BBA - General Subjects (v.1840, #7)
Editorial Board (i).
Serum thioredoxin reductase levels increase in response to chemically induced acute liver injury by Kang Sun; Sofi E. Eriksson; Yanping Tan; Le Zhang; Elias S.J. Arnér; Jinsong Zhang (2105-2111).
Mammalian thioredoxin reductases (TrxR) are selenoproteins with important roles in antioxidant defense and redox regulation, principally linked to functions of their main substrates thioredoxins (Trx). All major forms of TrxR are intracellular while levels in serum are typically very low.Serum TrxR levels were determined with immunoblotting using antibodies against mouse TrxR1 and total enzyme activity measurements were performed, with serum and tissue samples from mouse models of liver injury, as triggered by either thioacetamide (TAA) or carbon tetrachloride (CCl4).TrxR levels in serum increased upon treatment and correlated closely with those of alanine aminotransferase (ALT), an often used serum biomarker for liver damage. In contrast, Trx1, glutathione reductase, superoxide dismutase or selenium-containing glutathione peroxidase levels in serum displayed much lower increases than TrxR or ALT.Serum TrxR levels are robustly elevated in mouse models of chemically induced liver injury.The exaggerated TrxR release to serum upon liver injury may reflect more complex events than a mere passive release of hepatic enzymes to the extracellular milieu. It can also not be disregarded that enzymatically active TrxR in serum could have yet unidentified physiological functions.Display Omitted
Keywords: Thioredoxin reductase; Alanine aminotransferase; Hepatotoxicity; Serum;
Choline kinase beta is required for normal endochondral bone formation by Zhuo Li; Gengshu Wu; Roger B. Sher; Zohreh Khavandgar; Martin Hermansson; Gregory A. Cox; Michael R. Doschak; Monzur Murshed; Frank Beier; Dennis E. Vance (2112-2122).
Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb −/− mice display neonatal forelimb bone deformations.To understand the mechanisms underlying the bone deformations, we compared the biology and biochemistry of bone formation from embryonic to young adult wild-type (WT) and Chkb −/− mice.The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb −/− mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb −/− mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb −/− mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb −/− mice contained fewer osteoclasts along the cartilage/bone interface. Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice.Our data indicate that choline kinase beta plays an important role in endochondral bone formation by modulating growth plate physiology.
Keywords: Choline kinase; Endochondral bone formation; Growth plate; Chondrocyte; Radius; Ulna;
Inorganic phosphate uptake in unicellular eukaryotes by Claudia F. Dick; André L.A. Dos-Santos; José R. Meyer-Fernandes (2123-2127).
Inorganic phosphate (Pi) is an essential nutrient for all organisms. The route of Pi utilization begins with Pi transport across the plasma membrane.Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of Pi transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding Pi uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum.Pi uptake is the key step of Pi homeostasis and in the subsequent signaling event in eukaryotic microorganisms.Biochemical and structural studies are important for clarifying mechanisms of Pi homeostasis, as well as Pi sensor and downstream pathways, and raise possibilities for future studies in this field.
Keywords: Pi transporter; Saccharomyces cerevisiae; Neurospora crassa; Trypanosoma cruzi; Trypanosoma rangeli; Plasmodium falciparum;
Molecular modeling study on the dynamical structural features of human smoothened receptor and binding mechanism of antagonist LY2940680 by metadynamics simulation and free energy calculation by Qifeng Bai; Yulin Shen; Nengzhi Jin; Huanxiang Liu; Xiaojun Yao (2128-2138).
The smoothened (SMO) receptor, one of the Class F G protein coupled receptors (GPCRs), is an essential component of the canonical hedgehog signaling pathway which plays a key role in the regulation of embryonic development in animals. The function of the SMO receptor can be modulated by small-molecule agonists and antagonists, some of which are potential antitumour agents. Understanding the binding mode of an antagonist in the SMO receptor is crucial for the rational design of new antitumour agents.Molecular dynamics (MD) simulation and dynamical network analysis are used to study the dynamical structural features of SMO receptor. Metadynamics simulation and free energy calculation are employed to explore the binding mechanism between the antagonist and SMO receptor.The MD simulation results and dynamical network analysis show that the conserved KTXXXW motif in helix VIII has strong interaction with helix I. The α-helical extension of transmembrane 6 (TM6) is detected as part of the ligand-binding pocket and dissociation pathway of the antagonist. The metadynamics simulation results illustrate the binding mechanism of the antagonist in the pocket of SMO receptor, and free energy calculation shows the antagonist needs to overcome about 38 kcal/mol of energy barrier to leave the binding pocket of SMO receptor.The unusually long TM6 plays an important role on the binding behavior of the antagonist in the pocket of SMO receptor.The results can not only profile the binding mechanism between the antagonist and Class F GPCRs, but also supply the useful information for the rational design of a more potential small molecule antagonist bound to SMO receptor.
Keywords: Smoothened receptor; Molecular dynamics simulation; Metadynamics; Free energy calculation; GPCR;
Comparison of the effects of the repetition rate between microsecond and nanosecond pulses: Electropermeabilization-induced electro-desensitization? by A. Silve; A. Guimerà Brunet; B. Al-Sakere; A. Ivorra; L.M. Mir (2139-2151).
Applications of cell electropermeabilization are rapidly growing but basic concepts are still unclear. In particular, the impact of electric pulse repetition rate in the efficiency of permeabilization has not yet been understood.The impact of electric pulse repetition rate in the efficiency of permeabilization was analyzed in experiments performed on potato tissue and partially transposed on mice liver. On potato tissue, pulses with durations of 100 μs or 10 ns are applied. The intensity of permeabilization was quantified by means of bioimpedance changes and electric current measurements and a new index was defined.For the two pulse durations tested, very low repetition rates (below 0.1 Hz) are much more efficient to achieve cell permeabilization in potato tissue. In mice liver, using 100 μs pulses, the influence of the repetition rate is more complex. Indeed, repetition rates of 1 Hz and 10 Hz are more efficient than 100 Hz or 1 kHz, but not the repetition rate of 0.1 Hz for which there is an impact of the living mice organism response.We propose that the effects reported here might be caused by an electroporation-induced cell membrane ‘electro-desensitization’ which requires seconds to dissipate due to membrane resealing.This study not only reinforces previous observations, but moreover it sustains a new concept of ‘electro-desensitization’ which is the first unifying mechanism enabling to explain all the results obtained until now both in vitro and in vivo, with long and short pulses.
