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At least 415 records · Page 23Linked to original sources
PubMed2004

A screen for downstream effectors of Neurogenin2 in the embryonic neocortex.

Neurogenin (Ngn) 1 and Ngn2 encode basic-helix-loop-helix transcription factors expressed in the developing neocortex. Like other proneural genes, Ngns participate in the specification of neural fates and neuronal identities, but downstream effectors remain poorly defined. We set out to identify Ngn2 effectors in the cortex using a subtractive hybridization screen and identified several regionally expressed genes that were misregulated in Ngn2 and Ngn1;Ngn2 mutants. Included were genes down-regulated in germinal zone progenitors (e.g., Nlgn1, Unc5H4, and Dcc) and in postmitotic neurons in the cortical plate (e.g., Bhlhb5 and NFIB) and subplate (e.g., Mef2c, srGAP3, and protocadherin 9). Further analysis revealed that Ngn2 mutant subplate neurons were misspecified and that thalamocortical afferents (TCAs) that normally target this layer instead inappropriately projected towards the germinal zone. Strikingly, EphA5 and Sema3c, which encode repulsive guidance cues, were down-regulated in the Ngn2 and Ngn1;Ngn2 mutant germinal zones, providing a possible molecular basis for axonal targeting defects. Thus, we identified several new components of the differentiation cascade(s) activated downstream of Ngn1 and Ngn2 and provided novel insights into a new developmental process controlled by these proneural genes. Further analysis of the genes isolated in our screen should provide a fertile basis for understanding the molecular mechanisms underlying corticogenesis.

Animals↗
PubMed2004

Identification of downstream genes of the ascidian muscle determinant gene Ci-macho1.

Autonomous differentiation of primary muscle cells in ascidian embryos is triggered by a maternal determinant recently identified as the macho-1 gene. macho-1 encodes a transcription factor of the Zic family with five C2H2 zinc-finger motifs. In the present study, we firstly performed a screen, using a quantitative PCR method, of genes encoding transcription factors and components in major signaling pathways to identify those regulated downstream of Ci-macho1 in early embryos of Ciona intestinalis. The amount of transcripts for a total of 64 genes was altered at the 32-cell stage depending on the Ci-macho1 activity level. Whole-mount in situ hybridization assays revealed that the alteration of expression for at least 13 of them was adequately visualized to confirm the results of quantitative PCR. Second, we determined a possible binding sequence of Ciona macho1. macho1 recombinant proteins of both C. intestinalis and Ciona savignyi recognized a sequence, 5'-GCCCCCCGCTG-3', that resembles the mammalian Zic binding site. In addition, most of the genes identified as potential Ci-macho1 downstream genes, in particular Ci-Tbx6b and Ci-snail, possessed plausible Ci-macho1-binding sequences in their 5' upstream region, suggesting their direct activation by Ci-macho1. Furthermore, some of the genes including three Wnt genes noted in the quantitative analyses implied that Ci-macho1 is involved in the differentiation of endoderm and mesenchyme via intracellular communications.

Animals↗
PubMed2006

Long-range downstream enhancers are essential for Pax6 expression.

Pax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus. In this 420 kb YAC a tauGFP-IRES-Neomycin reporter cassette has been inserted into the PAX6 translational start site in exon 4. The YAC has been further engineered to insert LoxP sites flanking a 35 kb long, distant downstream regulatory region (DRR) containing previously described DNaseI hypersensitive sites, to allow direct comparison between the presence or absence of this region in the same genomic context. Five independent transgenic lines were obtained that vary in the extent of downstream PAX6 locus that has integrated. Analysis of transgenic embryos carrying full-length and truncated versions of the YAC indicates the location and putative function of several novel tissue-specific enhancers. Absence of these distal regulatory elements abolishes expression in specific tissues despite the presence of more proximal enhancers with overlapping specificity, strongly suggesting interaction between these control elements. Using plasmid-based reporter transgenic analysis we provide detailed characterization of one of these enhancers in isolation. Furthermore, we show that overexpression of a short PAX6 isoform derived from an internal promoter in a multicopy YAC transgenic line results in a microphthalmia phenotype. Finally, direct comparison of a single-copy line with the floxed DRR before and after Cre-mediated deletion demonstrates unequivocally the essential role of these long-range control elements for PAX6 expression.

