Inhibition of the formation of lipid-linked intermediates in normal and transformed cells by a purified tunicamycin homologue

Summary The deffects of a purified homologue of tunicamycin (B₂-tunicamycin) on the biosynthesis of lipid-linked intermediates participating in protein glycosylation in normal embryonic fibroblasts, 3T3 and virally transformed (simian virus 40 and polyoma virus) mouse fibroblasts grown in culture were investigated. Long incubations (20 h) with the antibiotic caused a higher degree of inhibition of sugar incorporation into glycoproteins in transformed cells. However, the formation of lipid-linked intermediates was inhibited to a similar level in both cell types. When time dependent inhibition experiments were carried out using transformed cells, an earlier and stronger inhibition of the formation of lipid-oligosaccharides occurred (70% inhibition at 30 min). In 3T3 cells, prolonged incubation (6–8 h) was necessary in order to reach a similar degree of inhibition. Formation of lipid-sugar was also inhibited to a greater extent by B₂-tunicamycin in transformed cells. This inhibition was not clearly time dependent. Analysis of the newly synthesized glycolipids in 3T3 and in transformed cells after B₂-tunicamycin treatment have shown reduction in dolichyl-P-P-sugars as well as in other glycolipids. Dimethylsulfoxide (10%) and linoleic acid (0.5 mg/ml) markedly increased the level of tunicamycin activity in 3T3 cells while phosphatidylcholine (2 mg/ml) partially reversed it. The stronger and faster inhibition of the formation of lipid intermediates of the dolichyl-phosphate cycle caused by B₂-tunicamycin in transformed cells, described here for the first time, may therefore be due to differences in penetration of the antibiotic into these cells.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethylsulfoxide - MF mouse fibroblasts from Balb/c mouse embryos - 3T3 Balb/3T3 mouse fibroblastic line - SV40 Simian virus 40 - PY polyoma virus - TLC thin layer chromatography     

Acetyltransferase p300 inhibitor reverses hypertension‐induced cardiac fibrosis

Epigenetic dysregulation plays a crucial role in cardiovascular diseases. Previously, we reported that acetyltransferase p300 (ATp300) inhibitor L002 prevents hypertension‐induced cardiac hypertrophy and fibrosis in a murine model. In this short communication, we show that treatment of hypertensive mice with ATp300‐specific small molecule inhibitor L002 or C646 reverses hypertension‐induced left ventricular hypertrophy, cardiac fibrosis and diastolic dysfunction, without reducing elevated blood pressures. Biochemically, treatment with L002 and C646 also reverse hypertension‐induced histone acetylation and myofibroblast differentiation in murine ventricles. Our results confirm and extend the role of ATp300, a major epigenetic regulator, in the pathobiology of cardiac hypertrophy and fibrosis. Most importantly, we identify the efficacies of ATp300 inhibitors C646 and L002 in reversing hypertension‐induced cardiac hypertrophy and fibrosis, and discover new anti‐hypertrophic and anti‐fibrotic candidates.     

Effects of Quercetin against Doxorubicin-Induced Testicular Toxicity in Male Rats

Biology Bulletin - This study investigated the protective effects of quercetin (QUE) in doxorubicin (DOX)-induced testicular toxicity in rats. In the present study, testicular histomorphometry, the...     

The distally pedicled peroneus brevis muscle flap: a new flap for the lower leg

Defects of the skin and soft tissue in the region of the lateral malleolus of the ankle and the Achilles tendon, resulting in exposed bone, tendons, or osteosynthetic material, cannot be covered with free skin transplants. Local or free flaps must be employed. The authors present the construction of a peroneus brevis muscle flap with a distal pedicle as a useful alternative. Between 1993 and 1999, distal pedicled peroneus brevis muscle flaps were used in 19 patients with various types of defects. During construction of the flap, both the long peroneal muscle and the peroneal artery remained intact. In the region of the distal third of the fibula, consistently arranged branches run from the artery into the muscle, and these form the distal pedicle. The proximal portion of the muscle can be transposed distally and easily extends to the tip of the fibula and the attachment of the Achilles tendon to the calcaneus. Primary healing occurred in 16 patients undergoing flap construction. Donor-site morbidity was mostly limited to the donor-site scar. The distally pedicled peroneus brevis muscle flap is a reliable means for covering defects in the lower leg. This form of muscle flap has not yet been described in the known literature. In the authors' opinion, this flap constitutes a logical and valuable extension of local flap procedures for plastic surgery in the distal leg region.     

Nitric Oxide Prevents Alveolar Senescence and Emphysema in a Mouse Model

N^ω-nitro-L-arginine methyl ester (L-NAME) treatment induces arteriosclerosis and vascular senescence. Here, we report that the systemic inhibition of nitric oxide (NO) production by L-NAME causes pulmonary emphysema. L-NAME-treated lungs exhibited both the structural (alveolar tissue destruction) and functional (increased compliance and reduced elastance) characteristics of emphysema development. Furthermore, we found that L-NAME-induced emphysema could be attenuated through both genetic deficiency and pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1). Because PAI-1 is an important contributor to the development of senescence both in vitro and in vivo, we investigated whether L-NAME-induced senescence led to the observed emphysematous changes. We found that L-NAME treatment was associated with molecular and cellular evidence of premature senescence in mice, and that PAI-1 inhibition attenuated these increases. These findings indicate that NO serves to protect and defend lung tissue from physiological aging.     

Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation

The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene) has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol’s anti-aging effects both in vitro and in vivo attributed to activation of a (NAD)-dependent histone deacetylase family member sirtuin-1 (SIRT1) protein. In mammals seven members (SIRT1-7) of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ) and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal) activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21^CIP1 and p16^INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21^CIP1 and p16^INK4A levels. Interestingly DNA damaging agent doxorubicin also induced senescence in BJ fibroblasts associated with decreased SIRT1/2 levels. In conclusion our data reveal that resveratrol induced premature senescence is associated with SIRT1 and SIRT2 down regulation in human dermal fibroblasts. Here we suggest that the concomitant decline in SIRT1/2 expression in response to resveratrol treatment may be a cause for induction of senescence, which is most likely mediated by a regulatory mechanism activated by DNA damage response.     

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and “pearls of wisdom”/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.     

Investigation of C5a receptor gene 450 C/T polymorphism in Turkish patients with familial Mediterranean fever

Familial Mediterranean fever (FMF) is a genetic disorder with acute inflammatory serosal attacks due to MEFV gene mutations which resides in chromosome 16. Lack of a C5a inhibitor activity in the peritoneum has previously been proposed in part to contribute in propagation of the serosal inflammation in FMF attacks. The aim of this study is to investigate C5a receptor (C5aR) gene polymorphism in patients with FMF and its relation to the main features of the disease. A polymorphism in the coding region of C5aR gene leading to C to T transition at nucleotide position 450 has been investigated in 85 non-related Turkish FMF patients and 160 non-related healthy controls by using PCR-RFLP. The frequencies of C5aR gene 450 CT genotype and T allele were not significantly different between Turkish FMF patients and healthy subjects (14.12 and 8.24% for FMF vs. 10 and 5% for controls, respectively). C5aR gene 450 CT genotype tended to associate with the presence Henoch-Schonlein purpura (OR: 1.25, 95% CI: 0.917–1.704, P = 0.017) but with no other clinical findings of the disease. C5aR polymorphism might be searched in populations having high prevalence of FMF.