Keywords: Bioimpedance; Repetition rate; Electropermeabilization; Potato; Liver; Electroporation;
Injured cardiomyocytes promote dental pulp mesenchymal stem cell homing by F. Di Scipio; A.E. Sprio; A. Folino; M.E. Carere; P. Salamone; Z. Yang; M. Berrone; M. Prat; G. Losano; R. Rastaldo; G.N. Berta (2152-2161).
The heart is unable to regenerate its tissues after severe injuries. Stem cell therapy appears to be one of the most promising approaches, though preclinical results are hitherto contradictory and clinical trials scanty and/or limited to phase-I. The limited knowledge about stem cell early homing in infarcted cardiac tissues can concur to this scenario.The stem cell migration was assessed in in-vitro and ex-vivo models of heart ischemia, employing a rat dental pulp stem cell line (MUR-1) that shares the same ontogenic progenitors with portions of the heart, expresses markers typical of cardiac/vascular-like progenitors and is able to differentiate into cardiomyocytes in-vitro.Here, we demonstrated that the MUR-1 can reach the injured cells/tissue and make contacts with the damaged cardiomyocytes, likely through Connexin 43, N-cadherin and von Willebrand Factor mediated cell–cell interactions, both in in-vitro and ex-vivo models. Furthermore, we found that SDF-1, FGF-2 and HGF, but not VEGF are involved as chemotactic factors in MUR-1 migration, notifying a similarity with neural crest cell behavior during the organogenesis of both the splanchnocranium and the heart.Herein we found a similarity between what happens during the heart organogenesis and the early migration and homing of MUR-1 cells in ischemic models.The comprehension of molecular aspects underlying the early phases of stem cell migration and interaction with damaged organ contributes to the future achievement of the coveted stem cell-mediated organ regeneration and function preservation in-vivo.
Keywords: Dental pulp stem cell; Cardiac ischemia; Cell migration; Hypoxic cardiomyocyte;
Degraded λ-carrageenan activates NF-κB and AP-1 pathways in macrophages and enhances LPS-induced TNF-α secretion through AP-1 by Haimin Chen; Feng Wang; Haihua Mao; Xiaojun Yan (2162-2170).
Carrageenan (CGN), a high molecular weight sulfated polysaccharide, is a traditional ingredient used in food industry. Its degraded forms have been identified as potential carcinogens, although the mechanism remains unclear.The effects of degraded λ-carrageenan (λ-dCGN) on murine RAW264.7 cells and human THP-1-derived macrophage cells were investigated by studying its actions on tumor necrosis factor alpha (TNF-α) secretion, Toll-like receptor 4 (TLR4) expression, and activation of nuclear factor-κb (NF-κB) and activation protein-1 (AP-1) pathways.We found that λ-dCGN was much stronger than native λ-CGN in the activation of macrophages to secrete TNF-α. Treatment of RAW264.7 cells with λ-dCGN resulted in the upregulation of TLR4, CD14 and MD-2 expressions, but it did not increase the binding of lipopolysacchride (LPS) with macrophages. Meanwhile, λ-dCGN treatment activated NF-κB via B-cell lymphoma/leukemia 10 (Bcl10) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation. In addition, λ-dCGN induced extracellular signal-regulated kinases/1/2/mitogen-activated protein kinases (ERK1/2/MAPK) and AP-1 activation. Interestingly, pretreatment of RAW264.7 cells with λ-dCGN markedly enhanced LPS-stimulated TNF-α secretion. This pretreatment resulted in the enhanced phosphorylation of ERK1/2 and c-Jun N-terminal kinase (JNK) and intensified activation of AP-1.λ-dCGN induced an inflammatory reaction via both NF-κB and AP-1, and enhanced the inflammatory effect of LPS through AP-1 activation.The study demonstrated the role of λ-dCGN to induce the inflammatory reaction and to aggravate the effect of LPS on macrophages, suggesting that λ-dCGN produced during food processing and gastric digestion may be a safety concern.
Keywords: Degraded λ-carrageenan (λ-dCGN); LPS; TLR4; NF-κB; AP-1;
p19INK4d is involved in the cellular senescence mechanism contributing to heterochromatin formation by Silvina V. Sonzogni; María Florencia Ogara; Laura M. Belluscio; Daniela S. Castillo; María E. Scassa; Eduardo T. Cánepa (2171-2183).
During evolution, organisms with renewable tissues have developed mechanisms to prevent tumorigenesis, including cellular senescence and apoptosis. Cellular senescence is characterized by a permanent cell cycle arrest triggered by both endogenous stress and exogenous stress. The p19INK4d, a member of the family of cyclin-dependent kinase inhibitors (INK4), plays an important role on cell cycle regulation and in the cellular DNA damage response. We hypothesize that p19INK4d is a potential factor involved in the onset and/or maintenance of the senescent state.Senescence was confirmed by measuring the cell cycle arrest and the senescence-associated β-galactosidase activity. Changes in p19INK4d expression and localization during senescence were determined by Western blot and immunofluorescence assays. Chromatin condensation was measured by microccocal nuclease digestion and histone salt extraction.The data presented here show for the first time that p19INK4d expression is up-regulated by different types of senescence. Changes in senescence-associated hallmarks were driven by modulation of p19 expression indicating a direct link between p19INK4d induction and the establishment of cellular senescence. Following a senescence stimulus, p19INK4d translocates to the nucleus and tightly associates with chromatin. Moreover, reduced levels of p19INK4d impair senescence-related global genomic heterochromatinization. Analysis of p19INK4d mRNA and protein levels in tissues from differently aged mice revealed an up-regulation of p19INK4d that correlates with age.We propose that p19INK4d participates in the cellular mechanisms that trigger senescence by contributing to chromatin compaction.This study provides novel insights into the dynamics process of cellular senescence, a central tumor suppressive mechanism.
