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At least 739 records · Page 41Linked to original sources
PubMed2004

A single magnetic field exposure system for sequential investigation of real time and downstream cellular responses.

To be able to correlate real time membrane potential or ion flux changes with further downstream gene transcription responses due to extremely low frequency (ELF) electromagnetic field (EMF) exposure, we devised an experimental system consisting of a pair of symmetric circular coils. This system can be used on an inverted microscope stage (real time signaling) as well as inside controlled environment incubators (gene transcription end points). The system includes a unique, custom made switch box for blinding the experimental staff and a power amplifier. We report herein the design and characterization of the system with respect to parameters considered important in in vitro ELF-EMF exposure studies, including linear magnetic field distribution, compensation for microscope objective lens interference, heating effects of the coils, and harmonic content of the signals.

Cell Culture Techniques↗
PubMed2004

LAT: a T lymphocyte adapter protein that couples the antigen receptor to downstream signaling pathways.

Adapter molecules in a variety of signal transduction systems link receptors to a limited number of commonly used downstream signaling pathways. During T-cell development and mature T-cell effector function, a multichain receptor (the pre-T-cell antigen receptor or the T-cell antigen receptor) activates several protein tyrosine kinases. Receptor and kinase activation is linked to distal signaling pathways (PLC-gamma1 activation, Ca2+ influx, PKC activation and Ras/Erk activation) via the adapter protein LAT (Linker for Activation of T cells). Structure/function studies of LAT including expression of selected LAT point mutations in vivo reveals that these multiple pathways are integrated at the level of the LAT adapter. These studies suggest that similar levels of control may be found in other systems where adapter molecules are known to have important functions.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Drosophila Hox complex downstream targets and the function of homeotic genes.

Hox complex genes are key developmental regulators highly conserved throughout evolution. The encoded proteins share a 60-amino-acid DNA-binding motif, the homeodomain, and function as transcription factors to control axial patterning. An important question concerns the nature and function of genes acting downstream of Hox proteins. This review focuses on Drosophila, as little is known about this question in other organisms. The noticeable progress gained in the field during the past few years has significantly improved our current understanding of how Hox genes control diversified morphogenesis. Here we summarise the strategies deployed to identify Hox target genes and discuss how their function contributes to pattern formation and morphogenesis. The regulation of target genes is also considered with special emphasis on the mechanisms underlying the specificity of action of Hox proteins in the whole animal.

Animals↗
PubMed2002

Isolation and characterization of Xenopus Hey-1: a downstream mediator of Notch signaling.

Regulation of Notch signaling likely occurs, at least in part, at the level of basic helix-loop-helix (bHLH) transcription factors that function downstream of Suppressor of Hairless (Su(H)) in the Notch pathway. To begin to characterize modulation of Notch signaling during organogenesis, we examined the bHLH transcription factor, XHey-1 (hairy related-1) in early Xenopus laevis embryos. XHey-1 is expressed in numerous tissues during early development including the somites, head, embryonic kidneys, and heart. Importantly, the expression of XHey-1 was significantly altered in response to perturbation of Notch signaling by means of inducible constructs that served to either activate or suppress Notch signaling through Su(H) in a temporally controlled manner.

Animals↗
PubMed2003

TALE class homeodomain gene Irx5 is an immediate downstream target for Hoxb4 transcriptional regulation.

The Hox genes are a family of homeodomain-containing transcription factors that determine anteroposterior identity early on in development. Although much is now known about their regulation and function, very little is known of their effector (downstream target) genes. Here, we show that the TALE class homeodomain transcription factor Irx5 is a direct, positively regulated target of Hoxb4.

Animals↗
PubMed2004

Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis.

Wnt4(-/-) XX gonads display features normally associated with testis differentiation, suggesting that WNT4 actively represses elements of the male pathway during ovarian development. Here, we show that follistatin (Fst), which encodes a TGFbeta superfamily binding protein, is a downstream component of Wnt4 signaling. Fst inhibits formation of the XY-specific coelomic vessel in XX gonads. In addition, germ cells in the ovarian cortex are almost completely lost in both Wnt4 and Fst null gonads before birth. Thus, we propose that WNT4 acts through FST to regulate vascular boundaries and maintain germ cell survival in the ovary.

Animals↗
PubMed2005

Microarray analysis of zebrafish cloche mutant using amplified cDNA and identification of potential downstream target genes.

