Changes in signalling molecule levels in 10-day hindlimb immobilized rat muscles

AIM: Hindlimb immobilization produces similar percentage decreases in muscle mass in the predominantly type I soleus and type II vastus lateralis muscles. Consequently we hypothesized that the percentage changes in potential regulatory molecules for atrophy would be similar in the two muscle fibre types. METHODS: Therefore, the purpose of the current study was to measure phosphorylated p38 MAPK and JNK, as well as the protein levels of p53, growth arrest and DNA damage-inducible 45 (GADD45), and full-length poly(ADP-ribose) polymerase (PARP) to determine whether their changes in expression in the soleus and vastus lateralis muscles were similar at 10th day of hindlimb immobilization in young rats. RESULTS: Unexpectedly, preferential increases in phosphorylation of p38 MAPK and JNK and in the protein levels of p53, GADD45, as well as decreases in full-length PARP occurred in the soleus muscle, while only p38 phosphorylation increased in the white portion of the vastus lateralis muscle at 10th day of hindlimb immobilization. CONCLUSION: Taken together, these results are interpreted to suggest that some of regulatory processes or kinetics in the atrophy of type I and II muscle fibres during limb immobilization may differ at the 10th day of limb immobilization.     

The efficiency of Poly(ADP‐Ribose) Polymerase (PARP) cleavage on detection of apoptosis in an experimental model of testicular torsion

The aim of this study was to evaluate the histopathological and apoptotic changes occurring in the rat ipsilateral and contralateral testes, after experimental spermatic cord torsion, and to explore and the role of poly(ADP‐ribose) polymerase (PARP) cleavage in testicular torsion–detorsion injury. A total of 37 Wistar albino rats were subjected to 720° unilateral spermatic cord torsion for 1, 2 and 4 h, followed by 4‐h reperfusion, or else to a sham operation (control group). Histology of the testicle was evaluated using haematoxylin–eosin (H&E) staining and Johnsen's scoring system. Germ cell apoptosis was evaluated via active caspase‐3 immunostaining, and PARP expression levels were evaluated via Western blotting. The mean Johnsen's tubular biopsy scores (JTBS) of the ipsilateral testicles were lower for all torsion groups than for the controls (P < 0.05), but the JTBS of the contralateral testicles were only lower in the 4‐h torsion group (P < 0.05). The mean apoptosis score (AS) of the ipsilateral and contralateral testicles was significantly higher in the torsion groups than in the sham group. AS increased correlatively with torsion time, in both testicles. The effect of testicular torsion on PARP cleavage was time dependent, with the highest effect observed after 4 h of testicular torsion (P < 0.05). Testicular torsion caused time‐dependent histological changes, apoptosis and increases in PARP cleavage. Our results suggest that testicular torsion–detorsion injury caused cell damage and germ cell apoptosis that apparently involved cleavage of PARP. Increased PARP cleavage could, in turn, lead to enhanced apoptosis.     

Bcl‐2 protects TK6 cells against hydroquinone‐induced apoptosis through PARP‐1 cytoplasm translocation and stabilizing mitochondrial membrane potential

B cell leukemia/lymphoma‐2 (Bcl‐2) suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating mitochondrial membrane potential (MMP). This study aimed to investigate the role of Bcl‐2 in controlling the mitochondrial pathway of apoptosis during hydroquinone (HQ)‐induced TK6 cytotoxicity. In this study, HQ, one metabolite of benzene, decreased the MMP in a concentration‐dependent manner and induced the generation of reactive oxygen species (ROS), the activation of the DNA damage marker γ‐H2AX, and production of the DNA damage‐responsive enzyme poly(ADP‐ribose)polymerase‐1 (PARP‐1). Exposure of TK6 cells to HQ leads to an increase in Bcl‐2 and co‐localization with PARP‐1 in the cytoplasm. Inhibition of Bcl‐2 using the BH3 mimetic, ABT‐737, suppressed the PARP‐1 nuclear to cytoplasm translocation and sensitized TK6 cells to HQ‐induced apoptosis through depolarization of the MMP. Western blot analysis indicated that ABT‐737 combined with HQ increased the levels of cleaved PARP and γ‐H2AX, but significantly decreased the level of P53. Thus, ABT‐737 can influence PARP‐1 translocation and induce apoptosis via mitochondria‐mediated apoptotic pathway, independently of P53. In addition, we found that knockdown of PARP‐1 attenuated the HQ‐induced production of cleaved PARP and P53. These results identify Bcl‐2 as a protective mediator of HQ‐induced apoptosis and show that upregulation of Bcl‐2 helps to localize PARP‐1 to the cytoplasm and stabilize MMP. Environ. Mol. Mutagen. 59:49–59, 2018. © 2017 Wiley Periodicals, Inc.     

