our results significantly enlarge the existing pool of CreERT2-driver lines in zebrafish and combined with Cre–dependent effector lines, the new CreERT2-driver lines will be important tools to manipulate the zebrafish genome. PMID:26083735

Generation of a non-leaky heat shock-inducible Cre line for conditional Cre/lox strategies in zebrafish.

PubMed

Hans, Stefan; Freudenreich, Dorian; Geffarth, Michaela; Kaslin, Jan; Machate, Anja; Brand, Michael

2011-01-01

Cre-mediated site-specific recombination has emerged as an indispensable tool for the precise manipulation of the mammalian genome. Recently, we showed that Cre is also highly efficient in zebrafish and temporal control of recombination can be achieved by using the ligand-inducible CreER(T2). Previous attempts have been made to control recombination by using the temperature inducible hsp70l promoter to conditionally drive the expression of Cre or EGFP-Cre, respectively. However, in this study we demonstrate that the hsp70l promoter possesses a basal leakiness resulting in Cre-mediated recombination even at permissive temperatures. In order to prevent non-conditional recombination, we combined the hsp70l promoter with a mCherry-tagged ligand-inducible CreER(T2). At permissive temperatures and in the absence of the ligand tamoxifen (TAM), no non-conditional recombination is observed indicating tight regulation of CreER(T2). Instead, comprehensive site-specific recombination is mediated following heat induction and administration of TAM. © 2010 Wiley-Liss, Inc.

Prx1 and 3.2 kb Col1a1 promoters target distinct bone cell populations in transgenic mice

PubMed Central

Ouyang, Zhufeng; Chen, Zhijun; Ishikawa, Masakazu; Yue, Xiuzhen; Kawanami, Aya; Leahy, Patrick; Greenfield, Edward M.; Murakami, Shunichi

2014-01-01

Bones consist of a number of cell types including osteoblasts and their precursor cells at various stages of differentiation. To analyze cellular organization within the bone, we generated Col1a1CreER-DsRed transgenic mice that express, in osteoblasts, CreER and DsRed under the control of a mouse 3.2 kb Col1a1 promoter. We further crossed Col1a1CreER-DsRed mice with Prx1CreER-GFP mice that express CreER and GFP in osteochondro progenitor cells under the control of a 2.4 kb Prx1 promoter. Since the 3.2 kb Col1a1 promoter becomes active in osteoblasts at early stages of differentiation, and Prx1CreER-GFP-expressing periosteal cells show endogenous Col1a1 expression, we expected to find a cell population in which both the 2.4 kb Prx1 promoter and the 3.2 kb Col1a1 promoter are active. However, our histological and flow cytometric analyses demonstrated that these transgenes are expressed in distinct cell populations. In the periosteum of long bones, Col1a1CreER-DsRed is expressed in the innermost layer directly lining the bone surface, while Prx1CreER-GFP-expressing cells are localized immediately outside of the Col1a1CreER-DsRed-expressing osteoblasts. In the calvaria, Prx1CreER-GFP-expressing cells are also localized in the cranial suture mesenchyme. Our experiments further showed that Col1a1CreER-DsRed-expressing cells lack chondrogenic potential, while the Prx1CreER-GFP-expressing cells show both chondrogenic and osteogenic potential. Our results indicate that Col1a1CreER-DsRed-expressing cells are committed osteoblasts, while Prx1CreER-GFP-expressing cells are osteochondro progenitor cells. The Prx1CreER-GFP and Col1a1CreER-DsRed transgenes will offer novel approaches for analyzing lineage commitment and early stages of osteoblast differentiation under physiologic and pathologic conditions. PMID:24513582

Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo

PubMed Central

Reinhardt, Robert; Centanin, Lázaro; Tavhelidse, Tinatini; Inoue, Daigo; Wittbrodt, Beate; Concordet, Jean-Paul; Martinez-Morales, Juan Ramón; Wittbrodt, Joachim

2015-01-01

Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina. PMID:25908840

Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo.

PubMed

Reinhardt, Robert; Centanin, Lázaro; Tavhelidse, Tinatini; Inoue, Daigo; Wittbrodt, Beate; Concordet, Jean-Paul; Martinez-Morales, Juan Ramón; Wittbrodt, Joachim

2015-06-03

Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina. © 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

The Epidemiology of Carbapenem-Resistant Enterobacteriaceae: The Impact and Evolution of a Global Menace

PubMed Central

Weinstein, Robert A.

2017-01-01

Abstract Carbapenem-resistant Enterobacteriaceae (CRE) are a serious public health threat. Infections due to these organisms are associated with significant morbidity and mortality. Mechanisms of drug resistance in gram-negative bacteria (GNB) are numerous; β-lactamase genes carried on mobile genetic elements are a key mechanism for the rapid spread of antibiotic-resistant GNB worldwide. Transmissible carbapenem-resistance in Enterobacteriaceae has been recognized for the last 2 decades, but global dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a more recent problem that, once initiated, has been occurring at an alarming pace. In this article, we discuss the evolution of CRE, with a focus on the epidemiology of the CPE pandemic; review risk factors for colonization and infection with the most common transmissible CPE worldwide, Klebsiella pneumoniae carbapenemase–producing K. pneumoniae; and present strategies used to halt the striking spread of these deadly pathogens. PMID:28375512

Expression of Genomic Functional Estrogen Receptor 1 in Mouse Sertoli Cells

PubMed Central

Lin, Jing; Zhu, Jia; Li, Xian; Li, Shengqiang; Lan, Zijian; Ko, Jay

2014-01-01

There is no consensus whether Sertoli cells express estrogen receptor 1 (Esr1). Reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence demonstrated that mouse Sertoli cell lines, TM4, MSC-1, and 15P-1, and purified primary mouse Sertoli cells (PSCs) contained Esr1 messenger RNA and proteins. Incubation of Sertoli cells with 17β-estradiol (E2) or ESR1 agonist stimulated the expression of an estrogen responsive gene Greb1, which was prevented by ESR inhibitor or ESR1 antagonist. Overexpression of Esr1 in MSC-1 enhanced E2-induced Greb1 expression, while knockdown of Esr1 by small interfering RNA in TM4 attenuated the response. Furthermore, E2-induced Greb1 expression was abolished in the PSCs isolated from Amh-Cre/Esr1-floxed mice in which Esr1 in Sertoli cells were selectively deleted. Chromatin immunoprecipitation assays indicated that E2-induced Greb1 expression in Sertoli cells was mediated by binding of ESR1 to estrogen responsive elements. In summary, ligand-dependent nuclear ESR1 was present in mouse Sertoli cells and mediates a classical genomic action of estrogens. PMID:24615934

R/L, a double reporter mouse line that expresses luciferase gene upon Cre-mediated excision, followed by inactivation of mRFP expression.

PubMed

Jia, Junshuang; Lin, Xiaolin; Lin, Xia; Lin, Taoyan; Chen, Bangzhu; Hao, Weichao; Cheng, Yushuang; Liu, Yu; Dian, Meijuan; Yao, Kaitai; Xiao, Dong; Gu, Weiwang

2016-10-01

The Cre/loxP system has become an important tool for the conditional gene knockout and conditional gene expression in genetically engineered mice. The applications of this system depend on transgenic reporter mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. To develop a sensitive assay for monitoring Cre-mediated DNA excisions in mice, we generated Cre-mediated excision reporter mice, designated R/L mice (R/L: mRFP(monomeric red fluorescent protein)/luciferase), express mRFP throughout embryonic development and adult stages, while Cre-mediated excision deletes a loxP-flanked mRFP reporter gene and STOP sequence, thereby activating the expression of the second reporter gene luciferase, as assayed by in vivo and ex vivo bioluminescence imaging. After germ line deletion of the floxed mRFP and STOP sequence in R/L mice by EIIa-Cre mice, the resulting luciferase transgenic mice in which the loxP-mRFP-STOP-loxP cassette is excised from all cells express luciferase in all tissues and organs examined. The expression of luciferase transgene was activated in liver of RL/Alb-Cre double transgenic mice and in brain of RL/Nestin-Cre double transgenic mice when R/L reporter mice were mated with Alb-Cre mice and Nestin-Cre mice, respectively. Our findings reveal that the double reporter R/L mouse line is able to indicate the occurrence of Cre-mediated excision from early embryonic to adult lineages. Taken together, these findings demonstrate that the R/L mice serve as a sensitive reporter for Cre-mediated DNA excision both in living animals and in organs, tissues, and cells following necropsy.

JMJD3 Is Crucial for the Female AVPV RIP-Cre Neuron-Controlled Kisspeptin-Estrogen Feedback Loop and Reproductive Function.

PubMed

Song, Anying; Jiang, Shujun; Wang, Qinghua; Zou, Jianghuan; Lin, Zhaoyu; Gao, Xiang

2017-06-01

The hypothalamic-pituitary-gonadal axis controls development, reproduction, and metabolism. Although most studies have focused on the hierarchy from the brain to the gonad, many questions remain unresolved concerning the feedback from the gonad to the central nervous system, especially regarding the potential epigenetic modifications in hypothalamic neurons. In the present report, we generated genetically modified mice lacking histone H3 lysine 27 (H3K27) demethylase Jumonji domain-containing 3 (JMJD3) in hypothalamic rat-insulin-promoter-expressing neurons (RIP-Cre neurons). The female mutant mice displayed late-onset obesity owing to reduced locomotor activity and decreased energy expenditure. JMJD3 deficiency in RIP-Cre neurons also results in delayed pubertal onset, an irregular estrous cycle, impaired fertility, and accelerated ovarian failure in female mice owing to the dysregulation of the hypothalamic-ovarian axis. We found that JMJD3 directly regulates Kiss1 gene expression by binding to the Kiss1 promoter and triggering H3K27me3 demethylation in the anteroventral periventricular (AVPV) nucleus. Further study confirmed that the aberrations arose from impaired kisspeptin signaling in the hypothalamic AVPV nucleus and subsequent estrogen deficiency. Estrogen replacement therapy can reverse obesity in mutant mice. Moreover, we demonstrated that Jmjd3 is an estrogen target gene in the hypothalamus. These results provide direct genetic and molecular evidence that JMJD3 is a key mediator for the kisspeptin-estrogen feedback loop. Copyright © 2017 Endocrine Society.

Chronic gonadotropin-releasing hormone inhibits activin induction of the ovine follicle-stimulating hormone beta-subunit: involvement of 3',5'-cyclic adenosine monophosphate response element binding protein and nitric oxide synthase type I.

PubMed

Shafiee-Kermani, Farideh; Han, Sang-oh; Miller, William L

2007-07-01

FSH is induced by activin, and this expression is modulated by GnRH through FSHB expression. This report focuses on the inhibitory effect of GnRH on activin-induced FSHB expression. Activin-treated primary murine pituitary cultures robustly express mutant ovine FSHBLuc-DeltaAP1, a luciferase transgene driven by 4.7 kb of ovine FSHB promoter. This promoter lacks two GnRH-inducible activator protein-1 sites, making it easier to observe GnRH-mediated inhibition. Luciferase expression from this transgene was decreased 94% by 100 nM GnRH with a half-time of approximately 4 h in pituitary cultures, and this inhibition was independent of follistatin. Activators of cAMP and protein kinase C like forskolin and phorbol 12-myristate 3-acetate (PMA), respectively, mimicked GnRH action. Kinetic studies of wild-type ovine FSHBLuc in LbetaT2 cells showed continuous induction by activin (4-fold) over 20 h. Most of this induction (78%) was blocked, beginning at 6 h. cAMP response element binding protein (CREB) was implicated in this inhibition because overexpression of its constitutively active mutant mimicked GnRH, and its inhibitor (inducible cAMP early repressor isoform II) reversed the inhibition caused by GnRH, forskolin, or PMA. In addition, GnRH, forskolin, or PMA increased the expression of a CREB-responsive reporter gene, 6xCRE-37PRL-Luc. Inhibition of nitric oxide type I (NOSI) by 7-nitroindazole also reversed GnRH-mediated inhibition by 60%. It is known that GnRH and CREB induce production of NOSI in gonadotropes and neuronal cells, respectively. These data support the concept that chronic GnRH inhibits activin-induced ovine FSHB expression by sequential activation of CREB and NOSI through the cAMP and/or protein kinase C pathways.

Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes.

PubMed

Koehler, Sybille; Brähler, Sebastian; Braun, Fabian; Hagmann, Henning; Rinschen, Markus M; Späth, Martin R; Höhne, Martin; Wunderlich, F Thomas; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T

2017-06-01

Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type -mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2 fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

[Toxicity of Coptis chinensis Rhizome Extracts to Green Algae].

PubMed

Chen, Ya-nan; Yuan, Ling

2015-05-01

Coptis chinensis contains antiseptic alkaloids and thus its rhizomes and preparations are widely used for the treatment of.fish diseases. In order to realize the risk of water ecosystems produced by this medical herb and preparations used in aquaculture, the present experiment was carried out to study the toxicity of Coptis chinensis rhizome extract (CRE) to Scenedesmus oblique and Chlorella pyrenoidosa grown in culture solution with 0.00 (CK), 0.088 (Tl), 0.44 (T2) and 1.76 mg · L(-1) (T3) of CRE, respectively. The results show that low concentration of CRE (T1) inhibited the growth rate of the alga and high CRE (T2 and T3) ceased growth and reproductions. CRE also decreased the chlorophyll and proteins in alga cells, indicating the inhibition of photosynthesis and protein biosynthesis, which could be direct reasons for the low growth rate and death of green alga. The efflux of protons and substances from alga cells led to pH reduction and conductivity increment in culture solution with CRE. Furthermore, the activity of superoxide dismutase in alga increased at the beginning of CRE in T1 and T2 treatments but decreased as time prolonged which was in contrast to high CRE treatment. And the long exposure to low CRE treatment behaved otherwise. This suggests that the low concentration of CRE could induce the resistant reactions in alga at initial time but high CRE concentration or long exposure even at low CRE concentration could inhibit the enzyme synthesis. Similarly, malondialdehyde in alga increased as CRE concentrations increased in culture solutions, implying the damage and high permeability of cell membrane. In general, Chlorella pyrenoidosa was more sensitive to CRE. The abuse of rhizomes and preparations in aquaculture and intensive cultivation of Coptis chinensis plants in a large scale might produce ecological risks to primary productivity of water ecosystems.

Cardiac-specific deletion of protein phosphatase 1β promotes increased myofilament protein phosphorylation and contractile alterations

PubMed Central

Liu, Ruijie; Correll, Robert N.; Davis, Jennifer; Vagnozzi, Ronald J.; York, Allen J.; Sargent, Michelle A.; Nairn, Angus C.; Molkentin, Jeffery D.

2015-01-01

There are 3 protein phosphatase 1 (PP1) catalytic isoforms (α, β and γ) encoded within the mammalian genome. These 3 gene products share ~90% amino acid homology within their catalytic domains but each has unique N- and C-termini that likely underlie distinctive subcellular localization or functionality. In this study, we assessed the effect associated with loss of each PP1 isoform in the heart using a conditional Cre-loxP targeting approach in mice. Ppp1ca-loxP, Ppp1cb-loxP and Ppp1cc-oxP alleles were crossed with either an Nkx2.5-Cre knock-in containing allele for early embryonic deletion or a tamoxifen inducible α-myosin heavy chain (αMHC)-MerCreMer transgene for adult and cardiac-specific deletion. We determined that while deletion of Ppp1ca (PP1α) or Ppp1cc (PP1γ) had little effect on the whole heart, deletion of Ppp1cb (PP1β) resulted in concentric remodeling of the heart, interstitial fibrosis and contractile dysregulation, using either the embryonic or adult-specific Cre-expressing alleles. However, myocytes isolated from Ppp1cb deleted hearts surprisingly showed enhanced contractility. Mechanistically we found that deletion of any of the 3 PP1 gene-encoding isoforms had no effect on phosphorylation of phospholamban, nor were Ca2+ handling dynamics altered in adult myocytes from Ppp1cb deleted hearts. However, loss of Ppp1cb from the heart, but not Ppp1ca or Ppp1cc, resulted in elevated phosphorylation of myofilament proteins such as myosin light chain 2 and cardiac myosin binding protein C, consistent with an enriched localization profile of this isoform to the sarcomeres. These results suggest a unique functional role for the PP1β isoform in affecting cardiac contractile function. PMID:26334248

Overexpression of Lin28b in Neural Stem Cells is Insufficient for Brain Tumor Formation, but Induces Pathological Lobulation of the Developing Cerebellum.

PubMed

Wefers, Annika K; Lindner, Sven; Schulte, Johannes H; Schüller, Ulrich

2017-02-01

LIN28B is a homologue of the RNA-binding protein LIN28A and regulates gene expression during development and carcinogenesis. It is strongly upregulated in a variety of brain tumors, such as medulloblastoma, embryonal tumor with multilayered rosettes (ETMR), atypical teratoid/rhabdoid tumor (AT/RT), or glioblastoma, but the effect of an in vivo overexpression of LIN28B on the developing central nervous system is unknown. We generated transgenic mice that either overexpressed Lin28b in Math1-positive cerebellar granule neuron precursors or in a broad range of Nestin-positive neural precursors. Sections of the cerebellar vermis from adult Math1-Cre::lsl-Lin28b mice had an additional subfissure in lobule IV. Vermes from p0 and p7 Nestin-Cre::lsl-Lin28b mice appeared normal, but we found a pronounced vermal hypersublobulation at p15 and p21 in these mice. Also, the external granule cell layer (EGL) was thicker at p15 than in controls, contained more proliferating cells, and persisted up to p21. Consistently, some Pax6- and NeuN-positive cells were present in the EGL of Nestin-Cre::lsl-Lin28b mice even at p21, and we detected more NeuN-positive granule neuron precursors in the molecular layer (ML) as compared to control. Finally, we found some residual Pax2-positive precursors of inhibitory interneurons in the ML of Nestin-Cre::lsl-Lin28b mice at p21, which have already disappeared in controls. We conclude that while overexpression of LIN28B in Nestin-positive cells does not lead to tumor formation, it results in a protracted development of granule cells and inhibitory interneurons and leads to a hypersublobulation of the cerebellar vermis.

Inducible targeting of CNS astrocytes in Aldh1l1-CreERT2 BAC transgenic mice

PubMed Central

Winchenbach, Jan; Düking, Tim; Berghoff, Stefan A.; Stumpf, Sina K.; Hülsmann, Swen; Nave, Klaus-Armin; Saher, Gesine

2016-01-01

Background: Studying astrocytes in higher brain functions has been hampered by the lack of genetic tools for the efficient expression of inducible Cre recombinase throughout the CNS, including the neocortex. Methods: Therefore, we generated BAC transgenic mice, in which CreERT2 is expressed under control of the Aldh1l1 regulatory region. Results: When crossbred to Cre reporter mice, adult Aldh1l1-CreERT2 mice show efficient gene targeting in astrocytes. No such Cre-mediated recombination was detectable in CNS neurons, oligodendrocytes, and microglia. As expected, Aldh1l1-CreERT2 expression was evident in several peripheral organs, including liver and kidney. Conclusions: Taken together, Aldh1l1-CreERT2 mice are a useful tool for studying astrocytes in neurovascular coupling, brain metabolism, synaptic plasticity and other aspects of neuron-glia interactions. PMID:28149504

Inducible targeting of CNS astrocytes in Aldh1l1-CreERT2 BAC transgenic mice.

PubMed

Winchenbach, Jan; Düking, Tim; Berghoff, Stefan A; Stumpf, Sina K; Hülsmann, Swen; Nave, Klaus-Armin; Saher, Gesine

2016-01-01

Background: Studying astrocytes in higher brain functions has been hampered by the lack of genetic tools for the efficient expression of inducible Cre recombinase throughout the CNS, including the neocortex. Methods: Therefore, we generated BAC transgenic mice, in which CreERT2 is expressed under control of the Aldh1l1 regulatory region. Results: When crossbred to Cre reporter mice, adult Aldh1l1-CreERT2 mice show efficient gene targeting in astrocytes. No such Cre-mediated recombination was detectable in CNS neurons, oligodendrocytes, and microglia. As expected, Aldh1l1-CreERT2 expression was evident in several peripheral organs, including liver and kidney. Conclusions: Taken together, Aldh1l1-CreERT2 mice are a useful tool for studying astrocytes in neurovascular coupling, brain metabolism, synaptic plasticity and other aspects of neuron-glia interactions.

System's flips in climate-related energy (CRE) systems

NASA Astrophysics Data System (ADS)

Ramos, Maria-Helena; Creutin, Jean-Dominique; Engeland, Kolbjørn; François, Baptiste; Renard, Benjamin

2014-05-01

Several modern environmental questions invite to explore the complex relationships between natural phenomena and human behaviour at a range of space and time scales. This usually involves a number of cause-effect (causal) relationships, linking actions and events. In lay terms, 'effect' can be defined as 'what happened' and 'cause', 'why something happened.' In a changing world or merely moving from one scale to another, shifts in perspective are expected, bringing some phenomena into the foreground and putting others to the background. Systems can thus flip from one set of causal structures to another in response to environmental perturbations and human innovations or behaviors, for instance, as space-time signatures are modified. The identification of these flips helps in better understanding and predicting how societies and stakeholders react to a shift in perspective. In this study, our motivation is to investigate possible consequences of the shift to a low carbon economy in terms of socio-technico systems' flips. The focus is on the regional production of Climate-Related Energy (CRE) (hydro-, wind- and solar-power). We search for information on historic shifts that may help defining the forcing conditions of abrupt changes and extreme situations. We identify and present a series of examples in which we try to distinguish the various tipping points, thresholds, breakpoints and regime shifts that are characteristic of complex systems in the CRE production domain. We expect that with these examples our comprehension of the question will be enriched, providing us the elements needed to better validate modeling attempts, to predict and manage flips of complex CRE production systems. The work presented is part of the FP7 project COMPLEX (Knowledge based climate mitigation systems for a low carbon economy; http://www.complex.ac.uk/).

Nutrient input through submarine groundwater discharge in two major Chinese estuaries: the Pearl River Estuary and the Changjiang River Estuary

NASA Astrophysics Data System (ADS)

Liu, Jianan; Du, Jinzhou; Wu, Ying; Liu, Sumei

2018-04-01

In this study, we used a 224Ra mass balance model to evaluate the importance of submarine groundwater discharge (SGD) for the budgets of biogenic elements in two major Chinese estuaries: the Pearl River Estuary (PRE) and the Changjiang River Estuary (CRE). The apparent water age in the PRE was estimated to be 4.8 ± 1.1 days in the dry season and 1.8 ± 0.6 days in the wet season using a physical model based on the tidal prism. In the dry season, the water age in the CRE was estimated to be 11.7 ± 3.0 days using the 224Ra/223Ra activities ratios apparent age model. By applying the 224Ra mass balance model, we obtained calculations of the SGD flow in the PRE of (4.5-10) × 108 m3 d-1 (0.23-0.50 m3 m-2 d-1) and (1.2-2.7) × 108 m3 d-1 (0.06-0.14 m3 m-2 d-1) in the dry season and wet season, respectively, and the estimated SGD flux was (4.6-11) × 109 m3 d-1 (0.18-0.45 m3 m-2 d-1) in the dry season of the CRE. In comparison with the nutrient fluxes from the rivers, the SGD-derived nutrient fluxes may play a vital role in controlling the nutrient budgets and stoichiometry in the study areas. The large amount of dissolved inorganic nitrogen and phosphorus fluxes together with high N: P ratios into the PRE and CRE would potentially contribute to eutrophication and the occurrence of red tides along the adjacent waters.

Fermented Citrus reticulata (ponkan) fruit squeezed draff that contains a large amount of 4'-demethylnobiletin prevents MK801-induced memory impairment.

PubMed

Kawahata, Ichiro; Suzuki, Tatsuya; Rico, Evelyn Gutiérrez; Kusano, Shuichi; Tamura, Hiroshi; Mimaki, Yoshihiro; Yamakuni, Tohru

2017-10-01

A previous study reported biotransformation of a citrus peel polymethoxyflavone, nobiletin, by Aspergillus enabling production of 4'-demethylnobiletin, and the product's antimutagenic activity. However, the effects of fermented citrus peel on the basal forebrain-hippocampal system remain unidentified. Citrus reticulata (ponkan) fruit squeezed draffs are generated as mass waste in beverage factories. In this study using PC12D cells and cultured central nervous system neurons, we therefore examined whether Aspergillus kawachii-fermented citrus fruit squeezed draff could affect cAMP response element (CRE)- and choline acetyltransferase gene (ChAT) promoter region-mediated transcriptional activities relevant to memory formation and cholinergic function. Our current fermentation yielded approximately 80% nobiletin bioconversion, and a sample of hot-water extract of the fermented fruit squeezed draff was stronger than that of the unfermented one in facilitating CRE-mediated transcription in cultured hippocampal neurons as well as in PC12D cells. A sample of 0-80% ethanol-eluted fraction of Diaion HP-20 column-adsorbed components of the preparation obtained by the fermentation concentration-dependently and more strongly facilitated CRE-mediated transcription than did the fraction of the unfermented one in both cell culture systems. In a separate study, this polymethoxyflavone-rich fraction of the fermented fruit squeezed draff showed a potent ability to facilitate CRE-mediated and ChAT transcription in a co-culture of hippocampal neurons and basal forebrain neurons. Repeated oral gavage of mice with the fermented fraction sample prevented MK801-impaired memory formation in mice. These findings suggest that the 4'-demethylnobiletin-rich fraction prepared from the Aspergillus-fermented ponkan squeezed draff has a potential anti-dementia effect.

Generation and characterization of Atoh1-Cre knock-in mouse line

PubMed Central

Yang, Hua; Xie, Xiaoling; Deng, Min; Chen, Xiaowei; Gan, Lin

2010-01-01

Summary Atoh1 encodes a basic helix-loop-helix (bHLH) transcription factor required for the development of the inner ear sensory epithelia, the dorsal spinal cord, brainstem, cerebellum, and intestinal secretory cells. In this study to create a genetic tool for the research on gene function in the ear sensory organs, we generated an Atoh1-Cre knock-in mouse line by replacing the entire Atoh1 coding sequences with the Cre coding sequences. Atoh1Cre/+mice were viable, fertile, and displayed no visible defects whereas the Atoh1Cre/Cremice died perinatally. The spatiotemporal activities of Cre recombinase were examined by crossing Atoh1-Cre mice with the R26R-lacZ conditional reporter mice. Atoh1-Cre activities were detected in the developing inner ear, the hindbrain, the spinal cord, and the intestine. In the inner ear, Atoh1-Cre activities were confined to the sensory organs in which lacZ expression is detected in nearly all of the hair cells and in many supporting cells. Thus, Atoh1-Cre mouse line serves as a useful tool for the functional study of genes in the inner ear. In addition, our results demonstrate that Atoh1 is expressed in the common progenitors destined for both hair and supporting cells. PMID:20533400

Preserved dopaminergic homeostasis and dopamine-related behaviour in hemizygous TH-Cre mice.

PubMed

Runegaard, Annika H; Jensen, Kathrine L; Fitzpatrick, Ciarán M; Dencker, Ditte; Weikop, Pia; Gether, Ulrik; Rickhag, Mattias

2017-01-01

Cre-driver mouse lines have been extensively used as genetic tools to target and manipulate genetically defined neuronal populations by expression of Cre recombinase under selected gene promoters. This approach has greatly advanced neuroscience but interpretations are hampered by the fact that most Cre-driver lines have not been thoroughly characterized. Thus, a phenotypic characterization is of major importance to reveal potential aberrant phenotypes prior to implementation and usage to selectively inactivate or induce transgene expression. Here, we present a biochemical and behavioural assessment of the dopaminergic system in hemizygous tyrosine hydroxylase (TH)-Cre mice in comparison to wild-type (WT) controls. Our data show that TH-Cre mice display preserved dopaminergic homeostasis with unaltered levels of TH and dopamine as well as unaffected dopamine turnover in striatum. TH-Cre mice also show preserved dopamine transporter expression and function supporting sustained dopaminergic transmission. In addition, TH-Cre mice demonstrate normal responses in basic behavioural paradigms related to dopaminergic signalling including locomotor activity, reward preference and anxiolytic behaviour. Our results suggest that TH-Cre mice represent a valid tool to study the dopamine system, though careful characterization must always be performed to prevent false interpretations following Cre-dependent transgene expression and manipulation of selected neuronal pathways. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillus oryzae

PubMed Central

Tanaka, Mizuki; Hiramoto, Tetsuya; Tada, Hinako; Shintani, Takahiro

2017-01-01

ABSTRACT Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB. The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae. IMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is mediated by their ubiquitination, and arrestin-like proteins act as adaptors of transporters and ubiquitin ligases. In this study, we showed that CreD, an arrestin-like protein, is involved in glucose-induced endocytosis of maltose permease and carbon catabolite derepression of amylase gene expression in Aspergillus oryzae. Dephosphorylation of CreD was required for CCR relief triggered by the disruption of creB, which encodes a deubiquitinating enzyme; a combination of the phosphorylation-defective mutation of CreD and creB disruption dramatically improved α-amylase production. This study shows the dual function of an arrestin-like protein and provides a novel approach for improving the production of amylolytic enzymes in A. oryzae. PMID:28455339

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillus oryzae.

PubMed

Tanaka, Mizuki; Hiramoto, Tetsuya; Tada, Hinako; Shintani, Takahiro; Gomi, Katsuya

2017-07-01

Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae IMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is mediated by their ubiquitination, and arrestin-like proteins act as adaptors of transporters and ubiquitin ligases. In this study, we showed that CreD, an arrestin-like protein, is involved in glucose-induced endocytosis of maltose permease and carbon catabolite derepression of amylase gene expression in Aspergillus oryzae Dephosphorylation of CreD was required for CCR relief triggered by the disruption of creB , which encodes a deubiquitinating enzyme; a combination of the phosphorylation-defective mutation of CreD and creB disruption dramatically improved α-amylase production. This study shows the dual function of an arrestin-like protein and provides a novel approach for improving the production of amylolytic enzymes in A. oryzae . Copyright © 2017 American Society for Microbiology.

The use of raw and acid-pretreated bivalve mollusk shells to remove metals from aqueous solutions.

PubMed

Liu, Yang; Sun, Changbin; Xu, Jin; Li, Youzhi

2009-08-30

Heavy metal removal from industrial wastewater is not only to protect living organisms in the environment but also to conserve resources such as metals and water by enabling their reuse. To overcome the disadvantage of high cost and secondary pollution by the conventional physico-chemical treatment techniques, environmentally benign and low-cost adsorbents are in demand. In this study, the use of raw and acid-pretreated bivalve mollusk shells (BMSs) to remove metals from aqueous solutions with single or mixed metal was evaluated at different BMSs doses, pH and temperatures in batch shaking experiments in laboratory conditions. When the BMSs were used to treat CuSO(4)x5H(2)O solution, the copper sorption capacities of the raw and acid-pretreated BMSs were approximately 38.93 mg/g and 138.95 mg/g, respectively. The copper removal efficiency (CRE) of the raw BMSs became greatly enhanced with increasing initial pH, reaching 99.51% at the initial pH 5. Conversely, the CRE of the acid-pretreated BMSs was maintained at 99.48-99.52% throughout the pH range of 1-5. Furthermore, the CRE values of the raw and acid-pretreated BMSs were not greatly changed when the temperature was varied from 15 degrees C to 40 degrees C. In addition, the CRE value of the raw BMSs was maintained for 12 cycles of sorption-desorption with a CRE of 98.4% being observed in the final cycle. Finally, when the BMSs were used to treat electroplating wastewater, the removal efficiencies (REs) of the raw BMSs were 99.97%, 98.99% and 87% for Fe, Zn and Cu, respectively, whereas the REs of the acid-pretreated BMSs were 99.98%, 99.43% and 92.13%, respectively. Ion exchange experiments revealed that one of mechanisms for metal sorption by the BMSs from aqueous solution is related to ion exchange, especially between the metal ions in the treated solution and Ca(2+) from BMSs. Infrared absorbance spectra analysis indicated that the acid pretreatment led to occurrence of the groups (i.e. -OH, -NH, C=O and S=O) of negative charge in treated BMSs. Scanning electron microscopy revealed that acid pretreatment enabled the used BMSs to form the flake-shaped structure with smooth surfaces that can supply a better interface for binding metal ions.

Germ-Line Recombination Activity of the Widely Used hGFAP-Cre and Nestin-Cre Transgenes

PubMed Central

Zhang, Jiong; Dublin, Pavel; Griemsmann, Stephanie; Klein, Alexandra; Brehm, Ralph; Bedner, Peter; Fleischmann, Bernd K.; Steinhäuser, Christian; Theis, Martin

2013-01-01

Herein we demonstrate with PCR, immunodetection and reporter gene approaches that the widely used human Glial Fibrillary Acidic Protein (hGFAP)-Cre transgene exhibits spontaneous germ-line recombination activity in leading to deletion in brain, heart and tail tissue with high frequency. The ectopic activity of hGFAP-Cre requires a rigorous control. We likewise observed that a second widely used nestin-Cre transgene shows germ-line deletion. Here we describe procedures to identify mice with germ-line recombination mediated by the hGFAP-Cre and nestin-Cre transgenes. Such control is essential to avoid pleiotropic effects due to germ-line deletion of loxP-flanked target genes and to maintain the CNS-restricted deletion status in transgenic mouse colonies. PMID:24349371

Risk Factors for Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae (CP-CRE) Acquisition Among Contacts of Newly Diagnosed CP-CRE Patients.

PubMed

Schwartz-Neiderman, Anat; Braun, Tali; Fallach, Noga; Schwartz, David; Carmeli, Yehuda; Schechner, Vered

2016-10-01

OBJECTIVE Carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) are extremely drug-resistant pathogens. Screening of contacts of newly identified CP-CRE patients is an important step to limit further transmission. We aimed to determine the risk factors for CP-CRE acquisition among patients exposed to a CP-CRE index patient. METHODS A matched case-control study was performed in a tertiary care hospital in Israel. The study population was comprised of patients who underwent rectal screening for CP-CRE following close contact with a newly identified CP-CRE index patient. Cases were defined as positive tests for CP-CRE. For each case patient, 2 matched controls were randomly selected from the pool of contacts who tested negative for CP-CRE following exposure to the same index case. Bivariate and multivariate analyses were conducted using conditional logistic regression. RESULTS In total, 53 positive contacts were identified in 40 unique investigations (896 tests performed on 735 contacts) between October 6, 2008, and June 7, 2012. bla KPC was the only carbapenemase identified. In multivariate analysis, risk factors for CP-CRE acquisition among contacts were (1) contact with an index patient for ≥3 days (odds ratio [OR], 9.8; 95% confidence interval [CI], 2.0-48.9), (2) mechanical ventilation (OR, 4.1; 95% CI, 1.4-11.9), and (3) carriage or infection with another multidrug-resistant organism (MDRO; OR, 2.6; 95% CI, 1.0-7.1). Among patients who received antibiotics, cephalosporins were associated with a lower risk of acquisition. CONCLUSIONS Patient characteristics (ventilation and carriage of another MDRO) as well as duration of contact are risk factors for CP-CRE acquisition among contacts. The role of cephalosporins requires further study. Infect Control Hosp Epidemiol 2016;1-7.

Different regulation of limb development by p63 transcript variants.

PubMed

Kawata, Manabu; Taniguchi, Yuki; Mori, Daisuke; Yano, Fumiko; Ohba, Shinsuke; Chung, Ung-Il; Shimogori, Tomomi; Mills, Alea A; Tanaka, Sakae; Saito, Taku

2017-01-01

The apical ectodermal ridge (AER), located at the distal end of each limb bud, is a key signaling center which controls outgrowth and patterning of the proximal-distal axis of the limb through secretion of various molecules. Fibroblast growth factors (FGFs), particularly Fgf8 and Fgf4, are representative molecules produced by AER cells, and essential to maintain the AER and cell proliferation in the underlying mesenchyme, meanwhile Jag2-Notch pathway negatively regulates the AER and limb development. p63, a transcription factor of the p53 family, is expressed in the AER and indispensable for limb formation. However, the underlying mechanisms and specific roles of p63 variants are unknown. Here, we quantified the expression of p63 variants in mouse limbs from embryonic day (E) 10.5 to E12.5, and found that ΔNp63γ was strongly expressed in limbs at all stages, while TAp63γ expression was rapidly increased in the later stages. Fluorescence-activated cell sorting analysis of limb bud cells from reporter mouse embryos at E11.5 revealed that all variants were abundantly expressed in AER cells, and their expression was very low in mesenchymal cells. We then generated AER-specific p63 knockout mice by mating mice with a null and a flox allele of p63, and Msx2-Cre mice (Msx2-Cre;p63Δ/fl). Msx2-Cre;p63Δ/fl neonates showed limb malformation that was more obvious in distal elements. Expression of various AER-related genes was decreased in Msx2-Cre;p63Δ/fl limb buds and embryoid bodies formed by p63-knockdown induced pluripotent stem cells. Promoter analyses and chromatin immunoprecipitation assays demonstrated Fgf8 and Fgf4 as transcriptional targets of ΔNp63γ, and Jag2 as that of TAp63γ. Furthermore, TAp63γ overexpression exacerbated the phenotype of Msx2-Cre;p63Δ/fl mice. These data indicate that ΔNp63 and TAp63 control limb development through transcriptional regulation of different target molecules with different roles in the AER. Our findings contribute to further understanding of the molecular network of limb development.

Mouse alpha1(I)-collagen promoter is the best known promoter to drive efficient Cre recombinase expression in osteoblast.

PubMed

Dacquin, Romain; Starbuck, Michael; Schinke, Thorsten; Karsenty, Gérard

2002-06-01

Cell- and time-specific gene inactivation should enhance our knowledge of bone biology. Implementation of this technique requires construction of transgenic mouse lines expressing Cre recombinase in osteoblasts, the bone forming cell. We tested several promoter fragments for their ability to drive efficient Cre expression in osteoblasts. In the first mouse transgenic line, the Cre gene was placed under the control of the 2.3-kb proximal fragment of the alpha1(I)-collagen promoter, which is expressed at high levels in osteoblasts throughout their differentiation. Transgenic mice expressing this transgene in bone were bred with the ROSA26 reporter (R26R) strain in which the ROSA26 locus is targeted with a conditional LacZ reporter cassette. In R26R mice, Cre expression and subsequent Cre-mediated recombination lead to expression of the LacZ reporter gene, an event that can be monitored by LacZ staining. LacZ staining was detected in virtually all osteoblasts of alpha1(I)-Cre;R26R mice indicating that homologous recombination occurred in these cells. No other cell type stained blue. In the second line studied, the 1.3-kb fragment of osteocalcin gene 2 (OG2) promoter, which is active in differentiated osteoblasts, was used to drive Cre expression. OG2-Cre mice expressed Cre specifically in bone. However, cross of OG2-Cre mice with R26R mice did not lead to any detectable LacZ staining in osteoblasts. Lastly, we tested a more active artificial promoter derived from the OG2 promoter. The artificial OG2-Cre transgene was expressed by reverse transcriptase-polymerase chain reaction in cartilage and bone samples. After cross of the artificial OG2-Cre mice with R26R mice, we detected a LacZ staining in articular chondrocytes but not in osteoblasts. Our data suggest that the only promoter able to drive Cre expression at a level sufficient to induce recombination in osteoblasts is the alpha1(I)-collagen promoter. Copyright 2002 Wiley-Liss, Inc.

Sacred Cows and Stubborn Mules: The Imperative to Reform the US Code

DTIC Science & Technology

2013-02-14

National Guard have also added specialized domestic military missions like the Chemical, Biological , Radiological and Nuclear (CBRN) Response Enterprise...lists cyber and EMP as exigent future threats, adding that the most predictable future megatrend is empowerment. “Individuals and small groups will...and include a Defense CBRN Response Force (DCRF) and two CBRN Response Elements (CRE). The US Marines also maintain two Chemical, Biological Incident

Gut Colonization with Carbapenem-resistant Enterobacteriaceae Adversely Impacts the Outcome in Patients with Hematological Malignancies: Results of A Prospective Surveillance Study.

PubMed

Jaiswal, Sarita Rani; Gupta, Satyanker; Kumar, Rekha Saji; Sherawat, Amit; Rajoreya, Ashok; Dash, Saroj K; Bhagwati, Gitali; Chakrabarti, Suparno

2018-01-01

Gut colonisation with carbapenem-resistant enterobacteriaceae (CRE) is a risk factor for CRE bacteremia and patients with haematological malignancies (HM) are at the highest risk of mortality. We conducted a prospective surveillance study of gut colonisation with CRE and its impact on the outcome of 225 consecutive patients of HM over 28 months. The median age of the cohort was 46 years, the majority with acute leukaemia. 48 (21%) patients were colonised with CRE on admission (CAD). Another 46 patients were colonised with CRE in the hospital (CIH). The risk factors for CAD and CIH were a diagnosis of acute leukaemia and duration of hospital stay respectively. CRE accounted for 77% of infection-related mortality (IRM). The incidence of CRE bacteremia in CRE positive patients was 18% (17/94), and mortality in those with CRE bacteremia was 100%. IRM was 35.3% in CIH group compared to 10.5% in the CAD group (p=0.0001). IRM was highest in those with acute myeloid leukaemia (AML) and CIH (54.9% p=0.0001). On multivariate analysis, CIH was the most important risk factor for IRM (HR-7.2). Our data demonstrate that a substantial proportion of patients with HM are colonised with CRE without prior hospitalisation, but those with nosocomial colonisation have the highest risk of mortality, particularly in those with AML.

Human renin 5'-flanking DNA to nucleotide-2750.

PubMed

Smith, D L; Jeyapalan, S; Lang, J A; Guo, X H; Sigmund, C D; Morris, B J

1995-01-01

Renin is one of the most important factors in blood pressure and electrolyte regulation in mammals and the renin locus has been implicated in hypertension. To assist studies of promoter control we therefore determined the 5'-flanking sequence of the human gene (REN) to residue -2750 relative to the transcription start site (+1). Sites of homology to consensus sequences for binding of trans-acting factors involved in transcriptional control of other genes were identified, and functionality for two of these (a CRE and Pit-1 site) have so far been demonstrated.

Phenotypically silent Cre recombination within the postnatal ventricular conduction system.

PubMed

Bhattacharyya, Samadrita; Bhakta, Minoti; Munshi, Nikhil Vilas

2017-01-01

The cardiac conduction system (CCS) is composed of specialized cardiomyocytes that initiate and maintain cardiac rhythm. Any perturbation to the normal sequence of electrical events within the heart can result in cardiac arrhythmias. To understand how cardiac rhythm is established at the molecular level, several genetically modified mouse lines expressing Cre recombinase within specific CCS compartments have been created. In general, Cre driver lines have been generated either by homologous recombination of Cre into an endogenous locus or Cre expression driven by a randomly inserted transgene. However, haploinsufficiency of the endogenous gene compromises the former approach, while position effects negatively impact the latter. To address these limitations, we generated a Cre driver line for the ventricular conduction system (VCS) that preserves endogenous gene expression by targeting the Contactin2 (Cntn2) 3' untranslated region (3'UTR). Here we show that Cntn23'UTR-IRES-Cre-EGFP/+ mice recombine floxed alleles within the VCS and that Cre expression faithfully recapitulates the spatial distribution of Cntn2 within the heart. We further demonstrate that Cre expression initiates after birth with preservation of native Cntn2 protein. Finally, we show that Cntn23'UTR-IRES-Cre-EGFP/+ mice maintain normal cardiac mechanical and electrical function. Taken together, our results establish a novel VCS-specific Cre driver line without the adverse consequences of haploinsufficiency or position effects. We expect that our new mouse line will add to the accumulating toolkit of CCS-specific mouse reagents and aid characterization of the cell-autonomous molecular circuitry that drives VCS maintenance and function.

Inhibition of JNK by pi class of glutathione S-transferase through PKA/CREB pathway is associated with carnosic acid protection against 6-hydroxydopamine-induced apoptosis.

PubMed

Lin, Chia-Yuan; Fu, Ru-Huei; Chou, Ruey-Hwang; Chen, Jing-Hsien; Wu, Chi-Rei; Chang, Shu-Wei; Tsai, Chia-Wen

2017-05-01

Pi class of glutathione S-transferase (GST) is known to suppress c-Jun N-terminal kinase (JNK)-related apoptosis through protein-protein interactions. Moreover, signaling by PKA/cAMP response element binding protein (CREB) is necessary for GSTP up-regulation. This study explored whether carnosic acid (CA) from rosemary prevents 6-hydroxydopamine (6-OHDA)-induced neurotoxicity by inhibition of JNK through GSTP via PKA/CREB signaling. Results indicated that the GSTP protein was increased in SH-SY5Y cells treated with CA for 18 and 24 h. However, CA had no significant effect on alpha or mu class of GST. Treatment of CA increased the induction of p-PKAα, nuclear p-CREB, and CRE-DNA binding activity. These effects of CA were attenuated in cells pretreated with the PKA inhibitor H89. CA pretreatment suppressed 6-OHDA-induced apoptosis by inhibition of JNK phosphorylation, poly(ADP)-ribose polymerase cleavage, and nuclear condensation. Pretreatment with H89 and GSTP siRNA attenuated the ability of CA to reverse 6-OHDA-induced apoptosis. By use of immunoprecipitation with JNK antibody to examine the interaction of GSTP-JNK with CA, we showed that CA pretreatment increased the immunoprecipitation of GSTP after 6-OHDA treatment, which suggests that CA promoted the interaction between GSTP and JNK. CA prevents 6-OHDA-induced apoptosis via inhibition of JNK by GSTP through the PKA/CREB pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination.

PubMed

Woodman, Andrew; Arnold, Jamie J; Cameron, Craig E; Evans, David J

2016-08-19

Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3' non-coding region we have further developed a cell-based assay (the 3'CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Temporally-Controlled Site-Specific Recombination in Zebrafish

PubMed Central

Hans, Stefan; Kaslin, Jan; Freudenreich, Dorian; Brand, Michael

2009-01-01

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreERT2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms. PMID:19247481

A Climatology of Surface Cloud Radiative Effects at the ARM Tropical Western Pacific Sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

McFarlane, Sally A.; Long, Charles N.; Flaherty, Julia E.

Cloud radiative effects on surface downwelling fluxes are investigated using long-term datasets from the three Atmospheric Radiation Measurement (ARM) sites in the Tropical Western Pacific (TWP) region. The Nauru and Darwin sites show significant variability in sky cover, downwelling radiative fluxes, and surface cloud radiative effect (CRE) due to El Niño and the Australian monsoon, respectively, while the Manus site shows little intra-seasonal or interannual variability. Cloud radar measurement of cloud base and top heights are used to define cloud types so that the effect of cloud type on the surface CRE can be examined. Clouds with low bases contributemore » 71-75% of the surface shortwave (SW) CRE and 66-74% of the surface longwave (LW) CRE at the three TWP sites, while clouds with mid-level bases contribute 8-9% of the SW CRE and 12-14% of the LW CRE, and clouds with high bases contribute 16-19% of the SW CRE and 15-21% of the LW CRE.« less

Building Cre Knockin Rat Lines Using CRISPR/Cas9.

PubMed

Ma, Yuanwu; Zhang, Lianfeng; Huang, Xingxu

2017-01-01

Conditional gene inactivation strategy helps researchers to study the gene functions that are critical in embryogenesis or in defined tissues of adulthood. The Cre/loxP system is widely used for conditional gene inactivation/activation in cells or organisms. Cre knockin animal lines are essential for gene expression or inactivation in a spatially and temporally restricted manner. However, to generate a Cre knockin line by traditional approach is laborious. Recently, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) has been proven as a simple and efficient genome-editing tool. We have used CRISPR/Cas9 system to generate rat strains that carry Cre genes in different targeted gene loci by direct delivery of gRNAs/Cas9/donors into fertilized eggs. Here, we described a stepwise procedure for the generation of Cre knockin rat, including target site selection, RNA preparation, the construction of the template donor, pronuclear injection, and the genotyping of precise Cre insertion in F 0 rats. Taken together, the establishment of Cre knockin line can be achieved within 6 weeks.

Disruption of Trichoderma reesei cre2, encoding an ubiquitin C-terminal hydrolase, results in increased cellulase activity

PubMed Central

2011-01-01

Background The filamentous fungus Trichoderma reesei (Hypocrea jecorina) is an important source of cellulases for use in the textile and alternative fuel industries. To fully understand the regulation of cellulase production in T. reesei, the role of a gene known to be involved in carbon regulation in Aspergillus nidulans, but unstudied in T. reesei, was investigated. Results The T. reesei orthologue of the A. nidulans creB gene, designated cre2, was identified and shown to be functional through heterologous complementation of a creB mutation in A. nidulans. A T. reesei strain was constructed using gene disruption techniques that contained a disrupted cre2 gene. This strain, JKTR2-6, exhibited phenotypes similar to the A. nidulans creB mutant strain both in carbon catabolite repressing, and in carbon catabolite derepressing conditions. Importantly, the disruption also led to elevated cellulase levels. Conclusions These results demonstrate that cre2 is involved in cellulase expression. Since the disruption of cre2 increases the amount of cellulase activity, without severe morphological affects, targeting creB orthologues for disruption in other industrially useful filamentous fungi, such as Aspergillus oryzae, Trichoderma harzianum or Aspergillus niger may also lead to elevated hydrolytic enzyme activity in these species. PMID:22070776

Silencing Inhibits Cre-Mediated Recombination of the Z/AP and Z/EG Reporters in Adult Cells

PubMed Central

Long, Michael A.; Rossi, Fabio M. V.

2009-01-01

Background The Cre-loxP system has been used to enable tissue specific activation, inactivation and mutation of many genes in vivo and has thereby greatly facilitated the genetic dissection of several cellular and developmental processes. In such studies, Cre-reporter strains, which carry a Cre-activated marker gene, are frequently utilized to validate the expression profile of Cre transgenes, to act as a surrogate marker for excision of a second allele, and to irreversibly label cells for lineage tracing experiments. Principal Findings We have studied three commonly used Cre-reporter strains, Z/AP, Z/EG and R26R-EYFP and have demonstrated that although each reporter can be reliably activated by Cre during early development, exposure to Cre in adult hematopoietic cells results in a much lower frequency of marker-positive cells in the Z/AP or Z/EG strains than in the R26R-EYFP strain. In marker negative cells derived from the Z/AP and Z/EG strains, the transgenic promoter is methylated and Cre-mediated recombination of the locus is inhibited. Conclusions These results show that the efficiency of Cre-mediated recombination is not only dependent on the genomic context of a given loxP-flanked sequence, but also on stochastic epigenetic mechanisms underlying transgene variegation. Furthermore, our data highlights the potential shortcomings of utilizing the Z/AP and Z/EG reporters as surrogate markers of excision or in lineage tracing experiments. PMID:19415111

Silencing inhibits Cre-mediated recombination of the Z/AP and Z/EG reporters in adult cells.

PubMed

Long, Michael A; Rossi, Fabio M V

2009-01-01

The Cre-loxP system has been used to enable tissue specific activation, inactivation and mutation of many genes in vivo and has thereby greatly facilitated the genetic dissection of several cellular and developmental processes. In such studies, Cre-reporter strains, which carry a Cre-activated marker gene, are frequently utilized to validate the expression profile of Cre transgenes, to act as a surrogate marker for excision of a second allele, and to irreversibly label cells for lineage tracing experiments. We have studied three commonly used Cre-reporter strains, Z/AP, Z/EG and R26R-EYFP and have demonstrated that although each reporter can be reliably activated by Cre during early development, exposure to Cre in adult hematopoietic cells results in a much lower frequency of marker-positive cells in the Z/AP or Z/EG strains than in the R26R-EYFP strain. In marker negative cells derived from the Z/AP and Z/EG strains, the transgenic promoter is methylated and Cre-mediated recombination of the locus is inhibited. These results show that the efficiency of Cre-mediated recombination is not only dependent on the genomic context of a given loxP-flanked sequence, but also on stochastic epigenetic mechanisms underlying transgene variegation. Furthermore, our data highlights the potential shortcomings of utilizing the Z/AP and Z/EG reporters as surrogate markers of excision or in lineage tracing experiments.

Improved α-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2014-01-01

In filamentous fungi, the expression of secretory glycoside hydrolase encoding genes, such as those for amylases, cellulases, and xylanases, is generally repressed in the presence of glucose. CreA and CreB have been observed to be regulating factors for carbon catabolite repression. In this study, we generated single and double deletion creA and/or creB mutants in Aspergillus oryzae. The α-amylase activities of each strain were compared under various culture conditions. For the wild-type strain, mRNA levels of α-amylase were markedly decreased in the later stage of submerged culture under inducing conditions, whereas this reduced expression was not observed for single creA and double creA/creB deletion mutants. In addition, α-amylase activity of the wild-type strain was reduced in submerged culture containing high concentrations of inducing sugars, whereas all constructed mutants showed higher α-amylase activities. In particular, the α-amylase activity of the double deletion mutant in a medium containing 5% starch was >10-fold higher than that of the wild-type strain under the same culture conditions. In solid-state cultures using wheat bran as a substrate, the α-amylase activities of single creA and double deletion mutants were >2-fold higher than that of the wild-type strain. These results suggested that deleting both creA and creB resulted in dramatic improvements in the production of secretory glycoside hydrolases in filamentous fungi.

Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice.

PubMed

Ichise, Hirotake; Hori, Akiko; Shiozawa, Seiji; Kondo, Saki; Kanegae, Yumi; Saito, Izumu; Ichise, Taeko; Yoshida, Nobuaki

2016-07-29

Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes.

Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

PubMed

Kawabe, Yoshinori; Shimomura, Takuya; Huang, Shuohao; Imanishi, Suguru; Ito, Akira; Kamihira, Masamichi

2016-07-01

Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre-based cell engineering. Biotechnol. Bioeng. 2016;113: 1600-1610. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Conditional Mesenchymal Disruption of Pkd1 Results in Osteopenia and Polycystic Kidney Disease

PubMed Central

Cao, Li; David, Valentin; Quarles, Leigh Darryl

2012-01-01

Conditional deletion of Pkd1 in osteoblasts using either Osteocalcin(Oc)-Cre or Dmp1-Cre results in defective osteoblast-mediated postnatal bone formation and osteopenia. Pkd1 is also expressed in undifferentiated mesenchyme that gives rise to the osteoblast lineage. To examine the effects of Pkd1 on prenatal osteoblast development, we crossed Pkd1 flox/flox and Col1a1(3.6)-Cre mice, which has been used to achieve selective inactivation of Pkd1 earlier in the osteoblast lineage. Control Pkd1 flox/flox and Pkd1 flox/+, heterozygous Col1a1(3.6)-Cre;Pkd1 flox/+ and Pkd1 flox/null, and homozygous Col1a1(3.6)-Cre;Pkd1 flox/flox and Col1a1(3.6)-Cre;Pkd1 flox/null mice were analyzed at ages ranging from E14.5 to 8-weeks-old. Newborn Col1a1(3.6)-Cre;Pkd1 flox/null mice exhibited defective skeletogenesis in association with a greater reduction in Pkd1 expression in bone. Conditional Col1a1(3.6)-Cre;Pkd1 flox/+ and Col1a1(3.6)-Cre;Pkd1 flox/flox mice displayed a gene dose-dependent decrease in bone formation and increase in marrow fat at 6 weeks of age. Bone marrow stromal cell and primary osteoblast cultures from homozygous Col1a1(3.6)-Cre;Pkd1 flox/flox mice showed increased proliferation, impaired osteoblast development and enhanced adipogenesis ex vivo. Unexpectedly, we found evidence for Col1a1(3.6)-Cre mediated deletion of Pkd1 in extraskeletal tissues in Col1a1(3.6)-Cre;Pkd1 flox/flox mice. Deletion of Pkd1 in mesenchymal precursors resulted in pancreatic and renal, but not hepatic, cyst formation. The non-lethality of Col1a1(3.6)-Cre;Pkd1 flox/flox mice establishes a new model to study abnormalities in bone development and cyst formation in pancreas and kidney caused by Pkd1 gene inactivation. PMID:23029375

Altered Baseline and Nicotine-Mediated Behavioral and Cholinergic Profiles in ChAT-Cre Mouse Lines.

