Comparison Genomics of Oryza rufipogon Chromosome Eight Centromere with Other Species Centromeres

Centromeres are playing a key role in proper segregation of sister chromatids during mitosis and meiosis. At the centromere region in the chromosome, spindle microtubules attached to the kinetochores that direct the chromosome segregation during mitosis and meiosis. Although centromeric function is conserved among different organisms, there is no conservation of centromeric DNA sequences ranging from budding yeast which has a consensus sequence of approximately 125 base pairs (bp) on each chromosome to Caenorhabditis elegans species which has a holocentric centromere that spans the entire chromosome. However, there are some common proteins that form the kinetochore, such as CENH3 and CENP-C. In this study, wild rice (Oryza ruflpogon) chromosome 8 centromere fragments were compared to all the domesticate rice {Oryza sativa ssp japónica) chromosome centromeres, Arabidopsis thaliana chromosome 5 centromere, Lycopersicon esculentum chromosome 12 centromere, and Dictyostelium discoideum chromosome centromere. Annotation results of all the genomes suggest that many of the structural aspects parallel in both the species of the rice centromeres. For example, both wild rice fragments and domesticated rice chromosome centromere sequences found to share their GC contents in between 40-45%. The Arabidopsis thaliana chromosome 5 centromere, the Lycopersicon esculentum chromosome 12 centromere, and the Dictyostelium discoideum chromosome centromere had less structural similarities with the Oryza rufipogon chromosome 8 centromere. Further, annotation of the wild rice fragments and the domesticated rice genome revealed that both the species had the cento satellite repeats and Ty3/gypsy retrotransposons

STAT3 inhibition induces apoptosis in cancer cells independent of STAT1 or STAT2

Signal transducers and activators of transcription (STATs) were originally discovered as mediators of signal transduction. Persistent aberrant activation of STAT3 is part of the malignant phenotype of hormone-refractory prostate cancer and pancreatic cancer; this is thought to be mediated by homodimers of phosphorylated STAT3, which translocate to the nucleus.  One consequence of persistently-activated STAT3 in malignant cells is that they depend upon it for survival.   STAT3 is observed to heterodimerize with STAT1 and STAT2; however the contributions of STAT3:STAT1  and STAT3:STAT2 heterodimers to the survival of malignant cells have not been investigated in detail. Previously we reported that single-stranded oligonucleotides containing consensus STAT3 binding sequences (13410 and 13411) were more effective for inducing apoptosis in prostate cancer cells than antisense STAT3 oligonucleotides. Control oligonucleotides (scrambled sequences) had no effect. STAT3-inhibiting oligonucleotide 13410, but not scrambled-sequence oligonucleotides, induced apoptosis in pancreatic cancer cells as well.  Here we report that 13410 and derivative olignucleotides induced apoptosis in STAT1-null and STAT2-null fibrosarcoma cell lines U3A and U6A, as well as in the parental fibrosarcoma cell line 2fTGH. The cell lines expressed constitutively-activated STAT3 and depended on its activity for survival.  Forty-eight hr after transfection of 13410 or related oligonucleotides, significant apoptosis was observed in 2fTGH, U3A and U6A cells. Scrambled-sequence oligonucleotides had no effect on survival.  These data indicate that neither STAT1 nor STAT2 play significant roles in the maintenance of these cells, and by extension that STAT3:STAT1 and STAT3:STAT2 heterodimers regulate a different set of genes from STAT3:STAT3 homodimers.  

Creation of a novel peptide with enhanced nuclear localization in prostate and pancreatic cancer cell lines

<p>Abstract</p> <p>Background</p> <p>For improved uptake of oligonucleotide-based therapy, the oligonucleotides often are coupled to peptides that facilitate entry into cells. To this end, novel cell-penetrating peptides (CPPs) were designed for mediating intracellular uptake of oligonucleotide-based therapeutics. The novel peptides were based on taking advantage of the nuclear localization properties of transcription factors in combination with a peptide that would bind putatively to cell surfaces. It was observed that adding a glutamate peptide to the N-terminus of the nuclear localization signal (NLS) of the Oct6 transcription factor resulted in a novel CPP with better uptake and better nuclear colocalization than any other peptide tested.</p> <p>Results</p> <p>Uptake of the novel peptide Glu-Oct6 by cancer cell lines was rapid (in less than 1 hr, more than 60% of DU-145 cells were positive for FITC), complete (by 4 hr, 99% of cells were positive for FITC), concentration-dependent, temperature-dependent, and inhibited by sodium azide (NaN₃). Substitution of Phe, Tyr, or Asn moieties for the glutamate portion of the novel peptide resulted in abrogation of novel CPP uptake; however none of the substituted peptides inhibited uptake of the novel CPP when coincubated with cells. Live-cell imaging and analysis by imaging flow cytometry revealed that the novel CPP accumulated in nuclei. Finally, the novel CPP was coupled to a carboxyfluorescein-labeled synthetic oligonucleotide, to see if the peptide could ferry a therapeutic payload into cells.</p> <p>Conclusions</p> <p>These studies document the creation of a novel CPP consisting of a glutamate peptide coupled to the N-terminus of the Oct6 NLS; the novel CPP exhibited nuclear colocalization as well as uptake by prostate and pancreatic cancer cell lines.</p