Key Roles for E2F1 in Signaling p53-Dependent Apoptosis and in Cell Division within Developing Tumors

AbstractApoptosis induced by the p53 tumor suppressor can attenuate cancer growth in preclinical animal models. Inactivation of the pRb proteins in mouse brain epithelium by the T121 oncogene induces aberrant proliferation and p53-dependent apoptosis. p53 inactivation causes aggressive tumor growth due to an 85% reduction in apoptosis. Here, we show that E2F1 signals p53-dependent apoptosis since E2F1 deficiency causes an 80% apoptosis reduction. E2F1 acts upstream of p53 since transcriptional activation of p53 target genes is also impaired. Yet, E2F1 deficiency does not accelerate tumor growth. Unlike normal cells, tumor cell proliferation is impaired without E2F1, counterbalancing the effect of apoptosis reduction. These studies may explain the apparent paradox that E2F1 can act as both an oncogene and a tumor suppressor in experimental systems

Long Tract of Untranslated CAG Repeats Is Deleterious in Transgenic Mice

The most frequent trinucleotide repeat found in human disorders is the CAG sequence. Expansion of CAG repeats is mostly found in coding regions and is thought to cause diseases through a protein mechanism. Recently, expanded CAG repeats were shown to induce toxicity at the RNA level in Drosophila and C. elegans. These findings raise the possibility that CAG repeats may trigger RNA-mediated pathogenesis in mammals. Here, we demonstrate that transgenic mice expressing EGFP transcripts with long CAG repeats in the 3′ untranslated region develop pathogenic features. Expression of the transgene was directed to the muscle in order to compare the resulting phenotype to that caused by the CUG expansion, as occurs in myotonic dystrophy. Transgenic mice expressing 200, but not those expressing 0 or 23 CAG repeats, showed alterations in muscle morphology, histochemistry and electrophysiology, as well as abnormal behavioral phenotypes. Expression of the expanded CAG repeats in testes resulted in reduced fertility due to defective sperm motility. The production of EGFP protein was significantly reduced by the 200 CAG repeats, and no polyglutamine-containing product was detected, which argues against a protein mechanism. Moreover, nuclear RNA foci were detected for the long CAG repeats. These data support the notion that expanded CAG repeat RNA can cause deleterious effects in mammals. They also suggest the possible involvement of an RNA mechanism in human diseases with long CAG repeats

The Relationship between Scabies and Stroke: A Population-Based Nationwide Study

Background: Scabies is a commonly occurring infectious skin infestation that substantially impacts the quality of life, while stroke, which consists of a neurological deficit resulting from a lack of blood flow to the brain, carries sizable economic costs. The pathophysiologic mechanisms underlying both diseases involve inflammatory processes that are mediated by the immune system; however, no prior research has been conducted to explore the relationship between the two conditions. Methods: This population-based nationwide study utilized data from the National Health Insurance Research Database (NHIRD) of Taiwan for a total of 6628 scabies patients, who comprised a scabies group, and a randomly selected cohort of 26,509 matching patients, who served as a control group. More specifically, the medical records for the patients in both groups were checked for seven years to identify any new cases of stroke within that seven-year follow-up period. The hazard ratio (HR) of stroke for the follow-up period was then calculated using Cox proportional hazards regressions, while comorbidities and demographic characteristics were likewise analyzed. Results: During the follow-up period, 2892 patients, or 8.7%, of the overall total of 33,137 patients included in the study were newly diagnosed with a stroke. Of those newly diagnosed stroke patients, 833 were from the scabies group, and 2059 were from the control group, accounting for 12.6% and 7.8%, respectively, of the individuals in each group. With a crude hazard ratio of 1.67, the patients in the scabies group had a significantly higher risk of subsequent stroke than those in the control group, although the adjusted hazard ratio (aHR) for the scabies patients, which was determined by adjusting for covariates, was only 1.32 (95% confidence interval (CI): 1.21–1.43). Conclusions: The results of the study indicated an elevated risk of stroke among scabies patients, an association that might be contributed to by immunopathological factors. This information could serve as a reminder to clinicians to remain alert to any indications of neurological impairment in patients previously infected with scabies

