Influence of age and physical exercise on sirtuin activity in humans.

Sirtuins are NAD+-dependent lysine deacetylases. Sirtuins acquired worldwide attention because of their ability to increase yeast, flies, worms and mice lifespan. Recently, this assumption has been challenged. However, their beneficial role on the quality of ageing is widely accepted. In this work we aimed to study how and if sirtuins expression and activity levels varies in function of age and, in the case of young subjects, of exercise. Fifteen blood donors of different ages and fifteen athletes of the Italian rowing male team were enrolled and peripheral blood mononuclear cells (PBMCs) isolated from blood samples. Our results show that sirtuins deacetylases activity measured in PBMCs increases from 18 to 40 years of age and then decreases during the following 20 years. Moreover, physical exercise in professional athletes can upregulate sirtuin activity. Thus, for the first time in humans, we demonstrate that sirtuin activity is a function of age and can be altered through physical exercise.

SIRT3 protects from hypoxia and staurosporine-mediated cell death by maintaining mitochondrial membrane potential and intracellular pH.

Mitochondrial sirtuin 3 (SIRT3) mediates cellular resistance toward various forms of stress. Here, we show that in mammalian cells subjected to hypoxia and staurosporine treatment SIRT3 prevents loss of mitochondrial membrane potential (ΔΨ(mt)), intracellular acidification and reactive oxygen species accumulation. Our results indicate that: (i) SIRT3 inhibits mitochondrial permeability transition and loss of membrane potential by preventing HKII binding to the mitochondria, (ii) SIRT3 increases catalytic activity of the mitochondrial carbonic anhydrase VB, thereby preventing intracellular acidification, Bax activation and apoptotic cell death. In conclusion we propose that, in mammalian cells, SIRT3 has a central role in connecting changes in ΔΨ(mt), intracellular pH and mitochondrial-regulated apoptotic pathways.

A new clinical approach: use of blood-derived stem cells (BDSCs) for superficial digital flexor tendon injuries in horses.

AIMS: In this study, we present an innovative therapy using stem cells that were obtained from the peripheral blood of racehorses affected by uninduced superficial digital flexor tendon (SDFT) injuries. MAIN METHODS: Blood-derived stem cells (BDSCs) were generated from the blood samples of three horses in the presence of macrophage colony-stimulating factor (M-CSF). The racehorses received a single autologous BDSC treatment, which resulted in the successful repair of the tendons injuries. KEY FINDINGS: The results demonstrated that the BDSCs injection into the damaged tendon stimulated the regeneration of normal tissue. Furthermore, a relationship may exist between the speed and the quality of new tissue formation and the welfare and management of the treated animals. SIGNIFICANCE: This study demonstrates that stem cell technology offers new tools for tissue repair that in many cases is considered incurable, and provides additional evidence that BDScs injections increase the speed and quality of the regeneration process in different animal tissues.

A multicancer-like syndrome in a dog characterized by p53 and cell cycle-checkpoint kinase 2 (CHK2) mutations and sirtuin gene (SIRT1) down-regulation.

INTRODUCTION: We have investigated SIRT1, p53 and cell cycle-checkpoint kinase 2 (CHK2) gene dysfunction in a dog with a multicancer syndrome-like in order to evaluate their potential role in the determinism of the disease and to establish a possible correlation between SIRT1 transcript level and p53 expression status. MATERIAL AND METHODS: Blood sample and tumour samples from a pure breed English Setter dog with different tumours were used for this study. Nucleotide sequence analysis was performed with a DNA autosequencer in order to examine p53 and CHK2 mutations. In addition, the expression level of SIRT1 was quantified by Southern Blot analysis of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). RESULTS: Cytological examination revealed five different tumours: a cutaneous sebaceous epithelioma, a cutaneous mast cell tumour, a testicular Sertoli cell tumour, an oral malignant melanoma, and a cutaneous squamous cell carcinoma. Sequencing analysis revealed the presence of a nucleotide substitution, (CGG>CAG) exon 7 of the p53 gene in DNA from peripheral blood mononuclear cells (PBMCs) as well as in the melanoma; whereas the other four cancers showed the loss of the wild-type allele. Furthermore, CHK2 mutation at codon 311 has been identified in the melanoma and sebaceous epithelioma. In addition, SIRT1 cDNA expression decreased in all tumour samples compared to cDNA SIRT1expression level in peripheral blood mononuclear cells (PBMCs) in the same dog. CONCLUSIONS: These results suggest that the germ line mutation of the p53 gene at codon 248 might be, at least, one cause of the multicancer syndrome-like in our dog; furthermore, we show a possible correlation between SIRT1 transcript level and p53 mutations status. The regulatory role of SIRT1 in tumour suppressor pathways suggests that the net effect seen may represent both direct and indirect downstream regulation and it is likely to depend on the presence or absence of functional p53.

Analysis of apoptosis induced by Caprine Herpesvirus 1 in vitro.

It is known that Caprine Herpesvirus 1 (CpHV-1) causes apoptosis in mitogen-stimulated as well as not stimulated caprine peripheral blood mononuclear cells (PBMC). Initial experiments in Madin Darby bovine kidney (MDBK) cells revealed that CpHV-1 infection induced apoptotic features like chromatin condensation and DNA laddering. Thus, to characterize in more detail this apoptotic process, activation of caspase-8, -9 and -3 in MDBK cells CpHV-1 infected was investigated and demonstrated. In addition, CpHV-1 infection resulted in disruption of mitochondrial membrane potential, cytochrome c release and alterations in the pro- and anti-apoptotic proteins of Bcl-2 family. Proteolytic cleavage of poly(ADP-ribose) polymerases (PARP), confirming the activation of downstream caspases, was also observed. Our data indicated that a "cross-talk" between the death-receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway occurred in CpHV-1-induced apoptosis in vitro.

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.

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 gamma34.5, 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 UL14 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.

Interference of bovine herpesvirus 1 (BHV-1) in sorbitol-Induced apoptosis.

In order to determine the ability of bovine herpesvirus type 1 (BHV-1) to suppress apoptosis, we examined the effects of BHV-1 infection on sorbitol-induced apoptosis on Madin-Darby bovine kidney (MDBK) cells. BHV-1 suppresses sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that BHV-1 has one or more anti-apoptotic genes. To elucidate the molecular mechanisms of apoptosis, expression of some genes encoding apoptosis-inhibiting and -promoting factors were analyzed on BHV-1 infected cells during the process of sorbitol-induced apoptosis. Our results revealed that the expression of bcl-2 and bcl-x(L) decreased after 5 and 3 h p.i., respectively; while bax and procaspase-3 expression increased with respect to control as a function of p.i. times and at 7 h p.i. they were not observed. We further show that the expression of p53 gene was also enhanced, suggesting that this apoptotic mechanism is p53 dependent. From these results, we propose that BHV-1 has one or more genes encoding apoptosis-inhibiting factors which interfere with the involvement of bcl-2 gene family members and apoptotic pathway, depending upon caspase-3, triggered by sorbitol.