PubMed

Chen, Edison; Lallai, Valeria; Sherafat, Yasmine; Grimes, Nickolas P; Pushkin, Anna N; Fowler, J P; Fowler, Christie D

2018-02-28

The recent development of transgenic rodent lines expressing cre recombinase in a cell-specific manner, along with advances in engineered viral vectors, has permitted in-depth investigations into circuit function. However, emerging evidence has begun to suggest that genetic modifications may introduce unexpected caveats. In the current studies, we sought to extensively characterize male and female mice from both the ChAT (BAC) -Cre mouse line, created with the bacterial artificial chromosome (BAC) method, and ChAT (IRES) -Cre mouse line, generated with the internal ribosome entry site (IRES) method. ChAT (BAC) -Cre transgenic and wild-type mice did not differ in general locomotor behavior, anxiety measures, drug-induced cataplexy, nicotine-mediated hypolocomotion, or operant food training. However, ChAT (BAC) -Cre transgenic mice did exhibit significant deficits in intravenous nicotine self-administration, which paralleled an increase in vesicular acetylcholine transporter and choline acetyltransferase (ChAT) hippocampal expression. For the ChAT (IRES) -Cre line, transgenic mice exhibited deficits in baseline locomotor, nicotine-mediated hypolocomotion, and operant food training compared with wild-type and hemizygous littermates. No differences among ChAT (IRES) -Cre wild-type, hemizygous, and transgenic littermates were found in anxiety measures, drug-induced cataplexy, and nicotine self-administration. Given that increased cre expression was present in the ChAT (IRES) -Cre transgenic mice, as well as a decrease in ChAT expression in the hippocampus, altered neuronal function may underlie behavioral phenotypes. In contrast, ChAT (IRES) -Cre hemizygous mice were more similar to wild-type mice in both protein expression and the majority of behavioral assessments. As such, interpretation of data derived from ChAT-Cre rodents must consider potential limitations dependent on the line and/or genotype used in research investigations. SIGNIFICANCE STATEMENT Altered baseline and/or nicotine-mediated behavioral profiles were discovered in transgenic mice from the ChAT (BAC) -Cre and ChAT (IRES) -Cre lines. Given that these cre-expressing mice have become increasingly used by the scientific community, either independently with chemicogenetic and optogenetic viral vectors or crossed with other transgenic lines, the current studies highlight important considerations for the interpretation of data from previous and future experimental investigations. Moreover, the current findings detail the behavioral effects of either increased or decreased baseline cholinergic signaling mechanisms on locomotor, anxiety, learning/memory, and intravenous nicotine self-administration behaviors. Copyright © 2018 the authors 0270-6474/18/382177-12$15.00/0.

The Specific Role of FAM20C in Dentinogenesis

PubMed Central

Wang, X.; Wang, J.; Liu, Y.; Yuan, B.; Ruest, L.B.; Feng, J.Q.

2015-01-01

FAM20C is an evolutionarily reserved molecule highly expressed in mineralized tissues. Previously we demonstrated that Sox2-Cre;Fam20Cfl/fl mice, in which Fam20C was ubiquitously inactivated, had dentin and enamel defects as well as hypophosphatemic rickets. We also showed that K14-Cre;Fam20Cfl/fl mice, in which Fam20C was specifically inactivated in the epithelium, had enamel defects but lacked hypophosphatemia and defects in the bone and dentin. These results indicated that the enamel defects in the Sox2-Cre;Fam20Cfl/fl mice were independent of dentin defects and hypophosphatemia. To determine if the dentin defects in the Sox2-Cre;Fam20Cfl/fl mice were associated with the enamel defects and hypophosphatemia, we crossed Fam20Cfl/fl mice with Wnt1-Cre and Osr2-Cre transgenic mice to inactivate Fam20C in the craniofacial mesenchymal cells that form dentin and alveolar bone. The resulting Wnt1-Cre;Fam20Cfl/fl and Osr2-Cre;Fam20Cfl/fl mice showed remarkable dentin and alveolar bone defects, while their enamel did not show apparent defects. The serum FGF23 levels in these mice were higher than normal but lower than those in the Sox2-Cre;Fam20Cfl/fl mice; they developed a mild type of hypophosphatemia that did not cause major defects in long bones. These results indicate that the dentin defects in the Sox2-Cre;Fam20Cfl/fl mice were independent of the enamel defects. PMID:25515778

Bacteremia due to carbapenem-resistant Enterobacteriaceae in neutropenic patients with hematologic malignancies.

PubMed

Satlin, Michael J; Cohen, Nina; Ma, Kevin C; Gedrimaite, Zivile; Soave, Rosemary; Askin, Gülce; Chen, Liang; Kreiswirth, Barry N; Walsh, Thomas J; Seo, Susan K

2016-10-01

To determine the prevalence, risk factors, treatments, and outcomes of bloodstream infections (BSIs) due to carbapenem-resistant Enterobacteriaceae (CRE) in adult neutropenic patients with hematologic malignancies. We reviewed all BSIs between 2008 and 2012 in this population at two New York City oncology centers. A case-control study was conducted to identify CRE BSI risk factors, using three controls of non-CRE BSIs per case. CRE caused 43 (2.2%) of 1992 BSIs overall and 4.7% of Gram-negative bacteremias. Independent risk factors for CRE BSI were prior β-lactam/β-lactamase inhibitor (adjusted odds ratio [aOR] 3.2; P = 0.03) or carbapenem (aOR 3.0; P = 0.05) use, current trimethoprim-sulfamethoxazole (aOR 24; P = 0.001) or glucocorticoid (aOR 5.4, P = 0.004) use, and having a prior CRE culture (aOR 12; P = 0.03). Patients with CRE bacteremia had a median of 52 h from culture collection until receipt of active therapy. They had a 51% BSI-related mortality rate, with a median of 4 days from bacteremia onset until death. CRE-active empirical therapy was associated with a lower 30-day mortality rate (17% vs. 59%; P = 0.08). CRE are lethal emerging causes of bacteremia in neutropenic patients. New strategies are needed to shorten the delay in administration of CRE-active agents and improve outcomes in this vulnerable population. Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

A systematic review of the epidemiology of carbapenem-resistant Enterobacteriaceae in the United States.

PubMed

Livorsi, Daniel J; Chorazy, Margaret L; Schweizer, Marin L; Balkenende, Erin C; Blevins, Amy E; Nair, Rajeshwari; Samore, Matthew H; Nelson, Richard E; Khader, Karim; Perencevich, Eli N

2018-01-01

Carbapenem-resistant Enterobacteriaceae (CRE) pose an urgent public health threat in the United States. An important step in planning and monitoring a national response to CRE is understanding its epidemiology and associated outcomes. We conducted a systematic literature review of studies that investigated incidence and outcomes of CRE infection in the US. We performed searches in MEDLINE via Ovid, CDSR, DARE, CENTRAL, NHS EED, Scopus, and Web of Science for articles published from 1/1/2000 to 2/1/2016 about the incidence and outcomes of CRE at US sites. Five studies evaluated incidence, but many used differing definitions for cases. Across the entire US population, the reported incidence of CRE was 0.3-2.93 infections per 100,000 person-years. Infection rates were highest in long-term acute-care (LTAC) hospitals. There was insufficient data to assess trends in infection rates over time. Four studies evaluated outcomes. Mortality was higher in CRE patients in some but not all studies. While the incidence of CRE infections in the United States remains low on a national level, the incidence is highest in LTACs. Studies assessing outcomes in CRE-infected patients are limited in number, small in size, and have reached conflicting results. Future research should measure a variety of clinical outcomes and adequately adjust for confounders to better assess the full burden of CRE.

The Epidemiology of Carbapenem-Resistant Enterobacteriaceae: The Impact and Evolution of a Global Menace.

PubMed

Logan, Latania K; Weinstein, Robert A

2017-02-15

Carbapenem-resistant Enterobacteriaceae (CRE) are a serious public health threat. Infections due to these organisms are associated with significant morbidity and mortality. Mechanisms of drug resistance in gram-negative bacteria (GNB) are numerous; β-lactamase genes carried on mobile genetic elements are a key mechanism for the rapid spread of antibiotic-resistant GNB worldwide. Transmissible carbapenem-resistance in Enterobacteriaceae has been recognized for the last 2 decades, but global dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a more recent problem that, once initiated, has been occurring at an alarming pace. In this article, we discuss the evolution of CRE, with a focus on the epidemiology of the CPE pandemic; review risk factors for colonization and infection with the most common transmissible CPE worldwide, Klebsiella pneumoniae carbapenemase-producing K. pneumoniae; and present strategies used to halt the striking spread of these deadly pathogens. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

The Sox17CreERT2 knock-in mouse line displays spatiotemporal activation of Cre recombinase in distinct Sox17 lineage progenitors.

PubMed

Engert, Silvia; Burtscher, Ingo; Kalali, Behnam; Gerhard, Markus; Lickert, Heiko

2013-11-01

The HMG-box transcription factor Sox17 is essential for endoderm formation, vascular development, and definitive hematopoiesis. To investigate the fate of distinct Sox17-expressing progenitor cells in a spatiotemporal manner, we generated a hormone-inducible CreERT2 knock-in mouse line. By homologous recombination we fused a codon improved, ligand-dependent estrogen receptor Cre recombinase by an intervening viral T2A sequence for co-translational cleavage to the 3' coding region of Sox17. Induction of Cre activity by administration of tamoxifen at defined time points of early mouse development and subsequent genetic lineage tracing confirmed the inducibility and tissue specificity of Cre recombination. Furthermore, Cre activity could be selectively induced in extra-embryonic and embryonic endoderm lineages, the primitive gut tube, and in endothelial cells of the vascular system as well as in the hemogenic endothelium of the dorsal aorta. The Sox17CreERT2 mouse line therefore represents a new tool for genetic lineage tracing in a tissue-specific manner and in addition enables lineage-restricted functional analysis. Copyright © 2013 Wiley Periodicals, Inc.

Molecular and genetic substrates linking stress and addiction.

PubMed

Briand, Lisa A; Blendy, Julie A

2010-02-16

Drug addiction is one of the top three health concerns in the United States in terms of economic and health care costs. Despite this, there are very few effective treatment options available. Therefore, understanding the causes and molecular mechanisms underlying the transition from casual drug use to compulsive drug addiction could aid in the development of treatment options. Studies in humans and animal models indicate that stress can lead to both vulnerability to develop addiction, and increased drug taking and relapse in addicted individuals. Exposure to stress or drugs of abuse results in long-term adaptations in the brain that are likely to involve persistent alterations in gene expression or activation of transcription factors, such as the cAMP Response Element Binding (CREB) protein. The signaling pathways controlled by CREB have been strongly implicated in drug addiction and stress. Many potential CREB target genes have been identified based on the presence of a CRE element in promoter DNA sequences. These include, but are not limited to CRF, BDNF, and dynorphin. These genes have been associated with initiation or reinstatement of drug reward and are altered in one direction or the other following stress. While many reviews have examined the interactions between stress and addiction, the goal of this review was to focus on specific molecules that play key roles in both stress and addiction and are therefore posed to mediate the interaction between the two. Focus on these molecules could provide us with new targets for pharmacological treatments for addiction. Copyright 2009 Elsevier B.V. All rights reserved.

High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting.

PubMed

Ackermann, Amanda M; Zhang, Jia; Heller, Aryel; Briker, Anna; Kaestner, Klaus H

2017-03-01

α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER "knock-in" mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreER T2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing. We utilized CRISPR-Cas9 technology to insert an IRES-CreER T2 sequence into the 3' UTR of the Glucagon ( Gcg ) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreER T2 mice. Recombination efficiency in GCG + pancreatic α-cells and glucagon-like peptide 1 positive (GLP1 + ) enteroendocrine L-cells was measured in Gcg-CreER T2 ; Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood. Tamoxifen injection of Gcg-CreER T2 ; Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the Gcg-CreER T2 allele were phenotypically normal. We successfully derived a Gcg-CreER T2 mouse line that expresses CreER T2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.

Differential Gene Dosage Effects of Diabetes-Associated Gene GLIS3 in Pancreatic β Cell Differentiation and Function

PubMed Central

Bush, Sean P.; Wen, Xianjie; Cao, Wei; Chan, Lawrence

2017-01-01

Mutations of GLI-similar 3 (GLIS3) underlie a neonatal diabetes syndrome. Genome-wide association studies revealed that GLIS3 variants are associated with both common type 1 and type 2 diabetes. Global Glis3-deficient (Glis3−/−) mice die of severe diabetes shortly after birth. GLIS3 controls islet differentiation by transactivating neurogenin 3 (Ngn3). To unravel the function of Glis3 in adults, we generated inducible global Glis3-deficient mice (Glis3fl/fl/RosaCreERT2). Tamoxifen (TAM)-treated Glis3fl/fl/RosaCreERT2 mice developed severe diabetes, which was reproduced in TAM-treated β cell–specific Glis3fl/fl/Pdx1CreERT mice, but not in TAM-treated Glis3fl/fl/MipCreERT mice. Furthermore, we generated constitutive β cell– or pancreas-specific Glis3-deficient mice using either RipCre (Glis3fl/fl/RipCre) or Pdx1Cre (Glis3fl/fl/Pdx1Cre) coexpressing mice. We observed that, remarkably, neither type of β cell– or pancreas-specific Glis3-deficient mice phenocopied the lethal neonatal diabetes observed in Glis3−/− mice. All Glis3fl/fl/RipCre mice survived to adulthood with normal glucose tolerance. Thirty percent of Glis3fl/fl/Pdx1Cre mice developed severe diabetes at 3 to 4 weeks of age, whereas 55% of them developed mild diabetes with age. In contrast to the >90% reduction of Ngn3 and near-total absence of insulin (Ins) in the embryonic pancreas of Glis3−/− mice, we found only 75%–80% reduction of Ngn3 and Ins messenger RNA or protein expression in the fetal pancreas of Glis3fl/fl/Pdx1Cre mice. The expression levels of Ngn3 and Ins correlated negatively with the extent of Cre-mediated Glis3 deletion. These mouse models are powerful tools to decipher Glis3 gene dosage effects and the role of GLIS3 mutations/variants in a spectrum of β cell dysfunction in people. PMID:27813676

Targeted deletion of RANKL in M cell inducer cells by the Col6a1-Cre driver.

PubMed

Nagashima, Kazuki; Sawa, Shinichiro; Nitta, Takeshi; Prados, Alejandro; Koliaraki, Vasiliki; Kollias, George; Nakashima, Tomoki; Takayanagi, Hiroshi

2017-11-04

The gut-associated lymphoid tissues (GALTs), including Peyer's patches (PPs), cryptopatches (CPs) and isolated lymphoid follicles (ILFs), establish a host-microbe symbiosis by the promotion of immune reactions against gut microbes. Microfold cell inducer (MCi) cells in GALTs are the recently identified mesenchymal cells that express the cytokine RANKL and initiate bacteria-specific immunoglobulin A (IgA) production via induction of microfold (M) cell differentiation. In the previous study, the Twist2-Cre driver was utilized for gene deletion in mesenchymal cells including MCi cells. In order to investigate MCi cells more extensively, it will be necessary to develop experimental tools in addition to the Twist2-Cre driver mice and characterize such drivers in specificity and efficiency. Here we show that M cell differentiation and IgA production are impaired in the targeted deletion of RANKL by the Col6a1-Cre driver. We compared Col6a1-Cre with Twist2-Cre in terms of the specificity for mesenchymal cells in GALTs. Col6a1-Cre CAG-CAT-EGFP mice exhibited EGFP expression in podoplanin + CD31 - cells including MCi cells, while Twist2-Cre mice were shown to target endothelial cells and podoplanin + CD31 - cells. Tnfsf11 fl/Δ Col6a1-Cre mice exhibited the absence of M cells and severe IgA reduction together with an alteration in gut microbial composition. Moreover, we analyzed germ free mice to test whether changes in the microbiota are the cause of M cell deficiency. M cell differentiation was normal in the CPs/ILFs of germ free mice, indicating that MCi cells induce M cells independently of microbial colonization. This study demonstrates that Col6a1-Cre driver mice are as useful as Twist2-Cre driver mice for functional analyses of GALT-resident mesenchymal cells, including MCi cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Rising Rates of Carbapenem-Resistant Enterobacteriaceae in Community Hospitals: A Mixed-Methods Review of Epidemiology and Microbiology Practices in a Network of Community Hospitals in the Southeastern United States

PubMed Central

Thaden, Joshua T.; Lewis, Sarah S.; Hazen, Kevin C.; Huslage, Kirk; Fowler, Vance G.; Moehring, Rebekah W.; Chen, Luke F.; Jones, Constance D.; Moore, Zack S.; Sexton, Daniel J.; Anderson, Deverick J.

2014-01-01

OBJECTIVE Describe the epidemiology of carbapenem-resistant Enterobacteriaceae (CRE) and examine the effect of lower carbapenem breakpoints on CRE detection. DESIGN Retrospective cohort. SETTING Inpatient care at community hospitals. PATIENTS All patients with CRE-positive cultures were included. METHODS CRE isolated from 25 community hospitals were prospectively entered into a centralized database from January 2008 through December 2012. Microbiology laboratory practices were assessed using questionnaires. RESULTS A total of 305 CRE isolates were detected at 16 hospitals (64%). Patients with CRE had symptomatic infection in 180 cases (59%) and asymptomatic colonization in the remainder (125 cases; 41%). Klebsiella pneumoniae (277 isolates; 91%) was the most prevalent species. The majority of cases were healthcare associated (288 cases; 94%). The rate of CRE detection increased more than fivefold from 2008 (0.26 cases per 100,000 patient-days) to 2012 (1.4 cases per 100,000 patient-days; incidence rate ratio (IRR), 5.3 [95% confidence interval (CI), 1.22–22.7]; P = .01). Only 5 hospitals (20%) had adopted the 2010 Clinical and Laboratory Standards Institute (CLSI) carbapenem breakpoints. The 5 hospitals that adopted the lower carbapenem breakpoints were more likely to detect CRE after implementation of breakpoints than before (4.1 vs 0.5 cases per 100,000 patient-days; P < .001; IRR, 8.1 [95% CI, 2.7–24.6]). Hospitals that implemented the lower carbapenem breakpoints were more likely to detect CRE than were hospitals that did not (3.3 vs 1.1 cases per 100,000 patientdays; P = .01). CONCLUSIONS The rate of CRE detection increased fivefold in community hospitals in the southeastern United States from 2008 to 2012. Despite this, our estimates are likely underestimates of the true rate of CRE detection, given the low adoption of the carbapenem breakpoints recommended in the 2010 CLSI guidelines. PMID:25026612

Insig proteins mediate feedback inhibition of cholesterol synthesis in the intestine.

PubMed

McFarlane, Matthew R; Liang, Guosheng; Engelking, Luke J

2014-01-24

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.

Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine*

PubMed Central

McFarlane, Matthew R.; Liang, Guosheng; Engelking, Luke J.

2014-01-01

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes. PMID:24337570

Plasma cholesterol-lowering and transient liver dysfunction in mice lacking squalene synthase in the liver[S

PubMed Central

Nagashima, Shuichi; Yagyu, Hiroaki; Tozawa, Ryuichi; Tazoe, Fumiko; Takahashi, Manabu; Kitamine, Tetsuya; Yamamuro, Daisuke; Sakai, Kent; Sekiya, Motohiro; Okazaki, Hiroaki; Osuga, Jun-ichi; Honda, Akira; Ishibashi, Shun

2015-01-01

Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality. PMID:25755092

SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation

PubMed Central

Matsuda, Morihiro; Korn, Bobby S.; Hammer, Robert E.; Moon, Young-Ah; Komuro, Ryutaro; Horton, Jay D.; Goldstein, Joseph L.; Brown, Michael S.; Shimomura, Iichiro

2001-01-01

In liver, the synthesis of cholesterol and fatty acids increases in response to cholesterol deprivation and insulin elevation, respectively. This regulatory mechanism underlies the adaptation to cholesterol synthesis inhibitors (statins) and high calorie diets (insulin). In nonhepatic cells, lipid synthesis is controlled by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose active domains are released proteolytically to enter the nucleus and activate genes involved in the synthesis and uptake of cholesterol and fatty acids. SCAP (SREBP cleavage-activating protein) is a sterol-regulated escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of cleavage in the Golgi. Here, we produced a conditional deficiency of SCAP in mouse liver by genomic recombination mediated by inducible Cre recombinase. SCAP-deficient mice showed an 80% reduction in basal rates of cholesterol and fatty acid synthesis in liver, owing to decreases in mRNAs encoding multiple biosynthetic enzymes. Moreover, these mRNAs failed to increase normally in response to cholesterol deprivation produced by a cholesterol synthesis inhibitor and to insulin elevation produced by a fasting–refeeding protocol. These data provide in vivo evidence that SCAP and the SREBPs are required for hepatic lipid synthesis under basal and adaptive conditions. PMID:11358865

Longwave Band-by-band Cloud Radiative Effect and its Application in GCM Evaluation

NASA Technical Reports Server (NTRS)

Huang, Xianglei; Cole, Jason N. S.; He, Fei; Potter, Gerald L.; Oreopoulos, Lazaros; Lee, Dongmin; Suarez, Max; Loeb, Norman G.

2012-01-01

The cloud radiative effect (CRE) of each longwave (LW) absorption band of a GCM fs radiation code is uniquely valuable for GCM evaluation because (1) comparing band-by-band CRE avoids the compensating biases in the broadband CRE comparison and (2) the fractional contribution of each band to the LW broadband CRE (f(sub CRE)) is sensitive to cloud top height but largely insensitive to cloud fraction, presenting thus a diagnostic metric to separate the two macroscopic properties of clouds. Recent studies led by the first author have established methods to derive such band ]by ]band quantities from collocated AIRS and CERES observations. We present here a study that compares the observed band-by-band CRE over the tropical oceans with those simulated by three different atmospheric GCMs (GFDL AM2, NASA GEOS-5, and CCCma CanAM4) forced by observed SST. The models agree with observation on the annual ]mean LW broadband CRE over the tropical oceans within +/-1W/sq m. However, the differences among these three GCMs in some bands can be as large as or even larger than +/-1W/sq m. Observed seasonal cycles of f(sub CRE) in major bands are shown to be consistent with the seasonal cycle of cloud top pressure for both the amplitude and the phase. However, while the three simulated seasonal cycles of f(sub CRE) agree with observations on the phase, the amplitudes are underestimated. Simulated interannual anomalies from GFDL AM2 and CCCma CanAM4 are in phase with observed anomalies. The spatial distribution of f(sub CRE) highlights the discrepancies between models and observation over the low-cloud regions and the compensating biases from different bands.

The nutritive value of cassava starch extraction residue for growing ducks.

PubMed

Abouelezz, Khaled; Yuan, Jianfeng; Wang, Guiping; Bian, Guozhi

2018-02-26

The cassava root meal (CRM) has been utilized as a cheap energy alternative to replace maize in poultry diets. Recently, the CRM in turn has an increasing demand for starch extraction industry, which renders large amounts of residues. This study evaluated the nutrient composition, amino acid profile, and feeding value of cassava starch extraction residue meal (CReM) for growing ducks. A total of 960, 11-day-old, ducklings were housed in 24 floor pens and allocated randomly into four dietary treatments: (i) 0CReM (control), (ii) 50 g CReM/kg, (iii) 100 g CReM/kg, and (iv) 150 g CReM/kg. The analyses (/kg) of CReM showed high gross energy (3306.88 kcal), ME (2109.54 kcal), and starch (514.0 g), with poor crude protein (20.9 g) and moderate crude fiber (140.0 g) and ash (60.0 g) contents. The total amino acid (AA) content amounted to 19.9 g/kg of CReM DM, in which the methionine, lysine, cystine, and isoleucine were present in low levels. The dietary inclusion of CReM up to 150 g/kg, between 11 and 42 days of age, had no significant effects (P > 0.05) on duck growth parameters, mortality, dressed weight, internal organs, or abdominal fat. Besides, the tested CReM levels did not show any significant effect on the blood proteins or liver enzymes. The results, therefore, revealed that the CReM contains a considerable amount of energy and could be incorporated successfully up to 150 g/kg in the diets of growing ducks.

Carbapenem-resistant Enterobacteriaceae colonization and infection in critically ill patients: a retrospective matched cohort comparison with non-carriers.

PubMed

Dickstein, Y; Edelman, R; Dror, T; Hussein, K; Bar-Lavie, Y; Paul, M

2016-09-01

To examine whether carbapenem-resistant Enterobacteriaceae (CRE) carriage is associated with incidence of clinical infection as a means of assessing whether the morbidity and mortality associated with these bacteria are mediated by underlying conditions or intrinsic properties of CRE. This retrospective matched cohort study compared the incidence of invasive infections in CRE-colonized patients and matched non-carriers in the intensive care unit (ICU). The primary outcome was infection caused by CRE of the same species as the colonizing strain among CRE carriers, and infections caused by carbapenem-sensitive strains of the same organism in non-carriers. Hospital discharge and death were considered as competing events. Competing-risks hazard analysis was performed for the entire cohort and for a nested cohort matched by Acute Physiology and Chronic Health Evaluation (APACHE) II scores, stratified by matching. In total, 146 CRE carriers were compared with 292 non-carriers. Patients were well matched for most risk factors for Enterobacteriaceae infection, including age, renal failure, previous invasive infection, previous hospitalization, APACHE II score, length of mechanical ventilation, length of hospitalization and CRE carriage. On regression analysis, colonization with CRE was independently associated with Enterobacteriaceae infection {cause-specific hazard ratio (CSHR) 2.06 [95% confidence interval (CI) 1.03-4.09]}. On regression analysis of the APACHE-II-matched cohort (N=284), colonization with CRE remained significantly associated with Enterobacteriaceae infection [CSHR 3.32 (95% CI 1.31-8.43)]. Colonization with CRE was associated with at least a two-fold increased risk of infection by the colonizing strain amongst ICU patients. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Pioglitazone Induces a Proadipogenic Antitumor Response in Mice with PAX8-PPARγ Fusion Protein Thyroid Carcinoma

PubMed Central

Dobson, Melissa E.; Diallo-Krou, Ericka; Grachtchouk, Vladimir; Yu, Jingcheng; Colby, Lesley A.; Wilkinson, John E.; Giordano, Thomas J.

2011-01-01

Approximately 35% of follicular thyroid carcinomas harbor a chromosomal translocation that results in expression of a paired box gene 8-peroxisome proliferator-activated receptor γ gene (PPARγ) fusion protein (PPFP). To better understand the oncogenic role of PPFP and its relationship to endogenous PPARγ, we generated a transgenic mouse model that combines Cre-dependent PPFP expression (PPFP;Cre) with homozygous deletion of floxed Pten (PtenFF;Cre), both thyroid specific. Although neither PPFP;Cre nor PtenFF;Cre mice develop thyroid tumors, the combined PPFP;PtenFF;Cre mice develop metastatic thyroid cancer, consistent with patient data that PPFP is occasionally found in benign thyroid adenomas and that PPFP carcinomas have increased phosphorylated AKT/protein kinase B. We then tested the effects of the PPARγ agonist pioglitazone in our mouse model. Pioglitazone had no effect on PtenFF;Cre mouse thyroids. However, the thyroids in pioglitazone-fed PPFP;PtenFF;Cre mice decreased 7-fold in size, and metastatic disease was prevented. Remarkably, pioglitazone caused an adipogenic response in the PPFP;PtenFF;Cre thyroids characterized by lipid accumulation and the induction of a broad array of adipocyte PPARγ target genes. These data indicate that, in the presence of pioglitazone, PPFP has PPARγ-like activity that results in trans-differentiation of thyroid carcinoma cells into adipocyte-like cells. Furthermore, the data predict that pioglitazone will be therapeutic in patients with PPFP-positive carcinomas. PMID:21952241

Aspm, a Key Element in Medulloblastoma Pathogenesis and a Novel Target for Treatment

DTIC Science & Technology

2013-10-01

pathogenesis ( Wechsler -Reya and Scott, 1999; Kenney and Rowitch, 2000; Zurawel et al., 2000; Ellison et al., 2011; Hatten and Roussel, 2011; Roussel...Laboratories, Bar Harbor, ME, USA) to generate Aspm floxed (Aspmf/+) mice. Math-1 cre mice were generously shared by David Rowitch, MD, PhD, UCSF and...Robert Wechsler -Reya, PhD, 10 Sanford-Burnham Medical Research Institute, La Jolla, CA and have been previously described (Matei et al., 2005

AGE (Argon Geochronology Experiment): An Instrument for Geochronology on the Surface of Mars

NASA Technical Reports Server (NTRS)

Swindle, T. D.; Bode, R.; Boynton, W. V.; Kring, D. A.; Williams, M.; Chutjian, A.; Darrach, M. R.; Cremers, D. A.; Wiens, R. C.; Baldwin, S. L.

2003-01-01

As our knowledge of the planet Mars continues to grow, one parameter that remains elusive is the absolute chronology of the planet s geological history. Although crater counts have provided a robust relative chronology, impactor fluxes are poorly enough known that there are places on Mars where the absolute age is uncertain by a factor of two or more. To resolve these uncertainties, it will be necessary to either analyze well-documented samples returned to the Earth from the Martian surface or to perform in situ measurements with sufficient precision. Sample return is still at least a decade away, and even then it might be from a biologically interesting area that might be geologically complex. Hence an in situ measurement, within an uncertainty of 20% or better, could greatly improve our knowledge of the history of Mars. With funding from the Planetary Instrument Definition and Development Program (PIDDP), we have been working on an instrument to perform potassium-argon (K-Ar) and cosmic-ray exposure (CRE) dating in situ on the surface of Mars. For either of these techniques, it is necessary to measure the abundance of one or more major or minor elements (K in the case of KAr; all majors and minors in the case of CRE) and the abundance and isotopes composition of a noble gas (Ar in the case of K-Ar; He, Ne and Ar for CRE dating). The technology for either of these types of measurements exists, but has never before been integrated for a spacecraft. We refer to the instrument as AGE, the Argon Geochronology Experiment (although we will measure the noble gases He and Ne as well for CRE ages). We report here on the basic components that go into such an instrument, both those that use existing technology and those that had to be developed to create the integrated package.

Utility of HoxB2 enhancer-mediated Cre activity for functional studies in the developing inner ear.

PubMed

Szeto, Irene Y Y; Leung, Keith K H; Sham, Mai Har; Cheah, Kathryn S E

2009-06-01

The rhombomere 4(r4)-restricted expression of the mouse Hoxb2 gene is regulated by a 1.4-kb enhancer-containing fragment. Here, we showthat transgenic mouse lines expressing cre driven by this fragment (B2-r4-Cre), activated the R26R Cre reporter in rhombomere 4 and the second branchial arch, the epithelium of the first branchial arch, apical ectodermal ridge of the limb buds and the tail region. Of particular interest is Cre activity in the developing inner ear. Cre activity was found in the preotic field and otic placode at E8.5 and otocyst at E9.5-E12.5, in the cochleovestibular and facio-acoustic ganglia at E10.5 and the vestibular and spiral ganglia and all the otic epithelia derived from the otocyst at E15.5 and P0. Our data suggest that the B2-r4-Cre transgenic mice provide an important tool for conditional gene manipulation and lineage tracing in the inner ear. In combination with other transgenic lines expressing cre exclusively in the otic vesicle, the relative contributions of the hindbrain, periotic mesenchyme and otic epithelium in otic development can be dissected. Copyright 2009 Wiley-Liss, Inc.

Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells.

PubMed

Jossin, Yves; Lee, Minhui; Klezovitch, Olga; Kon, Elif; Cossard, Alexia; Lien, Wen-Hui; Fernandez, Tania E; Cooper, Jonathan A; Vasioukhin, Valera

2017-06-05

Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1 fl/fl ), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1 fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Rce1, a novel transcriptional repressor, regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei.

PubMed

Cao, Yanli; Zheng, Fanglin; Wang, Lei; Zhao, Guolei; Chen, Guanjun; Zhang, Weixin; Liu, Weifeng

2017-07-01

Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1-mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1-encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy-efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei. © 2017 John Wiley & Sons Ltd.

Cre-mediated recombination in pituitary somatotropes

PubMed Central

Nasonkin, Igor O.; Potok, Mary Anne; Camper, Sally A.

2009-01-01

We report a transgenic line with highly penetrant cre recombinase activity in the somatotrope cells of the anterior pituitary gland. Expression of the cre transgene is under the control of the locus control region of the human growth hormone gene cluster and the rat growth hormone promoter. Cre recombinase activity was assessed with two different lacZ reporter genes that require excision of a floxed stop sequence for expression: a chick β-actin promoter with the CMV enhancer transgene and a ROSA26 knock-in. Cre activity is detectable in the developing pituitary after initiation of Gh transcription and persists through adulthood with high penetrance in Gh expressing cells and lower penetrance in lactotropes, a cell type that shares a common origin with somatotropes. This Gh-cre transgenic line is suitable for efficient, cell-specific deletion of floxed regions of genomic DNA in differentiated somatotropes and a subset of lactotrope cells of the anterior pituitary gland. PMID:19039787

Cell type-specific manipulation with GFP-dependent Cre recombinase.

PubMed

Tang, Jonathan C Y; Rudolph, Stephanie; Dhande, Onkar S; Abraira, Victoria E; Choi, Seungwon; Lapan, Sylvain W; Drew, Iain R; Drokhlyansky, Eugene; Huberman, Andrew D; Regehr, Wade G; Cepko, Constance L

2015-09-01

There are many transgenic GFP reporter lines that allow the visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. We developed a method that exploits GFP for gene manipulation, Cre recombinase dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, which led to effective recombination selectively in GFP-labeled cells. Furthermore, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP(+) cells, we found that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins.

Carbapenem resistance, inappropriate empiric treatment and outcomes among patients hospitalized with Enterobacteriaceae urinary tract infection, pneumonia and sepsis.

PubMed

Zilberberg, Marya D; Nathanson, Brian H; Sulham, Kate; Fan, Weihong; Shorr, Andrew F

2017-04-17

Drug resistance among gram-negative pathogens is a risk factor for inappropriate empiric treatment (IET), which in turn increases the risk for mortality. We explored the impact of carbapenem-resistant Enterobacteriaceae (CRE) on the risk of IET and of IET on outcomes in patients with Enterobacteriaceae infections. We conducted a retrospective cohort study in Premier Perspective database (2009-2013) of 175 US hospitals. We included all adult patients with community-onset culture-positive urinary tract infection (UTI), pneumonia, or sepsis as a principal diagnosis, or as a secondary diagnosis in the setting of respiratory failure, treated with antibiotics within 2 days of admission. We employed regression modeling to compute adjusted association of presence of CRE with risk of receiving IET, and of IET on hospital mortality, length of stay (LOS) and costs. Among 40,137 patients presenting to the hospital with an Enterobacteriaceae UTI, pneumonia or sepsis, 1227 (3.1%) were CRE. In both groups, the majority of the cases were UTI (51.4% CRE and 54.3% non-CRE). Those with CRE were younger (66.6+/-15.3 vs. 69.1+/-15.9 years, p < 0.001), and more likely to be African-American (19.7% vs. 14.0%, p < 0.001) than those with non-CRE. Both chronic (Charlson score 2.0+/-2.0 vs. 1.9+/-2.1, p = 0.009) and acute (by day 2: ICU 56.3% vs. 30.4%, p < 0.001, and mechanical ventilation 35.8% vs. 11.7%, p < 0.001) illness burdens were higher among CRE than non-CRE subjects, respectively. CRE patients were 3× more likely to receive IET than non-CRE (46.5% vs. 11.8%, p < 0.001). In a regression model CRE was a strong predictor of receiving IET (adjusted relative risk ratio 3.95, 95% confidence interval 3.5 to 4.5, p < 0.001). In turn, IET was associated with an adjusted rise in mortality of 12% (95% confidence interval 3% to 23%), and an excess of 5.2 days (95% confidence interval 4.8, 5.6, p < 0.001) LOS and $10,312 (95% confidence interval $9497, $11,126, p < 0.001) in costs. In this large US database, the prevalence of CRE among patients with Enterobacteriaceae UTI, pneumonia or sepsis was comparable to other national estimates. Infection with CRE was associated with a four-fold increased risk of receiving IET, which in turn increased mortality, LOS and costs.

AKI after Conditional and Kidney-Specific Knockdown of Stanniocalcin-1

PubMed Central

Huang, Luping; Belousova, Tatiana; Pan, Jenny Szu-Chin; Du, Jie; Ju, Huiming; Lu, Lianghao; Zhang, Pumin; Truong, Luan D.; Nuotio-Antar, Alli

2014-01-01

Stanniocalcin-1 is an intracrine protein; it binds to the cell surface, is internalized to the mitochondria, and diminishes superoxide generation through induction of uncoupling proteins. In vitro, stanniocalcin-1 inhibits macrophages and preserves endothelial barrier function, and transgenic overexpression of stanniocalcin-1 in mice protects against ischemia-reperfusion kidney injury. We sought to determine the kidney phenotype after kidney endothelium-specific expression of stanniocalcin-1 small hairpin RNA (shRNA). We generated transgenic mice that express stanniocalcin-1 shRNA or scrambled shRNA upon removal of a floxed reporter (phosphoglycerate kinase-driven enhanced green fluorescent protein) and used ultrasound microbubbles to deliver tyrosine kinase receptor-2 promoter-driven Cre to the kidney to permit kidney endothelium-specific shRNA expression. Stanniocalcin-1 mRNA and protein were expressed throughout the kidney in wild-type mice. Delivery of tyrosine kinase receptor-2 promoter-driven Cre to stanniocalcin-1 shRNA transgenic kidneys diminished the expression of stanniocalcin-1 mRNA and protein throughout the kidneys. Stanniocalcin-1 mRNA and protein expression did not change in similarly treated scrambled shRNA transgenic kidneys, and we observed no Cre protein expression in cultured and tyrosine kinase receptor-2 promoter-driven Cre–transfected proximal tubule cells, suggesting that knockdown of stanniocalcin-1 in epithelial cells in vivo may result from stanniocalcin-1 shRNA transfer from endothelial cells to epithelial cells. Kidney-specific knockdown of stanniocalcin-1 led to severe proximal tubule injury characterized by vacuolization, decreased uncoupling of protein-2 expression, greater generation of superoxide, activation of the unfolded protein response, initiation of autophagy, cell apoptosis, and kidney failure. Our observations suggest that stanniocalcin-1 is critical for tubular epithelial survival under physiologic conditions. PMID:24700878

Conditional Deletion of Murine Fgf23: Interruption of the Normal Skeletal Responses to Phosphate Challenge and Rescue of Genetic Hypophosphatemia.

PubMed

Clinkenbeard, Erica L; Cass, Taryn A; Ni, Pu; Hum, Julia M; Bellido, Teresita; Allen, Matthew R; White, Kenneth E

2016-06-01

The transgenic and knockout (KO) animals involving Fgf23 have been highly informative in defining novel aspects of mineral metabolism, but are limited by shortened lifespan, inability of spatial/temporal FGF23 control, and infertility of the global KO. To more finely test the role of systemic and genetic influences in FGF23 production, a mouse was developed that carried a floxed ("f")-Fgf23 allele (exon 2 floxed) which demonstrated in vivo recombination when bred to global-Cre transgenic mice (eIIa-cre). Mice homozygous for the recombined allele ("Δ") had undetectable serum intact FGF23, elevated serum phosphate (p < 0.05), and increased kidney Cyp27b1 mRNA (p < 0.05), similar to global Fgf23-KO mice. To isolate cellular FGF23 responses during phosphate challenge, Fgf23(Δ/f) mice were mated with early osteoblast type Iα1 collagen 2.3-kb promoter-cre mice (Col2.3-cre) and the late osteoblast/early osteocyte Dentin matrix protein-1-cre (Dmp1-cre). Fgf23(Δ/f) /Col2.3-cre(+) and Fgf23(Δ/f) /Dmp1-cre(+) exhibited reduced baseline serum intact FGF23 versus controls. After challenge with high-phosphate diet Cre(-) mice had 2.1-fold to 2.5-fold increased serum FGF23 (p < 0.01), but Col2.3-cre(+) mice had no significant increase, and Dmp1-cre(+) mice had only a 37% increase (p < 0.01) despite prevailing hyperphosphatemia in both models. The Fgf23(Δ/f) /Col2.3-cre was bred onto the Hyp (murine X-linked hypophosphatemia [XLH] model) genetic background to test the contribution of osteoblasts and osteocytes to elevated FGF23 and Hyp disease phenotypes. Whereas Hyp mice maintained inappropriately elevated FGF23 considering their marked hypophosphatemia, Hyp/Fgf23(Δ/f) /Col2.3-cre(+) mice had serum FGF23 <4% of Hyp (p < 0.01), and this targeted restriction normalized serum phosphorus and ricketic bone disease. In summary, deleting FGF23 within early osteoblasts and osteocytes demonstrated that both cell types contribute to baseline circulating FGF23 concentrations, and that targeting osteoblasts/osteocytes for FGF23 production can modify systemic responses to changes in serum phosphate concentrations and rescue the Hyp genetic syndrome. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for Bone and Mineral Research.

Mast cells promote melanoma colonization of lungs.

PubMed

Öhrvik, Helena; Grujic, Mirjana; Waern, Ida; Gustafson, Ann-Marie; Ernst, Nancy; Roers, Axel; Hartmann, Karin; Pejler, Gunnar

2016-10-18

Mast cells have been implicated in malignant processes, mainly through clinical correlative studies and by experiments performed using animals lacking mast cells due to defective c-kit signaling. However, mast cell-deficient mouse models based on c-kit defects have recently been questioned for their relevance. Here we addressed the effect of mast cells in a tumor setting by using transgenic Mcpt5-Cre+ R-DTA+ mice, in which the deficiency of mast cells is independent of c-kit defects. Melanoma cells (B16.F10) were administered either subcutaneously or intravenously into Mcpt5-Cre+ R-DTA+ mice or Mcpt5-Cre- R-DTA+ littermate controls, followed by the assessment of formed tumors. In the subcutaneous model, mast cells were abundant in the tumor stroma of control mice but were absent in Mcpt5-Cre+ R-DTA+ mice. However, the absence of mast cells did not affect tumor size. In contrast, after intravenous administration of B16.F10 cells, melanoma colonization of the lungs was markedly reduced in Mcpt5-Cre+ R-DTA+ vs. Mcpt5-Cre- R-DTA+ animals. Decreased melanoma colonization of the lungs in Mcpt5-Cre+ R-DTA+ animals was accompanied by increased inflammatory cell recruitment into the bronchoalveolar lavage fluid, suggesting that mast cells suppress inflammation in this setting. Further, qPCR analysis revealed significant alterations in the expression of Twist and E-cadherin in lungs of Mcpt5-Cre+ R-DTA+ vs. control Mcpt5-Cre- R-DTA+ animals, suggesting an impact of mast cells on epithelial-mesenchymal transition. In conclusion, this study reveals that mast cells promote melanoma colonization of the lung.

Fabp4-Cre-mediated Sirt6 deletion impairs adipose tissue function and metabolic homeostasis in mice.

PubMed

Xiong, Xiwen; Zhang, Cuicui; Zhang, Yang; Fan, Rui; Qian, Xinlai; Dong, X Charlie

2017-06-01

SIRT6 is a member of sirtuin family of deacetylases involved in diverse processes including genome stability, metabolic homeostasis and anti-inflammation. However, its function in the adipose tissue is not well understood. To examine the metabolic function of SIRT6 in the adipose tissue, we generated two mouse models that are deficient in Sirt6 using the Cre-lox approach. Two commonly used Cre lines that are driven by either the mouse Fabp4 or Adipoq gene promoter were chosen for this study. The Sirt6- knockout mice generated by the Fabp4-Cre line ( Sirt6 f/f : Fabp4-Cre) had a significant increase in both body weight and fat mass and exhibited glucose intolerance and insulin resistance as compared with the control wild-type mice. At the molecular levels, the Sirt6 f/f :Fabp4-Cre-knockout mice had increased expression of inflammatory genes including F4/80, TNFα, IL-6 and MCP-1 in both white and brown adipose tissues. Moreover, the knockout mice showed decreased expression of the adiponectin gene in the white adipose tissue and UCP1 in the brown adipose tissue, respectively. In contrast, the Sirt6 knockout mice generated by the Adipoq-Cre line ( Sirt6 f/f :Adipoq-Cre) only had modest insulin resistance. In conclusion, our data suggest that the function of SIRT6 in the Fabp4-Cre-expressing cells in addition to mature adipocytes plays a critical role in body weight maintenance and metabolic homeostasis. © 2017 Society for Endocrinology.

Conditional Deletion of Murine Fgf23: Interruption of the Normal Skeletal Responses to Phosphate Challenge and Rescue of Genetic Hypophosphatemia

PubMed Central

Clinkenbeard, Erica L.; Cass, Taryn A.; Ni, Pu; Hum, Julia M.; Bellido, Teresita; Allen, Matthew R.; White, Kenneth E.

2016-01-01

The transgenic and knock out (KO) animals involving Fgf23 have been highly informative in defining novel aspects of mineral metabolism, but are limited by shortened life span, inability of spatial/temporal FGF23 control, and infertility of the global KO. To more finely test the role of systemic and genetic influences in FGF23 production, a mouse was developed that carried a floxed (‘f’)-Fgf23 allele (exon 2 floxed) which demonstrated in vivo recombination when bred to global-Cre transgenic mice (eIIa-cre). Mice homozygous for the recombined allele (‘Δ’) had undetectable serum intact FGF23, elevated serum phosphate (p<0.05), and increased kidney Cyp27b1 mRNA (p<0.05) similar to global Fgf23-KO mice. To isolate cellular FGF23 responses during phosphate challenge Fgf23Δ/f mice were mated with early osteoblast type Iα1 collagen 2.3kb promoter-cre mice (Col2.3-cre) and the late osteoblast/early osteocyte Dentin matrix protein-1-cre (Dmp1-cre). Fgf23Δ/f/Col2.3-cre+ and Fgf23Δ/f/Dmp1-cre+ exhibited reduced baseline serum intact FGF23 versus controls. After challenge with high phosphate diet Cre− mice had 2.1–2.5 fold increased serum FGF23 (p<0.01), but Col2.3-cre+ mice had no significant increase, and Dmp1-cre+ mice had only a 37% increase (p<0.01) despite prevailing hyperphosphatemia in both models. The Fgf23Δ/f/Col2.3-cre was bred onto the Hyp (murine XLH model) genetic background to test the contribution of osteoblasts and osteocytes to elevated FGF23 and Hyp disease phenotypes. Whereas Hyp mice maintained inappropriately elevated FGF23 considering their marked hypophosphatemia, Hyp/Fgf23Δ/f/Col2.3-cre+ mice had serum FGF23 <4% of Hyp (p<0.01), and this targeted restriction normalized serum phosphorus and ricketic bone disease. In summary, deleting FGF23 within early osteoblasts and osteocytes demonstrated that both cell types contribute to baseline circulating FGF23 concentrations, and that targeting osteoblasts/osteocytes for FGF23 production can modify systemic responses to changes in serum phosphate concentrations and rescue the Hyp genetic syndrome. PMID:26792657

A Stabilizing Feedback Between Cloud Radiative Effects and Greenland Surface Melt: Verification From Multi-year Automatic Weather Station Measurements

NASA Astrophysics Data System (ADS)

Zender, C. S.; Wang, W.; van As, D.

2017-12-01

Clouds have strong impacts on Greenland's surface melt through the interaction with the dry atmosphere and reflective surfaces. However, their effects are uncertain due to the lack of in situ observations. To better quantify cloud radiative effects (CRE) in Greenland, we analyze and interpret multi-year radiation measurements from 30 automatic weather stations encompassing a broad range of climatological and topographical conditions. During melt season, clouds warm surface over most of Greenland, meaning the longwave greenhouse effect outweighs the shortwave shading effect; on the other hand, the spatial variability of net (longwave and shortwave) CRE is dominated by shortwave CRE and in turn by surface albedo, which controls the potential absorption of solar radiation when clouds are absent. The net warming effect decreases with shortwave CRE from high to low altitudes and from north to south (Fig. 1). The spatial correlation between albedo and net CRE is strong (r=0.93, p<<0.01). In the accumulation zone, the net CRE seasonal trend is controlled by longwave CRE associated with cloud fraction and liquid water content. It becomes stronger from May to July and stays constant in August. In the ablation zone, albedo determines the net CRE seasonal trend, which decreases from May to July and increases afterwards. On an hourly timescale, we find two distinct radiative states in Greenland (Fig. 2). The clear state is characterized by clear-sky conditions or thin clouds, when albedo and solar zenith angle (SZA) weakly correlates with CRE. The cloudy state is characterized by opaque clouds, when the combination of albedo and SZA strongly correlates with CRE (r=0.85, p<0.01). Although cloud properties intrinsically affect CRE, the large melt-season variability of these two non-cloud factors, albedo and solar zenith angle, explains the majority of the CRE variation in spatial distribution, seasonal trend in the ablation zone, and in hourly variability in the cloudy radiative state. Clouds warm the brighter and colder surfaces of Greenland, enhance snow melt, and tend to lower the albedo. Clouds cool the darker and warmer surfaces, inhibiting snow melt, which increases albedo, and thus stabilizes surface melt. This stabilizing mechanism may also occur over sea ice, helping to forestall surface melt as the Arctic becomes dimmer.

Redundancy in Kiss1 Expression Safeguards Reproduction in the Mouse

PubMed Central

Popa, Simina M.; Moriyama, Ryutaro M.; Caligioni, Claudia S.; Yang, Jasmine J.; Cho, Caroline M.; Concepcion, Tessa L.; Oakley, Amy E.; Lee, In Hae; Sanz, Elisenda; Amieux, Paul S.; Caraty, Alain; Palmiter, Richard D.; Navarro, Victor M.; Chan, Yee-Ming; Seminara, Stephanie B.; Clifton, Donald K.

2013-01-01

Kisspeptin (Kiss1) signaling to GnRH neurons is widely acknowledged to be a prerequisite for puberty and reproduction. Animals lacking functional genes for either kisspeptin or its receptor exhibit low gonadotropin secretion and infertility. Paradoxically, a recent study reported that genetic ablation of nearly all Kiss1-expressing neurons (Kiss1 neurons) does not impair reproduction, arguing that neither Kiss1 neurons nor their products are essential for sexual maturation. We posited that only minute quantities of kisspeptin are sufficient to support reproduction. If this were the case, animals having dramatically reduced Kiss1 expression might retain fertility, testifying to the redundancy of Kiss1 neurons and their products. To test this hypothesis and to determine whether males and females differ in the required amount of kisspeptin needed for reproduction, we used a mouse (Kiss1-CreGFP) that has a severe reduction in Kiss1 expression. Mice that are heterozygous and homozygous for this allele (Kiss1Cre/+ and Kiss1Cre/Cre) have ∼50% and 95% reductions in Kiss1 transcript, respectively. We found that although male Kiss1Cre/Cre mice sire normal-sized litters, female Kiss1Cre/Cre mice exhibit significantly impaired fertility and ovulation. These observations suggest that males require only 5% of normal Kiss1 expression to be reproductively competent, whereas females require higher levels for reproductive success. PMID:23736293

Performance evaluation of three automated identification systems in detecting carbapenem-resistant Enterobacteriaceae.

PubMed

He, Qingwen; Chen, Weiyuan; Huang, Liya; Lin, Qili; Zhang, Jingling; Liu, Rui; Li, Bin

2016-06-21

Carbapenem-resistant Enterobacteriaceae (CRE) is prevalent around the world. Rapid and accurate detection of CRE is urgently needed to provide effective treatment. Automated identification systems have been widely used in clinical microbiology laboratories for rapid and high-efficient identification of pathogenic bacteria. However, critical evaluation and comparison are needed to determine the specificity and accuracy of different systems. The aim of this study was to evaluate the performance of three commonly used automated identification systems on the detection of CRE. A total of 81 non-repetitive clinical CRE isolates were collected from August 2011 to August 2012 in a Chinese university hospital, and all the isolates were confirmed to be resistant to carbapenems by the agar dilution method. The potential presence of carbapenemase genotypes of the 81 isolates was detected by PCR and sequencing. Using 81 clinical CRE isolates, we evaluated and compared the performance of three automated identification systems, MicroScan WalkAway 96 Plus, Phoenix 100, and Vitek 2 Compact, which are commonly used in China. To identify CRE, the comparator methodology was agar dilution method, while the PCR and sequencing was the comparator one to identify CPE. PCR and sequencing analysis showed that 48 of the 81 CRE isolates carried carbapenemase genes, including 23 (28.4 %) IMP-4, 14 (17.3 %) IMP-8, 5 (6.2 %) NDM-1, and 8 (9.9 %) KPC-2. Notably, one Klebsiella pneumoniae isolate produced both IMP-4 and NDM-1. One Klebsiella oxytoca isolate produced both KPC-2 and IMP-8. Of the 81 clinical CRE isolates, 56 (69.1 %), 33 (40.7 %) and 77 (95.1 %) were identified as CRE by MicroScan WalkAway 96 Plus, Phoenix 100, and Vitek 2 Compact, respectively. The sensitivities/specificities of MicroScan WalkAway, Phoenix 100 and Vitek 2 were 93.8/42.4 %, 54.2/66.7 %, and 75.0/36.4 %, respectively. The MicroScan WalkAway and Viteck2 systems are more reliable in clinical identification of CRE, whereas additional tests are required for the Pheonix 100 system. Our study provides a useful guideline for using automated identification systems for CRE identification.

CDC Vital Signs: Making Health Care Safer -- Stop Infections from Lethal CRE Germs Now

MedlinePlus

... recommendations when treating patients with CRE. Dedicate rooms, staff, and equipment to patients with CRE. Prescribe antibiotics ... coordinated, and consistent effort by doctors, nurses, lab staff, medical facility leadership, health departments/states, policy makers, ...

A Comparison Study and Software Implementation of NORDA Ocean Models.

DTIC Science & Technology

1980-10-08

L01?C07 ’EEEEElshhhh A COMPARISON STUDY AND SOFTWARE IMPLEMENTATION OF NORDA OCEAN MODELS J&IEA m/ MST* f-....~ cre mx IRSD~I?( J 300 Unicorn Park...34- NOO-79- 741 9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT. PROJECT, TASK AREA 6 WORK UNIT NUMBERSJAYC0, 300 Unicorn Park Drive...before another execution of the energetics program, move them back to disk. Note that the outputs of the preprocessor reside on disk, they should not be

Neurometabolite Alterations Associated With Cognitive Performance in Perinatally HIV-Infected Children.

