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BIOCHEMISTRY (213 journals)                  1 2 3     

AAPS PharmSciTech     Hybrid Journal   (Followers: 8)
Acetic Acid Bacteria     Open Access   (Followers: 2)
ACS Chemical Biology     Full-text available via subscription   (Followers: 204)
ACS Chemical Neuroscience     Full-text available via subscription   (Followers: 16)
Acta Crystallographica Section D : Biological Crystallography     Hybrid Journal   (Followers: 11)
Acta Crystallographica Section F: Structural Biology Communications     Hybrid Journal   (Followers: 7)
Advances and Applications in Bioinformatics and Chemistry     Open Access   (Followers: 9)
Advances in Biological Chemistry     Open Access   (Followers: 5)
Advances in Carbohydrate Chemistry and Biochemistry     Full-text available via subscription   (Followers: 8)
Advances in Plant Biochemistry and Molecular Biology     Full-text available via subscription   (Followers: 7)
Advances in Protein Chemistry and Structural Biology     Full-text available via subscription   (Followers: 10)
African Journal of Biochemistry Research     Open Access   (Followers: 1)
African Journal of Chemical Education     Open Access   (Followers: 1)
Alkaloids: Chemical and Biological Perspectives     Full-text available via subscription   (Followers: 4)
American Journal of Biochemistry     Open Access   (Followers: 6)
American Journal of Biochemistry and Biotechnology     Open Access   (Followers: 98)
American Journal of Biochemistry and Molecular Biology     Open Access   (Followers: 11)
American Journal of Polymer Science     Open Access   (Followers: 20)
Amino Acids     Hybrid Journal   (Followers: 7)
Analytical Biochemistry     Hybrid Journal   (Followers: 99)
Annals of Clinical Biochemistry     Hybrid Journal   (Followers: 1)
Annual Review of Biochemistry     Full-text available via subscription   (Followers: 32)
Annual Review of Chemical and Biomolecular Engineering     Full-text available via subscription   (Followers: 10)
Applied Biochemistry and Biotechnology     Hybrid Journal   (Followers: 18)
Applied Biochemistry and Microbiology     Hybrid Journal   (Followers: 8)
Applied Organometallic Chemistry     Hybrid Journal   (Followers: 4)
Archives of Biochemistry and Biophysics     Hybrid Journal   (Followers: 9)
Archives of Insect Biochemistry and Physiology     Hybrid Journal   (Followers: 1)
Archives Of Physiology And Biochemistry     Hybrid Journal   (Followers: 1)
Asian Journal of Biochemistry     Open Access   (Followers: 1)
Asian Journal of Biomedical and Pharmaceutical Sciences     Open Access   (Followers: 2)
Avicenna Journal of Medical Biochemistry     Open Access  
Bangladesh Journal of Medical Biochemistry     Open Access   (Followers: 2)
BBA Clinical     Open Access  
BBR : Biochemistry and Biotechnology Reports     Open Access   (Followers: 3)
Biocatalysis     Open Access  
Biochemical and Biophysical Research Communications     Hybrid Journal   (Followers: 14)
Biochemical and Molecular Medicine     Full-text available via subscription   (Followers: 5)
Biochemical Compounds     Open Access  
Biochemical Engineering Journal     Hybrid Journal   (Followers: 9)
Biochemical Genetics     Hybrid Journal   (Followers: 3)
Biochemical Journal     Full-text available via subscription   (Followers: 18)
Biochemical Pharmacology     Hybrid Journal   (Followers: 6)
Biochemical Society Transactions     Full-text available via subscription   (Followers: 3)
Biochemical Systematics and Ecology     Hybrid Journal   (Followers: 4)
Biochemistry     Full-text available via subscription   (Followers: 169)
Biochemistry & Pharmacology : Open Access     Open Access   (Followers: 1)
Biochemistry & Physiology : Open Access     Open Access  
Biochemistry (Moscow)     Hybrid Journal   (Followers: 3)
Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology     Hybrid Journal   (Followers: 4)
Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry     Hybrid Journal   (Followers: 3)
Biochemistry and Cell Biology     Full-text available via subscription   (Followers: 10)
Biochemistry and Molecular Biology Education     Hybrid Journal   (Followers: 3)
Biochemistry and Molecular Biology of Fishes     Full-text available via subscription   (Followers: 1)
Biochemistry Research International     Open Access   (Followers: 5)
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids     Hybrid Journal   (Followers: 3)
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease     Hybrid Journal   (Followers: 16)
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research     Hybrid Journal   (Followers: 6)
Biochimie     Hybrid Journal   (Followers: 5)
Bioconjugate Chemistry     Full-text available via subscription   (Followers: 14)
BioDrugs     Full-text available via subscription   (Followers: 8)
Bioelectrochemistry     Hybrid Journal   (Followers: 2)
Biofuels     Hybrid Journal   (Followers: 10)
Biogeochemistry     Hybrid Journal   (Followers: 9)
BioInorganic Reaction Mechanisms     Hybrid Journal   (Followers: 1)
Biokemistri     Open Access  
Biological Chemistry     Partially Free   (Followers: 11)
Biomaterials Research     Open Access  
Biomedicines     Open Access   (Followers: 1)
BioMolecular Concepts     Hybrid Journal   (Followers: 2)
Bioscience, Biotechnology, and Biochemistry     Hybrid Journal   (Followers: 7)
Biosimilars     Open Access   (Followers: 1)
Biotechnology and Applied Biochemistry     Hybrid Journal   (Followers: 21)
BMC Biochemistry     Open Access   (Followers: 12)
Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca : Food Science and Technology     Open Access  
Carbohydrate Polymers     Hybrid Journal   (Followers: 9)
Cell Biochemistry and Biophysics     Hybrid Journal   (Followers: 6)
Cell Biochemistry and Function     Hybrid Journal   (Followers: 3)
Cellular Physiology and Biochemistry     Open Access   (Followers: 3)
Central European Journal of Chemistry     Hybrid Journal   (Followers: 5)
ChemBioChem     Hybrid Journal   (Followers: 2)
Chemical and Biological Technologies for Agriculture     Open Access  
Chemical Biology & Drug Design     Hybrid Journal   (Followers: 22)
Chemical Engineering Journal     Hybrid Journal   (Followers: 24)
Chemical Senses     Hybrid Journal   (Followers: 1)
Chemical Speciation and Bioavailability     Open Access   (Followers: 1)
Chemico-Biological Interactions     Hybrid Journal   (Followers: 2)
Chemistry & Biodiversity     Hybrid Journal   (Followers: 5)
Chemistry & Biology     Full-text available via subscription   (Followers: 17)
Chemistry and Ecology     Hybrid Journal   (Followers: 1)
ChemTexts     Hybrid Journal  
Clinical Biochemist Reviews     Full-text available via subscription   (Followers: 1)
Clinical Biochemistry     Hybrid Journal   (Followers: 4)
Clinical Chemistry and Laboratory Medicine     Hybrid Journal   (Followers: 10)
Clinical Lipidology     Full-text available via subscription  
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology     Hybrid Journal   (Followers: 3)
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology     Hybrid Journal   (Followers: 1)
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology     Hybrid Journal   (Followers: 5)
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics     Hybrid Journal   (Followers: 2)
Comprehensive Biochemistry     Full-text available via subscription   (Followers: 1)

        1 2 3     

Journal Cover   Archives of Biochemistry and Biophysics
  [SJR: 1.602]   [H-I: 124]   [9 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0003-9861 - ISSN (Online) 1096-0384
   Published by Elsevier Homepage  [2799 journals]
  • Mechanoreception at the cell membrane: More than the integrins
    • Abstract: Publication date: 15 November 2015
      Source:Archives of Biochemistry and Biophysics, Volume 586
      Author(s): Alexander N. Gasparski, Karen A. Beningo
      A cell receives mechanical cues from its surrounding microenvironment and transduces this mechanical information into a biochemical signal within the cell, ultimately resulting in physiological change. Several molecules within the plasma membrane have been identified that are capable of receiving and translating a mechanical signal. Although integrins are most often discussed as the cell's primary method of mechanoreception at the cell membrane, several non-integrin mechanoreceptors have emerged over the last decade. Specifically, multiple G-protein coupled receptors, the glycocalyx, ion channels, lipid rafts and receptor tyrosine kinases have been found to translate mechanical stimuli from the environment into cellular change. This review will discuss these non-integrin mechanoreceptors associated with the plasma membrane, and their impact on cell physiology.

      PubDate: 2015-10-06T22:08:00Z
  • PD150606 protects against ischemia/reperfusion injury by preventing
           μ-calpain-induced mitochondrial apoptosis
    • Abstract: Publication date: 15 November 2015
      Source:Archives of Biochemistry and Biophysics, Volume 586
      Author(s): Tao Luo, Rongchuan Yue, Houxiang Hu, Zhou Zhou, Kai Hang Yiu, Shuang Zhang, Lei Xu, Ke Li, Zhengping Yu
      Calpain plays an important role in myocardial ischemia/reperfusion (I/R) injury. PD150606, a nonpeptide, cell-permeable and noncompetitive calpain inhibitor, has been shown to have protective properties in ischemic disease. The aims of the present study were to investigate whether PD150606 could alleviate myocardial I/R injury and to examine the possible mechanisms involved. The I/R model was established in vivo in C57BL/6 mice and in vitro using neonatal mouse cardiomyocytes, respectively. To evaluate the protective effects of PD150606 on I/R injury, we measured the myocardial infarct area, apoptosis, and expression of cleaved caspase-3. We also investigated the underlying mechanisms by examining mitochondrial function as reflected by the ATP concentration, translocation of cytochrome c, dynamics of mPTP opening, and membrane potential (ΔΨm), coupled with calpain activity. Pretreatment with PD150606 significantly reduced the infarct area and apoptosis caused by I/R. PD150606 pretreatment also reduced mitochondrial dysfunction by inhibiting calpain activation. Moreover, we found that μ-calpain is the main contributor to I/R-induced calpain activation. Knockdown of μ-calpain with siRNA significantly reversed calpain activation, mitochondrial dysfunction, and cardiomyocyte apoptosis caused by I/R in vitro. Our results suggest that PD150606 may protect against I/R injury via preventing μ-calpain-induced mitochondrial apoptosis.

