Valproic acid inhibits the proliferation of SHSY5Y neuroblastoma cancer cells by downregulating URG4/URGCP and CCND1 gene expression

Valproic acid (VPA), used for the treatment of epilepsy and bipolar disorder, regulates several signaling pathways in brain cells. The up-regulated gene 4 (URG4/URGCP) is a novel gene located on 7p13. URG4/URGCP stimulates cyclin D1 (CCND1) mRNA expression, and URG4/URGCP silencing diminishes CCND1 mRNA expression in HepG2 cells. This study was performed to investigate the anti-cancer mechanism of action of VPA by analyzing the expression of novel gene URG4/URGCP, CCND1, p21, p53, p65 (RelA), Bax, and Bcl-2 in SHSY5Y neuroblastoma (NB) cancer cells. Cytotoxic effects of VPA in SHSY5Y were noticed in time and dose dependent manner with the IC₅₀ doses within the range of 0.5–10 mM. IC₅₀ doses in the SHSY5Y were detected as 7.5 mM. Expression profiles were determined by semi quantitative RT-PCR and URG4/URGCP protein change by western blot analysis. Our results suggest that VPA induces cell cycle arrest in SHSY5Y due to the decrease in URG4/URGCP, CCND1 gene expression and the increase in p65. To conclude, VPA may be a prospective agent for the treatment of NB as a single agent or in combination with other drugs. Thus, more studies should be designed to find a safe dose with the best effects of VPA.     

Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast

Nickel is an essential micronutrient due to its involvement in many enzymatic reactions as a cofactor. However, excess of this element is toxic to biological systems. Here, we constructed a cDNA library from Beta maritima and screened it in the yeast system to identify genes that confer resistance to toxic levels of nickel. A cDNA clone (NIC6), which encodes for a putative membrane protein with unknown function, was found to help yeast cells to tolerate toxic levels of nickel. A GFP fused form of Nic6 protein was localized to multivesicular structures in tobacco epidermal cells. Thus, our results suggest a possible role of Nic6 in nickel and intracellular ion homeostasis.     

Complications Following Endoscopic Retrograde Cholangiopancreatography: Minimal Invasive Surgical Recommendations

Background

ERCP has a complication rate ranging between 4% and 16% such as post-ERCP pancreatitis, hemorrhage, cholangitis and perforation. Perforation rate was reported as 0.08% to 1% and mortality rate up to 1.5%. Besides, injury related death rate is 16% to 18%. In this study we aimed to present a retrospective review of our experience with post ERCP-related perforations, reveal the type of injuries and management recommendations with the minimally invasive approaches.

Methods

Medical records of 28 patients treated for ERCP-related perforations in Okmeydani Training and Research Hospital between March 2007 and March 2013 were reviewed retrospectively. Patient age, gender, comorbidities, ERCP indication, ERCP findings and details were analyzed. All previous and current clinical history, laboratory and radiological findings were used to assess the evaluation of perforations.

Results

Between March 2007 and March 2013, 2972 ERCPs were performed, 28 (0.94%) of which resulted in ERCP-related perforations. 10 of them were men (35.8%) and 18 women (64.2%). Mean age was 53.36±14.12 years with a range of 28 to 78 years. 14 (50%) patients were managed conservatively, while 14 (50%) were managed surgically. In 6 patients, laparoscopic exploration was performed due to the failure of non-surgical management. In 6 of the patients that ERCP-related perforation was suspected during or within 2 hours after ERCP, underwent to surgery primarily. There were two mortalities. The mean length of hospitalization stay was 10.46±2.83 days. The overall mortality rate was 7.1%.

Conclusion

Successful management of ERCP-related perforation requires immediate diagnosis and early decision to decide whether to manage conservatively or surgically. Although traditionally conventional surgical approaches have been suggested for the treatment of perforations, laparoscopic techniques may be used in well-chosen cases especially in type II, III and IV perforations.     