PubMed

Van Dalen, Yvonne W; Blokhuis, Charlotte; Cohen, Sophie; Ter Stege, Jacqueline A; Teunissen, Charlotte E; Kuhle, Jens; Kootstra, Neeltje A; Scherpbier, Henriette J; Kuijpers, Taco W; Reiss, Peter; Majoie, Charles B L M; Caan, Matthan W A; Pajkrt, Dasja

2016-03-01

Despite treatment with combination antiretroviral therapy (cART), cognitive impairment is still observed in perinatally HIV-infected children. We aimed to evaluate potential underlying cerebral injury by comparing neurometabolite levels between perinatally HIV-infected children and healthy controls. This cross-sectional study evaluated neurometabolites, as measured by Magnetic Resonance Spectroscopy (MRS), in perinatally HIV-infected children stable on cART (n = 26) and healthy controls (n = 36).Participants were included from a cohort of perinatally HIV-infected children and healthy controls, matched group-wise for age, gender, ethnicity, and socio-economic status. N-acetylaspartate (NAA), glutamate (Glu), myo-inositol (mI), and choline (Cho) levels were studied as ratios over creatine (Cre). Group differences and associations with HIV-related parameters, cognitive functioning, and neuronal damage markers (neurofilament and total Tau proteins) were determined using age-adjusted linear regression analyses.HIV-infected children had increased Cho:Cre in white matter (HIV-infected = 0.29 ± 0.03; controls = 0.27 ± 0.03; P value = 0.045). Lower nadir CD4+ T-cell Z-scores were associated with reduced neuronal integrity markers NAA:Cre and Glu:Cre. A Centers for Disease Control and Prevention (CDC) stage C diagnosis was associated with higher glial markers Cho:Cre and mI:Cre. Poorer cognitive performance was mainly associated with higher Cho:Cre in HIV-infected children, and with lower NAA:Cre and Glu:Cre in healthy controls. There were no associations between neurometabolites and neuronal damage markers in blood or CSF.Compared to controls, perinatally HIV-infected children had increased Cho:Cre in white matter, suggestive of ongoing glial proliferation. Levels of several neurometabolites were associated with cognitive performance, suggesting that MRS may be a useful method to assess cerebral changes potentially linked to cognitive outcomes.

Col2-Cre and tamoxifen-inducible Col2-CreER target different cell populations in the knee joint

PubMed Central

Nagao, Masashi; Cheong, Chan Wook; Olsen, Bjorn

2015-01-01

Objective Collagen type 2 (Col2)-Cre or tamoxifen-inducible Col2-CreER transgenic mouse lines have been used for studies to explore the cellular and molecular pathogenesis of osteoarthritis (OA). The purpose of this study is to investigate whether the targeted cells are the same or different in the two mouse lines. Methods We crossed tamoxifen inducible Col2-CreER and Col2-Cre mice with Rosa tdTomato reporter mice and analyzed the labeling patterns at different time points. Results In the Col2-CreER mice, 90.8 [95% confidence interval (CI) (88.3, 93.2)] and 82.8 (77.4, 88.3) % of the articular surface cells are Tomato positive when tamoxifen was administered at 2 and 2.5 weeks of age and strong activity was observed even 4.5 months after injection. However, 46.0 (32.8, 59.1) and 22.2 (11.7, 32.6) % of the surface cells were Tomato positive when tamoxifen was administered at 3 and 4 weeks of age, respectively. Little to no Tomato activity in the articular surface cells was observed when tamoxifen was administered at 8 weeks of age. At any stage of tamoxifen injection, the Tomato activity was detected in growth plate and epiphyseal bone in addition to articular chondrocytes, but little in endothelium and not in the synovium and ligament. In contrast, the targeted tissues in the Col2-Cre mouse line were articular cartilage, growth plate, meniscus, endosteum, ligament, bone and synovium. Conclusions This study demonstrates that the pattern of targeted cells in the inducible Col2-CreER mice are partially overlapping with but different from that of targeted cells in Col2-Cre mice and the pattern varies dependent on when tamoxifen is administered. PMID:26256767

Control of blood pressure, appetite, and glucose by leptin in mice lacking leptin receptors in proopiomelanocortin neurons.

PubMed

do Carmo, Jussara M; da Silva, Alexandre A; Cai, Zhengwei; Lin, Shuying; Dubinion, John H; Hall, John E

2011-05-01

Although the central nervous system melanocortin system is an important regulator of energy balance, the role of proopiomelanocortin (POMC) neurons in mediating the chronic effects of leptin on appetite, blood pressure, and glucose regulation is unknown. Using Cre/loxP technology we tested whether leptin receptor deletion in POMC neurons (LepR(flox/flox)/POMC-Cre mice) attenuates the chronic effects of leptin to increase mean arterial pressure (MAP), enhance glucose use and oxygen consumption, and reduce appetite. LepR(flox/flox)/POMC-Cre, wild-type, LepR(flox/flox), and POMC-Cre mice were instrumented for MAP and heart rate measurement by telemetry and venous catheters for infusions. LepR(flox/flox)/POMC-Cre mice were heavier, hyperglycemic, hyperinsulinemic, and hyperleptinemic compared with wild-type, LepR(flox/flox), and POMC-Cre mice. Despite exhibiting features of metabolic syndrome, LepR(flox/flox)/POMC-Cre mice had normal MAP and heart rate compared with LepR(flox/flox) but lower MAP and heart rate compared with wild-type mice. After a 5-day control period, leptin was infused (2 μg/kg per minute, IV) for 7 days. In control mice, leptin increased MAP by ≈5 mm Hg despite decreasing food intake by ≈35%. In contrast, leptin infusion in LepR(flox/flox)/POMC-Cre mice reduced MAP by ≈3 mm Hg and food intake by ≈28%. Leptin significantly decreased insulin and glucose levels in control mice but not in LepR(flox/flox)/POMC-Cre mice. Leptin increased oxygen consumption in LepR(flox/flox)/POMC-Cre and wild-type mice. Activation of POMC neurons is necessary for the chronic effects of leptin to raise MAP and reduce insulin and glucose levels, whereas leptin receptors in other areas of the brain other than POMC neurons appear to play a key role in mediating the chronic effects of leptin on appetite and oxygen consumption.

Activation of hypothalamic RIP-Cre neurons promotes beiging of WAT via sympathetic nervous system.

PubMed

Wang, Baile; Li, Ang; Li, Xiaomu; Ho, Philip Wl; Wu, Donghai; Wang, Xiaoqi; Liu, Zhuohao; Wu, Kelvin Kl; Yau, Sonata Sy; Xu, Aimin; Cheng, Kenneth Ky

2018-04-01

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat-insulin-promoter-Cre (RIP-Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT Genetic ablation of APPL2 in RIP-Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP-Cre neurons, inactivation of VMH AMPK, or treatment with a β3-adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP-Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP-Cre neurons, in which the APPL2-AMPK signaling axis is crucial for this defending mechanism to cold and obesity. © 2018 The Authors.

Effects of Coffee and Caffeine Anhydrous Intake During Creatine Loading.

PubMed

Trexler, Eric T; Smith-Ryan, Abbie E; Roelofs, Erica J; Hirsch, Katie R; Persky, Adam M; Mock, Meredith G

2016-05-01

The purpose of this study was to determine the effect of 5 days of creatine (CRE) loading alone or in combination with caffeine anhydrous (CAF) or coffee (COF) on upper-body and lower-body strength and sprint performance. Physically active males (n = 54; mean ± SD; age = 20.1 ± 2.1 years; weight = 78.8 ± 8.8 kg) completed baseline testing, consisting of 1 repetition maximum (1RM) and repetitions to fatigue with 80% 1RM for bench press and leg press, followed by a repeated sprint test of five, 10-second sprints separated by 60-second rest on a cycle ergometer to determine peak power (PP) and total power (TP). At least 72 hours later, subjects were randomly assigned to supplement with CRE (5 g of CRE monohydrate, 4 times per day; n = 14), CRE + CAF (CRE +300 mg·d of CAF; n = 13), CRE + COF (CRE +8.9 g of COF, yielding 303 mg of CAF; n = 13), or placebo (PLA; n = 14) for 5 days. Serum creatinine (CRN) was measured before and after supplementation, and on day 6, participants repeated pretesting procedures. Strength measures were improved in all groups (p ≤ 0.05), with no significant time × treatment interactions. No significant interaction or main effects were observed for PP. For TP, a time × sprint interaction was observed (p ≤ 0.05), with no significant interactions among treatment groups. A time × treatment interaction was observed for serum CRN values (p ≤ 0.05) that showed increases in all groups except PLA. Four subjects reported mild gastrointestinal discomfort with CRE + CAF, with no side effects reported in other groups. These findings suggest that neither CRE alone nor in combination with CAF or COF significantly affected performance compared with PLA.

Presynaptic Inputs to Any CNS Projection Neuron Identified by Dual Recombinant Virus Infection

PubMed Central

Bráz, João M.; Wang, Fan; Basbaum, Allan I.

2015-01-01

Although neuroanatomical tracing studies have defined the origin and targets of major projection neurons (PN) of the central nervous system (CNS), there is much less information about the circuits that influence these neurons. Recently, genetic approaches that use Cre recombinase-dependent viral vectors have greatly facilitated such circuit analysis, but these tracing approaches are limited by the availability of Cre-expressing mouse lines and the difficulty in restricting Cre expression to discrete regions of the CNS. Here, we illustrate an alternative approach to drive Cre expression specifically in defined subsets of CNS projection neurons, so as to map both direct and indirect presynaptic inputs to these cells. The method involves a combination of Cre-dependent transneuronal viral tracers that can be used in the adult and that does not require genetically modified mice. To trigger Cre-expression we inject a Cre-expressing adenovirus that is retrogradely transported to the projection neurons of interest. The region containing the retrogradely labeled projection neurons is next injected with Cre-dependent pseudorabies or rabies vectors, which results in labeling of poly- and monosynaptic neuronal inputs, respectively. In proof-of-concept experiments, we used this novel tracing system to study the circuits that engage projection neurons of the superficial dorsal horn of the spinal cord and trigeminal nucleus caudalis, neurons of the parabrachial nucleus of the dorsolateral pons that project to the amygdala and cortically-projecting neurons of the lateral geniculate nucleus. Importantly, because this dual viral tracing method does not require genetically derived Cre-expressing mouse lines, inputs to almost any projection system can be studied and the analysis can be performed in larger animals, such as the rat. PMID:26470056

Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner.

PubMed

Hidema, Shizu; Fukuda, Tomokazu; Hiraoka, Yuichi; Mizukami, Hiroaki; Hayashi, Ryotaro; Otsuka, Ayano; Suzuki, Shingo; Miyazaki, Shinji; Nishimori, Katsuhiko

2016-05-01

The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR. © 2015 Wiley Periodicals, Inc.

Humidity trends imply increased sensitivity to clouds in a warming Arctic

DOE PAGES

Cox, Christopher J.; Walden, Von P.; Rowe, Penny M.; ...

2015-12-10

Infrared radiative processes are implicated in Arctic warming and sea-ice decline. The infrared cloud radiative effect (CRE) at the surface is modulated by cloud properties; however, CRE also depends on humidity because clouds emit at wavelengths that are semi-transparent to greenhouse gases, most notably water vapour. Here we show how temperature and humidity control CRE through competing influences between the mid- and far-infrared. At constant relative humidity, CRE does not decrease with increasing temperature/absolute humidity as expected, but rather is found to be approximately constant for temperatures characteristic of the Arctic. This stability is disrupted if relative humidity varies. Ourmore » findings explain observed seasonal and regional variability in Arctic CRE of order 10Wm 2. With the physical properties of Arctic clouds held constant, we calculate recent increases in CRE of 1–5Wm 2 in autumn and winter, which are projected to reach 5–15Wm 2 by 2050, implying increased sensitivity of the surface to clouds.« less

Humidity trends imply increased sensitivity to clouds in a warming Arctic.

PubMed

Cox, Christopher J; Walden, Von P; Rowe, Penny M; Shupe, Matthew D

2015-12-10

Infrared radiative processes are implicated in Arctic warming and sea-ice decline. The infrared cloud radiative effect (CRE) at the surface is modulated by cloud properties; however, CRE also depends on humidity because clouds emit at wavelengths that are semi-transparent to greenhouse gases, most notably water vapour. Here we show how temperature and humidity control CRE through competing influences between the mid- and far-infrared. At constant relative humidity, CRE does not decrease with increasing temperature/absolute humidity as expected, but rather is found to be approximately constant for temperatures characteristic of the Arctic. This stability is disrupted if relative humidity varies. Our findings explain observed seasonal and regional variability in Arctic CRE of order 10 W m(-2). With the physical properties of Arctic clouds held constant, we calculate recent increases in CRE of 1-5 W m(-2) in autumn and winter, which are projected to reach 5-15 W m(-2) by 2050, implying increased sensitivity of the surface to clouds.

Humidity trends imply increased sensitivity to clouds in a warming Arctic

PubMed Central

Cox, Christopher J.; Walden, Von P.; Rowe, Penny M.; Shupe, Matthew D.

2015-01-01

Infrared radiative processes are implicated in Arctic warming and sea-ice decline. The infrared cloud radiative effect (CRE) at the surface is modulated by cloud properties; however, CRE also depends on humidity because clouds emit at wavelengths that are semi-transparent to greenhouse gases, most notably water vapour. Here we show how temperature and humidity control CRE through competing influences between the mid- and far-infrared. At constant relative humidity, CRE does not decrease with increasing temperature/absolute humidity as expected, but rather is found to be approximately constant for temperatures characteristic of the Arctic. This stability is disrupted if relative humidity varies. Our findings explain observed seasonal and regional variability in Arctic CRE of order 10 W m−2. With the physical properties of Arctic clouds held constant, we calculate recent increases in CRE of 1–5 W m−2 in autumn and winter, which are projected to reach 5–15 W m−2 by 2050, implying increased sensitivity of the surface to clouds. PMID:26657324

Cell Type-Specific Manipulation with GFP-Dependent Cre Recombinase

PubMed Central

Tang, Jonathan C Y; Rudolph, Stephanie; Dhande, Onkar S; Abraira, Victoria E; Choi, Seungwon; Lapan, Sylvain; Drew, Iain R; Drokhlyansky, Eugene; Huberman, Andrew D; Regehr, Wade G; Cepko, Constance L

2016-01-01

Summary There are many transgenic GFP reporter lines that allow visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. Here we report the creation of a method that exploits GFP for gene manipulation, Cre Recombinase Dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, leading to effective recombination selectively in GFP-labeled cells. Further, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP+ cells, we demonstrate that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins. PMID:26258682

Selective oxidation of aliphatic C-H bonds in alkylphenols by a chemomimetic biocatalytic system.

PubMed

Du, Lei; Dong, Sheng; Zhang, Xingwang; Jiang, Chengying; Chen, Jingfei; Yao, Lishan; Wang, Xiao; Wan, Xiaobo; Liu, Xi; Wang, Xinquan; Huang, Shaohua; Cui, Qiu; Feng, Yingang; Liu, Shuang-Jiang; Li, Shengying

2017-06-27

Selective oxidation of aliphatic C-H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum The system consisting of CreHI (for installation of a phosphate directing/anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C-H bonds of p - and m -alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity.

Selective oxidation of aliphatic C–H bonds in alkylphenols by a chemomimetic biocatalytic system

PubMed Central

Du, Lei; Dong, Sheng; Zhang, Xingwang; Jiang, Chengying; Chen, Jingfei; Yao, Lishan; Wang, Xiao; Wan, Xiaobo; Liu, Xi; Wang, Xinquan; Huang, Shaohua; Cui, Qiu; Liu, Shuang-Jiang; Li, Shengying

2017-01-01

Selective oxidation of aliphatic C–H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum. The system consisting of CreHI (for installation of a phosphate directing/anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C–H bonds of p- and m-alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity. PMID:28607077

Spinal Hb9::Cre-derived excitatory interneurons contribute to rhythm generation in the mouse

PubMed Central

Caldeira, Vanessa; Dougherty, Kimberly J.; Borgius, Lotta; Kiehn, Ole

2017-01-01

Rhythm generating neurons are thought to be ipsilaterally-projecting excitatory neurons in the thoracolumbar mammalian spinal cord. Recently, a subset of Shox2 interneurons (Shox2 non-V2a INs) was found to fulfill these criteria and make up a fraction of the rhythm-generating population. Here we use Hb9::Cre mice to genetically manipulate Hb9::Cre-derived excitatory interneurons (INs) in order to determine the role of these INs in rhythm generation. We demonstrate that this line captures a consistent population of spinal INs which is mixed with respect to neurotransmitter phenotype and progenitor domain, but does not overlap with the Shox2 non-V2a population. We also show that Hb9::Cre-derived INs include the comparatively small medial population of INs which continues to express Hb9 postnatally. When excitatory neurotransmission is selectively blocked by deleting Vglut2 from Hb9::Cre-derived INs, there is no difference in left-right and/or flexor-extensor phasing between these cords and controls, suggesting that excitatory Hb9::Cre-derived INs do not affect pattern generation. In contrast, the frequencies of locomotor activity are significantly lower in cords from Hb9::Cre-Vglut2Δ/Δ mice than in cords from controls. Collectively, our findings indicate that excitatory Hb9::Cre-derived INs constitute a distinct population of neurons that participates in the rhythm generating kernel for spinal locomotion. PMID:28128321

[Allelopathic effects of extracts from fibrous roots of Coptis chinensis on two leguminous species].

PubMed

Li, Qian; Wu, Ye-Kuan; Yuan, Ling; Huang, Jian-Guo

2013-03-01

An experiment was carried out to study the allelopathic effects of Coptis chinensis fibrous root extracts (CRE) on the germination and seedling growth of Vicia faba and Pisum sativum in order to alleviate the allelopathic effects and increase land productivity. The seeds of both garden pea (P. sativum) and broad been (V. faba) were germinated in CRE solution of various concentrations, the germination rate, seedling growth and related physiological indexes were measured. The result indicated that there were no significant effects of CRE in low concentrations on seed germination, including both the rate and index, and seed vitality and membrane permeability. With the increment of CRE concentrations, however, the high seed membrane permeability and germination inhibition were observed. For example, the germination rates were reduced by 23.4% (P. sativum) and 9.5% (V. faba), respectively, in CRE solution with 800 mg . L-1. Simultaneously, soluble sugars and the free amino acids in the seeds were lower than those in the control (without CRE) after soaking seeds in CRE solutions. In addition, the seedling growth and nitrate reductase activity were stimulated by CRE at low concentrations in contrast to high concentrations which behaved otherwise and inhibited the nutrient utilization in endosperm. Therefore, the large amount of allelochemicals released from the roots and remains of C. chinensis in soils could inhibit the seed germination and seedling growth of legumes, which may lead to decrease even fail crop yields after growing this medical plant.

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification

PubMed Central

Cain-Hom, Carol; Splinter, Erik; van Min, Max; Simonis, Marieke; van de Heijning, Monique; Martinez, Maria; Asghari, Vida

2017-01-01

Abstract Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events. PMID:28053125

T-cell-specific deletion of Mof blocks their differentiation and results in genomic instability in mice

PubMed Central

Pandita, Tej K.

2013-01-01

Ataxia telangiectasia patients develop lymphoid malignancies of both B- and T-cell origin. Similarly, ataxia telangiectasia mutated (Atm)-deficient mice exhibit severe defects in T-cell maturation and eventually develop thymomas. The function of ATM is known to be influenced by the mammalian orthologue of the Drosophila MOF (males absent on the first) gene. Here, we report the effect of T-cell-specific ablation of the mouse Mof (Mof) gene on leucocyte trafficking and survival. Conditional Mof Flox/Flox (Mof F/F) mice expressing Cre recombinase under control of the T-cell-specific Lck proximal promoter (Mof F/F/Lck-Cre +) display a marked reduction in thymus size compared with Mof F/F/Lck-Cre – mice. In contrast, the spleen size of Mof F/F/Lck-Cre + mice was increased compared with control Mof F/F/Lck-Cre – mice. The thymus of Mof F/F/Lck-Cre + mice contained significantly reduced T cells, whereas thymic B cells were elevated. Within the T-cell population, CD4+CD8+ double-positive T-cell levels were reduced, whereas the immature CD4–CD8– double-negative (DN) population was elevated. Defective T-cell differentiation is also evident as an increased DN3 (CD44–CD25+) population, the cell stage during which T-cell receptor rearrangement takes place. The differentiation defect in T cells and reduced thymus size were not rescued in a p53-deficient background. Splenic B-cell distributions were similar between Mof F/F/Lck-Cre + and Mof F/F/Lck-Cre – mice except for an elevation of the κ light-chain population, suggestive of an abnormal clonal expansion. T cells from Mof F/F/Lck-Cre + mice did not respond to phytohaemagglutinin (PHA) stimulation, whereas LPS-stimulated B cells from Mof F/F/Lck-Cre + mice demonstrated spontaneous genomic instability. Mice with T-cell-specific loss of MOF had shorter lifespans and decreased survival following irradiation than did Mof F/F/Lck-Cre – mice. These observations suggest that Mof plays a critical role in T-cell differentiation and that depletion of Mof in T cells reduces T-cell numbers and, by an undefined mechanism, induces genomic instability in B cells through bystander mechanism. As a result, these mice have a shorter lifespan and reduced survival after irradiation. PMID:23386701

T-cell-specific deletion of Mof blocks their differentiation and results in genomic instability in mice.

PubMed

Gupta, Arun; Hunt, Clayton R; Pandita, Raj K; Pae, Juhee; Komal, K; Singh, Mayank; Shay, Jerry W; Kumar, Rakesh; Ariizumi, Kiyoshi; Horikoshi, Nobuo; Hittelman, Walter N; Guha, Chandan; Ludwig, Thomas; Pandita, Tej K

2013-05-01

Ataxia telangiectasia patients develop lymphoid malignancies of both B- and T-cell origin. Similarly, ataxia telangiectasia mutated (Atm)-deficient mice exhibit severe defects in T-cell maturation and eventually develop thymomas. The function of ATM is known to be influenced by the mammalian orthologue of the Drosophila MOF (males absent on the first) gene. Here, we report the effect of T-cell-specific ablation of the mouse Mof (Mof) gene on leucocyte trafficking and survival. Conditional Mof(Flox/Flox) (Mof (F/F)) mice expressing Cre recombinase under control of the T-cell-specific Lck proximal promoter (Mof(F/F)/Lck-Cre(+)) display a marked reduction in thymus size compared with Mof(F/F)/Lck-Cre(-) mice. In contrast, the spleen size of Mof(F/F)/Lck-Cre(+) mice was increased compared with control Mof(F/F)/Lck-Cre(-) mice. The thymus of Mof(F/F)/Lck-Cre(+) mice contained significantly reduced T cells, whereas thymic B cells were elevated. Within the T-cell population, CD4(+)CD8(+) double-positive T-cell levels were reduced, whereas the immature CD4(-)CD8(-) double-negative (DN) population was elevated. Defective T-cell differentiation is also evident as an increased DN3 (CD44(-)CD25(+)) population, the cell stage during which T-cell receptor rearrangement takes place. The differentiation defect in T cells and reduced thymus size were not rescued in a p53-deficient background. Splenic B-cell distributions were similar between Mof(F/F)/Lck-Cre(+) and Mof(F/F)/Lck-Cre(-) mice except for an elevation of the κ light-chain population, suggestive of an abnormal clonal expansion. T cells from Mof(F/F)/Lck-Cre(+) mice did not respond to phytohaemagglutinin (PHA) stimulation, whereas LPS-stimulated B cells from Mof(F/F)/Lck-Cre(+) mice demonstrated spontaneous genomic instability. Mice with T-cell-specific loss of MOF had shorter lifespans and decreased survival following irradiation than did Mof(F/F)/Lck-Cre(-) mice. These observations suggest that Mof plays a critical role in T-cell differentiation and that depletion of Mof in T cells reduces T-cell numbers and, by an undefined mechanism, induces genomic instability in B cells through bystander mechanism. As a result, these mice have a shorter lifespan and reduced survival after irradiation.

Fibroblast growth factor 2 and cyclic AMP synergistically regulate bone sialoprotein gene expression.

PubMed

Shimizu, Emi; Nakayama, Youhei; Nakajima, Yu; Kato, Naoko; Takai, Hideki; Kim, Dong-Soon; Arai, Masato; Saito, Ryoichiro; Sodek, Jaro; Ogata, Yorimasa

2006-07-01

Bone sialoprotein (BSP) is a noncollagenous protein of the mineralized bone extracellular matrix. We here report that FGF2 and cAMP act synergistically to stimulate BSP gene expression. Treatment of ROS 17/2.8 cells with either 10 ng/ml FGF2 or 1 microM FSK for 6 h resulted in 5.4- and 8.2-fold increases, respectively, in the levels of BSP mRNA. However, in the presence of both FGF2 and forskolin (FGF/FSK), BSP mRNA levels were increased synergistically by 20.4-fold. Using a luciferase reporter construct, encompassing BSP promoter nucleotides -116 to +60, transcription was also increased synergistically by 15.0-fold with FGF/FSK, compared to stimulations of 2.6- and 5.3-fold, respectively, for FGF2 and FSK alone. Transcriptional stimulation by FGF/FSK abrogated in constructs included 2 bp mutations in the inverted CCAAT, CRE, FRE and Pit-1 elements. Whereas the FRE-protein complex was increased by FGF2 and FGF/FSK, the Pit-1-protein complex was decreased by FSK and FGF/FSK. Notably, transcriptional activity induced by FGF/FSK was blocked by protein kinase A, tyrosine kinase and MEK inhibitors. These studies indicate that the combinatorial effects of FGF and FSK act through PKA, tyrosine kinase and MAP-kinase-dependent pathways, which target the inverted CCAAT, CRE, FRE and Pit-1 elements in the BSP gene to synergistically increase BSP expression.

Upstream CREs participate in the basal activity of minute virus of mice promoter P4 and in its stimulation in ras-transformed cells.

PubMed Central

Perros, M; Deleu, L; Vanacker, J M; Kherrouche, Z; Spruyt, N; Faisst, S; Rommelaere, J

1995-01-01

The activity of the P4 promoter of the parvovirus minute virus of mice (prototype strain MVMp) is stimulated in ras-transformed FREJ4 cells compared with the parental FR3T3 line. This activation may participate in the oncolytic effect of parvoviruses, given that P4 drives a transcriptional unit encoding cytotoxic nonstructural proteins. Our results suggest that the higher transcriptional activity of promoter P4 in FREJ4 cells is mediated at least in part by upstream CRE elements. Accordingly, mutations in the CRE motifs impair P4 function more strongly in the FREJ4 derivative than in its FR3T3 parent. Further evidence that these elements contribute to hyperactivity of the P4 promoter in the ras transformant is the fact that they form distinct complexes with proteins from FREJ4 and FR3T3 cell extracts. This difference can be abolished by treating the FREJ4 cell extracts with cyclic AMP-dependent protein kinase (PKA) or treating original cultures with a PKA activator. These findings can be linked with two previously reported features of ras-transformed cells: the activation of a PKA-inhibited protein kinase cascade and the reduction of PKA-induced protein phosphorylation. In keeping with these facts, P4-directed gene expression can be up- or downmodulated in vivo by exposing cells to known inhibitors or activators of PKA, respectively. PMID:7636996

Role of the human cytomegalovirus major immediate-early promoter's 19-base-pair-repeat cyclic AMP-response element in acutely infected cells.

PubMed

Keller, M J; Wheeler, D G; Cooper, E; Meier, J L

2003-06-01

Prior studies have suggested a role of the five copies of the 19-bp-repeat cyclic AMP (cAMP)-response element (CRE) in major immediate-early (MIE) promoter activation, the rate-limiting step in human cytomegalovirus (HCMV) replication. We used two different HCMV genome modification strategies to test this hypothesis in acutely infected cells. We report the following: (i) the CREs do not govern basal levels of MIE promoter activity at a high or low multiplicity of infection (MOI) in human foreskin fibroblast (HFF)- or NTera2-derived neuronal cells; (ii) serum and virion components markedly increase MIE promoter-dependent transcription at a low multiplicity of infection (MOI), but this increase is not mediated by the CREs; (iii) forskolin stimulation of the cAMP signaling pathway induces a two- to threefold increase in MIE RNA levels in a CRE-specific manner at a low MOI in both HFF- and NTera2-derived neuronal cells; and (iv) the CREs do not regulate basal levels of HCMV DNA replication at a high or low MOI in HFF. Their presence does impart a forskolin-induced increase in viral DNA replication at a low MOI but only when basal levels of MIE promoter activity are experimentally diminished. In conclusion, the 19-bp-repeat CREs add to the robust MIE promoter activity that occurs in the acutely infected stimulated cells, although the CREs' greater role may be in other settings.

Cis-regulatory Elements and Human Evolution

PubMed Central

Siepel, Adam

2014-01-01

Modification of gene regulation has long been considered an important force in human evolution, particularly through changes to cis-regulatory elements (CREs) that function in transcriptional regulation. For decades, however, the study of cis-regulatory evolution was severely limited by the available data. New data sets describing the locations of CREs and genetic variation within and between species have now made it possible to study CRE evolution much more directly on a genome-wide scale. Here, we review recent research on the evolution of CREs in humans based on large-scale genomic data sets. We consider inferences based on primate divergence, human polymorphism, and combinations of divergence and polymorphism. We then consider “new frontiers” in this field stemming from recent research on transcriptional regulation. PMID:25218861

Concerted formation of macromolecular Suppressor–mutator transposition complexes

PubMed Central

Raina, Ramesh; Schläppi, Michael; Karunanandaa, Balasulojini; Elhofy, Adam; Fedoroff, Nina

1998-01-01

Transposition of the maize Suppressor–mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA–DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein–protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range. PMID:9671711

Neuroprotective effects of α-iso-cubebene against glutamate-induced damage in the HT22 hippocampal neuronal cell line.

PubMed

Park, Sun Young; Jung, Won Jung; Kang, Jum Soon; Kim, Cheol-Min; Park, Geuntae; Choi, Young-Whan

2015-02-01

Since oxidative stress is critically involved in excitotoxic damage, we sought to determine whether the activation of the transcription factors, cAMP-responsive element binding protein (CREB) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2, also known as NFE2L2), by α-iso-cubebene is involved in its protective effects against glutamate-induced neuronal cell death. Pre-treatment with α-iso-cubebene significantly attenuated glutamate-induced cytotoxicity in mouse hippocampus-derived neuronal cells. α-iso-cubebene also reduced the glutamate-induced generation of reactive oxygen species and calcium influx, thus preventing apoptotic cell death. α-iso-cubebene inhibited glutamate-induced mitochondrial membrane depolarization and, consequently, inhibited the release of the apoptosis-inducing factor from the mitochondria. Immunoblot anlaysis revealed that the phosphorylation of extracellular signal-regulated kinase (ERK) by glutamate was reduced in the presence of α-iso-cubebene. α-iso-cubebene activated protein kinase A (PKA), CREB and Nrf2, which mediate the expression of the antioxidant enzymes, heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase [quinone] 1 (NQO1), involved in neuroprotection. In addition, α-iso-cubebene induced the expression of antioxidant responsive element and CRE transcriptional activity, thus conferring neuroprotection against glutamate-induced oxidative injury. α-iso-cubebene also induced the expression of Nrf2-dependent genes encoding HO-1 and NQO1. Furthermore, the knockdown of CREB and Nrf2 by small interfering RNA attenuated the neuroprotective effects of α-iso-cubebene. Taken together, our results indicate that α-iso-cubebene protects HT22 cells from glutamate-induced oxidative damage through the activation of Nrf2/HO-1/NQO-1, as well as through the PKA and CREB signaling pathways.

Tg(Th-Cre)FI172Gsat (Th-Cre) defines neurons that are required for full hypercapnic and hypoxic reflexes.

PubMed

Sun, Jenny J; Ray, Russell S

2017-08-15

The catecholaminergic (CA) system has been implicated in many facets of breathing control and offers an important target to better comprehend the underlying etiologies of both developmental and adult respiratory pathophysiologies. Here, we used a noninvasive DREADD-based pharmacogenetic approach to acutely perturb Tg(Th-Cre)FI172Gsat ( Th-Cre )-defined neurons in awake and unrestrained mice in an attempt to characterize CA function in breathing. We report that clozapine-N-oxide (CNO)-DREADD-mediated inhibition of Th-Cre -defined neurons results in blunted ventilatory responses under respiratory challenge. Under a hypercapnic challenge (5% CO 2 /21% O 2 /74% N 2 ), perturbation of Th-Cre neurons results in reduced f R , [Formula: see text] and [Formula: see text] Under a hypoxic challenge (10% O 2 /90% N 2 ), we saw reduced f R , [Formula: see text] and [Formula: see text], in addition to instability in both interbreath interval and tidal volume, resulting in a Cheyne-Stokes-like respiratory pattern. These findings demonstrate the necessity of Th-Cre -defined neurons for the hypercapnic and hypoxic ventilatory responses and breathing stability during hypoxia. However, given the expanded non-CA expression domains of the Tg(Th-Cre)FI172Gsat mouse line found in the brainstem, full phenotypic effect cannot be assigned solely to CA neurons. Nonetheless, this work identifies a key respiratory population that may lead to further insights into the circuitry that maintains respiratory stability in the face of homeostatic challenges. © 2017. Published by The Company of Biologists Ltd.

Transcription factor trapping by RNA in gene regulatory elements.

PubMed

Sigova, Alla A; Abraham, Brian J; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M; Guo, Yang Eric; Jangi, Mohini; Giallourakis, Cosmas C; Sharp, Phillip A; Young, Richard A

2015-11-20

Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs. Copyright © 2015, American Association for the Advancement of Science.

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

PubMed

Brent, G A; Williams, G R; Harney, J W; Forman, B M; Samuels, H H; Moore, D D; Larsen, P R

1992-04-01

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common feature which defines the capacity of these elements to confer T3 induction.

The Theory and Practice of Culturally Relevant Education: A Synthesis of Research across Content Areas

ERIC Educational Resources Information Center

Aronson, Brittany; Laughter, Judson

2016-01-01

Many teachers and educational researchers have claimed to adopt tenets of culturally relevant education (CRE). However, recent work describes how standardized curricula and testing have marginalized CRE in educational reform discourses. In this synthesis of research, we sought examples of research connecting CRE to positive student outcomes across…

Then and Now: Change for the Better?

ERIC Educational Resources Information Center

Clarke, Julian; Speeden, Stuart

This report investigates whether strategies employed by the United Kingdom's Commission for Racial Equality (CRE) have been effective in furthering racial equality. It examines the way that CRE strategies have responded to a range of changing conditions, and it looks at the value of this experience in shaping the CRE's future strategies. Section…

Gfi1-Cre knock-in mouse line: A tool for inner ear hair cell-specific gene deletion

PubMed Central

Yang, Hua; Gan, Jean; Xie, Xiaoling; Deng, Min; Feng, Liang; Chen, Xiaowei; Gao, Zhiqiang; Gan, Lin

2010-01-01

Summary Gfi1encodes a zinc-finger transcription factor essential for the development and maintenance of haematopoiesis and the inner ear. In mouse inner ear, Gfi1 expression is confined to hair cells during development and in adulthood. To construct a genetic tool for inner ear hair cell-specific gene deletion, we generated a Gfi1-Cre mouse line by knocking-in Cre coding sequences into the Gfi1 locus and inactivating the endogenous Gfi1. The specificity and efficiency of Gfi1-Cre recombinase-mediated recombination in the developing inner ear was revealed through the expression of the conditional R26R-lacZ reporter gene. The onset of lacZ expression in the Gfi1Cre/+ inner ear was first detected at E13.5 in the vestibule and at E15.5 in the cochlea, coinciding with the generation of hair cells. Throughout inner ear development, lacZ expression was detected only in hair cells. Thus, Gfi1-Cre knock-in mouse line provides a useful tool for gene manipulations specifically in inner ear hair cells. PMID:20533399

Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus.

PubMed

Togawa, Yoichiro; Nunoshiba, Tatsuo; Hiratsu, Keiichiro

2018-02-01

Markerless gene-disruption technology is particularly useful for effective genetic analyses of Thermus thermophilus (T. thermophilus), which have a limited number of selectable markers. In an attempt to develop a novel system for the markerless disruption of genes in T. thermophilus, we applied a Cre/lox system to construct a triple gene disruptant. To achieve this, we constructed two genetic tools, a loxP-htk-loxP cassette and cre-expressing plasmid, pSH-Cre, for gene disruption and removal of the selectable marker by Cre-mediated recombination. We found that the Cre/lox system was compatible with the proliferation of the T. thermophilus HB27 strain at the lowest growth temperature (50 °C), and thus succeeded in establishing a triple gene disruptant, the (∆TTC1454::loxP, ∆TTC1535KpnI::loxP, ∆TTC1576::loxP) strain, without leaving behind a selectable marker. During the process of the sequential disruption of multiple genes, we observed the undesired deletion and inversion of the chromosomal region between multiple loxP sites that were induced by Cre-mediated recombination. Therefore, we examined the effects of a lox66-htk-lox71 cassette by exploiting the mutant lox sites, lox66 and lox71, instead of native loxP sites. We successfully constructed a (∆TTC1535::lox72, ∆TTC1537::lox72) double gene disruptant without inducing the undesired deletion of the 0.7-kbp region between the two directly oriented lox72 sites created by the Cre-mediated recombination of the lox66-htk-lox71 cassette. This is the first demonstration of a Cre/lox system being applicable to extreme thermophiles in a genetic manipulation. Our results indicate that this system is a powerful tool for multiple markerless gene disruption in T. thermophilus.

Phenotypic and Molecular Characteristics of Carbapenem-Resistant Enterobacteriaceae in a Health Care System in Los Angeles, California, from 2011 to 2013

PubMed Central

Pollett, S.; Miller, S.; Hindler, J.; Uslan, D.; Carvalho, M.

2014-01-01

Carbapenem-resistant Enterobacteriaceae (CRE) are a concern for health care in the United States but remain relatively uncommon in California. We describe the phenotype, clonality, and carbapenemase-encoding genes present in CRE isolated from patients at a Californian tertiary health care system. CRE for this study were identified by evaluating the antibiograms of Enterobacteriaceae isolated in the UCLA Health System from 2011 to 2013 for isolates that were not susceptible to meropenem and/or imipenem. The identification of these isolates was subsequently confirmed by matrix-associated laser desorption ionization–time of flight, and broth microdilution tests were repeated to confirm the CRE phenotype. Real-time PCR for blaKPC, blaSME, blaIMP, blaNDM-1, blaVIM, and blaOXA-48 was performed. Clonality was assessed by repetitive sequence-based PCR (repPCR) and multilocus sequence typing (MLST). Of 15,839 nonduplicate clinical Enterobacteriaceae isolates, 115 (0.73%) met the study definition for CRE. This number increased from 0.5% (44/8165) in the first half of the study to 0.9% (71/7674) in the second (P = 0.004). The most common CRE species were Klebsiella pneumoniae, Enterobacter aerogenes, and Escherichia coli. A carbapenemase-encoding gene was found in 81.7% (94/115) of CRE and included blaKPC (78.3%), blaNDM-1 (0.9%), and blaSME (2.6%). The majority of blaKPC genes were in K. pneumoniae isolates, which fell into 14 clonal groups on typing. blaKPC was identified in more than one species of CRE cultured from the same patient in four cases. Three blaSME-carrying Serratia marcescens isolates and one blaNDM-1 carrying Providencia rettgeri isolate were detected. CRE are increasing in California, and carbapenemases, particularly KPC, are a common mechanism for carbapenem resistance in this region. PMID:25210072

Infection prevention and control measures and tools for the prevention of entry of carbapenem-resistant Enterobacteriaceae into healthcare settings: guidance from the European Centre for Disease Prevention and Control.

PubMed

Magiorakos, A P; Burns, K; Rodríguez Baño, J; Borg, M; Daikos, G; Dumpis, U; Lucet, J C; Moro, M L; Tacconelli, E; Simonsen, G Skov; Szilágyi, E; Voss, A; Weber, J T

2017-01-01

Infections with carbapenem-resistant Enterobacteriaceae (CRE) are increasingly being reported from patients in healthcare settings. They are associated with high patient morbidity, attributable mortality and hospital costs. Patients who are "at-risk" may be carriers of these multidrug-resistant Enterobacteriaceae (MDR-E).The purpose of this guidance is to raise awareness and identify the "at-risk" patient when admitted to a healthcare setting and to outline effective infection prevention and control measures to halt the entry and spread of CRE. The guidance was created by a group of experts who were functioning independently of their organisations, during two meetings hosted by the European Centre for Disease Prevention and Control. A list of epidemiological risk factors placing patients "at-risk" for carriage with CRE was created by the experts. The conclusions of a systematic review on the prevention of spread of CRE, with the addition of expert opinion, were used to construct lists of core and supplemental infection prevention and control measures to be implemented for "at-risk" patients upon admission to healthcare settings. Individuals with the following profile are "at-risk" for carriage of CRE: a) a history of an overnight stay in a healthcare setting in the last 12 months, b) dialysis-dependent or cancer chemotherapy in the last 12 months, c) known previous carriage of CRE in the last 12 months and d) epidemiological linkage to a known carrier of a CRE.Core infection prevention and control measures that should be considered for all patients in healthcare settings were compiled. Preliminary supplemental measures to be implemented for "at-risk" patients on admission are: pre-emptive isolation, active screening for CRE , and contact precautions. Patients who are confirmed positive for CRE will need additional supplemental measures. Strengthening the microbiological capacity, surveillance and reporting of new cases of CRE in healthcare settings and countries is necessary to monitor the epidemiological situation so that, if necessary, the implemented CRE prevention strategies can be refined in a timely manner. Creating a large communication network to exchange this information would be helpful to understand the extent of the CRE reservoir and to prevent infections in healthcare settings, by applying the principles outlined here.This guidance document offers suggestions for best practices, but is in no way prescriptive for all healthcare settings and all countries. Successful implementation will result if there is local commitment and accountability. The options for intervention can be adopted or adapted to local needs, depending on the availability of financial and structural resources.

Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system.

PubMed

Cao, Bihao; Huang, Zhiyin; Chen, Guoju; Lei, Jianjun

2010-04-01

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38') was transformed with Cre gene and the inbred/pure line ('E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T(0) -plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T (0) -plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification.

PubMed

Cain-Hom, Carol; Splinter, Erik; van Min, Max; Simonis, Marieke; van de Heijning, Monique; Martinez, Maria; Asghari, Vida; Cox, J Colin; Warming, Søren

2017-05-05

Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A SNP uncoupling Mina expression from the TGFβ signaling pathway.

PubMed

Lian, Shang L; Mihi, Belgacem; Koyanagi, Madoka; Nakayama, Toshinori; Bix, Mark

2018-03-01

Mina is a JmjC family 2-oxoglutarate oxygenase with pleiotropic roles in cell proliferation, cancer, T cell differentiation, pulmonary inflammation, and intestinal parasite expulsion. Although Mina expression varies according to cell-type, developmental stage and activation state, its transcriptional regulation is poorly understood. Across inbred mouse strains, Mina protein level exhibits a bimodal distribution, correlating with inheritance of a biallelic haplotype block comprising 21 promoter/intron 1-region SNPs. We previously showed that heritable differences in Mina protein level are transcriptionally regulated. Accordingly, we decided to test the hypothesis that at least one of the promoter/intron 1-region SNPs perturbs a Mina cis-regulatory element (CRE). Here, we have comprehensively scanned for CREs across a Mina locus-spanning 26-kilobase genomic interval. We discovered 8 potential CREs and functionally validated 4 of these, the strongest of which (E2), residing in intron 1, contained a SNP whose BALB/c-but not C57Bl/6 allele-abolished both Smad3 binding and transforming growth factor beta (TGFβ) responsiveness. Our results demonstrate the TGFβ signaling pathway plays a critical role in regulating Mina expression and SNP rs4191790 controls heritable variation in Mina expression level, raising important questions regarding the evolution of an allele that uncouples Mina expression from the TGFβ signaling pathway. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

A SNP uncoupling Mina expression from the TGFβ signaling pathway

PubMed Central

Lian, Shang L.; Mihi, Belgacem; Koyanagi, Madoka; Nakayama, Toshinori

2017-01-01

Abstract Introduction Mina is a JmjC family 2‐oxoglutarate oxygenase with pleiotropic roles in cell proliferation, cancer, T cell differentiation, pulmonary inflammation, and intestinal parasite expulsion. Although Mina expression varies according to cell‐type, developmental stage and activation state, its transcriptional regulation is poorly understood. Across inbred mouse strains, Mina protein level exhibits a bimodal distribution, correlating with inheritance of a biallelic haplotype block comprising 21 promoter/intron 1‐region SNPs. We previously showed that heritable differences in Mina protein level are transcriptionally regulated. Methods Accordingly, we decided to test the hypothesis that at least one of the promoter/intron 1‐region SNPs perturbs a Mina cis‐regulatory element (CRE). Here, we have comprehensively scanned for CREs across a Mina locus‐spanning 26‐kilobase genomic interval. Results We discovered 8 potential CREs and functionally validated 4 of these, the strongest of which (E2), residing in intron 1, contained a SNP whose BALB/c—but not C57Bl/6 allele—abolished both Smad3 binding and transforming growth factor beta (TGFβ) responsiveness. Conclusions Our results demonstrate the TGFβ signaling pathway plays a critical role in regulating Mina expression and SNP rs4191790 controls heritable variation in Mina expression level, raising important questions regarding the evolution of an allele that uncouples Mina expression from the TGFβ signaling pathway. PMID:28967702

Loss of p300 and CBP disrupts histone acetylation at the mouse Sry promoter and causes XY gonadal sex reversal

PubMed Central

Carré, Gwenn-Aël; Siggers, Pam; Xipolita, Marilena; Brindle, Paul; Lutz, Beat; Wells, Sara; Greenfield, Andy

2018-01-01

Abstract CREB-binding protein (CBP, CREBBP, KAT3A) and its closely related paralogue p300 (EP300, KAT3B), together termed p300/CBP, are histone/lysine acetyl-transferases that control gene expression by modifying chromatin-associated proteins. Here, we report roles for both of these chromatin-modifying enzymes in mouse sex determination, the process by which the embryonic gonad develops into a testis or an ovary. By targeting gene ablation to embryonic gonadal somatic cells using an inducible Cre line, we show that gonads lacking either gene exhibit major abnormalities of XY gonad development at 14.5 dpc, including partial sex reversal. Embryos lacking three out of four functional copies of p300/Cbp exhibit complete XY gonadal sex reversal and have greatly reduced expression of the key testis-determining genes Sry and Sox9. An analysis of histone acetylation at the Sry promoter in mutant gonads at 11.5 dpc shows a reduction in levels of the positive histone mark H3K27Ac. Our data suggest a role for CBP/p300 in testis determination mediated by control of histone acetylation at the Sry locus and reveal a novel element in the epigenetic control of Sry and mammalian sex determination. They also suggest possible novel causes of human disorders of sex development (DSD). PMID:29145650

Extracellular visfatin activates gluconeogenesis in HepG2 cells through the classical PKA/CREB-dependent pathway.

PubMed

Choi, Y J; Choi, S-E; Ha, E S; Kang, Y; Han, S J; Kim, D J; Lee, K W; Kim, H J

2014-04-01

Adipokines reportedly affect hepatic gluconeogenesis, and the adipokine visfatin is known to be related to insulin resistance and type 2 diabetes. However, whether visfatin contributes to hepatic gluconeogenesis remains unclear. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), modulates sirtuin1 (SIRT1) through the regulation of nicotinamide adenine dinucleotide (NAD). Therefore, we investigated the effect of extracellular visfatin on glucose production in HepG2 cells, and evaluated whether extracellular visfatin affects hepatic gluconeogenesis via an NAD+-SIRT1-dependent pathway. Treatment with visfatin significantly increased glucose production and the mRNA expression and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in HepG2 cells in a time- and concentration-dependent manner. Knockdown of SIRT1 had no remarkable effect on the induction of gluconeogenesis by visfatin. Subsequently, we evaluated if extracellular visfatin stimulates the production of gluconeogenic enzymes through the classical protein kinase A (PKA)/cyclic AMP-responsive element (CRE)-binding protein (CREB)-dependent process. The phosphorylation of CREB and PKA increased significantly in HepG2 cells treated with visfatin. Additionally, knockdown of CREB and PKA inhibited visfatin-induced gluconeogenesis in HepG2 cells. In summary, extracellular visfatin modulates glucose production in HepG2 cells through the PKA/CREB pathway, rather than via SIRT1 signaling. © Georg Thieme Verlag KG Stuttgart · New York.

Inducing Somatic Pkd1 Mutations in Vivo in a Mouse Model of Autosomal-Dominant Polycystic Kidney Disease

DTIC Science & Technology

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the worlds most common life threatening genetic diseases. Over 95 percent of diagnosed...several genetic models to induce mutations: two during embryogenesis (with Six2-cre and CVM-cre) and one in the adult (Villin-cre). One of the embryonic

Optimizing tamoxifen-inducible Cre/loxp system to reduce tamoxifen effect on bone turnover in long bones of young mice.

PubMed

Zhong, Zhendong A; Sun, Weihua; Chen, Haiyan; Zhang, Hongliang; Lay, Yu-An E; Lane, Nancy E; Yao, Wei

2015-12-01

For tamoxifen-dependent Cre recombinase, also known as CreER recombinase, tamoxifen (TAM) is used to activate the Cre to generate time- and tissue-specific mouse mutants. TAM is a potent CreER system inducer; however, TAM is also an active selective estrogen receptor modulator (SERM) that can influence bone homeostasis. The purpose of this study was to optimize the TAM dose for Cre recombinase activation while minimizing the effects of TAM on bone turnover in young growing mice. To evaluate the effects of TAM on bone turnover and bone mass, 1-month-old wild-type male and female mice were intraperitoneally injected with TAM at 0, 1, 10 or 100mg/kg/day for four consecutive days, or 100, 300 mg/kg/day for one day. The distal femurs were analyzed one month after the last TAM injection by microCT, mechanical test, and surface-based bone histomorphometry. Similar doses of TAM were used in Col1 (2.3 kb)-CreERT2; mT/mG reporter male mice to evaluate the dose-dependent efficacy of Cre-ER activation in bone tissue. A TAM dose of 100 mg/kg × 4 days significantly increased trabecular bone volume/total volume (BV/TV) of the distal femur, femur length, bone strength, and serum bone turnover markers compared to the 0mg control group. In contrast, TAM doses ≤ 10 mg/kg did not significantly change any of these parameters compared to the 0mg group, although a higher bone strength was observed in the 10mg group. Surface-based histomorphometry revealed that the 100mg/kg dose of TAM dose significantly increased trabecular bone formation and decreased periosteal bone formation at 1-week post-TAM treatment. Using the reporter mouse model Col1-CreERT2; mT/mG, we found that 10mg/kg TAM induced Col1-CreERT2 activity in bone at a comparable level to the 100mg/kg dose. TAM treatment at 100mg/kg/day × 4 days significantly affects bone homeostasis, resulting in an anabolic bone effect on trabecular bone in 1-month-old male mice. However, a lower dose of TAM at 10 mg/kg/day × 4 days can yield similar Col1-CreERT2 induction efficacy with minimum effects on bone turnover in young male mice. Copyright © 2015 Elsevier Inc. All rights reserved.

Cooperativity of E-cadherin and Smad4 loss to promote diffuse-type gastric adenocarcinoma and metastasis.

PubMed

Park, Jun Won; Jang, Seok Hoon; Park, Dong Min; Lim, Na Jung; Deng, Chuxia; Kim, Dae Yong; Green, Jeffrey E; Kim, Hark Kyun

2014-08-01

Loss of E-cadherin (CDH1), Smad4, and p53 has been shown to play an integral role in gastric, intestinal, and breast cancer formation. Compound conditional knockout mice for Smad4, p53, and E-cadherin were generated to define and compare the roles of these genes in gastric, intestinal, and breast cancer development by crossing with Pdx-1-Cre, Villin-Cre, and MMTV-Cre transgenic mice. Interestingly, gastric adenocarcinoma was significantly more frequent in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice than in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(+/+) mice, demonstrating that Cdh1 heterozygosity accelerates the development and progression of gastric adenocarcinoma, in combination with loss of Smad4 and p53. Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice developed gastric adenocarcinomas without E-cadherin expression. However, intestinal and mammary adenocarcinomas with the same genetic background retained E-cadherin expression and were phenotypically similar to mice with both wild-type Cdh1 alleles. Lung metastases were identified in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice, but not in the other genotypes. Nuclear β-catenin accumulation was identified at the invasive tumor front of gastric adenocarcinomas arising in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice. This phenotype was less prominent in mice with intact E-cadherin or Smad4, indicating that the inhibition of β-catenin signaling by E-cadherin or Smad4 downregulates signaling pathways involved in metastases in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice. Knockdown of β-catenin significantly inhibited the migratory activity of Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) cell lines. Thus, loss of E-cadherin and Smad4 cooperates with p53 loss to promote the development and metastatic progression of gastric adenocarcinomas, with similarities to human gastric adenocarcinoma. This study demonstrates that inhibition of β-catenin is a converging node for the antimetastatic signaling pathways driven by E-cadherin and Smad4 in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice, providing novel insights into mechanisms for gastric cancer metastasis. ©2014 American Association for Cancer Research.

Constitutive activation of IKK2/NF-κB impairs osteogenesis and skeletal development.

PubMed

Swarnkar, Gaurav; Zhang, Kaihua; Mbalaviele, Gabriel; Long, Fanxin; Abu-Amer, Yousef

2014-01-01

Pathologic conditions impair bone homeostasis. The transcription factor NF-κB regulates bone homeostasis and is central to bone pathologies. Whereas contribution of NF-κB to heightened osteoclast activity is well-documented, the mechanisms underlying NF-κB impact on chondrocytes and osteoblasts are scarce. In this study, we examined the effect of constitutively active IKK2 (IKK2ca) on chondrogenic and osteogenic differentiation. We show that retroviral IKK2ca but not GFP, IKK2WT, or the inactive IKK2 forms IKK2KM and IKK2SSAA, strongly suppressed osteogenesis and chondrogenesis, in vitro. In order to explore the effect of constitutive NF-κB activation on bone formation in vivo, we activated this pathway in a conditional fashion. Specifically, we crossed the R26StopIKK2ca mice with mice carrying the Col2-cre in order to express IKK2ca in osteoblasts and chondrocytes. Both chondrocytes and osteoblasts derived from Col2Cre/IKK2ca expressed IKK2ca. Mice were born alive yet died shortly thereafter. Histologically, newborn Col2Cre+/RosaIKK2ca heterozygotes (Cre+IKK2ca_w/f (het)) and homozygotes (Cre+IKK2ca_f/f (KI)) showed smaller skeleton, deformed vertebrate and reduced or missing digit ossification. The width of neural arches, as well as ossification in vertebral bodies of Cre+IKK2ca_w/f and Cre+IKK2ca_f/f, was reduced or diminished. H&E staining of proximal tibia from new born pups revealed that Cre+IKK2ca_f/f displayed disorganized hypertrophic zones within the smaller epiphysis. Micro-CT analysis indicated that 4-wk old Cre+IKK2ca_w/f has abnormal trabecular bone in proximal tibia compared to WT littermates. Mechanistically, ex-vivo experiments showed that expression of differentiation markers in calvarial osteoblasts derived from newborn IKK2ca knock-in mice was diminished compared to WT-derived cells. In situ hybridization studies demonstrated that the hypertrophic chondrocyte marker type-X collagen, the pre-hypertrophic chondrocyte markers Indian hedgehog and alkaline phosphatase, and the early markers Aggrecan and type-II collagen were reduced in Cre+IKK2ca_w/f and Cre+IKK2ca_f/f mice. Altogether, the in-vitro, in vivo and ex-vivo evidence suggest that IKK2ca perturbs osteoblast and chondrocyte maturation and impairs skeletal development.