Keywords: Cellular senescence; CDK inhibitor; DNA damage; Heterochromatin; Aging;
A comparison among the tissue-specific effects of aging and calorie restriction on TFAM amount and TFAM-binding activity to mtDNA in rat by Anna Picca; Vito Pesce; Flavio Fracasso; Anna-Maria Joseph; Christiaan Leeuwenburgh; Angela Maria Serena Lezza (2184-2191).
Mitochondrial Transcription Factor A (TFAM) is regarded as a histone-like protein of mitochondrial DNA (mtDNA), performing multiple functions for this genome. Aging affects mitochondria in a tissue-specific manner and only calorie restriction (CR) is able to delay or prevent the onset of several age-related changes also in mitochondria.Samples of the frontal cortex and soleus skeletal muscle from 6- and 26-month-old ad libitum-fed and 26-month-old calorie-restricted rats and of the livers from 18- and 28-month-old ad libitum-fed and 28-month-old calorie-restricted rats were used to detect TFAM amount, TFAM-binding to mtDNA and mtDNA content.We found an age-related increase in TFAM amount in the frontal cortex, not affected by CR, versus an age-related decrease in the soleus and liver, fully prevented by CR. The semi-quantitative analysis of in vivo binding of TFAM to specific mtDNA regions, by mtDNA immunoprecipitation assay and following PCR, showed a marked age-dependent decrease in TFAM-binding activity in the frontal cortex, partially prevented by CR. An age-related increase in TFAM-binding to mtDNA, fully prevented by CR, was found in the soleus and liver. MtDNA content presented a common age-related decrease, completely prevented by CR in the soleus and liver, but not in the frontal cortex.The modulation of TFAM expression, TFAM-binding to mtDNA and mtDNA content with aging and CR showed a trend shared by the skeletal muscle and liver, but not by the frontal cortex counterpart.Aging and CR appear to induce similar mitochondrial molecular mechanisms in the skeletal muscle and liver, different from those elicited in the frontal cortex.
Keywords: Mitochondrial Transcription Factor A; Mitochondrial Transcription Factor A–mitochondrial deoxyribonucleic acid binding; Tissue-specificity; Aging rat; Calorie restriction;
Time evolution of noise induced oxidation in outer hair cells: Role of NAD(P)H and plasma membrane fluidity by Giuseppe Maulucci; Diana Troiani; Sara Letizia Maria Eramo; Fabiola Paciello; Maria Vittoria Podda; Gaetano Paludetti; Massimiliano Papi; Alessandro Maiorana; Valentina Palmieri; Marco De Spirito; Anna Rita Fetoni (2192-2202).
Noise exposure impairs outer hair cells (OHCs). The common basis for OHC dysfunction and loss by acoustic over-stimulation is represented by reactive oxygen species (ROS) overload that may affect the membrane structural organization through generation of lipid peroxidation.Here we investigated in OHC different functional zones the mechanisms linking metabolic functional state (NAD(P)H intracellular distribution) to the generation of lipid peroxides and to the physical state of membranes by two photon fluorescence microscopy.In OHCs of control animals, a more oxidized NAD(P)H redox state is associated to a less fluid plasma membrane structure. Acoustic trauma induces a topologically differentiated NAD(P)H oxidation in OHC rows, which is damped between 1 and 6 h. Peroxidation occurs after ~ 4 h from noise insult, while ROS are produced in the first 0.2 h and damage cells for a period of time after noise exposure has ended (~ 7.5 h) when a decrease of fluidity of OHC plasma membrane occurs. OHCs belonging to inner rows, characterized by a lower metabolic activity with respect to other rows, show less severe metabolic impairment.Our data indicate that plasma membrane fluidity is related to NAD(P)H redox state and lipid peroxidation in hair cells.Our results could pave the way for therapeutic intervention targeting the onset of redox umbalance.Display Omitted
Keywords: Reactive oxygen species (ROS); Organ of Corti; Acoustic trauma; Two-photon microscopy; Laurdan; Lipid peroxidation;
A mechanistic and structural investigation of modified derivatives of the diaryltriazine class of NNRTIs targeting HIV-1 reverse transcriptase by Andrea C. Mislak; Kathleen M. Frey; Mariela Bollini; William L. Jorgensen; Karen S. Anderson (2203-2211).
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are vital in treating HIV-1 infection by inhibiting reverse transcriptase (RT). Drug toxicity and resistance drive the need for effective new inhibitors with improved physiochemical properties and potent antiviral activity. Computer-aided and structure-based drug design have guided the addition of solubilizing substituents to the diaryltriazine scaffold. These derivatives have markedly improved solubility and maintain low nanomolar antiviral activity against RT. The molecular and structural basis of inhibition for this series was determined to facilitate future inhibitor development with improved pharmacological profiles.The molecular mechanism of inhibition was investigated using transient-state kinetic analysis. Crystal structures of RT in complex with each inhibitor were obtained to investigate the structural basis of inhibition.The diaryltriazine and its morpholine derivative have RT inhibition constants of 9 ± 2 nM and 14 ± 4 nM, respectively. They adopt differential binding modes within the non-nucleoside inhibitor binding pocket to distort the catalytic site geometry and primer grip regions. The novel morpholinopropoxy substituent extends into the RT/solvent interface of the NNIBP.Kinetic and structural analyses show that these inhibitors behave as conventional NNRTIs and inhibit the polymerization step. This study confirms that appending solubilizing substituents on the azine ring of diaryltriazine class of NNRTIs that extend into the RT/solvent interface effectively maintains low nanomolar potency and improves physiochemical properties.The modification of NNRTI scaffolds with solubilizing substituents, which extend into the RT/solvent interface, yields potent antivirals and is an effective strategy for developing novel inhibitors with improved pharmacological properties.
