11 research outputs found
Predicted haplotype structure for <i>DKK</i>2.
<p>Predicted haplotype structure for <i>DKK</i>2.</p
DKK2 inhibits Wnt3a-induced Wnt/β-catenin signaling.
<p>HuH-7 cells were transiently transfected with a TCF reporter plasmid and were then incubated with various concentrations of Wnt3a and/or DKK2 (ng/ml). The presence of the Wnt3a ligands activated the Wnt signaling pathway in a dose-dependent manner, as measured by elevation in the luciferase reporter activity (A). Consistently, cell proliferation increased in HuH-7 cells when they were cultured in the presence of Wnt3a ligands (B). Nuclear translocation of β-catenin was analyzed in (C), and GAPDH and Histone H3 were used to indicate cytoplasmic or nuclear fractions, respectively. Note that DKK2 addition abrogated the effects of Wnt3a significantly. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 when compared to DKK2 stimulation in their respective Wnt3a levels.</p
<i>DKK2</i> expression is down-regulated in tumor tissues with chromosome 4q24-25 LOH and <i>DKK2</i> TAGC haplotype.
<p>(A) Luciferase activity of the various <i>DKK2</i> haplotypes in HuH-7 cells. A reporter assay was performed to test the sequences that cover the <i>DKK2</i> promoter region from 1,135 bp upstream to 667 bp downstream of the transcription initiation site. The coding sequence of the luciferase was fused to the <i>DKK</i>2 promoter and 5’UTR sequence. The promoter function of each <i>DKK</i>2 haplotype sequence was assessed using luciferase activity, and the data obtained was normalized against the expression level of the cotransfected β-galasctosidase gene. The position of the four SNPs (at -967, -923, -441, and +550) is shown relative to the transcription initiation site. The promoter activity of the TAGC haplotype was significantly reduced relative to the other haplotypes. No significant effect on cell viability post transfection was observed for the reporter plasmids containing different <i>DKK2</i> haplotypes. (B) Relative <i>DKK2</i> mRNA expression levels of the tumor (T) tissues and the non-tumor (N) counterparts (n = 30) were assayed by RT-qPCR and classified into three categories determined by the presence of chromosome 4q24-25 LOH and/or <i>DKK2</i> TAGC haplotype. The group with chromosome 4q24-25 LOH and <i>DKK2</i> TAGC haplotype (LOH with TAGC, n = 8) showed significantly lower <i>DKK2</i> expression in tumor tissue than the other two groups: without chromosome 4q24-25 LOH (non-LOH, n = 13) and with chromosome 4q24-25 LOH but no <i>DKK2</i> TAGC haplotype (LOH w/o TAGC, n = 9). *** <i>p</i> < 0.001</p
Haplotypes analysis of 16 HCC cases heterozygous for <i>DKK</i>2.
<p>Haplotypes analysis of 16 HCC cases heterozygous for <i>DKK</i>2.</p
A model of HCC tumorigenesis showing the selective retention of the low transcriptional activity of <i>DKK</i>2 haplotype 1 (TAGC) allele.
<p>The cell proliferation rate is elevated in the pre-cancerous liver tissues, and a DNA break occurs near the recombination hotspots in the <i>DKK</i>2 promoter region, which leads to loss of the <i>DKK</i>2 alleles. In this scheme, those cells with low <i>DKK</i>2 transcriptional activity are selected for clonal amplification as a result of unchecked Wnt signaling.</p
Polysomy of chromosome 4 and <i>DKK</i>2 deletion in a HCC case.
<p>The arrow indicates hybridization signals from a centromeric probe for chromosome 4. The arrowhead indicates hybridization signals from a 4q21 YAC probe. Note that there are three signals from the centromere but only two for 4q21.</p
Sequence analysis of KPX plasmids.
<p>Two circular sequences are shown for the organization of pKPX-1 (<b>A</b>) and pKPX-2 (<b>B</b>). Mapping shotgun sequencing reads of pECX-1 to the pKPX-1 is indicated by the red half-circle. A large part of the plasmid, corresponding to the nucleotide positions 23,125 to 145,377 of pKPX-1, was not found in pECX-1. Only the part on the left side, totaling 128,191-bp, is retained. Two genes encoding chloramphenicol and amikacin resistance were identified by functional library screening. Their positions in the deleted region are indicated. Nucleotides are numbered according to the replication origin. Genes are color coded: yellow, β-lactamase; red, antimicrobial resistance associated; blue, plasmid replication and partitioning; black, transposases or IS elements; and white, other coding sequences of miscellaneous features. The arrows on the open reading frames (ORFs) indicate the gene orientation. Gene clusters involved in gene transfer or mobility are marked in green. <i>Xba</i>I and <i>Avr</i>II restriction sites are shown inside the circle.</p
Tandem duplication of the <i>bla</i><sub>NDM-1</sub> gene in pKPX-1 and pECX-1.
<p>(<b>A</b>) Diagrammatic representation of the analysis of <i>bla</i><sub>NDM-1</sub> copy number by Southern blot. The probe is shown with a red arrow, and the tandem duplication of the 8588-bp repeat is indicated by the bracket. The asterisks indicate the methylated <i>Nru</i>I sites. The sizes of <i>Bam</i>HI or <i>Hind</i>III digested fragments depend on the copy number of the repeat. The pound sign indicates 79.1 kb and 71.8 kb for <i>Bam</i>HI and <i>Hind</i>III restrictions, respectively, when there are 8 copies of the tandem repeat, as in the case of pECX-1. (<b>B</b>) Sequencing read distribution and Southern analysis of the <i>bla</i><sub>NDM-1</sub> region for pECX-1. The upper panel shows the relative coverage depth of the repeat region and its flanking sequences. The average coverage of <i>bla</i><sub>NDM-1</sub> is 7–8 fold of those sequences of the immediately adjacent regions, suggesting that there are eight copies of the repeat. As shown in the lower panel, Southern analysis confirms this model of tandem duplication. (<b>C</b>) <i>Bla</i><sub>NDM-1</sub> copy number variation detected by the Southern analysis. Sequence depth of the region revealed an average of 3–4 copies of the repeat sequence in pKPX. <i>Bam</i>HI and <i>Hind</i>III digestion gave a series of ladder bands, corresponding to different copy numbers of the repeat. By contrast, <i>Avr</i>II and <i>Nru</i>I both deliberated a single major band of 8.6 kb, representing the unit length of the tandem repeats.</p
Restriction analysis of the KPX plasmids by PFGE.
<p>Plasmid DNA isolated from the KPX isolate was analyzed using pulsed field gel electrophoresis (PFGE). The restriction patterns of the plasmid DNA were compared to those of pECX-1 and pECX-2 in <i>Escherichia coli</i>. The size of the DNA markers is shown in kilobases (kb) on the left side.</p
Antimicrobial resistance determinants in pKPX-1 and pKPX-2.
*<p>Nonfunctional; <sup>†</sup> Deleted in pECX-1.</p><p>Abbreviations: AAC, aminoglycoside acetyltransferase; APH, aminoglycoside phosphotransferase; ANT, aminoglycoside nucleotdyltransferase; Rmt, 16S rRNA methyltransferase; Qnr, quinolone resistance protein; TetA, tetracycline efflux protein; Cat, chloramphenicol acetyltransferase; ARR-2, rifampin ADP-ribosyltransferase; DHPS, dihydropteroate synthase; DHFR, dihydrofolate reductase; Mph, macrolide phosphotransferase.</p