Effects of coffee and caffeine anhydrous intake during creatine loading

PubMed Central

Trexler, Eric T.; Smith-Ryan, Abbie E.; Roelofs, Erica J.; Hirsch, Katie R.; Persky, Adam M.; Mock, Meredith G.

2015-01-01

The purpose of this study was to determine the effect of 5 d of creatine (CRE) loading alone or in combination with caffeine anhydrous (CAF) or coffee (COF) on upper and lower body strength and sprint performance. Physically active males (n=54; Mean ± SD; Age = 20.1 ± 2.1 yrs; Weight = 78.8 ± 8.8 kg) completed baseline testing, consisting of one-repetition maximum (1RM) and repetitions to fatigue (RTF) with 80% 1RM for bench press (BP) and leg press (LP), followed by a repeated sprint test of five, 10 s sprints separated by 60 s rest on a cycle ergometer to determine peak power (PP) and total power (TP). At least 72 hr later, subjects were randomly assigned to supplement with CRE (5 g creatine monohydrate, 4 times*d−1; n=14), CRE+CAF (CRE + 300 mg*d−1 of CAF; n=13), CRE+COF (CRE + 8.9 g COF, yielding 303 mg caffeine; n=13), or placebo (PLA; n=14) for 5 d. Serum creatinine (CRN) was measured prior to and following supplementation and on day six, participants repeated pre-testing procedures. Strength measures were improved in all groups (p<0.05), with no significant time × treatment interactions. No significant interaction or main effects were observed for PP. For TP, a time × sprint interaction was observed (p<0.05), with no significant interactions between treatment groups. A time × treatment interaction was observed for serum CRN values (p<0.05) that showed increases in all groups except PLA. Four subjects reported mild gastrointestinal discomfort with CRE+CAF, with no side effects reported in other groups. These findings suggest that neither CRE alone, nor in combination with CAF or COF, significantly affected performance compared to PLA. PMID:26439785

Cosmic Ray Exposure Ages of Stony Meteorites: Space Erosion or Yarkovsky?

NASA Technical Reports Server (NTRS)

Rubincam, David Parry

2014-01-01

Space erosion from dust impacts may set upper limits on the cosmic ray exposure (CRE) ages of stony meteorites. A meteoroid orbiting within the asteroid belt is bombarded by both cosmic rays and interplanetary dust particles. Galactic cosmic rays penetrate only the first few meters of the meteoroid; deeper regions are shielded. The dust particle impacts create tiny craters on the meteoroid's surface, wearing it away by space erosion (abrasion) at a particular rate. Hence a particular point inside a meteoroid accumulates cosmic ray products only until that point wears away, limiting CRE ages. The results would apply to other regolith-free surfaces in the solar system as well, so that abrasion may set upper CRE age limits which depend on the dusty environment. Calculations based on N. Divine's dust populations and on micrometeoroid cratering indicate that stony meteoroids in circular ecliptic orbits at 2 AU will record 21Ne CRE ages of approx.176 x 10(exp 6) years if dust masses are in the range 10(exp -21) - 10(exp -3) kg. This is in broad agreement with the maximum observed CRE ages of approx. 100 x 10(exp 6) years for stones. High erosion rates in the inner solar system may limit the CRE ages of Near-Earth Asteroids (NEAs) to approx. 120 x 10(exp 6) years. If abrasion should prove to be approx. 6 times quicker than found here, then space erosion may be responsible for many of the measured CRE ages of main belt stony meteorites. In that case the CRE ages may not measure the drift time to the resonances due to the Yarkovsky effects as in the standard scenario, and that for some reason Yarkovsky is ineffective.

Adenovirus-mediated truncated Bid overexpression induced by the Cre/LoxP system promotes the cell apoptosis of CD133+ ovarian cancer stem cells.

PubMed

Long, Qifang; Yang, Ru; Lu, Weixian; Zhu, Weipei; Zhou, Jundong; Zheng, Cui; Zhou, Dongmei; Yu, Ling; Wu, Jinchang

2017-01-01

Cancer stem cells are a small subset of cancer cells that contribute to cancer progression, metastasis, chemoresistance and recurrence. CD133-positive (CD133+) ovarian cancer cells have been identified as ovarian cancer stem cells. Adenovirus-mediated gene therapy is an innovative therapeutic method for cancer treatment. In the present study, we aimed to develop a new gene therapy to specifically eliminate CD133+ ovarian cancer stem cells by targeting CD133. We used the Cre/LoxP system to augment the selective expression of the truncated Bid (tBid) gene as suicide gene therapy in CD133+ ovarian cancer stem cells. The adenovirus (Ad)-CD133-Cre expressing Cre recombinase under the control of the CD133 promoter and Ad-CMV-LoxP-Neo-LoxP-tBid expressing tBid under the control of the CMV promoter were successfully constructed using the Cre/LoxP switching system. The co-infection of Ad-CMV-LoxP-Neo-LoxP-tBid and Ad-CD133-Cre selectively induced tBid overexpression, which inhibited cell growth and triggered the cell apoptosis of CD133+ ovarian cancer stem cells. The Cre/LoxP system-mediated tBid overexpression activated the pro-apoptotic signaling pathway and augmented the cytotoxic effect of cisplatin in CD133+ ovarian cancer stem cells. Furthermore, in xenograft experiments, co-infection with the two recombinant adenoviruses markedly suppressed tumor growth in vivo and promoted cell apoptosis in tumor tissues. Taken together, the present study provides evidence that the adenovirus-mediated tBid overexpression induced by the Cre/LoxP system can effectively eliminate CD133+ ovarian cancer stem cells, representing a novel therapeutic strategy for the treatment of ovarian cancer.

Heme oxygenase-1 expression protects the heart from acute injury caused by inducible Cre recombinase

PubMed Central

Hull, Travis D.; Bolisetty, Subashini; DeAlmeida, Angela; Litovsky, Silvio H.; Prabhu, Sumanth D.; Agarwal, Anupam; George, James F.

2013-01-01

The protective effect of heme oxygenase-1 (HO-1) expression in cardiovascular disease has been previously demonstrated using transgenic animal models in which HO-1 is constitutively overexpressed in the heart. However, the temporal requirements for protection by HO-1 induction relative to injury have not been investigated, but are essential to employ HO-1 as a therapeutic strategy in human cardiovascular disease states. Therefore, we generated mice with cardiac-specific, tamoxifen (TAM)-inducible overexpression of a human HO-1 (hHO-1) transgene (MHC-HO-1 mice) by breeding mice with cardiac-specific expression of a TAM-inducible Cre recombinase (MHC-Cre mice) with mice containing an hHO-1 transgene preceded by a floxed stop signal (CBA-flox mice). MHC-HO-1 overexpress the HO-1 gene and enzymatically protein following TAM administration (40 mg/kg body weight on two consecutive days). In MHC-Cre controls, TAM administration leads to severe, acute cardiac toxicity, cardiomyocyte necrosis, and 80% mortality by day 3. This cardiac toxicity is accompanied by a significant increase in inflammatory cells in the heart that are predominantly neutrophils. In MHC-HO-1 mice, HO-1 overexpression ameliorates the depression of cardiac function and high mortality rate observed in MHC-Cre mice following TAM administration and attenuates cardiomyocyte necrosis and neutrophil infiltration. These results highlight that HO-1 induction is sufficient to prevent the depression of cardiac function observed in mice with TAM-inducible Cre recombinase expression by protecting the heart from necrosis and neutrophil infiltration. These findings are important because MHC-Cre mice are widely used in cardiovascular research despite the limitations imposed by Cre-induced cardiac toxicity and also because inflammation is an important pathological component of many human cardiovascular diseases. PMID:23732814

Heme oxygenase-1 expression protects the heart from acute injury caused by inducible Cre recombinase.

PubMed

Hull, Travis D; Bolisetty, Subhashini; DeAlmeida, Angela C; Litovsky, Silvio H; Prabhu, Sumanth D; Agarwal, Anupam; George, James F

2013-08-01

The protective effect of heme oxygenase-1 (HO-1) expression in cardiovascular disease has been previously demonstrated using transgenic animal models in which HO-1 is constitutively overexpressed in the heart. However, the temporal requirements for protection by HO-1 induction relative to injury have not been investigated, but are essential to employ HO-1 as a therapeutic strategy in human cardiovascular disease states. Therefore, we generated mice with cardiac-specific, tamoxifen (TAM)-inducible overexpression of a human HO-1 (hHO-1) transgene (myosin heavy chain (MHC)-HO-1 mice) by breeding mice with cardiac-specific expression of a TAM-inducible Cre recombinase (MHC-Cre mice), with mice containing an hHO-1 transgene preceded by a floxed-stop signal. MHC-HO-1 mice overexpress HO-1 mRNA and the enzymatically active protein following TAM administration (40 mg/kg body weight on 2 consecutive days). In MHC-Cre controls, TAM administration leads to severe, acute cardiac toxicity, cardiomyocyte necrosis, and 80% mortality by day 3. This cardiac toxicity is accompanied by a significant increase in inflammatory cells in the heart that are predominantly neutrophils. In MHC-HO-1 mice, HO-1 overexpression ameliorates the depression of cardiac function and high mortality rate observed in MHC-Cre mice following TAM administration and attenuates cardiomyocyte necrosis and neutrophil infiltration. These results highlight that HO-1 induction is sufficient to prevent the depression of cardiac function observed in mice with TAM-inducible Cre recombinase expression by protecting the heart from necrosis and neutrophil infiltration. These findings are important because MHC-Cre mice are widely used in cardiovascular research despite the limitations imposed by Cre-induced cardiac toxicity, and also because inflammation is an important pathological component of many human cardiovascular diseases.

The ENSO Effects on Tropical Clouds and Top-of-Atmosphere Cloud Radiative Effects in CMIP5 Models

NASA Technical Reports Server (NTRS)

Su, Wenying; Wang, Hailan

2015-01-01

The El Nino-Southern Oscillation (ENSO) effects on tropical clouds and top-of-atmosphere (TOA) cloud radiative effects (CREs) in Coupled Model Intercomparison Project Phase5 (CMIP5) models are evaluated using satellite-based observations and International Satellite Cloud Climatology Project satellite simulator output. Climatologically, most CMIP5 models produce considerably less total cloud amount with higher cloud top and notably larger reflectivity than observations in tropical Indo-Pacific (60 degrees East - 200 degrees East; 10 degrees South - 10 degrees North). During ENSO, most CMIP5 models considerably underestimate TOA CRE and cloud changes over western tropical Pacific. Over central tropical Pacific, while the multi-model mean resembles observations in TOA CRE and cloud amount anomalies, it notably overestimates cloud top pressure (CTP) decreases; there are also substantial inter-model variations. The relative effects of changes in cloud properties, temperature and humidity on TOA CRE anomalies during ENSO in the CMIP5 models are assessed using cloud radiative kernels. The CMIP5 models agree with observations in that their TOA shortwave CRE anomalies are primarily contributed by total cloud amount changes, and their TOA longwave CRE anomalies are mostly contributed by changes in both total cloud amount and CTP. The model biases in TOA CRE anomalies particularly the strong underestimations over western tropical Pacific are, however, mainly explained by model biases in CTP and cloud optical thickness (tau) changes. Despite the distinct model cloud biases particularly in tau regime, the TOA CRE anomalies from cloud amount changes are comparable between the CMIP5 models and observations, because of the strong compensations between model underestimation of TOA CRE anomalies from thin clouds and overestimation from medium and thick clouds.

Functional Effects of TGF-beta1 on Mesenchymal Stem Cell Mobilization in Cockroach Allergen Induced Asthma

PubMed Central

Xian, Lingling; Li, Changjun; Xu, Ting; Plunkett, Beverly; Huang, Shau-Ku; Wan, Mei; Cao, Xu

2014-01-01

Mesenchymal stem cells (MSCs) have been suggested to participate in immune regulation and airway repair/remodeling. Transforming growth factor β1 (TGFβ1) is critical in the recruitment of stem/progenitor cells for tissue repair, remodeling and cell differentiation. In this study, we sought to investigate the role of TGFβ1 in MSC migration in allergic asthma. We examined nestin expression (a marker for MSCs) and TGFβ1 signaling activation in airways in cockroach allergen (CRE) induced mouse models. Compared with control mice, there were increased nestin+ cells in airways, and higher levels of active TGFβ1 in serum and p-Smad2/3 expression in lungs of CRE-treated mice. Increased activation of TGFβ1 signaling was also found in CRE-treated MSCs. We then assessed MSC migration induced by conditioned medium (ECM) from CRE-challenged human epithelium in air/liquid interface (ALI) culture in Transwell assays. MSC migration was stimulated by ECM, but was significantly inhibited by either TGFβ1 neutralizing antibody or TβR1 inhibitor. Intriguingly, increased migration of MSCs from blood and bone marrow to the airway was also observed after systemic injection of GFP+-MSCs, and from bone marrow of Nes-GFP mice following CRE challenge. Furthermore, TGFβ1 neutralizing antibody inhibited the CRE-induced MSC recruitment, but promoted airway inflammation. Finally, we investigated the role of MSCs in modulating CRE induced T cell response, and found that MSCs significantly inhibited CRE-induced inflammatory cytokine secretion (IL-4, IL13, IL17 and IFN-γ) by CD4+ T cells. These results suggest that TGFβ1 may be a key pro-migratory factor in recruiting MSCs to the airways in mouse models of asthma. PMID:24711618

Caveats and considerations for performing pancreas-specific gene manipulations in the mouse.

PubMed

Magnuson, M A; Burlison, J S

2007-11-01

Conditional gene targeting using the Cre/loxP strategy has proven to be very useful for studies of glucose homeostasis, tissue function and dysfunction in diabetes, and pancreas development. However, use of this strategy over the past decade has revealed a variety of experimental caveats, many of which are a direct consequence of the procedures used to generate Cre-driver lines. We discuss frequently encountered experimental artefacts, the advantages of using bacterial artificial chromosome-derived transgenes or performing a Cre knockin for improving the specificity of expression, and systems for regulating Cre activity. In addition, recent studies indicate that high amounts of Cre in the pancreatic beta-cell may cause glucose intolerance and impaired insulin secretion. However, these findings, while serving as a reminder for simple experimental controls, are unlikely to diminish utilization of this very powerful and useful technology.

Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site.

PubMed

Means, A L; Farnham, P J

1990-02-01

We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).

Is faecal microbiota transplantation an option to eradicate highly drug-resistant enteric bacteria carriage?

PubMed

Davido, B; Batista, R; Michelon, H; Lepainteur, M; Bouchand, F; Lepeule, R; Salomon, J; Vittecoq, D; Duran, C; Escaut, L; Sobhani, I; Paul, M; Lawrence, C; Perronne, C; Chast, F; Dinh, A

2017-04-01

Carbapenem-resistant Enterobacteriaceae (CRE) or vancomycin-resistant enterococci (VRE) carriage present a major public health challenge. Decolonization strategies are lacking. We aimed to evaluate the impact of faecal microbiota transplantation (FMT) on a cohort of patients with digestive tract colonization by CRE or VRE. Eight patients were included: six carrying CRE and two colonized by VRE. One month after FMT, two patients were free from CRE carriage, and another patient was free from VRE after three months. In our experience, this strategy is safe. Copyright © 2017 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Developmentally induced Mll1 loss reveals defects in postnatal haematopoiesis.

PubMed

Gan, T; Jude, C D; Zaffuto, K; Ernst, P

2010-10-01

The mixed lineage leukemia (MLL) gene is disrupted by chromosomal translocations in acute leukemia, producing a fusion oncogene with altered properties relative to the wild-type gene. Murine loss-of-function studies have shown an essential role for Mll in developing the haematopoietic system, yet studies using different conditional knockout models have yielded conflicting results regarding the requirement for Mll during adult steady-state haematopoiesis. In this study, we used a loxP-flanked Mll allele (Mll(F)) and a developmentally regulated, haematopoietic-specific VavCre transgene to reassess the consequences of Mll loss in the haematopoietic lineage, without the need for inducers of Cre recombinase. We show that VavCre;Mll mutants exhibit phenotypically normal fetal haematopoiesis, but rarely survive past 3 weeks of age. Surviving animals are anemic, thrombocytopenic and exhibit a significant reduction in bone marrow haematopoietic stem/progenitor populations, consistent with our previous findings using the inducible Mx1Cre transgene. Furthermore, the analysis of VavCre mutants revealed additional defects in B-lymphopoiesis that could not be assessed using Mx1Cre-mediated Mll deletion. Collectively, these data support the conclusion that Mll has an essential role in sustaining postnatal haematopoiesis.

Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors.

PubMed

Vega, Juan M; Yu, Weichang; Han, Fangpu; Kato, Akio; Peters, Eric M; Zhang, Zhanyuan J; Birchler, James A

2008-04-01

The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

Molecular switch of Cre/loxP for radiation modulated gene therapy on hepatoma

NASA Astrophysics Data System (ADS)

Hsieh, Ya-Ju; Chen, Fu-Du; Wang, Fu Hui; Ke, Chien Chih; Wang, Hsin-Ell; Liu, Ren-Shyan

2007-02-01

For the purpose of enhancement of AFP promoter for the use of radiation modulated gene therapy for hepatocellular carcinoma (HCC), we combined hepatitis B virus (HBV) enhancer II with AFP promoter which shows the selectivity to the target cells to control the Cre/loxP system. Different gene constructs, pE4luc, pE4Tk, EIIAPA-Cre, E4CMV-STOP-Tk and chimeric promoters combined with HBV enhancer were constructed and transfected into HepG2, HeLa and NIH-3T3 cell lines. Cell experiments revealed that E4 enhancer responses to radiation best after 60 h irradiation at a dose range of 5-7 Gy in HepG2 stable clone. The EIIAPA promoter provided high specificity to hepatoma and activated the Cre downstream and removed the stop cassette only in hepatoma cells. After removal of the stop cassette, the E4 response to radiation could encode more Tk protein and kill more tumor cells. In summary, the chimeric EIIAPA promoter can stringently control the expression of Cre recombinase only in HCC. The radiation effect of the EIIAPA-Cre and E4CMV-STOP-Tk system shows promising results in terms of cell survival of HCC.

Factors Associated to Prevalence and Incidence of Carbapenem-Resistant Enterobacteriaceae Fecal Carriage: A Cohort Study in a Mexican Tertiary Care Hospital

PubMed Central

Torres-Gonzalez, Pedro; Cervera-Hernandez, Miguel Enrique; Niembro-Ortega, María Dolores; Leal-Vega, Francisco; Cruz-Hervert, Luis Pablo; García-García, Lourdes; Galindo-Fraga, Arturo; Martinez-Gamboa, Areli; Bobadilla-del Valle, Miriam; Sifuentes-Osornio, Jose; Ponce-de-Leon, Alfredo

2015-01-01

Background Carbapenem-resistant Enterobacteriaceae (CRE) infections have emerged as a serious threat to health worldwide. They are associated with increased morbidity and mortality and are capable of silently colonizing the gastrointestinal tract. Because of this, there is great interest to characterize the epidemiology of CRE carriage and acquisition in healthcare facilities. The aim of this study was to determine the prevalence and factors associated with CRE fecal carriage (CRE-fc), and risk factors for incident cases. Methods/Results A cohort study was conducted at a tertiary care hospital from January 1st to April 30th, 2014 during a CRE outbreak. Weekly rectal swabs were performed in patients considered at risk until discharge. CRE-fc prevalence was 10.9% (CI 95% 7.7–14.7) among 330 patients. Treatment with carbapenems (OR 2.54, CI 95% 1.15–5.62); transfer from an institution (OR 2.16, CI 95% 1.02–4.59); multi-drug resistant infection within the previous six months (OR 2.81, CI 95% 1.47–5.36); intensive care unit admission (OR 0.42, CI 95% 0.20–0.88); hematologic malignancy (OR 4.02, CI 95% 1.88–8.06); invasive procedures (OR 2.18, CI 95% 1.10–4.32); and sharing a room with a known CRE carrier (OR 3.0, CI 95% 1.43–6.31) were independently associated factors for CRE-fc. Risk factors associated with CRE-fc incidence were determined for 87 patients initially negative and with subsequent screening; the incidence rate was 2.5 cases, per 1000 person-years (CI 95% 1.5–3.9). Independently associated risk factors were carbapenem treatment (HR 2.68, CI 95% 1.03–6.98), hematologic malignancy (HR 5.74, 95% CI 2.46–13.4) and a mean daily colonization pressure ≥10% (HR 5.03, IC 95% 1.77–14.28). OXA-48-like (OXA-232) and CTX-M-15 were the predominantly identified mechanisms of resistance. Conclusions We found an elevated incidence and prevalence of CRE-fc in our hospital. Hematologic patients need to be considered a population at risk, and antibiotic stewardship along with infection control programs need to be improved to avoid nosocomial spread. PMID:26431402

A Dreissena Risk Assessment for the Colorado River Ecosystem

USGS Publications Warehouse

Kennedy, Theodore A.

2007-01-01

Executive Summary Nonnative zebra and quagga mussels (Dreissena polymorpha and Dreissena bugensis, respectively; see photo above) were accidentally introduced to the Great Lakes in the 1980s and subsequently spread to watersheds of the Eastern United States (Strayer and others, 1999). The introduction of Dreissena mussels has been economically costly and has had large and far-reaching ecological impacts on these systems. Quagga mussels were found in Lakes Mead and Havasu in January 2007. Given the likelihood that quagga mussels and, eventually, zebra mussels will be introduced to Lake Powell and the Colorado River at Lees Ferry, it is important to assess the risks that introduction of Dreissena mussels pose to the Colorado River ecosystem (here defined as the segment of river from just below Glen Canyon Dam to Diamond Creek; hereafter CRE). In this report, I assess three different types of risks associated with Dreissena and the CRE: (1) the risk that Dreissena will establish at high densities in the CRE, (2) the risk of ecological impacts should Dreissena establish at high densities in the CRE or in Lake Powell, and (3) the risk that Dreissena will be introduced to tributaries of the CRE. The risk of Dreissena establishing within the CRE is low, except for the Lees Ferry tailwater reach where the risk appears high. Dreissena are unlikely to establish at high densities within the CRE or its tributaries because of high suspended sediment, high ratios of suspended inorganic:organic material, and high water velocities, all of which interfere with the ability of Dreissena to effectively filter feed. The rapids of Grand Canyon may represent a large source of mortality to larval Dreissena, which would limit their ability to disperse and colonize downstream reaches of the CRE. In contrast, conditions within the Lees Ferry tailwater generally appear suitable for Dreissena establishment, with the exception of high average water velocity. If Dreissena establish within the CRE, the risks of negative ecological impacts appear low. If Dreissena are able to attain moderate densities in Lees Ferry, estimates of filtration capacity indicate they are unlikely to substantially alter the composition (e.g., nutrient concentrations, suspended organic matter concentrations) of water exported from Lees Ferry. Further, a moderate density of Dreissena within Lees Ferry may actually increase food available to fishes by increasing habitat complexity and stimulating benthic production. If Dreissena attain moderate densities in the CRE mainstem, which seems unlikely, ecological impacts will probably be comparable to Lees Ferry-an increase in benthic production. Dreissena may have ecological impacts on the CRE, if they become established in Lake Powell and substantially alter the composition of water released from Glen Canyon Dam; however, it is unclear whether changes in the composition of water released from Glen Canyon Dam will have a net positive or negative impact on food availability in the CRE mainstem. The risk of Dreissena introduction to tributaries appears low. None of the tributaries have upstream lakes or reservoirs that could actually serve as a source population for Dreissena; reservoirs on the Little Colorado River may eventually support Dreissena, but they are far up in the watershed and the segment of river connecting them with the mainstem CRE is intermittent. If the CRE mainstem is colonized by Dreissena, there are no significant vectors for transporting them upstream into the tributaries. In addition, lethally high summer water temperatures make it unlikely that Dreissena will establish in many tributaries. Lake Powell is a logical focus for management and research efforts, given that maintenance of Dreissena populations within the CRE will require an upriver source population and the uncertainty associated with the downstream impact of changes in Lake Powell water quality.

Out of Place, Out of Mind: Schema-Driven False Memory Effects for Object-Location Bindings

ERIC Educational Resources Information Center

Lew, Adina R.; Howe, Mark L.

2017-01-01

Events consist of diverse elements, each processed in specialized neocortical networks, with temporal lobe memory systems binding these elements to form coherent event memories. We provide a novel theoretical analysis of an unexplored consequence of the independence of memory systems for elements and their bindings, 1 that raises the paradoxical…

Assisting couples to develop healthy relationships: effects of couples relationship education on cortisol.

PubMed

Ditzen, Beate; Hahlweg, Kurt; Fehm-Wolfsdorf, Gabriele; Baucom, Don

2011-06-01

Couple conflict in unhappy marriages is suggested to impair individual health via chronic psychophysiological stress reactions in couples' everyday lives. As a consequence, we hypothesized that standard couples relationship education (CRE) would decrease psychophysiological stress, namely salivary cortisol levels, during couple conflict in the laboratory as compared to a standard psychological stress paradigm. We considered cortisol to be of particular interest in this context, as it mediates endocrine and immune responses to stress, and thus might influence couples' health. Salivary cortisol was repeatedly investigated in 61 couples during (a) a standard psychological stress test with no relevance for the couples, and (b) a standard couple conflict discussion in the laboratory before and after CRE. In addition, increases in self-evaluated relationship quality were analyzed with regard to their influence on salivary cortisol. Data were analyzed using multilevel modeling. Cortisol responses to the couple-external psychological stress test were unaffected by CRE, but specifically cortisol responses during the couple conflict discussion were significantly reduced following CRE compared to pre-intervention levels. Moreover, cortisol decreases during conflict were partially mediated by increases in self-reported relationship quality following CRE. These data suggest that CRE might buffer the harmful effects of repeated conflict in close relationships. Rather than ameliorating overall stress resilience, CRE might thus specifically improve individual health through increased relationship quality and reduced HPA axis activity during couple conflict. Copyright © 2010 Elsevier Ltd. All rights reserved.

Nestin-Cre Mice Are Affected by Hypopituitarism, Which Is Not Due to Significant Activity of the Transgene in the Pituitary Gland

PubMed Central

Galichet, Christophe; Lovell-Badge, Robin; Rizzoti, Karine

2010-01-01

Nestin-Cre mice express Cre recombinase under control of the rat nestin promoter and central nervous system (CNS) enhancer. While endogenous Nestin is expressed in some other tissues including the pituitary gland, Nestin-Cre mice induce recombination predominantly in the CNS. For this reason, they have been widely used to explore gene function or cell fate in the latter. Pituitary hormonal deficiencies, or hypopituitarism, are associated with a wide range of symptoms and with a significant morbidity. These can have a neural and/or a pituitary origin as the gland's secretions are controlled by the hypothalamus. We report here that Nestin-Cre mice themselves are affected by mild hypopituitarism. Hence, physiological consequences are expected, especially in combination with defects resulting from Cre mediated deletion of any gene under investigation. To further investigate the origin of this phenotype, we re-examined the activity of the transgene. We compared it with expression of Nestin itself in the context of the hypothalamo-pituitary axis, especially in the light of a recent report showing pituitary Nestin-Cre activity, which contrasts with previous data. Our results disagree with those of this recent study and do not support the claim that Nestin positive cells are present in the pituitary anlagen, the Rathke's pouch (RP). Moreover we did not observe any significant activity in the post-natal pituitary, in agreement with the initial report. PMID:20625432

Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

PubMed

Langouet-Astrie, Christophe J; Yang, Zhiyong; Polisetti, Sraavya M; Welsbie, Derek S; Hauswirth, William W; Zack, Donald J; Merbs, Shannath L; Enke, Raymond A

2016-10-01

Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Conditional Deletion of the Pten Gene in the Mouse Prostate Induces Prostatic Intraepithelial Neoplasms at Early Ages but a Slow Progression to Prostate Tumors

PubMed Central

Zhu, Chunfang; Lee, Suk Hyung; Ye, Ding-Wei; Luong, Richard; Sun, Zijie

2013-01-01

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, PtenLoxP:Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of PtenLoxP/LoxP:Osr1-Cre mice as early as 2 weeks of age. Intriguingly, PtenLoxP/LoxP:Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. PtenLoxP/+:Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of PtenLoxP/LoxP:Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated PtenLoxP/LoxP:Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate. PMID:23308230

Carbapenem-Resistant Enterobacteriaceae Transmission in Health Care Facilities - Wisconsin, February-May 2015.

PubMed

Elbadawi, Lina I; Borlaug, Gwen; Gundlach, Kristin M; Monson, Timothy; Warshauer, David; Walters, Maroya S; Kallen, Alexander; Gulvik, Christopher A; Davis, Jeffrey P

2016-09-02

Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug-resistant gram-negative bacilli that can cause infections associated with high case fatality rates, and are emerging as epidemiologically important health care-associated pathogens in the United States (1). Prevention of CRE transmission in health care settings is dependent on recognition of cases, isolation of colonized and infected patients, effective use of infection control measures, and the correct use of antibiotics. The use of molecular technologies, including polymerase chain reaction (PCR) testing, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing (WGS), can lead to detection of transmission events and interruption of transmission. In Wisconsin, acute care and critical access hospitals report laboratory-identified CRE to the Wisconsin Division of Public Health (WDPH), and clinical laboratories submit CRE isolates to the Wisconsin State Laboratory of Hygiene (WSLH) for molecular testing. During February-May 2015, a total of 49 CRE isolates from 46 patients were submitted to WSLH. On June 8, WSLH informed WDPH of five carbapenemase-producing CRE isolates with closely related PFGE patterns identified among four inpatients at two hospitals in southeastern Wisconsin. An investigation revealed a high degree of genetic relatedness among the patients' isolates, but did not identify the mechanism of transmission between the two facilities. No breaches in recommended practices were identified; after reviewing respiratory care procedures, no further cases were identified. Routine hospital- and laboratory-based surveillance can detect and prevent health care transmission of CRE.

A pink mouse reports the switch from red to green fluorescence upon Cre-mediated recombination.

PubMed

Hartwich, Heiner; Satheesh, Somisetty V; Nothwang, Hans Gerd

2012-06-14

Targeted genetic modification in the mouse becomes increasingly important in biomedical and basic science. This goal is most often achieved by use of the Cre/loxP system and numerous Cre-driver mouse lines are currently generated. Their initial characterization requires reporter mouse lines to study the in vivo spatiotemporal activity of Cre. Here, we report a dual fluorescence reporter mouse line, which switches expression from the red fluorescent protein mCherry to eGFP after Cre-mediated recombination. Both fluorescent proteins are expressed from the ubiquitously active and strong CAGGS promoter. Among the founders, we noticed a pink mouse line, expressing high levels of the red fluorescent protein mCherry throughout the entire body. Presence of mCherry in the living animal as well as in almost all organs was clearly visible without optical equipment. Upon Cre-activity, mCherry expression was switched to eGFP, demonstrating functionality of this reporter mouse line. The pink mouse presented here is an attractive novel reporter line for fluorescence-based monitoring of Cre-activity. The high expression of mCherry, which is visible to the naked eye, facilitates breeding and crossing, as no genotyping is required to identify mice carrying the reporter allele. The presence of two fluorescent proteins allows in vivo monitoring of recombined and non-recombined cells. Finally, the pink mouse is an eye-catching animal model to demonstrate the power of transgenic techniques in teaching courses.

Atorvastatin inhibits insulin synthesis by inhibiting the Ras/Raf/ERK/CREB pathway in INS-1 cells

PubMed Central

Sun, Hongxi; Li, Yu; Sun, Bei; Hou, Ningning; Yang, Juhong; Zheng, Miaoyan; Xu, Jie; Wang, Jingyu; Zhang, Yi; Zeng, Xianwei; Shan, Chunyan; Chang, Bai; Chen, Liming; Chang, Baocheng

2016-01-01

Abstract Backround: Type 2 diabetes has become a global epidemic disease. Atorvastatin has become a cornerstone in the prevention and treatment of atherosclerosis. However, increasing evidence showed that statins can dose-dependently increase the risk of diabetes mellitus. The mechanism is not clear. Objective: The Ras complex pathway (Ras/Raf/extracellular signal-regulated kinase [ERK]/cAMP response element-binding protein [CREB]) is the major pathway that regulates the gene transcription. Except for the inhibition of cholesterol synthesis by inhibiting the 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-COA) reductase, statins can also downregulate the phosphorylation of a series of downstream substrates including the key proteins of the Ras complex pathway, therefore may inhibit the insulin syntheses in pancreatic beta cells. In our study, we investigated the inhibitory effect and the underlying mechanism of atorvastatin on insulin synthesis in rat islets. Methods: Islets were isolated from Wistar rats and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. The insulin content in the medium was measured by radioimmunoassay before and after the treatment of 50 μM atorvastatin. Effect of atorvastatin on the expression of insulin message Ribonucleic acid (mRNA) in pancreatic islet beta cells was also detected using quantitative real-time polymerase chain reaction. Western blotting was used to explore the possible role of the Ras complex pathway (Ras/Raf/ERK/CREB) in atorvastatin-inhibited insulin synthesis. The effects of atorvastatin on the binding of nuclear transcription factor p-CREB with CRE in INS-1 cells were examined via chromatin immunoprecipitation assay. Results: Compared with the control group, the insulin level decreased by 27.1% at 24 hours after atorvastatin treatment. Atorvastatin inhibited insulin synthesis by decreasing insulin mRNA expression of pancreatic islet beta cells. The activities of Ras, Raf-1, and p-CREB in the Ras complex pathway were inhibited by 50 μM atorvastatin in INS-1 cells in vitro. Moreover, 50 μM atorvastatin reduced the binding of p-CREB with deoxyribonucleic acid (DNA) in INS-1 cells in vitro. Conclusion: Atorvastatin inhibits insulin synthesis in beta cells by inhibiting the activation of the Ras complex pathway. PMID:27684825

Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator.

PubMed

Zhang, Xiaoyue; Li, Yonghao; Zhao, Xinqing; Bai, Fengwu

2017-01-01

The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved. Copyright © 2016. Published by Elsevier Ltd.

Out of place, out of mind: Schema-driven false memory effects for object-location bindings.

PubMed

Lew, Adina R; Howe, Mark L

2017-03-01

Events consist of diverse elements, each processed in specialized neocortical networks, with temporal lobe memory systems binding these elements to form coherent event memories. We provide a novel theoretical analysis of an unexplored consequence of the independence of memory systems for elements and their bindings, 1 that raises the paradoxical prediction that schema-driven false memories can act solely on the binding of event elements despite the superior retrieval of individual elements. This is because if 2, or more, schema-relevant elements are bound together in unexpected conjunctions, the unexpected conjunction will increase attention during encoding to both the elements and their bindings, but only the bindings will receive competition with evoked schema-expected bindings. We test our model by examining memory for object-location bindings in recognition (Study 1) and recall (Studies 2 and 3) tasks. After studying schema-relevant objects in unexpected locations (e.g., pan on a stool in a kitchen scene), participants who then viewed these objects in expected locations (e.g., pan on stove) at test were more likely to falsely remember this object-location pairing as correct, compared with participants that viewed a different unexpected object-location pairing (e.g., pan on floor). In recall, participants were more likely to correctly remember individual schema-relevant objects originally viewed in unexpected, as opposed to expected locations, but were then more likely to misplace these items in the original room scene to expected places, relative to control schema-irrelevant objects. Our theoretical analysis and novel paradigm provide a tool for investigating memory distortions acting on binding processes. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus.

PubMed Central

Marciniak, R A; Garcia-Blanco, M A; Sharp, P A

1990-01-01

Human immunodeficiency virus type 1 RNAs contain a sequence, trans-activation-response (TAR) element, which is required for tat protein-mediated trans-activation of viral gene expression. We have identified a nuclear protein from extracts of HeLa cells that binds to the TAR element RNA in a sequence-specific manner. The binding of this 68-kDa polypeptide was detected by UV cross-linking proteins to TAR element RNA transcribed in vitro. Competition experiments were performed by using a partially purified preparation of the protein to quantify the relative binding affinities of TAR element RNA mutants. The binding affinity of the TAR mutants paralleled the reported ability of those mutants to support tat trans-activation in vivo. We propose that this cellular protein moderates TAR activity in vivo. Images PMID:2333305

The role of sensory organs and the forebrain for the development of the craniofacial shape as revealed by Foxg1-cre mediated microRNA loss

PubMed Central

Kersigo, Jennifer; D’Angelo, Alex; Gray, Brian; Soukup, Garrett A.; Fritzsch, Bernd

2011-01-01

Cranial development is critically influenced by the relative growth of distinct elements. Previous studies have shown the transcription factor Foxg1 to be expressed is essential for development of telencephalon, olfactory epithelium, parts of the eye and the ear. Here we investigate the effects of a Foxg1-cre mediated conditional deletion of Dicer1 and microRNA (miRNA) on mouse embryos. We report the rapid and complete loss of the telencephalon and cerebellum as well as severe reduction in the ears and loss of the anterior half of the eyes. These losses result in unexpectedly limited malformations of anterodorsal aspects of the skull. We investigated the progressive disappearance of these initially developing structures and found a specific miRNA of nervous tissue, miR-124, to disappear prior to reduction in growth of the specific neurosensory areas. Correlated with the absence of miR-124, these areas showed numerous apoptotic cells that stained positive for anti-cleaved caspase 3 and the phosphatidylserine stain PSVue prior to the near or complete loss of those brain and sensory areas (forebrain, cerebellum, anterior retina, ear). We conclude that Foxg1-cre mediated conditional deletion of Dicer1 leads to absence of functional miRNA followed by complete or nearly complete loss of neurons. Embryonic neurosensory development therefore depends critically on miRNA. Our data suggest that loss of a given neuronal compartment can be triggered using early deletion of Dicer1 and thus provides a novel means to genetically remove specific neurosensory areas to investigate loss of their function on morphology (this study) or signal processing within the brain. PMID:21225654

Modified Carbapenem Inactivation Method for Phenotypic Detection of Carbapenemase Production among Enterobacteriaceae

PubMed Central

Simner, Patricia J.; Lonsway, David R.; Roe-Carpenter, Darcie E.; Johnson, J. Kristie; Brasso, William B.; Bobenchik, April M.; Lockett, Zabrina C.; Charnot-Katsikas, Angella; Ferraro, Mary Jane; Thomson, Richard B.; Jenkins, Stephen G.; Limbago, Brandi M.; Das, Sanchita

2017-01-01

ABSTRACT The ability of clinical microbiology laboratories to reliably detect carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) is an important element of the effort to prevent and contain the spread of these pathogens and an integral part of antimicrobial stewardship. All existing methods have limitations. A new, straightforward, inexpensive, and specific phenotypic method for the detection of carbapenemase production, the carbapenem inactivation method (CIM), was recently described. Here we describe a two-stage evaluation of a modified carbapenem inactivation method (mCIM), in which tryptic soy broth was substituted for water during the inactivation step and the length of this incubation was extended. A validation study was performed in a single clinical laboratory to determine the accuracy of the mCIM, followed by a nine-laboratory study to verify the reproducibility of these results and define the zone size cutoff that best discriminated between CP-CRE and members of the family Enterobacteriaceae that do not produce carbapenemases. Bacterial isolates previously characterized through whole-genome sequencing or targeted PCR as to the presence or absence of carbapenemase genes were tested for carbapenemase production using the mCIM; isolates with Ambler class A, B, and D carbapenemases, non-CP-CRE isolates, and carbapenem-susceptible isolates were included. The sensitivity of the mCIM observed in the validation study was 99% (95% confidence interval [95% CI], 93% to 100%), and the specificity was 100% (95% CI, 82% to 100%). In the second stage of the study, the range of sensitivities observed across nine laboratories was 93% to 100%, with a mean of 97%; the range of specificities was 97% to 100%, with a mean of 99%. The mCIM was easy to perform and interpret for Enterobacteriaceae, with results in less than 24 h and excellent reproducibility across laboratories. PMID:28381609

Muscle-specific deletion of exons 2 and 3 of the IL15RA gene in mice: effects on contractile properties of fast and slow muscles.

PubMed

O'Connell, Grant; Guo, Ge; Stricker, Janelle; Quinn, LeBris S; Ma, Averil; Pistilli, Emidio E

2015-02-15

Interleukin-15 (IL-15) is a putative myokine hypothesized to induce an oxidative skeletal muscle phenotype. The specific IL-15 receptor alpha subunit (IL-15Rα) has also been implicated in specifying this contractile phenotype. The purposes of this study were to determine the muscle-specific effects of IL-15Rα functional deficiency on skeletal muscle isometric contractile properties, fatigue characteristics, spontaneous cage activity, and circulating IL-15 levels in male and female mice. Muscle creatine kinase (MCK)-driven IL-15Rα knockout mice (mIl15ra(fl/fl)/Cre(+)) were generated using the Cre-loxP system. We tested the hypothesis that IL-15Rα functional deficiency in skeletal muscle would increase resistance to contraction-induced fatigue, cage activity, and circulating IL-15 levels. There was a significant effect of genotype on the fatigue curves obtained in extensor digitorum longus (EDL) muscles from female mIl15ra(fl/fl)/Cre(+) mice, such that force output was greater during the repeated contraction protocol compared with mIl15ra(fl/fl)/Cre(-) control mice. Muscles from female mIl15ra(fl/fl)/Cre(+) mice also had a twofold greater amount of the mitochondrial genome-specific COXII gene compared with muscles from mIl15ra(fl/fl)/Cre(-) control mice, indicating a greater mitochondrial density in these skeletal muscles. There was a significant effect of genotype on the twitch:tetanus ratio in EDL and soleus muscles from mIl15ra(fl/fl)/Cre(+) mice, such that the ratio was lower in these muscles compared with mIl15ra(fl/fl)/Cre(-) control mice, indicating a pro-oxidative shift in muscle phenotype. However, spontaneous cage activity was not different and IL-15 protein levels were lower in male and female mIl15ra(fl/fl)/Cre(+) mice compared with control. Collectively, these data support a direct effect of muscle IL-15Rα deficiency in altering contractile properties and fatigue characteristics in skeletal muscles.

Inhibition of IKKβ in enterocytes exacerbates sepsis-induced intestinal injury and worsens mortality.

PubMed

Dominguez, Jessica A; Samocha, Alexandr J; Liang, Zhe; Burd, Eileen M; Farris, Alton B; Coopersmith, Craig M

2013-10-01

Nuclear factor-κB is a critical regulator of cell-survival genes and the host inflammatory response. The purpose of this study was to investigate the role of enterocyte-specific NF-kB in sepsis through selective ablation of IkB kinase. Prospective, randomized controlled study. Animal laboratories in university medical centers. Mice lacking functional NF-kB in their intestinal epithelium (Vil-Cre/Ikkβ) and wild-type mice were subjected to sham laparotomy or cecal ligation and puncture. Animals were killed at 24 hours or followed 7 days for survival. Septic wild-type mice had decreased villus length compared with sham mice, whereas villus atrophy was further exacerbated in septic Vil-Cre/Ikkβ mice. Sepsis induced an increase in intestinal epithelial apoptosis compared with sham mice, which was further exacerbated in Vil-Cre/Ikkβ mice. Sepsis induced intestinal hyperpermeability in wild-type mice compared with sham mice, which was further exacerbated in septic Vil-Cre/Ikkβ mice. This was associated with increased intestinal expression of claudin-2 in septic wild-type mice, which was further increased in septic Vil-Cre/Ikkβ mice. Both, pro-inflammatory and anti-inflammatory cytokines were increased in serum following cecal ligation and puncture, and interleukin 10 and monocyte chemoattractant protein-1 levels were higher in septic Vil-Cre/Ikkβ mice than in septic wild-type mice. All septic mice were bacteremic, but no differences in bacterial load were identified between wild-type and Vil-Cre/Ikkβ mice. To determine the functional significance of these results, animals were followed for survival. Septic wild-type mice had lower mortality than septic Vil-Cre/Ikkβ mice (47% vs 80%, p<0.05). Antitumor necrosis factor administration decreased intestinal apoptosis, permeability, and mortality in wild-type septic mice, and a similar improvement in intestinal integrity and survival were seen when antitumor necrosis factor was given to Vil-Cre/Ikkβ mice. Enterocyte-specific NF-kB has a beneficial role in sepsis by partially preventing sepsis-induced increases in apoptosis and permeability, which are associated with worsening mortality.

MnSOD deficiency results in elevated oxidative stress and decreased mitochondrial function but does not lead to muscle atrophy during aging.

PubMed

Lustgarten, Michael S; Jang, Youngmok C; Liu, Yuhong; Qi, Wenbo; Qin, Yuejuan; Dahia, Patricia L; Shi, Yun; Bhattacharya, Arunabh; Muller, Florian L; Shimizu, Takahiko; Shirasawa, Takuji; Richardson, Arlan; Van Remmen, Holly

2011-06-01

In a previous study, we reported that a deficiency in MnSOD activity (approximately 80% reduction) targeted to type IIB skeletal muscle fibers was sufficient to elevate oxidative stress and to reduce muscle function in young adult mice (TnIFastCreSod2(fl/fl) mice). In this study, we used TnIFastCreSod2(fl/fl) mice to examine the effect of elevated oxidative stress on mitochondrial function and to test the hypothesis that elevated oxidative stress and decreased mitochondrial function over the lifespan of the TnIFastCreSod2(fl/fl) mice would be sufficient to accelerate muscle atrophy associated with aging. We found that mitochondrial function is reduced in both young and old TnIFastCreSod2(fl/fl) mice, when compared with control mice. Complex II activity is reduced by 47% in young and by approximately 90% in old TnIFastCreSod2(fl/fl) mice, and was found to be associated with reduced levels of the catalytic subunits for complex II, SDHA and SDHB. Complex II-linked mitochondrial respiration is reduced by approximately 70% in young TnIFastCreSod2(fl/fl) mice. Complex II-linked mitochondrial Adenosine-Tri-Phosphate (ATP) production is reduced by 39% in young and was found to be almost completely absent in old TnIFastCreSod2(fl/fl) mice. Furthermore, in old TnIFastCreSod2(fl/fl) mice, aconitase activity is almost completely abolished; mitochondrial superoxide release remains > 2-fold elevated; and oxidative damage (measured as F(2) - isoprostanes) is increased by 30% relative to age-matched controls. These data show that despite elevated skeletal muscle-specific mitochondrial oxidative stress, oxidative damage, and complex II-linked mitochondrial dysfunction, age-related muscle atrophy was not accelerated in old TnIFastCreSod2(fl/fl) mice, suggesting mitochondrial oxidative stress may not be causal for age-related muscle atrophy. No claim to original US government works. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

The Effects of Androgens on Murine Cortical Bone Do Not Require AR or ERα Signaling in Osteoblasts and Osteoclasts

PubMed Central

Ucer, Serra; Iyer, Srividhya; Bartell, Shoshana M; Martin-Millan, Marta; Han, Li; Kim, Ha-Neui; Weinstein, Robert S; Jilka, Robert L; O’Brien, Charles A; Almeida, Maria; Manolagas, Stavros C

2016-01-01

In men, androgens are critical for the acquisition and maintenance of bone mass in both the cortical and cancellous bone compartment. Male mice with targeted deletion of the androgen receptor (AR) in mature osteoblasts or osteocytes have lower cancellous bone mass, but no cortical bone phenotype. We have investigated the possibility that the effects of androgens on the cortical compartment result from AR signaling in osteoprogenitors or cells of the osteoclast lineage; or via estrogen receptor alpha (ERα) signaling in either or both of these two cell types upon conversion of testosterone to estradiol. To this end, we generated mice with targeted deletion of an AR or an ERα allele in the mesenchymal (ARf/y;Prx1-Cre or ERαf/f;Osx1-Cre) or myeloid cell lineage (ARf/y; LysM-Cre or ERαf/f;LysM-Cre) and their descendants. Male ARf/y;Prx1-Cre mice exhibited decreased bone volume and trabecular number, and increased osteoclast number in the cancellous compartment. Moreover, they did not undergo the loss of cancellous bone volume and trabecular number caused by orchidectomy (ORX) in their littermate controls. In contrast, ARf/y;LysM-Cre, ERαf/f; Osx1-Cre, or ERαf/f;LysM-Cre mice had no cancellous bone phenotype at baseline and lost the same amount of cancellous bone as their controls following ORX. Most unexpectedly, adult males of all four models had no discernible cortical bone phenotype at baseline, and lost the same amount of cortical bone as their littermate controls after ORX. Recapitulation of the effects of ORX by AR deletion only in the ARf/y;Prx1-Cre mice indicates that the effects of androgens on cancellous bone result from AR signaling in osteoblasts—not on osteoclasts or via aromatization. The effects of androgens on cortical bone mass, on the other hand, do not require AR or ERα signaling in any cell type across the osteoblast or osteoclast differentiation lineage. Therefore, androgens must exert their effects indirectly by actions on some other cell type(s) or tissue(s). PMID:25704845

Development of selectable marker free, insect resistant, transgenic mustard (Brassica juncea) plants using Cre/lox mediated recombination

PubMed Central

2013-01-01

Background Antibiotic/ herbicide resistant marker genes have been proven to be very useful in plant transformation for the initial selection of desired transgenic events. However, presence of these genes in the genetically modified crops may render the crop less acceptable to the consumers. Among several different approaches, the effectiveness of Cre/lox mediated recombination strategy for selectable marker gene (SMG) elimination has previously been demonstrated by different groups in several plants including Brassica. In the present study exploiting Cre/lox mediated recombination strategy, attempt has been made for selectable marker gene elimination from Allium sativum leaf agglutinin (ASAL) expressing Brassica plants with hemipteran insect resistant phenotype. Results Allium sativum leaf agglutinin (ASAL) linked with lox flanked hygromycin resistant (hpt) gene was introduced in mustard. Cre recombinase gene cassette was also integrated in separate event. A Cre/lox mediated recombination using crossing strategy was adopted to remove the hpt gene from the subsequent generation of selected hybrid events. Reciprocal crosses were made between T1ASAL-lox-hpt-lox and cre-bar plants. Marker gene elimination was confirmed in the resulting F1 hybrid progenies by PCR analysis, using hpt, cre and ASAL specific primers followed by Southern hybridization. In marker free plants, expression of ASAL was also confirmed by western blotting and ELISA analysis. Retention of functionality of expressed ASAL was investigated by agglutination assay using rabbit erythrocytes. Expressed ASAL was also found to be thermo-sensitive. In planta insect bioassay on F1 hybrid progenies exhibited detrimental effect on the performance of devastating target pest, Lipaphis erysimi. The F1 hybrid hpt negative, ASAL positive plants were allowed to self- fertilize to obtain F2 progeny plants. In some of these plants cre gene was found to be segregated out of the ASAL gene by genetic segregation yielding completely marker free plants. Conclusions The present study establishes the efficient expression of the newly introduced insect resistant ASAL gene even after Cre/lox mediated recombination resulting in elimination of selectable marker gene. PMID:24144281

Dual catenin loss in murine liver causes tight junctional deregulation and progressive intrahepatic cholestasis.

PubMed

Pradhan-Sundd, Tirthadipa; Zhou, Lili; Vats, Ravi; Jiang, An; Molina, Laura; Singh, Sucha; Poddar, Minakshi; Russell, Jacquelyn; Stolz, Donna B; Oertel, Michael; Apte, Udayan; Watkins, Simon; Ranganathan, Sarangarajan; Nejak-Bowen, Kari N; Sundd, Prithu; Monga, Satdarshan Pal

2018-06-01

β-Catenin, the downstream effector of the Wnt signaling, plays important roles in hepatic development, regeneration, and tumorigenesis. However, its role at hepatocyte adherens junctions (AJ) is relatively poorly understood, chiefly due to spontaneous compensation by γ-catenin. We simultaneously ablated β- and γ-catenin expression in mouse liver by interbreeding β-catenin-γ-catenin double-floxed mice and Alb-Cre transgenic mice. Double knockout mice show failure to thrive, impaired hepatocyte differentiation, cholemia, ductular reaction, progressive cholestasis, inflammation, fibrosis, and tumorigenesis, which was associated with deregulation of tight junctions (TJ) and bile acid transporters, leading to early morbidity and mortality, a phenotype reminiscent of progressive familial intrahepatic cholestasis (PFIC). To address the mechanism, we specifically and temporally eliminated both catenins from hepatocytes using adeno-associated virus 8 carrying Cre-recombinase under the thyroid-binding globulin promoter (AAV8-TBG-Cre). This led to a time-dependent breach of the blood-biliary barrier associated with sequential disruption of AJ and TJ verified by ultrastructural imaging and intravital microscopy, which revealed unique paracellular leaks around individual hepatocytes, allowing mixing of blood and bile and leakage of blood from one sinusoid to another. Molecular analysis identified sequential losses of E-cadherin, occludin, claudin-3, and claudin-5 due to enhanced proteasomal degradation, and of claudin-2, a β-catenin transcriptional target, which was also validated in vitro. We report partially redundant function of catenins at AJ in regulating TJ and contributing to the blood-biliary barrier. Furthermore, concomitant hepatic loss of β- and γ-catenin disrupts structural and functional integrity of AJ and TJ via transcriptional and posttranslational mechanisms. Mice with dual catenin loss develop progressive intrahepatic cholestasis, providing a unique model to study diseases such as PFIC. (Hepatology 2018;67:2320-2337). © 2017 by the American Association for the Study of Liver Diseases.

Fission yeast Tup1-like repressors repress chromatin remodeling at the fbp1+ promoter and the ade6-M26 recombination hotspot.

PubMed Central

Hirota, Kouji; Hoffman, Charles S; Shibata, Takehiko; Ohta, Kunihiro

2003-01-01

Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1(+) gene and recombination at the meiotic recombination hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ATF-type transcription factor Atf1*Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1(+) transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1(+) promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1(+) was remodeled under derepressed conditions in concert with the robust activation of fbp1(+) transcription. Strains with tup11delta tup12delta double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingly, deletion of rst2(+), encoding a transcription factor controlled by the cAMP-dependent kinase, alleviated the tup11delta tup12delta defects in chromatin regulation but not in transcription repression. The chromatin at the M26 site in mitotic cultures of a tup11delta tup12delta mutant resembled that of wild-type meiotic cells. These observations suggest that these fission yeast Tup1-like corepressors repress chromatin remodeling at CRE-related sequences and that Rst2 antagonizes this function. PMID:14573465

Using ISCCP Weather States to Decompose Cloud Radiative Effects

NASA Technical Reports Server (NTRS)

Oreopoulos, L.; Rossow, W. B.