Keywords: HIV-1; Reverse transcriptase; NNRTI; Crystallography; Transient kinetics;
Azoxystrobin, a mitochondrial complex III Qo site inhibitor, exerts beneficial metabolic effects in vivo and in vitro by An-Hui Gao; Yan-Yun Fu; Kun-Zhi Zhang; Mei Zhang; Hao-Wen Jiang; Li-Xia Fan; Fa-Jun Nan; Chong-Gang Yuan; Jia Li; Yu-Bo Zhou; Jing-Ya Li (2212-2221).
Several anti-diabetes drugs exert beneficial effects against metabolic syndrome by inhibiting mitochondrial function. Although much progress has been made toward understanding the role of mitochondrial function inhibitors in treating metabolic diseases, the potential effects of these inhibitors on mitochondrial respiratory chain complex III remain unclear.We investigated the metabolic effects of azoxystrobin (AZOX), a Qo inhibitor of complex III, in a high-fat diet-fed mouse model with insulin resistance in order to elucidate the mechanism by which AZOX improves glucose and lipid metabolism at the metabolic cellular level.Acute administration of AZOX in mice increased the respiratory exchange ratio. Chronic treatment with AZOX reduced body weight and significantly improved glucose tolerance and insulin sensitivity in high-fat diet-fed mice. AZOX treatment resulted in decreased triacylglycerol accumulation and down-regulated the expression of genes involved in liver lipogenesis. AZOX increased glucose uptake in L6 myotubes and 3T3-L1 adipocytes and inhibited de novo lipogenesis in HepG2 cells. The findings indicate that AZOX-mediated alterations to lipid and glucose metabolism may depend on AMP-activated protein kinase (AMPK) signaling.AZOX, a Qo inhibitor of mitochondrial respiratory complex III, exerts whole-body beneficial effects on the regulation of glucose and lipid homeostasis in high-fat diet-fed mice.These findings provide evidence that a Qo inhibitor of mitochondrial respiratory complex III could represent a novel approach for the treatment of obesity.
Keywords: Azoxystrobin; Mitochondria complex III; Metabolic diseases; AMP-activated protein kinase;
G-quadruplex-mediated regulation of telomere binding protein POT1 gene expression by Qingqing He; Ping Zeng; Jia-Heng Tan; Tian-Miao Ou; Lian-Quan Gu; Zhi-Shu Huang; Ding Li (2222-2233).
Telomere is protected by its G-quadruplex, T-loop structure, telomerase, and binding protein complex. Protein POT1 (protection of telomeres 1) is one subunit of telomere binding protein complex Shelterin. POT1 acts as a regulator of telomerase-dependent telomere length, and it can help telomere to form D-loop structure to stabilize telomere. POT1 protects telomere ends from ATR-dependent DNA damage response as well.Extensive methods were used, including CD, EMSA, ITC, PCR stop assay, luciferase reporter assay, quantitative real-time PCR, Western blot, chromatin immunoprecipitation (Ch-IP), cloning, expression and purification of proteins.We found a new G-rich 30-base-pair long sequence (P-pot1 G18) located from − 165 to − 136 base pairs upstream of the translation starting site of protein POT1. This sequence in the promoter region of pot1 gene formed G-quadruplex resulting in down-regulation of pot1 gene transcription. This G-rich sequence is close to a binding site “TCCC” for transcription factor hnRNP K (heterogeneous nuclear ribonucleoprotein K), and its conversion to G-quadruplex prevented the access of hnRNP K to this binding site. The binding of hnRNP K could up-regulate pot1 gene transcription. TMPyP4 (meso-tetra(N-methyl-4-pyridyl)porphine) has been widely used as G-quadruplex binding ligand, which stabilized the G-quadruplex in vitro and in cellulo, resulting in down-regulation of pot1 gene transcription.This G-quadruplex might become a potentially new drug target for antitumor agents.Our results first demonstrated that G-quadruplex formation can affect the binding of transcription factor to its nearby binding site, and thus making additional influence to gene transcription.
Keywords: POT1; Shelterin; Telomere; G-quadruplex; hnRNP K; Cancer;
Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell by Li Xing; Meijuan Niu; Xia Zhao; Lawrence Kleiman (2234-2243).
RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNALys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.
Keywords: HIV-1; RNA helicase A; Double-stranded RNA binding domain; Lysine; Helicase activity;
Cellular nucleic acid binding protein suppresses tumor cell metastasis and induces tumor cell death by downregulating heterogeneous ribonucleoprotein K in fibrosarcoma cells by Jun Qiu; Siqi Chen; Lijuan Su; Jinggong Liu; Nannan Xiao; Tian-Miao Ou; Jia-Heng Tan; Lian-Quan Gu; Zhi-Shu Huang; Ding Li (2244-2252).
Cellular nucleic acid binding protein (CNBP) has been implicated in vertebrate craniofacial development and in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human diseases by controlling cell proliferation and survival to mediate neural crest expansion. CNBP has been found to bind single-stranded nucleic acid and promote rearrangements of nucleic acid secondary structure in an ATP-independent manner, acting as a nucleic acid chaperone.A variety of methods were used, including cell viability assays, wound-scratch assays, chemotaxis assays, invasion assays, circular dichroic (CD) spectroscopy, NMR spectroscopy, chromatin immunoprecipitation, expression and purification of recombinant human CNBP, electrophoretic mobility shift assay (EMSA), surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET) analyses, luciferase reporter assay, Western blotting, and isothermal titration calorimetry (ITC).Up-regulation of CNBP induced human fibrosarcoma cell death and suppressed fibrosarcoma cell motility and invasiveness. It was found that CNBP transcriptionally down-regulated the expression of heterogeneous ribonucleoprotein K (hnRNP K) through its conversion of a G-rich sequence into G-quadruplex in the promoter of hnRNP K. G-quadruplex stabilizing ligand tetra-(N-methyl-4-pyridyl) porphyrin (TMPyP4) could interact with and stabilize the G-quadruplex, resulting in downregulation of hnRNP K transcription.CNBP overexpression caused increase of cell death and suppression of cell metastasis through its induction of G-quadruplex formation in the promoter of hnRNP K resulting in hnRNP K down-regulation.The present result provided a new solution for controlling hnRNP K expression, which should shed light on new anticancer drug design and development.Display Omitted
Keywords: Metastasis; Cell death; CNBP; hnRNP K; G-quadruplex; Transcriptional regulation;
Cold-inducible RNA-binding protein mediates neuroinflammation in cerebral ischemia by Mian Zhou; Weng-Lang Yang; Youxin Ji; Xiaoling Qiang; Ping Wang (2253-2261).