The protective role of topical propolis on experimental keratitis via nitric oxide levels in rabbits

The aim of this study was to investigate antioxidant, anti-inflammatory, and antibacterial properties of propolis in the treatment of experimental Staphylococcus aureus keratitis. Twenty young New Zealand white rabbits were used in this experiment. Staphylococcus aureus were given by intrastromal injection to 16 rabbits and 4 rabbits were used as control group (Group 1). Group 2 was treated with phosphate-buffered solution drops; Group 3 was administered ethanolic extract of propolis drops; Group 4 received topical ciprofloxacin drops; Group 5 was treated with topical ciprofloxacin drops along with ethanolic extract of propolis drops. The eyes were examined by slit lamp to assess corneal opacity. And then, corneas were removed to determine nitric oxide (NO) levels and count bacteria. Corneas were also evaluated histologically. Corneal NO concentration in gruop 5, treated with a combination of propolis and ciprofloxacin was determined significantly lower (10.0± 1.8 μmol/g wet tissue) than in Group 4, treated with ciprofloxacin (24.0± 3.1 μmol/g wet tissue), from Group 3, treated with propolis (15.6± 1.8 μmol/g wet tissue), and treated with PBS (44.7± 7.8 μmol/g wet tissue). There were significantly fewer bacteria in eyes that received propolis plus ciprofloxacin than in eyes treated with ciprofloxacin (p = 0.0001) or propolis (p = 0.0001) or eyes treated with PBS (p = 0.0001). The light microscopic examination revealed that the control group showed normal corneal morphology. In the nontreated group, sections of the stromal infiltration revealed the presence of inflammatory cells, which were diffusely distributed (p < 0.05). Administrations of ciprofloxacin plus propolis resulted in a significantly reduced histological damage with fewer bacterial inoculation of the corneal stroma in comparison with the other groups (p < 0.05). Based on these findings, we suggest that ethanolic extract of propolis has antioxidant, anti-inflammatory, and antibacterial properties for S. aureus keratitis in rabbits.     

Sensitivity of cells to apoptosis induced by iron deprivation can be reversibly changed by iron availability

We tested the effect of iron deprivation on cell death induction in human Raji cells pre-adapted to differing availability of extracellular iron. Iron deprivation was achieved by incubation in a defined iron-free medium. Original Raji cells have previously been adapted to long-term culture in a defined medium with 5 microg/ml of iron-saturated human transferrin as a source of iron. Raji/lowFe cells were derived from original Raji cells by subsequent adaptation to culture in the medium with 50 microm ferric citrate as a source of iron. Raji/lowFe-re cells were derived from Raji/lowFe cells by re-adaptation to the transferrin-containing (5 microg/ml) medium. Iron deprivation induced cell death in both Raji cells and Raji/lowFe-re cells; that is, cells pre-adapted to a near optimum source of extracellular iron (5 microg/ml of transferrin). However, Raji/lowFe cells preadapted to a limited source of extracellular iron (50 microm ferric citrate) became resistant to the induction of cell death by iron deprivation. We demonstrated that cell death induction by iron deprivation in Raji cells correlates with the activation of executioner caspase-3 and the cleavage of caspase-3 substrate, poly-ADP ribose polymerase. Two other executioner caspases, caspase-7 and caspase-6, were not activated. Taken together, we suggest that in human Raji cells, iron deprivation induces apoptotic cell death related to caspase-3 activation. However, the sensitivity of the cells to death induction by iron deprivation can be reversibly changed by extracellular iron availability. The cells pre-adapted to a limited source of extracellular iron became resistant.     

Functional Analysis of Free Methionine-R-sulfoxide Reductase from Saccharomyces cerevisiae