Zebrafish is an excellent model organism for studying vertebrate development and human disease. With the availability of increased numbers of zebrafish mutants and microarray chips, gene expression profiling has become a powerful tool for identification of downstream target genes perturbed by a specific mutation. One of the obstacles often encountered, however, is to isolate large numbers of zebrafish mutant embryos that are indistinguishable in morphology from the wild-type siblings for microarray analysis. Here, we report a method using amplified cDNA derived from five embryos for gene expression profiling of the 18-somite zebrafish cloche (clo) mutant, in which development of hematopoietic and endothelial lineages is severely impaired. In total, 31 differentially expressed target genes are identified, of which 13 have not been reported previously. We further determine that of these 13 new targets, 8 genes, including coproporphyrinogen oxidase (cpo), carbonic anhydrase (cahz), claudin g (cldn g), zinc-finger-like gene 2 (znfl2), neutrophil cytosol factor 1 (ncf1), matrix metalloproteinase 13 (mmp13), dual specificity phosphatase 5 (dusp5), and a novel gene referred as zebrafish vessel-specific gene 1 (zvsg1) are predominantly expressed in hematopoietic and endothelial cells. Comparative analysis demonstrates that this method is comparable and complementary to that of the conventional approach using unamplified sample. Our study provides valuable information for studying hematopoiesis and vessel formation. The method described here offers a powerful tool for gene expression profiling of zebrafish mutants in general.

Animals↗
PubMed2006

A mutation in the zebrafish Na,K-ATPase subunit atp1a1a.1 provides genetic evidence that the sodium potassium pump contributes to left-right asymmetry downstream or in parallel to nodal flow.

While there is a good conceptual framework of dorsoventral and anterioposterior axes formation in most vertebrate groups, understanding of left-right axis initiation is fragmentary. Diverse mechanisms have been implied to contribute to the earliest steps of left-right asymmetry, including small molecule signals, gap junctional communication, membrane potential, and directional flow of extracellular liquid generated by monocilia in the node region. Here we demonstrate that a mutation in the zebrafish Na,K-ATPase subunit atp1a1a causes left-right defects including isomerism of internal organs at the anatomical level. The normally left-sided Nodal signal spaw as well as its inhibitor lefty are expressed bilaterally, while pitx2 may appear random or bilateral. Monocilia movement and fluid circulation in Kupffer's vesicle are normal in atp1a1a(m883) mutant embryos. Therefore, the Na,K-ATPase is required downstream or in parallel to monocilia function during initiation of left-right asymmetry in zebrafish.

Animals↗
PubMed1987

Provision of the immunoglobulin heavy chain enhancer downstream of a test gene is sufficient to confer lymphoid-specific expression in transgenic mice.

To test whether the immunoglobulin heavy chain enhancer is sufficient to direct lymphoid specific gene expression in vivo, we have incorporated into the mouse germ line a test gene (chloramphenicol-acetyl transferase) whose transcription is potentiated by the enhancer. Analysis of transgenic mice bearing single-copy integrations of the injected DNA indicates that provision of the enhancer downstream of the test gene (beyond the polyadenylation site) is sufficient to direct lymphoid-specific expression. The tissue specificity is considerable as judged by the fact that transgene expression is at least one thousandfold higher in spleen than in liver. Within the lymphoid family, expression of the transgene is much higher in surface IgM+ spleen cells than in thymocytes high in Thy-1 expression, suggesting that the enhancer preferentially directs expression to cells of the B lineage.

Acetyltransferases↗
PubMed2005

Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4.

Phosphoinositide 3-kinases (PI3K) are known to regulate Toll-like receptor (TLR)-mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta (TRIF)-dependent signaling, which induces IFN-beta gene expression downstream of TLR3 and TLR4. First, treatment of monocyte-derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN-beta expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA-binding activity of NF-kappaB, but not interferon response factor (IRF)-3, the key transcription factors required for TLR-mediated IFN-beta synthesis. In parallel, wortmannin-treated DC exhibited enhanced levels of IkappaB kinase (IKK)-alpha/beta phosphorylation and IkappaB-alpha degradation with a concomitant increase in NF-kappaB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF-kappaB-dependent promoters as well as IFN-beta promoter activities without interfering with transcription at the positive regulatory domain III-I. Furthermore, wortmannin enhanced NF-kappaB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF-mediated NF-kappaB transcriptional activity. Finally, in co-immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF-dependent NF-kappaB activity, and thereby increases IFN-beta synthesis elicited by TLR3 or TLR4 ligands.

Adaptor Proteins, Vesicular Transport↗
PubMed2006

BLIMP-1 is a target of cellular stress and downstream of the unfolded protein response.