      PubDate: 2015-10-03T13:53:08Z
  • Glutathione modifies the oxidation products of 2’-deoxyguanosine by
           singlet molecular oxygen
    • Abstract: Publication date: Available online 30 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Patrícia S. Peres, Andressa Valerio, Silvia M.S.C. Cadena, Sheila M.B. Winnischofer, Alexsandra C. Scalfo, Paolo Di Mascio, Glaucia R. Martinez
      The oxidation of the free nucleoside 2’-deoxyguanosine (dGuo) by singlet molecular oxygen (1O2) has been studied over the three last decades due to the major role of DNA oxidation products in process such as ageing, mutation and carcinogenesis. In the present work we investigated the dGuo oxidation by 1O2 in the presence of the important low molecular antioxidant, glutathione, in its reduced (GSH) and oxidized (GSSG) forms. There were applied different conditions of concentration, pH, time of incubation, and the use of a [18O]-labeled thermolabile endoperoxide naphthalene derivative as a source of [18O]-labeled 1O2. Data was obtained through high performance liquid chromatography (HPLC) and HPLC coupled to micrOTOFQ-II analysis of the main oxidation products: the diastereomers of spiroiminodihydantoin-2′-deoxyribonucleosides (dSp) and 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodGuo). An intriguing result was that 8-oxodGuo levels increased by 100 fold when dGuo was oxidized by 1O2 in the presence of GSH and by 2 fold in the presence of GSSG, while dSp levels dropped to zero for both conditions. All data from dGuo, 8-oxodGuo and dSp quantification together with the analysis of residual GSH/GSSG content in each sample strongly suggest that glutathione modifies the mechanism of dGuo oxidation by 1O2 by disfavoring the pathway of dSp formation.

      PubDate: 2015-10-03T13:53:08Z
  • Functional structure and physiological functions of mammalian wild-type
    • Abstract: Publication date: Available online 30 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Tomoya Okamoto, Ryuichi Ishida, Hiroshi Yamamoto, Masako Tanabe-Ishida, Asami Haga, Hiroki Takahashi, Kyosuke Takahashi, Daisuke Goto, Ewa Grave, Hideaki Itoh
      The Chaperonins comprise a family of molecular chaperones having a double-ring structure and similar sequence homology. These proteins play an essential role in biological reactions that mediate the folding of newly synthesized polypeptides and partially denatured proteins. In the prokaryotic group I chaperonins, structural and reaction cycle analyses of GroEL and its co-chaperone GroES have been performed in detail. While in eukaryotes, there have been limited reports analying the group I chaperonin HSP60 and its co-chaperone HSP10. In the present study, we purified the wild type HSP60 from porcine liver and investigated the interaction between HSP60 and HSP10, including conformation and physiological relationships. Based on the results of transmission electron microscopy, native PAGE, and gel filtration column chromatography, the wild type HSP60 displayed a heptameric single-ring structure in the absence of ATP. In contrast, HSP60 formed mainly a “football-type” complex with HSP10 in the presence of ATP and mediated the refolding of denatured substrate protein. The functional conformation cycle of the purified mammalian HSP60 is distinct from the cycle of the prokaryotic GroEL/GroES chaperonin.

      PubDate: 2015-10-03T13:53:08Z
  • Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits
           polyIC induced PKR threonine phosphorylation
    • Abstract: Publication date: 1 November 2015
      Source:Archives of Biochemistry and Biophysics, Volume 585
      Author(s): Medchalmi Swetha, Kolluru V.A. Ramaiah
      Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser51 phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells.
      Graphical abstract image

      PubDate: 2015-09-29T13:46:23Z
  • The structural basis of substrate promiscuity in UDP-hexose
           4-epimerase from the hyperthermophilic Eubacterium Thermotoga
    • Abstract: Publication date: 1 November 2015
      Source:Archives of Biochemistry and Biophysics, Volume 585
      Author(s): Sun-Mi Shin, Jin Myung Choi, Eric di Luccio, Yong-Jik Lee, Sang-Jae Lee, Sang Jun Lee, Sung Haeng Lee, Dong-Woo Lee
      UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80 °C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism.
      Graphical abstract image

      PubDate: 2015-09-25T13:44:26Z
  • Crystal structure and tartrate inhibition of Legionella pneumophila
           histidine acid phosphatase
    • Abstract: Publication date: 1 November 2015
      Source:Archives of Biochemistry and Biophysics, Volume 585
      Author(s): Richa Dhatwalia, Harkewal Singh, Thomas J. Reilly, John J. Tanner
      Histidine acid phosphatases (HAPs) utilize a nucleophilic histidine residue to catalyze the transfer of a phosphoryl group from phosphomonoesters to water. HAPs function as protein phosphatases and pain suppressors in mammals, are essential for Giardia lamblia excystation, and contribute to virulence of the category A pathogen Francisella tularensis. Herein we report the first crystal structure and steady-state kinetics measurements of the HAP from Legionella pneumophila (LpHAP), also known as Legionella major acid phosphatase. The structure of LpHAP complexed with the inhibitor l(+)-tartrate was determined at 2.0 Å resolution. Kinetics assays show that l(+)-tartrate is a 50-fold more potent inhibitor of LpHAP than of other HAPs. Electrostatic potential calculations provide insight into the basis for the enhanced tartrate potency: the tartrate pocket of LpHAP is more positive than other HAPs because of the absence of an ion pair partner for the second Arg of the conserved RHGXRXP HAP signature sequence. The structure also reveals that LpHAP has an atypically expansive active site entrance and lacks the nucleotide substrate base clamp found in other HAPs. These features imply that nucleoside monophosphates may not be preferred substrates. Kinetics measurements confirm that AMP is a relatively inefficient in vitro substrate of LpHAP.
      Graphical abstract image

      PubDate: 2015-09-25T13:44:26Z
  • Understanding of real alternative redox partner of Streptomyces peucetius
           DoxA: Prediction and validation using in silico and in vitro analyses
    • Abstract: Publication date: 1 November 2015
      Source:Archives of Biochemistry and Biophysics, Volume 585
      Author(s): Hemraj Rimal, Seung-Won Lee, Joo-Ho Lee, Tae-Jin Oh
      Streptomyces peucetius ATCC27952 contains the cytochrome P450 monoxygenase DoxA that is responsible for the hydroxylation of daunorubicin into doxorubicin. Although S. peucetius ATCC27952 contains several potential redox partners, the most suitable endogenous electron-transport system is still unclear; therefore, we conducted a study of potential redox partners using Accelrys Discovery Studio 3.5. Recombinant DoxA along with its redox partners from S. peucetius FDX1, FDR2, and FDX3, and the putidaredoxin and putidaredoxin reductase from Pseudomonas putida that are essential equivalents of the class I type of bacterial electron-transport system were over-expressed and purified. The successful development of an efficient redox system was achieved by an in vitro enzymatic catalysis reaction with DoxA. The optimal pH for the activation of the heme was 7.6 and the optimal temperature was 30 °C. Our findings suggest a two-fold increase of DoxA activity via the NADH → FDR2 → FDX1 → DoxA pathway for the hydroxylation of the daunorubicin, and indicate that the usage of a native redox partner may increase daunorubicin-derived doxorubicin production due to the inclusion of DoxA.

      PubDate: 2015-09-25T13:44:26Z
  • Enhanced heme accessibility in horse heart mini-myoglobin: Insights from
           molecular modelling and reactivity studies
    • Abstract: Publication date: 1 November 2015
      Source:Archives of Biochemistry and Biophysics, Volume 585
      Author(s): Fabio Polticelli, Veranika Zobnina, Chiara Ciaccio, Giampiero de Sanctis, Paolo Ascenzi, Massimo Coletta
      Mini-myoglobin (mini-HHMb) is a fragment of horse-heart myoglobin (HHMb) considered to be the prototype of the product encoded by the central exon of the HHMb gene. For this reason, mini-HHMb has been studied extensively showing that carbonylation and oxygenation properties of the ferrous form are similar to those of the full-length protein, while kinetics and thermodynamics of azide binding to the ferric form are significantly different from those of HHMb. To analyze the structure–function relationships in mini-HHMb and the role of conformational fluctuations in ligand accessibility, the molecular model of mini-HHMb has been built and refined by molecular dynamics simulations, and analyzed in parallel with that of full length HHMb. Moreover, imidazole binding parameters of ferric mini-HHMb and HHMb have been determined. Furthermore, structural data of ferric mini-HHMb and HHMb have been correlated with the imidazole and previously determined azide binding properties. Present results indicate that, despite the extensive trimming, the heme-α-helices E-F substructure is essentially unaltered in mini-HHMb with respect to HHMb. However, the heme-Fe atom displays an enhanced accessibility in mini-HHMb, which may affect both ligand association and dissociation kinetics.
      Graphical abstract image

      PubDate: 2015-09-21T13:31:13Z
  • Phosphatidylglycerol-Derived Phospholipids Have a Universal,
           Domain-Crossing Role in Stress Responses
    • Abstract: Publication date: Available online 21 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Luis Alberto Luévano-Martínez, Alicia J. Kowaltowski
      Phosphatidylglycerol and phospholipids derived from it are widely distributed throughout the three domains of life. Cardiolipin is the best characterized of these phospholipids, and plays a key role in the response to environmental variations. Phosphatidylglycerol-derived phospholipids confer cell membranes with a wide range of responses, including changes in surface charge, fluidity, flexibility, morphology, biosynthesis and remodeling, that adapt the cell to these situations. Furthermore, the synthesis and remodeling of these phospholipids is finely regulated, highlighting the importance of these lipids in cell homeostasis and responses during stressful situations. In this article, we review the most important roles of these anionic phospholipids across domains, focusing on the biophysical basis by which these phospholipids are used in stress responses.
      Graphical abstract image

      PubDate: 2015-09-21T13:31:13Z
  • Relationship between calcium mobilization and platelet α- and
           δ-granule secretion. A role for TRPC6 in thrombin-evoked
           δ-granule exocytosis
    • Abstract: Publication date: Available online 18 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): E. Lopez, N. Bermejo, A. Berna-Erro, N. Alonso, G.M. Salido, P.C. Redondo, J.A. Rosado
      Changes in cytosolic Ca2+ concentration ([Ca2+]c) regulate granule secretion in different cell types. Thrombin activates PAR1 and PAR4 receptors and promotes release of Ca2+ from distinct intracellular stores, which, in turn, activates store-operated Ca2+ entry (SOCE). A crucial step during platelet function is the release of physiological agonists stored in secretory granules to the extracellular compartment during activation. We aim to study the role of Ca2+ mobilization from the extracellular compartment or from different intracellular stores in platelet granule secretion. By using flow cytometry, we have found that α- and δ-granules are secreted in thrombin-stimulated platelets in the absence of extracellular Ca2+, and in a concentration-dependent manner. Our findings show that thrombin-stimulated granule secretion depends on Ca2+ mobilization from intracellular stores. Analysis of the kinetics of granule secretion reveals that platelet stimulation with thrombin results in rapid release of α-granules which precedes the secretion of δ-granules. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 573-586 of TRPC6, inhibited thrombin-evoked δ-granule exocytosis. Our results indicate that the mechanisms underlying thrombin-induced α- and δ-granule secretion show differences in dependency on Ca2+ mobilization.