2012-01-01

The presentation will examine the shortwave (SW) and longwave (LW) cloud radiative effect CRE (aka "cloud radiative forcing") at the top-of-the-atmosphere and surface of ISCCP weather states (aka "cloud regimes") in three distinct geographical zones, one tropical and two mid-latitude. Our goal is to understand and quantify the contribution of the different cloud regimes to the planetary radiation budget. In the tropics we find that the three most convectively active states are the ones with largest SW, LW and net TOA CRE contributions to the overall daytime tropical CRE budget. They account for 59%, 71% and 55% of the total CRE, respectively. The boundary layer-dominated weather states account for only 34% of the total SW CRE and 41% of the total net CRE, so to focus only on them in cloud feedback studies may be imprudent. We also find that in both the northern and southern midlatitude zones only two weather states, the first and third most convectively active with large amounts of nimbostratus-type clouds, contribute ",40% to both the SW and net TOA CRE budgets, highlighting the fact that cloud regimes associated with frontal systems are not only important for weather (precipitation) but also for climate (radiation budget). While all cloud regimes in all geographical zones have a slightly larger SFC than TOA SW CRE, implying cooling of the surface and slight warming of the atmosphere, their LW radiative effects are more subtle: in the tropics the weather states with plentiful high clouds warm the atmosphere while those with copious amounts of low clouds cool the atmosphere. In both midlatitude zones only the weather states with peak cloud fractions at levels above 440 mbar warm the atmosphere while all the rest cool it. These results make the connection of the contrasting CRE effects to the atmospheric dynamics more explicit - "storms" tend to warm the atmosphere whereas fair weather clouds cool it, suggesting a positive feedback of clouds on weather systems. The breakdown of CRE by cloud regime are however not entirely similar between the two midlatitude zones. Despite the existence of an additional state in the nort!lern midlatitudes, only four weather states have net daytime CREs with absolute values above 100 Watts per square meter compared to six in the south. This reminds us that the environment where clouds occur also has a crucial role in determining their radiative effects. All the above make evident that reproducing grand averages of current CRE by climate models in only part of the challenge. If existing cloud regimes and shifts in their distributions and frequency of occurrence in a changed climate are not properly simulated, the radiative role of clouds will not be adequately predicted.

Single-neuron labeling with inducible cre-mediated knockout in transgenic mice

PubMed Central

Young, Paul; Qiu, Li; Wang, Dongqing; Zhao, Shengli; Gross, James; Feng, Guoping

2011-01-01

To facilitate functional analysis of neuronal connectivity in a mammalian nervous system tightly packed with billions of cells, we developed a new technique that allows inducible genetic manipulations within fluorescently labeled single neurons in mice. We term this technique SLICK for Single-neuron Labeling with Inducible Cre-mediated Knockout. SLICK is achieved by co-expressing a drug-inducible form of cre recombinase and a fluorescent protein within the same small subsets of neurons. Thus, SLICK combines the powerful cre recombinase system for conditional genetic manipulation and the fluorescent labeling of single neurons for imaging. We demonstrate efficient inducible genetic manipulation in several types of neurons using SLICK. Furthermore, we apply SLICK to eliminate synaptic transmission in a small subset of neuromuscular junctions. Our results provide evidence for the long-term stability of inactive neuromuscular synapses in adult animals. More broadly, these studies demonstrate a cre-LoxP compatible system for dissecting gene functions in single identifiable neurons. PMID:18454144

Impact of conditional deletion of the pro-apoptotic BCL-2 family member BIM in mice.

PubMed

Herold, M J; Stuchbery, R; Mérino, D; Willson, T; Strasser, A; Hildeman, D; Bouillet, P

2014-10-09

The pro-apoptotic BH3-only BCL-2 family member BIM is a critical determinant of hematopoietic cell development and homeostasis. It has been argued that the striking hematopoietic abnormalities of BIM-deficient mice (accumulation of lymphocytes and granulocytes) may be the result of the loss of the protein throughout the whole animal rather than a consequence intrinsic to the loss of BIM in hematopoietic cells. To address this issue and allow the deletion of BIM in specific cell types in future studies, we have developed a mouse strain with a conditional Bim allele as well as a new Cre transgenic strain, Vav-CreER, in which the tamoxifen-inducible CreER recombinase (fusion protein) is predominantly expressed in the hematopoietic system. We show that acute loss of BIM in the adult mouse rapidly results in the hematopoietic phenotypes previously observed in mice lacking BIM in all tissues. This includes changes in thymocyte subpopulations, increased white blood cell counts and resistance of lymphocytes to BIM-dependent apoptotic stimuli, such as cytokine deprivation. We have validated this novel conditional Bim knockout mouse model using established and newly developed CreER strains (Rosa26-CreER and Vav-CreER) and will make these exciting new tools for studies on cell death and cancer available.

Generation of knock-in mice that express nuclear enhanced green fluorescent protein and tamoxifen-inducible Cre recombinase in the notochord from Foxa2 and T loci.

PubMed

Imuta, Yu; Kiyonari, Hiroshi; Jang, Chuan-Wei; Behringer, Richard R; Sasaki, Hiroshi

2013-03-01

The node and the notochord are important embryonic signaling centers that control embryonic pattern formation. Notochord progenitor cells present in the node and later in the posterior end of the notochord move anteriorly to generate the notochord. To understand the dynamics of cell movement during notochord development and the molecular mechanisms controlling this event, analyses of cell movements using time-lapse imaging and conditional manipulation of gene activities are required. To achieve this goal, we generated two knock-in mouse lines that simultaneously express nuclear enhanced green fluorescent protein (EGFP) and tamoxifen-inducible Cre, CreER(T2) , from two notochord gene loci, Foxa2 and T (Brachury). In Foxa2(nEGFP-CreERT2/+) and T(nEGFP-CreERT2/+) embryos, nuclei of the Foxa2 or T-expressing cells, which include the node, notochord, and endoderm (Foxa2) or wide range of posterior mesoderm (T), were labeled with EGFP at intensities that can be used for live imaging. Cre activity was also induced in cells expressing Foxa2 and T 1 day after tamoxifen administration. These mice are expected to be useful tools for analyzing the mechanisms of notochord development. Copyright © 2013 Wiley Periodicals, Inc.

Role of newer and re-emerging older agents in the treatment of infections caused by carbapenem-resistant Enterobacteriaceae.

PubMed

Thaden, Joshua T; Pogue, Jason M; Kaye, Keith S

2017-05-19

Antimicrobial resistance has been identified by the World Health Organization as "one of the three greatest threats to human health." Gram negative bacteria in particular drive this alarming trend. Carbapenem-resistant Enterobacteriaceae (CRE) such as Escherichia coli, Klebsiella pneumoniae, and Enterobacter species are of particular importance as they are associated with poor clinical outcomes and are common causes for a variety of infections including bacteremia, urinary tract infection, intra-abdominal infections and pneumonia. CRE are difficult to treat as carbapenem resistance is often accompanied by resistance to additional drug classes. For example, CRE may be extensively drug resistant or even pandrug resistant. Unfortunately, CRE infections have increased over the past 15 y while new and effective antibiotics have not kept pace. Recently, however, new agents have become available to help treat CRE infection, and several more are under development. This article reviews the efficacy, safety, and pharmacokinetic issues around 4 emerging agents to treat CRE - ceftazidime-avibactam, fosfomycin, tigecycline, and minocycline. In addition, an overview of agents in the antibiotic pipeline - meropenem-vaborbactam, imipenem-relebactam, plazomicin, and eravacycline is provided. More established agents, such as those in the polymyxin class and aminoglycoside class (other than the pipeline agent plazomicin), are not addressed here.

Bacteroides cellulosilyticus sp. nov., a cellulolytic bacterium from the human gut microbial community.

PubMed

Robert, Céline; Chassard, Christophe; Lawson, Paul A; Bernalier-Donadille, Annick

2007-07-01

A strictly anaerobic cellulolytic bacterium, strain CRE21(T), was isolated from a human faecal sample. Cells were Gram-negative non-motile rods that were about 1.7 microm in length and 0.9 microm in width. Strain CRE21(T) degraded different types of cellulose and was able to grow on a variety of carbohydrates. Cellulose and sugars were mainly converted to acetate, propionate and succinate. The G+C content of the DNA was 41.1 mol%. 16S rRNA gene sequence analysis revealed that the isolate belonged to the genus Bacteroides with highest sequence similarity to the type strain of Bacteroides intestinalis (98 %). DNA-DNA hybridization results revealed that strain CRE21(T) was distinct from B. intestinalis (40 % DNA-DNA relatedness). Strain CRE21(T) also showed several characteristics distinct from B. intestinalis. In particular, it exhibited different capacity to degrade polysaccharides such as cellulose. On the basis of phylogenetic analysis and the morphological, physiological and biochemical data presented in this study, strain CRE21(T) can be readily differentiated from recognized species of the genus Bacteroides. The name Bacteroides cellulosilyticus sp. nov. is proposed to accommodate this organism. The type strain is CRE21(T) (=DSM 14838(T)=CCUG 44979(T)).

Anything but Shadowing! Early Clinical Reasoning in Emergency Department Improves Clinical Skills

PubMed Central

Royan, Regina; Wu, Christine; Theyyunni, Nik; Montas, Sacha; Cranford, James A.; House, Joseph B.; Lukela, Michael P.; Santen, Sally A.

2018-01-01

Introduction Transitioning from the pre-clinical environment to clerkships poses a challenge to students and educators alike. Students along with faculty developed the Clinical Reasoning Elective (CRE) to provide pre-clinical students exposure to patients in the emergency department and the opportunity to build illness scripts and practice clinical skills with longitudinal mentorship in a low-stakes environment before entering clerkships. It is a voluntary program. Each year, the CRE has received overwhelming positive feedback from students. The objective of this study is to determine if the CRE improved students’ clinical skills and reported comfort in their skills. Methods We examined the relationships between students’ self-reported participation in the CRE and their individual scores on a comprehensive clinical assessment (CCA) at the end of the pre-clerkship period. A total of 178 students took the CCA exam in 2016. Of these, 113 participated in the CRE and 65 did not. Seven students who participated in CRE did not complete the exit survey and were omitted from analysis. We performed regression analysis and dichotomous (participants/nonparticipants) comparisons of means with t-tests. Survey of student reactions was collected. Results Participants completed an average of 10 sessions over the course of the program (range=1–20). Involvement in the CRE was associated with significantly increased scores on Abdominal History; Pulmonary Physical Exam; Overall History-Taking; Overall Communication; and Overall Physical Exam (p<0.05). Nearly all students (97%) reported that the program offered opportunities to enhance clinical skills, increased their comfort with patients, and better prepared them for their clinical years. Conclusion There were measurable improvements in clinical skills performance for students who participated in CRE. As many schools seek to incorporate early clinical exposure to their curricula, this program provides a successful framework to provide meaningful clinical exposure to real patients that also shows objective benefits to students’ clinical skills. PMID:29383078

Associations of serum leptin, ghrelin and peptide YY levels with physical activity and cardiorespiratory fitness in adolescent boys with different BMI values

PubMed Central

Tillmann, Vallo; Purge, Priit; Lätt, Evelin; Jürimäe, Jaak

2017-01-01

The aim of this study was to investigate the differences in associations of serum acylated and des-acylated ghrelin, peptide YY (PYY) and leptin levels with physical activity (PA) and cardiorespiratory fitness (CReF) in adolescent boys (mean age of 14.0 years) with overweight (OWB; n=55) and with normal weight (NWB; n=154). Methods: Total PA was measured by 7-day accelerometry (counts/min) and CReF by peak oxygen consumption (VO2peak/kg). Results: No differences were found in serum PYY, acylated ghrelin or des-acyl ghrelin levels, whereas mean leptin (11.6±10.6 vs. 2.0±2.7 ng/ml; p<0.05) and insulin (18.1±8.7 vs. 11.0±6.2 mU/l; p<0.05) levels were significantly higher in OWB compared to NWB. Mean CReF was significantly lower in OWB compared to NWB (39.7±8.7 vs. 50.5±6.8 ml/min/kg; p<0.05). Leptin was negatively correlated with CReF in both groups (r=-0.43; p<0.05), des-acylated ghrelin with CReF only in OWB (r =-0.36; p<0.05). In OWB leptin was negatively correlated with total PA (r=-0.32; p<0.05) and positively with sedentary time of PA (r=0.35; p<0.05). In NWB 28.1% of the variability of CReF was determined by leptin and insulin resistance index (HOMA-IR), whereas in OWB 71.9% was determined by trunk FM and BMI. Conclusions: Leptin concentration was inversely associated with CReF in adolescent boys independently of BMI in both groups, while des-acylated ghrelin was associated with CReF only in OWB. Low PA in OWB was associated with high serum leptin level. PMID:29472737

Level of tissue differentiation influences the activation of a heat-inducible flower-specific system for genetic containment in poplar (Populus tremula L.).

PubMed

Hoenicka, Hans; Lehnhardt, Denise; Nunna, Suneetha; Reinhardt, Richard; Jeltsch, Albert; Briones, Valentina; Fladung, Matthias

2016-02-01

Differentiation level but not transgene copy number influenced activation of a gene containment system in poplar. Heat treatments promoted CRE gene body methylation. The flower-specific transgene deletion was confirmed. Gene flow between genetic modified trees and their wild relatives is still motive of concern. Therefore, approaches for gene containment are required. In this study, we designed a novel strategy for achieving an inducible and flower-specific transgene removal from poplar trees but still expressing the transgene in the plant body. Hence, pollen carrying transgenes could be used for breeding purposes under controlled conditions in a first phase, and in the second phase genetic modified poplars developing transgene-free pollen grains could be released. This approach is based on the recombination systems CRE/loxP and FLP/frt. Both gene constructs contained a heat-inducible CRE/loxP-based spacer sequence for in vivo assembling of the flower-specific FLP/frt system. This allowed inducible activation of gene containment. The FLP/frt system was under the regulation of a flower-specific promoter, either CGPDHC or PTD. Our results confirmed complete CRE/loxP-based in vivo assembling of the flower-specific transgene excision system after heat treatment in all cells for up to 30 % of regenerants derived from undifferentiated tissue cultures. Degradation of HSP::CRE/loxP spacer after recombination but also persistence as extrachromosomal DNA circles were detected in sub-lines obtained after heat treatments. Furthermore, heat treatment promoted methylation of the CRE gene body. A lower methylation level was detected at CpG sites in transgenic sub-lines showing complete CRE/loxP recombination and persistence of CRE/loxP spacer, compared to sub-lines with incomplete recombination. However, our results suggest that low methylation might be necessary but not sufficient for recombination. The flower-specific FLP/frt-based transgene deletion was confirmed in 6.3 % of flowers.

Potential economic burden of carbapenem-resistant Enterobacteriaceae (CRE) in the United States.

PubMed

Bartsch, S M; McKinnell, J A; Mueller, L E; Miller, L G; Gohil, S K; Huang, S S; Lee, B Y

2017-01-01

The Centers for Disease Control and Prevention considers carbapenem-resistant Enterobacteriaceae (CRE) an urgent public health threat; however, its economic burden is unknown. We developed a CRE clinical and economics outcomes model to determine the cost of CRE infection from the hospital, third-party payer, and societal, perspectives and to evaluate the health and economic burden of CRE to the USA. Depending on the infection type, the median cost of a single CRE infection can range from $22 484 to $66 031 for hospitals, $10 440 to $31 621 for third-party payers, and $37 778 to $83 512 for society. An infection incidence of 2.93 per 100 000 population in the USA (9418 infections) would cost hospitals $275 million (95% CR $217-334 million), third-party payers $147 million (95% CR $129-172 million), and society $553 million (95% CR $303-1593 million) with a 25% attributable mortality, and would result in the loss of 8841 (95% CR 5805-12 420) quality-adjusted life years. An incidence of 15 per 100 000 (48 213 infections) would cost hospitals $1.4 billion (95% CR $1.1-1.7 billion), third-party payers $0.8 billion (95% CR $0.6-0.8 billion), and society $2.8 billion (95% CR $1.6-8.2 billion), and result in the loss of 45 261 quality-adjusted life years. The cost of CRE is higher than the annual cost of many chronic diseases and of many acute diseases. Costs rise proportionally with the incidence of CRE, increasing by 2.0 times, 3.4 times, and 5.1 times for incidence rates of 6, 10, and 15 per 100 000 persons. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Differential tissue-specific function of the Adora2b in cardio-protection

PubMed Central

Seo, Seong-wook; Koeppen, Michael; Bonney, Stephanie; Gobel, Merit; Thayer, Molly; Harter, Patrick N.; Ravid, Katya; Eltzschig, Holger K.; Mittelbronn, Michel; Walker, Lori; Eckle, Tobias

2015-01-01

The adenosine A2b-receptor (Adora2b) has been implicated in cardio-protection from myocardial ischemia. As such the Adora2b was found to be critical in ischemic preconditioning (IP) or ischemia reperfusion (IR) injury of the heart. While the Adora2b is present on various cells types, the tissue specific role of the Adora2b in cardio-protection is still unknown. To study the tissue specific role of Adora2b signaling on inflammatory cells, endothelia or myocytes during myocardial ischemia in vivo, we intercrossed floxed Adora2b mice with Lyz2-Cre+, VE-Cadherin-Cre+ or Myosin-Cre+ transgenic mice, respectively. Mice were exposed to 60 minutes of myocardial ischemia with or without IP (4×5min) followed by 120 minutes of reperfusion. Cardio-protection by IP was abolished in Adora2bf/f-VE-Cadherin-Cre+ or Adora2bf/f-Myosin-Cre+, indicating that Adora2bs signaling on endothelia or myocytes mediates IP. In contrast, primarily Adora2b signaling on inflammatory cells was necessary to provide cardio-protection in IR injury, indicated by significantly larger infarcts and higher troponin levels in Adora2bf/f-Lyz2-Cre+ mice only. Cytokine profiling of IR injury in Adora2bf/f-Lyz2-Cre+ mice pointed towards PMNs. Analysis of PMNs from Adora2bf/f-Lyz2-Cre+ confirmed PMNs as one source of identified tissue cytokines. Finally, adoptive transfer of Ador2b−/− PMNs revealed a critical role of the Adorab2 on PMNs in cardio-protection from IR-injury. Adora2b signaling mediates different types of cardio-protection in a tissue specific manner. These findings have implications for the use of Adora2b agonists in the treatment or prevention of myocardial injury by ischemia. PMID:26136425

Mouse model of proximal colon-specific tumorigenesis driven by microsatellite instability-induced Cre-mediated inactivation of Apc and activation of Kras.

PubMed

Kawaguchi, Yasuo; Hinoi, Takao; Saito, Yasufumi; Adachi, Tomohiro; Miguchi, Masashi; Niitsu, Hiroaki; Sasada, Tatsunari; Shimomura, Manabu; Egi, Hiroyuki; Oka, Shiro; Tanaka, Shinji; Chayama, Kazuaki; Sentani, Kazuhiro; Oue, Naohide; Yasui, Wataru; Ohdan, Hideki

2016-05-01

KRAS gene mutations are found in 40-50% of colorectal cancer cases, but their functional contribution is not fully understood. To address this issue, we generated genetically engineered mice with colon tumors expressing an oncogenic Kras(G12D) allele in the context of the Adenomatous polyposis coli (Apc) deficiency to compare them to tumors harboring Apc deficiency alone. CDX2P9.5-G22Cre (referred to as G22Cre) mice showing inducible Cre recombinase transgene expression in the proximal colon controlled under the CDX2 gene promoter were intercrossed with Apc (flox/flox) mice and LSL-Kras (G12D) mice carrying loxP-flanked Apc and Lox-Stop-Lox oncogenic Kras(G12D) alleles, respectively, to generate G22Cre; Apc(flox/flox); Kras(G12D) and G22Cre; Apc(flox/flox); KrasWT mice. Gene expression profiles of the tumors were analyzed using high-density oligonucleotide arrays. Morphologically, minimal difference in proximal colon tumor was observed between the two mouse models. Consistent with previous findings in vitro, Glut1 transcript and protein expression was up-regulated in the tumors of G22Cre;Apc (flox/flox) ; Kras(G12D) mice. Immunohistochemical staining analysis revealed that GLUT1 protein expression correlated with KRAS mutations in human colorectal cancer. Microarray analysis identified 11 candidate genes upregulated more than fivefold and quantitative PCR analysis confirmed that Aqp8, Ttr, Qpct, and Slc26a3 genes were upregulated 3.7- to 30.2-fold in tumors with mutant Kras. These results demonstrated the validity of the G22Cre; Apc(flox/flox) ;Kras (G12D) mice as a new mouse model with oncogenic Kras activation. We believe that this model can facilitate efforts to define novel factors that contribute to the pathogenesis of human colorectal cancer with KRAS mutations.

Carbapenem-resistant Enterobacteriaceae (CRE) or Delayed Appropriate Therapy (DAT)—Does One Affect Outcomes More Than the Other Among Patients With Serious Infections Due to Enterobacteriaceae?

PubMed Central

Lodise, Thomas; Berger, Ariel; Altincatal, Arman; Wang, Rosa; Bhagnani, Tarun; Gillard, Patrick; Bonine, Nicole G

2017-01-01

Abstract Background While CRE and DAT are both associated with worse outcomes, their relative impact to the clinical and economic burden among patients with infections due to Enterobacteriaceae is not well understood. This study assessed the independent and combined effect of these two items on selected outcomes among patients with these infections. Methods Hospitalized adults between July 2011 and September 2014 were identified from Premier Hospital Database. Patients were diagnosed with complicated urinary tract infection, complicated intra-abdominal infection, hospital-associated pneumonia, or bloodstream infection, and had a positive culture for Enterobacteriaceae from a site consistent with infection type (date of culture draw was index date). Patients were required to receive antibiotics on this date or ≤2 days after. Delayed therapy was defined as no receipt of an antibiotic with microbiologic activity during this period. CRE was defined as resistant to ≥1 carbapenems. Inverse probability weighting and multivariate regression analyses were used to estimate the associations between CRE status, DAT and outcomes. Logistic models were used for composite mortality (in-hospital death or discharge to hospice), in-hospital mortality, and discharge to home (reference group was timely therapy plus non-CRE); generalized linear models, for post-index duration of antibiotic therapy, hospital length of stay (LOS), and costs. Results A total of 50,069 patients were included in the analyses; 514 had CRE and 16,414 received DAT. A gradient effect was observed across strata as the burden of serious infections was least among the reference group, and greatest among patients with CRE infection who received DAT (Figure). For example, as compared with the reference group, the risk of composite mortality increased nearly fourfold in patients with CRE infection who received DAT; total in-hospital costs more than doubled. Conclusion DAT has a stronger association than CRE on outcomes, and their effects are synergistic. Given these findings, better methods of early pathogen identification (especially organisms such as CRE) should reduce time to appropriate therapy, thereby improving outcomes in this patient population. Disclosures T. Lodise, Allergan plc: Consultant, Scientific Advisor and Speaker’s Bureau, Consulting fee and Speaker honorarium; A. Berger, Allergan plc: Consultant, Consulting fee; A. Altincatal, Evidera: Consultant, Salary; R. Wang, Evidera: Employee, Salary; T. Bhagnani, Allergan plc: Consultant, Consulting fee; P. Gillard, Allergan plc: Employee, Salary; N. G. Bonine, Allergan plc: Employee, Salary

Evolutionarily Conserved, Growth Plate Zone-Specific Regulation of the Matrilin-1 Promoter: L-Sox5/Sox6 and Nfi Factors Bound near TATA Finely Tune Activation by Sox9 ▿

PubMed Central

Nagy, Andrea; Kénesi, Erzsébet; Rentsendorj, Otgonchimeg; Molnár, Annamária; Szénási, Tibor; Sinkó, Ildikó; Zvara, Ágnes; Thottathil Oommen, Sajit; Barta, Endre; Puskás, László G.; Lefebvre, Veronique; Kiss, Ibolya

2011-01-01

To help uncover the mechanisms underlying the staggered expression of cartilage-specific genes in the growth plate, we dissected the transcriptional mechanisms driving expression of the matrilin-1 gene (Matn1). We show that a unique assembly of evolutionarily conserved cis-acting elements in the Matn1 proximal promoter restricts expression to the proliferative and prehypertrophic zones of the growth plate. These elements functionally interact with distal elements and likewise are capable of restricting the domain of activity of a pancartilaginous Col2a1 enhancer. The proximal elements include a Pe1 element binding the chondrogenic L-Sox5, Sox6, and Sox9 proteins, a SI element binding Nfi proteins, and an initiator Ine element binding the Sox trio and other factors. Sox9 binding to Pe1 is indispensable for functional interaction with the distal promoter. Binding of L-Sox5/Sox6 to Ine and Nfib to SI modulates Sox9 transactivation in a protein dose-dependent manner, possibly to enhance Sox9 activity in early stages of chondrogenesis and repress it at later stages. Hence, our data suggest a novel model whereby Sox and Nfi proteins bind to conserved Matn1 proximal elements and functionally interact with each other to finely tune gene expression in specific zones of the cartilage growth plate. PMID:21173167

Ternary Complex Factors and Cofactors Are Essential for Human T-Cell Leukemia Virus Type 1 Tax Transactivation of the Serum Response Element

PubMed Central

Shuh, Maureen; Derse, David

2000-01-01

The human T-cell leukemia virus type 1 Tax protein activates the expression of cellular immediate early genes controlled by the serum response element (SRE), which contains both the serum response factor (SRF) binding element (CArG box) and the ternary complex factor (TCF) binding element (Ets box). We show that TCF binding is necessary for Tax activation of the SRE and that Tax directly interacts with TCFs in vitro. In addition, Tax interactions with CREB binding protein (CBP) and p300- and CBP-associated factor were found to be essential for Tax activation of SRF-mediated transcription. PMID:11070040

Activation of liver X receptor decreases atherosclerosis in Ldlr⁻/⁻ mice in the absence of ATP-binding cassette transporters A1 and G1 in myeloid cells.

PubMed

Kappus, Mojdeh S; Murphy, Andrew J; Abramowicz, Sandra; Ntonga, Vusisizwe; Welch, Carrie L; Tall, Alan R; Westerterp, Marit

2014-02-01

Liver X receptor (LXR) activators decrease atherosclerosis in mice. LXR activators (1) directly upregulate genes involved in reverse cholesterol transport and (2) exert anti-inflammatory effects mediated by transrepression of nuclear factor-κB target genes. We investigated whether myeloid cell deficiency of ATP-binding cassette transporters A1 and G1 (ABCA1/G1), principal targets of LXR that promote macrophage cholesterol efflux and initiate reverse cholesterol transport, would abolish the beneficial effects of LXR activation on atherosclerosis. LXR activator T0901317 substantially reduced inflammatory gene expression in macrophages lacking ABCA1/G1. Ldlr(-/-) mice were transplanted with Abca1(-/-)Abcg1(-/-) or wild-type bone marrow (BM) and fed a Western-type diet for 6 weeks with or without T0901317 supplementation. Abca1/g1 BM deficiency increased atherosclerotic lesion complexity and inflammatory cell infiltration into the adventitia and myocardium. T0901317 markedly decreased lesion area, complexity, and inflammatory cell infiltration in the Abca1(-/-)Abcg1(-/-) BM-transplanted mice. To investigate whether this was because of macrophage Abca1/g1 deficiency, Ldlr(-/-) mice were transplanted with LysmCreAbca1(fl/fl)Abcg1(fl/fl) or Abca1(fl/fl)Abcg1(fl/fl) BM and fed Western-type diet with or without the more specific LXR agonist GW3965 for 12 weeks. GW3965 decreased lesion size in both groups, and the decrease was more prominent in the LysmCreAbca1(fl/fl)Abcg1(fl/fl) group. The results suggest that anti-inflammatory effects of LXR activators are of key importance to their antiatherosclerotic effects in vivo independent of cholesterol efflux pathways mediated by macrophage ABCA1/G1. This has implications for the development of LXR activators that lack adverse effects on lipogenic genes while maintaining the ability to transrepress inflammatory genes.

Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

PubMed

Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J

2000-12-01

The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

Skeletal Muscle Satellite Cells Are Committed to Myogenesis and Do Not Spontaneously Adopt Nonmyogenic Fates

PubMed Central

Starkey, Jessica D.; Yamamoto, Masakazu; Yamamoto, Shoko; Goldhamer, David J.

2011-01-01

The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoDiCre/+;R26REYFP/+ muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate. PMID:21339173

NMDA receptor agonists reverse impaired psychomotor and cognitive functions associated with hippocampal Hbegf-deficiency in mice.

PubMed

Sasaki, Keita; Omotuyi, Olaposi Idowu; Ueda, Mutsumi; Shinohara, Kazuyuki; Ueda, Hiroshi

2015-12-04

Structural and functional changes of the hippocampus are correlated with psychiatric disorders and cognitive dysfunctions. Genetic deletion of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is predominantly expressed in cortex and hippocampus, also causes similar psychiatric and cognitive dysfunctions, accompanying down-regulated NMDA receptor signaling. However, little is known of such dysfunctions in hippocampus-specific Hbegf cKO mice. We successfully developed hippocampus-specific cKO mice by crossbreeding floxed Hbegf and Gng7-Cre knock-in mice, as Gng7 promoter-driven Cre is highly expressed in hippocampal neurons as well as striatal medium spiny neurons. In mice lacking hippocampus Hbegf gene, there was a decreased neurogenesis in the subgranular zone (SGZ) of the dentate gyrus as well as down-regulation of PSD-95/NMDA-receptor-NR1/NR2B subunits and related NMDA receptor signaling. Psychiatric, social-behavioral and cognitive abnormalities were also observed in hippocampal cKO mice. Interestingly, D-cycloserine and nefiracetam, positive allosteric modulators (PAMs) of NMDA receptor reversed the apparent reduction in NMDA receptor signaling and most behavioral abnormalities. Furthermore, decreased SGZ neurogenesis in hippocampal cKO mice was reversed by nefiracetam. The present study demonstrates that PAMs of NMDA receptor have pharmacotherapeutic potentials to reverse down-regulated NMDA receptor signaling, neuro-socio-cognitive abnormalities and decreased neurogenesis in hippocampal cKO mice.

Utilizing GCaMP transgenic mice to monitor endogenous Gq/11-coupled receptors

PubMed Central

Partridge, John G.

2015-01-01

The family of GCaMPs are engineered proteins that contain Ca2+ binding motifs within a circularly permutated variant of the Aequorea Victoria green fluorescent protein (cp-GFP). The rapidly advancing field of utilizing GCaMP reporter constructs represents a major step forward in our ability to monitor intracellular Ca2+ dynamics. With the use of these genetically encoded Ca2+ sensors, investigators have studied activation of endogenous Gq types of G protein-coupled receptors (GPCRs) and subsequent rises in intracellular calcium. Escalations in intracellular Ca2+ from GPCR activation can be faithfully monitored in space and time as an increase in fluorescent emission from these proteins. Further, transgenic mice are now commercially available that express GCaMPs in a Cre recombinase dependent fashion. These GCaMP reporter mice can be bred to distinct Cre recombinase driver mice to direct expression of this sensor in unique populations of cells. Concerning the central nervous system (CNS), sources of calcium influx, including those arising from Gq activation can be observed in targeted cell types like neurons or astrocytes. This powerful genetic method allows simultaneous monitoring of the activity of dozens of cells upon activation of endogenous Gq-coupled GPCRs. Therefore, in combination with pharmacological tools, this strategy of monitoring GPCR activation is amenable to analysis of orthosteric and allosteric ligands of Gq-coupled receptors in their endogenous environments. PMID:25805995

Visual impairment in FOXG1-mutated individuals and mice.

PubMed

Boggio, E M; Pancrazi, L; Gennaro, M; Lo Rizzo, C; Mari, F; Meloni, I; Ariani, F; Panighini, A; Novelli, E; Biagioni, M; Strettoi, E; Hayek, J; Rufa, A; Pizzorusso, T; Renieri, A; Costa, M

2016-06-02

The Forkead Box G1 (FOXG1 in humans, Foxg1 in mice) gene encodes for a DNA-binding transcription factor, essential for the development of the telencephalon in mammalian forebrain. Mutations in FOXG1 have been reported to be involved in the onset of Rett Syndrome, for which sequence alterations of MECP2 and CDKL5 are known. While visual alterations are not classical hallmarks of Rett syndrome, an increasing body of evidence shows visual impairment in patients and in MeCP2 and CDKL5 animal models. Herein we focused on the functional role of FOXG1 in the visual system of animal models (Foxg1(+/Cre) mice) and of a cohort of subjects carrying FOXG1 mutations or deletions. Visual physiology of Foxg1(+/Cre) mice was assessed by visually evoked potentials, which revealed a significant reduction in response amplitude and visual acuity with respect to wild-type littermates. Morphological investigation showed abnormalities in the organization of excitatory/inhibitory circuits in the visual cortex. No alterations were observed in retinal structure. By examining a cohort of FOXG1-mutated individuals with a panel of neuro-ophthalmological assessments, we found that all of them exhibited visual alterations compatible with high-level visual dysfunctions. In conclusion our data show that Foxg1 haploinsufficiency results in an impairment of mouse and human visual cortical function. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

N-3 polyunsaturated fatty acid regulation of hepatic gene transcription

PubMed Central

Jump, Donald B.

2009-01-01

Purpose of review The liver plays a central role in whole body lipid metabolism and adapts rapidly to changes in dietary fat composition. This adaption involves changes in the expression of genes involved in glycolysis, de-novo lipogenesis, fatty acid elongation, desaturation and oxidation. This review brings together metabolic and molecular studies that help explain n-3 (omega-3) polyunsaturated fatty acid regulation of hepatic gene transcription. Recent findings Dietary n-3 polyunsaturated fatty acid regulates hepatic gene expression by targeting three major transcriptional regulatory networks: peroxisome proliferator-activated receptor α, sterol regulatory element binding protein-1 and the carbohydrate regulatory element binding protein/Max-like factor X heterodimer. 22 : 6,n-3, the most prominent n-3 polyunsaturated fatty acid in tissues, is a weak activator of peroxisome proliferator-activated receptor α. Hepatic metabolism of 22 : 6,n-3, however, generates 20 : 5,n-3, a strong peroxisome proliferator-activated receptor α activator. In contrast to peroxisome proliferator-activated receptor α, 22 : 6,n-3 is the most potent fatty acid regulator of hepatic sterol regulatory element binding protein-1. 22 : 6,n-3 suppresses sterol regulatory element binding protein-1 gene expression while enhancing degradation of nuclear sterol regulatory element binding protein-1 through 26S proteasome and Erk1/2-dependent mechanisms. Both n-3 and n-6 polyunsaturated fatty acid suppress carbohydrate regulatory element binding protein and Max-like factor X nuclear abundance and interfere with glucose-regulated hepatic metabolism. Summary These studies have revealed unique mechanisms by which specific polyunsaturated fatty acids control peroxisome proliferator activated receptor α, sterol regulatory element binding protein-1 and carbohydrate regulatory element binding protein/Max-like factor X function. As such, specific metabolic and signal transduction pathways contribute significantly to the fatty acid regulation of these transcription factors and their corresponding regulatory networks. PMID:18460914

Inhibition of IKKß in enterocytes exacerbates sepsis-induced intestinal injury and worsens mortality

PubMed Central

Dominguez, Jessica A.; Samocha, Alexandr J.; Liang, Zhe; Burd, Eileen M.; Farris, Alton B.; Coopersmith, Craig M.

2013-01-01

Objective NF-kB is a critical regulator of cell survival genes and the host inflammatory response. The purpose of this study was to investigate the role of enterocyte-specific NF-kB in sepsis through selective ablation of IkB kinase (IKK)-ß. Design Prospective, randomized, controlled study. Setting Animal laboratories in university medical centers. Subjects and Interventions Mice lacking functional NF-kB in their intestinal epithelium (Vil-Cre/Ikkßf/Δ) and wild type (WT) mice were subjected to sham laparotomy or cecal ligation and puncture (CLP). Animals were sacrified at 24 hours or followed seven days for survival. Measurements and Main Results Septic WT mice had decreased villus length compared to sham mice while villus atrophy was further exacerbated in septic Vil-Cre/Ikkßf/Δ mice. Sepsis induced an increase in intestinal epithelial apoptosis compared to sham mice which was further exacerbated in Vil-Cre/Ikkßf/Δ mice. Sepsis induced intestinal hyperpermeability in WT mice compared to sham mice, which was further exacerbated in septic Vil-Cre/Ikkßf/Δ mice. This was associated with increased intestinal expression of claudin-2 in septic WT mice, which was further increased in septic Vil-Cre/Ikkßf/Δ mice. Both, pro-inflammatory and anti-inflammatory cytokines were increased in serum following CLP, and IL-10 and MCP-1 levels were higher in septic Vil-Cre/Ikkßf/Δ mice than septic WT mice. All septic mice were bacteremic, but no differences in bacterial load were identified between WT and Vil-Cre/Ikkßf/Δ mice. To determine the functional significance of these results, animals were followed for survival. Septic WT mice had lower mortality than septic Vil-Cre/Ikkßf/Δ mice (47% vs. 80%, p<0.05). Anti-TNF administration decreased intestinal apoptosis, permeability and mortality in WT septic mice and a similar improvement in intestinal integrity and survival were seen when anti-TNF was given to Vil-Cre/Ikkßf/Δ mice. Conclusions Enterocyte-specific NF-kB has a beneficial role in sepsis by partially preventing sepsis-induced increases in apoptosis and permeability, which are associated with worsening mortality. PMID:23939348

A high-fat diet activates oncogenic Kras and COX2 to induce development of pancreatic ductal adenocarcinoma in mice.

PubMed

Philip, Bincy; Roland, Christina L; Daniluk, Jaroslaw; Liu, Yan; Chatterjee, Deyali; Gomez, Sobeyda B; Ji, Baoan; Huang, Haojie; Wang, Huamin; Fleming, Jason B; Logsdon, Craig D; Cruz-Monserrate, Zobeida

2013-12-01

Obesity is a risk factor for pancreatic ductal adenocarcinoma (PDAC), but it is not clear how obesity contributes to pancreatic carcinogenesis. The oncogenic form of KRAS is expressed during early stages of PDAC development and is detected in almost all of these tumors. However, there is evidence that mutant KRAS requires an additional stimulus to activate its full oncogenic activity and that this stimulus involves the inflammatory response. We investigated whether the inflammation induced by a high-fat diet, and the accompanying up-regulation of cyclooxygenase-2 (COX2), increases Kras activity during pancreatic carcinogenesis in mice. We studied mice with acinar cell-specific expression of KrasG12D (LSL-Kras/Ela-CreERT mice) alone or crossed with COX2 conditional knockout mice (COXKO/LSL-Kras/Ela-CreERT). We also studied LSL-Kras/PDX1-Cre mice. All mice were fed isocaloric diets with different amounts of fat, and a COX2 inhibitor was administered to some LSL-Kras/Ela-CreERT mice. Pancreata were collected from mice and analyzed for Kras activity, levels of phosphorylated extracellular-regulated kinase, inflammation, fibrosis, pancreatic intraepithelial neoplasia (PanIN), and PDACs. Pancreatic tissues from LSL-Kras/Ela-CreERT mice fed high-fat diets (HFDs) had increased Kras activity, fibrotic stroma, and numbers of PanINs and PDACs than LSL-Kras/Ela-CreERT mice fed control diets; the mice fed the HFDs also had shorter survival times than mice fed control diets. Administration of a COX2 inhibitor to LSL-Kras/Ela-CreERT mice prevented these effects of HFDs. We also observed a significant reduction in survival times of mice fed HFDs. COXKO/LSL-Kras/Ela-CreERT mice fed HFDs had no evidence for increased numbers of PanIN lesions, inflammation, or fibrosis, as opposed to the increases observed in LSL-Kras/Ela-CreERT mice fed HFDs. In mice, an HFD can activate oncogenic Kras via COX2, leading to pancreatic inflammation and fibrosis and development of PanINs and PDAC. This mechanism might be involved in the association between risk for PDAC and HFDs. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

The Effects of Androgens on Murine Cortical Bone Do Not Require AR or ERα Signaling in Osteoblasts and Osteoclasts.

PubMed

Ucer, Serra; Iyer, Srividhya; Bartell, Shoshana M; Martin-Millan, Marta; Han, Li; Kim, Ha-Neui; Weinstein, Robert S; Jilka, Robert L; O'Brien, Charles A; Almeida, Maria; Manolagas, Stavros C

2015-07-01

In men, androgens are critical for the acquisition and maintenance of bone mass in both the cortical and cancellous bone compartment. Male mice with targeted deletion of the androgen receptor (AR) in mature osteoblasts or osteocytes have lower cancellous bone mass, but no cortical bone phenotype. We have investigated the possibility that the effects of androgens on the cortical compartment result from AR signaling in osteoprogenitors or cells of the osteoclast lineage; or via estrogen receptor alpha (ERα) signaling in either or both of these two cell types upon conversion of testosterone to estradiol. To this end, we generated mice with targeted deletion of an AR or an ERα allele in the mesenchymal (AR(f/y);Prx1-Cre or ERα(f/f);Osx1-Cre) or myeloid cell lineage (AR(f/y);LysM-Cre or ERα(f/f);LysM-Cre) and their descendants. Male AR(f/y);Prx1-Cre mice exhibited decreased bone volume and trabecular number, and increased osteoclast number in the cancellous compartment. Moreover, they did not undergo the loss of cancellous bone volume and trabecular number caused by orchidectomy (ORX) in their littermate controls. In contrast, AR(f/y);LysM-Cre, ERα(f/f);Osx1-Cre, or ERα(f/f);LysM-Cre mice had no cancellous bone phenotype at baseline and lost the same amount of cancellous bone as their controls following ORX. Most unexpectedly, adult males of all four models had no discernible cortical bone phenotype at baseline, and lost the same amount of cortical bone as their littermate controls after ORX. Recapitulation of the effects of ORX by AR deletion only in the AR(f/y);Prx1-Cre mice indicates that the effects of androgens on cancellous bone result from AR signaling in osteoblasts-not on osteoclasts or via aromatization. The effects of androgens on cortical bone mass, on the other hand, do not require AR or ERα signaling in any cell type across the osteoblast or osteoclast differentiation lineage. Therefore, androgens must exert their effects indirectly by actions on some other cell type(s) or tissue(s). © 2015 American Society for Bone and Mineral Research.

Self-excising Cre/mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae.

PubMed

Zhang, Silai; Ban, Akihiko; Ebara, Naoki; Mizutani, Osamu; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2017-04-01

In this study, we developed a self-excising Cre/loxP-mediated marker recycling system with mutated lox sequences to introduce a number of biosynthetic genes into Aspergillus oryzae. To construct the self-excising marker cassette, both the selectable marker, the Aspergillus nidulans adeA gene, and the Cre recombinase gene (cre), conditionally expressed by the xylanase-encoding gene promoter, were designed to be located between the mutant lox sequences, lox66 and lox71. However, construction of the plasmid failed, possibly owing to a slight expression of cre downstream of the fungal gene promoter in Escherichia coli. Hence, to avoid the excision of the cassette in E. coli, a 71-bp intron of the A. oryzae xynG2 gene was inserted into the cre gene. The A. oryzae adeA deletion mutant was transformed with the resulting plasmid in the presence of glucose, and the transformants were cultured in medium containing xylose as the sole carbon source. PCR analysis of genomic DNA from resultant colonies revealed the excision of both the marker and Cre expression construct, indicating that the self-excising marker cassette was efficient at removing the selectable marker. Using the marker recycling system, hyperproduction of kojic acid could be achieved in A. oryzae by the introduction of two genes that encode oxidoreductase and transporter. Furthermore, we also constructed an alternative marker recycling cassette bearing the A. nidulans pyrithiamine resistant gene (ptrA) as a dominant selectable marker. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Down, But Not Out: Partial Elimination of Androgen Receptors in the Male Mouse Brain Does Not Affect Androgenic Regulation of Anxiety or HPA Activity.

PubMed

Chen, Chieh V; Brummet, Jennifer L; Jordan, Cynthia L; Breedlove, S Marc

2016-02-01

We previously found that androgen receptor (AR) activity mediates two effects of T in adult male mice: reduction of anxiety-like behaviors and dampening of the hypothalamic-pituitary-adrenal response to stress. To determine whether brain ARs mediate these effects, we used the Cre/loxP technology seeking to disable AR throughout the central nervous system (CNS). Female mice carrying the floxed AR allele (ARlox) were crossed with males carrying cre recombinase transgene controlled by the nestin promoter (NesCre), producing cre in developing neurons and glia. Among male offspring, four genotypes resulted: males carrying ARlox and NesCre (NesARko), and three control groups (wild types, NesCre, and ARlox). Reporter mice indicated ubiquitous Cre expression throughout the CNS. Nevertheless, AR immunocytochemistry in NesARko mice revealed efficient knockout (KO) of AR in some brain regions (hippocampus and medial prefrontal cortex [mPFC]), but not others. Substantial AR protein was seen in the amygdala and hypothalamus among other regions, whereas negligible AR remained in others like the bed nucleus of the stria terminalis and dorsal periaqueductal gray. This selective KO allowed for testing the role of AR in hippocampus and mPFC. Males were castrated and implanted with T at postnatal day 60 before testing on postnatal day 90-100. In contrast with males with global KO of AR, T still modulated anxiety-related behavior and hypothalamic-pituitary-adrenal activity in NesARko males. These results leave open the possibility that AR acting in the CNS mediates these effects of T, but demonstrate that AR is not required in the hippocampus or mPFC for T's anxiolytic effects.

Inactivation of the Progesterone Receptor in Mx1+ Cells Potentiates Osteogenesis in Calvaria but Not in Long Bone.

PubMed

Zhong, Zhendong A; Sun, Weihua; Chen, Haiyan; Zhang, Hongliang; Lane, Nancy E; Yao, Wei

2015-01-01

The effect of progesterone on bone remains elusive. We previously reported that global progesterone receptor (PR) knockout mice displayed high bone mass phenotype, suggesting that PR influences bone growth and modeling. Recently, Mx1+ cells were characterized to be mesenchymal stem cell-like pluripotent Cells. The aim of this study was to evaluate whether the PR in Mx1+ cells regulates osteogenesis. Using the Mx1-Cre;mT/mG reporter mouse model, we found that the calvarial cells exhibited minimal background Mx1-Cre activity prior to Cre activation by IFNα treatment as compared to the bone marrow stromal cells. IFNα treatment significantly activated Mx1-Cre in the calvarial cells. When the PR gene was deleted in the Mx1-Cre;PR-flox calvarial cells in vitro, significantly higher levels of expression of osteoblast maturation marker genes (RUNX2, Osteocalcin, and Dmp1) and osteogenic potential were detected. The PR-deficient calvariae exhibited greater bone volume, especially in the males. Although Mx1-Cre activity could be induced on the bone surface in vivo, the Mx1+ cells did not differentiate into osteocytes in long bones. Bone volumes at the distal femurs and the bone turnover marker serum Osteocalcin were similar between the Mx1-Cre;PR-flox mutant mice and the corresponding wild types in both sexes. In conclusion, our data demonstrates that blocking progesterone signaling via PRs in calvarial Mx1+ cells promoted osteoblast differentiation in the calvaria. Mx1+ was expressed by heterogeneous cells in bone marrow and did not differentiate into osteocyte during long bone development in vivo. Selectively inactivating the PR gene in Mx1+ cells affected the membrane bone formation but did not affect peripheral skeletal homeostasis.

Cost-Utility of Competing Strategies to Prevent Endoscopic Transmission of Carbapenem-Resistant Enterobacteriaceae

PubMed Central

Almario, Christopher V.; May, Folasade P.; Shaheen, Nicholas J.; Murthy, Rekha; Gupta, Kapil; Jamil, Laith H.; Lo, Simon K.; Spiegel, Brennan M.R.

2015-01-01

OBJECTIVES Prior reports have linked patient transmission of carbapenem-resistant Enterobacteriaceae (CRE, or “superbug”) to endoscopes used during endoscopic retrograde cholangiopancreatography (ERCP). We performed a decision analysis to measure the cost-effectiveness of four competing strategies for CRE risk management. METHODS We used decision analysis to calculate the cost-effectiveness of four approaches to reduce the risk of CRE transmission among patients presenting to the hospital for symptomatic common bile duct stones. The strategies included: (1) perform ERCP followed by U.S. Food and Drug Administration (FDA)-recommended endoscope reprocessing procedures; (2) perform ERCP followed by “endoscope culture and hold”; (3) perform ERCP followed by ethylene oxide (EtO) sterilization of the endoscope; and (4) stop performing ERCP in lieu of laparoscopic cholecystectomy (LC) with common bile duct exploration (CBDE). Our outcome was incremental cost per quality-adjusted life year (QALY) gained. RESULTS In the base-case scenario, ERCP with FDA-recommended endoscope reprocessing was the most cost-effective strategy. Both the ERCP with culture and hold ($4,228,170/QALY) and ERCP with EtO sterilization ($50,572,348/QALY) strategies had unacceptable incremental costs per QALY gained. LC with CBDE was dominated, being both more costly and marginally less effective versus the alternatives. In sensitivity analysis, ERCP with culture and hold became the most cost-effective approach when the pretest probability of CRE exceeded 24%. CONCLUSIONS In institutions with a low CRE prevalence, ERCP with FDA-recommended reprocessing is the most cost-effective approach for mitigating CRE transmission risk. Only in settings with an extremely high CRE prevalence did ERCP with culture and hold become cost-effective. PMID:26526083

Down, But Not Out: Partial Elimination of Androgen Receptors in the Male Mouse Brain Does Not Affect Androgenic Regulation of Anxiety or HPA Activity

PubMed Central

Brummet, Jennifer L.; Jordan, Cynthia L.; Breedlove, S. Marc

2016-01-01

We previously found that androgen receptor (AR) activity mediates two effects of T in adult male mice: reduction of anxiety-like behaviors and dampening of the hypothalamic-pituitary-adrenal response to stress. To determine whether brain ARs mediate these effects, we used the Cre/loxP technology seeking to disable AR throughout the central nervous system (CNS). Female mice carrying the floxed AR allele (ARlox) were crossed with males carrying cre recombinase transgene controlled by the nestin promoter (NesCre), producing cre in developing neurons and glia. Among male offspring, four genotypes resulted: males carrying ARlox and NesCre (NesARko), and three control groups (wild types, NesCre, and ARlox). Reporter mice indicated ubiquitous Cre expression throughout the CNS. Nevertheless, AR immunocytochemistry in NesARko mice revealed efficient knockout (KO) of AR in some brain regions (hippocampus and medial prefrontal cortex [mPFC]), but not others. Substantial AR protein was seen in the amygdala and hypothalamus among other regions, whereas negligible AR remained in others like the bed nucleus of the stria terminalis and dorsal periaqueductal gray. This selective KO allowed for testing the role of AR in hippocampus and mPFC. Males were castrated and implanted with T at postnatal day 60 before testing on postnatal day 90–100. In contrast with males with global KO of AR, T still modulated anxiety-related behavior and hypothalamic-pituitary-adrenal activity in NesARko males. These results leave open the possibility that AR acting in the CNS mediates these effects of T, but demonstrate that AR is not required in the hippocampus or mPFC for T's anxiolytic effects. PMID:26562258

Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

PubMed

Shuda, Masahiro; Guastafierro, Anna; Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M; Lukianov, Stefan; Jenkins, Frank J; Honda, Kord; Maricich, Stephen M; Moore, Patrick S; Chang, Yuan

2015-01-01

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

Transient Cnp expression by early progenitors causes Cre-Lox-based reporter lines to map profoundly different fates.

PubMed

Tognatta, Reshmi; Sun, Wenjing; Goebbels, Sandra; Nave, Klaus-Armin; Nishiyama, Akiko; Schoch, Susanne; Dimou, Leda; Dietrich, Dirk

2017-02-01

NG2 expressing oligodendroglial precursor cells are ubiquitous in the central nervous system and the only cell type cycling throughout life. Previous fate mapping studies have remained inconsistent regarding the question whether NG2 cells are capable of generating certain types of neurons. Here, we use CNP-Cre mice to map the fate of a sub-population of NG2 cells assumed to be close to differentiation. When crossing these mice with the ROSA26/YFP Cre-reporter line we discovered large numbers of reporter-expressing pyramidal neurons in the piriform and dorsal cortex. In contrast, when using Z/EG reporter mice to track the fate of Cnp-expressing NG2 cells only oligodendroglial cells were found reporter positive. Using BrdU-based birth dating protocols and inducible NG2CreER:ROSA26/YFP mice we show that YFP positive neurons are generated from radial glial cells and that these radial glial cells display temporary and low level activity of certain oligodendroglial genes sufficient to recombine the Cre-inducible reporter gene in ROSA26/YFP but not in Z/EG mice. Taken together, we did not obtain evidence for generation of neurons from NG2 cells. Our results suggest that with an appropriate reporter system Cnp activity can be used to define a proliferative subpopulation of NG2 cells committed to generate oligodendrocytes. However, the strikingly different results obtained from ROSA26/YFP versus Z/EG mice demonstrate that the choice of Cre-reporter line can be of crucial importance for fate mapping studies and other applications of the Cre-lox technology. GLIA 2017;65:342-359. © 2016 Wiley Periodicals, Inc.

Mu-opioid receptors in nociceptive afferents produce a sustained suppression of hyperalgesia in chronic pain.

PubMed

Severino, Amie; Chen, Wenling; Hakimian, Joshua K; Kieffer, Brigitte L; Gaveriaux-Ruff, Claire; Walwyn, Wendy; Marvizon, Juan Carlos

2018-04-17

The latent sensitization model of chronic pain reveals that recovery from some types of long-term hyperalgesia is an altered state in which nociceptive sensitization persists but is suppressed by the ongoing activity of analgesic receptors such as µ-opioid receptors (MORs). To determine whether these MORs are the ones present in nociceptive afferents, we bred mice expressing Cre-recombinase under the Nav1.8 channel promoter (Nav1.8cre) with MOR-floxed mice (flMOR). These Nav1.8cre/flMOR mice had reduced MOR expression in primary afferents, as revealed by quantitative PCR, in situ hybridization and immunofluorescence colocalization with the neuropeptide CGRP. We then studied the recovery from chronic pain of these mice and their flMOR littermates. When Nav1.8cre/flMOR mice were injected in the paw with complete Freund's adjuvant they developed mechanical hyperalgesia that persisted for over two months, whereas the responses of flMOR mice returned to baseline after three weeks. We then used the inverse agonist naltrexone to assess ongoing MOR activity. Naltrexone produced a robust reinstatement of hyperalgesia in control flMOR mice, but produced no effect in the Nav1.8/flMOR males and a weak reinstatement of hyperalgesia in Nav1.8/flMOR females. Naltrexone also reinstated swelling of the hind paw in flMOR mice and female Nav1.8cre/flMOR mice, but not male Nav1.8cre/flMOR mice. The MOR agonist DAMGO inhibited substance P release in flMOR mice but not Nav1.8cre/flMOR mice, demonstrating a loss of MOR function at the central terminals of primary afferents. We conclude that MORs in nociceptive afferents mediate an ongoing suppression of hyperalgesia to produce remission from chronic pain.

Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

PubMed

Hunter, A J; Morris, T A; Jin, B; Saint, C P; Kelly, J M

2013-09-01

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

Deletion of creB in Aspergillus oryzae Increases Secreted Hydrolytic Enzyme Activity

PubMed Central

Hunter, A. J.; Morris, T. A.; Jin, B.; Saint, C. P.

2013-01-01

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes. PMID:23835170

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

PubMed Central

Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T.; Hocart, Charles H.; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

2015-01-01

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation. PMID:26253705

Region-specific deletions of RIM1 reproduce a subset of global RIM1α−/− phenotypes

PubMed Central

Haws, M E; Kaeser, P S; Jarvis, D L; Südhof, T C; Powell, C M

2012-01-01

The presynaptic protein RIM1α mediates multiple forms of presynaptic plasticity at both excitatory and inhibitory synapses. Previous studies of mice lacking RIM1α (RIM1α−/− throughout the brain showed that deletion of RIM1α results in multiple behavioral abnormalities. In an effort to begin to delineate the brain regions in which RIM1 deletion mediates these abnormal behaviors, we used conditional (floxed) RIM1 knockout mice (fRIM1). By crossing these fRIM1 mice to previously characterized transgenic cre lines, we aimed to delete RIM1 selectively in the dentate gyrus (DG), using a specific preproopiomelanocortin promoter driving cre recombinase (POMC-cre) line , and in pyramidal neurons of the CA3 region of hippocampus, using the kainate receptor subunit 1 promoter driving cre recombinase (KA-cre). Neither of these cre driver lines was uniquely selective to the targeted regions. In spite of this, we were able to reproduce a subset of the global RIM1α−/− behavioral abnormalities, thereby narrowing the brain regions in which loss of RIM1 is sufficient to produce these behavioral differences. Most interestingly, hypersensitivity to the pyschotomimetic MK-801 was shown in mice lacking RIM1 selectively in the DG, arcuate nucleus of the hypothalamus and select cerebellar neurons, implicating novel brain regions and neuronal subtypes in this behavior. PMID:22103334

Deficiency of PDK1 in liver results in glucose intolerance, impairment of insulin-regulated gene expression and liver failure

PubMed Central

2004-01-01

The liver plays an important role in insulin-regulated glucose homoeostasis. To study the function of the PDK1 (3-phosphoinositide-dependent protein kinase-1) signalling pathway in mediating insulin's actions in the liver, we employed CRE recombinase/loxP technology to generate L(liver)-PDK1−/− mice, which lack expression of PDK1 in hepatocytes and in which insulin failed to induce activation of PKB in liver. The L-PDK1−/− mice were not insulin-intolerant, possessed normal levels of blood glucose and insulin under normal feeding conditions, but were markedly glucose-intolerant when injected with glucose. The L-PDK1−/− mice also possessed 10-fold lower levels of hepatic glycogen compared with control littermates, and were unable to normalize their blood glucose levels within 2 h after injection of insulin. The glucose intolerance of the L-PDK1−/− mice may be due to an inability of glucose to suppress hepatic glucose output through the gluconeogenic pathway, since the mRNA encoding hepatic PEPCK (phosphoenolpyruvate carboxykinase), G6Pase (glucose-6-phosphatase) and SREBP1 (sterol-regulatory-element-binding protein 1), which regulate gluconeogenesis, are no longer controlled by feeding. Furthermore, three other insulin-controlled genes, namely IGFBP1 (insulin-like-growth-factor-binding protein-1), IRS2 (insulin receptor substrate 2) and glucokinase, were regulated abnormally by feeding in the liver of PDK1-deficient mice. Finally, the L-PDK1−/− mice died between 4–16 weeks of age due to liver failure. These results establish that the PDK1 signalling pathway plays an important role in regulating glucose homoeostasis and controlling expression of insulin-regulated genes. They suggest that a deficiency of the PDK1 pathway in the liver could contribute to development of diabetes, as well as to liver failure. PMID:15554902

Evaluation of dementia by acrolein, amyloid-β and creatinine.

PubMed

Igarashi, Kazuei; Yoshida, Madoka; Waragai, Masaaki; Kashiwagi, Keiko

2015-10-23

Plasma, urine and cerebrospinal fluid (CSF) were examined for biochemical markers of dementia. Protein-conjugated acrolein (PC-Acro) and the amyloid-β (Aβ)40/42 ratio in plasma can be used to detect mild cognitive impairment (MCI) and Alzheimer's disease (AD). In plasma, PC-Acro and the Aβ40/42 ratio in MCI and AD were significantly higher relative to non-demented subjects. Furthermore, urine acrolein metabolite, 3-hydroxypropyl mercapturic acid (3-HPMA)/creatinine (Cre) and amino acid-conjugated acrolein (AC-Acro)/Cre in AD were significantly lower than MCI. It was also shown that reduced urine 3-HPMA/Cre correlated with increased plasma Aβ40/42 ratio in dementia. The Aβ40/PC-Acro ratio in CSF, together with Aβ40 and Aβ40/42 ratio, was lower in AD than MCI. Increased plasma PC-Acro and Aβ40/42 ratio and decreased urine 3-HPMA/Cre correlated with cognitive ability (MMSE). These results indicate that the measurements of acrolein derivatives together with Aβ and Cre in biologic fluids is useful to estimate severity of dementia. Copyright © 2015 Elsevier B.V. All rights reserved.

Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2.

PubMed

Fukuda, Tomokazu; Scott, Gregory; Komatsu, Yoshihiro; Araya, Runa; Kawano, Masako; Ray, Manas K; Yamada, Masahisa; Mishina, Yuji

2006-04-01

BMP signaling plays pleiotropic roles in various tissues. Transgenic mouse lines that overexpress BMP signaling in a tissue-specific manner would be beneficial; however, production of each tissue-specific transgenic mouse line is labor-intensive. Here, using a Cre-loxP system, we generated a conditionally overexpressing mouse line for BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI, ActRI, or ActRIA). By mating this line with Cre-expression mouse lines, Cre-mediated recombination removes an intervening floxed lacZ expression cassette and thereby permits the expression of a constitutively active form of Alk2 (caAlk2) driven by a ubiquitous promoter, CAG. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 in the areas where Cre recombination had occurred. The conditional overexpression system described here provides versatility in investigating gene functions in a tissue-specific manner without having to generate independent tissue-specific transgenic lines. Published 2006 Wiley-Liss, Inc.

Cre recombinase-mediated site-specific recombination between plant chromosomes.

PubMed Central

Qin, M; Bayley, C; Stockton, T; Ow, D W

1994-01-01

We report the use of the bacteriophage P1 Cre-lox system for generating conservative site-specific recombination between tobacco chromosomes. Two constructs, one containing a promoterless hygromycin-resistance gene preceded by a lox site (lox-hpt) and the other containing a cauliflower mosaic virus 35S promoter linked to a lox sequence and the cre coding region (35S-lox-cre), were introduced separately into tobacco plants. Crosses between plants harboring either construct produced plants with the two constructs situated on different chromosomes. Plants with recombination events were identified by selecting for hygromycin resistance, a phenotype expressed upon recombination. Molecular analysis showed that these recombination events occurred specifically at the lox sites and resulted in the reciprocal exchange of flanking host DNA. Progenies of these plants showed 67-100% cotransmission of the new transgenes, 35S-lox-hpt and lox-cre, consistent with the preferential cosegregation of translocated chromosomes. These results illustrate that site-specific recombination systems can be useful tools for the large-scale manipulation of eukaryotic chromosomes in vivo. Images PMID:8127869

Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.

PubMed

Feyerabend, Thorsten B; Weiser, Anne; Tietz, Annette; Stassen, Michael; Harris, Nicola; Kopf, Manfred; Radermacher, Peter; Möller, Peter; Benoist, Christophe; Mathis, Diane; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2011-11-23

Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy. Copyright © 2011 Elsevier Inc. All rights reserved.

Generation and Characterization of Transgenic Mice Expressing Mouse Ins1 Promoter for Pancreatic β-Cell-Specific Gene Overexpression and Knockout.

PubMed

Cheng, Yulong; Su, Yutong; Shan, Aijing; Jiang, Xiuli; Ma, Qinyun; Wang, Weiqing; Ning, Guang; Cao, Yanan

2015-07-01

The technologies for pancreatic β-cell-specific gene overexpression or knockout are fundamental for investigations of functional genes in vivo. Here we generated the Ins1-Cre-Dsred and Ins1-rtTA mouse models, which expressed the Cre recombinase or reverse tetracycline regulatable transactivator (rtTA) without hGH minigene under the control of mouse Ins1 promoter. Our data showed that the Cre-mediated recombination and rtTA-mediated activation could be efficiently detected at embryonic day 13.5 when these models were crossed with the reporter mice (ROSA(mT/mG) or tetO-HIST1H2BJ/GFP). The Cre and rtTA expression was restricted to β-cells without leakage in the brain and other tissues. Moreover, both the transgenic lines showed normal glucose tolerance and insulin secretion. These results suggested that the Ins1-Cre-Dsred and Ins1-rtTA mice could be used to knock out or overexpress target genes in embryos and adults to facilitate β-cell researches.

The STAT3 HIES mutation is a gain-of-function mutation that activates genes via AGG-element carrying promoters.

PubMed

Xu, Li; Ji, Jin-Jun; Le, Wangping; Xu, Yan S; Dou, Dandan; Pan, Jieli; Jiao, Yifeng; Zhong, Tianfei; Wu, Dehong; Wang, Yumei; Wen, Chengping; Xie, Guan-Qun; Yao, Feng; Zhao, Heng; Fan, Yong-Sheng; Chin, Y Eugene

2015-10-15

Cytokine or growth factor activated STAT3 undergoes multiple post-translational modifications, dimerization and translocation into nuclei, where it binds to serum-inducible element (SIE, 'TTC(N3)GAA')-bearing promoters to activate transcription. The STAT3 DNA binding domain (DBD, 320-494) mutation in hyper immunoglobulin E syndrome (HIES), called the HIES mutation (R382Q, R382W or V463Δ), which elevates IgE synthesis, inhibits SIE binding activity and sensitizes genes such as TNF-α for expression. However, the mechanism by which the HIES mutation sensitizes STAT3 in gene induction remains elusive. Here, we report that STAT3 binds directly to the AGG-element with the consensus sequence 'AGG(N3)AGG'. Surprisingly, the helical N-terminal region (1-355), rather than the canonical STAT3 DBD, is responsible for AGG-element binding. The HIES mutation markedly enhances STAT3 AGG-element binding and AGG-promoter activation activity. Thus, STAT3 is a dual specificity transcription factor that promotes gene expression not only via SIE- but also AGG-promoter activity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Risk of transmission of carbapenem-resistant Enterobacteriaceae and related “superbugs” during gastrointestinal endoscopy

PubMed Central

Muscarella, Lawrence F

2014-01-01

To evaluate the risk of transmission of carbapenem-resistant Enterobacteriaceae (CRE) and their related superbugs during gastrointestinal (GI) endoscopy. Reports of outbreaks linked to GI endoscopes contaminated with different types of infectious agents, including CRE and their related superbugs, were reviewed. Published during the past 30 years, both prior to and since CRE’s emergence, these reports were obtained by searching the peer-reviewed medical literature (via the United States National Library of Medicine’s “MEDLINE” database); the Food and Drug Administration’s Manufacturer and User Facility Device Experience database, or “MAUDE”; and the Internet (via Google’s search engine). This review focused on an outbreak of CRE in 2013 following the GI endoscopic procedure known as endoscopic retrograde cholangiopancreatography, or ERCP, performed at “Hospital X” located in the suburbs of Chicago (IL; United States). Part of the largest outbreak of CRE in United States history, the infection and colonization of 10 and 28 of this hospital’s patients, respectively, received considerable media attention and was also investigated by the Centers for Disease Control and Prevention (CDC), which published a report about this outbreak in Morbidity and Mortality Weekly Report (MMWR), in 2014. This report, along with the results of an independent inspection of Hospital X’s infection control practices following this CRE outbreak, were also reviewed. While this article focuses primarily on the prevention of transmissions of CRE and their related superbugs in the GI endoscopic setting, some of its discussion and recommendations may also apply to other healthcare settings, to other types of flexible endoscopes, and to other types of transmissible infectious agents. This review found that GI endoscopy is an important risk factor for the transmission of CRE and their related superbugs, having been recently associated with patient morbidity and mortality following ERCP. The CDC reported in MMWR that the type of GI endoscope, known as an ERCP endoscope, that Hospital X used to perform ERCP in 2013 on the 38 patients who became infected or colonized with CRE might be particularly challenging to clean and disinfect, because of the complexity of its physical design. If performed in strict accordance with the endoscope manufacturer’s labeling, supplemented as needed with professional organizations’ published guidelines, however, current practices for reprocessing GI endoscopes, which include high-level disinfection, are reportedly adequate for the prevention of transmission of CRE and their related superbugs. Several recommendations are provided to prevent CRE transmissions in the healthcare setting. CRE transmissions are not limited to contaminated GI endoscopes and also have been linked to other reusable flexible endoscopic instrumentation, including bronchoscopes and cystoscopes. In conclusion, contaminated GI endoscopes, particularly those used during ERCP, have been causally linked to outbreaks of CRE and their related superbugs, with associated patient morbidity and mortality. Thorough reprocessing of these complex reusable instruments is necessary to prevent disease transmission and ensure patient safety during GI endoscopy. Enhanced training and monitoring of reprocessing staffers to verify the proper cleaning and brushing of GI endoscopes, especially the area around, behind and near the forceps elevator located at the distal end of the ERCP endoscope, are recommended. If the ERCP endoscope features a narrow and exposed channel that houses a wire connecting the GI endoscope’s control head to this forceps elevator, then this channel’s complete reprocessing, including its flushing with a detergent using a procedure validated for effectiveness, is also emphasized. PMID:25324917

Transcriptional regulation of human MUC4 gene: identification of a novel inhibitory element and its nuclear binding protein.

PubMed

Zhang, Jing-Jing; Zhu, Yi; Zhang, Xiong-Fei; Liang, Wen-Biao; Xie, Kun-Ling; Tao, Jin-Qiu; Peng, Yun-Peng; Xu, Ze-Kuan; Miao, Yi

2013-08-01

The human mucin 4 (MUC4) is aberrantly expressed in pancreatic adenocarcinoma and tumor cell lines, while remaining undetectable in normal pancreas, indicating its important role in pancreatic cancer development. Although its transcriptional regulation has been investigated in considerable detail, some important elements remain unknown. The aim of the present study was to demonstrate the existence of a novel inhibitory element in the MUC4 promoter and characterize some of its binding proteins. By luciferase reporter assay, we located the inhibitory element between nucleotides -2530 and -2521 in the MUC4 promoter using a series of deletion and mutant reporter constructs. Electrophoretic mobility shift assay (EMSA) with Bxpc-3 cell nuclear extracts revealed that one protein or protein complex bind to this element. The proteins binding to this element were purified and identified as Yin Yang 1 (YY1) by mass spectrometry. Supershift assay and chromatin immunoprecipitation (ChIP) assay confirmed that YY1 binds to this element in vitro and in vivo. Moreover, transient YY1 overexpression significantly inhibited MUC4 promoter activity and endogenous MUC4 protein expression. In conclusion, we reported here a novel inhibitory element in the human MUC4 promoter. This provides additional data on MUC4 gene regulation and indicates that YY1 may be a potential target for abnormal MUC4 expression.

Stress-Induced Anxiety- and Depressive-Like Phenotype Associated with Transient Reduction in Neurogenesis in Adult Nestin-CreERT2/Diphtheria Toxin Fragment A Transgenic Mice

PubMed Central

Yun, Sanghee; Donovan, Michael H.; Ross, Michele N.; Richardson, Devon R.; Reister, Robin; Farnbauch, Laure A.; Fischer, Stephanie J.; Riethmacher, Dieter; Gershenfeld, Howard K.; Lagace, Diane C.; Eisch, Amelia J.

2016-01-01

Depression and anxiety involve hippocampal dysfunction, but the specific relationship between these mood disorders and adult hippocampal dentate gyrus neurogenesis remains unclear. In both humans with MDD and rodent models of depression, administration of antidepressants increases DG progenitor and granule cell number, yet rodents with induced ablation of DG neurogenesis typically do not demonstrate depressive- or anxiety-like behaviors. The conflicting data may be explained by the varied duration and degree to which adult neurogenesis is reduced in different rodent neurogenesis ablation models. In order to test this hypothesis we examined how a transient–rather than permanent–inducible reduction in neurogenesis would alter depressive- and anxiety-like behaviors. Transgenic Nestin-CreERT2/floxed diphtheria toxin fragment A (DTA) mice (Cre+DTA+) and littermates (Cre+DTA-; control) were given tamoxifen (TAM) to induce recombination and decrease nestin-expressing stem cells and their progeny. The decreased neurogenesis was transient: 12 days post-TAM Cre+DTA+ mice had fewer DG proliferating Ki67+ cells and fewer DCX+ neuroblasts/immature neurons relative to control, but 30 days post-TAM Cre+DTA+ mice had the same DCX+ cell number as control. This ability of DG neurogenesis to recover after partial ablation also correlated with changes in behavior. Relative to control, Cre+DTA+ mice tested between 12–30 days post-TAM displayed indices of a stress-induced anxiety phenotype–longer latency to consume highly palatable food in the unfamiliar cage in the novelty-induced hypophagia test, and a depression phenotype–longer time of immobility in the tail suspension test, but Cre+DTA+ mice tested after 30 days post-TAM did not. These findings suggest a functional association between adult neurogenesis and stress induced anxiety- and depressive-like behaviors, where induced reduction in DCX+ cells at the time of behavioral testing is coupled with stress-induced anxiety and a depressive phenotype, and recovery of DCX+ cell number corresponds to normalization of these behaviors. PMID:26795203

The Influence of Hyperlipidemia on Endothelial Function of FPN1 Tek-Cre Mice and the Intervention Effect of Tetramethylpyrazine.

PubMed

Sun, Ming-Yue; Zhang, Miao; Chen, Shui-Ling; Zhang, Shu-Ping; Guo, Chun-Yu; Wang, Jing-Shang; Liu, Xin; Miao, Yang; Yin, Hui-Jun

2018-01-01

Systemic iron homeostasis is strictly governed in mammals; however, disordered iron metabolism (such as excess iron burden) is recognized as a risk factor for various types of diseases including AS (Atherosclerosis). The hepcidin-ferroportin axis plays the key role in regulation of iron homeostasis and modulation of this signaling could be a potential therapeutic strategy in the treatment of these diseases. TMP (Tetramethylpyrazine) has been reported to have therapeutical effect on AS. Here, we aimed to investigate the effect of iron overload under hyperlipidemia condition on the endothelial injury, inflammation and oxidative stress by employing FPN1 Tek-cre mouse model with or without TMP intervention. Subjects for this study were 80 FPN1 Tek-cre mice and 40 C57BL/6 mice and we randomly divided them into six groups: Group N: C57BL/6 mice with normal diet, Group M: C57BL/6 mice with high-fat diet, Group FN: FPN1 Tek-cre mice with normal diet, Group FNT: FPN1 Tek-cre mice with normal diet and TMP injection, Group FM: FPN1 Tek-cre mice with high-fat diet, Group FMT: FPN1 Tek-cre mice with high-fat diet and TMP injection. After seven days of treatment, blood samples were obtained to detect the levels of blood lipids, Hepcidin, NO, ET-1, ROS, MDA, SOD, IL-1, IL-6 and TNF-α respectively. The liver and aorta were used for testing the lipid deposition by using hematoxylin and eosin(HE). Hyperlipidemia could cause iron overload in the aorta and increased serum hepcidin level, particularly in FPN1 Tek-cre mice, and can be reversed by TMP intervention. Knockout of Fpn1 induced increase of serum hepcidin, exacerbated endothelial dysfunction, oxidative stress and inflammatory response, particularly under hyperlipidemia condition. TMP intervention attenuated these processes. Our study signifies the potential application of certain natural compounds to ameliorating iron disorders induced by hyperlipidemia and protecting on endothelial function through modulation of hepcidin-ferroportin signaling. © 2018 The Author(s). Published by S. Karger AG, Basel.

Reliable Genetic Labeling of Adult-Born Dentate Granule Cells Using Ascl1 CreERT2 and Glast CreERT2 Murine Lines.

PubMed

Yang, Sung M; Alvarez, Diego D; Schinder, Alejandro F

2015-11-18

Newly generated dentate granule cells (GCs) are relevant for input discrimination in the adult hippocampus. Yet, their precise contribution to information processing remains unclear. To address this question, it is essential to develop approaches to precisely label entire cohorts of adult-born GCs. In this work, we used genetically modified mice to allow conditional expression of tdTomato (Tom) in adult-born GCs and characterized their development and functional integration. Ascl1(CreERT2);CAG(floxStopTom) and Glast(CreERT2);CAG(floxStopTom) mice resulted in indelible expression of Tom in adult neural stem cells and their lineage upon tamoxifen induction. Whole-cell recordings were performed to measure intrinsic excitability, firing behavior, and afferent excitatory connectivity. Developing GCs were also staged by the expression of early and late neuronal markers. The slow development of adult-born GCs characterized here is consistent with previous reports using retroviral approaches that have revealed that a mature phenotype is typically achieved after 6-8 weeks. Our findings demonstrate that Ascl1(CreERT2) and Glast(CreERT2) mouse lines enable simple and reliable labeling of adult-born GC lineages within restricted time windows. Therefore, these mice greatly facilitate tagging new neurons and manipulating their activity, required for understanding adult neurogenesis in the context of network remodeling, learning, and behavior. Our study shows that Ascl1(CreERT2) and Glast(CreERT2) mice lines can be used to label large cohorts of adult-born dentate granule cells with excellent time resolution. Neurons labeled in this manner display developmental and functional profiles that are in full agreement with previous findings using thymidine analogs and retroviral labeling, thus providing an alternative approach to tackle fundamental questions on circuit remodeling. Because of the massive neuronal targeting and the simplicity of this method, genetic labeling will contribute to expand research on adult neurogenesis. Copyright © 2015 the authors 0270-6474/15/3515379-12$15.00/0.

Cell-Specific Actions of a Human LHX3 Gene Enhancer During Pituitary and Spinal Cord Development

PubMed Central

Park, Soyoung; Mullen, Rachel D.

2013-01-01

The LIM class of homeodomain protein 3 (LHX3) transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie complex pediatric syndromes featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. Here we characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in α-glycoprotein subunit -expressing cells secreting the TSHβ, LHβ, or FSHβ hormones and expressing the GATA2 and steroidogenic factor 1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module active in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression. Furthermore, these studies revealed significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice also provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. PMID:24100213

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

PubMed

Zhang, Jun; Li, Jing; Craig, Theodore A; Kumar, Rajiv; Gross, Michael L

2017-07-18

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca 2+ -binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca 2+ but detaches from DRE under Ca 2+ stimulation, allowing gene expression. The Ca 2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca 2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca 2+ . Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca 2+ -binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca 2+ signal to CREB-mediated gene transcription.

HCN1 Channels Contribute to the Effects of Amnesia and Hypnosis But Not Immobility of Volatile Anesthetics

PubMed Central

Liu, Jin; Ke, Bowen; Wang, Xiaojia; Li, Fengshan; Li, Tao; Bayliss, Douglas A.; Chen, Xiangdong

2015-01-01

Background HCN1 channels have been identified as targets of ketamine to produce hypnosis. Volatile anesthetics also inhibit HCN1 channels. However, the effects of HCN1 channels on volatile anesthetics in vivo is still elusive. This study uses global and conditional HCN1 knockout mice to evaluate how HCN1 channels affect the actions of volatile anesthetics. Methods Minimum alveolar concentrations (MAC) of isoflurane and sevoflurane that induced immobility (MAC of immobility) and/or hypnosis (MAC of hypnosis) were determined in wild-type (WT) mice, global HCN1 channel knockout mice (HCN1−/−), floxed HCN1 channel gene (HCN1f/f) mice and forebrain-selective HCN1 channel knockout (HCN1f/f: cre) mice. Immobility of mice was defined as no purposeful reactions to tail-clamping stimulus and hypnosis was defined as loss of righting reflex (LORR). The amnestic effects of isoflurane and sevoflurane were evaluated by fear-potentiated startle in these four strains of mice. Results All MAC values were expressed as mean ± SEM. For MAC of immobility of isoflurane, no significant difference was found among wild-type, HCN1−/−, HCN1f/f and HCN1f/f: cre mice (all ~1.24-1.29% isoflurane). For both HCN1−/− and HCN1f/f: cre mice, the MAC of hypnosis for isoflurane (each ~1.05% isoflurane) were significantly increased over their nonknockout controls: HCN1−/− vs. wild-type (0.86±0.03%, P<0.001) and HCN1f/f: cre vs. HCN1f/f mice (0.84±0.03%, P<0.001); no significant difference was found between HCN1−/− and HCN1f/f: cre mice. For MAC of immobility of sevoflurane, no significant difference was found among wild-type, HCN1−/−, HCN1f/f and HCN1f/f: cre mice (all ~2.6-2.7% sevoflurane). For both HCN1−/− and HCN1f/f: cre mice, the MAC of hypnosis for sevoflurane (each ~1.90% sevoflurane) was significantly increased over their nonknockout controls: HCN1−/− vs. wild-type (1.58±0.05%, P<0.001) and HCN1f/f: cre vs. HCN1f/f mice (1.56±0.05%, P<0.001). No significant difference was found between HCN1−/− and HCN1f/f: cre mice. By fear-potentiated startle experiments, amnestic effects of isoflurane and sevoflurane were significantly attenuated in HCN1−/− and HCN1f/f: cre mice (both P<0.002 vs. wild-type or HCN1f/f mice). No significant difference was found between HCN1−/− and HCN1f/f: cre mice. Conclusions Forebrain HCN1 channels contribute to hypnotic and amnestic effects of volatile anesthetics, but HCN1 channels are not involved in the immobilizing actions of volatile anesthetics. PMID:26287296

Site-specific DNA excision in transgenic rice with a cell-permeable cre recombinase.

PubMed

Cao, Ming-Xia; Huang, Jian-Qiu; Yao, Quan-Hong; Liu, Sheng-Jun; Wang, Cheng-Long; Wei, Zhi-Ming

2006-01-01

The removal of selected marker genes from transgenic plants is necessary to address biosafety concerns and to carry out further experiments with transgenic organisms. In the present study, the 12-amino-acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF)-4 was used as a carrier to deliver enzymatically active Cre proteins into living plant cells, and to produce a site-specific DNA excision in transgenic rice plants. The process, which made cells permeable to Cre recombinase-mediated DNA recombination, circumvented the need to express Cre under spatiotemporal control and was proved to be a simple and efficient system to achieve marker-free transgenic plants. The ultimate aim of the present study is to develop commercial rice cultivars free from selected marker genes to hasten public acceptance of transgenic crops.

Calcium-creatinine ratio in a morning urine sample for the estimation of hypercalciuria associated with non-glomerular hematuria observed in children and adolescents.

PubMed

Quiñones-Vázquez, Susana; Liriano-Ricabal, María Del Rosario; Santana-Porbén, Sergio; Salabarría-González, José Reinaldo

2018-01-01

Hypercalciuria might be revealed during the differential diagnosis of hematuria accompanying renal lithiasis (RL). In spite of this, diagnostic accuracy of calcium urinary excretion might be affected by incomplete 24-hour urine collections. In the present study, the diagnostic utility of calcium/creatinine (ICaCre) index for determining hypercalciuria associated with non-glomerular hematuria (NGH) and RL was assessed. ICaCre (mg/mg) index was calculated from calcium (mmol/l) and creatinine (µmol/l) concentrations in an aliquot from a 24-hour urine collection in 169 children and adolescents with NGH or RL. Calciuria values > 4.0 mg/kg in 24 hours were distributed according to the presence of NGH or RL. Mean ICaCre index was 0.2 ± 0.1 mg/mg. Calciuria values estimated from ICaCre were statistically higher to those from 24-hour urine collection (p < 0.05). The frequency of hypercalciuria was independent from the measurement method (estimated from ICaCre 39.5% vs. 24 h collection 32.1%; p > 0.05). Hypercalciuria distribution was as follows: no NGH + no RL: 59.0%; no NGH + RL: 60.0% (∆ = +1.0%); NGH + no RL: 68.2% (∆ = +9.2%); NGH + RL: 73.3% (∆ = +14.4%). The use of ICaCre index for determining calcium urine excretion might be effective in the study of hypercalciuria associated with NGH and RL. Copyright: © 2018 Permanyer.

Selective deletion of leptin receptors in adult hippocampus induces depression-related behaviors

PubMed Central

Guo, Ming; Huang, Tung-Yi; Garza, Jacob C.; Chua, Streamson C.; Lu, Xin-Yun

2013-01-01

Previous studies have demonstrated that leptin and its receptors (LepRb) in the central nervous system play an important role in regulating depression- and anxiety-related behaviors. However, the physiological functions of LepRb in specific brain regions for mediating different emotional behaviors remain to be defined. In this study, we examined the behavioral effects of LepRb ablation in the adult hippocampus using a series of behavioral paradigms for assessing depression- and anxiety-related behaviors. Targeted deletion of LepRb was achieved using the Cre/loxP site-specific recombination system through bilateral stereotaxic delivery of an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the dentate gyrus of adult mice homozygous for a floxed leptin receptor allele. AAV-Cre-mediated deletion of the floxed region of LepRb was detected 2 weeks after injection. In accordance with this, leptin-stimulated phophorylation of Akt was attenuated in the hippocampus of AAV-Cre injected mice. Mice injected with AAV-Cre displayed normal locomotor activity and anxiety-like behavior, as determined in the elevated plus maze, light dark box and open field tests, but showed increased depression-like behaviors in the tail suspension, sucrose preference and learned helplessness tests. Taken together, this data suggests that deletion of LepRb in the adult hippocampus is sufficient to induce depression-like behaviors. Our results support the view that leptin signaling in the hippocampus may be essential for maintaining positive mood states and active coping to stress. PMID:22932068

Soluble Adenylyl Cyclase Is Required for Retinal Ganglion Cell and Photoreceptor Differentiation

PubMed Central

Shaw, Peter X.; Fang, Jiahua; Sang, Alan; Wang, Yan; Kapiloff, Michael S.; Goldberg, Jeffrey L.

2016-01-01

Purpose We have previously demonstrated that soluble adenylyl cyclase (sAC) is necessary for retinal ganglion cell (RGC) survival and axon growth. Here, we further investigate the role of sAC in neuronal differentiation during retinal development. Methods Chx10 or Math5 promoter-driven Cre-Lox recombination were used to conditionally delete sAC from early and intermediate retinal progenitor cells during retinal development. We examined cell type–specific markers expressed by retinal cells to estimate their relative numbers and characterize retinal laminar morphology by immunofluorescence in adult and newborn mice. Results Retinal ganglion cell and amacrine cell markers were significantly lower in the retinas of adult Math5cre/sACfl/fl and Chx10cre/sACfl/fl mice than in those of wild-type controls. The effect on RGC development was detectable as early as postnatal day 1 and deleting sAC in either Math5- or Chx10-expressing retinal progenitor cells also reduced nerve fiber layer thickness into adulthood. The thickness of the photoreceptor layer was slightly but statistically significantly decreased in both the newborn Chx10cre/sACfl/fl and Math5cre/sACfl/fl mice, but this reduction and abnormal morphology persisted in the adults in only the Chx10cre/sACfl/fl mice. Conclusions sAC plays an important role in the early retinal development of RGCs as well as in the development of amacrine cells and to a lesser degree photoreceptors. PMID:27679853

Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

PubMed

Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

2017-05-01

Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p < 0.0001). Sterol regulatory element binding protein-1 gene expression was positively correlated with body mass index (r = 0.017, p = 0.921) and waist-hip ratio (r = 0.023, p = 0.544) in polycystic ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression was significantly positively correlated with triglyceride in both polycystic ovary syndrome and endometrial cancer (p = 0.028 and p = 0.027, respectively). Quantitative serum sterol regulatory element binding protein-1 gene correlated with endometrial gene expression (p < 0.05). Sterol regulatory element binding protein-1 gene expression is significantly increased in the endometrium of women with polycystic ovary syndrome and women with endometrial cancer compared with controls and positively correlates with serum triglyceride in both polycystic ovary syndrome and endometrial cancer. © 2017 Nordic Federation of Societies of Obstetrics and Gynecology.

Cosmic-ray Exposure Ages of Meteorites

NASA Astrophysics Data System (ADS)

Herzog, G. F.

2003-12-01

The classic idea of a cosmic-ray exposure (CRE) age for a meteorite is based on a simple but useful picture of meteorite evolution, the one-stage irradiation model. The precursor rock starts out on a parent body, buried under a mantle of material many meters thick that screens out cosmic rays. At a time ti, a collision excavates a precursor rock - a "meteoroid." The newly liberated meteoroid, now fully exposed to cosmic rays, orbits the Sun until a time tf, when it strikes the Earth, where the overlying blanket of air (and possibly of water or ice) again shuts out almost all cosmic rays (cf. Masarik and Reedy, 1995). The quantity tf-ti is called the CRE age, t. To obtain the CRE age of a meteorite, we measure the concentrations in it of one or more cosmogenic nuclides (Table 1), which are nuclides that cosmic rays produce by inducing nuclear reactions. Many shorter-lived radionuclides excluded from Table 1 such as 22Na (t1/2=2.6 yr) and 60Co (t1/2=5.27 yr) can also furnish valuable information, but can be measured only in meteorites that fell within the last few half-lives of those nuclides (see, e.g., Leya et al. (2001) and references therein). Table 1. Cosmogenic nuclides used for calculating exposure ages NuclideHalf-lifea (Myr) Radionuclides 14C0.005730 59Ni0.076 41Ca0.1034 81Kr0.229 36Cl0.301 26Al0.717 10Be1.51 53Mn3.74 129I15.7 Stable nuclides 3He 21Ne 38Ar 83Kr 126Xe a http://www2.bnl.gov/ton. CRE ages have implications for several interrelated questions. From how many different parent bodies do meteorites come? How well do meteorites represent the population of the asteroid belt? How many distinct collisions on each parent body have created the known meteorites of each type? How often do asteroids collide? How big and how energetic were the collisions that produced meteoroids? What factors control the CRE age of a meteorite and how do meteoroid orbits evolve through time? We will touch on these questions below as we examine the data.By 1975, the CRE ages of hundreds of meteorites had been estimated from noble gas measurements. Histograms of the CRE age distributions pointed to several important observations.(i) The CRE ages of meteorites increase in the order stones

Transcriptional switches in the control of macronutrient metabolism.

PubMed

Wise, Alan

2008-06-01

This review shows how some transcription factors respond to alterations in macronutrients. Carbohydrates induce enzymes for their metabolism and fatty acid synthesis. Fatty acids reduce carbohydrate processing, induce enzymes for their metabolism, and increase both gluconeogenesis and storage of fat. Fat stores help control carbohydrate uptake by other cells. The following main transcription factors are discussed: carbohydrate response element-binding protein; sterol regulatory element-binding protein-1c, cyclic AMP response element-binding protein, peroxisome proliferator-activated receptor-alpha, and peroxisome proliferator-activated receptor-gamma.

Cre-driver lines used for genetic fate mapping of neural crest cells in the mouse: An overview.

PubMed

Debbache, Julien; Parfejevs, Vadims; Sommer, Lukas

2018-04-19

The neural crest is one of the embryonic structures with the broadest developmental potential in vertebrates. Morphologically, neural crest cells emerge during neurulation in the dorsal folds of the neural tube before undergoing an epithelial-to-mesenchymal transition (EMT), delaminating from the neural tube, and migrating to multiple sites in the growing embryo. Neural crest cells generate cell types as diverse as peripheral neurons and glia, melanocytes, and so-called mesectodermal derivatives that include craniofacial bone and cartilage and smooth muscle cells in cardiovascular structures. In mice, the fate of neural crest cells has been determined mainly by means of transgenesis and genome editing technologies. The most frequently used method relies on the Cre-loxP system, in which expression of Cre-recombinase in neural crest cells or their derivatives genetically enables the expression of a Cre-reporter allele, thus permanently marking neural crest-derived cells. Here, we provide an overview of the Cre-driver lines used in the field and discuss to what extent these lines allow precise neural crest stage and lineage-specific fate mapping. © 2018 The Authors Genesis: The Journal of Genetics and Development Published by Wiley Periodicals, Inc.

Manipulating the Coffee-Ring Effect: Interactions at Work.

PubMed

Anyfantakis, Manos; Baigl, Damien

2015-07-31

The evaporation of a drop of colloidal suspension pinned on a substrate usually results in a ring of particles accumulated at the periphery of the initial drop. Intense research has been devoted to understanding, suppressing and ultimately controlling this so-called coffee-ring effect (CRE). Although the crucial role of flow patterns in the CRE has been thoroughly investigated, the effect of interactions on this phenomenon has been largely neglected. This Concept paper reviews recent works in this field and shows that the interactions of colloids with (and at) liquid-solid and liquid-gas interfaces as well as bulk particle-particle interactions drastically affect the morphology of the deposit. General rules are established to control the CRE by tuning these interactions, and guidelines for the rational physicochemical formulation of colloidal suspensions capable of depositing particles in desirable patterns are provided. This opens perspectives for the reliable control of the CRE in real-world formulations and creates new paradigms for flexible particle patterning at all kinds of interfaces as well for the exploitation of the CRE as a robust and inexpensive diagnostic tool. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Promiscuous Foxp3-cre activity reveals a differential requirement for CD28 in Foxp3⁺ and Foxp3⁻ T cells.

PubMed

Franckaert, Dean; Dooley, James; Roos, Evelyne; Floess, Stefan; Huehn, Jochen; Luche, Herve; Fehling, Hans Joerg; Liston, Adrian; Linterman, Michelle A; Schlenner, Susan M

2015-04-01

Costimulatory signals by CD28 are critical for thymic regulatory T-cell (Treg) development. To determine the functional relevance of CD28 for peripheral Treg post thymic selection, we crossed the widely used Forkhead box protein 3 (Foxp3)-CreYFP mice to mice bearing a conditional Cd28 allele. Treg-specific CD28 deficiency provoked a severe autoimmune syndrome as a result of a strong disadvantage in competitive fitness and proliferation of CD28-deficient Tregs. By contrast, Treg survival and lineage integrity were not affected by the lack of CD28. This data demonstrate that, even after the initial induction requirement, Treg maintain a higher dependency on CD28 signalling than conventional T cells for homeostasis. In addition, we found the Foxp3-CreYFP allele to be a hypomorph, with reduced Foxp3 protein levels. Furthermore, we report here the stochastic activity of the Foxp3-CreYFP allele in non-Tregs, sufficient to recombine some conditional alleles (including Cd28) but not others (including R26-RFP). This hypomorphism and 'leaky' expression of the Foxp3-CreYFP allele should be considered when analysing the conditionally mutated Treg.

The FEM simulation of continuous rotary extrusion (CRE) of aluminum alloy AA3003

NASA Astrophysics Data System (ADS)

Rajendran, Nijenthan; Valberg, Henry; Misiolek, Wojciech Z.

2017-10-01

Continuous Rotary Extrusion (CRE) process is also known in literature under Conform TM name and it is mainly used for the continuous extrusion of Aluminum and Copper alloys. CRE use a feedstock in the form of rod, powders and chips, which are fed into the groove of the rotating wheel. As the wheel rotates the feedstock moves along with it due to friction with the wheel. Once the feedstock reaches the abutment the material deforms plastically and it is extruded through the die. CRE has lot to offer when compared to other more conventional extrusion processes such as low energy input, no limit in billet length as it is a continuous process as well as improved material physical properties due to plastic deformation under constant parameters. In this work a FEM model has been developed using Deform TM 3D, to study the metal flow and state variables of AA3003 CRE extrusion. The effect of extrusion wheel velocity has been investigated. The results show that increase in wheel velocity will heat up the feedstock metal due to high shear deformation and higher friction, which significantly changes metal flow conditions at the die exit.

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

PubMed Central

Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

1992-01-01

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

Nonlocal symmetry and explicit solutions from the CRE method of the Boussinesq equation

NASA Astrophysics Data System (ADS)

Zhao, Zhonglong; Han, Bo

2018-04-01

In this paper, we analyze the integrability of the Boussinesq equation by using the truncated Painlevé expansion and the CRE method. Based on the truncated Painlevé expansion, the nonlocal symmetry and Bäcklund transformation of this equation are obtained. A prolonged system is introduced to localize the nonlocal symmetry to the local Lie point symmetry. It is proved that the Boussinesq equation is CRE solvable. The two-solitary-wave fusion solutions, single soliton solutions and soliton-cnoidal wave solutions are presented by means of the Bäcklund transformations.

Expression of ESR1 in Glutamatergic and GABAergic Neurons Is Essential for Normal Puberty Onset, Estrogen Feedback, and Fertility in Female Mice.

PubMed

Cheong, Rachel Y; Czieselsky, Katja; Porteous, Robert; Herbison, Allan E

2015-10-28

Circulating estradiol exerts a profound influence on the activity of the gonadotropin-releasing hormone (GnRH) neuronal network controlling fertility. Using genetic strategies enabling neuron-specific deletion of estrogen receptor α (Esr1), we examine here whether estradiol-modulated GABA and glutamate transmission are critical for the functioning of the GnRH neuron network in the female mouse. Using Vgat- and Vglut2-ires-Cre knock-in mice and ESR1 immunohistochemistry, we demonstrate that subpopulations of GABA and glutamate neurons throughout the limbic forebrain express ESR1, with ESR1-GABAergic neurons being more widespread and numerous than ESR1-glutamatergic neurons. We crossed Vgat- and Vglut2-ires-Cre mice with an Esr1(lox/lox) line to generate animals with GABA-neuron-specific or glutamate-neuron-specific deletion of Esr1. Vgat-ires-Cre;Esr1(lox/lox) mice were infertile, with abnormal estrous cycles, and exhibited a complete failure of the estrogen positive feedback mechanism responsible for the preovulatory GnRH surge. However, puberty onset and estrogen negative feedback were normal. Vglut2-ires-Cre;Esr1(lox/lox) mice were also infertile but displayed a wider range of deficits, including advanced puberty onset, abnormal negative feedback, and abolished positive feedback. Whereas <25% of preoptic kisspeptin neurons expressed Cre in Vgat- and Vglut2-ires-Cre lines, ∼70% of arcuate kisspeptin neurons were targeted in Vglut2-ires-Cre;Esr1(lox/lox) mice, possibly contributing to their advanced puberty phenotype. These observations show that, unexpectedly, ESR1-GABA neurons are only essential for the positive feedback mechanism. In contrast, we reveal the key importance of ESR1 in glutamatergic neurons for multiple estrogen feedback loops within the GnRH neuronal network required for fertility in the female mouse. Copyright © 2015 the authors 0270-6474/15/3514533-11$15.00/0.

All-cause mortality increased by environmental cadmium exposure in the Japanese general population in cadmium non-polluted areas.

PubMed

Suwazono, Yasushi; Nogawa, Kazuhiro; Morikawa, Yuko; Nishijo, Muneko; Kobayashi, Etsuko; Kido, Teruhiko; Nakagawa, Hideaki; Nogawa, Koji

2015-07-01

The aim of the present study was to evaluate the effect of environmental cadmium (Cd) exposure indicated by urinary Cd on all-cause mortality in the Japanese general population. A 19-year cohort study was conducted in 1067 men and 1590 women aged 50 years or older who lived in three cadmium non-polluted areas in Japan. The subjects were divided into four quartiles based on creatinine adjusted U-Cd (µg g(-1) cre). The hazard ratio (HR) and 95% confidence interval (CI) for continuous U-Cd or the quartiles of U-Cd were estimated for all-cause mortality using a proportional hazards regression.The all-cause mortality rates per 1000 person years were 31.2 and 15.1 in men and women, respectively. Continuous U-Cd (+1 µg g(-1) cre) was significantly related to the all-cause mortality in men (HR 1.05, 95% CI: 1.02-1.09) and women (HR 1.04, 95% CI: 1.01-1.07). Furthermore in men, the third (1.96-3.22 µg g(-1) cre) and fourth quartile (≥3.23 µg g(-1) cre) of U-Cd showed a significant, positive HR (third: HR 1.35, 95% CI: 1.03-1.77, fourth: HR 1.64, 95% CI: 1.26-2.14) for all-cause mortality compared with the first quartile (<1.14 µg g(-1) cre). In women, the fourth quartile of U-Cd (≥4.66 µg g(-1) cre) also showed a significant HR (1.49, 95% CI 1.11-2.00) for all-cause mortality compared with the first quartile (<1.46 µg g(-1) cre).In the present study, U-Cd was significantly associated with increased mortality in the Japanese general population, indicating that environmental Cd exposure adversely affects the life prognosis in Cd non-polluted areas in Japan. Copyright © 2014 John Wiley & Sons, Ltd.

High-dose HOOK effect in urinary DcR2 assay in patients with chronic kidney disease.

PubMed

Chen, Jia; Chen, Ke-Hong; Wang, Li-Ming; Zhang, Wei-Wei; Feng, Lei; Dai, Huan-Zi; He, Ya-Ni

2018-06-05

Urinary DcR2 (uDcR2) is a biomarker for the early detection the tubulointerstitial injury (TII) in patients with chronic kidney disease (CKD), but the high-dose hook effect may lead to falsely low or even negative results when using an enzyme-linked immunosorbent assay (ELISA). This study aimed to investigate if the high-dose hook effect exists with ELISA testing, and to uncover a potential approach for reducing this effect. 72 CKD patients were recruited and categorized into four groups based on TII scores. uDcR2 was measured in undiluted and serially diluted (two-, four-, eight- and 16-fold dilutions) urine using an ELISA kit. The results from the assay were normalized to urinary creatinine. We evaluated the correlation between uDcR2/cre levels at different dilutions and renal histological parameters. Receiver operating characteristic (ROC) curves were generated to examine the value of uDcR2/cre for predicting TII. uDcR2/cre levels in the undiluted urine were significantly higher in patients with CKD than those in the control. However, higher TII scores did not yield higher levels of uDcR2/cre in the undiluted urine. After serial dilution, uDcR2/cre levels were highest with the four-fold dilution. A positive correlation was found between uDcR2/cre levels at different dilutions and TII scores, with the highest correlation coefficient and the largest AUC being observed at the four-fold dilution. The high-dose hook effect was apparent during ELISA testing of uDcR2 in CKD patients, yet dilution of the urine samples neutralized this effect. However, the use of a four-fold dilution of urine for uDcR2/cre testing may eliminate the high-dose hook effect and make it possible to effectively monitor the severity of TII in CKD patients. Copyright © 2018. Published by Elsevier Inc.

Type 1 Deiodinase Regulates ApoA-I Gene Expression and ApoA-I Synthesis Independent of Thyroid Hormone Signaling.

PubMed

Liu, Jing; Hernandez-Ono, Antonio; Graham, Mark J; Galton, Valerie Anne; Ginsberg, Henry N

2016-07-01

Plasma levels of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (ApoA-I) are reduced in individuals with defective insulin signaling. Initial studies using liver-specific insulin receptor (InsR) knockout mice identified reduced expression of type 1 deiodinase (Dio1) as a potentially novel link between defective hepatic insulin signaling and reduced expression of the ApoA-I gene. Our objective was to examine the regulation of ApoA-I expression by Dio1. Acute inactivation of InsR by adenoviral delivery of Cre recombinase to InsR floxed mice reduced HDL-C and expression of both ApoA-I and Dio1. Overexpression of Dio1 in InsR knockout mice restored HDL-C and ApoA-I levels and increased the expression of ApoA-I. Dio1 knockout mice had low expression of ApoA-I and reduced serum levels of HDL-C and ApoA-I. Treatment of C57BL/6J mice with antisense to Dio1 reduced ApoA-I mRNA, HDL-C, and serum ApoA-I. Hepatic 3,5,3'-triiodothyronine content was normal or elevated in InsR knockout mice or Dio1 knockout mice. Knockdown of either InsR or Dio1 by siRNA in HepG2 cells decreased the expression of ApoA-I and ApoA-I synthesis and secretion. siRNA knockdown of InsR or Dio1 decreased activity of a region of the ApoA-I promoter lacking thyroid hormone response elements (region B). Electrophoretic mobility shift assay demonstrated that reduced Dio1 expression decreased the binding of nuclear proteins to region B. Reductions in Dio1 expression reduce the expression of ApoA-I in a 3,5,3'-triiodothyronine-/thyroid hormone response element-independent manner. © 2016 American Heart Association, Inc.

Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway in depressive disorder☆

PubMed Central

Wang, Hongyan; Zhang, Yingquan; Qiao, Mingqi

2013-01-01

The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression. PMID:25206732

Immunologic applications of conditional gene modification technology in the mouse.

PubMed

Sharma, Suveena; Zhu, Jinfang

2014-04-02

Since the success of homologous recombination in altering mouse genome and the discovery of Cre-loxP system, the combination of these two breakthroughs has created important applications for studying the immune system in the mouse. Here, we briefly summarize the general principles of this technology and its applications in studying immune cell development and responses; such implications include conditional gene knockout and inducible and/or tissue-specific gene over-expression, as well as lineage fate mapping. We then discuss the pros and cons of a few commonly used Cre-expressing mouse lines for studying lymphocyte development and functions. We also raise several general issues, such as efficiency of gene deletion, leaky activity of Cre, and Cre toxicity, all of which may have profound impacts on data interpretation. Finally, we selectively list some useful links to the Web sites as valuable mouse resources. Copyright © 2014 John Wiley & Sons, Inc.

Constraints on dark matter models from a Fermi LAT search for high-energy cosmic-ray electrons from the Sun

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ajello, M.; Atwood, W. B.; Baldini, L.

During its first year of data taking, the Large Area Telescope (LAT) onboard the Fermi Gamma-Ray Space Telescope has collected a large sample of high-energy cosmic-ray electrons and positrons (CREs). We present the results of a directional analysis of the CRE events, in which we searched for a flux excess correlated with the direction of the Sun. Two different and complementary analysis approaches were implemented, and neither yielded evidence of a significant CRE flux excess from the Sun. Here, we derive upper limits on the CRE flux from the Sun’s direction, and use these bounds to constrain two classes ofmore » dark matter models which predict a solar CRE flux: (1) models in which dark matter annihilates to CREs via a light intermediate state, and (2) inelastic dark matter models in which dark matter annihilates to CREs.« less

Constraints on dark matter models from a Fermi LAT search for high-energy cosmic-ray electrons from the Sun

DOE PAGES

Ajello, M.; Atwood, W. B.; Baldini, L.; ...

2011-08-15

During its first year of data taking, the Large Area Telescope (LAT) onboard the Fermi Gamma-Ray Space Telescope has collected a large sample of high-energy cosmic-ray electrons and positrons (CREs). We present the results of a directional analysis of the CRE events, in which we searched for a flux excess correlated with the direction of the Sun. Two different and complementary analysis approaches were implemented, and neither yielded evidence of a significant CRE flux excess from the Sun. Here, we derive upper limits on the CRE flux from the Sun’s direction, and use these bounds to constrain two classes ofmore » dark matter models which predict a solar CRE flux: (1) models in which dark matter annihilates to CREs via a light intermediate state, and (2) inelastic dark matter models in which dark matter annihilates to CREs.« less

Multivalent DNA-binding properties of the HMG-1 proteins.

PubMed Central

Maher, J F; Nathans, D

1996-01-01

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692884

The disulfide isomerase ERp57 is required for fibrin deposition in vivo.

PubMed

Zhou, J; Wu, Y; Wang, L; Rauova, L; Hayes, V M; Poncz, M; Essex, D W

2014-11-01

ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57(fl/fl) mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57(fl/fl) mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57(fl/fl) mice, Tie2-Cre/ERp57(fl/fl) mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. ERp57 regulates thrombosis via multiple targets. © 2014 International Society on Thrombosis and Haemostasis.

Rates of gastrointestinal tract colonization of carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa in hospitals in Saudi Arabia

PubMed Central

Abdalhamid, B.; Elhadi, N.; Alabdulqader, N.; Alsamman, K.; Aljindan, R.

2016-01-01

Carbapenem-resistant Enterobacteriaceae (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPAE) are globally a major medical issue, especially in intensive care units. The digestive tract is the main reservoir for these isolates; therefore, rectal swab surveillance is highly recommended. The purpose of this study was to detect the prevalence of gastrointestinal tract colonization of CRE and CRPAE in patients admitted to intensive care units in Saudi Arabia. This project also aimed to characterize carbapenem-hydrolyzing enzyme production in these isolates. From February to May 2015, 200 rectal swab specimens were screened by CHROMagar KPC. Organism identification and susceptibility testing were performed using the Vitek 2 system. One CRE and 13 CRPAE strains were identified, for a prevalence of 0.5% (1/200) and 6.5% (13/200) respectively. Strains showed high genetic diversity using enterobacterial repetitive intergenic consensus sequence-based PCR. NDM type and VIM type were detected by PCR in four and one CRPAE isolates respectively. ampC overexpression was detected in eight CRPAE isolates using Mueller-Hinton agar containing 1000 μg/mL cloxacillin. CTX-M-15 type was detected in 1 CRE by PCR. The prevalence of CRE strain colonization was lower than that of CRPAE isolates. The detection of NDM and VIM in the colonizing CRPAE strains is a major infection control concern. To our knowledge, this is the first study in Saudi Arabia and the gulf region focusing on digestive tract colonization of CRE and CRPAE organisms and characterizing the mechanisms of carbapenem resistance. PMID:26933499

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

PubMed

Barata, Lidia; Miwa, Takashi; Sato, Sayaka; Kim, David; Mohammed, Imran; Song, Wen-Chao

2013-03-15

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

Identification of high-confidence RNA regulatory elements by combinatorial classification of RNA-protein binding sites.

PubMed

Li, Yang Eric; Xiao, Mu; Shi, Binbin; Yang, Yu-Cheng T; Wang, Dong; Wang, Fei; Marcia, Marco; Lu, Zhi John

2017-09-08

Crosslinking immunoprecipitation sequencing (CLIP-seq) technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein (RBP) with high resolution. We apply a soft-clustering method, RBPgroup, to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites. Such combinatorial clustering of RBPs helps interpret CLIP-seq data and suggests functional RNA regulatory elements. Furthermore, we validate two RBP-RBP interactions in cell lines. Our approach links proteins and RNA motifs known to possess similar biochemical and cellular properties and can, when used in conjunction with additional experimental data, identify high-confidence RBP groups and their associated RNA regulatory elements.

Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

PubMed Central

McNeill, Eileen; Crabtree, Mark J.; Sahgal, Natasha; Patel, Jyoti; Chuaiphichai, Surawee; Iqbal, Asif J.; Hale, Ashley B.; Greaves, David R.; Channon, Keith M.

2015-01-01

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1fl/flTie2cre). Macrophages from Gch1fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation. PMID:25451639

Viral nanoparticle-encapsidated enzyme and restructured DNA for cell delivery and gene expression

PubMed Central

Liu, Jinny L.; Dixit, Aparna Banerjee; Robertson, Kelly L.; Qiao, Eric; Black, Lindsay W.

