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At least 631 records · Page 35Linked to original sources
PubMed1985

Stimulation of sea urchin H2B histone gene transcription by a chromatin-associated protein fraction depends on gene sequences downstream of the transcription start site.

We isolated a chromosomal protein fraction derived from chromatin of sea urchin embryos which specifically stimulated the expression of the histone H2B gene by a factor of 5- to 10-fold when the complete sea urchin histone gene repeat h22 was injected in Xenopus laevis oocyte nuclei. Gene manipulation experiments revealed the existence of two different target sites in the H2B gene which appear to mediate the response to injection of the stimulatory sea urchin chromatin-associated proteins; both are located downstream of the transcription initiation site. The first sequence element which is shown to be implicated is within, or at least includes, the H2B 5' untranslated leader sequence between nucleotides 11 and 76. The second element resides within an H2B DNA segment located near the 3' end of the gene, extending from 90 base pairs upstream of the mRNA 3' terminus to 140 base pairs in the spacer sequences downstream.

Animals↗
PubMed1985

ATTAAA as well as downstream sequences are required for RNA 3'-end formation in the E3 complex transcription unit of adenovirus.

We mapped the location of the E3A RNA 3' end site in the E3 transcription unit of adenovirus 2. The procedure used was nuclease-gel analysis with 32P-labeled RNA probes. The poly(A) addition sites were microheterogeneous and were located approximately 17 to 29 nucleotides downstream from an ATTAAA sequence. To identify the sequences that make up the E3A RNA 3' end signal, we constructed five viable virus mutants with deletions in or near the E3A RNA 3' end site. The mutants were analyzed for E3A RNA 3' end formation in vivo. No effect was observed from a 47-base-pair (bp) deletion (dl716) or a 72-bp deletion (dl714) located 22 and 19 nucleotides, respectively, upstream of the ATTAAA. In contrast, E3A RNA 3' end formation was abolished by a 554-bp deletion (dl708) that removes both the ATTAAA and the poly(A) addition sites, a 124-bp deletion (dl713) that removes the ATTAAA but leaves the poly(A) addition sites, and a 65-bp deletion (dl719) that leaves the ATTAAA but removes the poly(A) addition sites. These results indicate that the ATTAAA, as well as downstream sequences, including the poly(A) addition sites, are required for E3A RNA 3' end formation.

Adenoviruses, Human↗
PubMed1986

In vitro cleavage of the simian virus 40 early polyadenylation site adjacent to a required downstream TG sequence.

Exogenous RNA containing the simian virus 40 early polyadenylation site was efficiently and accurately polyadenylated in in vitro nuclear extracts. Correct cleavage required ATP. In the absence of ATP, nonpoly(A)+ products accumulated which were 18 to 20 nucleotides longer than the RNA generated by correct cleavage; the longer RNA terminated adjacent to the downstream TG element required for polyadenylation. In the presence of ATP analogs, alternate cleavage was not observed; instead, correct cleavage without poly(A) addition occurred. ATP-independent cleavage of simian virus 40 early RNA had many of the same properties as correct cleavage including requirements for an intact AAUAAA element, a proximal 3' terminus, and extract small nuclear ribonucleoproteins. This similarity in reaction parameters suggested that ATP-independent cleavage is an activity of the normal polyadenylation machinery. The ATP-independent cleavage product, however, did not behave as an intermediate in polyadenylation. The alternate RNA did not preferentially chase into correctly cleaved material upon readdition of ATP; instead, poly(A) was added to the 3' terminus of the cleaved RNA during a chase. Purified ATP-independent cleavage RNA, however, was a substrate for correct cleavage when reintroduced into the nuclear extract. Thus, alternate cleavage of polyadenylation sites adjacent to a required downstream sequence element is directed by the polyadenylation machinery in the absence of ATP.

Adenosine Triphosphate↗
PubMed1989

Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly.

To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.

Adenoviridae↗
PubMed2007

NSAIDs and cancer prevention: targets downstream of COX-2.