Calcium content of different compositions of gallstones and pathogenesis of calcium carbonate gallstones

Our aim was to investigate the calcium content of different gallstone compositions and the pathogenic mechanisms of calcium carbonate gallstones. Between August 2001 and July 2007, gallstones from 481 patients, including 68 calcium carbonate gallstones, were analyzed for total calcium content. Gallbladder bile samples from 33 cases and six controls were analyzed for pH, carbonate anion level, free-ionized calcium concentration and saturation index for calcium carbonate. Total calcium content averaged 75.6 %, 11.8 %, and 4.2 % for calcium carbonate, calcium bilirubinate and cholesterol gallstones. In 29.4 % of patients, chronic and/or intermittent cystic duct obstructions were caused by polypoid lesions in the neck region and 70.6 % were caused by stones. A total of 82 % of patients had chronic low-grade inflammation of the gallbladder wall and 18.0 % had acute inflammatory exacerbations. In the bile, we found the mean pH, mean carbonate anion, free-ionized calcium concentrations, and mean saturation index for calcium carbonate to be elevated in comparison to controls. From our study, we found chronic and/or intermittent cystic duct obstructions and low-grade GB wall inflammation lead to GB epithelium hydrogen secretion dysfunction. Increased calcium ion efflux into the GB lumen combined with increased carbonate anion presence increases SI_CaCO3 from 1 to 22.4. Thus, in an alkaline milieu with pH 7.8, calcium carbonate begins to aggregate and precipitate

Calcium content of different compositions of gallstones and pathogenesis of calcium carbonate gallstones

Background/Objectives: Our aim was to investigate the calcium content of different gallstone compositions and the pathogenic mechanisms of calcium carbonate gallstones. Methods: Between August 2001 and July 2007, gallstones from 481 patients, including 68 calcium carbonate gallstones, were analyzed for total calcium content. Gallbladder bile samples from 33 cases and six controls were analyzed for pH, carbonate anion level, free-ionized calcium concentration and saturation index for calcium carbonate. Results: Total calcium content averaged 75.6 %, 11.8 %, and 4.2 % for calcium carbonate, calcium bilirubinate and cholesterol gallstones. In 29.4 % of patients, chronic and/or intermittent cystic duct obstructions were caused by polypoid lesions in the neck region and 70.6 % were caused by stones. A total of 82 % of patients had chronic low-grade inflammation of the gallbladder wall and 18.0 % had acute inflammatory exacerbations. In the bile, we found the mean pH, mean carbonate anion, free-ionized calcium concentrations, and mean saturation index for calcium carbonate to be elevated in comparison to controls. Conclusion: From our study, we found chronic and/or intermittent cystic duct obstructions and low-grade GB wall inflammation lead to GB epithelium hydrogen secretion dysfunction. Increased calcium ion efflux into the GB lumen combined with increased carbonate anion presence increases SI_CaCO3 from 1 to 22.4. Thus, in an alkaline milieu with pH 7.8, calcium carbonate begins to aggregate and precipitate

The Critical Role of Protein Arginine Methyltransferase <em>prmt8</em> in Zebrafish Embryonic and Neural Development Is Non-Redundant with Its Paralogue <em>prmt1</em>

