The modulation of sirtuins and apoptotic proteins in rats after exhaustive exercise

A large body of evidence shows that a single bout of strenuous exercise induces oxidative stress in circu- lating human lymphocytes leading to lipid peroxide- tion, DNA damage, mitochondrial perturbations, and protein oxidation. In a training experiment, Wistar rats were divided into control group (CG) and exer- cise group (EG). After a running level exercise until exhaustion, we observed an increase in the mRNA content and protein expression of SIRT1 and SIRT7 in the EG compared to the CG. Moreover, such train- ing exercise did not change mRNA transcripts and protein expression of FOXO3A and GADD45. We also observed an increase of pro-apoptotic protein bax and a decrease of the anti-apoptotic protein bcl-2 in the EG. Accordingly, we observed a caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage only in EG rats. Statistical analysis of the data showed a significant correlation between SIRT1 and SIRT7 expression and apoptotic proteins such as bax, bcl-2 in both tissues. We conclude that, in both muscle, such exercise activates both a damaging apoptotic mecha- nism with bax increase and bcl-2 decrease and a counterbalancing protective mechanism with SIRT1 and SIRT7 increase

Degenerate PCR method for identification of an antiapoptotic gene in BHV-1

To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type-1) BHV-1 DNA. An alignment of carboxyl-terminal domains belonging to three proteins encoded by gamma 34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type-1 (BHV-1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that ULI 4 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The protein's anti-apoptotic function may derive from its heat shock protein-like properties

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Blood-derived stem cells (BDSCs) plasticity: In vitro hepatic differentiation.

8noThe limited availability of hepatic tissue suitable for the treatment of liver disease and drug research encourages the generation of hepatic-like cells from alternative sources as support for the regenerative medicine. Human Blood Derived Stem Cells (BDSCs) express surface markers and genes characteristic of pluripotent stem cells and have the ability to differentiate into different cell types, including tissues of endodermal origin (i.e. liver). Therefore they can represent a valuable source of hepatocytes for medicine. In this investigation we exploited a fast hepatic differentiation protocol to generate hepatocyte-like cells from human BDSCs using only Hepatocyte Growth Factor (HGF) and Fibroblast Growth Factor- 4 (FGF-4) as growth factors. The resulting cell population exhibited hepatic cell-like morphology and it was characterized with a variety of biological endpoint analyses. Here, we demonstrate how human BDSCs can be reprogrammed in hepatocyte-like cells by morphological, functional analysis, Reverse Transcriptase (RT)-PCR and Western Blot assay. This study defines a fast and easy reprogramming strategy that facilitates the differentiation of human BDSCs along a hepatic lineage and provides a framework for a helpful source in the stem cells therapy and liver disorders. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.reservedmixedAlaimo G; Cozzoli E; Marfe G; Esposito L; Ranalli M; Hmada D; Giordano, Antonio; Gambacurta AAlaimo, G; Cozzoli, E; Marfe, G; Esposito, L; Ranalli, M; Hmada, D; Giordano, Antonio; Gambacurta, A

The interference of rosmarinic acid in the DNA fragmentation induced by osmotic shock

The induction of cell death in human erythroleukemic cells ( K562) by sorbitol shows the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation and nuclear membrane breakdown.. Using a cytofluorimetric approach, we found that sorbitol induced production of reactive oxygen species ( ROS) followed by DNA fragmentation in leukemic cells. In this study, we investigated effect of curcumin and rosmarinic acid on cell viability in three different cell lines: erythroleukemia K562, papillary NPA, and anaplastic ARO thyroid cancers. Curcumin was able to induce apoptosis in a concentration- and time dependent manner in three cell lines, while rosmarinic acid was less effective on this process. To examine this possibility in cellular system, this study evaluated the capacities of both compounds acting as antioxidant inhibiting sorbitolinduced apoptosis. K562, NPA and ARO cells were preincubated with 25 mu M rosmarinic acid to allow the uptake and then the cell lines were treated with 1 M sorbitol. Afterwards, the cells were subjected to agarose gel electrophoresis to assess the DNA fragmentation. In conclusion, the antioxidant activity of rosmarinic acid is able to inhibit sorbitol- induced apoptosis

Antiapoptotic activity of bovine herpesvirus type-1 (BHV-1) UL14 protein

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. One previous study showed that transient transfection of bovine herpesvirus type-1 (BHV-1) UL14 protein is efficient in protecting Madin Darby kidney (MDBK) and human chronic myelogenous leukemia (K562) cells from sorbitol-induced apoptosis. This protein corresponds to a putative protein of BHV-1, which shares aminoacid sequence with a part of the peptide-binding domain conserved in human heat shock protein (HSP70) family. The pBK-CMV-UL14 plasmid transfected MDBK cells treated with sorbitol did not show caspase-3 and caspase-9 activation with respect to non-transfected MDBK cells (UL14 negative). Furthermore, we report that the expression of the full length sequence of BHV-1 UL14 is evident after 7 h of infection of BHV-1 on MDBK cells which were then treated with sorbitol. These results indicate that UL14 gene product has important implications to enhance cell survival in response to apoptotic stimuli

EFFECT OF SIMILAR PHENOLIC PHYTOCHEMICALS AS INHIBITORS OF SORBITOL-INDUCED APOPTOSIS

BERGAM