BBA - General Subjects (v.1820, #2)

The aim of this work was to evaluate the hepatoprotective ability of allopurinol to prevent the liver injury induced by carbon tetrachloride (CCl4).Acute liver damage was induced with CCl4 (4 g/kg, by gavage); allopurinol (50 mg/kg, by gavage) was given 1 h before and 1 h after CCl4 intoxication and two daily doses for the previous three days. Cirrhosis was established by CCl4 administration (0.4 g/kg, i. p. three times a week, eight weeks); allopurinol was administered (100 mg/kg, by gavage, daily) during the long-term of CCl4 treatment. Alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), xanthine oxidase (XO), lipid peroxidation, reduced and oxidized glutathione (GSH, GSSG, respectively), hydroxyproline and histopathologycal analysis were performed. Nuclear factor-κB (NF-κB), pro-inflammatory and anti-inflammatory cytokines, transforming growth factor-β (TGF-β) and metalloproteinase-13 (MMP-13) were analyzed by Western blots.Acute injury increased ALT and γ-GTP activities, additionally enhanced NF-κB nuclear translocation and cytokines production such as tumor necrosis factor-α, interleukine-1β, and interleukine-6. Allopurinol partially prevented these effects, while increased interleukine-10. Acute and chronic CCl4 treatments altered the levels of XO activity, lipid peroxidation, and GSH/GSSG ratio, while these remained within normal range with allopurinol administration. Necrosis, fibrosis and TGF-β production induced in chronic injury were partially prevented by allopurinol, interestingly, this drug induced MMP-13 activity.Allopurinol possesses antioxidant, anti-inflammatory and antifibrotic properties, probably by its capacity to reduce NF-κB nuclear translocation and TGF-β expression, as well as to induce MMP-13.General significanceAllopurinol might be effective treatment of liver diseases.► Allopurinol protects against necrosis regardless of dose and exposure time with CCl4. ► Allopurinol maintained redox balance both hydrophilic and lipophilic induced by CCl4. ► Allopurinol inhibited partially the nuclear translocation of NF-κB. ► Allopurinol showed immunomodulatory effects on pro- and anti-inflammatory cytokines. ► Allopurinol reduces fibrosis by decreased TGF-β expression couple induce MMP-13.
Keywords: Allopurinol; liver damage; oxidative stress; NF-κB; cytokines; carbon tetrachloride;

Microphthalmia-associated transcription factor is required for mature myotube formation by Ryo Ooishi; Mitsuyuki Shirai; Masayuki Funaba; Masaru Murakami (76-83).
The roles of microphthalmia-associated transcription factor (Mitf) in the skeletal muscle and during myogenesis are unclear.Expression of Mitf in mouse tissues and during myogenesis was evaluated. Effects of Mitf knockdown on myogenesis and gene expression related to myogenesis were subsequently explored. Furthermore, effects of p21, a cyclin-dependent kinase inhibitor, and integrin α9 (Itga9) were examined.Mitf was highly expressed in the skeletal muscle; Mitf-A and -J were expressed. Mitf expression increased after differentiation stimulation in C2C12 myogenic cells. Down-regulation of Mitf expression by transfection of siRNA for common Mitf inhibited myotube formation, which was reproduced by Mitf-A knockdown. Morphometric analyses indicated that both multinucleated cell number and the proportion of myotubes with more than 6 nuclei were decreased in Mitf-knockdown cells, suggesting that Mitf is required for not only the formation of nascent myotubes but also their maturation. Searching for genes positively regulated by Mitf revealed p21 and Itga9; decreasing Mitf expression inhibited up-regulation of p21 expression after differentiation stimulation and blocked the induction of Itga9 expression in response to differentiation. Knockdown of p21 decreased the number of multinucleated cells, whereas Itga9 knockdown did not affect the myotube number. Both p21 knockdown and Itga9 knockdown decreased the proportion of myotubes with more than 6 nuclei.Mitf positively regulates skeletal muscle formation; Mitf is significantly expressed during myogenesis, and is required for efficient myotube formation through expression of p21 and Itga9.► A isoform of Mitf mRNA is abundant in the skeletal muscle. ► Mitf-A expression increased with progression of myogenesis. ► Knockdown of Mitf mRNA disrupts myotube formation. ► Expression of p21 and Itga9 is regulated by Mitf for appropriate myogenesis. ► p21 and Itga9 are required for myoblast fusion and myotube maturation, respectively.
Keywords: Mitf; Myogenesis; Integrin; p21; Myotube;