Animals↗
PubMed2004

Determination of downstream targets of FGF signalling using gene trap and cDNA subtractive approaches.

Signalling through the fibroblast growth factor family (FGF) of ligands is essential for normal mammalian embryonic development. At a cellular level, many details of the molecular basis of the signal transduction process have been uncovered, but our knowledge of the identity of the downstream effectors of the FGF signal in the developing embryo remains limited. We have used two independent approaches to begin to identify downstream targets of FGF signalling in the embryo: (1). a gene trap approach and (2). cDNA subtraction, using mouse embryonic stem (ES) cells as a cellular system representative of an early window on the developing embryo. Both approaches led to the identification of a number of targets of FGF signalling, and we provide data to show that the chaperone Mrj, the tumour antigen Tum, collapsin mediator response protein Crmp, a novel transcriptional repressor Nac1 and ribophorin are all differentially regulated following FGF signalling. Independent gene trapping of Mrj previously indicated a role for the gene in embryogenesis [Development 126 (1999) 1247], and we present transcript data implicating a number of the newly isolated FGF target genes in different embryonic processes.

Animals↗
PubMed2004

P27 expression is regulated by separate signaling pathways, downstream of Ras, in each cell cycle phase.

The cyclin inhibitory protein p27Kip1 (p27) plays a vital role in regulating cell proliferation in response to the extracellular growth environment. Active proliferation requires the suppression of p27 levels throughout the cell cycle. Late in the cell cycle, p27 degradation requires phosphorylation of Thr 187 by cyclin dependent kinase 2, leading to recognition by the SCF ubiquitin ligase containing the Skp2 F-box protein. Suppression of p27 is also essential for cell proliferation early in the cell cycle, but this occurs independently of Skp2, whose expression is suppressed during G1 phase. In this study, we use a time lapse and quantitative imaging approach to study the connection between proliferative signaling and the degradation of p27 during each cell cycle period in actively cycling cells. Ras activity was required for the suppression of p27 levels throughout the cell cycle, but separate pathways downstream of Ras signaling were required in different cell cycle periods. For example, inhibitors of MEK and phosphatidylinositol-3-kinase induced p27 expression primarily in G1 phase, while inhibitors of AKT activity stimulated these levels primarily in S phase. Skp2 was expressed in a Ras-dependent manner at higher levels late in the cell cycle. Its ablation resulted in higher p27 levels primarily in G2 phase as expected. The fact that separate signaling pathways downstream of Ras function in each cell cycle phase to suppress p27 levels helps explain the vital connection between proliferative signaling, cell cycle control, and p27 expression.

Animals↗
PubMed1999

Downstream resistance effects of intracoronary thrombosis in the stenosed canine coronary artery.

OBJECTIVE: The presence is well established in unstable angina of intracoronary thrombosis in a stenosed epicardial coronary artery. The effects of the thrombus formation on the distal microcirculation are however still unclear. METHODS: We adapted the Folts canine model of left circumflex coronary arterial stenosis and intracoronary thrombosis by the insertion of a pressure catheter distal to the stenosis and by the use of 15 microns radioactive microspheres for measurement of regional myocardial blood flow. This permitted measurement during circumflex artery occlusion of collateral flow, downstream vascular resistance and collateral resistance. RESULTS: Distal circumflex resistance, obtained by dividing the distal circumflex coronary pressure gradient by the collateral flow, significantly increased with thrombosis (94.47 +/- 35.72 to 120.06 +/- 34.47; p = 0.0018) mmHg/ml/min/g. Changes in collateral flow and resistance in the presence of thrombosis, during maximum ischaemic vasodilatation, were inconsistent. CONCLUSION: Thrombosis causes increased vascular resistance in the microcirculation distal to the site of injury. This may be of clinical relevance in unstable angina, characterised by episodes of thrombus growth and embolization, in which ischaemic episodes may be worsened by generalised downstream vascular changes.