2014-01-01

Packaging specific exogenous active proteins and DNAs together within a single viral-nanocontainer is challenging. The bacteriophage T4 capsid (100 × 70 nm) is well suited for this purpose, because it can hold a single long DNA or multiple short pieces of DNA up to 170 kb packed together with more than 1,000 protein molecules. Any linear DNA can be packaged in vitro into purified procapsids. The capsid-targeting sequence (CTS) directs virtually any protein into the procapsid. Procapsids are assembled with specific CTS-directed exogenous proteins that are encapsidated before the DNA. The capsid also can display on its surface high-affinity eukaryotic cell-binding peptides or proteins that are in fusion with small outer capsid and head outer capsid surface-decoration proteins that can be added in vivo or in vitro. In this study, we demonstrate that the site-specific recombinase cyclic recombination (Cre) targeted into the procapsid is enzymatically active within the procapsid and recircularizes linear plasmid DNA containing two terminal loxP recognition sites when packaged in vitro. mCherry expression driven by a cytomegalovirus promoter in the capsid containing Cre-circularized DNA is enhanced over linear DNA, as shown in recipient eukaryotic cells. The efficient and specific packaging into capsids and the unpackaging of both DNA and protein with release of the enzymatically altered protein–DNA complexes from the nanoparticles into cells have potential in numerous downstream drug and gene therapeutic applications. PMID:25161284

The Prrx1 homeodomain transcription factor plays a central role in pancreatic regeneration and carcinogenesis

PubMed Central

Reichert, Maximilian; Takano, Shigetsugu; von Burstin, Johannes; Kim, Sang-Bae; Lee, Ju-Seog; Ihida-Stansbury, Kaori; Hahn, Christopher; Heeg, Steffen; Schneider, Günter; Rhim, Andrew D.; Stanger, Ben Z.; Rustgi, Anil K.

2013-01-01

Pancreatic exocrine cell plasticity can be observed during development, pancreatitis with subsequent regeneration, and also transformation. For example, acinar–ductal metaplasia (ADM) occurs during acute pancreatitis and might be viewed as a prelude to pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC) development. To elucidate regulatory processes that overlap ductal development, ADM, and the progression of normal cells to PanIN lesions, we undertook a systematic approach to identify the Prrx1 paired homeodomain Prrx1 transcriptional factor as a highly regulated gene in these processes. Prrx1 annotates a subset of pancreatic ductal epithelial cells in Prrx1creERT2-IRES-GFP mice. Furthermore, sorted Prrx1+ cells have the capacity to self-renew and expand during chronic pancreatitis. The two isoforms, Prrx1a and Prrx1b, regulate migration and invasion, respectively, in pancreatic cancer cells. In addition, Prrx1b is enriched in circulating pancreatic cells (Pdx1cre;LSL-KrasG12D/+;p53fl/+;R26YFP). Intriguingly, the Prrx1b isoform, which is also induced in ADM, binds the Sox9 promoter and positively regulates Sox9 expression. This suggests a new hierarchical scheme whereby a Prrx1–Sox9 axis may influence the emergence of acinar–ductal metaplasia and regeneration. Furthermore, our data provide a possible explanation of why pancreatic cancer is skewed toward a ductal fate. PMID:23355395

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagata, Takayuki; Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575; Murata, Kazuko, E-mail: murata-k@iwakimu.ac.jp

Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examinedmore » the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs{sup flox/flox};mb1{sup cre/+}:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.« less

Neuronal expression of a thyroid hormone receptor α mutation alters mouse behaviour.

PubMed

Richard, S; Aguilera, N; Thévenet, M; Dkhissi-Benyahya, O; Flamant, F

2017-03-15

In humans, alterations in thyroid hormone signalling are associated with mood and anxiety disorders, but the neural mechanisms underlying such association are poorly understood. The present study investigates the involvement of neuronal thyroid hormone receptor α (TRα) in anxiety, using mouse genetics and Cre/loxP technology to specifically alter TRα signalling in neurons. We evaluated the behaviour of mice expressing a dominant negative, neuron-specific mutation of TRα (TRα AMI /Cre3 mice), using the elevated-plus maze, light-dark box and open-field tests. In a first experiment, mice were housed individually, and the behaviour of TRα AMI /Cre3 mice differed significantly from that of control littermates in these 3 tests, suggesting heightened anxiety. In a second experiment, designed to evaluate the robustness of the results with the same 3 tests, mice were housed in groups. In these conditions, the behaviour of TRα AMI /Cre3 mice differed from that of control littermates only in the light-dark box. Thus, TRα AMI /Cre3 mice appear to be more likely to develop anxiety under stressful housing conditions than control mice. These results suggest that in adult mice, thyroid hormone signalling in neurons, via TRα, is involved in the control of anxiety behaviour. Copyright © 2016 Elsevier B.V. All rights reserved.

Predictors for gut colonization of carbapenem-resistant Enterobacteriaceae in neonates in a neonatal intensive care unit.

PubMed

Singh, Narendra Pal; Choudhury, Debapriya Das; Gupta, Kavita; Rai, Sumit; Batra, Prerna; Manchanda, Vikas; Saha, Rituparna; Kaur, I R

2018-06-01

With the emergence of carbapenem-resistant isolates, the therapeutic alternatives have become limited. Various factors are responsible for carbapenem-resistant Enterobacteriaceae (CRE) gut colonization. This study was conducted to determine predictors for CRE gut colonization in neonates who were hospital delivered and admitted in a neonatal intensive care unit (NICU). Three rectal swabs were collected from 300 hospital-delivered and NICU-admitted neonates (likely to stay for >3 days). The data collected for the possible risk factors for CRE gut colonization were namely mode of delivery, prolonged rupture of membrane >18 hours, period of gestation, birth weight, meconium-stained liquor, ventilation, intravenous catheter, nasogastric (NG) tube, NG feeding, breastfeeding, katori spoon feeding, top feeding, expressed breastmilk, antibiotics administration, and duration of hospitalization. P < .05 was considered as statistically significant. A total of 26 cases of CRE were isolated from 300 neonates. Statistically significant risk factors were found to be NG tube, breastfeeding, NG feeding, top feeding, expressed breastmilk, ventilation, antibiotic administration, and duration of hospitalization. Top feeding and antibiotics administration were identified as 2 independent risk factors by multiple logistic regression. Active surveillance of cultures from hospitalized patients and implementation of preventive efforts can reduce the risk of CRE. Copyright © 2018. Published by Elsevier Inc.

Induced Pluripotent Stem Cells Generated from P0-Cre;Z/EG Transgenic Mice

PubMed Central

Ogawa, Yasuhiro; Eto, Akira; Miyake, Chisato; Tsuchida, Nana; Miyake, Haruka; Takaku, Yasuhiro; Hagiwara, Hiroaki; Oishi, Kazuhiko

2015-01-01

Neural crest (NC) cells are a migratory, multipotent cell population that arises at the neural plate border, and migrate from the dorsal neural tube to their target tissues, where they differentiate into various cell types. Abnormal development of NC cells can result in severe congenital birth defects. Because only a limited number of cells can be obtained from an embryo, mechanistic studies are difficult to perform with directly isolated NC cells. Protein zero (P0) is expressed by migrating NC cells during the early embryonic period. In the P0-Cre;Z/EG transgenic mouse, transient activation of the P0 promoter induces Cre-mediated recombination, indelibly tagging NC-derived cells with enhanced green fluorescent protein (EGFP). Induced pluripotent stem cell (iPSC) technology offers new opportunities for both mechanistic studies and development of stem cell-based therapies. Here, we report the generation of iPSCs from the P0-Cre;Z/EG mouse. P0-Cre;Z/EG mouse-derived iPSCs (P/G-iPSCs) exhibited pluripotent stem cell properties. In lineage-directed differentiation studies, P/G-iPSCs were efficiently differentiated along the neural lineage while expressing EGFP. These results suggest that P/G-iPSCs are useful to study NC development and NC-associated diseases. PMID:26382630

Smad 1/5 and Smad 4 Expression Are Important for Osteoclast Differentiation

PubMed Central

Tasca, Amy; Stemig, Melissa; Broege, Aaron; Huang, Brandon; Davydova, Julia; Zwijsen, An; Umans, Lieve; Jensen, Eric D.; Gopalakrishnan, Raj; Mansky, Kim C.

2015-01-01

To investigate the necessity of the canonical BMP pathway during osteoclast differentiation, we created osteoclasts with a conditional gene deletion for Smad1 and Smad5 (SMAD1/5), or Smad4 using adenovirus expressing CRE recombinase (Ad-CRE). Reduction of either Smad4 or Smad1/5 expression resulted in fewer and smaller multinuclear cells compared to control cells. We also detected changes in osteoclast enriched genes, demonstrated by decreased Dc-stamp and cathepsin K expression in both Smad4 and Smad1/5 Ad-CRE osteoclasts, and changes in c-fos and Nfatc1 expression in only Smad4 Ad-CRE cells. Lastly we also detected a significant decrease in resorption pits and area resorbed in both the Smad4 and Smad1/5 Ad-CRE osteoclasts. Because we inhibited osteoclast differentiation with loss of either Smad4 or Smad1/5 expression, we assessed whether BMPs affected osteoclast activity in addition to BMP’s effects on differentiation. Therefore, we treated mature osteoclasts with BMP2 or with dorsomorphin, a chemical inhibitor that selectively suppresses canonical BMP signaling. We demonstrated that BMP2 stimulated resorption in mature osteoclasts whereas treatment with dorsomorphin blocks osteoclast resorption. These results indicate that the BMP canonical signaling pathway is important for osteoclast differentiation and activity. PMID:25711193

Induced Pluripotent Stem Cells Generated from P0-Cre;Z/EG Transgenic Mice.

PubMed

Ogawa, Yasuhiro; Eto, Akira; Miyake, Chisato; Tsuchida, Nana; Miyake, Haruka; Takaku, Yasuhiro; Hagiwara, Hiroaki; Oishi, Kazuhiko

2015-01-01

Neural crest (NC) cells are a migratory, multipotent cell population that arises at the neural plate border, and migrate from the dorsal neural tube to their target tissues, where they differentiate into various cell types. Abnormal development of NC cells can result in severe congenital birth defects. Because only a limited number of cells can be obtained from an embryo, mechanistic studies are difficult to perform with directly isolated NC cells. Protein zero (P0) is expressed by migrating NC cells during the early embryonic period. In the P0-Cre;Z/EG transgenic mouse, transient activation of the P0 promoter induces Cre-mediated recombination, indelibly tagging NC-derived cells with enhanced green fluorescent protein (EGFP). Induced pluripotent stem cell (iPSC) technology offers new opportunities for both mechanistic studies and development of stem cell-based therapies. Here, we report the generation of iPSCs from the P0-Cre;Z/EG mouse. P0-Cre;Z/EG mouse-derived iPSCs (P/G-iPSCs) exhibited pluripotent stem cell properties. In lineage-directed differentiation studies, P/G-iPSCs were efficiently differentiated along the neural lineage while expressing EGFP. These results suggest that P/G-iPSCs are useful to study NC development and NC-associated diseases.

Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti.

PubMed

Häcker, Irina; Harrell Ii, Robert A; Eichner, Gerrit; Pilitt, Kristina L; O'Brochta, David A; Handler, Alfred M; Schetelig, Marc F

2017-03-07

Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

Simultaneous determination of creatinine, creatine, and UV-absorbing amino acids using dual-mode gradient low-capacity cation-exchange chromatography.

PubMed

Yokoyama, Yukio; Tsuji, Sachiyo; Sato, Hisakuni

2005-08-26

A simple and versatile cation-exchange chromatography technique for the simultaneous determination of urinary creatinine (Cre), creatine (Crn), methionine (Met), tyrosine (Tyr), phenylalanine (Phe), histidine (His), and tryptophan (Trp) was developed. A novel low-capacity cation-exchange column packed with a newly developed sulfoacylated hypercross-linked macroreticular polystyrene-divinylbenzene resin, referred to as TMR-A/75 (capacity: 75 microequiv/column), was successfully used with a binary dual-mode gradient eluting system. Two solvents, (A) 25 mM phosphoric acid-methanol (30:70, v/v) and (B) 25 mM disodium hydrogenphosphate-methanol (30:70, v/v) were pumped through the column by programming solvent delivery ratios as 0 to 5 min: A-B (55:45, pH 3.6); 5-21 min: A-B (49:51, pH 5.3); and 21-35 min: A-B (55:45, pH 3.6). The flow rate was simultaneously time-programmed to be 0.6 mL/min from 0 to 19 min and to be 1.0 mL/min from 19 to 35 min. This eluting system could permit the use of the UV detection at 210 nm. The analytes, Crn, Met, Tyr, His, Cre, Phe, and Trp, were well separated in this order in 27 min with minimum resolution of approximately 2, and the cycle time was about 35 min. Retention time of each analyte was very reproducible with relative standard deviations (RSDs) between 0.05 and 0.38% (n = 5). The peak area responses were also reproducible with RSDs between 0.74 and 2.24% (n = 5). Calibration lines based on area data were linear from 1 to 1000 microM with r2 values of 0.9998 (Crn), 0.9998 (Met), 0.9999 (Tyr), 0.9999 (His), 1.0000 (Cre), 1.0000 (Phe), and 0.9999 (Trp). The method was applicable to the screening and/or chemical diagnosis of inherited metabolic disorders such as phenylketonuria (PKU), tyrosinemia, and Lowe syndrome. The creatinine ratios of diagnostic markers (microM/microM Cre) were easily determined. The Phe/Cre ratios for five urines from patients with PKU ranged from 0.162 to 0.521, and the Tyr/Cre ratio for tyrosinemia was 0.147. The ratios of Tyr/Cre, Phe/Cre, and Trp/Cre for Lowe syndrome were 0.497, 0.321, and 0.495, respectively. In contrast, the creatinine ratios for healthy newborns showed one digit lower than those for patients did. The developed method is very practical and can provide useful information and results for the clinical or biomedical researches with low analytical run costs.

Targeted Deletion of the Antisilencer/Enhancer (ASE) Element from Intron 1 of the Myelin Proteolipid Protein Gene (Plp1) in Mouse Reveals that the Element Is Dispensable for Plp1 Expression in Brain during Development and Remyelination

PubMed Central

Pereira, Glauber B.; Meng, Fanxue; Kockara, Neriman T.; Yang, Baoli; Wight, Patricia A.

2012-01-01

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. While removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is nonfunctional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene. PMID:23157328

Cryptic glucocorticoid receptor-binding sites pervade genomic NF-κB response elements.

PubMed

Hudson, William H; Vera, Ian Mitchelle S de; Nwachukwu, Jerome C; Weikum, Emily R; Herbst, Austin G; Yang, Qin; Bain, David L; Nettles, Kendall W; Kojetin, Douglas J; Ortlund, Eric A

2018-04-06

Glucocorticoids (GCs) are potent repressors of NF-κB activity, making them a preferred choice for treatment of inflammation-driven conditions. Despite the widespread use of GCs in the clinic, current models are inadequate to explain the role of the glucocorticoid receptor (GR) within this critical signaling pathway. GR binding directly to NF-κB itself-tethering in a DNA binding-independent manner-represents the standing model of how GCs inhibit NF-κB-driven transcription. We demonstrate that direct binding of GR to genomic NF-κB response elements (κBREs) mediates GR-driven repression of inflammatory gene expression. We report five crystal structures and solution NMR data of GR DBD-κBRE complexes, which reveal that GR recognizes a cryptic response element between the binding footprints of NF-κB subunits within κBREs. These cryptic sequences exhibit high sequence and functional conservation, suggesting that GR binding to κBREs is an evolutionarily conserved mechanism of controlling the inflammatory response.

Probing a 2-aminobenzimidazole library for binding to RNA internal loops via two-dimensional combinatorial screening.

PubMed

Velagapudi, Sai Pradeep; Pushechnikov, Alexei; Labuda, Lucas P; French, Jonathan M; Disney, Matthew D

2012-11-16

There are many potential RNA drug targets in bacterial, viral, and human transcriptomes. However, there are few small molecules that modulate RNA function. This is due, in part, to a lack of fundamental understanding about RNA-ligand interactions including the types of small molecules that bind to RNA structural elements and the RNA structural elements that bind to small molecules. In an effort to better understand RNA-ligand interactions, we diversified the 2-aminobenzimidazole core (2AB) and probed the resulting library for binding to a library of RNA internal loops. We chose the 2AB core for these studies because it is a privileged scaffold for binding RNA based on previous reports. These studies identified that N-methyl pyrrolidine, imidazole, and propylamine diversity elements at the R1 position increase binding to internal loops; variability at the R2 position is well tolerated. The preferred RNA loop space was also determined for five ligands using a statistical approach and identified trends that lead to selective recognition.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sungsoo, E-mail: sungsoo.lee@utsouthwestern.edu; Wang, Ping-Yuan; Jeong, Yangsik

Oxysterol binding protein related protein 1S (ORP1S) is a member of a family of sterol transport proteins. Here we present evidence that ORP1S translocates from the cytoplasm to the nucleus in response to sterol binding. The sterols that best promote nuclear import of ORP1S also activate the liver X receptor (LXR) transcription factors and we show that ORP1S binds to LXRs, promotes binding of LXRs to LXR response elements (LXREs) and specifically enhances LXR-dependent transcription via the ME.1 and ME.2 enhancer elements of the apoE gene. We propose that ORP1S is a cytoplasmic sterol sensor, which transports sterols to themore » nucleus and promotes LXR-dependent gene transcription through select enhancer elements. -- Highlights: Black-Right-Pointing-Pointer ORP1S translocates to the nucleus in response to sterol binding. Black-Right-Pointing-Pointer The sterols that best promote nuclear import of ORP1S are LXR agonists. Black-Right-Pointing-Pointer ORP1S binds to LXRs, enhances binding of LXRs to LXREs and promotes LXR-dependent transcription of apoE.« less

Cell Autonomous Phosphoinositide 3-Kinase Activation in Oocytes Disrupts Normal Ovarian Function Through Promoting Survival and Overgrowth of Ovarian Follicles

PubMed Central

Ebbert, Katherine; Cordeiro, Marilia H.; Romero, Megan; Zhu, Jie; Serna, Vanida Ann; Whelan, Kelly A.; Woodruff, Teresa K.

2015-01-01

In this study, we explored the effects of oocytic phosphoinositide 3-kinase (PI3K) activation on folliculogensis by generating transgenic mice, in which the oocyte-specific Cre-recombinase induces the expression of constitutively active mutant PI3K during the formation of primordial follicles. The ovaries of neonatal transgenic (Cre+) mice showed significantly reduced apoptosis in follicles, which resulted in an excess number of follicles per ovary. Thus, the elevation of phosphatidylinositol (3,4,5)-trisphosphate levels within oocytes promotes the survival of follicles during neonatal development. Despite the increase in AKT phosphorylation, primordial follicles in neonatal Cre+ mice remained dormant demonstrating a nuclear accumulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These primordial follicles containing a high level of nuclear PTEN persisted in postpubertal females, suggesting that PTEN is the dominant factor in the maintenance of female reproductive lifespan through the regulation of primordial follicle recruitment. Although the oocytic PI3K activity and PTEN levels were elevated, the activation of primordial follicles and the subsequent accumulation of antral follicles with developmentally competent oocytes progressed normally in prepubertal Cre+ mice. However, mature Cre+ female mice were anovulatory. Because postnatal day 50 Cre+ mice released cumulus-oocyte complexes with developmentally competent oocytes in response to super-ovulation treatment, the anovulatory phenotype was not due to follicular defects but rather endocrine abnormalities, which were likely caused by the excess number of overgrown follicles. Our current study has elucidated the critical role of oocytic PI3K activity in follicular function, as well as the presence of a PTEN-mediated mechanism in the prevention of immature follicle activation. PMID:25594701

Serum and urine chromium as indices of chromium status in tannery workers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Randall, J.A.; Gibson, R.S.

Serum and urinary Cr levels of a selected group of men exposed to CrIII in four Southern Ontario tanneries were compared with those of men not exposed to Cr. Fasted blood samples were obtained from 72 tannery workers (TW; mean age +/- SD = 36 +/- 12 years) and from 52 controls (CS; mean age +/- SD = 41 +/- 13 years). Serum Cr levels as determined by graphite furnace atomic absorption spectrophotometry were significantly higher (P = 0.0001) for TW (median 0.49 ng/ml, range 0.37-0.81) than for CS (median 0.15 ng/ml, range 0.12-0.20). Urine samples were collected from 49more » TW and 43 CS on a Friday pm and from 42 TW on a Monday am. Urinary creatine (Cre) was determined by the Jaffe reaction. For Friday samples, the median urinary Cr/Cre ratio was significantly higher (P = 0.0001) for TW (median 0.83 ng/mg, range 0.48-1.82) than for CS (median 0.18 ng/mg, range 0.13-0.26). For TW, Cr/Cre was correlated with serum Cr (r = 0.72, P = 0.0001). Neither urinary Cr/Cre nor serum Cr was correlated with length of employment in the tanning industry. There were significant differences in serum Cr levels and urinary Cr/Cre ratios among TW employed in different areas of the tanneries. For TW, the median urinary Cr/Cre ratio for Monday morning samples was significantly lower than for Friday afternoon samples (P = 0.03). These data indicate that CrIII is absorbed and that serum and urine Cr in tannery workers may be indices of Cr exposure and status.« less

Serum and urine chromium as indices of chromium status in tannery workers.

PubMed

Randall, J A; Gibson, R S

1987-05-01

Serum and urinary Cr levels of a selected group of men exposed to CrIII in four Southern Ontario tanneries were compared with those of men not exposed to Cr. Fasted blood samples were obtained from 72 tannery workers (TW; mean age +/- SD = 36 +/- 12 years) and from 52 controls (CS; mean age +/- SD = 41 +/- 13 years). Serum Cr levels as determined by graphite furnace atomic absorption spectrophotometry were significantly higher (P = 0.0001) for TW (median 0.49 ng/ml, range 0.37-0.81) than for CS (median 0.15 ng/ml, range 0.12-0.20). Urine samples were collected from 49 TW and 43 CS on a Friday pm and from 42 TW on a Monday am. Urinary creatine (Cre) was determined by the Jaffe reaction. For Friday samples, the median urinary Cr/Cre ratio was significantly higher (P = 0.0001) for TW (median 0.83 ng/mg, range 0.48-1.82) than for CS (median 0.18 ng/mg, range 0.13-0.26). For TW, Cr/Cre was correlated with serum Cr (r = 0.72, P = 0.0001). Neither urinary Cr/Cre nor serum Cr was correlated with length of employment in the tanning industry. There were significant differences in serum Cr levels and urinary Cr/Cre ratios among TW employed in different areas of the tanneries. For TW, the median urinary Cr/Cre ratio for Monday morning samples was significantly lower than for Friday afternoon samples (P = 0.03). These data indicate that CrIII is absorbed and that serum and urine Cr in tannery workers may be indices of Cr exposure and status.

Impaired clearance of influenza A virus in obese, leptin receptor deficient mice is independent of leptin signaling in the lung epithelium and macrophages.

PubMed

Radigan, Kathryn A; Morales-Nebreda, Luisa; Soberanes, Saul; Nicholson, Trevor; Nigdelioglu, Recep; Cho, Takugo; Chi, Monica; Hamanaka, Robert B; Misharin, Alexander V; Perlman, Harris; Budinger, G R Scott; Mutlu, Gökhan M

2014-01-01

During the recent H1N1 outbreak, obese patients had worsened lung injury and increased mortality. We used a murine model of influenza A pneumonia to test the hypothesis that leptin receptor deficiency might explain the enhanced mortality in obese patients. We infected wild-type, obese mice globally deficient in the leptin receptor (db/db) and non-obese mice with tissue specific deletion of the leptin receptor in the lung epithelium (SPC-Cre/LepR fl/fl) or macrophages and alveolar type II cells (LysM-Cre/Lepr fl/fl) with influenza A virus (A/WSN/33 [H1N1]) (500 and 1500 pfu/mouse) and measured mortality, viral clearance and several markers of lung injury severity. The clearance of influenza A virus from the lungs of mice was impaired in obese mice globally deficient in the leptin receptor (db/db) compared to normal weight wild-type mice. In contrast, non-obese, SP-C-Cre+/+/LepR fl/fl and LysM-Cre+/+/LepR fl/fl had improved viral clearance after influenza A infection. In obese mice, mortality was increased compared with wild-type mice, while the SP-C-Cre+/+/LepR fl/fl and LysM-Cre+/+/LepR fl/fl mice exhibited improved survival. Global loss of the leptin receptor results in reduced viral clearance and worse outcomes following influenza A infection. These findings are not the result of the loss of leptin signaling in lung epithelial cells or macrophages. Our results suggest that factors associated with obesity or with leptin signaling in non-myeloid populations such as natural killer and T cells may be associated with worsened outcomes following influenza A infection.

A Novel Mgp-Cre Knock-In Mouse Reveals an Anticalcification/Antistiffness Candidate Gene in the Trabecular Meshwork and Peripapillary Scleral Region.

PubMed

Borrás, Teresa; Smith, Matthew H; Buie, LaKisha K

2015-04-01

Soft tissue calcification is a pathological condition. Matrix Gla (MGP) is a potent mineralization inhibitor secreted by cartilage chondrocytes and arteries' vascular smooth muscle cells. Mgp knock-out mice die at 6 weeks due to massive arterial calcification. Arterial calcification results in arterial stiffness and higher systolic blood pressure. Intriguingly, MGP was highly abundant in trabecular meshwork (TM). Because tissue stiffness is relevant to glaucoma, we investigated which additional eye tissues use Mgp's function using knock-in mice. An Mgp-Cre-recombinase coding sequence (Cre) knock-in mouse, containing Mgp DNA plus an internal ribosomal entry site (IRES)-Cre-cassette was generated by homologous recombination. Founders were crossed with Cre-mediated reporter mouse R26R-lacZ. Their offspring expresses lacZ where Mgp is transcribed. Eyes from MgpCre/+;R26RlacZ/+ (Mgp-lacZ knock-in) and controls, 1 to 8 months were assayed for β-gal enzyme histochemistry. As expected, Mgp-lacZ knock-in's TM was intensely blue. In addition, this mouse revealed high specific expression in the sclera, particularly in the peripapillary scleral region (ppSC). Ciliary muscle and sclera above the TM were also positive. Scleral staining was located immediately underneath the choroid (chondrocyte layer), began midsclera and was remarkably high in the ppSC. Cornea, iris, lens, ciliary body, and retina were negative. All mice exhibited similar staining patterns. All controls were negative. Matrix Gla's restricted expression to glaucoma-associated tissues from anterior and posterior segments suggests its involvement in the development of the disease. Matrix Gla's anticalcification/antistiffness properties in the vascular tissue, together with its high TM and ppCS expression, place this gene as a strong candidate for TM's softness and sclera's stiffness regulation in glaucoma.

Presynaptic NCAM Is Required for Motor Neurons to Functionally Expand Their Peripheral Field of Innervation in Partially Denervated Muscles

PubMed Central

Chipman, Peter H.; Schachner, Melitta

2014-01-01

The function of neural cell adhesion molecule (NCAM) expression in motor neurons during axonal sprouting and compensatory reinnervation was explored by partially denervating soleus muscles in mice lacking presynaptic NCAM (Hb9creNCAMflx). In agreement with previous studies, the contractile force of muscles in wild-type (NCAM+/+) mice recovered completely 2 weeks after 75% of the motor innervation was removed because motor unit size increased by 2.5 times. In contrast, similarly denervated muscles in Hb9creNCAMflx mice failed to recover the force lost due to the partial denervation because motor unit size did not change. Anatomical analysis indicated that 50% of soleus end plates were completely denervated 1–4 weeks post-partial denervation in Hb9creNCAMflx mice, while another 25% were partially reinnervated. Synaptic vesicles (SVs) remained at extrasynaptic regions in Hb9creNCAMflx mice rather than being distributed, as occurs normally, to newly reinnervated neuromuscular junctions (NMJs). Electrophysiological analysis revealed two populations of NMJs in partially denervated Hb9creNCAMflx soleus muscles, one with high (mature) quantal content, and another with low (immature) quantal content. Extrasynaptic SVs in Hb9creNCAMflx sprouts were associated with L-type voltage-dependent calcium channel (L-VDCC) immunoreactivity and maintained an immature, L-VDCC-dependent recycling phenotype. Moreover, acute nifedipine treatment potentiated neurotransmission at newly sprouted NMJs, while chronic intraperitoneal treatment with nifedipine during a period of synaptic consolidation enhanced functional motor unit expansion in the absence of presynaptic NCAM. We propose that presynaptic NCAM bridges a critical link between the SV cycle and the functional expansion of synaptic territory through the regulation of L-VDCCs. PMID:25100585

Endothelial fibroblast growth factor receptor signaling is required for vascular remodeling following cardiac ischemia-reperfusion injury

PubMed Central

Castro, Angela M.; Lupu, Traian S.; Weinheimer, Carla; Smith, Craig; Kovacs, Attila

2016-01-01

Fibroblast growth factor (FGF) signaling is cardioprotective in various models of myocardial infarction. FGF receptors (FGFRs) are expressed in multiple cell types in the adult heart, but the cell type-specific FGFR signaling that mediates different cardioprotective endpoints is not known. To determine the requirement for FGFR signaling in endothelium in cardiac ischemia-reperfusion injury, we conditionally inactivated the Fgfr1 and Fgfr2 genes in endothelial cells with Tie2-Cre (Tie2-Cre, Fgfr1f/f, Fgfr2f/f DCKO mice). Tie2-Cre, Fgfr1f/f, Fgfr2f/f DCKO mice had normal baseline cardiac morphometry, function, and vessel density. When subjected to closed-chest, regional cardiac ischemia-reperfusion injury, Tie2-Cre, Fgfr1f/f, Fgfr2f/f DCKO mice showed a significantly increased hypokinetic area at 7 days, but not 1 day, after reperfusion. Tie2-Cre, Fgfr1f/f, Fgfr2f/f DCKO mice also showed significantly worsened cardiac function compared with controls at 7 days but not 1 day after reperfusion. Pathophysiological analysis showed significantly decreased vessel density, increased endothelial cell apoptosis, and worsened tissue hypoxia in the peri-infarct area at 7 days following reperfusion. Notably, Tie2-Cre, Fgfr1f/f, Fgfr2f/f DCKO mice showed no impairment in the cardiac hypertrophic response. These data demonstrate an essential role for FGFR1 and FGFR2 in endothelial cells for cardiac functional recovery and vascular remodeling following in vivo cardiac ischemia-reperfusion injury, without affecting the cardiac hypertrophic response. This study suggests the potential for therapeutic benefit from activation of endothelial FGFR pathways following ischemic injury to the heart. PMID:26747503

Potent activity of nobiletin-rich Citrus reticulata peel extract to facilitate cAMP/PKA/ERK/CREB signaling associated with learning and memory in cultured hippocampal neurons: identification of the substances responsible for the pharmacological action.

PubMed

Kawahata, Ichiro; Yoshida, Masaaki; Sun, Wen; Nakajima, Akira; Lai, Yanxin; Osaka, Naoya; Matsuzaki, Kentaro; Yokosuka, Akihito; Mimaki, Yoshihiro; Naganuma, Akira; Tomioka, Yoshihisa; Yamakuni, Tohru

2013-10-01

cAMP/PKA/ERK/CREB signaling linked to CRE-mediated transcription is crucial for learning and memory. We originally found nobiletin as a natural compound that stimulates this intracellular signaling and exhibits anti-dementia action in animals. Citrus reticulata or C. unshiu peels are employed as "chinpi" and include a small amount of nobiletin. We here provide the first evidence for beneficial pharmacological actions on the cAMP/PKA/ERK/CREB cascade of extracts from nobiletin-rich C.reticulata peels designated as Nchinpi, the nobiletin content of which was 0.83 ± 0.13% of the dry weight or 16-fold higher than that of standard chinpi extracts. Nchinpi extracts potently facilitated CRE-mediated transcription in cultured hippocampal neurons, whereas the standard chinpi extracts showed no such activity. Also, the Nchinpi extract, but not the standard chinpi extract, stimulated PKA/ERK/CREB signaling. Interestingly, treatment with the Nchinpi extract at the concentration corresponding to approximately 5 μM nobiletin more potently facilitated CRE-mediated transcriptional activity than did 30 μM nobiletin alone. Consistently, sinensetin, tangeretin, 6-demethoxynobiletin, and 6-demethoxytangeretin were also identified as bioactive substances in Nchinpi that facilitated the CRE-mediated transcription. Purified sinensetin enhanced the transcription to a greater degree than nobiletin. Furthermore, samples reconstituted with the four purified compounds and nobiletin in the ratio of each constituent's content in the extract showed activity almost equal to that of the Nchinpi extract to stimulate CRE-mediated transcription. These findings suggest that above four compounds and nobiletin in the Nchinpi extract mainly cooperated to facilitate potently CRE-mediated transcription linked to the upstream cAMP/PKA/ERK/CREB pathway in hippocampal neurons.

An intact Pms2 ATPase domain is not essential for male fertility

PubMed Central

Fischer, Jared M; Dudley, Sandra; Miller, Ashleigh J; Liskay, R Michael

2016-01-01

The DNA mismatch repair (MMR) machinery in mammals plays critical roles in both mutation avoidance and spermatogenesis. Meiotic analysis of knockout mice of two different MMR genes, Mlh1 and Mlh3, revealed both male and female infertility associated with a defect in meiotic crossing over. In contrast, another MMR gene knockout, Pms2 (Pms2ko/ko), which contained a deletion of a portion of the ATPase domain, produced animals that were male sterile but female fertile. However, the meiotic phenotype of Pms2ko/ko males was less clear-cut than for Mlh1- or Mlh3-deficient meiosis. More recently, we generated a different Pms2 mutant allele (Pms2cre), which results in deletion of the same portion of the ATPase domain. Surprisingly, Pms2cre/cre male mice were completely fertile, suggesting that the ATPase domain of Pms2 is not required for male fertility. To explore the difference in male fertility, we examined the Pms2 RNA and found that alternative splicing of the Pms2cre allele results in a predicted Pms2 containing the C-terminus, which contains the Mlh1-interaction domain, a possible candidate for stabilizing Mlh1 levels. To study further the basis of male fertility, we examined Mlh1 levels in testes and found that whereas Pms2 loss in Pms2ko/ko mice results in severely reduced levels of Mlh1 expression in the testes, Mlh1 levels in Pms2cre/cre testes were reduced to a lesser extent. Thus, we propose that a primary function of Pms2 during spermatogenesis is to stabilize Mlh1 levels prior to its critical crossing over function with Mlh3. PMID:26753533

An intact Pms2 ATPase domain is not essential for male fertility.

PubMed

Fischer, Jared M; Dudley, Sandra; Miller, Ashleigh J; Liskay, R Michael

2016-03-01

The DNA mismatch repair (MMR) machinery in mammals plays critical roles in both mutation avoidance and spermatogenesis. Meiotic analysis of knockout mice of two different MMR genes, Mlh1 and Mlh3, revealed both male and female infertility associated with a defect in meiotic crossing over. In contrast, another MMR gene knockout, Pms2 (Pms2(ko/ko)), which contained a deletion of a portion of the ATPase domain, produced animals that were male sterile but female fertile. However, the meiotic phenotype of Pms2(ko/ko) males was less clear-cut than for Mlh1- or Mlh3-deficient meiosis. More recently, we generated a different Pms2 mutant allele (Pms2(cre)), which results in deletion of the same portion of the ATPase domain. Surprisingly, Pms2(cre/cre) male mice were completely fertile, suggesting that the ATPase domain of Pms2 is not required for male fertility. To explore the difference in male fertility, we examined the Pms2 RNA and found that alternative splicing of the Pms2(cre) allele results in a predicted Pms2 containing the C-terminus, which contains the Mlh1-interaction domain, a possible candidate for stabilizing Mlh1 levels. To study further the basis of male fertility, we examined Mlh1 levels in testes and found that whereas Pms2 loss in Pms2(ko/ko) mice results in severely reduced levels of Mlh1 expression in the testes, Mlh1 levels in Pms2(cre/cre) testes were reduced to a lesser extent. Thus, we propose that a primary function of Pms2 during spermatogenesis is to stabilize Mlh1 levels prior to its critical crossing over function with Mlh3. Copyright © 2015 Elsevier B.V. All rights reserved.

Endothelial fibroblast growth factor receptor signaling is required for vascular remodeling following cardiac ischemia-reperfusion injury.

PubMed

House, Stacey L; Castro, Angela M; Lupu, Traian S; Weinheimer, Carla; Smith, Craig; Kovacs, Attila; Ornitz, David M

2016-03-01

Fibroblast growth factor (FGF) signaling is cardioprotective in various models of myocardial infarction. FGF receptors (FGFRs) are expressed in multiple cell types in the adult heart, but the cell type-specific FGFR signaling that mediates different cardioprotective endpoints is not known. To determine the requirement for FGFR signaling in endothelium in cardiac ischemia-reperfusion injury, we conditionally inactivated the Fgfr1 and Fgfr2 genes in endothelial cells with Tie2-Cre (Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice). Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice had normal baseline cardiac morphometry, function, and vessel density. When subjected to closed-chest, regional cardiac ischemia-reperfusion injury, Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice showed a significantly increased hypokinetic area at 7 days, but not 1 day, after reperfusion. Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice also showed significantly worsened cardiac function compared with controls at 7 days but not 1 day after reperfusion. Pathophysiological analysis showed significantly decreased vessel density, increased endothelial cell apoptosis, and worsened tissue hypoxia in the peri-infarct area at 7 days following reperfusion. Notably, Tie2-Cre, Fgfr1(f/f), Fgfr2(f/f) DCKO mice showed no impairment in the cardiac hypertrophic response. These data demonstrate an essential role for FGFR1 and FGFR2 in endothelial cells for cardiac functional recovery and vascular remodeling following in vivo cardiac ischemia-reperfusion injury, without affecting the cardiac hypertrophic response. This study suggests the potential for therapeutic benefit from activation of endothelial FGFR pathways following ischemic injury to the heart. Copyright © 2016 the American Physiological Society.

Insights from a Regime Decomposition Approach on CERES and CloudSat-inferred Cloud Radiative Effects

NASA Astrophysics Data System (ADS)

Oreopoulos, L.; Cho, N.; Lee, D.

2015-12-01

Our knowledge of the Cloud Radiative Effect (CRE) not only at the Top-of-the-Atmosphere (TOA), but also (with the help of some modeling) at the surface (SFC) and within the atmospheric column (ATM) has been steadily growing in recent years. Not only do we have global values for these CREs, but we can now also plot global maps of their geographical distribution. The next step in our effort to advance our knowledge of CRE is to systematically assess the contributions of prevailing cloud systems to the global values. The presentation addresses this issue directly. We identify the world's prevailing cloud systems, which we call "Cloud Regimes" (CRs) via clustering analysis of MODIS (Aqua-Terra) daily joint histograms of Cloud Top Pressure and Cloud Optical Thickness (TAU) at 1 degree scales. We then composite CERES diurnal values of CRE (TOA, SFC, ATM) separately for each CR by averaging these values for each CR occurrence, and thus find the contribution of each CR to the global value of CRE. But we can do more. We can actually decompose vertical profiles of inferred instantaneous CRE from CloudSat/CALIPSO (2B-FLXHR-LIDAR product) by averaging over Aqua CR occurrences (since A-Train formation flying allows collocation). Such an analysis greatly enhances our understanding of the radiative importance of prevailing cloud mixtures at different atmospheric levels. We can, for example, in addition to examining whether the CERES findings on which CRs contribute to radiative cooling and warming of the atmospheric column are consistent with CloudSat, also gain insight on why and where exactly this happens from the shape of the full instantaneous CRE vertical profiles.

The bZIP dimer localizes at DNA full-sites where each basic region can alternately translocate and bind to subsites at the half-site

PubMed Central

Chan, I-San; Al-Sarraj, Taufik; Shahravan, S. Hesam; Fedorova, Anna V.; Shin, Jumi A.

2012-01-01

Crystal structures of the GCN4 bZIP (basic region/leucine zipper) with the AP-1 or CRE site show how each GCN4 basic region binds to a 4-bp cognate half-site as a single DNA target; however, this may not always fully describe how bZIP proteins interact with their target sites. Previously, we showed that the GCN4 basic region interacts with all 5 bp in half-site TTGCG (termed 5H-LR), and that 5H-LR comprises two 4-bp subsites, TTGC and TGCG, which individually are also target sites of the basic region. In this work, we explored how the basic region interacts with 5H-LR when the bZIP dimer localizes to full-sites. Using AMBER molecular modeling, we simulated GCN4 bZIP complexes with full-sites containing 5H-LR to investigate in silico the interface between the basic region and 5H-LR. We also performed in vitro investigation of bZIP–DNA interactions at a number of full-sites that contain 5H-LR vs. either subsite: we analyzed results from DNase I footprinting and electrophoretic mobility shift assay (EMSA) and from EMSA titrations to quantify binding affinities. Our computational and experimental results together support a highly dynamic DNA-binding model: when a bZIP dimer localizes to its target full-site, the basic region can alternately recognize either subsite as a distinct target at 5H-LR and translocate between the subsites, potentially by sliding and hopping. This model provides added insights into how α-helical DNA-binding domains of transcription factors can localize to their gene regulatory sequences in vivo. PMID:22856882

The bZIP dimer localizes at DNA full-sites where each basic region can alternately translocate and bind to subsites at the half-site.

PubMed

Chan, I-San; Al-Sarraj, Taufik; Shahravan, S Hesam; Fedorova, Anna V; Shin, Jumi A

2012-08-21

Crystal structures of the GCN4 bZIP (basic region/leucine zipper) with the AP-1 or CRE site show how each GCN4 basic region binds to a 4 bp cognate half-site as a single DNA target; however, this may not always fully describe how bZIP proteins interact with their target sites. Previously, we showed that the GCN4 basic region interacts with all 5 bp in half-site TTGCG (termed 5H-LR) and that 5H-LR comprises two 4 bp subsites, TTGC and TGCG, which individually are also target sites of the basic region. In this work, we explore how the basic region interacts with 5H-LR when the bZIP dimer localizes to full-sites. Using AMBER molecular modeling, we simulated GCN4 bZIP complexes with full-sites containing 5H-LR to investigate in silico the interface between the basic region and 5H-LR. We also performed in vitro investigation of bZIP-DNA interactions at a number of full-sites that contain 5H-LR versus either subsite: we analyzed results from DNase I footprinting and electrophoretic mobility shift assay (EMSA) and from EMSA titrations to quantify binding affinities. Our computational and experimental results together support a highly dynamic DNA-binding model: when a bZIP dimer localizes to its target full-site, the basic region can alternately recognize either subsite as a distinct target at 5H-LR and translocate between the subsites, potentially by sliding and hopping. This model provides added insights into how α-helical DNA-binding domains of transcription factors can localize to their gene regulatory sequences in vivo.

Chemical Reaction Engineering: Current Status and Future Directions.

ERIC Educational Resources Information Center

Dudukovic, M. P.

1987-01-01

Describes Chemical Reaction Engineering (CRE) as the discipline that quantifies the interplay of transport phenomena and kinetics in relating reactor performance to operating conditions and input variables. Addresses the current status of CRE in both academic and industrial settings and outlines future trends. (TW)

Mice expressing GFP and CreER in osteochondro progenitor cells in the periosteum.

PubMed

Kawanami, Aya; Matsushita, Takehiko; Chan, Yuk Yu; Murakami, Shunichi

2009-08-28

We generated Prx1CreER-GFP transgenic mice that express tamoxifen-inducible Cre recombinase and GFP under the control of a 2.4 kb Prx1 promoter. The transgene is expressed in osteochondro progenitor cells in the developing limb buds and in a subpopulation of periosteal cells that is closely associated with the cortical bone. GFP-expressing cells isolated from the diaphyses of long bones by cell sorting express multiple markers of periosteal cells, including Prx1, Fgf18, Tenascin-W, Periostin, and Thrombospondin 2. In addition, these cells undergo chondrogenic and osteogenic differentiation in culture upon induction. Cell fate analysis using the Rosa26 LacZ reporter indicated that transgene-expressing cells give rise to some of the chondrocytes and osteoblasts in the fracture callus. Collectively, these observations strongly suggest that the transgene-expressing cells are osteochondro progenitor cells in the periosteum. The established Prx1CreER-GFP mice would offer novel approaches for analyzing the functions of periosteal cells in vitro and in vivo.

Microglia promote learning-dependent synapse formation through BDNF

PubMed Central

Parkhurst, Christopher N.; Yang, Guang; Ninan, Ipe; Savas, Jeffrey N.; Yates, John R.; Lafaille, Juan J.; Hempstead, Barbara L.; Littman, Dan R.; Gan, Wen-Biao

2014-01-01

SUMMARY Microglia are the resident macrophages of the central nervous system and their functions have been extensively studied in various brain pathologies. The physiological roles of microglia in brain plasticity and function, however, remain unclear. To address this question, we generated CX3CR1CreER mice expressing tamoxifen-inducible Cre recombinase that allow for specific manipulation of gene function in microglia. Using CX3CR1CreER to drive diphtheria toxin receptor expression in microglia, we found that microglia could be specifically depleted from the brain upon diphtheria toxin administration. Mice depleted of microglia show deficits in multiple learning tasks and a significant reduction in motor learning-dependent synapse formation. Furthermore, Cre-dependent removal of brain-derived neurotrophic factor (BDNF) from microglia largely recapitulated the effects of microglia depletion. Microglial BDNF increases neuronal TrkB phosphorylation, a key mediator of synaptic plasticity. Together, our findings reveal important physiological functions of microglia in learning and memory by promoting learning-related synapse formation through BDNF signaling. PMID:24360280

Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival

PubMed Central

Kaul, Sunil; Mittal, Sharad K.; Feigenbaum, Lionel; Kruhlak, Michael J.; Roche, Paul A.

2015-01-01

Members of the SNARE-family of proteins are known to be key regulators of the membrane-membrane fusion events required for intracellular membrane traffic. The ubiquitously expressed SNARE protein SNAP-23 regulates a wide variety of exocytosis events and is essential for mouse development. Germline deletion of SNAP-23 results in early embryonic lethality in mice, and for this reason we now describe mice and cell lines in which SNAP-23 can be conditionally-deleted using Cre-lox technology. Deletion of SNAP-23 in CD19-Cre expressing mice prevents B lymphocyte development and deletion of SNAP-23 using a variety of T lymphocyte-specific Cre mice prevents T lymphocyte development. Acute depletion of SNAP-23 in mouse fibroblasts leads to rapid apoptotic cell death. These data highlight the importance of SNAP-23 for cell survival and describe a mouse in which specific cell types can be eliminated by expression of tissue-specific Cre-recombinase. PMID:25706117

Using the Textpresso Site-Specific Recombinases Web server to identify Cre expressing mouse strains and floxed alleles.

PubMed

Condie, Brian G; Urbanski, William M

2014-01-01

Effective tools for searching the biomedical literature are essential for identifying reagents or mouse strains as well as for effective experimental design and informed interpretation of experimental results. We have built the Textpresso Site Specific Recombinases (Textpresso SSR) Web server to enable researchers who use mice to perform in-depth searches of a rapidly growing and complex part of the mouse literature. Our Textpresso Web server provides an interface for searching the full text of most of the peer-reviewed publications that report the characterization or use of mouse strains that express Cre or Flp recombinase. The database also contains most of the publications that describe the characterization or analysis of strains carrying conditional alleles or transgenes that can be inactivated or activated by site-specific recombinases such as Cre or Flp. Textpresso SSR complements the existing online databases that catalog Cre and Flp expression patterns by providing a unique online interface for the in-depth text mining of the site specific recombinase literature.

Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice.

PubMed

Hu, Zhan; Ding, Xuezhi; Hu, Shengbiao; Sun, Yunjun; Xia, Liqiu

2013-12-01

Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops.

Cosmogenic Samarium-150 and Calcium-41 in Norton County

NASA Technical Reports Server (NTRS)

Fink, D.; Klein, J.; Middleton, R.; Albrecht, A.; Ma, P.; Herzog, G. F.; Bogard, D. D.; Nyquist, L. E.; Shih, C.-Y.; Reese, Y.;

Atoh1-lineal neurons are required for hearing and for the survival of neurons in the spiral ganglion and brainstem accessory auditory nuclei

PubMed Central

Maricich, Stephen M.; Xia, Anping; Mathes, Erin L.; Wang, Vincent Y.; Oghalai, John S.; Fritzsch, Bernd; Zoghbi, Huda Y.

2009-01-01

Atoh1 is a basic helix-loop-helix transcription factor necessary for the specification of inner ear hair cells and central auditory system neurons derived from the rhombic lip. We used the Cre-loxP system and two Cre-driver lines (Egr2Cre and Hoxb1Cre) to delete Atoh1 from different regions of the cochlear nucleus (CN) and accessory auditory nuclei (AAN). Adult Atoh1-conditional knockout mice (Atoh1CKO) are behaviorally deaf, have diminished auditory brainstem evoked responses and disrupted CN and AAN morphology and connectivity. In addition, Egr2; Atoh1CKO mice lose spiral ganglion neurons in the cochlea and AAN neurons during the first 3 days of life, revealing a novel critical period in the development of these neurons. These new mouse models of predominantly central deafness illuminate the importance of the CN for support of a subset of peripheral and central auditory neurons. PMID:19741118

Using HSV-TK/GCV suicide gene therapy to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification

PubMed Central

Jiang, Yong-Xiang; Liu, Tian-Jing; Yang, Jin; Chen, Yan; Fang, Yan-Wen

2011-01-01

Purpose To establish a novel, targeted lentivirus-based HSV-tk (herpes simplex virus thymidine kinase)/GCV (ganciclovir) gene therapy system to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification (PCO) after cataract surgery. Methods An enhanced Cre recombinase (Cre/loxP) system with a lentiviral vector expressing Cre under the control of the lens-specific promoter LEP503 (Lenti-LEP503-HSVtk-Cre [LTKCRE]) was constructed, as well as another lentiviral vector containing a switching unit. The latter vector contains a stuffer sequence encoding EGFP (Lenti-hPGK-Loxp-EGFP-pA-Loxp-HSVtk [PGFPTK]) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-tk) gene, both under the control of the human posphoglycerate kinase (hPGK) promoter. Expression of the downstream gene (HSV-tk) is activated by co-expression of Cre. Human lens epithelial cells (HLECs) or retinal pigmental epithelial cells (RPECs) were co-infected with LTKCRE and PGFPTK. The inhibitory effects on HLECs and RPECs infected by the enhanced specific lentiviral vector combination at the concentration of 20 µg/ml GCV were assayed and compared. Results The specific gene expression of Cre and HSV-tk in HLECs is activated by the LEP503 promoter. LTKCRE and PGFPTK co-infected HLECs, but not RPECs, expressed high levels of the HSV-tk protein. After 96 h of GCV treatment, the percentage of apoptotic HLECs infected by the enhanced specific lentiviral vector combination was 87.23%, whereas that of apoptotic RPECs was only 10.12%. Electron microscopy showed that GCV induced apoptosis and necrosis of the infected HLECs. Conclusions The enhanced specific lentiviral vector combination selectively and effectively expressed HSV-tk in HLECs. A concentration of 20 µg/ml, GCV is effective against the proliferation of HLECs in vitro. This cell-type-specific gene therapy using a Cre/loxP lentivirus system may be a feasible treatment strategy to prevent PCO. PMID:21283526

Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination.

PubMed

Betz, U A; Vosshenrich, C A; Rajewsky, K; Müller, W

1996-10-01

The analysis of gene function based on the generation of mutant mice by homologous recombination in embryonic stem cells is limited if gene disruption results in embryonic lethality. Mosaic mice, which contain a certain proportion of mutant cells in all organs, allow lethality to be circumvented and the potential of mutant cells to contribute to different cell lineages to be analyzed. To generate mosaic animals, we used the bacteriophage P1-derived Cre-loxP recombination system, which allows gene alteration by Cre-mediated deletion of loxP-flanked gene segments. We generated nestin-cre transgenic mouse lines, which expressed the Cre recombinase under the control of the rat nestin promoter and its second intron enhancer. In crosses to animals carrying a loxP-flanked target gene, partial deletion of the loxP-flanked allele occurred before day 10.5 post coitum and was detectable in all adult organs examined, including germ-line cells. Using this approach, we generated mosaic mice containing cells deficient in the gamma-chain of the interleukin-2 receptor (IL-2R gamma); in these animals, the IL-2R gamma-deficient cells were underrepresented in the thymus and spleen. Because mice deficient in DNA polymerase beta die perinatally, we studied the effects of DNA polymerase beta deficiency in mosaic animals. We found that some of the mosaic polymerase beta-deficient animals were viable, but were often reduced in size and weight. The fraction of DNA polymerase beta-deficient cells in mosaic embryos decreased during embryonic development, presumably because wild-type cells had a competitive advantage. The nestin-cre transgenic mice can be used to generate mosaic animals in which target genes are mutated by Cre-mediated recombination of loxP-flanked target genes. By using mosaic animals, embryonic lethality can be bypassed and cell lineages for whose development a given target gene is critical can be identified. In the case of DNA polymerase beta, deficient cells are already selected against during embryonic development, demonstrating the general importance of this protein in multiple cell types.

Associations between polycyclic aromatic hydrocarbon (PAH) exposure and oxidative stress in people living near e-waste recycling facilities in China.

PubMed

Lu, Shao-You; Li, Yan-Xi; Zhang, Jian-Qing; Zhang, Tao; Liu, Gui-Hua; Huang, Ming-Zhi; Li, Xiao; Ruan, Ju-Jun; Kannan, Kurunthachalam; Qiu, Rong-Liang

2016-09-01

Emission of polycyclic aromatic hydrocarbons (PAHs) from e-waste recycling activities in China is known. However, little is known on the association between PAH exposure and oxidative damage to DNA and lipid content in people living near e-waste dismantling sites. In this study, ten hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) and two biomarkers [8-hydroxy-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA)] of oxidative stress were investigated in urine samples collected from people living in and around e-waste dismantling facilities, and in reference population from rural and urban areas in China. The urinary levels of ∑10OH-PAHs determined in e-waste recycling area (GM: 25.4μg/g Cre) were significantly higher (p<0.05) than those found in both rural (11.7μg/g Cre) and urban (10.9μg/g Cre) reference areas. The occupationally exposed e-waste workers (36.6μg/g Cre) showed significantly higher (p<0.01) urinary Σ10OH-PAHs concentrations than non-occupationally exposed people (23.2μg/g Cre) living in the e-waste recycling site. The differences in urinary Σ10OH-PAHs levels between smokers (23.4μg/g Cre) and non-smokers (24.7μg/g Cre) were not significant (p>0.05) in e-waste dismantling sites, while these differences were significant (p<0.05) in rural and urban reference areas; this indicated that smoking is not associated with elevated levels of PAH exposure in e-waste dismantling site. Furthermore, we found that urinary concentrations of Σ10OH-PAHs and individual OH-PAHs were significantly associated with elevated 8-OHdG, in samples collected from e-waste dismantling site; the levels of urinary 1-hydroxypyrene (1-PYR) (r=0.284, p<0.01) was significantly positively associated with MDA. Our results indicate that the exposure to PAHs at the e-waste dismantling site may have an effect on oxidative damage to DNA among selected participants, but this needs to be validated in large studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of Blood Pressure, Appetite, and Glucose by Leptin After Inactivation of Insulin Receptor Substrate 2 Signaling in the Entire Brain or in Proopiomelanocortin Neurons.