Effect of insulin deprivation on metabolism and metabolism-associated gene transcript levels of in vitro cultured human Sertoli cells by P.F. Oliveira; M.G. Alves; L. Rato; S. Laurentino; J. Silva; R. Sá; A. Barros; M. Sousa; R.A. Carvalho; J.E. Cavaco; S. Socorro (84-89).
Sertoli cells metabolize glucose producing lactate for developing germ cells. As insulin regulates glucose uptake and its disturbance/insensitivity is associated with diabetes mellitus, we aimed to determine the effect of insulin deprivation in human Sertoli cell (hSC) metabolism and metabolism-associated gene expression.hSC-enriched primary cultures were maintained in the absence/presence of insulin and metabolite variations were determined by 1H-NMR. mRNA expression levels of glucose transporters (GLUT1, GLUT3), lactate dehydrogenase (LDHA) and monocarboxylate transporter (MCT4) were determined by RT-PCR.Insulin deprivation resulted in decreased lactate production and in decrease of glucose consumption that was completely reverted after 6 h. Cells of both groups consumed similar amounts of glucose. In insulin-deprived cells, transcript levels of genes associated to lactate metabolism (LDHA and MCT4) were decreased. Transcript levels of genes involved in glucose uptake exhibited a divergent variation: GLUT3 levels were decreased while GLUT1 levels increased.Insulin-deprived hSCs presented: 1) altered glucose consumption and lactate secretion; 2) altered expression of metabolism-associated genes involved in lactate production and export; 3) an adaptation of glucose uptake by modulating the expression of GLUT1 and GLUT3.This is the first report regarding the effect of insulin-deprivation on hSC metabolism.Display Omitted► The first hours of insulin deprivation are critical in hSCs in vitro. ► Insulin deprivation affects glucose uptake and lactate production/export. ► Insulin-deprived hSCs present altered expression of metabolism-associated genes. ► GLUT1 and GLUT3 expression levels are modulated by insulin-deprivation in hSCs.
Keywords: Human Sertoli cell; Energy metabolism; Insulin; Lactate; Glucose;

Structure-based discovery of anti-influenza virus A compounds among medicines by Mayuko Fukuoka; Moeko Minakuchi; Atsushi Kawaguchi; Kyosuke Nagata; Yuji O. Kamatari; Kazuo Kuwata (90-95).
Influenza A virus (IAV) infection is nowadays a major public health concern, in particular since the 2009 H1N1 flu pandemic. The outbreak of IAV strains resistant to currently available drugs, such as oseltamivir or zanamivir targeting the neuraminidase, is a real threat for humans as well as for animals. Thus the development of anti-IAV drugs with a novel action mechanism may be an urgent theme.We performed a docking simulation targeting the interface of PA interacting with PB1 using a drug database including ~ 4000 compounds. We then conducted cell viability assay and plaque assay using IAV/WSN/33. Finally we examined their anti-IAV mechanism by surface plasmon resonance and IAV replicon assay.We found that benzbromarone, diclazuril, and trenbolone acetate had strong anti-IAV activities. We confirmed that benzbromarone and diclazuril bound with PA subunit, and decreased the transcriptional activity of the viral RNA polymerase.Benzbromarone and diclazuril had strong anti-IAV activities with novel action mechanism, i.e. inhibition of viral RNA polymerase.Since benzbromarone and diclazuril are already used in public as medicines, these could be the candidates for alternatives in case of an outbreak of IAV which is resistant to pre-existing anti-IAV drugs.► We performed structure-based drug discovery targeting PA of the influenza A virus. ► We discovered novel anti-influenza A virus compounds among medicines. ► We found benzbromarone and diclazuril bound with PA using SPR. ► We found that benzbromarone and diclazuril inhibit the viral RNA polymerase activity. ► These could be alternatives if influenza A virus is resistant to pre-existing drugs.
Keywords: Structure-based drug design; Influenza A virus; H1N1; RNA polymerase; PA; Surface plasmon resonance;