Angina, Unstable↗
PubMed2003

Activation of Gbetagamma signaling downstream of Wnt-11/Xfz7 regulates Cdc42 activity during Xenopus gastrulation.

Wnt-11/Xfz7 signaling plays a major role in the regulation of convergent extension movements affecting the dorsal marginal zone (DMZ) of gastrulating Xenopus embryos. In order to provide data concerning the molecular targets of Wnt-11/Xfz7 signals, we have analyzed the regulation of the Rho GTPase Cdc42 by Wnt-11. In animal cap ectoderm, Cdc42 activity increases as a response to Wnt-11 expression. This increase is inhibited by pertussis toxin, or sequestration of free Gbetagamma subunits by exogenous Galphai2 or Galphat. Activation of Cdc42 is also produced by the expression of bovine Gbeta1 and Ggamma2. This process is abolished by a PKC inhibitor, while phorbol esther treatment of ectodermal explants activates Cdc42 in a PKC-dependent way, implicating PKC downstream of Gbetagamma. In activin-treated animal caps and in the embryo, interference with Gbetagamma signaling rescues morphogenetic movements inhibited by Wnt-11 hyperactivation, thus phenocopying the dominant negative version of Cdc42 (N(17)Cdc42). Conversely, expression of Gbeta1gamma2 blocks animal cap elongation. This effect is reversed by N(17)Cdc42. Together, our results strongly argue for a role of Gbetagamma signaling in the regulation of Cdc42 activity downstream of Wnt-11/Xfz7 in mesodermal cells undergoing convergent extension. This idea is further supported by the observation that expression of Galphat in the DMZ causes severe gastrulation defects.

Activins↗
PubMed2003

The CBP coactivator functions both upstream and downstream of Dpp/Screw signaling in the early Drosophila embryo.

The CBP histone acetyltransferase plays important roles in development and disease by acting as a transcriptional coregulator. A small reduction in the amount of Drosophila CBP (dCBP) leads to a specific loss of signaling by the TGF-beta molecules Dpp and Screw in the early embryo. We show that the expression of Screw itself, and that of two regulators of Dpp/Screw activity, Twisted-gastrulation and the Tolloid protease, is compromised in dCBP mutant embryos. This prevents Dpp/Screw from initiating a signal transduction event in the receiving cell. Smad proteins, the intracellular transducers of the signal, fail to become activated by phosphorylation in dCBP mutants, leading to diminished Dpp/Screw-target gene expression. At a slightly later stage of development, Dpp/Screw-signaling recovers in dCBP mutants, but without a restoration of Dpp/Screw-target gene expression. In this situation, dCBP acts downstream of Smad protein phosphorylation, presumably via direct interactions with the Drosophila Smad protein Mad. It appears that a major function of dCBP in the embryo is to regulate upstream components of the Dpp/Screw pathway by Smad-independent mechanisms, as well as acting as a Smad coactivator on downstream target genes. These results highlight the exceptional sensitivity of components in the TGF-beta signaling pathway to a decline in CBP concentration.

Acetyltransferases↗
PubMed2001

Neurophilin-1 is a downstream target of transcription factor Ets-1 in human umbilical vein endothelial cells.

Transcription factor Ets-1 expressed in endothelial cells promotes angiogenesis. Here, we transiently overexpressed Ets-1 in human umbilical vein endothelial cells (HUVECs) and comprehensively searched for potential downstream targets of Ets-1 by cDNA microarray analysis. The expression of several angiogenesis-related genes including neuropilin-1 was augmented by the overexpression of Ets-1. Quantitative real-time RT-PCR and Western blotting confirmed the increase in the levels of neuropilin-1 mRNA and protein. In contrast, dominant negative ets-1 decreased the levels of neuropilin-1 mRNA and protein. These results indicate that neuropilin-1 is a downstream target of Ets-1 in HUVECs.

Base Sequence↗
PubMed1998

The effect of retinoic acid on the activation of the human H19 promoter by a 3' downstream region.