      PubDate: 2015-09-21T13:31:13Z
  • Regulation of Ca2+/calmodulin-dependent protein kinase phosphatase
           (CaMKP/PPM1F) by protocadherin-γC5 (Pcdh-γC5)
    • Abstract: Publication date: Available online 18 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Takashi Onouchi, Yoshimi Kishino-Kaneko, Isamu Kameshita, Atsuhiko Ishida, Noriyuki Sueyoshi
      Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. It is important to identify an endogenous regulator of CaMKP. Using an Escherichia coli two-hybrid screening method, we identified the C-terminal cytoplasmic fragment of protocadherin γ subfamily C5 (Pcdh-γC5), which was generated by intracellular processing, as a CaMKP-binding protein. Dephosphorylation of phosphorylated Ca2+/calmodulin-dependent protein kinase I (CaMKI) by CaMKP was significantly activated by the C-terminal cytoplasmic fragment, Pcdh-γC5(715-944), both in vitro and in cells, suggesting that the C-terminal fragment functions as an endogenous activator of CaMKP. The nuclear translocation of the fragment was blocked by its binding to cytoplasmic CaMKP to form a ternary complex with CaMKI. Taken together, these results strongly suggest that the C-terminal cytoplasmic fragment of Pcdh-γC5 acts as a scaffold for CaMKP and CaMKI to regulate CaMKP activity. These findings may provide new insights into the reversible regulation of CaMKP in cells.

      PubDate: 2015-09-21T13:31:13Z
  • Selective antagonization of activated Nrf2 and inhibition of cancer cell
           proliferation by procyanidins from Cinnamomi Cortex extract
    • Abstract: Publication date: 1 November 2015
      Source:Archives of Biochemistry and Biophysics, Volume 585
      Author(s): Tomokazu Ohnuma, Eri Anzai, Yohei Suzuki, Mai Shimoda, Shin Saito, Takahito Nishiyama, Kenichiro Ogura, Akira Hiratsuka
      Nuclear factor-E2-related factor 2 (Nrf2) is an important transcription factor and plays a central role in inducible expression of many cytoprotective genes. Recent studies have reported that various cancer cells having unrestrained Nrf2 due to its overexpression exhibit increased proliferation and resistance to chemotherapy. Suppression of abnormal Nrf2 activation is needed for a new therapeutic approach against these cancers. Our previous study found that procyanidins prepared from Cinnamomi Cortex extract (CCE) have an ability to suppress Nrf2-regulated enzyme activity and Nrf2 expression in human lung cancer A549 cells. In the present study, we investigated the effect of CCE procyanidins on Nrf2 activity and cell proliferation in several cancer cells, which have normal or constitutively active Nrf2. Interestingly, CCE procyanidin treatment selectively reduced Nrf2 expression and inhibited cell proliferation in cancer cells that overexpress Nrf2, but these phenomena were not seen in cells with low Nrf2 expression. Moreover, transfection assay demonstrated that CCE procyanidins had selective inhibition of activated Nrf2. These results suggest that CCE procyanidins might be an effective cancer therapeutic agent to selectively suppress abnormal Nrf2 activation responsible for enhanced proliferation.

      PubDate: 2015-09-21T13:31:13Z
  • Bisphenol A stimulates the epithelial mesenchymal transition of estrogen
           negative breast cancer cells via FOXA1 signals
    • Abstract: Publication date: 1 November 2015
      Source:Archives of Biochemistry and Biophysics, Volume 585
      Author(s): Xiao-Lin Zhang, Hong-Sheng Wang, Na Liu, Li-Chen Ge
      Estrogen receptor negative (ER-) breast cancer are associated with increased risks for metastasis and high rates of recurrence. Our present study revealed that nanomolar bisphenol A (BPA), a typical endocrine disrupting chemical, promoted the in vitro migration and induced mesenchymal transition (EMT) of ER-breast cancer cells. PCR array revealed that BPA can down regulate 12 and up regulate 2 genes involved in regulation of signal transduction and biological pathways of breast cancer. The down regulated genes included FOXA1, which is a key determinant of endocrine response and down regulated by BPA via a time dependent manner. Silencing of FOXA1 by siRNA triggered the EMT of SkBr3 cells. While over expression of FOXA1 abolished BPA induced EMT. Further, 10−8 M BPA significantly increased the phosphorylation of ERK1/2, p38-MAPK, and Akt in SkBr3 cells, while only PI3K/Akt inhibitor LY294002 attenuated the BPA induced down regulation of FOXA1 and E-Cadherin (E-Cad). Over expression of Akt also suppressed FOXA1 expression in SkBr3 cells. It suggested that PI3K/Akt mediated, at least partially, BPA induced EMT of ER-breast cancer cells. In summary, our data provided the first evidence that BPA can promote the EMT of ER-breast cancer cells through down regulation of FOXA1.

      PubDate: 2015-09-21T13:31:13Z
  • Interaction of the chlorite-based drug WF10 and chlorite with hemoglobin,
           methemoglobin and ferryl hemoglobin
    • Abstract: Publication date: Available online 21 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Annelie Pichert, Jürgen Arnhold
      The interaction of the chlorite-based drug solution WF10 with human oxyhemoglobin and oxidized hemoglobin forms was investigated monitoring the corresponding spectral changes in heme states. The chlorite component of WF10 converts oxyhemoglobin into methemoglobin with a rate of 35.4 M-1s-1. Methemoglobin is also formed upon the interaction of ferryl hemoglobin and WF10/chlorite. The rate of this interconversion depends on the oxidation state of ferryl hemoglobin. This rate is 114 M-1s-1, when ferryl hemoglobin was generated upon reaction of oxyhemoglobin and hydrogen peroxide. A considerable higher rate (6600 M-1s-1) is measured between the chlorite components of WF10 and ferryl hemoglobin after formation of the latter species from methemoglobin. WF10/chlorite inactivates also methemoglobin as evidenced by the continuous decrease of the Soret band and all other absorbances with a rate of 8.3 M-1s-1. In all interconversions, the chlorite component of WF10 was the active principle as shown in experiments applying pure chlorite at the same concentration as in WF10. Thus, WF10 is able to diminish efficiently the yield of cytotoxic hemoglobin species that might appear after excessive hemolysis of red blood cells under pathologic situations.
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      PubDate: 2015-09-21T13:31:13Z
  • Analysis of the pH-Dependent Stability and Millisecond Folding Kinetics of
           Horse Cytochrome c
    • Abstract: Publication date: Available online 16 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Rishu Jain, Rajesh Kumar, Sandeep Kumar, Ritika Chhabra, Mukesh Chand Agarwal, Rajesh Kumar
      This paper analyzes the effect of pH on thermodynamic stability and folding kinetics of horse cytochrome c (cyt c). Analysis of equilibrium unfolding transitions of Ferricyt c and Ferrocyt c measured between pH 3.0 and pH 13.0 reveal that these proteins have maximum thermodynamic stability between pH 8.0 and pH 9.5. Theoretically predicted pH-dependent electrostatic unfolding energy of Ferricyt c also supports this result. Unfolded Ferrocyt c in refolding buffer at pH 7.0 and pH 12.7 refolds rapidly to native state. Between pH 7.0 and pH 12.7, the activation free energy barrier for folding of Ferrocyt c varies by <1.0 kcal mol-1 while the folding free energy, which is measured by two-state analysis of equilibrium unfolding transitions of Ferrocyt c varies by 8.0 kcal mol-1. This finding reveals that the large disparity in thermodynamic stability between pH 7.0 and pH 12.7 is not strongly reflected in the refolding rates. The Wyman Tanford linkage relation was used to calculate the βpH-value for folding of Ferrocyt c, which is <0.1 between pH 7.0 and pH 12.7, indicating that the electrostatic interactions are weakly formed in transition state and exhibit a very small effect on the folding kinetics.
      Graphical abstract image