Preclinical and clinical studies have clearly shown a benefit of nonsteroidal anti-inflammatory drug (NSAID) use in reducing cancer risk. However, the adverse gastrointestinal and cardiovascular side effects associated with NSAIDs and COX-2 selective inhibitors (coxibs) have provoked more scrutiny of the precise role of specific downstream mediators in the prostaglandin (PG) signaling cascade. NSAIDs and coxibs inhibit PG biosynthesis. One of the PGs produced at high levels in the tumor microenvironment is PGE(2), which is thought to play a major role in cancer progression. Thus, a better understanding of PGE(2) signaling could enable identification of novel and safer therapeutic targets downstream of the cyclooxygenase enzymes. We review the emerging molecular mechanisms by which COX-2-derived PGE(2) is involved in cancer progression and delineate potential opportunities for development of novel pharmacologic approaches utilizing this pathway.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1999

Effect of downstream cross-sectional area of an arterial bed on the resistive index and the early systolic acceleration.

PURPOSE: To evaluate the effect of the increase in total cross-sectional area of arteries as they branch beyond the main trunks on the resistive index (RI) and early systolic acceleration (ESA). MATERIALS AND METHODS: An essentially noncompliant in vitro model that used a pulsatile pump, blood-mimicking fluid, and a branching tubing network that could be configured to produce a downstream cross-sectional area one, two, four, or eight times that of the feeding vessel was used to investigate the relationship, if any, between arterial bed cross-sectional area and the RI and ESA. RESULTS: The mean ESA in the branching network was inversely proportional to cross-sectional area, decreasing by approximately a factor of two for every doubling of the cross-sectional area. The mean RI in the branching network decreased with increasing cross-sectional area, but not as greatly as the ESA did; the mean RI in the bed with eight times the upstream cross-sectional area had an RI that was approximately three-fourths the upstream RI. These relationships are real, as the slopes of the plots (ESA vs cross-sectional area, P = .001; RI vs cross-sectional area, P < .02) are significantly different from zero. CONCLUSION: RI and ESA decrease as a result of increasing downstream cross-sectional diameter of the arterial bed.

Arteries↗
PubMed2005

Proteomic analysis on insulin signaling in human hematopoietic cells: identification of CLIC1 and SRp20 as novel downstream effectors of insulin.

Insulin/IGF-I-dependent signals play important roles for the regulation of proliferation, differentiation, metabolism, and autophagy in various cells, including hematopoietic cells. Although the early protein kinase activation cascade has been intensively studied, the whole picture of intracellular signaling events has not yet been clarified. To identify novel downstream effectors of insulin-dependent signals in relatively early phases, we performed high-resolution two-dimensional electrophoresis (2-DE)-based proteomic analysis using human hematopoietic cells 1 h after insulin stimulation. We identified SRp20, a splicing factor, and CLIC1, an intracellular chloride ion channel, as novel downstream effectors besides previously reported effectors of Rho-guanine nucleotide dissociation inhibitor 2 and glutathione S-transferase-pi. Reduction in SRp20 was confirmed by one-dimensional Western blotting. Moreover, MG-132, a proteasome inhibitor, prevented this reduction. By contrast, upregulation of CLIC1 was not observed in one-dimensional Western blotting, unlike the 2-DE results. As hydrophilic proteins were predominantly recovered in 2-DE, the discrepancy between the 1-DE and 2-DE results may indicate a certain qualitative change of the protein. Indeed, the nuclear localization pattern of CLIC1 was remarkably changed by insulin stimulation. Thus insulin induces the proteasome-dependent degradation of SRp20 as well as the subnuclear relocalization of CLIC1.

Amino Acid Sequence↗
PubMed2006

Downstream regulatory element antagonistic modulator regulates islet prodynorphin expression.