B lymphocyte-induced maturation protein-1 (BLIMP-1) acts during differentiation of B cells and monocytes, but was originally identified as a repressor of the IFN-beta promoter induced during viral infection. A central regulator of the intracellular response to viral infection is the interferon-inducible double-stranded RNA activated protein kinase (PKR). PKR belongs to a family of kinases that phosphorylate the eukaryotic translation initiation factor 2-alpha (eIF2alpha) and activate common downstream signaling pathways. PERK, the endoplasmic reticulum resident PKR-homologue, is activated during the unfolded protein response (UPR), a stress response involved in both macrophage activation and terminal B-cell differentiation. This suggested that BLIMP-1 might be a target of stress responses involving PERK. We demonstrate that BLIMP-1 is rapidly up-regulated during the UPR in human myeloid and B-cell lines. This response is conserved in murine B-cells and murine macrophages, in which mimics of physiological stress and classical activation stimuli also induce Blimp-1. During the UPR, BLIMP-1 mRNA is induced at the level of transcription. This response is dependent on an intact PERK signaling pathway, independent of new protein synthesis and blocked by an inhibitor of NF-kappaB. Our data provide evidence for a novel pathway linking cellular stress to BLIMP-1, a regulator of differentiation in macrophages and B cells.

Animals↗
PubMed1992

The translocation (1;14)(p34;q11) in human T-cell leukemia: chromosome breakage 25 kilobase pairs downstream of the TAL1 protooncogene.

Nearly 30 percent of patients with T-cell acute lymphoblastic leukemia (T-ALL) exhibit a tumor-specific rearrangement of the TAL1 gene (also called TCL5 or SCL). These rearrangements are generated by either local DNA deletion or a (1;14)(p34;q11) chromosome translocation, and they typically result in structural alterations of the TAL1 transcription unit. In this report we present a molecular characterization of the t(1;14)(p34;q11) from a T-ALL patient. As a consequence of the translocation, TAL1 is transposed from its normal position on chromosome 1 into the T-cell receptor alpha/delta chain locus on chromosome 14. Unlike previous cases, the chromosome 1 breakpoint in this patient did not disrupt the continuity of the TAL1 transcription unit, but instead occurred approximately 25 kilobase pairs (kb) downstream of TAL1. This observation suggests that malignant alteration of TAL1 can be mediated by long-range cis-activating mechanisms that are triggered by DNA rearrangement at a distant site. Genes Chrom Cancer 4:211-216 (1992).

Base Sequence↗
PubMed2005

Dll1 is a downstream target of Tbx6 in the paraxial mesoderm.

Tbx6 is a member of the T-box family of transcription factors. In the mouse, Tbx6 is expressed in the primitive streak, tail bud, and presomitic mesoderm and is essential for the specification of posterior paraxial mesoderm; in its absence, posterior somites are replaced by ectopic neural tubes. Analysis of embryos expressing reduced levels of Tbx6 also revealed that it is required for the correct patterning of the somites as well as their initial specification. As a first step toward identifying downstream targets of Tbx6, we examined the DNA binding properties of Tbx6 and identified a Tbx6 consensus binding site. Previously, we have shown that expression of Dll1, which encodes a Notch ligand, is lost in the Tbx6 mutant and that Tbx6 and Dll1 genetically interact, indicating that Dll1 may be a direct target of Tbx6 in the paraxial mesoderm. We uncovered four putative Tbx6 binding sites within a Dll1 paraxial mesoderm enhancer and show that Tbx6 can bind two of these sites in vitro. Altogether, these results lend further support for Dll1 being a direct target of Tbx6 in the presomitic mesoderm.

Animals↗
PubMed1998

p21Ras downstream effectors are increased in activity or expression in mouse liver tumors but do not differ between ras-mutated and ras-wild-type lesions.

Mouse liver tumors frequently harbor activating ras gene mutations. Downstream effector molecules of p21Ras include Raf-1 kinase which mediates external signals via kinase signaling pathways to nuclear transcription factors including c-Fos and c-Jun. Mouse liver tumors with differing ras-mutational status were analyzed for alterations in Ras/Raf-1 signal transduction. Tumors were characterized with respect to the presence of base substitutions in the 3 known hot-spot positions at codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras. Ha-ras codon 61 or Ki-ras codon 13 mutations, but no N-ras mutations, were detected in 23 out of 33 tumors analyzed, while no ras-mutations were found in 10 of the tumors. There was no significant difference in the expression of p21RaS proteins between ras-mutated tumors and tumors without detectable ras mutations. To allow for determination of Raf-1 kinase activity in tumors, a sensitive and specific assay was developed for measurements with tissue homogenates. Raf-1 kinase activity was increased about four-fold in liver tumors as compared with normal liver tissue. No significant differences in kinase activity, however, were evident between ras-mutated and ras-wild-type tumors. The same was true with respect to the levels of c-fos and c-jun mRNAs. Moreover, there were no significant differences in cell division (5-bromo-2'-deoxyuridine-labeling indices) of hepatocytes from ras-mutated and ras-wild-type tumors. The similar degree of constitutive activation of the Ras/Raf-1 signaling pathway in liver tumors, with and without detectable ras mutations, suggests that other molecules within the signaling pathway may substitute for ras-mutations during oncogenic conversion of ras-wild-type hepatocytes.

