Inhibition of Poly(ADP-ribose)polymerase impairs Epstein Barr Virus lytic cycle progression

<p>Abstract</p> <p>Background</p> <p>Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins involved in several cellular events as well as in processes that characterize the infective cycle of some viruses. In the present study, we investigated the role of poly(ADP-ribosylation) on Epstein-Barr Virus (EBV) lytic cycle activation.</p> <p>Results</p> <p>Inhibition of PARP-1 by 3-aminobenzamide (3-ABA) during EBV induction, diminished cell damage and apoptosis in the non-productive Raji cell line while markedly reducing the release of viral particles in the productive Jijoye cells. Furthermore, incubation with 3-ABA up-regulated the levels of LMP1 and EBNA2 latent viral proteins. At the same time, it slightly affected the expression of the immediate early BZLF1 gene, but largely down-regulated the levels of the early BFRF1 protein. The modulation of the expression of both latent and lytic EBV genes appeared to be post-transcriptionally regulated.</p> <p>Conclusion</p> <p>Taken together the data indicate that PARP-1 plays a role in the progression of EBV lytic cycle and therefore, PARP inhibitors might represent suitable pharmacological adjuncts to control viral spread in EBV productive infection.</p

Antisense to Epstein Barr virus-encoded LMP1 does not affect the transcription of viral and cellular proliferation-related genes, but induces phenotypic effects on EBV-transformed B lymphocytes

It is generally accepted that Epstein-Barr virus (EBV) latent genes EBNA-2, EBNA-3A, -3C, EBNA-LP and LMP1 are essential for growth transformation and immortalization of B lymphocytes. Among these genes, LMP1 plays a key role in the survival and dissemination of the infected B cells by inducing anti-apoptotic genes and surface expression of several activation antigens and adhesion molecules. We have previously shown that antisense oligodeoxynucleotides directed to LMP1 mRNA, effectively suppress LMP1 gene expression and substantially reduce B95.8 cell proliferation. In this study, we have used antisense LMP1 oligomers to investigate whether LMP1 suppression might influence the expression of latent EBV genes with oncogenic potential, anti-apoptotic genes, or affect the phenotype of EBV-infected B95.8 cells. Our data show that LMP1 suppression does not affect the transcription of EBNA-2, EBNA-3A, -3B and -3C genes, or that of bcl-2 and mcl-1 anti-apoptotic genes. In contrast, consistent modifications in the expression of CD39, CD54, CD23, CD11 and CD10 molecules were observed in B95.8 cells after treatment with antisense LMP1. Our findings support the possibility for using LMP1 antisense oligomers as therapeutics in EBV-associated tumors

The ubiquitin–proteasome system in cardiac dysfunction

Abstract Since proteins play crucial roles in all biological processes, the finely tuned equilibrium between their synthesis and degradation regulates cellular homeostasis. Controlling the quality of proteome informational content is essential for cell survival and function. After initial synthesis, membrane and secretory proteins are modified, folded, and assembled in the endoplasmic reticulum, whereas other proteins are synthesized and processed in the cytosol. Cells have different protein quality control systems, the molecular chaperones, which help protein folding and stabilization, and the ubiquitin–proteasome system (UPS) and lysosomes, which degrade proteins. It has generally been assumed that UPS and lysosomes are regulated independently and serve distinct functions. The UPS degrades both cytosolic, nuclear proteins, and myofibrillar proteins, whereas the lysosomes degrade most membrane and extracellular proteins by endocytosis as well as cytosolic proteins and organelles via autophagy. Over the last two decades, the UPS has been increasingly recognized as a major system in several biological processes including cell proliferation, adaptation to stress and cell death. More recently, activation or impairment of the UPS has been reported in cardiac disease and recent evidence indicate that autophagy is a key mechanism to maintain cardiac structure and function. This review mainly focuses on the UPS and its various components in healthy and diseased heart, but also summarizes recent data suggesting parallel activation of the UPS and autophagy in cardiac disease

Analysis of Contractile Function of Permeabilized Human Hypertrophic Cardiomyopathy Multicellular Heart Tissue

Background: Many forms of hypertrophic cardiomyopathy (HCM) show an increased myofilament Ca2+ sensitivity. This observation has been mainly made in HCM mouse models, myofilament systems, and cardiomyocytes. Studies of multicellular tissues from patients with different HCM-associated gene mutations are scarce. We investigated Ca2+ sensitivity in multicellular cardiac muscle strips of HCM patients. We furthermore evaluated the use of epigallocatechin-3-gallate (EGCg), a Ca2+ desensitizer.Methods: After strip isolation from cardiac tissues with single (MYBPC3, MYH7) or double heterozygous mutations (MYBPC3/FLNC, MYH7/LAMP2, MYBPC3/MYH7) and permeabilization, we performed contractility measurements ±EGCg. We furthermore evaluated gene expression with a customized heart failure gene panel using the NanoString technology.Results: Fmax tended to be higher in HCM than in non-failing (NF) control strips and in single than in double heterozygous strips. Ca2+ sensitivity was higher by trend in most HCM vs. NF strips and by trend in tissues with double vs. single heterozygous mutations. EGCg desensitized myofilaments to Ca2+ in most of the strips and tended to induce a more pronounced shift in strips with truncating than missense or single than double heterozygous mutations. Gene expression analysis revealed lower ATP2A2, PPP1R1A, and FHL2 and higher NPPA, NPPB, COL1A1, CTGF, and POSTN marker levels in HCM than in NF tissues. NPPA, NPPB, ACTA1, CTGF, COL1A1, and POSTN levels were higher in tissues with missense than truncating mutations.Conclusion: We report an increased myofilament Ca2+ sensitivity in native multicellular cardiac HCM strips, which by trend was more pronounced in samples with double heterozygous mutations. EGCg could have differential effects depending on the underlying genetic status (single vs. double heterozygous) and type (missense vs. truncating)