      PubDate: 2015-09-17T13:19:09Z
  • Blue Copper Proteins: A rigid machine for efficient electron transfer, a
           flexible device for metal uptake
    • Abstract: Publication date: 15 October 2015
      Source:Archives of Biochemistry and Biophysics, Volume 584
      Author(s): Sergio Alejo Pérez-Henarejos, Luis A. Alcaraz, Antonio Donaire
      Blue Copper Proteins (BCPs) are small and generally soluble copper-containing proteins which participate in monoelectron transfer processes in biological systems. An overview of their electronic and tertiary structure is detailed here. The well-established entatic/rack-induced mechanism is explained by comparing thermodynamic parameters between the folded (tense) and the unfolded (relaxed) forms of the BCP rusticyanin. Recently, NMR solution data have shown that the active sites of BCPs in absence of the metal ion, i.e. in the apoforms, are flexible in the micro-to-second timescale. The rigidity proposed by the entatic/rack-induced mechanism is an imperative for the holoprotein to perform electron transfer; while the flexibility of the apocupredoxin is necessary to uptake the metal ion from the metallochaperones. These apparently contradictory requirements are discussed in the present work. Finally, the role of azurin and some peptides derived from it in anticancer therapy are also described.
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      PubDate: 2015-09-17T13:19:09Z
  • Contribution to catalysis of ornithine binding residues in ornithine
    • Abstract: Publication date: Available online 12 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Reeder Robinson, Insaf A. Qureshi, Catherine A. Klancher, Pedro J. Rodriguez, John J. Tanner, Pablo Sobrado
      The SidA ornithine N5-monooxygenase from A. fumigatus is a flavin monooxygenase that catalyzes the NADPH-dependent hydroxylation of ornithine. Herein we report a mutagenesis study targeting four residues that contact ornithine in crystal structures of SidA: Lys107, Asn293, Asn323, and Ser469. Mutation of Lys107 to Ala abolishes activity as measured in steady-state oxygen consumption and ornithine hydroxylation assays, indicating that the ionic interaction of Lys107 with the carboxylate of ornithine is essential for catalysis. Mutation of Asn293, Asn323, or Ser469 individually to Ala results in >14-fold increases in Km values for ornithine. Asn323 to Ala also increases the rate constant for flavin reduction by NADPH by 18-fold. Asn323 is unique among the four ornithine binding residues in that it also interacts with NADPH by forming a hydrogen bond with the nicotinamide ribose. The crystal structure of N323A complexed with NADP+ and ornithine shows that the nicontinamide riboside group of NADP+ is disordered. This result suggests that the increase in flavin reduction rate results from an increase in conformational space available to the enzyme-bound NADP(H). Asn323 thus facilitates ornithine binding at the expense of hindering flavin reduction, which demonstrates the delicate balance that exists within protein-ligand interaction networks in enzyme active sites.
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      PubDate: 2015-09-13T13:13:37Z
  • Metformin ameliorates lipotoxicity-induced mesangial cell apoptosis partly
           via upregulation of glucagon like peptide-1 receptor (GLP-1R)
    • Abstract: Publication date: 15 October 2015
      Source:Archives of Biochemistry and Biophysics, Volume 584
      Author(s): Dong-il Kim, Min-jung Park, Young-ran Heo, Soo-hyun Park
      Glucagon like peptide-1 receptor (GLP-1R), known to be expressed in pancreatic beta cells, is also expressed in glomerular mesangial cells and its agonist has protective effects in diabetic nephropathy. However, its regulatory mechanisms by lipotoxicity in glomerular mesangial cells are not understood. We found that palmitate-mediated lipotoxicity increased apoptosis and decreased GLP-1R expression in a rat mesangial cell line. Silencing GLP-1R expression also increased mesangial cell apoptosis. Interestingly, metformin, one of the biguanide drugs that has anti-diabetic effects, attenuated lipotoxicity-induced mesangial cell apoptosis and restored GLP-1R expression. Moreover, this treatment alleviated GLP-1R knockdown-induced mesangial cell apoptosis. To further evaluate in vivo, diabetic obese db/db mice were administered metformin. Glomerular GLP-1R expression was diminished in db/db mice, as compared with db/m control mice. However, this decrease significantly recovered on metformin administration. Together, these data provide novel evidence that lipotoxicity decreases the mesangial GLP-1R expression in intact cells and in vivo. The decrease induced mesangial cell apoptosis. Furthermore, we provided the evidence that metformin treatment has a renal protective effect partly via increased mesangial GLP-1R expression. Our data suggested that regulation of GLP-1R expression could be a promising approach to treat diabetic nephropathy and the novel mechanism of metformin mediated GLP-1R regulation.

      PubDate: 2015-09-13T13:13:37Z
  • Carotenoid binding to proteins: Modeling pigment transport to lipid
    • Abstract: Publication date: Available online 8 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Emilia Reszczynska, Renata Welc, Wojciech Grudzinski, Kazimierz Trebacz, Wieslaw I. Gruszecki
      Carotenoid pigments play numerous important physiological functions in human organism. Very special is a role of lutein and zeaxanthin in the retina of an eye and in particular in its central part, the macula lutea. In the retina, carotenoids can be directly present in the lipid phase of the membranes or remain bound to the protein-pigment complexes. In this work we address a problem of binding of carotenoids to proteins and possible role of such structures in pigment transport to lipid membranes. Interaction of three carotenoids, beta-carotene, lutein and zeaxanthin with two proteins: bovine serum albumin and glutathione S-transferase (GST) was investigated with application of molecular spectroscopy techniques: UV-Vis absorption, circular dichroism and Fourier transform infrared spectroscopy (FTIR). Interaction of pigment-protein complexes with model lipid bilayers formed with egg yolk phosphatidylcholine was investigated with application of FTIR, Raman imaging of liposomes and electrophysiological technique, in the planar lipid bilayer models. The results show that in all the cases of protein and pigment studied, carotenoids bind to protein and that the complexes formed can interact with membranes. This means that protein-carotenoid complexes are capable of playing physiological role in pigment transport to biomembranes.
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      PubDate: 2015-09-09T13:08:33Z
  • Proteoliposomes with the ability to transport Ca2+ into the vesicles and
           hydrolyze phosphosubstrates on their surface
    • Abstract: Publication date: 15 October 2015
      Source:Archives of Biochemistry and Biophysics, Volume 584
      Author(s): Maytê Bolean, Ana Maria S. Simão, Tina Kiffer-Moreira, Marc F. Hoylaerts, José Luis Millán, Rosangela Itri, Pietro Ciancaglini
      We describe the production of stable DPPC and DPPC:DPPS-proteoliposomes harboring annexin V (AnxA5) and tissue-nonspecific alkaline phosphatase (TNAP) and their use to investigate whether the presence of AnxA5 impacts the kinetic parameters for hydrolysis of TNAP substrates at physiological pH. The best catalytic efficiency was achieved in DPPS 10%-proteoliposomes (molar ratio), conditions that also increased the specificity of TNAP hydrolysis of PPi. Melting behavior of liposomes and proteoliposomes was analyzed via differential scanning calorimetry. The presence of 10% DPPS in DPPC-liposomes causes a broadening of the transition peaks, with AnxA5 and TNAP promoting a decrease in ΔH values. AnxA5 was able to mediate Ca2+-influx into the DPPC and DPPC:DPPS 10%-vesicles at physiological Ca2+ concentrations (∼2 mM). This process was not affected by the presence of TNAP in the proteoliposomes. However, AnxA5 significantly affects the hydrolysis of TNAP substrates. Studies with GUVs confirmed the functional reconstitution of AnxA5 in the mimetic systems. These proteoliposomes are useful as mimetics of mineralizing cell-derived matrix vesicles, known to be responsible for the initiation of endochondral ossification, as they successfully transport Ca2+ and possess the ability to hydrolyze phosphosubstrates in the lipid–water interface.
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      PubDate: 2015-09-09T13:08:33Z
  • Kinetic Analysis of Electron Flux in Cytochrome P450 Reductases Reveals
           Differences in Rate-determining Steps in Plant and Mammalian Enzymes
    • Abstract: Publication date: Available online 8 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Douglas Whitelaw, Rochelle Tonkin, Carla Meints, Kirsten R. Wolthers
      Herein, we compare the kinetic properties of CPR from Arabidopsis thaliana (ATR2), with CPR from Artemisia annua (aaCPR) and human CPR (hCPR). While all three CPR forms elicit comparable rates for cytochrome c 3+ turnover, NADPH reduction of the FAD cofactor is ∼50-fold faster in aaCPR and ATR2 compared to hCPR, with a k obs of ∼500 s-1 (6 °C). Stopped-flow analysis of the isolated FAD-domains reveals that NADP+-FADH2 charge-transfer complex formation is also significantly faster in the plant enzymes, but the rate of its decay is comparable for all three proteins. In hCPR, transfer of a hydride ion from NADPH to FAD is tightly coupled to subsequent FAD to FMN electron transfer, indicating that the former catalytic event is slow relative to the latter. In contrast, interflavin electron transfer is slower than NADPH hydride transfer in aaCPR and ATR2, occurring with an observed rate constant of ∼50 s-1. Finally, the transfer of electrons from FMN to cytochrome c 3+ is rapid (>103 s-1) in all three enzymes and does not limit catalytic turnover. In combination, the data reveal differences in rate-determining steps between plant CPR and their mammalian equivalent in mediating the flux of reducing equivalents from NADPH to external electron acceptors.
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      PubDate: 2015-09-09T13:08:33Z
  • Secreted lipoxygenase from Pseudomonas aeruginosa exhibits biomembrane
           oxygenase activity and induces hemolysis in human red blood cells
    • Abstract: Publication date: Available online 8 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Swathi Banthiya, Mária Pekárová, Hartmut Kuhn, Dagmar Heydeck
      Pseudomonas aeruginosa (PA) expresses a secreted lipoxygenase (LOX), which oxygenates free arachidonic acid predominantly to 15S-H(p)ETE. The enzyme is capable of binding phospholipids at its active side and physically interacts with model membranes. However, its membrane oxygenase activity has not been quantified. To address this question, we overexpressed PA-LOX as intracellular his-tag fusion protein in E. coli, purified it to electrophoretic homogeneity and compared its biomembrane oxygenase activity with that of rabbit ALOX15. We found that both enzymes were capable of oxygenating mitochondrial membranes to specific oxygenation products and 13S-H(p)ODE and 15S-H(p)ETE esterified to phosphatidylcholine and phosphatidylethanolamine were identified as major oxygenation products. When normalized to similar linoleic acid oxygenase activity, the rabbit enzyme exhibited a much more effective mitochondrial membrane oxygenase activity. In contrast, during long-term incubations (24 h) with red blood cells PA-LOX induced significant (50%) hemolysis whereas rabbit ALOX15 was more or less ineffective. These data indicate the principle capability of PA-LOX of oxygenating membrane bound phospholipids altering the barrier function of the biomembranes. Although the membrane oxygenase activity was lower than the fatty acid oxygenase activity of PA-LOX red blood cell membrane oxygenation might be of biological relevance for P. aeruginosa septicemia.

      PubDate: 2015-09-09T13:08:33Z
  • Solution structure and base specificity of cytotoxic RC-RNase 2 from Rana
    • Abstract: Publication date: 15 October 2015
      Source:Archives of Biochemistry and Biophysics, Volume 584
      Author(s): Chun-Hua Hsu, Chi-Fon Chang, You-Di Liao, Shih-Hsiung Wu, Chinpan Chen
      Cytotoxic ribonucleases found in the oocytes and early embryos of frogs with antitumor activity are well-documented. RC-RNase 2, a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana, consists of 105 residues linked with 4 disulfide bridges and belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Among the RC-RNases, the base preference for RNase 2 is UpG but CpG for RC-RNase 4; while RC-RNase possesses the base specificity of both UpG and CpG. Interestingly, RC-RNase 2 or 4 has much lower catalytic activity but only three-fold less cytotoxicity than RC-RNase. Here, we report the NMR solution structure of rRC-RNase 2, comprising three alpha-helices and two sets of antiparallel beta-sheets. The differences of side-chain conformations of subsite residues among RNase A, RC-RNase, RC-RNase 4 and rRNase 2 are related to their distinct catalytic activities and base preferences. Furthermore, the substrate-related residues in the base specificity among native RC-RNases are derived using the chemical shift perturbation on ligand binding.