Role of Cytotoxic Protease Granzyme‐b in Neuronal Degeneration During Human Stroke

Infiltration of leukocytes into post‐ischemic cerebrum is a well‐described phenomenon in stroke injury. Because CD‐8⁺ T‐lymphocytes secrete cytotoxic proteases, including granzyme‐b (Gra‐b) that exacerbates post‐ischemic brain damage, we investigated roles of Gra‐b in human stroke. To study the role of Gra‐b in stroke, ischemic and non‐ischemic tissues (from post‐mortem stroke patients) were analyzed using immunoblotting, co‐immunoprecipitation, terminal deoxy uridine nick end labeling (TUNEL) and Annexin–V immunostaining, and in vitro neuron survival assays. Activated CG‐SH cells and supernatants were used to model leukocyte‐dependent injury. Non‐ischemic brain tissues were used as non‐pathological controls. Non‐activated CG‐SH cells and supernatants were used as controls for in vitro experiments. Human stroke (ischemic) samples contained significantly higher levels of Gra‐b and interferon‐gamma inducible protein‐10 (IP‐10/CXCL10) than non‐ischemic controls. In stroke, poly (ADP‐ribose) polymerase‐1 and heat shock protein‐70 were cleaved to canonical proteolytic “signature” fragments by Gra‐b. Gra‐b was also found to bind to Bid and caspase‐3. Gra‐b also co‐localized with Annexin‐V⁺/TUNEL⁺ in degenerating neurons. Importantly, Gra‐b inhibition protected both normal and ischemia‐reperfused neurons against in vitro neurotoxicity mediated by activated CG‐SH cells and supernatants. These results suggest that increased leukocyte infiltration and elevated Gra‐b levels in the post‐stroke brain can induce contact‐dependent and independent post‐ischemic neuronal death to aggravate stroke injury.     

Polychlorinated dibenzo‐p‐dioxins and dibenzofurans levels in piglet liver with various diseases

This study deals with the levels of toxic polychlorinated dibenzo‐p‐dioxin and furan congeners (PCDD/Fs) in the livers of piglets affected by infectious diseases using isotope dilution high‐resolution gas chromatography/high‐resolution mass spectrometry (HRGC/HRMS). Seventeen toxic congeners in the liver samples infected with bacterial and viral diseases were compared. For porcine reproductive and respiratory syndrome virus (PRRSV) samples, the North American‐ and European‐type PRRS diseases were observed. This study shows that there are significantly different levels of PCDD/Fs, present, which vary according to the types of diseases as evidenced by our analysis of the piglet liver samples.     

