28 research outputs found
Two alternative promoters distinctly control RCAN1 expression.
<p>(<b>A</b>) Genomic organization of the <i>RCAN1</i> gene. <i>RCAN1</i> gene has seven exons and six introns. The first four exons are alternatively spliced and the last three exons are constitutive. There are two promoters and two translation initiation codons in the <i>RCAN1</i> gene, in the 5′UTR of exon 1 and 5′UTR of exon 4 respectively. TSS, transcription start site. E stands for exon. (<b>B</b>) Human <i>RCAN1</i> isoform 4 promoter sequence. A 1200 bp fragment of the 5′ flanking region of human RCAN1 exon 4 was amplified from a human genomic library. Thymine +1 represents the major transcription start site. Positions of some of the unique and common restriction enzymes are indicated in italics and boldface. Putative transcription factor binding sites are underlined in boldface. The codon of the first nine amino acids of exon 4 is indicated. (<b>C</b>) 1200-bp fragment upstream of the exon 4 had significant promoter activity. 1200-bp fragment upstream of the exon 4 was cloned into pGL3-Basic to generate the pDE4Luc luciferase reporter plasmid. pDE4Luc was transfected into HEK293 cells. pGL3-Basic was used as negative control. Luciferase activity was measured 24 hours after transfection. Values represent means ± SE (n = 4), *P<0.05 by student's t-test. (<b>D</b>) Compared to the RCAN1 exon 1 promoter pRCAN1Luc-A, RCAN1 exon 4 promoter pDE4Luc had a higher promoter activity in C6 cells but a lower activity in N2A cells. RCAN1 promoter constructs pDE4Luc and pRCAN1Luc-A were transfected into C6 and N2A cells. pGL3-Basic was used as negative control. Luciferase activity was measured 24 hours after transfection by a luminometer. <i>Renilla</i> luciferase activity was used to normalize transfection efficiency. Values represent means ± SE (n = 4), *P<0.05 by student's t-test. (<b>E</b>) Calcium ionophore A23187 significantly increased pDE4Luc activity. HEK293 cells transfected with pDE4Luc were treated with 2.5 µM A23187 for 12 hours. Luciferase assay was used to measure the promoter activity. pGL3-Basic was used as negative control. <i>Renilla</i> luciferase activity was used to normalize transfection efficiency. Values represent means ± SE (n = 4), *P<0. 05 by student's t-test.</p
RCAN1.4 overexpression exacerbates calcium overloading-induced neuronal apoptosis.
<p>(<b>A</b>) RCAN1.4 overexpression reduced cell survival. SH-SY5Y cells transfected with empty vector pcDNA4mychisA or pcDNA4-RCAN1.4mychis were treated with 2.5 uM A23187. MTS assay was used to indicate the viability of SH-SY5Y cells. Values represent mean ± SEM, n = 3, *P<0. 05 by ANOVA. (<b>B</b>) and (<b>C</b>) RCAN1.4 overexpression exacerbated cell apoptosis. SH-SY5Y cells transfected with empty vector pcDNA4mychisA and pcDNA4-RCAN1.4mychis were treated with vehicle control solution (B) or 2.5 uM A23187and (C). TUNEL staining was used to indicate cell apoptosis (green color). Nuclei were counterstained with DAPI (blue color). (<b>D</b>) Quantification of (B) and (C). Values represent means ± SE (n = 3), *P<0. 05 by ANOVA.</p
Caspase-3 mediates the neurotoxic effect of RCAN1.4 and calcium overloading.
<p>(<b>A</b>) RCAN1.4 overexpression increased caspase-3 activation. SH-SY5Y cells transfected with empty vector pcDNA4mychisA (Vector) and pcDNA4-RCAN1.4mychis (RCAN1) were treated with 2.5 uM A23187 for 12 hours. 100 µg cell lysates were separated in a 16% tricine SDS-PAGE gel. Procaspase-3 and cleaved caspase-3 were detected with anti-caspase-3 antibody from Sigma. Myc-tagged RCAN1.4 was detected by 9E10 antibody. β-actin served as loading control. (<b>B</b>) Quantification of (A). The ratio of cleaved caspase-3 to procaspase-3 was calculated. Values represent means ± SE (n = 3), *P<0. 05 by ANOVA.</p
Folding of the 5′ and 3′ NCRs of the DpCPV-MC genome segments.