Neuroinflammation is a key cascade after cerebral ischemia. Excessive production of proinflammatory mediators in ischemia exacerbates brain injury. Cold-inducible RNA-binding protein (CIRP) is a newly discovered proinflammatory mediator that can be released into the circulation during hemorrhage or septic shock. Here, we examine the involvement of CIRP in brain injury during ischemic stroke.Stroke was induced by middle cerebral artery occlusion (MCAO). In vitro hypoxia was conducted in a hypoxia chamber containing 1% oxygen. CIRP and tumor necrosis factor-α (TNF-α) levels were assessed by RT-PCR and Western blot analysis.CIRP is elevated along with an upregulation of TNF-α expression in mouse brain after MCAO. In CIRP-deficient mice, the brain infarct volume, induction of TNF-α, and activation of microglia are markedly reduced after MCAO. Using microglial BV2 cells, we demonstrate that hypoxia induces the expression, translocation, and release of CIRP, which is associated with an increase of TNF-α levels. Addition of recombinant murine (rm) CIRP directly induces TNF-α release from BV2 cells and such induction is inhibited by neutralizing antisera to CIRP. Moreover, rmCIRP activates the NF-κB signaling pathway in BV2 cells. The conditioned medium from BV2 cells exposed to hypoxia triggers the apoptotic cascade by increasing caspase activity and decreasing Bcl-2 expression in neural SH-SY5Y cells, which is inhibited by antisera to CIRP.Extracellular CIRP is a detrimental factor in stimulating inflammation to cause neuronal damage in cerebral ischemia.Development of an anti-CIRP therapy may benefit patients with brain ischemia.
Keywords: Cerebral ischemia; Hypoxia; Cold shock protein; Inflammation; Microglia; Neural cell;
Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells by Mariana C.C. Silva; Cláudia A.A. de Paula; Joana G. Ferreira; Edgar J. Paredes-Gamero; Angela M.S.F. Vaz; Misako U. Sampaio; Maria Tereza S. Correia; Maria Luiza V. Oliva (2262-2271).
Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity.MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting.BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α1, α6 and β1 integrin subunit expression, and increases α5 subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21.BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells.Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer.Display Omitted
Keywords: Bauhinia forficata lectin; Breast cancer; Cell adhesion; Cell death; Integrin;
Impact of subdomain D1 of the short form S1b of the human prolactin receptor on its inhibitory action on the function of the long form of the receptor induced by prolactin by J.-H. Kang; S.A. Hassan; P. Zhao; C.H. Tsai-Morris; M.L. Dufau (2272-2280).
Long-form (LF) homodimers of the human prolactin receptor (PRLR) mediate prolactin's diverse actions. Short form S1b inhibits the LF function through heterodimerization. Reduced S1b/LF-ratio in breast cancer could contribute to tumor development/progression. Current work defines the structural and functional relevance of the D1 domain of S1b on its inhibitory function on prolactin-induced LF function.Studies were conducted using mutagenesis, promoter/signaling analyses, bioluminescence resonance energy transfer (BRET) and molecular modeling approaches.Mutation of E69 in D1 S1b or adjacent residues at the receptor surface near to the binding pocket (S) causes loss of its inhibitory effect while mutations away from this region (A) or in the D2 domain display inhibitory action as the wild-type. All S1b mutants preserved prolactin-induced Jak2 activation. BRET reveals an increased affinity in D1 mutated S1b (S) homodimers in transfected cells stably expressing LF. In contrast, affinity in S1b homodimers with either D1 (A) or D2 mutations remained unchanged. This favors LF mediated signaling induced by prolactin. Molecular dynamics simulations show that mutations (S) elicit major conformational changes that propagate downward to the D1/D2 interface and change their relative orientation in the dimers.These findings demonstrate the essential role of D1 on the S1b structure and its inhibitory action on prolactin-induced LF-mediated function.Major changes in receptor conformation and dimerization affinity are triggered by single mutations in critical regions of D1. Our structure–function/simulation studies provide a basis for modeling and design of small molecules to enhance inhibition of LF activation for potential use in breast cancer treatment.
Keywords: Human prolactin receptor; Short and long form; Subdomain D1; Structure/function;
Molecular basis of thermal stability in truncated (2/2) hemoglobins by Juan P. Bustamante; Alessandra Bonamore; Alejandro D. Nadra; Natascia Sciamanna; Alberto Boffi; Darío A. Estrin; Leonardo Boechi (2281-2288).
Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures.We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position.The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible.This gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability.These results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins.
Keywords: Truncated hemoglobin; Thermostability; Thermobifida fusca; Mycobacterium tuberculosis; Molecular dynamics; Folding;
Ascorbic acid deficiency affects genes for oxidation–reduction and lipid metabolism in livers from SMP30/GNL knockout mice by Keita Takahashi; Yuki Kishimoto; Tomokazu Konishi; Yasunori Fujita; Masafumi Ito; Kentaro Shimokado; Naoki Maruyama; Akihito Ishigami (2289-2298).
We sought to elucidate the effect of an ascorbic acid (AA) deficiency on gene expression, because the water soluble antioxidant AA is an important bioactive substance in vivo.We performed microarray analyses of the transcriptome in the liver from senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which are unable to synthesize AA in vivo.Our microarray analysis revealed that the AA deficiency increased gene expression related to the oxidation–reduction process, i.e., the nuclear factor, erythroid derived 2, like 2 (Nrf2) gene, which is a reactive oxygen species-sensitive transcriptional factor. Moreover, this AA deficiency increased the expression of genes for lipid metabolism including the cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1), which is a late-limiting enzyme of the primary bile acid biosynthesis pathway. Although an AA deficiency increased the Cyp7a1 protein level, bile acid levels in the liver and gallbladder decreased. Since Cyp7a1 has a heme iron at the active site, AA must function as a reductant of the iron required for the continuous activation of Cyp7a1.This experimental evidence strongly supports a role for AA in the physiologic oxidation–reduction process and lipid metabolism including bile acid biosynthesis.Although many effects of AA supplementation have been reported, no microarray analysis of AA deficiency in vivo is available. Results from using this unique model of AA deficiency, the SMP30/GNL-KO mouse, now provide new information about formerly unknown AA functions that will implement further study of AA in vivo.