Methionine sulfoxide reductases (Msrs) are oxidoreductases that catalyze thiol-dependent reduction of oxidized methionines. MsrA and MsrB are the best known Msrs that repair methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO) residues in proteins, respectively. In addition, an Escherichia coli enzyme specific for free Met-R-SO, designated fRMsr, was recently discovered. In this work, we carried out comparative genomic and experimental analyses to examine occurrence, evolution, and function of fRMsr. This protein is present in single copies and two mutually exclusive subtypes in about half of prokaryotes and unicellular eukaryotes but is missing in higher plants and animals. A Saccharomyces cerevisiae fRMsr homolog was found to reduce free Met-R-SO but not free Met-S-SO or dabsyl-Met-R-SO. fRMsr was responsible for growth of yeast cells on Met-R-SO, and the double fRMsr/MsrA mutant could not grow on a mixture of methionine sulfoxides. However, in the presence of methionine, even the triple fRMsr/MsrA/MsrB mutant was viable. In addition, fRMsr deletion strain showed an increased sensitivity to oxidative stress and a decreased life span, whereas overexpression of fRMsr conferred higher resistance to oxidants. Molecular modeling and cysteine residue targeting by thioredoxin pointed to Cys¹⁰¹ as catalytic and Cys¹²⁵ as resolving residues in yeast fRMsr. These residues as well as a third Cys, resolving Cys⁹¹, clustered in the structure, and each was required for the catalytic activity of the enzyme. The data show that fRMsr is the main enzyme responsible for the reduction of free Met-R-SO in S. cerevisiae.Among the 20 common amino acids in proteins, Met and Cys are the residues most susceptible to oxidation by reactive oxygen species (ROS).³ Upon oxidation, Met forms a diastereomeric mixture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO). Met-S-SO and Met-R-SO can be reduced back to Met by MsrA (Met-S-SO reductase) and MsrB (Met-R-SO reductase), respectively (1). These enzymes have been reported to play important roles in the protection of cells and proteins against oxidative stress (2–8). Reversible Met oxidation has also been proposed to scavenge ROS, thereby protecting cells from oxidative damage (9–11). Increased expression of MsrA and MsrB can extend the life span of yeast cells and fruit flies, whereas deletion of the MsrA gene leads to the reduction in life span in mice and yeast (12–14).Previously, three MsrB isozymes and a single MsrA were found in mammals. MsrB1 (also known as SelR or SelX) is a selenoprotein, which contains selenocysteine (Sec) in the active site and is localized to cytosol and nucleus. MsrB2 and MsrB3 are Cys-containing homologs of MsrB1. MsrB2 resides in mitochondria, whereas human MsrB3 has two alternative splice forms, wherein MsrB3A localizes to the endoplasmic reticulum and MsrB3B is targeted to mitochondria (15).The catalytic mechanism of MsrA involves a sulfenic acid intermediate at the catalytic Cys followed by the formation of a disulfide bond between the catalytic and resolving Cys. A third Cys may then form a disulfide with the resolving Cys (16, 17). The resulting disulfide is reduced by thioredoxin or other oxidoreductases, generating the initial, reduced form of the protein. X-ray structures of MsrAs from several organisms have been solved (17, 18).Cys-containing MsrBs (e.g. mammalian MsrB2 and MsrB3) follow the same mechanism, although the two Msr types have no homology and are characterized by different structural folds (19–21). Sec-containing mammalian MsrB1 has also been characterized and compared with Cys-containing MsrBs (20). Interestingly, Cys-containing MsrBs share some active site features (e.g. conserved residues His⁷⁷, Val⁸¹, and Asn⁹⁷, numbering based on mouse MsrB1 sequence), which are absent in selenoprotein MsrB1s. When these three residues were introduced into the Sec-containing MsrB1, the enzyme was inactive. However, when the three residues were introduced into the Cys mutant form of MsrB1, the activity was partially recovered (20). This evidence supports the idea that catalytic Cys and Sec require different active site features.In addition to MsrA and MsrB functions, previous studies suggested the presence of additional Msr activities in Escherichia coli and yeast cells, which were especially evident in cells deficient in both enzymes (14, 21–23). Recently, Lowther and colleagues (24) discovered a new enzyme, designated fRMsr (free Met-R-SO reductase), which catalyzes the reduction of free Met-R-SO in E. coli. They showed that this activity is associated with a GAF-like-domain-containing protein. Homologs of this enzyme were found in other bacteria as well as in eukaryotes, suggesting that these proteins also could function as fRMsrs. However, none of these other proteins have been functionally characterized.In this work, we cloned a yeast homolog of bacterial fRMsr and functionally characterized it with regard to the in vivo function and catalytic mechanism. In addition, we carried out comparative genomic analyses to examine evolution of this protein family. The data show that fRMsr is the main enzyme responsible for the reduction of free Met-R-SO in both prokaryotes and unicellular eukaryotes.     

