Depletion of Tip60/Kat5 affects the hepatic antioxidant system in mice

Tat-interactive protein 60 kDa (TIP60, also known as lysine acetyltransferase 5 [KAT5]) is a member of the MYST protein family with histone acetyltransferase activity. Recent studies have reported that TIP60 has multiple functions in many signal transduction mechanisms, especially p53-mediated apoptosis. Although the activation of apoptosis signaling pathways requires the presence of cellular reactive oxygen species (ROS) at a certain level, an imbalance between the production and consumption of ROS in cells results in oxidative stress (OS). In this study, we investigated for the first time how the absence of the Tip60 gene in the liver affects gene expression, enzyme activity, and protein expression of the hepatic antioxidant members localized in the cytoplasm, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST). First, we successfully generated liver-specific Tip60 knockout mice (mutants) using Cre/LoxP recombination. The reduced glutathione level and nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, a marker of OS, increased significantly in the Tip60 mutant liver. Gene expression, activity, and protein expression of the enzymatic antioxidant system, including SOD, CAT, GR, GPx, and GST were investigated in mutants and control groups. Despite a significant correlation between the gene, enzyme activity, and protein content for CAT and GR, this was not true for SOD and GPx. The overall results suggest that TIP60 acts on the hepatic antioxidant system both at the gene and protein levels, but the actual effect of the deletion of Tip60 is observed at the protein level, especially for SOD and GPx.     

Propolis Enhances 5-Fluorouracil Mediated Antitumor Efficacy and Reduces Side Effects in Colorectal Cancer: An in Vitro and in Vivo Study

In this study, we investigated the combined treatment of 5-fluorouracil (5-FU) and Anatolian propolis extract (PE) on colorectal cancer (CRC)using in vitro and in vivo studies. We exposed luciferase-transfected (Lovo-Luc CRC) cells and healthy colon cells (CCD-18Co) to varying concentrations of 5-FU and PE to assess their genotoxic, apoptotic, and cytotoxic effects, as well as their intracellular reactive oxygen species (iROS) levels. We also developed a xenograft model in nude mice and evaluated the anti-tumor effects of PE and 5-FU using various methods. Our findings showed that the combination of PE and 5-FU had selectivity against cancer cells, particularly at higher doses, and enhanced the anti-tumor effectiveness of 5-FU against colon CRC. The results suggest that PE can reduce side effects and increase the effectiveness of 5-FU through iROS generation in a dose-dependent manner.     

Inositol‐requiring enzyme‐1 regulates phosphoinositide signaling lipids and macrophage growth

The ER‐bound kinase/endoribonuclease (RNase), inositol‐requiring enzyme‐1 (IRE1), regulates the phylogenetically most conserved arm of the unfolded protein response (UPR). However, the complex biology and pathology regulated by mammalian IRE1 cannot be fully explained by IRE1’s one known, specific RNA target, X box‐binding protein‐1 (XBP1) or the RNA substrates of IRE1‐dependent RNA degradation (RIDD) activity. Investigating other specific substrates of IRE1 kinase and RNase activities may illuminate how it performs these diverse functions in mammalian cells. We report that macrophage IRE1 plays an unprecedented role in regulating phosphatidylinositide‐derived signaling lipid metabolites and has profound impact on the downstream signaling mediated by the mammalian target of rapamycin (mTOR). This cross‐talk between UPR and mTOR pathways occurs through the unconventional maturation of microRNA (miR) 2137 by IRE1’s RNase activity. Furthermore, phosphatidylinositol (3,4,5) phosphate (PI(3,4,5)P₃) 5‐phosphatase‐2 (INPPL1) is a direct target of miR‐2137, which controls PI(3,4,5)P₃ levels in macrophages. The modulation of cellular PI(3,4,5)P₃/PIP₂ ratio and anabolic mTOR signaling by the IRE1‐induced miR‐2137 demonstrates how the ER can provide a critical input into cell growth decisions.     

Biotization of Arabidopsis thaliana with Pseudomonas putida and assessment of its positive effect on in vitro growth

Determination of the optimal inoculation method and concentration to use for plant-bacteria interaction studies is important in many cases, such as the phytoremediation of heavy metals and other toxic compounds in contaminated areas. The aim of this study was to compare different concentrations and times of inoculation of Pseudomonas putida with various growth stages of Arabidopsis thaliana in 14-d in vitro cultures. A significant beneficial impact of the bacterium was detected in the shoot length and root weight of seedlings. The highest shoot length and root fresh and dry weights were detected in 14-d and 2 × 10³ cfu mL⁻¹ inoculated samples. In addition, the increase in root weight could be visualized with crystal violet staining, as relatively more root hair and lateral root formation occurred in seedlings inoculated with moderate concentrations of bacteria, possibly due to the ability of P. putida to produce indole-acetic acid. Moreover, the highest photosynthetic pigment accumulation was obtained with the highest bacterial inoculum (2 × 10⁶ cfu mL⁻¹), which was tested in 0- or 3-d-old seedlings. Rhizospheric bacterial colonization was also visualized with GFP-labeled bacteria by confocal microscopy. These results showed that biotization of A. thaliana with P. putida KT2440 did not cause severe oxidative stress in seedlings, because H₂O₂ accumulation levels together with CAT and POX activities were not significantly induced. Therefore, this strain could be used for several applications based on plant-bacteria interactions.

