Blocking the Cleavage of Filamin A by Calpain Inhibitor Decreases Tumor Cell Growth.

BACKGROUND/AIM: Filamin A (FLNA) is the most abundant and widely expressed isoform of filamin in human tissues. It is cleaved by calpain at the hinge 1 and 2 domains, producing a 90-kDa carboxyl-terminal fragment (FLNACT). Recently, it has been shown that FLNACT mediates cell signaling and transports transcription factors into the cell nucleus. However, the significance of cleavage of FLNA by calpain has not been studied in cancer cell growth. Calpeptin is a chemical inhibitor of both calpain 1 and 2 that cleaves FLNA. In this study, we questioned if inhibiting calpain using calpeptin would decrease tumor cell proliferation, migration, invasion, and colony formation. MATERIALS AND METHODS: Human melanoma (A7), prostate cancer (PC3), mouse fibrosarcoma (T241) and endothelial (MS1) cells were assayed for proliferation, migration, invasion and colony formation after treatment with calpeptin. Cell lysates were immunoblotted for FLNA and FLNACT Results: Calpeptin treatment of these cells resulted in a decreased production of FLNACT Calpeptin-treated human and mouse tumor cells displayed impaired proliferation, migration, and colony formation. CONCLUSION: These data suggest that the cleavage of FLNA by calpain is an important cellular event in the regulation of tumor cell growth.

Use and Evaluation of Newly Synthesized Fluorescence Probes to Detect Generated OH• Radicals in Fibroblast Cells.

Reactive oxygen species (ROS) are pro-oxidant molecules synthesized in body with various functions and are essential for life. Increasing in reactive oxygen species or decreasing in antioxidants level cause oxidative stress which is very harmful. OH• radical is one of ROS's, with tendency to bind to lipids, DNA and proteins which cause irreversible damage in cells. The most devastating consequences related to excess OH• radicals occur via direct binding to nucleic acids and proteins. Quantification of this high reactive radical with short life time is difficult. Electron Spin Resonance, Fluorescence, and Luminescence Spectroscopy are commonly used to determine the level of ROS. Fluorescence Probes have higher specificity and sensitivity with their excellent sensors to detect ROS's compare to the other methods. Also, there are different probes specifically designed for each radical. The purpose of this study was to identify the probe better suiting for detection of OH• radical levels. The two most recommended fluorescence probes, 2-[6-(4 V-Hydroxy) phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and coumarin-3-carboxylic acid (3-CCA) to determine OH• radical levels were compared. Following the formation of OH• radical with Fenton reaction, HPF and 3-CCA probes were added to cells and spectrofluorometric measurements were performed in their respective wavelengths. The mean amplitude of fluorescence for HPF was 32.72 ± 2.37 F.I (n = 40) and for 3-CCA was 52.11 ± 0.5 F.I (n = 40). This difference was statistically significant. 3-CCA also demonstrated more stable measurements at different days compered to HPF.

Effect of hydroxyethyl starch 130/0.4 on ischemia-reperfusion determinants in minor lower extremity surgery with tourniquet application.

STUDY OBJECTIVE: To investigate the effect of 6% hydroxyethyl starch (HES) (130/0.4) infusion on ischemia-reperfusion determinants in minor lower extremity operations with tourniquet application. DESIGN: Prospective, randomized, clinical trial. SETTING: University hospital operation room. PATIENTS: American Society of Anesthesiologists I and II 40 patients between 18 and 65 years of age who were scheduled to undergo knee arthroscopy and below-knee minor orthopedic surgery. INTERVENTIONS: Patients were randomized into 2 groups (normal saline [NS] group and HES group). Unilateral spinal anesthesia with 2-mL 0.5% hyperbaric bupivacaine after 10 mL/kg NS intravenous infusion to NS group and 10 mL/kg 6% HES intravenous infusions to HES group. MAIN OUTCOME MEASURES: Blood samples were obtained from antecubital vein for malondialdehyde, xanthine oxidase, and hypoxanthine before tourniquet inflation and after 10 minutes of tourniquet deflation. RESULTS: There was no significant difference between groups with respect of hemodynamic data. There were no significant differences between 2 groups in terms of malondialdehyde values before tourniquet inflation and after tourniquet deflation. In the HES group, xanthine oxidase levels after tourniquet deflation were significantly lower than xanthine oxidase levels before tourniquet inflation (P < .05). In the HES group, hypoxanthine levels after tourniquet deflation were similar with the basal levels; however, they were significantly higher than levels obtained before tourniquet inflation in the SF group (P < .05). CONCLUSION: HES infusion may be beneficial for reduction of tourniquet-associated ischemia-reperfusion injury; however, further large-scale studies are needed to fully elucidate its mechanism.

Resveratrol reduces matrix metalloproteinase-2 activity induced by oxygen-glucose deprivation and reoxygenation in human cerebral microvascular endothelial cells.

The main pathophysiology in cerebral ischemia is the structural alteration in the neurovascular unit, coinciding with neurovascular matrix degradation. Among the human matrix metalloproteinases (MMPs), MMP-2 and -9, known as gelatinases, are the key enzymes for degrading type IV collagen, which is the major component of the basal membrane that surrounds the cerebral blood vessel. In the present study, we investigated the effects of resveratrol on cytotoxicity, reactive oxygen species (ROS), and gelatinases (MMP-2 and -9) in human cerebral microvascular endothelial cells exposed to 6 hours of oxygen-glucose deprivation and a subsequent 24 hours of reoxygenation with glucose (OGD/R), to mimic ischemia/reperfusion in vivo. Lactate dehydrogenase increased significantly, in comparison to that in the normoxia group. ROS was markedly increased in the OGD/R group, compared to normoxia. Correspondingly, ROS was significantly reduced with 50 µM of resveratrol. The proMMP-2 activity in the OGD/R group showed a statistically significant increase from the control cells. Resveratrol preconditioning decreased significantly the proMMP-2 in the cells exposed to OGD/R in comparison to that in the OGD/R group. Our results indicate that resveratrol regulates MMP-2 activity induced by OGD/R via its antioxidant effect, implying a possible mechanism related to the neuroprotective effect of resveratrol.

Special-study modules in a problem-based learning medical curriculum: an innovative laboratory research practice supporting introduction to research methodology in the undergraduate curriculum.

We describe the organization of wet-lab special-study modules (SSMs) in the Central Research Laboratory of Dokuz Eylül Medical School, Izmir, Turkey with the aim of discussing the scientific, laboratory, and pedagogical aspects of this educational activity. A general introduction to the planning and functioning of these SSMs is given, along with specific examples. The wet-lab SSMs incorporate several innovative pedagogies: problem-based learning, research-based learning, practical laboratory education, team-based learning, and project-based learning. Oral and written evaluations show that the students find this activity rewarding. The wet-lab SSM model applied in the Research-Lab of Dokuz Eylül School of Medicine represents a format which is effective in training the students in research methodology, practical laboratory work, and independent learning.