      PubDate: 2015-09-09T13:08:33Z
  • Microbial oxidative stress response: Novel insights from environmental
           facultative anaerobic bacteria
    • Abstract: Publication date: 15 October 2015
      Source:Archives of Biochemistry and Biophysics, Volume 584
      Author(s): Huihui Fu, Jie Yuan, Haichun Gao
      Facultative bacteria can grow under either oxic or anoxic conditions. While oxygen provides substantial advantages in energy yield by respiration, it can become life-threatening because of reactive oxygen species that derive from the molecule naturally. Thus, to survive and thrive in a given niche, these bacteria have to constantly regulate physiological processes to make maximum benefits from oxygen respiration while restraining oxidative stress. Molecular mechanisms and physiological consequences of oxidative stress have been under extensive investigation for decades, mostly on research model Escherichia coli, from which our understanding of bacterial oxidative stress response is largely derived. Nevertheless, given that bacteria live in enormously diverse environments, to cope with oxidative stress different strategies are conceivably developed.

      PubDate: 2015-09-04T13:05:29Z
  • Calpain-dependent regulation of the skeletal muscle atrophy following
    • Abstract: Publication date: 15 October 2015
      Source:Archives of Biochemistry and Biophysics, Volume 584
      Author(s): Boris S. Shenkman, Svetlana P. Belova, Yulia N. Lomonosova, Tatiana Y. Kostrominova, Tatyana L. Nemirovskaya
      Unloading causes rapid skeletal muscle atrophy due to increased protein degradation via activation of calpains and decreased protein synthesis. Our study elucidated role of calpain-1 in the regulation of ubiquitin proteasome pathway (UPP) and anabolic processes mediated by Akt-mTOR-p70S6K and MAPK-Erk (p90RSK) signaling. We hypothesized that blocking calpain will inhibit activation of UPP and decrease protein degradation resulting in reduction of unloading-induced skeletal muscle atrophy. Rats were divided into three groups: non-treated control (C), three day hindlimb suspension with (HSPD) or without (HS) treatment with calpain inhibitor PD150606. When compared with control PD150606 treatment during unloading: 1) attenuated loss of muscle mass, 2) prevented accumulation of calpain-1 (1.8-fold in HS vs 1.3-fold in HSPD) and ubiquitin (2.3-fold in HS vs 0.7-fold in HSPD) mRNA and ubiquitinated proteins (1.6-fold in HS vs 0.8-fold in HSPD), 3) prevented decrease in the pAkt (0.4-fold in HS vs 1-fold in HSPD) and pFOXO3 (0.2-fold in HS vs 1.2-fold in HSPD) levels, 4) prevented increase in MAFbx (3.8-fold in HS vs 1.3-fold in HSPD) and eEF2k (1.8-fold in HS vs 0.6-fold in HSPD) mRNA. Our study indicates that blocking of calpain during unloading decreases skeletal muscle atrophy by inhibiting UPP activation and preserving anabolic signaling.

      PubDate: 2015-09-04T13:05:29Z
  • Probing the Chemical Mechanism of Saccharopine Reductase from
           Saccharomyces cerevisiae Using Site-Directed Mutagenesis
    • Abstract: Publication date: Available online 3 September 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Ashwani K. Vashishtha, Ann H. West, Paul F. Cook
      Saccharopine reductase catalyzes the reductive amination of L-α-aminoadipate-δ-semialdehyde with L-glutamate to give saccharopine. Two mechanisms have been proposed for the reductase, one that makes use of enzyme side chains as acid-base catalytic groups, and a second, in which the reaction is catalyzed by enzyme-bound reactants. Site-directed mutagenesis was used to change acid-base candidates in the active site of the reductase to eliminate their ionizable side chain. Thus, the D126A, C154S and Y99F and several double mutant enzymes were prepared. Kinetic parameters in the direction of glutamate formation exhibited modest decreases, inconsistent with the loss of an acid-base catalyst. The pH-rate profiles obtained with all mutant enzymes decrease at low and high pH, suggesting acid and base catalytic groups are still present in all enzymes. Solvent kinetic deuterium isotope effects are all larger than those observed for wild type enzyme, and approximately equal to one another, suggesting the slow step is the same as that of wild type enzyme, a conformational change to open the site and release products (in the direction of saccharopine formation). Overall, the acid-base chemistry is likely catalyzed by bound reactants, with the exception of deprotonation of the α-amine of glutamate, which likely requires an enzyme residue.

      PubDate: 2015-09-04T13:05:29Z
  • Plasma-activated medium-induced intracellular zinc liberation causes death
           of SH-SY5Y cells
    • Abstract: Publication date: 15 October 2015
      Source:Archives of Biochemistry and Biophysics, Volume 584
      Author(s): Hirokazu Hara, Miko Taniguchi, Mari Kobayashi, Tetsuro Kamiya, Tetsuo Adachi
      Plasma is an ionized gas consisting of ions, electrons, free radicals, neutral particles, and photons. Plasma-activated medium (PAM), which is prepared by the irradiation of cell-free medium with non-thermal atmospheric pressure plasma, induces cell death in various types of cancer cell. Since PAM contains reactive oxygen species (ROS), its anti-cancer effects are thought to be attributable to oxidative stress. Meanwhile, oxidative stress has been shown to induce the liberation of zinc (Zn2+) from intracellular Zn2+ stores and to provoke Zn2+-dependent cell death. In this study, we thus examined whether Zn2+ is involved in PAM-induced cell death using human neuroblastoma SH-SY5Y cells. Exposure to PAM triggered cell death in SH-SY5Y cells. The cell-permeable Zn2+ chelator N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) protected against PAM-induced cell death. Zn2+ imaging using the fluorescent Zn2+ probe FluoZin-3 revealed that PAM elicited a rise of intracellular free Zn2+. In addition, PAM stimulated PARP-1 activation, mitochondrial ROS generation, and the depletion of intracellular NAD+ and ATP. These findings suggest that PAM-induced PARP-1 activation causes energy supply exhaustion. Moreover, TPEN suppressed all of these events elicited by PAM. Taken together, we demonstrated here that Zn2+ released from intracellular Zn2+ stores serves as a key mediator of PAM-induced cell death in SH-SY5Y cells.

      PubDate: 2015-09-04T13:05:29Z
  • Key roles of Tyr 10 in Cu bound Aβ complexes and its relevance to
           Alzheimer's disease
    • Abstract: Publication date: 15 October 2015
      Source:Archives of Biochemistry and Biophysics, Volume 584
      Author(s): Naihao Lu, Jiayu Li, Zhonghong Gao
      Recent studies show that the accumulation of redox-active Cu mediates the aggregation of amyloid β-peptide (Aβ) and conspicuous oxidative damage to the brain in Alzheimer's disease (AD). However, the key roles for Tyr 10 in Aβ–Cu(II) complex and its potential biological relevance to AD etiology under oxidative stress, were not stressed enough. Interestingly, our results indicated that Aβ40 (not Aβ16)–Cu(II) complex showed obviously enhanced peroxidase activity than free Cu(II). Although Tyr 10 was not the residue binding Cu(II), the mutation of Tyr 10 residue in Aβ40 decreased the peroxidase activity of Aβ40–Cu(II) complex, and the mutation of Tyr 10 could inhibit Aβ40 aggregation. Under oxidative and nitrative stress conditions, the Aβ–Cu(II) complex caused oxidation and nitration of the Aβ Tyr 10 residue through peroxidase-like reactions, where the formation of Cu(I) and hydroxyl radical (OH) was proposed as a chemical mechanism. We also showed that, when Aβ40 aggregates were bound to Cu(II), they retained peroxidase-like activity. Therefore, Tyr 10 residue is pivotal in Aβ–Cu(II) complex and shows important relevance to oxidative stress, implicating the novel significance of Tyr 10 residue as well as Aβ–Cu(II) complex in the pathology of AD.

      PubDate: 2015-08-31T13:54:55Z
  • Relationship between protein stability and functional activity in the
           presence of macromolecular crowding agents alone and in mixture: an
           insight into stability-activity trade-off
    • Abstract: Publication date: Available online 29 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Sumra Shahid, Faizan Ahmad, Md. Imtaiyaz Hassan, Asimul Islam
      The cellular environment is crowded with different kinds of molecules with varying sizes, shapes and compositions. Most of the experiments studying the nature and behaviour of a protein have been done on the isolated protein in dilute buffer solutions which actually do not imitate the in vivo situation. To understand the consequences of such crowded environment, we investigated the effect of macromolecular crowding on the stability and activity of hen egg white lysozyme. Two crowding agents, dextran 70 and ficoll 70 which have different shapes and composition, have been employed in this study. To mimic the cellular condition from physiological point of view, the effect of mixtures of both the crowding agents has been also studied. The results indicate that owing to volume exclusion, lysozyme is stabilized while its activity decays with the increasing concentration of both the crowders elucidating the hypothesis of stability-activity trade-off. Mixed macromolecular crowding exerts greater effect than the sum of constituent crowding agents (dextran 70 and ficoll 70).

      PubDate: 2015-08-31T13:54:55Z
  • Ameliorative effect of riboflavin on hyperglycemia, oxidative stress and
           DNA damage in type-2 diabetic mice: Mechanistic and therapeutic strategies
    • Abstract: Publication date: 15 October 2015
      Source:Archives of Biochemistry and Biophysics, Volume 584
      Author(s): Md. Maroof Alam, Sarah Iqbal, Imrana Naseem
      Increasing evidence in both experimental and clinical studies suggests that oxidative stress play a major role in the pathogenesis of type-2 diabetes mellitus (T2DM). Abnormally high levels of free radicals and the simultaneous decline of antioxidant defence mechanisms can lead to damage of cellular organelles and enzymes. Riboflavin constitutes an essential nutrient for humans and is also an important food additive for animals. It is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which serves as a coenzyme for several enzymes. The aim of this study was to observe the effects of illuminated and non-illuminated riboflavin in a diabetic mice model. The protocol included treatment of diabetic mice with illuminated RF and a control set without light. To our surprise, group receiving RF without light gave better results in a dose dependent manner. Significant amelioration of oxidative stress was observed with an increased glucose uptake in skeletal muscles and white adipose tissue. Histological studies showed recovery in the liver and kidney tissue injury. Cellular DNA damage was also recovered. Therefore, it is suggested that supplementation with dietary riboflavin might help in the reduction of diabetic complications. A possible mechanism of action is also proposed.