Calcium-binding proteins regulate transcription and secretion of pancreatic islet hormones. Here, we demonstrate neuroendocrine expression of the calcium-binding downstream regulatory element antagonistic modulator (DREAM) and its role in glucose-dependent regulation of prodynorphin (PDN) expression. DREAM is distributed throughout beta- and alpha-cells in both the nucleus and cytoplasm. As DREAM regulates neuronal dynorphin expression, we determined whether this pathway is affected in DREAM(-/-) islets. Under low glucose conditions, with intracellular calcium concentrations of <100 nM, DREAM(-/-) islets had an 80% increase in PDN message compared with controls. Accordingly, DREAM interacts with the PDN promoter downstream regulatory element (DRE) under low calcium (<100 nM) conditions, inhibiting PDN transcription in beta-cells. Furthermore, beta-cells treated with high glucose (20 mM) show increased cytoplasmic calcium (approximately 200 nM), which eliminates DREAM's interaction with the DRE, causing increased PDN promoter activity. As PDN is cleaved into dynorphin peptides, which stimulate kappa-opioid receptors expressed predominantly in alpha-cells of the islet, we determined the role of dynorphin A-(1-17) in glucagon secretion from the alpha-cell. Stimulation with dynorphin A-(1-17) caused alpha-cell calcium fluctuations and a significant increase in glucagon release. DREAM(-/-) islets also show elevated glucagon secretion in low glucose compared with controls. These results demonstrate that PDN transcription is regulated by DREAM in a calcium-dependent manner and suggest a role for dynorphin regulation of alpha-cell glucagon secretion. The data provide a molecular basis for opiate stimulation of glucagon secretion first observed over 25 years ago.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed2006

Hop functions downstream of Nkx2.1 and GATA6 to mediate HDAC-dependent negative regulation of pulmonary gene expression.

Hop is an unusual homeodomain protein that was first identified in the developing heart where it functions downstream of Nkx2.5 to modulate cardiac gene expression. Hop functions through interactions with histone deacetylase (HDAC) 2 to mediate repression of cardiac-specific genes, and recent studies show that HDAC activity and HDAC2 expression are decreased in people with chronic obstructive pulmonary disease. Here, we show that Hop is expressed in airway epithelium coincident with HDAC2, and expression is induced by the combination of dexamethasone and cAMP in parallel with induction of surfactant protein gene expression. Hop functions in the developing pulmonary airway, acting downstream of Nkx2.1 and GATA6, to negatively regulate surfactant protein expression. Loss of Hop expression in vivo results in defective type 2 pneumocyte development with increased surfactant production and disrupted alveolar formation. Thus Hop represents a novel regulator of pulmonary maturation that is induced by glucocorticoids to mediate functionally important HDAC-dependent negative feedback regulation.

Animals↗
PubMed2006

Selective upregulation of the ADP-ribosyl cyclases CD38 and CD157 by TNF but not by RANK-L reveals differences in downstream signaling.

In macrophages and osteoclast precursors, the cytokines TNF and RANK-L induce similar downstream pathways and share some of the same adaptor molecules. However, despite these similarities, no defined signaling schematic has emerged to show how each cytokine favors particular pathways. In this report, we investigate whether TNF and RANK-L differentially regulate ADP-ribosyl cyclases-enzymes that are unique in being crucial for immunological function yet detrimental to osteoclastogenesis. TNF but not RANK-L led to the sustained upregulation of both CD38 and CD157 as demonstrated by real-time PCR and flow cytometry. Further investigation demonstrated that this upregulation was a result of continuous, direct TNF signaling and involved JNK, and more critically PKC and NF-kappaB. Using this approach allowed us to highlight the relative importance of the PKC, NF-kappaB, and JNK pathways in actualizing proper outcomes of TNF signaling. Albeit speculative, we believe that differences between TNF- and RANK-l-induced activation of downstream signaling pathways, in particular PKC, are crucial for determining whether progenitor cells become geared for immunity or bone resorption.

ADP-ribosyl Cyclase↗
PubMed2003

Contribution of signals downstream from adaptation to saccade programming.