Keywords: Ascorbic acid; Bile acid; Cyp7a1; Lipid metabolism; Microarray; Nrf2;
Dual anti-oxidant and anti-inflammatory actions of the electrophilic cyclooxygenase-2-derived 17-oxo-DHA in lipopolysaccharide- and cigarette smoke-induced inflammation by Chiara Cipollina; Serena Di Vincenzo; Stefania Gerbino; Liboria Siena; Mark Gjomarkaj; Elisabetta Pace (2299-2309).
17-Oxo-DHA is an endogenous electrophilic derivative of the omega-3 fatty acid docosahexaenoic acid (DHA) which is generated in activated macrophages by the action of cyclooxygenase-2.The ability of 17-oxo-DHA to control inflammation and oxidative stress was tested in human macrophages (THP-1) and bronchial epithelial cell line (16HBE) stimulated with cigarette smoke extract (CSE) and lipopolysaccharide (LPS). All data were further confirmed using primary bronchial epithelial cells, alveolar macrophages and peripheral blood mononuclear cells.17-Oxo-DHA was a strong inducer of the anti-oxidant response promoting Nrf2 nuclear accumulation, leading to the expression of heme oxygenase 1 and more than doubling glutathione levels. This resulted in suppression of CSE-induced ROS generation in macrophages. In macrophages, 17-oxo-DHA potently suppressed TNFα release in response to LPS, CSE and IL-1β acting at transcriptional level via a mechanism independent of Nrf2. Externally supplemented 17-oxo-DHA displayed the same effects in the presence of the Cox-inhibitor indomethacin. The non-electrophilic 17-oxo-DHA precursor DHA did not show any biological actions, indicating that the electrophilic moiety was required for this compound to become bioactive.17-Oxo-DHA promotes cytoprotective actions both in immune and structural cells. In immune cells, 17-oxo-DHA is effective in contrasting CSE- and LPS-induced oxidative damage and inflammation acting via multiple independent pathways.Herein we provide insights on how the novel endogenous electrophilic DHA-derivative 17-oxo-DHA promotes anti-oxidant and anti-inflammatory actions. Data herein reported indicate that 17-oxo-DHA is an attractive lead compound for the development of new treatments for cigarette smoke-related airway inflammatory disorders.
Keywords: Omega-3 fatty acid; Electrophile; Cigarette smoke; Inflammation; Oxidative stress;
Cell death caused by quinazolinone HMJ-38 challenge in oral carcinoma CAL 27 cells: dissections of endoplasmic reticulum stress, mitochondrial dysfunction and tumor xenografts by Chi-Cheng Lu; Jai-Sing Yang; Jo-Hua Chiang; Mann-Jen Hour; Kuei-Li Lin; Tsung-Han Lee; Jing-Gung Chung (2310-2320).
This investigation clearly clarified the synthesized and antimitotic compound, 2-(3′-methoxyphenyl)-6-pyrrolidinyl-4-quinazolinone (HMJ-38), addressing its target and precise mechanism of action. We hypothesized that HMJ-38 might sensitize apoptotic death of human oral carcinoma CAL 27 cells in vitro and inhibit xenograft tumor growth in vivo.Cell viability was assessed utilizing MTT assay. HMJ-38-treated cells represented DNA fragmentation using agarose gel electrophoresis as further evidenced using TUNEL staining. Flow cytometric analyses, immunoblotting and quantitative RT-PCR were applied for protein and gene expression. Antitumor xenograft study was employed.HMJ-38 concentration- and time-dependently reduced viability of CAL 27 cells. The effect of intrinsic molecules was signalized during HMJ-38 exposure with disruption of ΔΨm, MPT pore opening and the release of various events from mitochondria undergoing cell apoptosis. HMJ-38 also markedly facilitated G2/M phase arrest. HMJ-38 stimulated the activation of CDK1 activity that modulated phosphorylation on Ser70 of Bcl-2-mediated mitotic arrest and apoptosis. HMJ-38 triggered intracellular Ca2 + release and activated related pivotal hallmarks of ER stress. HMJ-38 in nude mice bearing CAL 27 tumor xenografts decreased tumor growth. Furthermore, HMJ-38 enhanced caspase-3 gene expression and protein level in xenotransplanted tumors.Early roles of mitotic arrest, unfolded protein response and mitochondria-dependent signaling contributed to apoptotic CAL 27 cell demise induced by HMJ-38. In in vivo experiments, HMJ-38 also efficaciously suppressed tumor volume in a xenotransplantation model.This finding might fully support a critical event for HMJ-38 via induction of apoptotic machinery and ER stress against human oral cancer cells.Display Omitted
Keywords: HMJ-38; Human oral carcinoma cells; Endoplasmic reticulum stress; Mitochondrial dysfunction; Tumor xenografts;
Neuroprotective effect of PEP-1-peroxiredoxin2 on CA1 regions in the hippocampus against ischemic insult by Hoon Jae Jeong; Dae Young Yoo; Dae Won Kim; Hyeon Ji Yeo; Su Bin Cho; Jiye Hyeon; Jung Hwan Park; Jinseu Park; Won Sik Eum; Hyun Sook Hwang; Moo-Ho Won; In Koo Hwang; Soo Young Choi (2321-2330).