<div><p>Protein arginine methyltransferase (PRMT) 1 is the most conserved and widely distributed PRMT in eukaryotes. PRMT8 is a vertebrate-restricted paralogue of PRMT1 with an extra N-terminal sequence and brain-specific expression. We use zebrafish (<i>Danio rerio</i>) as a vertebrate model to study PRMT8 function and putative redundancy with PRMT1. The transcripts of zebrafish <i>prmt8</i> were specifically expressed in adult zebrafish brain and ubiquitously expressed from zygotic to early segmentation stage before the neuronal development. Whole-mount <i>in situ</i> hybridization revealed ubiquitous <i>prmt8</i> expression pattern during early embryonic stages, similar to that of <i>prmt1.</i> Knockdown of <i>prmt8</i> with antisense morpholino oligonucleotide phenocopied <i>prmt1-</i>knockdown, with convergence/extension defects at gastrulation. Other abnormalities observed later include short body axis, curled tails, small and malformed brain and eyes. Catalytically inactive <i>prmt8</i> failed to complement the morphants, indicating the importance of methyltransferase activity. Full-length <i>prmt8</i> but not <i>prmt1</i> cRNA can rescue the phenotypic changes. Nevertheless, cRNA encoding Prmt1 fused with the N-terminus of Prmt8 can rescue the <i>prmt8</i> morphants. In contrast, N-terminus- deleted but not full-length <i>prmt8</i> cRNA can rescue the <i>prmt1</i> morphants as efficiently as <i>prmt1</i> cRNA. Abnormal brain morphologies illustrated with brain markers and loss of fluorescent neurons in a transgenic fish upon <i>prmt8</i> knockdown confirm the critical roles of <i>prmt8</i> in neural development. In summery, our study is the first report showing the expression and function of <i>prmt8</i> in early zebrafish embryogenesis. Our results indicate that <i>prmt8</i> may play important roles non-overlapping with <i>prmt1</i> in embryonic and neural development depending on its specific N-terminus.</p> </div

N-terminal amino acid sequence and critical regions in the 5’-region of zebrafish <i>prmt8</i>.

<p>(A) Comparison of the N-terminal amino acid sequences of human and zebrafish Prmt1 and Prmt8. The N-terminal amino acid sequences of human PRMT1 (H1; NP_938074.2), zebrafish Prmt1 (Z1; NP_956944.2), human PRMT8 (H8; NP_956944.2) and zebrafish Prmt8 (Z8; Q5RGQ2.2) were aligned by COBALT (<a href="http://www.ncbi.nlm.nih.gov/tools/cobalt/" target="_blank">http://www.ncbi.nlm.nih.gov/tools/cobalt/</a>). The amino acids encoded by different exons are marked by boxes. The grey shading shows identical amino acid sequence in the four entries. The conserved sequence in human and zebrafish PRMT8 before the PRMT1/PRMT8 conserved region are underlined. The glutamine-rich and proline/histidine-rich sequences are shown in italics. The second glycyl residue that might be myristoylated, the second methionine residue that corresponds to the third and alternative initiating methionine proposed in rat PRMT8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055221#pone.0055221-Kousaka1" target="_blank">[14]</a>, and the SGT at the conserved AdoMet-binding site that were mutated to AAA to make catalytic-inactive methyltransferase are highlighted. (<b>B</b>) Schematic representation of part of the zebrafish <i>prmt8</i> gene encompassing the 5’-flanking region, exon I to II including intron I. The accession number for zebrafish <i>prmt8</i> gene is Q5RGQ2.2. The region displayed comprises genome coordinates of the zebrafish chromosome 4 (in Jul. 2010 Zv9/danRer7 assembly). The potential NRSEs encompassed 21 nucleotides (10,230,373-10,230,353, and 10,229,127-10,229,107 complementary strand) were aligned by the consensus NRSE motif derived from the “core” motif (5′-NNCAGCACCNNGCACAGNNNC-3′). The red boxes show the NRSEs located within the 5′-flanking and the first intron of the <i>prmt8</i> gene. The MO1 and the MO2 binding site to pair with the <i>prmt8</i> initiating ATG and the second ATG are indicated.</p

Expression analysis of <i>prmt8</i> RNA in zebrafish adult tissues and embryos.

<p>(A) Expression of <i>prmt8</i> in different adult tissues was analyzed by RT-PCR. EF indicates the RT-PCR product of elongation factor. (B: brain; SP: spleen; G: gill; O: ovary; H: heart; SB: swim bladder; S: skin; E: eye; M: muscle; T: testis) (B) RT-PCR of <i>prmt8</i> RNA prepared from 0.25 hpf to 72 hpf embryos. (C) The spatial and temporal expression of <i>prmt8</i> RNA from 1 cell to 96 hpf by WISH in zebrafish embryonic development. (m: midbrain; Mhb: mid-hindbrain boundary; som: somites).</p