Information about ongoing behavior is necessary for stable perception and subsequent motor planning. Although many recurrent networks are known in the motor systems, the pathways that transmit the signals for internal monitoring of behavior are not specified. The present study reports that the pathways originating from sites downstream of cerebellar adaptation provide internal signals that are used for subsequent eye-movement programming. When monkeys made two successive saccades toward the locations of previously flashed targets or initial fixation, the second saccade compensated for the adaptive changes in the primary saccade. The use of signals downstream from adaptation for saccade programming contrasts with recent findings that signals upstream from adaptation control the perceptual localization of visual stimuli presented around the time of saccade, suggesting that separate recurrent networks provide behavioral information for perception versus movement programming.

Adaptation, Physiological↗
PubMed2005

ABL oncogenes and phosphoinositide 3-kinase: mechanism of activation and downstream effectors.

The BCR-ABL oncogene is responsible for most cases of chronic myelogenous leukemia and some acute lymphoblastic leukemias. The fusion protein encoded by BCR-ABL possesses an aberrantly regulated tyrosine kinase activity. Imatinib mesylate (Gleevec, STI-571) is an inhibitor of ABL tyrosine kinase activity that has been remarkably effective in slowing disease progression in patients with chronic phase chronic myelogenous leukemia, but the emergence of imatinib resistance underscores the need for additional therapies. Targeting signaling pathways activated by BCR-ABL is a promising approach for drug development. The study of signaling components downstream of BCR-ABL and the related murine oncogene v-Abl has revealed a complex web of signals that promote cell division and survival. Of these, activation of phosphoinositide 3-kinase (PI3K) has emerged as one of the essential signaling mechanisms in ABL leukemogenesis. This review describes molecular mechanisms by which PI3K is activated and the downstream PI3K effectors that propagate the signal to promote myeloid and lymphoid transformation. Of particular recent interest is the mammalian target of rapamycin, a PI3K-regulated kinase that regulates protein synthesis and contributes to leukemogenesis.

Animals↗
PubMed2005

Uncoupling between epidermal growth factor receptor and downstream signals defines resistance to the antiproliferative effect of Gefitinib in bladder cancer cells.

Activation of the epidermal growth factor receptor (EGFR) and downstream signaling pathways, such as phosphatidylinositol-3 kinase/Akt and Ras/mitogen-activated protein kinase (MAPK), have been implicated in causing resistance to EGFR-targeted therapy in solid tumors, including the urogenital tumors. To investigate the mechanism of resistance to EGFR inhibition in bladder cancer, we compared EGFR tyrosine kinase inhibitor (Gefitinib, Iressa, ZD1839) with respect to its inhibitory effects on three kinases situated downstream of EGFR: MAPK, Akt, and glycogen synthase kinase-3beta (GSK-3beta). We found that the resistance to the antiproliferative effects of gefitinib, in vitro as well as in vivo in nude mice models, was associated with uncoupling between EGFR and MAPK inhibition, and that GSK-3beta activation and degradation of its target cyclin D1 were indicators of a high cell sensitivity to gefitinib. Further analysis of one phenotypic sensitive (253J B-V) and resistant (UM-UC13) cell lines revealed that platelet-derived growth factor receptor-beta (PDGFRbeta) activation was responsible for short circuiting the EGFR/MAPK pathway for mitogenic stimuli. However, invasion as well as actin dynamics were efficiently reduced by EGFR inhibition in UM-UC13. Chemical disruption of signaling pathways or of PDGFR kinase activity significantly reduced the inactive pool of cellular GSK-3beta in UM-UC13 cells. In conclusion, our data show that the uncoupling of EGFR with mitogenic pathways can cause resistance to EGFR inhibition in bladder cancer. Although this uncoupling may arise through different mechanisms, we suggest that the resistance of bladder cancer cells to EGFR blockade can be predicted early in the course of treatment by measuring the activation of GSK-3beta and of nuclear cyclin D1.