      PubDate: 2015-08-31T13:54:55Z
  • Crystal structure and activity of protein
           L-isoaspartyl-O-methyltransferase from Vibrio cholerae, and the effect of
           AdoHcy binding
    • Abstract: Publication date: 1 October 2015
      Source:Archives of Biochemistry and Biophysics, Volume 583
      Author(s): Tanaya Chatterjee, Debadrita Mukherjee, Mousumi Banerjee, Barun K. Chatterjee, Pinak Chakrabarti
      The repair enzyme Protein L-isoaspartyl-O-methyltransferase (PIMT) is widely distributed in various organisms. PIMT catalyzes S-adenosylmethionine (AdoMet) dependent methylation of abnormal L-isoaspartyl residues, formed by the deamidation of asparagines and isomerization of aspartates. We report the crystal structure of PIMT of Vibrio cholerae (VcPIMT), the aetiological agent for cholera, complexed with the demethylated cofactor S-adenosyl-L-homocysteine (AdoHcy) to 2.05 Å resolution. A stretch of residues (39–58), lining the substrate-binding site, is disordered. Urea-induced unfolding free energy for apo and VcPIMT-AdoHcy complex reveals greater stability for the cofactor-bound protein. The kinetic parameters for the methyltransferase activity of the recombinant VcPIMT was determined using a continuous spectrophotometric color-based assay using the peptide substrate [VYP(L-isoD)HA]. The enzyme exhibited activity higher than the Escherichia coli enzyme and closer to those from thermophilic bacteria and the mammalian source. The association constant for substrate binding is 2.29 × 106 M−1, quite similar to that for AdoHcy. The crystal structure and the model of the peptide-bound structure indicate that the majority of the interactions used for cofactor/substrate binding are provided by the main-chain atoms. Evolutionary relationships derived based on a phylogenetic tree constructed using the PIMT sequences are in conformity with the crystal structures of nine AdoHcy-bound PIMTs.
      Graphical abstract image

      PubDate: 2015-08-31T13:54:55Z
  • Evidence for an Induced Conformational Change in the Catalytic Mechanism
           of Homoisocitrate Dehydrogenase for Saccharomyces cerevisiae:
           Characterization of the D271N Mutant Enzyme
    • Abstract: Publication date: Available online 29 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Chaonan Hsu, Ann H. West, Paul F. Cook
      Homoisocitrate dehydrogenase (HIcDH) catalyzes the NAD+-dependent oxidative decarboxylation of HIc to α-ketoadipate, the fourth step in the α-aminoadipate pathway responsible for the de novo synthesis of L-lysine in fungi. A mechanism has been proposed for the enzyme that makes use of a Lys-Tyr pair as acid-base catalysts, with Lys acting as a base to accept a proton from the α-hydroxyl of homoisocitrate, and Tyr acting as an acid to protonate the C3 of the enol of α-ketoadipate in the enolization reaction. Three conserved aspartate residues, D243, D267 and D271, coordinate Mg2+, which is also coordinated to the α-carboxylate and α-hydroxyl of homoisocitrate. On the basis of kinetic isotope effects, it was proposed that a conformational change to close the active site and organize the active site for catalysis contributed to rate limitation of the overall reaction of the Saccharomyces cerevisiae HIcDH (Lin, Y., Volkman, J., Nicholas, K. M., Yamamoto, T., Eguchi, T., Nimmo, S. L., West, A. H., and Cook, P. F. (2008) Biochemistry 47, 4169–4180.). In order to test this hypothesis, site-directed mutagenesis was used to change D271, a metal ion ligand and binding determinant for MgHIc, to N. The mutant enzyme was characterized using initial rate studies. A decrease of 520-fold was observed in V and V/K MgHIc , suggesting the same step(s) limit the reaction at limiting and saturating MgHIc concentrations. Solvent kinetic deuterium isotope effects (SKIE) and viscosity effects are consistent with a rate-limiting pre-catalytic conformational change at saturating reactant concentrations. In addition, at limiting MgHIc, an inverse (SKIE) of 0.7 coupled to a significant normal effect of viscosogen (2.1) indicates equilibrium binding of MgHIc prior to the rate-limiting conformational change. The maximum rate exhibits a small partial change at high pH suggesting a pH-dependent conformational change, while V/K MgHIc exhibits the same partial change observed in V, and a decrease at low pH with a pKa of 6 reflecting the requirement for the unprotonated form of MgHIc to bind to enzyme. However, neither parameter reflects the pH dependence of the chemical reaction. This pH independence of the chemical reaction over the range 5.5-9.5 is consistent with the much slower conformational change that would effectively perturb the observed pK values for catalytic groups to lower and higher pH. In other words, the pH dependence of the chemical reaction will only be observed when chemistry becomes slower than the rate of the conformational change. Data support the hypothesis of the existence of a pre-catalytic conformational change coupled to the binding of MgHIc.

      PubDate: 2015-08-31T13:54:55Z
  • Functional Importance of a Peripheral Pocket in Mammalian Cytochrome P450
           2B Enzymes
    • Abstract: Publication date: Available online 28 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Hyun-Hee Jang, Jingbao Liu, Ga-Young Lee, James R. Halpert, P. Ross Wilderman
      The functional importance of a peripheral pocket found in previously published X-ray crystal structures of CYP2B4 and CYP2B6 was probed using a biophysical approach. Introduction of tryptophan within the pocket of CYP2B4 at F202 or I241 leads to marked impairment of 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) or 7-benzyloxyresorufin O-dealkylation efficiency; a similar substitution at F195, near the surface access to the pocket, does not affect these activities. The analogous CYP2B6 F202W mutant is inactive in the 7-EFC O-dealkylation assay. The stoichiometry of 7-EFC deethylation suggested that the decreased activity of F202W and I241W in CYP2B4 and lack of activity of F202W in CYP2B6 coincided with a sharp increase in the flux of reducing equivalents through the oxidase shunt to produce excess water. The results indicate that the chemical identity of residues within this peripheral pocket, but not at the mouth of the pocket, is important in substrate turnover and redox coupling, likely through effects on active site topology.

      PubDate: 2015-08-31T13:54:55Z
  • Quercetin reduced inflammation and increased antioxidant defense in rat
           adjuvant arthritis
    • Abstract: Publication date: Available online 20 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): C. Gardi, K. Bauerova, B. Stringa, V. Kuncirova, L. Slovak, S. Ponist, F. Drafi, L. Bezakova, I. Tedesco, A. Acquaviva, S. Bilotto, G.L. Russo
      Novel therapies for rheumatoid arthritis also include the use of naturally occurring compounds possessing antioxidant properties. In the present work, the effects of oral administration of quercetin were investigated in a rat model of adjuvant arthritis. Arthritis was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund’s adjuvant. The experimental groups were treated with an oral daily dose of 150 mg/kg b.w. of quercetin for 28 days. Results indicated that quercetin was able to ameliorate all markers of inflammation and oxidative stress measured. Quercetin lowered levels of interleukin-1β, C-reactive protein, and monocyte chemotactic protein-1 and restored plasma antioxidant capacity. In addition, quercetin inhibited the enzymatic activity of pro-inflammatory 12/15-lipoxygenase in lung and liver and increased the expression of heme oxygenase-1 in joint and lung of arthritic rats. Finally, quercetin inhibited the 2-fold increase of NF-қB activity observed in lung, liver and joint after induction of arthritis.

      PubDate: 2015-08-22T13:19:09Z
  • 60S ribosomal protein L35 regulates β-casein translational elongation
           and secretion in bovine mammary epithelial cells
    • Abstract: Publication date: Available online 19 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Nan Jiang, Lijun Hu, Chaonan Liu, Xueli Gao, Shimin Zheng
      60S ribosomal protein L35 (RPL35) is an important component of the 60S ribosomal subunit and has a role in protein translation and endoplasmic reticulum (ER) docking. However, few studies have investigated RPL35 in eukaryotes and much remains to be learned. Here, we analyzed the function of RPL35 in β-casein (CSN2) synthesis and secretion in bovine mammary epithelial cells (BMECs). We found that methionine (Met) could promote the expressions of CSN2 and RPL35. Analysis of overexpression and inhibition of RPL35 confirmed that it could mediate the Met signal and regulate CSN2 expression. The mechanism of CSN2 regulation by RPL35 was analyzed by coimmunoprecipitation (Co-IP), colocalization, and fluorescence resonance energy transfer (FRET) and gene mutation. We found that RPL35 could control ribosome translational elongation during synthesis of CSN2 by interacting with eukaryotic translational elongation factor 2 (eEF2), and that eEF2 was the signaling molecule downstream of RPL35 controlling this process. RPL35 could also control the secretion of CSN2 by locating it to the ER. Taken together, these results revealed that, RPL35 was an important positive regulatory factor involving in the Met-mediated regulation of CSN2 translational elongation and secretion.

      PubDate: 2015-08-19T13:16:00Z
  • The Busulfan Metabolite EdAG Irreversibly Glutathionylates Glutaredoxins
    • Abstract: Publication date: Available online 14 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Michele Scian, William M. Atkins
      The DNA alkylating agent busulfan is used to ‘precondition’ patients with leukemia, lymphomas and other hematological disorders prior to hematopoietic stem cell transplants. Busulfan is metabolized via conjugation with glutathione (GSH) followed by intramolecular rearrangement to the GSH analog γ−glutamyl-dehydroalanyl -glycine (EdAG). EdAG contains the electrophilic dehydroalanine, which is expected to react with protein nucleophiles, particularly proteins with GSH binding sites such as glutaredoxins (Grx’s). Incubation of EdAG with human Grx-1 or Grx-2 results in facile adduction of cys-23 and cys-77, respectively, as determined by ESI-MS/MS. The resulting modified proteins are catalytically inactive. In contrast, the glutathione transferase A1-1 includes a GSH binding site with a potentially reactive tyrosinate (Tyr-9) but it does not react with EdAG. Similarly, Cys-112 of GSTA1-1, which lies outside the active site and is known to form disulfides with GSH, does not react with EdAG. The results provide the first demonstration of the reactivity of any busulfan metabolites with intact proteins, and they suggest that GSH-binding sites containing thiolates are most susceptible. The adduction of Grx’s by EdAG suggests the possible alteration of proteins that are normally regulated via Grx-dependent reversible glutathionylation or deglutathionylation. Dysregulation of Grx-dependent processes could contribute to cellular toxicity of busulfan.