The human H19 is paternally imprinted (maternally expressed). It is transcribed by RNA pol II, but has no protein product. Its function is unknown. We showed that the transcription of the human H19 gene is under the simultaneous control of both a 5' upstream (promoter) region and a 3' downstream region in cell lines derived from human choriocarcinomas. Moreover, the activation of the H19 promoter by retinoic acid in cells derived from human testicular germ cell tumors is dependent upon the 3' downstream region. The possibility that the action of retinoic acid on the H19 promoter is an indirect one and involves a member of the AP2 transcription factor family is discussed.

Antineoplastic Agents↗
PubMed2000

Suspended bed chromatography, a new approach in downstream processing.

A new technique in downstream processing, suspended bed chromatography has been developed. This hybrid technique exploiting the benefits of batch adsorption and the process advantages of an enclosed column system can be carried out using established contactors and adsorbents. A 44 cm I.D. IsoPak column and the anion-exchange cellulose Express-Ion Exchanger Q were used in the purification of ovalbumin from hen-egg white. After suspension of 16.25 kg Express-Ion Q in 500 l of feedstock containing 5 g protein/l, adsorption was effected by recirculation of the suspension using the IsoPak slurry preparation station. Protein-loaded adsorbent was collected in the IsoPak column unit, where it was washed and protein desorbed using gradient elution at a flow-rate of 300 cm/h. The entire process was complete in under 3 h. With the introduction of pump-packed column systems and the availability of mechanically strong adsorbents suitable for column separations, suspended bed chromatography offers a new approach to downstream processing and provides a less challenging alternative to batch separations.

Adsorption↗
PubMed2002

A mechanistic framework for co-transcriptional folding of the HDV genomic ribozyme in the presence of downstream sequence.

Hepatitis delta virus (HDV) is a circular pathogenic RNA that uses self-cleavage by closely related 84 nt genomic and antigenomic ribozymes to facilitate the replication of its genome. Downstream of each ribozyme is a stretch of nucleotides termed the attenuator that functions to base-pair with and unfold the ribozyme into a rod-like fold. The competing rates of RNA synthesis, ribozyme folding and cleavage, and rod folding are therefore likely to affect the efficiency of co-transcriptional self-cleavage. In these studies, co-transcriptional folding of the genomic ribozyme was assayed in vitro by monitoring co-transcriptional self-cleavage of transcripts having variable lengths of sequence downstream of the ribozyme. Co-transcriptional cleavage data were simulated successfully only with kinetic models in which cleavage-inactive channels were populated during transcription. Partitioning to and escape from these channels was influenced, in part, by whether the available attenuator sequence could form structures with the ribozyme, and by the stability of such structures. Surprisingly, only 23 nt of attenuator were needed for strong inactivation of cleavage. Self-cleavage of certain 3'-virus-containing sequences could be restored, partially, by renaturation; however, self-cleavage of transcripts with a full-length attenuator could not be restored efficiently by renaturation in vitro. This suggests that in the presence of the attenuator, the cleavage-active ribozyme fold is not the thermodynamically most stable species. In accordance with this model, the efficiency of self-cleavage of the ribozyme followed by a full-length attenuator was increased by decreasing the rate of transcription. These results suggest that, in the absence of additional factors, efficient co-transcriptional cleavage of the full-length genomic HDV RNA may require cleavage to occur prior to synthesis of the attenuator.

Base Sequence↗
PubMed1996

Bypass conduit vessel wall biology substantially influences downstream myocardial contractile response to injury from ischemia and reperfusion.