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C

Disease modeling of a mutation in α‐actinin 2 guides clinical therapy in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease accompanied by structural and contractile alterations. We identified a rare c.740C>T (p.T247M) mutation in ACTN2, encoding α‐actinin 2 in a HCM patient, who presented with left ventricular hypertrophy, outflow tract obstruction, and atrial fibrillation. We generated patient‐derived human‐induced pluripotent stem cells (hiPSCs) and show that hiPSC‐derived cardiomyocytes and engineered heart tissues recapitulated several hallmarks of HCM, such as hypertrophy, myofibrillar disarray, hypercontractility, impaired relaxation, and higher myofilament Ca2+ sensitivity, and also prolonged action potential duration and enhanced L‐type Ca2+ current. The L‐type Ca2+ channel blocker diltiazem reduced force amplitude, relaxation, and action potential duration to a greater extent in HCM than in isogenic control. We translated our findings to patient care and showed that diltiazem application ameliorated the prolonged QTc interval in HCM‐affected son and sister of the index patient. These data provide evidence for this ACTN2 mutation to be disease‐causing in cardiomyocytes, guiding clinical therapy in this HCM family. This study may serve as a proof‐of‐principle for the use of hiPSC for personalized treatment of cardiomyopathies

Localization and dynamics of small circular DNA in live mammalian nuclei

While genomic DNA, packaged into chromatin, is known to be locally constrained but highly dynamic in the nuclei of living cells, little is known about the localization and dynamics of small circular DNA molecules that invade cells by virus infection, application of gene therapy vectors or experimental transfection. To address this point, we have created traceable model substrates by direct labeling of plasmid DNA with fluorescent peptide nucleic acids, and have investigated their fate after microinjection into living cells. Here, we report that foreign DNA rapidly undergoes interactions with intranuclear structural sites that strongly reduce its mobility and restrict the DNA to regions excluding nucleoli and nuclear bodies such as PML bodies. The labeled plasmids partially co-localize with SAF-A, a well characterized marker protein for the nuclear ‘scaffold’ or ‘matrix’, and are resistant towards extraction by detergent and, in part, elevated salt concentrations. We show that the localization and the low mobility of plasmids is independent of the plasmid sequence, and does not require the presence of either a scaffold attachment region (SAR) DNA element or a functional promoter

Stability Assessment of Candidate Reference Genes in Urine Sediment of Prostate Cancer Patients for miRNA Applications

We aimed at assessing the stability of candidate reference genes in urine sediments of men subjected to digital rectal examination for suspected prostate cancer (PCa). Two microRNAs (miR-191 and miR-25) and 1 small nucleolar RNA (SNORD48) were assayed in 35 post-DRE urine sediments of men with PCa and in 26 subjects with histologically confirmed benign prostatic hyperplasia (BPH). The stability of candidate reference genes was assessed through BestKeeper algorithm and equivalence test. miR-200b and miR-452 were used to test for the effect of normalization on target genes. Our results proved miR-191 to be the most stable gene, showing the lowest degree of variation and the highest stability value. miR-25 and SNORD48 values fell beyond the cutoff of acceptability. In conclusion, we recommend the use of miR-191 for normalization purposes in post-DRE urine sediments

Circulating microRNAs and Kallikreins before and after Radical Prostatectomy: Are They Really Prostate Cancer Markers?

The aim of our study was to monitor serum levels of two miRNAs (miR-21 and miR-141) and three KLKs (hK3/PSA, hK11, and hK13) before and 1, 5, and 30 days after radical prostatectomy, in order to characterize their fluctuations after surgery. 38 patients with prostate cancer were included. miR-21 and miR-141 were quantified through real-time PCR, while ELISA assays were used to quantify hK3 (PSA), hK11, and hK13. Both miR-21 and miR-141 showed a significant increase at the 5th postoperative day, after which a gradual return to the preoperative levels was recorded. These findings suggest that miR-21 and miR-141 could be involved in postsurgical inflammatory processes and that radical prostatectomy does not seem to alter their circulating levels. Postoperative serum kallikreins showed a significant decrease, highlighting the potential usefulness of kallikreins apart from PSA as potential prostate cancer markers