Localization of nerve growth factor (NGF) receptors in the mitochondrial compartment: Characterization and putative role by Valentina Carito; Attilio Pingitore; Erika Cione; Ida Perrotta; Domenico Mancuso; Antonio Russo; Giuseppe Genchi; Maria Cristina Caroleo (96-103).
The neurotrophin NGF receptors trkA and p75NTR are expressed in the central and peripheral nervous system as well as in non-neuronal tissues; originally described to localize to the plasma membrane, recent studies have suggested other intracellular localizations for both NGF receptors.In order to determine whether NGF receptors localize to the mitochondrial compartment mitochondria isolated from human kidney, rat tissues and a human podocyte as cell line before and after differentiation were used.Our results demonstrate that NGF receptors are localized in the mitochondrial compartment of undifferentiated human podocytes and in all tissues analyzed including rat central nervous system. In mitochondria p75NTR, but not trkA, co-immunoprecipitates with the adenine nucleotide translocator (ANT) and the phosphodiesterase 4 isoform A5 (PDE4A5). Moreover, NGF, via trkA, protects isolated mitochondria of rat brain cortex from mitochondrial permeability transition induced by Ca2+.Although NGF receptors have been described as mainly citoplasmatic so far, we proved evidence of their expression at the mitochondrial level and their interaction with specific proteins. Our results demonstrating the expression of NGF receptors in the mitochondria provide new insights into the role of NGF at subcellular level, in different areas of the organism, including CNS.► p75NTR and trkA are NGF cellular receptors known to localize in the plasma membrane. ► We found trkA and p75NTR in Central Nervous System and other tissues mitochondria. ► In mitochondria p75NTR interacts with ANT and PDE4A5. ► NGF protects rat brain cortex mitochondria from Ca2+induced swelling.
Keywords: Nerve growth factor; Mitochondria; Receptor;

Trichostatin A and sirtinol suppressed survivin expression through AMPK and p38MAPK in HT29 colon cancer cells by Ya-Fen Hsu; Joen-Rong Sheu; Chien-Huang Lin; De-Shin Yang; George Hsiao; George Ou; Pei-Ting Chiu; Yu-Han Huang; Wen-Hsin Kuo; Ming-Jen Hsu (104-115).
Elevated levels of survivin and histone deacetylases (HDACs) are often found over-expressed in human cancers, including colorectal cancer, and have been implicated in tumorigenesis. HDAC inhibition induces growth arrest and cell death in various transformed cell; however, the mechanisms by which this reduces cell viability in colorectal cancer cells remain unexplained.We explored the actions of two HDAC inhibitors, trichostatin A (TSA) and sirtinol, in HT29 colon cancer cells.TSA and sirtinol induced apoptosis and inhibited cell proliferation in HT29 cells. These results are associated with the modulation of survivin. Survivin promoter luciferase activity and Sp1, a transcription factor that contributes to survivin expression, were suppressed in cells exposed to TSA or sirtinol. TSA and sirtinol also activated p38 mitogen-activated protein kinase (p38MAPK) and AMP-activated protein kinase (AMPK). Inhibitors of p38MAPK or AMPK signaling abrogated TSA and sirtinol's effects of decreasing cell viability. Survivin promoter luciferase activity in the presence of TSA or sirtinol was restored by AMPK dominant negative mutant or p38MAPK inhibitor. Furthermore, Sp1 binding to the survivin promoter region decreased while p63 binding to the promoter region increased after TSA or sirtinol exposure.We report a p38MAPK- and AMPK-mediated downregulation of survivin, and its functional correlation with decreased colon cancer cell viability in the presence of HDAC inhibitor. p63 and Sp1 may also contribute to TSA and sirtinol actions.This study delineates, in part, the underlying mechanisms of TSA and sirtinol in decreasing survivin expression and subsequent colon cancer cell viability.Display Omitted► TSA and sirtinol suppress survivin expression through activating AMPK and p38MAPK. ► TSA and sirtinol decrease cell viability through activating AMPK and p38MAPK. ► TSA and sirtinol recruit p63 to the survivin promoter region.
Keywords: Trichostatin A; Sirtinol; Histone deacetylase; Survivin; Colon cancer;

Prion diseases are associated with a conformational switch for PrP from PrPC to PrPSc. Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD.MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native β-sheet and the conversion of S2–H2 loop to a 310 helix.Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrPC to PrPSc.General significanceOur present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrPSc assemblies.► The misfolding of prion protein is a key event at the origin of prion diseases. ► MD simulations were performed to explore the effects of T188K/R/A mutations on PrPC. ► The mutations have diverse effects on the structural properties of PrPC.
Keywords: Familial Creutzfeldt - Jakob disease; Familial prion disease; Molecular dynamics simulation; T188K/R/A mutant;

Inhibiting effect of αs1-casein on Aβ1–40 fibrillogenesis by R. Carrotta; C. Canale; A. Diaspro; A. Trapani; P.L. San Biagio; D. Bulone (124-132).
αs1-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of αs1-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of αs1-casein on the fibrillogenesis of 1–40 β-amyloid peptide, involved in Alzheimer's disease.The aggregation kinetics of β-peptide in presence and absence of αs1-casein was followed under shear at 37 °C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of αs1-casein–Aβ1–40 interaction.αs1-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. αs1-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism.Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.► αs1-Casein, a natively unfolded protein, as molecular chaperone. ► Effect of αs1-casein on amyloid fibrillization. ► αs1-Casein reduces the content of amyloid fibers. ► Amyloid peptide is recruited on casein hydrophobic surface. ► Colloidal inhibition of the fibrillization process.
Keywords: Casein; Chaperone activity; Unstructured protein; Colloidal inhibition; Beta peptide; Amyloid fibril;