Coronary vascular intraluminal release of endogenous endothelium-derived substances, such as prostacyclin, may affect downstream cardiac myocyte contractile function. With a "chronic" canine model of endothelialized and deendothelialized internal thoracic artery coronary grafts, we tested the hypothesis that higher basal release of endothelium-derived prostacyclin in internal thoracic artery bypass conduit effluent accelerates functional recovery of postischemic stunned myocardium in the intact circulation. Eleven dogs underwent left internal thoracic artery-left circumflex artery bypass, and the proximal circumflex artery was then ligated. Internal thoracic artery conduit endothelium was denuded by balloon catheter in five dogs before grafting and left intact in six dogs. After 7 days, awake dogs were studied to measure myocardial segment length in the circumflex region with ultrasonic dimension transducers, left ventricular pressure with micromanometers, and circumflex artery flow with an ultrasonic flow probe. Regional contractile function was quantified by the area beneath the linear preload recruitable stroke work relationship at baseline and at intervals after a 15-minute circumflex graft occlusion followed by 3 hours of reperfusion. Heart rate, left ventricular peak pressure, left ventricular end-diastolic pressure, left ventricular peak first derivative of pressure (dP/dt), and circumflex flow were similar (all p not significant) in endothelialized and nonendothelialized dogs during ischemia and reperfusion. Ischemia reduced the preload recruitable stroke work relationship to 44% +/- 35% of control values (p < 0.01) in endothelialized dogs and to 47% +/- 18% of control values in nonendothelialized dogs (p < 0.01) at 15 minutes of reperfusion, indicating a similar (p not significant) initial degree of injury. During 3 hours of reperfusion, the preload recruitable stroke work relationship returned to 51% +/- 17% of control values in endothelialized dogs but to only 35% +/- 20% of control values in nonendothelialized dogs (p < 0.02). Basal intraluminal release of endogenous prostanoids in excised internal thoracic artery conduits was subsequently quantified by ex vivo bioassay of vasoactive properties of conduit effluent on normal coronary artery smooth muscle. Endothelialized conduits induced greater smooth muscle relaxation than did nonendothelialized conduits (67% vs 23%), and this increased relaxation by endothelialized conduits was eliminated by indomethacin, a blocker of prostanoid synthesis. These data indicate that coronary bypass conduit endothelium-derived substances, such as prostacyclin, significantly influence downstream myocardial contractile response to ischemia and reperfusion, independent of alterations in coronary flow in the intact circulation.

Animals↗
PubMed2000

Adenosine and its receptor agonists regulate nitric oxide production and RAW 264.7 macrophages via both receptor binding and its downstream metabolites-inosine.

Adenosine and its receptor agonists enhanced the production of nitric oxide (NO) in lipopolysaccharide (LPS)-treated RAW 264.7 cells. The enhancement of LPS-induced NO production by adenosine, as represented by the amount of its oxidation products, nitrite and nitrate, was inhibited by adenosine uptake inhibitors, such as dipyridamole, S(4-nitrobenzyl)-6-thioinosine (NBTI) and S(4-nitrobenzyl)-6-thioguanosine (NBTG). These indicate that the uptake of adenosine by macrophages is a prerequisite for the enhancement effects observed. A downstream metabolite of adenosine, inosine, also potentiated the LPS-induced NO production in a dose-dependent manner while its enhancement effect was also inhibited by dipyridamole. However, the degree of enhancement by inosine on NO production and nitric oxide synthase (NOS) activity in LPS-treated RAW 264.7 was weaker than the effect of adenosine. Furthermore, adenosine agonists also enhanced the NO production in a dose-dependent manner, but were not specific for A1, A2 nor A3 adenosine receptor. Adenosine uptake inhibitors had no effects on the enhancement activity of the adenosine receptor agonists. Thus, extracellular receptor/s may also play an important role in the observed enhancement responses. The results of this study indicate that the enhancement effects of adenosine on NO production in macrophages could be mediated by the extracellular adenosine receptors as well as the downstream metabolites of adenosine.

Adenine↗
PubMed2000

Downstream change in leucine aminopeptidase activity and leucine assimilation by epilithic microbiota along the River Swale, northern England.