Changes in calsequestrin,TNF‐α, TGF‐β and MyoD levels during the progression of skeletal muscle dystrophy in mdx mice: a comparative analysis of the quadriceps,diaphragm and intrinsic laryngeal muscles

In Duchenne muscular dystrophy (DMD), the search for new biomarkers to follow the evolution of the disease is of fundamental importance in the light of the evolving gene and pharmacological therapies. In addition to the lack of dystrophin, secondary events including changes in calcium levels, inflammation and fibrosis greatly contribute to DMD progression and the molecules involved in these events may represent potential biomarkers. In this study, we performed a comparative evaluation of the progression of dystrophy within muscles that are differently affected by dystrophy (diaphragm; DIA and quadriceps; QDR) or spared (intrinsic laryngeal muscles) using the mdx mice model of DMD. We assessed muscle levels of calsequestrin (calcium‐related protein), tumour necrosis factor (TNF‐α; pro‐inflammatory cytokine), tumour growth factor (TGF‐β; pro‐fibrotic factor) and MyoD (muscle proliferation) vs. histopathology at early (1 and 4 months of age) and late (9 months of age) stages of dystrophy. Fibrosis was the primary feature in the DIA of mdx mice (9 months: 32% fibrosis), which was greater than in the QDR (9 months: 0.6% fibrosis). Muscle regeneration was the primary feature in the QDR (9 months: 90% of centrally nucleated fibres areas vs. 33% in the DIA). The QDR expressed higher levels of calsequestrin than the DIA. Laryngeal muscles showed normal levels of TNF‐α, TGF‐β and MyoD. A positive correlation between histopathology and cytokine levels was observed only in the diaphragm, suggesting that TNF‐α and TGF‐β serve as markers of dystrophy primarily for the diaphragm.     

B type CpG-DNA suppresses poly(I:C)-induced BLyS expression and production in human tonsillar fibroblasts

Although B lymphocyte stimulator (BLyS) has potent costimulatory effects on B cells, the details of BLyS-expression in tonsillar fibroblasts remain unexplored. We examined the effect of the Toll-like receptor (TLR) ligands on BLyS-expression in human tonsillar fibroblasts as well as the crosstalk that occurs among different TLR ligands. The expression of BLyS mRNA by tonsillar fibroblasts was strongly induced in the presence of polyinosinic–polycytidylic acid (poly(I:C)) that is a ligand, of TLR3. We also revealed that DNA containing CpG motifs (CpG-DNA), coding for a TLR9 ligand, markedly suppressed the poly(I:C)-induced mRNA expression and protein production of BLyS. B type CpG-DNA decreased the poly(I:C)-induced phosphorylation of inhibitor kappa B alpha (IκBα) and its degradation. Pre-incubation with nuclear factor kappa B (NF-κB) signaling inhibitors reduced the poly(I:C)-induced BLyS-expression. These results indicate that human tonsillar fibroblasts strongly induce BLyS-expression and production that can be inhibited by CpG-DNA and regulated through NF-κB signaling.     

Reversion of the ErbB malignant phenotype and the DNA damage response

The ErbB or HER family is a group of membrane bound tyrosine kinase receptors that initiate signal transduction cascades, which are critical to a wide range of biological processes. When over-expressed or mutated, members of this kinase family form homomeric or heteromeric kinase assemblies that are involved in certain human malignancies. Targeted therapy evolved from studies showing that monoclonal antibodies to the ectodomain of ErbB2/neu would reverse the malignant phenotype. Unfortunately, tumors develop resistance to targeted therapies even when coupled with genotoxic insults such as radiation.     

The ubiquitin proteasome system in neurodegenerative diseases: Culprit,accomplice or victim?

A shared hallmark for many neurodegenerative disorders is the accumulation of toxic protein species which is assumed to be the cause for these diseases. Since the ubiquitin proteasome system (UPS) is the most important pathway for selective protein degradation it is likely that it is involved in the aetiology neurodegenerative disorders. Indeed, impairment of the UPS has been reported to occur during neurodegeneration. Although accumulation of toxic protein species (amyloid β) are in turn known to impair the UPS the relationship is not necessarily causal. We provide an overview of the most recent insights in the roles the UPS plays in protein degradation and other processes. Additionally, we discuss the role of the UPS in clearance of the toxic proteins known to accumulate in the hallmarks of neurodegenerative diseases. The present paper will focus on critically reviewing the involvement of the UPS in specific neurodegenerative diseases and will discuss if UPS impairment is a cause, a consequence or both of the disease.