Perturbation of energy homeostasis in skeletal muscle and liver resulting from a transient inhibition of mitochondrial energy transduction can produce effects of relevance for the control of hyperglycemia through activation of the AMP-activated protein kinase, as exemplified by the antidiabetic drug metformin. The present study focuses on uncoupling of oxidative phosphorylation rather than its inhibition as a trigger for such effects.The reference weak uncoupler 2,4-dinitrophenol, fourteen naturally-occurring phenolic compounds identified as uncouplers in isolated rat liver mitochondria, and fourteen related compounds with little or no uncoupling activity were tested for enhancement of glucose uptake in differentiated C2C12 skeletal muscle cells following 18 h of treatment at 25–100 μM. A subset of compounds were tested for suppression of glucose-6-phosphatase (G6Pase) activity in H4IIE hepatocytes following 16 h at 12.5–25 μM. Metformin (400 μM) was used as a standard in both assays.Dinitrophenol and nine of eleven compounds that induced 50% or more uncoupling at 100 μM in isolated mitochondria enhanced basal glucose uptake by 53 to 269%; the effect of the 4′-hydroxychalcone butein was more than 6-fold that of metformin; negative control compounds increased uptake by no more than 25%. Dinitrophenol and four 4′-hydroxychalconoids also suppressed hepatocyte G6Pase as well as, or more effectively than metformin, whereas the unsubstituted parent compound chalcone, devoid of uncoupling activity, had no effect.Activities key to glycemic control can be induced by a wide range of weak uncouplers, including compounds free of difficult-to-metabolize groups typically associated with uncouplers.Uncoupling represents a valid and possibly more efficient alternative to inhibition for triggering cytoprotective effects of therapeutic relevance to insulin resistance in both muscle and liver. Identification of actives of natural origin and the insights into their structure–activity relationship reported herein may lead to alternatives to metformin.► Phenolic compounds of natural origin were screened for mitochondrial uncoupling. ► Structures and properties conducive to uncoupling activity were identified. ► Identified uncouplers enhanced muscle cell glucose uptake more than metformin. ► Identified uncouplers suppressed hepatocyte glucose output as much as metformin. ► Weak uncouplers may be well-suited to the treatment of insulin resistance.
Keywords: Mitochondrial energy transduction; Chalconoids; Flavonoids; Skeletal muscle cell glucose uptake; Hepatocyte glucose-6-phosphatase activity; Insulin resistance;

Cellular retinaldehyde-binding protein (CRALBP) is a direct downstream target of transcription factor Pax6 by Sridhar Boppana; Alexander Scheglov; Robert Geffers; Victor Tarabykin (151-156).
Transcription factor Pax6 plays an essential role in the expression of other transcription factors, cell adhesion molecules and is crucial for neurogenesis in the developing forebrain. Analysis of gene expression profiles through microarray experiments in Pax6 mutants allowed us to focus on CRALBP, one of the many genes that were downregulated.We studied the expression of CRALBP in wt and Pax6–/– mutants through in situ hybridization and immunohistochemistry. ChIP assay and luciferase reporter assay were performed to show the regulatory role of Pax6 on CRALBP promoter.RNA and protein expression data show that CRALBP expression was completely abolished in Pax6 mutants. In vivo binding assays and in vitro reporter assays indicate that Pax6 not only binds the promoter of CRALBP but also positively regulates protein expression.This work provides evidence supporting that CRALBP is a direct downstream target of Pax6. However, the role of CRALBP in the cortex is yet to be elucidated.Pax6 is a marker expressed on neural stem cells and progenitor cells. Understanding Pax6-dependent gene regulatory mechanisms unravels signaling cascades that occur early during development.► Pax6 and CRALBP are co-expressed in several regions of the CNS. ► CRALBP gene expression was abolished in Pax6−/− mutant embryos. ► Pax6 protein was detected on CRALBP promoter region in Chromatin IP experiments. ► In the eye, Pax6 is expressed in the NR whereas CRALBP is expressed in the RPE.
Keywords: Ventricular zone; Expression; Immunoprecipitation; Pallial–subpallial boundary; Cortex; Transcription factor;