     

Effect of pioglitazone on the expression of ubiquitin proteasome system and autophagic proteins in rat pancreas with metabolic syndrome

The metabolic syndrome (MetS) and pathologies associated with metabolic dysregulations a worldwide growing problem. Our previous study demonstrated that pioglitazone (PGZ) has beneficial effects on metabolic syndrome associated disturbances in the heart. However, mechanism mediating the molecular alterations of Ubiquitin proteasome system (UPS) and autophagy has not been investigated in rat pancreas with metabolic syndrome. For this reason, we first aimed to detect whether MetS effects on the expression of UPS (p97/VCP, SVIP, Ubiquitin) and autophagic (p62, LC3) proteins in rat pancreas. The second aim of the study was to find impact of pioglitazone on the expression of UPS and autophagic proteins in MetS rat pancreas. To answer these questions, metabolic syndrome induced rats were used as a model and treated with pioglitazone for 2 weeks. Pancreatic tissue injuries, fibrosis and lipid accumulation were evaluated histopathologically in control, MetS and MetS-PGZ groups. Apoptosis and cell proliferation of pancreatic islet cells were assessed in all groups. UPS and autophagic protein expressions of pancreas in all groups were detected by using immunohistochemistry, double-immunfluorescence and Western blotting. Compared with the controls, the rat fed with high sucrose exhibited signs of metabolic syndrome, such as higher body weight, insulin resistance, higher triglyceride level and hyperglycaemia. MetS rats showed pancreatic tissue degeneration, fibrosis and lipid accumulation when their pancreas were examined with Hematoxilen-eozin and Mallory trichrome staining. Metabolic, histopathologic parameters and cell proliferation showed greater improvement in MetS-PGZ rats and pioglitazone decreased apoptosis of islet cells. Moreover, SVIP, ubiquitin, LC3 and p62 expressions were significantly increased while only p97/VCP expression was significantly decreased in MetS-rat pancreas compared to control. PGZ treatment significantly decreased the MetS-induced increases in autophagy markers. Additionally, UPS and autophagy markers were found to colocalizated with insulin and glucagon. Colocalization ratio of UPS markers with insulin showed significant decrease in MetS rats and PGZ increased this ratio, whereas LC3-insulin colocalization displayed significant increase in MetS rats and PGZ reversed this effect. In conclusion, PGZ improved the pancreatic tissue degeneration by increasing the level of p97/VCP and decreasing autophagic proteins, SVIP and ubiquitin expressions in MetS-rats. Moreover, PGZ has an effect on the colocalization ratio of UPS and autophagy markers with insulin.

     

Rational Design of a Fibroblast Growth Factor 21-Based Clinical Candidate,LY2405319

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.     

K+ Efflux and Retention in Response to NaCl Stress Do Not Predict Salt Tolerance in Contrasting Genotypes of Rice (Oryza sativa L.)

Sudden elevations in external sodium chloride (NaCl) accelerate potassium (K⁺) efflux across the plasma membrane of plant root cells. It has been proposed that the extent of this acceleration can predict salt tolerance among contrasting cultivars. However, this proposal has not been considered in the context of plant nutritional history, nor has it been explored in rice (Oryza sativa L.), which stands among the world’s most important and salt-sensitive crop species. Using efflux analysis with ⁴²K, coupled with growth and tissue K⁺ analyses, we examined the short- and long-term effects of NaCl exposure to plant performance within a nutritional matrix that significantly altered tissue-K⁺ set points in three rice cultivars that differ in salt tolerance: IR29 (sensitive), IR72 (moderate), and Pokkali (tolerant). We show that total short-term K⁺ release from roots in response to NaCl stress is small (no more than 26% over 45 min) in rice. Despite strong varietal differences, the extent of efflux is shown to be a poor predictor of plant performance on long-term NaCl stress. In fact, no measure of K⁺ status was found to correlate with plant performance among cultivars either in the presence or absence of NaCl stress. By contrast, shoot Na⁺ accumulation showed the strongest correlation (a negative one) with biomass, under long-term salinity. Pharmacological evidence suggests that NaCl-induced K⁺ efflux is a result of membrane disintegrity, possibly as result of osmotic shock, and not due to ion-channel mediation. Taken together, we conclude that, in rice, K⁺ status (including efflux) is a poor predictor of salt tolerance and overall plant performance and, instead, shoot Na⁺ accumulation is the key factor in performance decline on NaCl stress.     

Effect of Cu,Fe, Mn,Ni, and Zn and Bioaccessibilities in the Hazelnuts Growing in Sakarya,Turkey using In-Vitro Gastrointestinal Extraction Method

Biological Trace Element Research - Total concentrations of heavy metals such as Cu, Fe, Mn, Ni, and Zn in different hazelnut samples obtained by different regions of Sakarya city, Turkey were...     

FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.