Antineoplastic Agents↗
PubMed2006

p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation.

The role of the cyclin-dependent kinase (CDK) inhibitor p21 as a mediator of p53-induced growth arrest is well established. In addition, recent data provide strong evidence for new emerging functions of p21, including a role as a modulator of apoptosis. The mechanisms, however, by which p21 interferes with the death machinery, especially following ionizing radiation (IR), are largely unknown. Here, we report that IR induced caspase-9 and caspase-3 activation and subsequent apoptosis only in p21-deficient colon carcinoma cells, whereas similar treated wild-type cells were permanently arrested in the G(2)-M phase, correlating with the induction of cellular senescence. Interestingly, activation of the mitochondrial pathway, including caspase-2 processing, depolarization of the outer mitochondrial membrane, and cytochrome c release, was achieved by IR in both cell lines, indicating that p21 inhibits an event downstream of mitochondria but preceding caspase-9 activation. IR-induced p21 protein expression was restricted to the nucleus, and no evidence for a mitochondrial or cytoplasmic association was found. In addition, p21 did neither interact with caspase-3 or caspase-9, suggesting that these events are not required for the observed protection. Consistent with this assumption, we found that CDK inhibitors potently abrogated IR-induced caspase processing and activation without affecting mitochondrial events. In addition, in vitro caspase activation assays yielded higher caspase-3 activities in extracts of irradiated p21-deficient cells compared with extracts of similar treated wild-type cells. Thus, our results strongly indicate that p21 protects cells from IR-induced apoptosis by suppression of CDK activity that seems to be required for activation of the caspase cascade downstream of the mitochondria.

Apoptosis↗
PubMed2006

Integrin-linked kinase functions as a downstream mediator of endothelin-1 to promote invasive behavior in ovarian carcinoma.

The endothelin-1 (ET-1) axis represents a novel target in several malignancies, including ovarian carcinoma. Upon being activated, the endothelin A receptor (ET(A)R) mediates multiple tumor-promoting activities, including mitogenesis, escape from apoptosis, angiogenesis, metastasis-related protease activation, epithelial-mesenchymal transition, and invasion. Integrin-linked kinase (ILK) is a multidomain focal adhesion protein that conveys intracellular signaling elicited by beta1-integrin and growth factor receptors. In this study, we investigate whether the signaling triggered by ET(A)R leading to an aggressive phenotype is mediated by an ILK-dependent mechanism. In HEY and OVCA 433 ovarian carcinoma cell lines, activation of ET(A)R by ET-1 enhances the expression of alpha2beta1 and alpha3beta1 integrins. ILK activity increases as ovarian cancer cells adhere to type I collagen through beta1 integrin signaling, and do so to a greater extent on ET-1 stimulation. ET-1 increases ILK mRNA and protein expression and activity in a time- and concentration-dependent manner. An ILK small-molecule inhibitor (KP-392) or transfection with a dominant-negative ILK mutant effectively blocks the phosphorylation of downstream signals, Akt and glycogen synthase kinase-3beta. The blockade of ET-1/ET(A)R-induced ILK activity results in an inhibition of matrix metalloproteinase activation as well as of cell motility and invasiveness in a phosphoinositide 3 kinase-dependent manner. In ovarian carcinoma xenografts, ABT-627, a specific ET(A)R antagonist, suppresses ILK expression, Akt and glycogen synthase kinase-3beta phosphorylation, and tumor growth. These data show that ILK functions as a downstream mediator of the ET-1/ET(A)R axis to potentiate aggressive cellular behavior. Thus, the ILK-related signaling cascade can be efficiently targeted by pharmacologic blockade of ET(A)R.

Animals↗
PubMed2003

Enhancement of translation initiation by A/T-rich sequences downstream of the initiation codon in Escherichia coli.