Animals↗
PubMed2004

DFold: PCR design that minimizes secondary structure and optimizes downstream genotyping applications.

Secondary structures in polymerase chain reaction (PCR) target sequences have a negative impact on amplification success rates and on downstream uses of PCR products. For example, signal strength and allele discrimination in single nucleotide polymorphism (SNP) genotyping methods can be compromised by allele-biased amplification and/or by PCR product folding that limits access of interrogating probes. To increase the fidelity and robustness of PCR, and to aid follow-on applications, we have developed DFold (http://dfold.cgb.ki.se)-a generalized software solution that creates PCR oligonucleotide primer designs devoid of stable secondary structures. We demonstrate the effectiveness of the tool by applying it to a range of dynamic allele-specific hybridization (DASH) assay designs, many of which we evaluate in the laboratory. We further consider how the system throughput may be made sufficiently high for use upon millions of target sequences in order to support whole-genome analyses.

Algorithms↗
PubMed2006

The 185delAG mutation (c.68_69delAG) in the BRCA1 gene triggers translation reinitiation at a downstream AUG codon.

The 185delAG mutation (c.68_69delAG; ter39) in the BRCA1 gene is a founder Jewish Ashkenazi mutation that is carried by 1% of this population and has been identified in thousands of breast or ovarian cancer patients. We have previously described that transcripts bearing this mutation, as well as transcripts bearing the 188del11 mutation (c.71_81del; ter36), are not degraded by nonsense-mediated mRNA decay (NMD), contrary to our observations of other truncating mutations that introduce premature termination codons (PTCs) farther downstream in the coding sequence [Perrin-Vidoz et al., 2002]. To test the hypothesis that these two mutations fail to trigger NMD because of translation reinitiation, we have constructed BRCA1 minigenes and studied their protein expression after transient expression in HeLa cells. We show here that in the presence of a (PTC) at position 36 or 39, translation reinitiation occurs in the BRCA1 minigenes at position 128.

BRCA1 Protein↗
PubMed2003

N-Myc downstream-regulated gene 2 (NDRG2) inhibits glioblastoma cell proliferation.

The most severe form of brain glioma, glioblastoma (GBM), is highly malignant and usually resistant to chemotherapy. Therefore, discovery of new targets for gene therapy is important. Using subtraction cloning, we identified the human N-Myc downstream-regulated gene 2 (hNDRG2), located at chromosome 14q11.2, as a gene that is significantly suppressed in GBM tissues. Semiquantitative RT-PCR showed that the hNDRG2 gene transcript is expressed in normal brain tissue and low-grade gliomas but is present at low levels in 15 of 27 (56%) human GBM tissues and all of the 6 human glioblastoma cell lines examined. Furthermore, transfection of human glioblastoma U373 and U138 cells with a cDNA encoding hNDRG2 markedly reduced the cell proliferation. Our findings provide the first evidence to suggest that hNDRG2 may play a role in glioblastoma carcinogenesis.

Apoptosis↗
PubMed2004

Loss of cyclin E requirement in cell growth of an oral squamous cell carcinoma cell line implies deregulation of its downstream pathway.

Cyclin E and Cdk2 have been shown to play an important role in G1/S transition of the cell cycle. Two E-type cyclins (E1 and E2) have been identified to date and share functionally similarities. Upregulation of these cyclins has been observed frequently in human cancers. We examined the expression profile of cyclin E1 and E2 in cell lines derived from human oral squamous cell carcinoma (SCC), and found that the expression of cyclin E1 protein was hardly detected in HSC-2 cells. Although cyclin E2 was abundantly expressed, histone H1 kinase activities of both E-type cyclins were virtually undetectable in this cell line. Inhibition of cyclin E1, but not that of E2, by using vectors expressing antisense-oriented their cDNAs induced drastic growth suppression on HOC313 cells that express both E-type cyclins. Inhibition of neither cyclin E1 nor E2 suppressed the growth of HSC-2 cells, and compensatory elevation of cyclin E1 was not evident in cyclin E2-inhibited HSC-2 cells. In contrast, HSC-2 cells expressed cyclin D1 and hyperphosphorylated forms of Rb family proteins, and were arrested in G1 by overexpression of p16(INK4), a specific inhibitor against D-type cyclin activity. These results indicate that HSC-2 cells lost proper growth control specifically mediated by cyclin E and suggest that deregulation of its downstream pathway may contribute to tumorigenesis of oral SCC.

Cell Division↗