      PubDate: 2015-08-14T19:57:49Z
  • 4-O-methylascochlorin suppresses differentiation of 3T3-L1 preadipocytes
           by inhibiting PPARγ expression through regulation of AMPK/mTOR
           signaling pathways
    • Abstract: Publication date: Available online 10 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Mihyun Kim, Hyun-Ji Cho, Yun-Jeong Jeong, Il-Kyung Chung, Junji Magae, Young-Chae Chang
      Obesity increases the risk of developing many chronic diseases, including type 2 diabetes and certain cancers, and is thereby associated with premature death. The present study was conducted to identify the inhibitory effect of the ascochlorin derivative 4-O-methylascochlorin (MAC) on the differentiation of 3T3-L1 preadipocytes. MAC suppressed the differentiation of 3T3-L1 preadipocytes and inhibited the expression of adipocyte differentiation marker genes, FABP4, PPARγ and C/EBPα. In addition, we found that the inhibitory effects of MAC on differentiation of 3T3-L1 preadipocytes were caused by suppression of mTORC1 via inhibition of mTOR/p70S6K/4E-BP1 phosphorylation and activation of Raptor phosphorylation. MAC also regulated the PPARγ expression and the mTORC1 activation by increasing AMPK phosphorylation and inhibiting PI3K/Akt, which suggest that MAC suppresses the differentiation of 3T3-L1 adipocytes by regulating the AMPK- and PI3K-mTOR-PPARγ signaling pathways. Furthermore, animal model results showed that the phosphorylation of AMPK was enhanced in the liver of C57BL/6 mice intraperitoneally injected with MAC. These results indicate that MAC could be a therapeutic agent for obesity involving PPARγ and AMPK.

      PubDate: 2015-08-14T19:57:49Z
  • Open challenges in structure-based virtual screening: receptor modeling,
           target flexibility consideration and active site water molecules
    • Abstract: Publication date: Available online 10 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Francesca Spyrakis, Claudio N. Cavasotto
      Structure-based virtual screening is currently an established tool in drug lead discovery projects. Although in the last years the field saw an impressive progress in terms of algorithm development, computational performance, and retrospective and prospective applications in ligand identification, there are still long-standing challenges where further improvement is needed. In this review, we consider the conceptual frame, state-of-the-art and recent developments of three critical “structural” issues in structure-based drug lead discovery: the use of homology modeling to accurately model the binding site when no experimental structures are available, the necessity of accounting for the dynamics of intrinsically flexible systems as proteins, and the importance of considering active site water molecules in lead identification and optimization campaigns.
      Graphical abstract image

      PubDate: 2015-08-14T19:57:49Z
  • Rate-limiting steps of a stereochemistry retaining β-d-xylosidase
           from Geobacillus stearothermophilus acting on four substrates
    • Abstract: Publication date: Available online 10 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Douglas B. Jordan, Jay D. Braker
      Kinetic experiments of GSXynB2, a GH52 retaining β-xylosidase, acting on 2-nitrophenyl-β-d-xylopyranoside (2NPX), 4-nitrophenyl-β-d-xylopyranoside (4NPX), 4-methylumbelliferyl-β-d-xylopyanoside (MuX) and xylobiose (X2) were conducted at pH 7.0 and 25 °C. Catalysis proceeds in two steps (xylodidation followed by dexylosidation): E + substrate TO E-xylose + leaving group TO E + xylose. k cat falls into two groups: 4NPX (1.95 s-1) and 2NPX, MuX and X2 (15.8 s-1, 12.6 s-1, 12.8 s-1, respectively). Dexylosylation (E-xylose to E + xylose), the common step for the enzymatic hydrolysis of the four substrates, must exceed 15.8 s-1. k cat of 4NPX would seem mainly limited by xylosylation (step 1) and the other three substrates would seem mainly limited by dexylosylation (step 2) – a conclusion that critically lacks chemical justification (compare 4NPX and 2NPX). Presteady-state rates indicate rapid xylosidation rates for all substrates so a later step (not dexylosidation) is rate-limiting for 4NPX. That 2NPX is an onlier and 4NPX is an outlier (both leaving group pK a of 7.2) of the Brønsted plot pattern (logk cat vs pK a of phenol leaving group) is thus possibly explained by 4NP release. The pH dependency of k cat 2NPX encompasses 2 bell-shaped curves with peaks of pH 3 and pH 7.
      Graphical abstract image

      PubDate: 2015-08-14T19:57:49Z
  • Exploring biochemical and functional features of Leishmania major
           phosphoenolpyruvate carboxykinase
    • Abstract: Publication date: Available online 11 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Máximo Hernán Sosa, Lucila Giordana, Cristina Nowicki
      This work reports the first functional characterization of leishmanial PEPCK. The recombinant L. major enzyme (Lmj_PEPCK) exhibits equivalent K cat values for the phosphoenolpyruvate (PEP) and oxaloacetate (OAA) forming reactions. The apparent K m towards OAA is 10-fold lower than that for PEP, while the K m values for ADP and ATP are equivalent. Mutagenesis studies showed that D241, D242 and H205 of Lmj_PEPCK like the homologous residues of all known PEPCKs are implicated in metal ions binding. In contrast, the replacement of R43 for Q nearly abolishes Lmj_PEPCK activity. Moreover, the Y180F variant exhibits unchanged K m values for PEP, Mn2+, and HCO3 -, being the K cat for PEP- but not that for OAA- forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the K m value towards Mn2+. Therefore in Lmj_PEPCK, Y180 seems to exert different functions to those of the analogous residue in ATP- and GTP-dependant enzymes. Besides, the guanidinium group of R43 appears to play an essential but yet unknown role. These findings promote the need for further structural studies to disclose whether Y180 and R43 participate in the catalytic mechanism or/and in the transitions between the open and the catalytically competent (closed) forms of Lmj_PEPCK.
      Graphical abstract image

      PubDate: 2015-08-14T19:57:49Z
  • High-Mobility Group Box 1 Enhances the Inflammatory Process in Diabetic
    • Abstract: Publication date: Available online 6 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Raluca Maria Boteanu, Elena Uyy, Viorel Iulian Suica, Felicia Antohe
      Diabetes mellitus generates metabolic changes associated with inflammatory events that may eventually affect all body tissues. Both high-mobility group box 1 (HMGB1) and β-catenin are active players in inflammation. The study aimed to determine whether HMGB1 modulates the β-catenin activity in supporting inflammation, using an experimental type 1 diabetes mouse model. The protein and gene expression of HMGB1 were significantly increased (2-fold) in the diabetic lung compared to control and were positively correlated with the HMGB1 levels detected in serum. Co-immunoprecipitation of HMGB1 with RAGE co-exists with activation of PI3K/ AKT1 and NF-kB signaling pathways. At the same time β-catenin was increased in nuclear fraction (3.5 fold) while it was down-regulated in diabetic plasma membrane (2-fold). There was no difference of β-catenin gene expression between the control and diabetic mice. β-Catenin phosphorylation at Ser552 was higher in diabetic nuclear fraction, suggesting that AKT1 activation promotes β-catenin nuclear translocation. In addition, c-Jun directly binds β-catenin indicating the transcriptional activity of β-catenin in diabetes, sustained by significantly COX2 increase by 6-fold in the cytosolic extract of diabetic lung compared to control. Taken together, the data support the new concept that HMGB1 maintains the inflammation through RAGE/ AKT1/ β-catenin pathway in the diabetic lung.
      Graphical abstract image

      PubDate: 2015-08-10T15:44:31Z
  • SOD1 deficiency decreases proteasomal function, leading to the
           accumulation of ubiquitinated proteins in erythrocytes.
    • Abstract: Publication date: Available online 8 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Takujiro Homma, Toshihiro Kurahashi, Jaeyong Lee, Eun Sil Kang, Junichi Fujii
      We previously demonstrated that elevated levels of ROS in red blood cells (RBCs) are responsible for anemia in SOD1-deficient mice, suggesting that the oxidative stress-induced massive destruction of RBCs is an underlying mechanism for autoimmune hemolytic anemia. In the current study, we examined the issue of how elevated ROS are involved in the destruction of RBCs and the onset of anemia from the view point of the proteolytic removal of oxidatively-damaged proteins. We found that poly-ubiquitinated proteins had accumulated and had undergone aggregation in RBCs from SOD1-deficient mice and from phenylhydrazine-induced anemic mice. Although the protein levels of the three catalytic components of the proteasome, β1, β2, and β5, were not significantly altered, their proteolytic activities were decreased in the SOD1-deficient RBCs. These data suggest that oxidative-stress triggers the dysfunction of the proteasomal system, which results in the accumulation of the aggregation of poly-ubiquitinated proteins. We conclude that an oxidative stress-induced malfunction in the scavenging activity of proteasomes accelerates the accumulation of damaged proteins, leading to a shortened life span of RBCs and, hence, anemia.

      PubDate: 2015-08-10T15:44:31Z
  • Expression and characterization of manganese lipoxygenase of the rice
           blast fungus reveals prominent sequential lipoxygenation of
           α-linolenic acid
    • Abstract: Publication date: Available online 8 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Anneli Wennman, Fredrik Jernerén, Ann Magnuson, Ernst H. Oliw
      Magnaporthe oryzae causes rice blast disease and has become a model organism of fungal infections. M. oryzae can oxygenate fatty acids by 7,8-linoleate diol synthase, 10R-dioxygenase-epoxy alcohol synthase, and by a putative manganese lipoxygenase (Mo-MnLOX). The latter two are transcribed during infection. The open reading frame of Mo-MnLOX was deduced from genome and cDNA analysis. Recombinant Mo-MnLOX was expressed in Pichia pastoris and purified to homogeneity. The enzyme contained protein-bound Mn and oxidized 18:2n-6 and 18:3n-3 to 9S-, 11-, and 13R-hydroperoxy metabolites by suprafacial hydrogen abstraction and oxygenation. The 11-hydroperoxides were subject to β-fragmentation with formation of 9S- and 13R-hydroperoxy fatty acids. Oxygen consumption indicated apparent kcat values of 2.8 s-1 (18:2n-6) and 3.9 s-1 (18:3n-3), and UV analysis yielded apparent Km values of 8 and 12 μM, respectively, for biosynthesis of cis-trans conjugated metabolites. 9S-Hydroperoxy-10E,12Z,15Z-octadecatrienoic acid was rapidly further oxidized to a triene, 9S,16S-dihydroperoxy-10E,12Z,14E-octadecatrienoic acid. In conclusion, we have expressed, purified and characterized a new MnLOX from M. oryzae. The pathogen likely secretes Mo-MnLOX and phospholipases to generate oxylipins and to oxidize lipid membranes of rice cells and the cuticle.