PubMed

do Carmo, Jussara M; da Silva, Alexandre A; Wang, Zhen; Freeman, Nathan J; Alsheik, Ammar J; Adi, Ahmad; Hall, John E

2016-02-01

Insulin receptor substrate 2 (IRS2) is one of the 3 major leptin receptor signaling pathways, but its role in mediating the chronic effects of leptin on blood pressure, food intake, and glucose regulation is unclear. We tested whether genetic inactivation of IRS2 in the entire brain (IRS2/Nestin-cre mice) or specifically in proopiomelanocortin (POMC) neurons (IRS2/POMC-cre mice) attenuates the chronic cardiovascular, metabolic, and antidiabetic effects of leptin. Mice were instrumented with telemetry probes for measurement of blood pressure and heart rate and with venous catheters for intravenous infusions. After a 5-day control period, mice received leptin infusion (2 μg/kg per minute) for 7 days. Compared with control IRS2(flox/flox) mice, IRS2/POMC-cre mice had similar body weight and food intake (33±1 versus 35±1 g and 3.6±0.5 versus 3.8±0.2 g per day) but higher mean arterial pressure (MAP) and heart rate (110±2 versus 102±2 mm Hg and 641±9 versus 616±5 bpm). IRS2/Nestin-cre mice were heavier (38±2 g), slightly hyperphagic (4.5±1.0 g per day), and had higher MAP and heart rate (108±2 mm Hg and 659±9 bpm) compared with control mice. Leptin infusion gradually increased MAP despite decreasing food intake by 31% in IRS2(flox/flox) and in Nestin-cre control mice. In contrast, leptin infusion did not change MAP in IRS2/Nestin-cre or IRS2/POMC-cre mice. The anorexic and antidiabetic effects of leptin, however, were similar in all 3 groups. These results indicate that IRS2 signaling in the central nervous system, and particularly in POMC neurons, is essential for the chronic actions of leptin to raise MAP but not for its anorexic or antidiabetic effects. © 2015 American Heart Association, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cherukuri, Durga Prasad; Chen, Xiao B.O.; Goulet, Anne-Christine

Accumulating evidence indicates that elevated levels of prostaglandin E{sub 2} (PGE{sub 2}) can increase intestinal epithelial cell proliferation, and thus play a role in colorectal tumorigenesis. PGE{sub 2} exerts its effects through four G-protein-coupled PGE receptor (EP) subtypes, named the EP1, EP2, EP3, and EP4. Increased phosphorylation of extracellular regulated kinases (ERK1/2) is required for PGE{sub 2} to stimulate cell proliferation of human colon cancer cells. However, the EP receptor(s) that are involved in this process remain unknown. We provide evidence that L-161,982, a selective EP4 receptor antagonist, completely blocks PGE{sub 2}-induced ERK phosphorylation and cell proliferation of HCA-7 cells.more » In order to identify downstream target genes of ERK1/2 signaling, we found that PGE{sub 2} induces expression of early growth response gene-1 (EGR-1) downstream of ERK1/2 and regulates its expression at the level of transcription. PGE{sub 2} treatment induces phosphorylation of cyclic AMP response element binding protein (CREB) at Ser133 residue and CRE-mediated luciferase activity in HCA-7 cells. Studies with dominant-negative CREB mutant (ACREB) provide clear evidence for the involvement of CREB in PGE{sub 2} driven egr-1 transcription in HCA-7 cells. In conclusion, this study reveals that egr-1 is a target gene of PGE{sub 2} in HCA-7 cells and is regulated via the newly identified EP4/ERK/CREB pathway. Finally our results support the notion that antagonizing EP4 receptors may provide a novel therapeutic approach to the treatment of colon cancer.« less

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia.

PubMed

Müller-Tidow, Carsten; Klein, Hans-Ulrich; Hascher, Antje; Isken, Fabienne; Tickenbrock, Lara; Thoennissen, Nils; Agrawal-Singh, Shuchi; Tschanter, Petra; Disselhoff, Christine; Wang, Yipeng; Becker, Anke; Thiede, Christian; Ehninger, Gerhard; zur Stadt, Udo; Koschmieder, Steffen; Seidl, Matthias; Müller, Frank U; Schmitz, Wilhelm; Schlenke, Peter; McClelland, Michael; Berdel, Wolfgang E; Dugas, Martin; Serve, Hubert

2010-11-04

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

β1-adrenergic receptors activate two distinct signaling pathways in striatal neurons

PubMed Central

Meitzen, John; Luoma, Jessie I.; Stern, Christopher M.; Mermelstein, Paul G.

2010-01-01

Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by β-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of β1-adrenergic receptors leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. Norepinephrine-mediated CREB phosphorylation requires β1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of β1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of β1-adrenergic receptors produces cAMP production, but surprisingly, β1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of β1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which norepinephrine and β1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how norepinephrine and β1-adrenergic receptors may affect striatal physiology. PMID:21143600

Adenovirus Vector E4 Gene Regulates Connexin 40 and 43 Expression in Endothelial Cells via PKA and PI3K Signal Pathways

PubMed Central

Zhang, Fan; Cheng, Joseph; Lam, George; Jin, David K.; Vincent, Loïc; Hackett, Neil R.; Wang, Shiyang; Young, Lauren M.; Hempstead, Barbara; Crystal, Ronald G.; Rafii, Shahin

2010-01-01

Connexins (Cxs) provide a means for intercellular communication and play important roles in the pathophysiology of vascular cardiac diseases. Infection of endothelial cells (ECs) with first-generation E1/E3-deleted E4+ adenovirus (AdE4+) selectively modulates the survival and angiogenic potential of ECs by as of yet unrecognized mechanisms. We show here that AdE4+ vectors potentiate Cx expression in ECs in vitro and in mouse heart tissue. Infection of ECs with AdE4+, but not AdE4−, resulted in a time- and dose-dependent induction of junctional Cx40 expression and suppression of Cx43 protein and mRNA expression. Treatment of ECs with PKA inhibitor H89 or PI3K inhibitor LY294002 prevented the AdE4+-mediated regulation of Cx40 and Cx43 that was associated with diminished AdE4+-mediated survival of ECs. Moreover, both PKA activity and cAMP-response element (CRE)-binding activity were enhanced by treatment of ECs with AdE4+. However, there is no causal evidence of a cross-talk between the 2 modulatory pathways, PKA and PI3K. Remarkably, Cx40 immunostaining was markedly increased and Cx43 was decreased in the heart tissue of mice treated with intratracheal AdE4+. Taken together, these results suggest that AdE4+ may play an important role in the regulation of Cx expression in ECs, and that these effects are mediated by both the PKA/CREB and PI3K signaling pathways. PMID:15831817

Hallucinogenic 5-HT2AR agonists LSD and DOI enhance dopamine D2R protomer recognition and signaling of D2-5-HT2A heteroreceptor complexes.

PubMed

Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Narvaez, Manuel; Oflijan, Julia; Agnati, Luigi F; Fuxe, Kjell

2014-01-03

Dopamine D2LR-serotonin 5-HT2AR heteromers were demonstrated in HEK293 cells after cotransfection of the two receptors and shown to have bidirectional receptor-receptor interactions. In the current study the existence of D2L-5-HT2A heteroreceptor complexes was demonstrated also in discrete regions of the ventral and dorsal striatum with in situ proximity ligation assays (PLA). The hallucinogenic 5-HT2AR agonists LSD and DOI but not the standard 5-HT2AR agonist TCB2 and 5-HT significantly increased the density of D2like antagonist (3)H-raclopride binding sites and significantly reduced the pKiH values of the high affinity D2R agonist binding sites in (3)H-raclopride/DA competition experiments. Similar results were obtained in HEK293 cells and in ventral striatum. The effects of the hallucinogenic 5-HT2AR agonists on D2R density and affinity were blocked by the 5-HT2A antagonist ketanserin. In a forskolin-induced CRE-luciferase reporter gene assay using cotransfected but not D2R singly transfected HEK293 cells DOI and LSD but not TCB2 significantly enhanced the D2LR agonist quinpirole induced inhibition of CRE-luciferase activity. Haloperidol blocked the effects of both quinpirole alone and the enhancing actions of DOI and LSD while ketanserin only blocked the enhancing actions of DOI and LSD. The mechanism for the allosteric enhancement of the D2R protomer recognition and signalling observed is likely mediated by a biased agonist action of the hallucinogenic 5-HT2AR agonists at the orthosteric site of the 5-HT2AR protomer. This mechanism may contribute to the psychotic actions of LSD and DOI and the D2-5-HT2A heteroreceptor complex may thus be a target for the psychotic actions of hallunicogenic 5-HT2A agonists. Copyright © 2013 Elsevier Inc. All rights reserved.

Information filtering based on corrected redundancy-eliminating mass diffusion.

PubMed

Zhu, Xuzhen; Yang, Yujie; Chen, Guilin; Medo, Matus; Tian, Hui; Cai, Shi-Min

2017-01-01

Methods used in information filtering and recommendation often rely on quantifying the similarity between objects or users. The used similarity metrics often suffer from similarity redundancies arising from correlations between objects' attributes. Based on an unweighted undirected object-user bipartite network, we propose a Corrected Redundancy-Eliminating similarity index (CRE) which is based on a spreading process on the network. Extensive experiments on three benchmark data sets-Movilens, Netflix and Amazon-show that when used in recommendation, the CRE yields significant improvements in terms of recommendation accuracy and diversity. A detailed analysis is presented to unveil the origins of the observed differences between the CRE and mainstream similarity indices.

Regulation of Aspergillus nidulans CreA-Mediated Catabolite Repression by the F-Box Proteins Fbx23 and Fbx47.

PubMed

de Assis, Leandro José; Ulas, Mevlut; Ries, Laure Nicolas Annick; El Ramli, Nadia Ali Mohamed; Sarikaya-Bayram, Ozlem; Braus, Gerhard H; Bayram, Ozgur; Goldman, Gustavo Henrique

2018-06-19

The attachment of one or more ubiquitin molecules by SCF ( S kp- C ullin- F -box) complexes to protein substrates targets them for subsequent degradation by the 26S proteasome, allowing the control of numerous cellular processes. Glucose-mediated signaling and subsequent carbon catabolite repression (CCR) are processes relying on the functional regulation of target proteins, ultimately controlling the utilization of this carbon source. In the filamentous fungus Aspergillus nidulans , CCR is mediated by the transcription factor CreA, which modulates the expression of genes encoding biotechnologically relevant enzymes. Although CreA-mediated repression of target genes has been extensively studied, less is known about the regulatory pathways governing CCR and this work aimed at further unravelling these events. The Fbx23 F-box protein was identified as being involved in CCR and the Δ fbx23 mutant presented impaired xylanase production under repressing (glucose) and derepressing (xylan) conditions. Mass spectrometry showed that Fbx23 is part of an SCF ubiquitin ligase complex that is bridged via the GskA protein kinase to the CreA-SsnF-RcoA repressor complex, resulting in the degradation of the latter under derepressing conditions. Upon the addition of glucose, CreA dissociates from the ubiquitin ligase complex and is transported into the nucleus. Furthermore, casein kinase is important for CreA function during glucose signaling, although the exact role of phosphorylation in CCR remains to be determined. In summary, this study unraveled novel mechanistic details underlying CreA-mediated CCR and provided a solid basis for studying additional factors involved in carbon source utilization which could prove useful for biotechnological applications. IMPORTANCE The production of biofuels from plant biomass has gained interest in recent years as an environmentally friendly alternative to production from petroleum-based energy sources. Filamentous fungi, which naturally thrive on decaying plant matter, are of particular interest for this process due to their ability to secrete enzymes required for the deconstruction of lignocellulosic material. A major drawback in fungal hydrolytic enzyme production is the repression of the corresponding genes in the presence of glucose, a process known as carbon catabolite repression (CCR). This report provides previously unknown mechanistic insights into CCR through elucidating part of the protein-protein interaction regulatory system that governs the CreA transcriptional regulator in the reference organism Aspergillus nidulans in the presence of glucose and the biotechnologically relevant plant polysaccharide xylan. Copyright © 2018 de Assis et al.

Loss of Activin Receptor Type 1B Accelerates Development of Intraductal Papillary Mucinous Neoplasms in Mice With Activated KRAS.

PubMed

Qiu, Wanglong; Tang, Sophia M; Lee, Sohyae; Turk, Andrew T; Sireci, Anthony N; Qiu, Anne; Rose, Christian; Xie, Chuangao; Kitajewski, Jan; Wen, Hui-Ju; Crawford, Howard C; Sims, Peter A; Hruban, Ralph H; Remotti, Helen E; Su, Gloria H

2016-01-01

Activin, a member of the transforming growth factor-β (TGFB) family, might be involved in pancreatic tumorigenesis, similar to other members of the TGFB family. Human pancreatic ductal adenocarcinomas contain somatic mutations in the activin A receptor type IB (ACVR1B) gene, indicating that ACVR1B could be a suppressor of pancreatic tumorigenesis. We disrupted Acvr1b specifically in pancreata of mice (Acvr1b(flox/flox);Pdx1-Cre mice) and crossed them with LSL-KRAS(G12D) mice, which express an activated form of KRAS and develop spontaneous pancreatic tumors. The resulting Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice were monitored; pancreatic tissues were collected and analyzed by histology and immunohistochemical analyses. We also analyzed p16(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice and Cre-negative littermates (controls). Genomic DNA, total RNA, and protein were isolated from mouse tissues and primary pancreatic tumor cell lines and analyzed by reverse-transcription polymerase chain reaction, sequencing, and immunoblot analyses. Human intraductal papillary mucinous neoplasm (IPMN) specimens were analyzed by immunohistochemistry. Loss of ACVR1B from pancreata of mice increased the proliferation of pancreatic epithelial cells, led to formation of acinar to ductal metaplasia, and induced focal inflammatory changes compared with control mice. Disruption of Acvr1b in LSL-KRAS(G12D);Pdx1-Cre mice accelerated the growth of pancreatic IPMNs compared with LSL-KRAS(G12D);Pdx1-Cre mice, but did not alter growth of pancreatic intraepithelial neoplasias. We associated perinuclear localization of the activated NOTCH4 intracellular domain to the apical cytoplasm of neoplastic cells with the expansion of IPMN lesions in Acvr1b(flox/flox);LSL-KRAS(G12D);Pdx1-Cre mice. Loss of the gene that encodes p16 (Cdkn2a) was required for progression of IPMNs to pancreatic ductal adenocarcinomas in Acvr1b(flox/flox);LSL-Kras(G12D);Pdx1-Cre mice. We also observed progressive loss of p16 in human IPMNs of increasing grades. Loss of ACVR1B accelerates growth of mutant KRAS-induced pancreatic IPMNs in mice; this process appears to involve NOTCH4 and loss of p16. ACVR1B suppresses early stages of pancreatic tumorigenesis; the activin signaling pathway therefore might be a therapeutic target for pancreatic cancer. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Nucleo-cytoplasmic shuttling dynamics of the transcriptional regulators XYR1 and CRE1 under conditions of cellulase and xylanase gene expression in Trichoderma reesei

PubMed Central

Lichius, Alexander; Seidl-Seiboth, Verena; Seiboth, Bernhard; Kubicek, Christian P

2014-01-01

Trichoderma reesei is a model for investigating the regulation of (hemi-)cellulase gene expression. Cellulases are formed adaptively, and the transcriptional activator XYR1 and the carbon catabolite repressor CRE1 are main regulators of their expression. We quantified the nucleo-cytoplasmic shuttling dynamics of GFP-fusion proteins of both transcription factors under cellulase and xylanase inducing conditions, and correlated their nuclear presence/absence with transcriptional changes. We also compared their subcellular localization in conidial germlings and mature hyphae. We show that cellulase gene expression requires de novo biosynthesis of XYR1 and its simultaneous nuclear import, whereas carbon catabolite repression is regulated through preformed CRE1 imported from the cytoplasmic pool. Termination of induction immediately stopped cellulase gene transcription and was accompanied by rapid nuclear degradation of XYR1. In contrast, nuclear CRE1 rapidly decreased upon glucose depletion, and became recycled into the cytoplasm. In mature hyphae, nuclei containing activated XYR1 were concentrated in the colony center, indicating that this is the main region of XYR1 synthesis and cellulase transcription. CRE1 was found to be evenly distributed throughout the entire mycelium. Taken together, our data revealed novel aspects of the dynamic shuttling and spatial bias of the major regulator of (hemi-)cellulase gene expression, XYR1, in T. reesei. PMID:25302561

Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect.

PubMed

Chakraborti, Dipankar; Sarkar, Anindya; Mondal, Hossain A; Schuermann, David; Hohn, Barbara; Sarmah, Bidyut K; Das, Sampa

2008-10-01

A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.

Clinical outcomes for patients finished with the SureSmile™ method compared with conventional fixed orthodontic therapy.

PubMed

Alford, Timothy J; Roberts, W Eugene; Hartsfield, James K; Eckert, George J; Snyder, Ronald J

2011-05-01

Utilize American Board of Orthodontics (ABO) cast/radiographic evaluation (CRE) to compare a series of 63 consecutive patients, finished with manual wire bending (conventional) treatment, vs a subsequent series of 69 consecutive patients, finished by the same orthodontist using the SureSmile™ (SS) method. Records of 132 nonextraction patients were scored by a calibrated examiner blinded to treatment mode. Age and discrepancy index (DI) between groups were compared by t-tests. A chi-square test was used to compare for differences in sex and whether the patient was treated using braces only (no orthopedic correction). Analysis of covariance tested for differences in CRE outcomes and treatment times, with sex and DI included as covariates. A logarithmic transformation of CRE outcomes and treatment times was used because their distributions were skewed. Significance was defined as P < .05. Compared with conventional finishing, SS patients had significantly lower DI scores, less treatment time (∼7 months), and better CRE scores for first-order alignment-rotation and interproximal space closure; however, second-order root angulation (RA) was inferior. SS patients were treated in less time to better CRE scores for first-order rotation (AR) and interproximal space closure (IC) but on the average, malocclusions were less complex and second order root alignment was inferior, compared with patients finished with manual wire bending.

Clinical outcomes for patients finished with the SureSmile™ method compared with conventional fixed orthodontic therapy

PubMed Central

Alford, Timothy J.; Roberts, W. Eugene; Hartsfield, James K.; Eckert, George J.; Snyder, Ronald J.

2016-01-01

Objective Utilize American Board of Orthodontics (ABO) cast/radiographic evaluation (CRE) to compare a series of 63 consecutive patients, finished with manual wire bending (conventional) treatment, vs a subsequent series of 69 consecutive patients, finished by the same orthodontist using the SureSmile™ (SS) method. Materials and Methods Records of 132 nonextraction patients were scored by a calibrated examiner blinded to treatment mode. Age and discrepancy index (DI) between groups were compared by t-tests. A chi-square test was used to compare for differences in sex and whether the patient was treated using braces only (no orthopedic correction). Analysis of covariance tested for differences in CRE outcomes and treatment times, with sex and DI included as covariates. A logarithmic transformation of CRE outcomes and treatment times was used because their distributions were skewed. Significance was defined as P < .05. Results Compared with conventional finishing, SS patients had significantly lower DI scores, less treatment time (~7 months), and better CRE scores for first-order alignment-rotation and interproximal space closure; however, second-order root angulation (RA) was inferior. Conclusion SS patients were treated in less time to better CRE scores for first-order rotation (AR) and interproximal space closure (IC) but on the average, malocclusions were less complex and second order root alignment was inferior, compared with patients finished with manual wire bending. PMID:21261488

New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism.

PubMed

Meng, Fantao; Han, Yong; Srisai, Dollada; Belakhov, Valery; Farias, Monica; Xu, Yong; Palmiter, Richard D; Baasov, Timor; Wu, Qi

2016-03-29

Currently available inducible Cre/loxP systems, despite their considerable utility in gene manipulation, have pitfalls in certain scenarios, such as unsatisfactory recombination rates and deleterious effects on physiology and behavior. To overcome these limitations, we designed a new, inducible gene-targeting system by introducing an in-frame nonsense mutation into the coding sequence of Cre recombinase (nsCre). Mutant mRNAs transcribed from nsCre transgene can be efficiently translated into full-length, functional Cre recombinase in the presence of nonsense suppressors such as aminoglycosides. In a proof-of-concept model, GABA signaling from hypothalamic neurons expressing agouti-related peptide (AgRP) was genetically inactivated within 4 d after treatment with a synthetic aminoglycoside. Disruption of GABA synthesis in AgRP neurons in young adult mice led to a dramatic loss of body weight due to reduced food intake and elevated energy expenditure; they also manifested glucose intolerance. In contrast, older mice with genetic inactivation of GABA signaling by AgRP neurons had only transient reduction of feeding and body weight; their energy expenditure and glucose tolerance were unaffected. These results indicate that GABAergic signaling from AgRP neurons plays a key role in the control of feeding and metabolism through an age-dependent mechanism. This new genetic technique will augment current tools used to elucidate mechanisms underlying many physiological and neurological processes.

New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism

PubMed Central

Meng, Fantao; Han, Yong; Srisai, Dollada; Belakhov, Valery; Farias, Monica; Xu, Yong; Palmiter, Richard D.; Baasov, Timor; Wu, Qi

2016-01-01

Currently available inducible Cre/loxP systems, despite their considerable utility in gene manipulation, have pitfalls in certain scenarios, such as unsatisfactory recombination rates and deleterious effects on physiology and behavior. To overcome these limitations, we designed a new, inducible gene-targeting system by introducing an in-frame nonsense mutation into the coding sequence of Cre recombinase (nsCre). Mutant mRNAs transcribed from nsCre transgene can be efficiently translated into full-length, functional Cre recombinase in the presence of nonsense suppressors such as aminoglycosides. In a proof-of-concept model, GABA signaling from hypothalamic neurons expressing agouti-related peptide (AgRP) was genetically inactivated within 4 d after treatment with a synthetic aminoglycoside. Disruption of GABA synthesis in AgRP neurons in young adult mice led to a dramatic loss of body weight due to reduced food intake and elevated energy expenditure; they also manifested glucose intolerance. In contrast, older mice with genetic inactivation of GABA signaling by AgRP neurons had only transient reduction of feeding and body weight; their energy expenditure and glucose tolerance were unaffected. These results indicate that GABAergic signaling from AgRP neurons plays a key role in the control of feeding and metabolism through an age-dependent mechanism. This new genetic technique will augment current tools used to elucidate mechanisms underlying many physiological and neurological processes. PMID:26976589

The Specific Role of FAM20C in Amelogenesis

PubMed Central

Wang, X.; Jung, J.; Liu, Y.; Yuan, B.; Lu, Y.; Feng, J.Q.; Qin, C.

2013-01-01

Previously, we showed that Sox2-Cre;Fam20Cfl/fl mice in which Fam20C was ubiquitously inactivated had severe defects in dentin, enamel, and bone, along with hypophosphatemia. It remains to be determined if the enamel defects in the mice with universal inactivation of Family with sequence similarity 20-C (FAM20C) were associated with the dentin defects and whether hypophosphatemia in the knockout mice contributed to the enamel defects. In this study, we crossed Fam20Cfl/fl mice with keratin 14-Cre (K14-Cre) transgenic mice to specifically inactivate Fam20C in the epithelial cells, including the dental epithelial cells that are responsible for forming tooth enamel. X-ray, backscattered scanning electron microscopic, and histological analyses showed that the K14-Cre;Fam20Cfl/fl mice had severe enamel and ameloblast defects, while their dentin and alveolar bone were not significantly affected. Accordingly, serum biochemistry of the K14-Cre;Fam20Cfl/fl mice showed normal phosphate and FGF23 levels in the circulation. Analysis of these data indicates that, while FAM20C is a molecule essential to amelogenesis, its inactivation in the dental epithelium does not significantly affect dentinogenesis. Hypophosphatemia makes no significant contribution to the enamel defects in the mice with the ubiquitous deletion of Fam20C. PMID:24026952

The specific role of FAM20C in amelogenesis.

PubMed

Wang, X; Jung, J; Liu, Y; Yuan, B; Lu, Y; Feng, J Q; Qin, C

2013-11-01

Previously, we showed that Sox2-Cre;Fam20C(fl/fl) mice in which Fam20C was ubiquitously inactivated had severe defects in dentin, enamel, and bone, along with hypophosphatemia. It remains to be determined if the enamel defects in the mice with universal inactivation of Family with sequence similarity 20-C (FAM20C) were associated with the dentin defects and whether hypophosphatemia in the knockout mice contributed to the enamel defects. In this study, we crossed Fam20C(fl/fl) mice with keratin 14-Cre (K14-Cre) transgenic mice to specifically inactivate Fam20C in the epithelial cells, including the dental epithelial cells that are responsible for forming tooth enamel. X-ray, backscattered scanning electron microscopic, and histological analyses showed that the K14-Cre;Fam20C(fl/fl) mice had severe enamel and ameloblast defects, while their dentin and alveolar bone were not significantly affected. Accordingly, serum biochemistry of the K14-Cre;Fam20C(fl/fl) mice showed normal phosphate and FGF23 levels in the circulation. Analysis of these data indicates that, while FAM20C is a molecule essential to amelogenesis, its inactivation in the dental epithelium does not significantly affect dentinogenesis. Hypophosphatemia makes no significant contribution to the enamel defects in the mice with the ubiquitous deletion of Fam20C.

Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.

PubMed

Tetteh, Paul W; Kretzschmar, Kai; Begthel, Harry; van den Born, Maaike; Korving, Jeroen; Morsink, Folkert; Farin, Henner; van Es, Johan H; Offerhaus, G Johan A; Clevers, Hans

2016-10-18

Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1 CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1 CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1 CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.

BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice.

PubMed

Crispino, Giulia; Di Pasquale, Giovanni; Scimemi, Pietro; Rodriguez, Laura; Galindo Ramirez, Fabian; De Siati, Romolo Daniele; Santarelli, Rosa Maria; Arslan, Edoardo; Bortolozzi, Mario; Chiorini, John A; Mammano, Fabio

2011-01-01

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26(Sox10Cre) mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10-Cre line. Cx26(Sox10Cre) mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26(Sox10Cre) mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.

Suppressing the Coffee-Ring Effect in Semitransparent MnO2 Film for a High-Performance Solar-Powered Energy Storage Window.

PubMed

Jin, Huanyu; Qian, Jiasheng; Zhou, Limin; Yuan, Jikang; Huang, Haitao; Wang, Yu; Tang, Wing Man; Chan, Helen Lai Wa

2016-04-13

We introduce a simple and effective method to deposit a highly uniform and semitransparent MnO2 film without coffee-ring effect (CRE) by adding ethanol into MnO2 ink for transparent capacitive energy storage devices. By carefully controlling the amount of ethanol added in the MnO2 droplet, we could significantly reduce the CRE and thus improve the film uniformity. The electrochemical properties of supercapacitor (SC) devices using semitransparent MnO2 film electrodes with or without CRE were measured and compared. The SC device without CRE shows a superior capacitance, high rate capability, and lower contact resistance. The CRE-free device could achieve a considerable volumetric capacitance of 112.2 F cm(-3), resulting in a high volumetric energy density and power density of 10 mWh cm(-3) and 8.6 W cm(-3), respectively. For practical consideration, both flexible SC and large-area rigid SC devices were fabricated to demonstrate their potential for flexible transparent electronic application and capacitive energy-storage window application. Moreover, a solar-powered energy storage window which consists of a commercial solar cell and our studied semitransparent MnO2-film-based SCs was assembled. These SCs could be charged by the solar cell and light up a light emitting diode (LED), demonstrating their potential for self-powered systems and energy-efficient buildings.

STIM1fl/fl Ksp-Cre Mouse has Impaired Renal Water Balance

PubMed Central

Cebotaru, Liudmila; Cebotaru, Valeriu; Wang, Hua; Arend, Lois J.; Guggino, William B.

2016-01-01

Background/AIM STIM1 is as an essential component in store operated Ca2+entry. However give the paucity of information on the role of STIM1 in kidney, the aim was to study the function of STIM1 in the medulla of the kidney. Methods we crossed a Ksp-cre mouse with another mouse containing two loxP sites flanking Exon 6 of STIM1. The Ksp-cre mouse is based upon the Ksp-cadherin gene promoter which expresses cre recombinase in developing nephrons, collecting ducts (SD) and thick ascending limbs (TAL) of the loop of Henle. Results The offspring of these mice are viable without gross morphological changes, however, we noticed that the STIM1 Ksp-cre knockout mice produced more urine compared to control. To examine this more carefully, we fed mice low (LP) and high protein (HP) diets respectively. When mice were fed HP diet STIM1 ko mice had significantly increased urinary volume and lower specific gravity compared to wt mice. In STIM1 ko mice fed HP diet urine creatinine and urea were significantly lower compared to wt mice fed HP diet, however the fractional excretion was the same. Conclusion These data support the idea that STIM1 ko mice have impaired urinary concentrating ability when challenged with HP diet is most likely caused by impaired Ca2+-dependent signal transduction through the vasopressin receptor cascade. PMID:27336410

Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle.

PubMed

Collins, Christine R; Das, Sujaan; Wong, Eleanor H; Andenmatten, Nicole; Stallmach, Robert; Hackett, Fiona; Herman, Jean-Paul; Müller, Sylke; Meissner, Markus; Blackman, Michael J

2013-05-01

Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes. © 2013 John Wiley & Sons Ltd.

Decreased the creatinine to cystatin C ratio is a surrogate marker of sarcopenia in patients with type 2 diabetes.

PubMed

Osaka, Takafumi; Hamaguchi, Masahide; Hashimoto, Yoshitaka; Ushigome, Emi; Tanaka, Muhei; Yamazaki, Masahiro; Fukui, Michiaki

2018-05-01

Sarcopenia has recently been shown to affect quality of life and mortality in patients with diabetes. However, early detection requires an expensive equipment. We hypothesized that the ratio of the creatinine/cystatin (Cre/CysC) could be used as a marker for sarcopenia. We investigated the association between the Cre/CysC ratio and sarcopenia in a cross-sectional study of patients with type 2 diabetes. Skeletal muscle mass (SMM) was estimated by bioelectrical impedance. The skeletal muscle index (SMI) was defined as the appendicular SMM divided by the square of the height. Sarcopenia was defined with SMI and a grip strength. We identified 285 patients with type 2 diabetes, of whom 25 (8.8%) had sarcopenia. The Cre/CysC ratio was associated with an increased risk of sarcopenia [odds ratio per 0.01 increment, 1.05; 95% confidence interval (CI), 1.01-1.09] after adjusting for covariates. Receiver operating characteristic curve analysis indicated that the optimal the Cre/CysC ratio cut-off point for identifying sarcopenia was 0.9, with an area under the curve, sensitivity, and specificity of 0.683 (95% CI, 0.573-0.793), 0.80, and 0.48, respectively. We recommend the Cre/CysC ratio as a practical screening marker for sarcopenia in patients with type 2 diabetes. Copyright © 2018 Elsevier B.V. All rights reserved.

Leptin induces CREB-dependent aromatase activation through COX-2 expression in breast cancer cells.

PubMed

Kim, Hyung Gyun; Jin, Sun Woo; Kim, Yong An; Khanal, Tilak; Lee, Gi Ho; Kim, Se Jong; Rhee, Sang Dal; Chung, Young Chul; Hwang, Young Jung; Jeong, Tae Cheon; Jeong, Hye Gwang

2017-08-01

Leptin plays a key role in the control of adipocyte formation, as well as in the associated regulation of energy intake and expenditure. The goal of this study was to determine if leptin-induced aromatase enhances estrogen production and induces tumor cell growth stimulation. To this end, breast cancer cells were incubated with leptin in the absence or presence of inhibitor pretreatment, and changes in aromatase and cyclooxygenase-2 (COX-2) expression were evaluated at the mRNA and protein levels. Transient transfection assays were performed to examine the aromatase and COX-2 gene promoter activities and immunoblot analysis was used to examine protein expression. Leptin induced aromatase expression, estradiol production, and promoter activity in breast cancer cells. Protein levels of phospho-STAT3, PKA, Akt, ERK, and JNK were increased by leptin. Leptin also significantly increased cAMP levels, cAMP response element (CRE) activation, and CREB phosphorylation. In addition, leptin induced COX-2 expression, promoter activity, and increased the production of prostaglandin E 2 . Finally, a COX-2 inhibitor and aromatase inhibitor suppressed leptin-induced cell proliferation in MCF-7 breast cancer cells. Together, our data show that leptin increased aromatase expression in breast cancer cells, which was correlated with COX-2 upregulation, mediated through CRE activation and cooperation among multiple signaling pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Targeted disruption of Pten in ovarian granulosa cells enhances ovulation and extends the life span of luteal cells.

PubMed

Fan, Heng-Yu; Liu, Zhilin; Cahill, Nicola; Richards, JoAnne S

2008-09-01

FSH activates the phosphatidylinositol-3 kinase (PI3K)/acute transforming retrovirus thymoma protein kinase pathway and thereby enhances granulosa cell differentiation in culture. To identify the physiological role of the PI3K pathway in vivo we disrupted the PI3K suppressor, Pten, in developing ovarian follicles. To selectively disrupt Pten expression in granulosa cells, Ptenfl/fl mice were mated with transgenic mice expressing cAMP response element recombinase driven by Cyp19 promoter (Cyp19-Cre). The resultant Pten mutant mice were fertile, ovulated more oocytes, and produced moderately more pups than control mice. These physiological differences in the Pten mutant mice were associated with hyperactivation of the PI3K/acute transforming retrovirus thymoma protein kinase pathway, decreased susceptibility to apoptosis, and increased proliferation of mutant granulosa cells. Strikingly, corpora lutea of the Pten mutant mice persisted longer than those of control mice. Although the follicular and luteal cell steroidogenesis in Ptenfl/fl;Cyp19-Cre mice was similar to controls, viable nonsteroidogenic luteal cells escaped structural luteolysis. These findings provide the novel evidence that Pten impacts the survival/life span of granulosa/luteal cells and that its loss not only results in the facilitated ovulation but also in the persistence of nonsteroidogenic luteal structures in the adult mouse ovary.

Cannabinoid inhibition of adenylate cyclase-mediated signal transduction and interleukin 2 (IL-2) expression in the murine T-cell line, EL4.IL-2.

PubMed

Condie, R; Herring, A; Koh, W S; Lee, M; Kaminski, N E

1996-05-31

Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

The Fate of Visible Features of Invisible Elements

PubMed Central

Herzog, Michael H.; Otto, Thomas U.; Ögmen, Haluk

2012-01-01

To investigate the integration of features, we have developed a paradigm in which an element is rendered invisible by visual masking. Still, the features of the element are visible as part of other display elements presented at different locations and times (sequential metacontrast). In this sense, we can “transport” features non-retinotopically across space and time. The features of the invisible element integrate with features of other elements if and only if the elements belong to the same spatio-temporal group. The mechanisms of this kind of feature integration seem to be quite different from classical mechanisms proposed for feature binding. We propose that feature processing, binding, and integration occur concurrently during processes that group elements into wholes. PMID:22557985

Identification of cis-elements for ethylene and circadian regulation of the Solanum melongena gene encoding cysteine proteinase.

PubMed

Rawat, Reetika; Xu, Zeng-Fu; Yao, Kwok-Ming; Chye, Mee-Len

2005-03-01

We have previously shown that the expression of SmCP which encodes Solanum melongena cysteine proteinase is ethylene-inducible and is under circadian control. To understand the regulation of SmCP, a 1.34-kb SmCP 5'-flanking region and its deletion derivatives were analyzed for cis-elements using GUS and luc fusions and by in vitro binding assays. Analysis of transgenic tobacco transformed with SmCP promoter-GUS constructs confirmed that the promoter region -415/+54 containing Ethylene Responsive Element ERE(-355/-348) conferred threefold ethylene-induction of GUS expression, while -827/+54 which also contains ERE(-683/-676), produced fivefold induction. Using gel mobility shift assays, we demonstrated that each ERE binds nuclear proteins from both ethephon-treated and untreated 5-week-old seedlings, suggesting that different transcriptions factors bind each ERE under varying physiological conditions. Binding was also observed in extracts from senescent, but not young, fruits. The variation in binding at the EREs in fruits and seedlings imply that organ-specific factors may participate in binding. Analysis of transgenic tobacco expressing various SmCP promoter-luc constructs containing wild-type or mutant Evening Elements (EEs) confirmed that both conserved EEs at -795/-787 and -785/-777 are important in circadian control. We confirmed the binding of total nuclear proteins to EEs in gel mobility shift assays and in DNase I footprinting. Our results suggest that multiple proteins bind the EEs which are conserved in plants other than Arabidopsis and that functional EEs and EREs are present in the 5'-flanking region of a gene encoding cysteine proteinase.

Genome-Wide Profiling of p63 DNA–Binding Sites Identifies an Element that Regulates Gene Expression during Limb Development in the 7q21 SHFM1 Locus

PubMed Central

Oti, Martin; Dutilh, Bas E.; Alonso, M. Eva; de la Calle-Mustienes, Elisa; Smeenk, Leonie; Rinne, Tuula; Parsaulian, Lilian; Bolat, Emine; Jurgelenaite, Rasa; Huynen, Martijn A.; Hoischen, Alexander; Veltman, Joris A.; Brunner, Han G.; Roscioli, Tony; Oates, Emily; Wilson, Meredith; Manzanares, Miguel; Gómez-Skarmeta, José Luis; Stunnenberg, Hendrik G.; Lohrum, Marion; van Bokhoven, Hans; Zhou, Huiqing

2010-01-01

Heterozygous mutations in p63 are associated with split hand/foot malformations (SHFM), orofacial clefting, and ectodermal abnormalities. Elucidation of the p63 gene network that includes target genes and regulatory elements may reveal new genes for other malformation disorders. We performed genome-wide DNA–binding profiling by chromatin immunoprecipitation (ChIP), followed by deep sequencing (ChIP–seq) in primary human keratinocytes, and identified potential target genes and regulatory elements controlled by p63. We show that p63 binds to an enhancer element in the SHFM1 locus on chromosome 7q and that this element controls expression of DLX6 and possibly DLX5, both of which are important for limb development. A unique micro-deletion including this enhancer element, but not the DLX5/DLX6 genes, was identified in a patient with SHFM. Our study strongly indicates disruption of a non-coding cis-regulatory element located more than 250 kb from the DLX5/DLX6 genes as a novel disease mechanism in SHFM1. These data provide a proof-of-concept that the catalogue of p63 binding sites identified in this study may be of relevance to the studies of SHFM and other congenital malformations that resemble the p63-associated phenotypes. PMID:20808887

Probing a 2-Aminobenzimidazole Library for Binding to RNA Internal Loops via Two-Dimensional Combinatorial Screening

PubMed Central

Velegapudi, Sai Pradeep; Pushechnikov, Alexei; Labuda, Lucas P.; French, Jonathan M.; Disney, Matthew D.

2012-01-01

There are many potential RNA drug targets in bacterial, viral, and the human transcriptomes. However, there are few small molecules that modulate RNA function. This is due, in part, to a lack of fundamental understanding about RNA-ligand interactions including the types of small molecules that bind to RNA structural elements and the RNA structural elements that bind to small molecules. In an effort to better understand RNA-ligand interactions, we diversified the 2-aminobenzimidazole core (2AB) and probed the resulting library for binding to a library of RNA internal loops. We chose the 2AB core for these studies because it is a privileged scaffold for binding RNA based on previous reports. These studies identified that N-methyl pyrrolidine, imidazole, and propylamine diversity elements at the R1 position increase binding to internal loops; variability at the R2 position is well tolerated. The preferred RNA loop space was also determined for five ligands using a statistical approach and identified trends that lead to selective recognition. PMID:22958065

The structure of distractor-response bindings: Conditions for configural and elemental integration.

PubMed

Moeller, Birte; Frings, Christian; Pfister, Roland

2016-04-01

Human action control is influenced by bindings between perceived stimuli and responses carried out in their presence. Notably, responses given to a target stimulus can also be integrated with additional response-irrelevant distractor stimuli that accompany the target (distractor-response binding). Subsequently reencountering such a distractor then retrieves the associated response. Although a large body of evidence supports the existence of this effect, the specific structure of distractor-response bindings is still unclear. Here, we test the predictions derived from 2 possible assumptions about the structure of bindings between distractors and responses. According to a configural approach, the entire distractor object is integrated with a response, and only upon repetition of the entire distractor object the associated response would be retrieved. According to an elemental approach, one would predict integration of individual distractor features with the response and retrieval due to the repetition of an individual distractor feature. Four experiments indicate that both, configural and elemental bindings exist and specify boundary conditions for each type of binding. These findings provide detailed insights into the architecture of bindings between response-irrelevant stimuli and actions and thus allow for specifying how distractor stimuli influence human behavior. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae.

PubMed

Koibuchi, K; Nagasaki, H; Yuasa, A; Kataoka, J; Kitamoto, K

2000-07-01

A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of L-glutamine but also of D-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137-143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5'-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae.

A Missense Mutation in PPP1R15B Causes a Syndrome Including Diabetes, Short Stature, and Microcephaly

PubMed Central

Abdulkarim, Baroj; Igoillo-Esteve, Mariana; Daures, Mathilde; Romero, Sophie; Philippi, Anne; Senée, Valérie; Lopes, Miguel; Cunha, Daniel A.; Harding, Heather P.; Derbois, Céline; Bendelac, Nathalie; Hattersley, Andrew T.; Eizirik, Décio L.; Ron, David

2015-01-01

Dysregulated endoplasmic reticulum stress and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) are associated with pancreatic β-cell failure and diabetes. Here, we report the first homozygous mutation in the PPP1R15B gene (also known as constitutive repressor of eIF2α phosphorylation [CReP]) encoding the regulatory subunit of an eIF2α-specific phosphatase in two siblings affected by a novel syndrome of diabetes of youth with short stature, intellectual disability, and microcephaly. The R658C mutation in PPP1R15B affects a conserved amino acid within the domain important for protein phosphatase 1 (PP1) binding. The R658C mutation decreases PP1 binding and eIF2α dephosphorylation and results in β-cell apoptosis. Our findings support the concept that dysregulated eIF2α phosphorylation, whether decreased by mutation of the kinase (EIF2AK3) in Wolcott-Rallison syndrome or increased by mutation of the phosphatase (PPP1R15B), is deleterious to β-cells and other secretory tissues, resulting in diabetes associated with multisystem abnormalities. PMID:26159176

SOCS3

PubMed Central

White, Christine A; Nicola, Nicos A

2013-01-01

SOCS3 is an inducible negative feedback inhibitor of cytokine signaling. Conditional deletion of SOCS3 in mice using the Cre-lox system has now been applied to a range of cell types in the steady-state and under inflammatory, pathogenic, or tumorigenic stress, with the resulting phenotypes demonstrating the effects of SOCS3 in physiological and disease contexts. Together with recent structural and biochemical studies on the mechanisms of SOCS3 binding to cytokine receptors and associated kinases, we now have a better understanding of the non-redundant roles of SOCS3 in the inhibition of cytokine signaling via the receptors gp130, G-CSFR, leptinR, and IL-12Rβ. This review discusses the known functional activities of SOCS3 in fertility and development, inflammation, innate and adaptive immunity, and malignancy as determined by genetic studies in mice. PMID:24416642

Mining the protein data bank with CReF to predict approximate 3-D structures of polypeptides.

PubMed

Dorn, Márcio; de Souza, Osmar Norberto

2010-01-01

n this paper we describe CReF, a Central Residue Fragment-based method to predict approximate 3-D structures of polypeptides by mining the Protein Data Bank (PDB). The approximate predicted structures are good enough to be used as starting conformations in refinement procedures employing state-of-the-art molecular mechanics methods such as molecular dynamics simulations. CReF is very fast and we illustrate its efficacy in three case studies of polypeptides whose sizes vary from 34 to 70 amino acids. As indicated by the RMSD values, our initial results show that the predicted structures adopt the expected fold, similar to the experimental ones.

Information filtering based on corrected redundancy-eliminating mass diffusion

PubMed Central

Zhu, Xuzhen; Yang, Yujie; Chen, Guilin; Medo, Matus; Tian, Hui

2017-01-01

Methods used in information filtering and recommendation often rely on quantifying the similarity between objects or users. The used similarity metrics often suffer from similarity redundancies arising from correlations between objects’ attributes. Based on an unweighted undirected object-user bipartite network, we propose a Corrected Redundancy-Eliminating similarity index (CRE) which is based on a spreading process on the network. Extensive experiments on three benchmark data sets—Movilens, Netflix and Amazon—show that when used in recommendation, the CRE yields significant improvements in terms of recommendation accuracy and diversity. A detailed analysis is presented to unveil the origins of the observed differences between the CRE and mainstream similarity indices. PMID:28749976

GRP78 plays an essential role in adipogenesis and postnatal growth in mice

PubMed Central

Zhu, Genyuan; Ye, Risheng; Jung, Dae Young; Barron, Ernesto; Friedline, Randall H.; Benoit, Vivian M.; Hinton, David R.; Kim, Jason K.; Lee, Amy S.

2013-01-01

To investigate the role of GRP78 in adipogenesis and metabolic homeostasis, we knocked down GRP78 in mouse embryonic fibroblasts and 3T3-L1 preadipocytes induced to undergo differentiation into adipocytes. We also created an adipose Grp78-knockout mouse utilizing the aP2 (fatty acid binding protein 4) promoter-driven Cre-recombinase. Adipogenesis was monitored by molecular markers and histology. Tissues were analyzed by micro-CT and electron microscopy. Glucose homeostasis and cytokine analysis were performed. Our results indicate that GRP78 is essential for adipocyte differentiation in vitro. aP2-cre-mediated GRP78 deletion leads to lipoatrophy with ∼90% reduction in gonadal and subcutaneous white adipose tissue and brown adipose tissue, severe growth retardation, and bone defects. Despite severe abnormality in adipose mass and function, adipose Grp78-knockout mice showed normal plasma triglyceride levels, and plasma glucose and insulin levels were reduced by 40-60% compared to wild-type mice, suggesting enhanced insulin sensitivity. The endoplasmic reticulum is grossly expanded in the residual mutant white adipose tissue. Thus, these studies establish that GRP78 is required for adipocyte differentiation, glucose homeostasis, and balanced secretion of adipokines. Unexpectedly, the phenotypes and metabolic parameters of the mutant mice, which showed early postnatal mortality, are uniquely distinct from previously characterized lipodystrophic mouse models.—Zhu, G., Ye, R., Jung, D. Y., Barron, E., Friedline, R. H., Benoit, V. M., Hinton, D. R., Kim, J. K., Lee, A. S. GRP78 plays an essential role in adipogenesis and postnatal growth in mice. PMID:23180827

Bisphenol A differentially activates protein kinase C isoforms in murine placental tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Wenjuan; Huang, Hui; Wang, Yanfei

2013-06-01

Bisphenol A is utilized to make polycarbonate plastics and is an environmental pollutant. Recent research has indicated that it is an endocrine disruptor and may interfere with reproductive processes. Our lab has previously shown that bisphenol A could regulate corticotrophin releasing hormone and aromatase in cultured placental cells. In the present study, the effect of bisphenol A on these two genes in the placenta was investigated in mice. Pregnant ICR mice were gavaged with bisphenol A at 2, 20 and 200 mg/kg body weight/day from E13 to E16 and were euthanized at E17. Compared to the control mice, increased plasmamore » estrogen and corticotrophin releasing hormone were observed in bisphenol A-treated mice. Messenger RNA quantification indicated that placental crh but not cyp19 was induced in mice treated with bisphenol A. Tracking the related signaling pathway, we found that protein kinase C ζ/λ and δ were activated in the placentas of bisphenol A-treated mice. As the gene promoter of crh contains CRE and the half site of ERE, either phospho-PKC or estrogen could stimulate the gene transactivation. These results indicate that bisphenol A might increase plasma concentrations of estradiol, testosterone, corticotrophin releasing hormone and placental phospho-PKC ζ/λ and δ in mice. Ultimately, the incidence of premature birth in these mice could increase. - Highlights: • The pollutant bisphenol A differentially activated PKC isoforms in the placenta. • CRE-binding activity in the nuclear protein of placenta was increased. • Bisphenol A induces CRH mRNA expression in mice.« less

An ABA-responsive DRE-binding protein gene from Setaria italica, SiARDP, the target gene of SiAREB, plays a critical role under drought stress.

PubMed

Li, Cong; Yue, Jing; Wu, Xiaowei; Xu, Cong; Yu, Jingjuan

2014-10-01

The DREB (dehydration-responsive element binding)-type transcription factors regulate the expression of stress-inducible genes by binding the DRE/CRT cis-elements in promoter regions. The upstream transcription factors that regulate the transcription of DREB transcription factors have not been clearly defined, although the function of DREB transcription factors in abiotic stress is known. In this study, an abscisic acid (ABA)-responsive DREB-binding protein gene (SiARDP) was cloned from foxtail millet (Setaria italica). The transcript level of SiARDP increased not only after drought, high salt, and low temperature stresses, but also after an ABA treatment in foxtail millet seedlings. Two ABA-responsive elements (ABRE1: ACGTGTC; ABRE2: ACGTGGC) exist in the promoter of SiARDP. Further analyses showed that two ABA-responsive element binding (AREB)-type transcription factors, SiAREB1 and SiAREB2, could physically bind to the ABRE core element in vitro and in vivo. The constitutive expression of SiARDP in Arabidopsis thaliana enhanced drought and salt tolerance during seed germination and seedling development, and overexpression of SiARDP in foxtail millet improved drought tolerance. The expression levels of target genes of SiARDP were upregulated in transgenic Arabidopsis and foxtail millet. These results reveal that SiARDP, one of the target genes of SiAREB, is involved in ABA-dependent signal pathways and plays a critical role in the abiotic stress response in plants. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Comparing anterior and posterior Hox complex formation reveals guidelines for predicting cis-regulatory elements

PubMed Central

Uhl, Juli D.; Cook, Tiffany A.; Gebelein, Brian

2010-01-01

Hox transcription factors specify numerous cell fates along the anterior-posterior axis by regulating the expression of downstream target genes. While expression analysis has uncovered large numbers of de-regulated genes in cells with altered Hox activity, determining which are direct versus indirect targets has remained a significant challenge. Here, we characterize the DNA binding activity of Hox transcription factor complexes on eight experimentally verified cis-regulatory elements. Hox factors regulate the activity of each element by forming protein complexes with two cofactor proteins, Extradenticle (Exd) and Homothorax (Hth). Using comparative DNA binding assays, we found that a number of flexible arrangements of Hox, Exd, and Hth binding sites mediate cooperative transcription factor complexes. Moreover, analysis of a Distal-less regulatory element (DMXR) that is repressed by abdominal Hox factors revealed that suboptimal binding sites can be combined to form high affinity transcription complexes. Lastly, we determined that the anterior Hox factors are more dependent upon Exd and Hth for complex formation than posterior Hox factors. Based upon these findings, we suggest a general set of guidelines to serve as a basis for designing bioinformatics algorithms aimed at identifying Hox regulatory elements using the wealth of recently sequenced genomes. PMID:20398649

Extended HSR/CARD domain mediates AIRE binding to DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maslovskaja, Julia, E-mail: julia.maslovskaja@ut.ee; Saare, Mario; Liiv, Ingrid

Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved inmore » AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.« less

An experimental investigation of fractionation by sputter deposition. [application to solar wind irradiation of lunar soil

NASA Technical Reports Server (NTRS)

Paruso, D. M.; Cassidy, W. A.; Hapke, B. W.

1978-01-01

Artificial glass targets composed of elements varying widely in atomic weight were irradiated at an angle of incidence of 45 deg by 2-keV hydrogen ions at a current density of .33 mA/sq cm, and sputtered atoms were caught on a molybdenum film. Analyses of the sputter-deposited films and unsputtered target glasses were carried out by electron microprobe. The backward-sputtered component was found to be enriched in elements of low atomic weight, while the forward-sputtered component was enriched in heavy atoms. These results indicate that at the lunar surface lighter elements and isotopes would tend to be ejected in backward directions, escaping directly through the openings which admit bombarding ions without first striking an adjacent grain surface; heavy elements and isotopes would be forward-sputtered deeper into the soil and be preferentially retained, contributing to the reported enrichments of heavy elements and isotopes. Additional results show that the binding energy of an element in its oxide form influences the sticking coefficient of a sputtered atom; elements of low binding energy are likely to desorb, while elements of high binding energy tend to stick to the first bounce surface.

Does Couple and Relationship Education Work for Individuals in Stepfamilies? A Meta-Analytic Study

ERIC Educational Resources Information Center

Lucier-Greer, Mallory; Adler-Baeder, Francesca

2012-01-01

Recent meta-analytic efforts have documented how couple and relationship education (CRE) programs promote healthy relationship and family functioning. The current meta-analysis contributes to this body of literature by examining stepfamily couples, an at-risk, subpopulation of participants, and assessing the effectiveness of CRE programs for…

Normalizing Effective Conflict Management through Academic Curriculum Integration: The Example of Workable Peace

ERIC Educational Resources Information Center

Smith, Stacie Nicole; Fairman, David

2004-01-01

Conflict resolution education (CRE) grew out of several parallel efforts: integrating social justice into schools, concerns about safety and youth violence, and desires to enhance responsible citizenship. Today, CRE encompasses, or is a component of, a broad range of initiatives in schools: violence prevention programs, diversity and tolerance…

The Terminator mouse is a diphtheria toxin-receptor knock-in mouse strain for rapid and efficient enrichment of desired cell lineages.

PubMed

Guo, Jian-Kan; Shi, Hongmei; Koraishy, Farrukh; Marlier, Arnaud; Ding, Zhaowei; Shan, Alan; Cantley, Lloyd G

2013-11-01

Biomedical research often requires primary cultures of specific cell types, which are challenging to obtain at high purity in a reproducible manner. Here we engineered the murine Rosa26 locus by introducing the diphtheria toxin receptor flanked by loxP sites. The resultant strain was nicknamed the Terminator mouse. This approach results in diphtheria toxin-receptor expression in all non-Cre expressing cell types, making these cells susceptible to diphtheria toxin exposure. In primary cultures of kidney cells derived from the Terminator mouse, over 99.99% of cells were dead within 72 h of diphtheria toxin treatment. After crossing the Terminator with the podocin-Cre (podocyte specific) mouse or the Ggt-Cre (proximal tubule specific) mouse, diphtheria toxin treatment killed non-Cre expressing cells but spared podocytes and proximal tubule cells, respectively, enriching the primary cultures to over 99% purity, based on both western blotting and immunostaining of marker proteins. Thus, the Terminator mouse can be a useful tool to selectively and reproducibly obtain even low-abundant cell types at high quantity and purity.

Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation

PubMed Central

Ng, Ashley P.; Kauppi, Maria; Metcalf, Donald; Hyland, Craig D.; Josefsson, Emma C.; Lebois, Marion; Zhang, Jian-Guo; Baldwin, Tracey M.; Di Rago, Ladina; Hilton, Douglas J.; Alexander, Warren S.

2014-01-01

Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (MplPF4cre/PF4cre). MplPF4cre/PF4cre mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool. PMID:24711413