The region located downstream of the initiation codon constitutes part of the translation initiation signal, significantly affecting the level of protein expression in E. coli. In order to determine its influence on translation initiation, we inserted random 12-base sequences downstream of the initiation codon of the lacZ gene. A total of 119 random clones showing higher beta-galactosidase activities than the control lacZ gene were isolated and subsequently sequenced. Analysis of these clones revealed that their insertion sequences are strikingly rich in A and T, but poor in G, with no consensus sequences among them. Toeprinting experiments and polysome profile analysis confirmed that the A/T-rich sequences enhance translation at the level of initiation. Collectively, the present data demonstrate that A/T richness of the region following the initiation codon plays a significant role in E. coli gene expression.

AT Rich Sequence↗
PubMed2004

Down syndrome candidate region 1,a downstream target of VEGF, participates in endothelial cell migration and angiogenesis.

Vascular endothelial growth factor (VEGF) is a principal stimulator of angiogenesis. However, the downstream targets of VEGF in endothelial cells (ECs) are not entirely clarified. Survey of downstream targets of VEGF in human ECs identified a number of genes, including Down syndrome candidate region 1 (DSCR1). Here, we confirmed the inducible expression of DSCR1 in ECs by Northern and Western blottings. Moreover, VEGF-stimulated induction of DSCR1 was blocked by anti-VEGF receptor-2 monoclonal antibody (mAb), or the specific calcineurin inhibitors cyclosporin A and FK506. The expression of DSCR1 in ECs of neovessels was further shown by immunohistochemical analysis. We therefore examined whether DSCR1 played any roles in angiogenesis. The specific downregulation of DSCR1 expression by antisense oligonucleotide (AS-ODN) inhibited VEGF-stimulated migration of ECs as well as angiogenesis in vivo. AS-ODN inhibited the spreading of ECs on vitronectin, as well as on the immobilized anti-alphavbeta3 mAb, but not on anti-alphavbeta5 mAb. Moreover, AS-ODN inhibited tyrosine phosphorylation of focal adhesion kinase when ECs were plated on a vitronectin-coated dish. Immunoprecipitation followed by Western blotting showed the coimmunoprecipitation of DSCR1 and integrin alphavbeta3. These results suggest that DSCR1 is involved in angiogenesis by regulating adhesion and migration of ECs via the interaction with integrin alphavbeta3.

Cell Movement↗
PubMed2003

Fluorescence imaging analysis of upstream regulators and downstream targets of STAT3 in melanoma precursor lesions obtained from patients before and after systemic low-dose interferon-alpha treatment.

Atypical nevi are the precursors and risk markers of melanoma. Apart from persistently monitoring these nevocytic lesions and resecting them at the earliest signs of clinical changes, there is as yet no systemic clinical treatment available to interfere with their progression to melanoma. To explore clinical treatments that might interfere with and possibly prevent atypical nevus progression, a previous study documented that 3 months systemic low-dose interferon-alpha (IFN-alpha) treatment of patients with a clinical history of melanoma and numerous atypical nevi, led to inactivation of the STAT1 and STAT3 transcription factors in atypical nevi. Based upon this finding, we initiated a second study to determine whether systemic low-dose IFN-alpha treatment also impairs the expression of upstream regulators and downstream targets of STAT1 and STAT3 in atypical nevi. Using cyanine dye-conjugated antibodies, fluorescence imaging analysis revealed expression of JAK2, JNK1, AKT1, NF-kappa B, and IFN-alpha/beta receptor in benign and atypical nevi, and early- and advanced-stage melanomas. To determine possible changes in the level of expression of these molecules in atypical nevi, excised before and after 3 months of systemic low-dose IFN-alpha treatment, newly designed optical imaging software was used to quantitate the captured fluorescent hybridization signals on a cell-by-cell basis and across an entire nevus section. The results of this analysis did not provide evidence that systemic low-dose IFN-alpha treatment alters the level of expression of upstream regulators or downstream targets of STAT1 and STAT3.

DNA-Binding Proteins↗