      PubDate: 2015-08-10T15:44:31Z
  • Role of miR-148a in Cutaneous Squamous Cell Carcinoma by Repression of
           MAPK pathway
    • Abstract: Publication date: Available online 5 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Quan Luo, Wei Li, Tian Zhao, Xin Tian, Yumei Liu, Xibao Zhang
      Dysregulation of miRNAs is a common feature in human cancers, but there are lack of studies on roles of miRNAs in cutaneous squamous cell carcinoma (CSCC). miR-148a, a member of the miR-148/152 family, has been found to be downregulated in different types of cancer and its role in CSCC remains unknown. The study was aimed to investigate the expression and cellular function of miR-148a in CSCC. We found that miR-148a was underexpressed in CSCC tissues and cell lines. MAP3K2, MAP3K4 and MAP3K9 were predicted as the target genes of miR-148a and the latter two genes were verified as the target genes of miR-148a in CSCC cells. Importantly, we demonstrated that the overexpression of miR-148a significantly inhibited CSCC cell proliferation and metastasis via down-regulation of MAP3K9 and MAP3K4 expression. MAP3K4 and MAP3K9 were negatively associated with the expression of miR-148a in CSCC tissues. Our results suggested indicated that miR-148a acts as a tumor suppressor of CSCC via inhibiting MAPK pathway. These results may provide a promising alterative strategy for CSCC therapy.

      PubDate: 2015-08-06T10:32:52Z
  • The effect of TGF-beta-induced epithelial-mesenchymal transition on the
           expression of intracellular calcium-handling proteins in T47D and MCF-7
           human breast cancer cells
    • Abstract: Publication date: Available online 3 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Shah H.A. Mahdi, Huanyi Cheng, Jinfeng Li, Renqing Feng
      The contribution of Ca2+ in TGF-β-induced EMT is poorly understood. We aimed to confirm the effect of TGF-β on the gene expression of intracellular calcium-handling proteins and to investigate the potential underlying mechanisms in TGF-β-induced EMT. T47D and MCF-7 cells were cultured in vitro and treated with TGF-β. The mRNA expression of EMT marker genes and intracellular calcium-handling proteins were quantified by qRT-PCR. qRT-PCR and Western blot analysis results verified the changes of EMT marker gene expression. Furthermore, we found that TGF-β induced cell morphological changes significantly with an increase of cell surface area and cell length. These results indicated that TGF-β induced EMT. The mRNA expression levels of SPCA1, SPCA2 and MCU were not influenced by TGF-β treatment, while NCX1 expression was decreased in T47D cells. In addition, the mRNA levels of SERCAs and IP3Rs were significantly changed due to TGF-β-induced EMT. The TGF-β-treated T47D cells exhibited markedly greater response to ATP than the control cells, and the descent velocity of cytosolic calcium concentration was faster in TGF-β-treated cells than in control cells. This is the first report to demonstrate that TGF-β-induced EMT in human breast cancer cells is associated with alterations in endoplasmic reticulum calcium homeostasis.

      PubDate: 2015-08-06T10:32:52Z
  • Chronic Cadmium Exposure in Rats Produces Pancreatic Impairment and
           Insulin Resistance in Multiple Peripheral Tissues
    • Abstract: Publication date: Available online 5 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Samuel Treviño, Michael P. Waalkes, José Angel Flores Hernández, Bertha Alicia León-Chavez, Patricia Aguilar-Alonso, Eduardo Brambila
      Previous studies have linked cadmium exposure to disturbances in carbohydrate and lipid metabolism. In this study we investigate the effects in Wistar rats of an oral cadmium exposure in drinking water on carbohydrates, lipids and insulin release. Also, using mathematical models we studied the effect of cadmium on insulin resistance and sensitivity in liver, muscle, adipose and cardiovascular tissue. Cadmium exposure induced hyperglycemia, increased insulin release after a glucose load, and caused increases in serum triglycerides, cholesterol, LDL-C and VLDL-C, and a decrease of HDL-C. In addition, there was an accumulation of cadmium in pancreas and an increase of insulin. After exposure, HOMA-IR was increased, while the HOMA-S%, QUICKI and Matsuda-DeFronzo indexes showed decreases. A decrease of insulin sensitivity was shown in muscle and liver. Additionally, cadmium increases insulin resistance in the liver, adipose tissue and cardiovascular system. Finally, β-cell functioning was evaluated by HOMA-B% index and insulin disposition index, which were decreased, while insulin generation index increased. In conclusion, cadmium increases insulin release, induces hyperglycemia and alters lipid metabolism. These changes likely occur as a consequence of reduced sensitivity and increased insulin resistance in multiple insulin-dependent and non-dependent tissues, producing a biochemical phenotype similar to metabolic syndrome and diabetes.

      PubDate: 2015-08-06T10:32:52Z
  • Identification of rice Os4BGlu13 as a β-glucosidase which hydrolyzes
           gibberellin A4 1-O-β-d-glucosyl ester, in addition to tuberonic acid
           glucoside and salicylic acid derivative glucosides
    • Abstract: Publication date: Available online 1 August 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Yanling Hua, Watsamon Ekkhara, Sompong Sansenya, Chantragan Srisomsap, Sittiruk Roytrakul, Wataru Saburi, Ryosuke Takeda, Hideyuki Matsuura, Haruhide Mori, James R. Ketudat Cairns
      Gibberellin 1-O-β-d-glucose ester hydrolysis activity has been detected in rice seedling extracts, but no enzyme responsible for this activity has ever been purified and identified. Therefore, gibberellin A4 glucosyl ester (GA4-GE) β-d-glucosidase activity was purified from ten-day rice seedling stems and leaves. The family 1 glycoside hydrolase Os4BGlu13 was identified in the final purification fraction. The Os4BGlu13 cDNA was amplified from rice seedlings and expressed as an N-terminal thioredoxin-tagged fusion protein in E. coli. The purified recombinant Os4BGlu13 protein (rOs4BGlu13) had an optimum pH of 4.5, for hydrolysis of p-nitrophenyl β-d-glucopyranoside (pNPGlc), which was the best substrate identified, with a k cat/K m of 637 mM-1s-1. rOs4BGlu13 hydrolyzed helicin best among natural glycosides tested (k cat/K m of 74.4 mM-1s-1). Os4BGlu13 was previously designated tuberonic acid glucoside (TAG) β-glucosidase (TAGG), and here the k cat/K m of rOsBGlu13 for TAG was 6.68 mM-1s-1, while that for GA4-GE was 3.63 mM-1s-1 and for salicylic acid glucoside (SAG) is 0.88 mM-1s-1. rOs4BGlu13 also hydrolyzed oligosaccharides, with preference for short β-(1→3)-linked over β-(1→4)-linked glucooligosaccharides. The enzymatic data suggests that Os4BGlu13 may contribute to TAG, SAG, oligosaccharide and GA4-GE hydrolysis in the rice plant, although helicin or a similar compound may be its primary target.
      Graphical abstract image

      PubDate: 2015-08-02T03:21:11Z
  • Physicochemical constraints of elevated pH affect efficient membrane
           interaction and arrest an abortive membrane-bound oligomeric intermediate
           of the beta-barrel pore-forming toxin Vibrio cholerae cytolysin
    • Abstract: Publication date: Available online 31 July 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Anand Kumar Rai, Nidhi Kundu, Kausik Chattopadhyay
      Vibrio cholerae cytolysin (VCC) is a potent membrane-damaging cytotoxic protein. VCC causes permeabilization of the target cell membranes by forming transmembrane oligomeric beta-barrel pores. Membrane pore formation by VCC involves following key steps: (i) membrane binding, (ii) formation of a pre-pore oligomeric intermediate, (iii) membrane insertion of the pore-forming motifs, and (iv) formation of the functional transmembrane pore. Membrane binding, oligomerization, and subsequent pore-formation process of VCC appear to be facilitated by multiple regulatory mechanisms that are only partly understood. Here, we have explored the role(s) of the physicochemical constraints, specifically imposed by the elevated pH conditions, on the membrane pore-formation mechanism of VCC. Elevated pH abrogates efficient interaction of VCC with the target membranes, and blocks its pore-forming activity. Under the elevated pH conditions, membrane-bound fractions of VCC remain trapped in the form of abortive oligomeric species that fail to generate the functional transmembrane pores. Such an abortive oligomeric assembly appears to represent a distinct, more advanced intermediate state than the pre-pore state. The present study offers critical insights regarding the implications of the physicochemical constraints for regulating the efficient membrane interaction and pore formation by VCC.

      PubDate: 2015-08-02T03:21:11Z
  • Nanometric features of myosin filaments extracted from a single muscle
           fiber to uncover the mechanisms underlying organized motility
    • Abstract: Publication date: Available online 23 June 2015
      Source:Archives of Biochemistry and Biophysics
      Author(s): Meishan Li , Takahiro Deguchi , Tuomas Näreoja , Bhanu P. Jena , Pekka Hänninen , Lars Larsson
      The single muscle fiber in vitro motility assay (SF-IVMA) is characterized by organized linear motility of actin filaments, i.e., actin filaments motility showing a parallel or anti-parallel direction with similar speed independent of direction in the central part of the flow-cell where density of myosin is high. In contrast, the low myosin density region in the flow-cell exhibits random filament movements, but the mechanisms underlying the organized motility remain unknown. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) imaging techniques have been combined to investigate the morphological features of myosin extracted from single muscle fiber segments in the flow cell. Nanometric scale imaging of myosin filaments in the SF-IVMA showed intact spatial distances between myosin heads being essential for myosin filament function. However, angular spectrum analyses of myosin filaments in the high myosin density region showed organized myosin filament orientation only in small areas, while unorganized filament orientation were dominantly presented when larger areas were analyzed. Thus, parallel myosin filament organization is a less likely mechanism underlying the organized motility of actin filaments and the high myosin density per se is therefore forwarded as the primary “driver” that promotes organized linear motility.

      PubDate: 2015-07-08T13:01:50Z
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