Oxidative stress is a leading cause of various diseases, including ischemia and inflammation. Peroxiredoxin2 (PRX2) is one of six mammalian isoenzymes (PRX1–6) that can reduce hydrogen peroxide (H2O2) and organic hydroperoxides to water and alcohols.We produced PEP-1-PRX2 transduction domain (PTD)-fused protein and investigated the effect of PEP-1-PRX2 on oxidative stress-induced neuronal cell death by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western blot, immunofluorescence microscopy, and immunohistochemical analysis.Our data showed that PEP-1-PRX2, which can effectively transduce into various types of cells and brain tissues, could be implicated in suppressing generation of reactive oxygen species, preventing depolarization of the mitochondrial membrane, and inhibiting the apoptosis pathway in H2O2-stimulated HT22, murine hippocampal neuronal cells, likely resulting in protection of HT22 cells against H2O2-induced toxicity. In addition, we found that in a transient forebrain ischemia model, PEP-1-PRX2 inhibited the activation of astrocytes and microglia in the CA1 region of the hippocampus and lipid peroxidation and also prevented neuronal cell death against ischemic damage.These findings suggest that the transduced PEP-1-PRX2 has neuroprotective functions against oxidative stress-induced cell death in vitro and in vivo.PEP-1-PRX2 could be a potential therapeutic agent for oxidative stress-induced brain diseases such as ischemia.
Keywords: Peroxiredoxin2; Ischemic insult; Oxidative stress; Protein transduction domain; Apoptosis pathway;
Tracing the pathway of compositional changes in bone mineral with age: Preliminary study of bioapatite aging in hypermineralized dolphin's bulla by Zhen Li; Jill D. Pasteris (2331-2339).
Studies of mineral compositional effects during bone aging are complicated by the presence of collagen.Hypermineralized bullae of Atlantic bottlenose dolphins of < 3 months, 2.5 years, and 20 years underwent micrometer-scale point analysis by Raman spectroscopy and electron microprobe in addition to bulk analysis for carbon.Bulla central areas have a mineral content of ~ 96 wt.% and 9–10 wt.% carbonate in their bioapatite, which is ~ 2 wt.% more than edge areas. Ca/P atomic ratios (~ 1.8) and concentrations of Mg, S, and other minor/trace elements are almost constant in central areas over time. Maturity brings greater over-all homogeneity in mineral content, stoichiometry, and morphology throughout the central and edge areas of the bullae. During aging, edge areas become less porous, whereas the concentration of organics in the edge is reduced. Enhancement of coupled substitutions of CO3 2 − for PO4 3 − and Na for Ca during aging increases carbonate content up to ~ 10 wt.% in the adult bulla.1) Changes in physical properties during aging did not occur simultaneously with changes in chemical properties of the bone mineral. 2) Compositional changes in bone mineral were minor during the neonatal to sub-adult stage, but significant during later maturity. 3) Na and CO3 concentrations co-vary in a 1:1 molar proportion during aging. 4) The mineral's crystallinity did not decrease as CO3 concentration increased during aging.Hypermineralized dolphin's bulla, due to extreme depletion in collagen, is an ideal material for investigating mineralogical changes in bioapatite during bone aging.
Keywords: Bulla; Aging; Hypermineralization; Bone mineral; Carbonated hydroxylapatite;
Knocking down 10-formyltetrahydrofolate dehydrogenase increased oxidative stress and impeded zebrafish embryogenesis by obstructing morphogenetic movement by Wen-Ni Chang; Gang-Hui Lee; Tseng-Ting Kao; Cha-Ying Lin; Tsun-Hsien Hsiao; Jen-Ning Tsai; Bing-Hung Chen; Yau-Hung Chen; Hsin-Ru Wu; Huai-Jen Tsai; Tzu-Fun Fu (2340-2350).
Folate is an essential nutrient for cell survival and embryogenesis. 10-Formyltetrahydrofolate dehydrogenase (FDH) is the most abundant folate enzyme in folate-mediated one-carbon metabolism. 10-Formyltetrahydrofolate dehydrogenase converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2, the only pathway responsible for formate oxidation in methanol intoxication. 10-Formyltetrahydrofolate dehydrogenase has been considered a potential chemotherapeutic target because it was down-regulated in cancer cells. However, the normal physiological significance of 10-Formyltetrahydrofolate dehydrogenase is not completely understood, hampering the development of therapeutic drug/regimen targeting 10-Formyltetrahydrofolate dehydrogenase.10-Formyltetrahydrofolate dehydrogenase expression in zebrafish embryos was knocked-down using morpholino oligonucleotides. The morphological and biochemical characteristics of fdh morphants were examined using specific dye staining and whole-mount in-situ hybridization. Embryonic folate contents were determined by HPLC.The expression of 10-formyltetrahydrofolate dehydrogenase was consistent in whole embryos during early embryogenesis and became tissue-specific in later stages. Knocking-down fdh impeded morphogenetic movement and caused incorrect cardiac positioning, defective hematopoiesis, notochordmalformation and ultimate death of morphants. Obstructed F-actin polymerization and delayed epiboly were observed in fdh morphants. These abnormalities were reversed either by adding tetrahydrofolate or antioxidant or by co-injecting the mRNA encoding 10-formyltetrahydrofolate dehydrogenase N-terminal domain, supporting the anti-oxidative activity of 10-formyltetrahydrofolate dehydrogenase and the in vivo function of tetrahydrofolate conservation for 10-formyltetrahydrofolate dehydrogenase N-terminal domain.10-Formyltetrahydrofolate dehydrogenase functioned in conserving the unstable tetrahydrofolate and contributing to the intracellular anti-oxidative capacity of embryos, which was crucial in promoting proper cell migration during embryogenesis.These newly reported tetrahydrofolate conserving and anti-oxidative activities of 10-formyltetrahydrofolate dehydrogenase shall be important for unraveling 10-formyltetrahydrofolate dehydrogenase biological significance and the drug development targeting 10-formyltetrahydrofolate dehydrogenase.
Keywords: Zebrafish; ROS; Cell migration; Folate metabolism; FDH;
Hemopexin-dependent heme uptake via endocytosis regulates the Bach1 transcription repressor and heme oxygenase gene activation by Hiroshi Hada; Takuma Shiraki; Miki Watanabe-Matsui; Kazuhiko Igarashi (2351-2360).