<p>The RNAfold program was used to predict the secondary structures formed by the 16 DpCPV-MC genome segments. The panhandle structure was formed by base-pairing between the 5′ and 3′ ends, and the stem–loop structure was formed by either the 5′ or 3′ terminal sequence. The panhandle structure and the stem–loop structure are marked with long brackets.</p
Median lethal concentrations (LC<sub>50</sub>) of DpCPV-MC and DpCPV-1 against second-instar <i>S. exigua</i> larvae.
a<p>Potency ratio was calculated by dividing the LC<sub>50</sub> of DpCPV-MC by that of DpCPV-1. Significant difference was based on whether the 95% confidence interval (CI) of the potency ratio included the value 1.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113201#pone.0113201-Robertson2" target="_blank">[47]</a>.</p><p>Median lethal concentrations (LC<sub>50</sub>) of DpCPV-MC and DpCPV-1 against second-instar <i>S. exigua</i> larvae.</p
Electron micrographs of OBs of DpCPV-MC.
<p>(A) Transmission electron micrograph of ultrathin sections of OBs of DpCPV-MC. (B) Transmission electron micrograph of purified virions of DpCPV-MC.</p
Neighbor-joining tree constructed from polyhedrin amino acid sequences of representative CPVs.
<p>Sequences were aligned with the multiple sequence alignment program ClustalX2. The neighbor-joining method was used to construct the phylogenetic tree of the derived polyhedrin protein sequences with the MEGA software version 5.2. The black triangle shows the position of DpCPV-MC. Bootstrap percentage values are indicated on the left. All reference sequences used for the construction of this tree were retrieved from GenBank with their corresponding accession numbers: <i>Antheraea assamensis cypovirus 4</i>, AY212275; <i>Antheraea mylitta cypovirus 4</i>, AY212273; <i>Antheraea proylei cypovirus 4</i>, AY212276; <i>Dendrolimus punctatus cypovirus 1</i>, AY204879; <i>Bombyx mori cypovirus 1</i>, D37770; <i>Culex restuans cypovirus 17</i>, DQ212785; <i>Choristoneura occidentalis cypovirus 16</i>, EU201043; <i>Euxoa scandens cypovirus 5</i>, J04338; <i>Heliothis assulta cypovirus 14</i>, DQ077914; <i>Heliothis armigera cypovirus 5</i>, DQ077912; <i>Lymantria dispar cypovirus 1</i>, AF389471; <i>Operophtera brumata cypovirus 18</i>, DQ192250; <i>Operophtera brumata cypovirus 19</i>, DQ192254; <i>Simulium ubiquitum cypovirus 20</i>, DQ834386; <i>Trichoplusia ni cypovirus 15</i>, NC_002565; <i>Uranotaenia sapphirina cypovirus 17</i>, AY876384; <i>Lymantria dispar cypovirus 14</i>, AF389461; <i>Choristoneura fumiferana cypovirus 16</i>, U95954.</p
NFATc1 mutants abolished the effects of DYRK1A.
<p><b>A.</b> Nine mutant constructs of NFATc1 were co-transfected with pCMV-DYRK1A in HEK293 cells. <b>B.</b> HEK293 cells co-transfected with NFATc1 and nine mutant constructs of NFATc1 were treated with proteasome inhibitor lactacystin (5uM) for 24 hours after transfection. Values represent means±SD, n = 3. DY1A means DYRK1A. ubi means ubiqutin. pSer means phosphorylated serine.</p
DYRK1A affected NFATc2 protein phosphorylation.
<p><b>A.</b> DYRK1A inhibitor harmine (2ug/ml) decreased NFATc1 expression (the image was flipped horizontally from the original picture). <b>B.</b> Anti-pSer antibody was used to detect the serine phosphorylated NFATc1 and rabbit anti-FLAG antibody was used to detect the total NFATc1 in the mouse anti-FLAG antibody immunoprecipitates. Mouse anti-DYRK1A antibody and mouse anti-NFATc1 antibody were used to detect total DYRK1A and total NFATc1, respectively in transfected cells. <b>C.</b> Co-IP assay showed the cross-interaction between NFATc1 and DYRK1A. <b>D.</b> Anti-ubiquitin antibody was used to detect the ubiquitination of NFATc1 and rabbit anti-FLAG antibody was used to detect the total NFATc1 in mouse anti-FLAG antibody immunoprecipitates. Mouse anti-DYRK1A antibody and mouse anti-NFATc1 antibody were used to detect total DYRK1A and total NFATc1, respectively in transfected cells, Values represent means±SD, n = 3. Ubi means ubiqutin. pSer means phosphorylation of serine.</p