Effect of black cumin (Nigella sativa) on heart rate, some hematological values, and pancreatic β-cell damage in cadmium-treated rats

This study was designed to investigate the effect of Nigella sativa (NS) on the heart rate, some hematological values, and pancreatic β-cell damage in cadmium (Cd)-treated rats. The rats were randomly grouped into one of three experimental groups: Control, Cd treated, and Cd+NS treated. Each group contained 10 animals. The Cd-treated and Cd+NS-treated groups were injected subcutaneously daily with CdCl₂ dissolved in isotonic NaCl in the amount of 2 mL/kg for 30 d, resulting in a dosage of 0.49 mg Cd/kg/d. The control group was injected with only isotonic NaCl (2 mL/kg/d) throughout the experiment (for 30 d). Three days prior to administration of CdCl₂, the Cd+NS-treated group received the daily intraperitoneal (ip) injection of 2 mL/kg NS until the end of the study; animals in all three groups were fasted for 12 h and blood samples were taken for the determination of the glucose and insulin levels, red blood cell (RBC) and white blood cell (WBC) counts, packet cell volume (PCV), and hemoglobin (Hb) concentration. The heart rates of rats were also measured by a direct writing electrocardiograph before the blood withdrawals. It was found that NS treatment increased the lowered insulin levels, RBC and WBC counts, PCV, and neutrophil percentage in Cd-treated rats. However, the WBC count of Cd-treated rats with NS treatment was still lower than those of control values. NS treatment also decreased the elevated heart rate and glucose concentration of Cd-treated rats. Pancreatic tissues were also harvested from the sacrificed animals for morphological and immunohistochemical examinations. Cd exposure alone caused a degeneration, necrosis, and weak degranulation in the β-cells of the pancreatic islets. In Cd+NS-treated rats, increased staining of insulin and preservation of islet cells were apparent. It is concluded that NS treatment might decrease the Cd-treated disturbances on heart rate, some hematological values, and pancreatic β-cell.     

Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes

In humans the mitochondrial inner membrane protein Oxa1L is involved in the biogenesis of membrane proteins and facilitates the insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane. The C-terminal ∼100-amino acid tail of Oxa1L (Oxa1L-CTT) binds to mitochondrial ribosomes and plays a role in the co-translational insertion of mitochondria-synthesized proteins into the inner membrane. Contrary to suggestions made for yeast Oxa1p, our results indicate that the C-terminal tail of human Oxa1L does not form a coiled-coil helical structure in solution. The Oxa1L-CTT exists primarily as a monomer in solution but forms dimers and tetramers at high salt concentrations. The binding of Oxa1L-CTT to mitochondrial ribosomes is an enthalpy-driven process with a K_d of 0.3–0.8 μm and a stoichiometry of 2. Oxa1L-CTT cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins L13, L20, and L28 and to mammalian mitochondrial specific ribosomal proteins MRPL48, MRPL49, and MRPL51. Oxa1L-CTT does not cross-link to proteins decorating the conventional exit tunnel of the bacterial large ribosomal subunit (L22, L23, L24, and L29).     

Linking Pharmacokinetics and Biomarker Data to Mechanism of Action in Risk Assessment

Incorporating information on metabolism, pharmacokinetics, and DNA and protein biomarkers provides a means to integrate these important factors into the risk assessment process. Such data are useful for species to species extrapolation, high- to low-dose extrapolation and PBPK modeling. In addition, these data are critical for understanding the mode of action for chemical carcinogens. Through the use of mass spectrometry, stable isotopes can be used to unequivocally demonstrate pathways of formation of biomarkers and relationships between exogenous and endogenous processes. This paper reviews what has been learned for two carcinogens, vinyl chloride and butadiene. It is clear that such data play major roles in improving the understanding of how chemicals cause cancer, extending the range of data on exposure-response relationships, and examining interspecies differences and inter-individual differences that may affect susceptibility. As such, it is also clear that these data play a critical role in improving the accuracy of risk assessments.     

An Ethnobotanical survey of the beypazari, ayas, and Güdül district towns of Ankara Province (Turkey)

Dietary, therapeutical, and other ethnobotanical uses of the wild plants grown in the Beypazari, Aya§, and Güdül district towns of Ankara were investigated. Information was collected by oral interviews, with 400 individuals participating in 25 selected sites. The demographic characteristics of the informants were cross-linked with the recorded plant data for purposes of statistical analysis by SPSS software. The results indicated that 82% of the informants recognized the use of wild plants for food and home remedies. Both the breadth and scope of knowledge on the use of wild plants increased significantly with the advancing age of the informants, but there was no significant correlation between the knowledge of the informants and their educational status. Altogether, the authors recorded 192 uses for wild plants in the surveyed area; these emanating from 85 plant species belonging to 31 plant families. Among the most popular uses for wild plants were for medicines (115 citations) or food (70 citations). Only 7 plants fell in the miscellaneous category.