Intracellular heme plays versatile roles in a variety of physiological processes including mitochondrial respiration. Heme also induces the expression of genes such as heme oxygenase-1 (HO-1) by inactivating the transcription repressor Bach1 through direct binding. However, the source of heme for the regulation of the Bach1–HO-1 axis has been unclear. Considering that extracellular heme exists as a complex with hemopexin (Hx) in serum under the physiological conditions, heme–Hx complex may deliver heme for the gene regulation.Using a mammalian expression system, high secretory recombinant Hx (rHx) was developed. We examined the effects of rHx-bound heme on HO-1 expression and Bach1 in Hepa-1c1c7 liver cells and THP-1 macrophage cells. We investigated the uptake pathway of rHx-bound heme by treating cells with chlorpromazine (CPZ).rHx-bound heme induced the expression of HO-1 and decreased the level of Bach1 protein. CPZ inhibited the induction of the HO-1 expression by rHx-bound heme.rHx-bound heme was internalized into the cells via endocytosis, resulting in HO-1 expression and inactivation of Bach1.The Bach1-dependent repression of the HO-1 expression is under the control of the Hx-dependent uptake of extracellular heme. Heme may regulate Bach1 as an extracellular signaling molecule.
Keywords: Heme; Hemopexin; Bach1; Heme oxygenase-1; Oxidative stress; Endocytosis;
Cladosporol A, a new peroxisome proliferator-activated receptor γ (PPARγ) ligand, inhibits colorectal cancer cells proliferation through β-catenin/TCF pathway inactivation by Diana Zurlo; Gemma Assante; Salvatore Moricca; Vittorio Colantuoni; Angelo Lupo (2361-2372).
Cladosporol A, a secondary metabolite from Cladosporium tenuissimum, exhibits antiproliferative properties in human colorectal cancer cells by modulating the expression of some cell cycle genes (p21waf1/cip1, cyclin D1).PPARγ activation by cladosporol A was studied by overexpression and RNA interference assays. The interactions between PPARγ and Sp1 were investigated by co-immunoprecipitation and ChIp assays. β-Catenin subcellular distribution and β-catenin/TCF pathway inactivation were analyzed by western blot and RTqPCR, respectively. Cladosporol A-induced β-catenin proteasomal degradation was examined in the presence of the specific inhibitor MG132.Cladosporol A inhibits cell growth through upregulation of p21waf1/cip1 gene expression mediated by Sp1-PPARγ interaction. Exposure of HT-29 cells to cladosporol A causes β-catenin nuclear export, proteasome degradation and reduced expression of its target genes. Upon treatment, PPARγ also activates E-cadherin gene at the mRNA and protein levels.In this work we provide evidence that PPARγ mediates the anti-proliferative action of cladosporol A in colorectal cancer cells. Upon ligand activation, PPARγ interacts with Sp1 and stimulates p21waf1/cip1 gene transcription. PPARγ activation causes degradation of β-catenin and inactivation of the downstream target pathway and, in addition, upregulates E-cadherin expression reinforcing cell–cell interactions and a differentiated phenotype.We elucidated the molecular mechanisms by which PPARγ mediates the anticancer activity of cladosporol A.
Keywords: Cladosporol A; Cell proliferation; p21waf1/cip1; Transcription; β-catenin; E-cadherin;
Kolaviron, a natural flavonoid from the seeds of Garcinia kola, reduces LPS-induced inflammation in macrophages by combined inhibition of IL-6 secretion, and inflammatory transcription factors, ERK1/2, NF-κB, p38, Akt, p-c-JUN and JNK by Sunny O. Abarikwu (2373-2381).
Kolaviron (Kol-v), an important component of Garcinia kola seed has a variety of biologic activities, including anti-inflammatory properties.We tested the ability of Kol-v to block signalling pathways implicated in lipopolysaccharide (LPS)-induced inflammatory gene expression in RAW 264.7 macrophage cell line.When macrophages pre-treated with Kol-v (15 and 25 μM) were activated with LPS, phosphorylation of p38 and p-c-JUN but not IκBα degradation and phosphorylation of NF-κB (p65), ERK1/2, and IκBα were blocked. Furthermore, Kol-v suppressed LPS-induced increase in the expression of IL-18 gene and LPS-induced decrease in the mRNA expression of IP-10 but it had no effect on the LPS-induced decrease in the gene expression levels of IL-1α, IL-33, IL-1β, and IFNβ1-1. When macrophages pre-treated with Kol-v (50 and 100 μM) were activated with LPS, phosphorylation of Akt, ERK1/2, IκBα, and NFκB (p65) but not that of CREB was blocked by Kol-v. The protective effect of Kol-v on the LPS-induced phosphorylation of the mitogen activated protein kinase (MAPK) family member JNK was only observed at 100 μM. At all concentrations of Kol-v (0–100 μM) tested in this study, there was no effect of Kol-v on LPS-induced secretion of the pro-inflammatory cytokine TNF-α but a concentration dependent inhibition of Kol-v on IL-6 secretion was observed.Kol-v interferes with LPS signalling by reducing the activation of several inflammatory transcription factors and that its inhibitory action on IL-6 secretion correlates with inhibition of ERK1/2, p38, Akt, p-c-JUN and JNK signalling pathways.The anti-inflammatory potential of Kol-v via inhibition of IL-6 secretion in RAW macrophage was established in this study.Anti-inflammatory activity of Garcinia kola extract in LPS-induced RAW 264.7 macrophages.Display Omitted
Keywords: Kolaviron; RAW macrophage; Lipopolysaccharide; IL-6; MAPK; Inflammation;
Corrigendum to “Determination of the warfarin inhibition constant Ki for vitamin K 2,3-epoxide reductase complex subunit-1 (VKORC1) using an in vitro DTT-driven assay” [BBAGEN (2013) 4202–4210] by Carville G. Bevans; Christoph Krettler; Christoph Reinhart; Hélène Tran; Katja Koßmann; Matthias Watzka; Johannes Oldenburg (2382-2384).