Parallel determinations of epilithic extracellular leucine aminopeptidase activity and leucine assimilation were made at five sites along 112 km of the River Swale and also in two tributaries, the River Wiske and Cod Beck. Epilithic leucine aminopeptidase activity along the Swale increased with distance downstream; this increase was gradual, rather than stepwise in response to specific sewage-works outfalls. Epilithic leucine assimilation, in contrast, did not consistently increase along the river. Epilithic leucine aminopeptidase activity and leucine assimilation were both potentially controlled by epilithic microbial variables (bacterial abundance and chlorophyll a) while leucine aminopeptidase activity was also strongly related to water-quality variables, especially temperature, pH and conductivity. Epilithic leucine aminopeptidase activity and leucine assimilation were coupled, but the magnitude of aminopeptidase activity was always substantially greater than that of leucine assimilation. Arguments are presented, however, which suggest that this did not necessarily indicate the constant availability of excess leucine, and by inference amino-acid nitrogen, to epilithic bacteria. Values of epilithic leucine aminopeptidase activity and leucine assimilation, expressed relative to rates in overlying water, suggested that most activity and assimilation was epilithic rather than planktonic, although the planktonic contribution was proportionately greater at the deeper, more downstream, sites. In the tributaries, River Wiske and Cod Beck, values of epilithic leucine aminopeptidase activity and epilithic microbial abundance, as well as those of many water-quality variables, resembled values in the middle and lower Swale. Thus, these tributaries were essentially lowland, enriched watercourses being very different from the headstreams of the main river.

Animals↗
PubMed2001

Downstream changes in the transport and storage of sediment-associated contaminants (P, Cr and PCBs) in agricultural and industrialized drainage basins.

Samples of suspended, floodplain and channel bed sediment have been used to examine downstream changes in ediment-associated contaminant transport and storage in contrasting rivers in Yorkshire, UK. The concentrations of hosphorus, chromium and selected PCBs associated with sediment in the River Aire and its main tributary, the River Calder, which drain an urbanized and industrialized catchment, are considerably higher than those in the relatively unpolluted River Swale, which drains an agricultural catchment. Concentrations of sediment-associated contaminants in the Aire/Calder system increase downstream, reflecting the location of urban and industrial areas in the middle and lower reaches, and the location of point source inputs, such as sewage treatment works. The ontaminant concentrations associated with floodplain and channel bed sediment in the Rivers Aire and Calder are high, particularly in the lower reaches. This, combined with measurements of sediment storage on the floodplain and channel bed, indicate that significant storage of sediment-associated contaminants occurs in the Rivers Aire and Calder.

Agriculture↗
PubMed2001

Biogeochemical removal of Zn and Cd in the Coeur D'Alene River (Idaho, USA), downstream of a mining district.

The conservative biogeochemical behavior of dissolved Zn and Cd in a 17-km, free-flowing reach of the Coeur d'Alene River downstream of a mining district is typical of watersheds in which suspended matter concentrations are low. For watersheds impacted by acid-rock drainage (ARD), low suspended matter concentrations are more likely to be found when acid rock drainage travels through soils because much of the Fe and Al that could form adsorbing surfaces is retained within the soils. In the absence of additional sources of solid substrates, metals can be transported great distances downstream once this Fe- and Al-poor ARD seeps into surface waters. In a 46-km backwater reach of the Coeur d'Alene River, it appears that biological activity increased pH and provided the organic matter solid substrate which removed approximately 50% of the Zn and Cd. Zn removal was partially reversible as pH decreased. These observations reinforce the concept that both inorganic and organic carbon chemistry must be measured if significant. advances in our understanding of the attenuation of Zn and Cd from ARD sources are to be made.

Adsorption↗
PubMed2000

Enhancement of memory-related long-term facilitation by ApAF, a novel transcription factor that acts downstream from both CREB1 and CREB2.

The memory for sensitization of the gill withdrawal reflex in Aplysia is reflected in facilitation of the monosynaptic connection between the sensory and motor neurons of the reflex. The switch from short- to long-term facilitation requires activation of CREB1, derepression of ApCREB2, and induction of ApC/EBP. In search for genes that act downstream from CREB1, we have identified a transcription activator, ApAF, which is stimulated by protein kinase A and can dimerize with both ApC/EBP and ApCREB2. ApAF is necessary for long-term facilitation induced by five pulses of serotonin, by activation of CREB1, or by derepression of ApCREB2. Overexpression of ApAF enhances the long-term facilitation further. Thus, ApAF is a candidate memory enhancer gene downstream from both CREB1 and ApCREB2.

Amino Acid Sequence↗