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BIOCHEMISTRY (222 journals)                  1 2     

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AAPS PharmSciTech     Hybrid Journal   (Followers: 4)
Acetic Acid Bacteria     Open Access   (Followers: 1)
ACS Central Science     Hybrid Journal   (Followers: 3)
ACS Chemical Biology     Full-text available via subscription   (Followers: 149)
ACS Chemical Neuroscience     Full-text available via subscription   (Followers: 15)
Acta Crystallographica Section D : Biological Crystallography     Hybrid Journal   (Followers: 10)
Acta Crystallographica Section F: Structural Biology Communications     Hybrid Journal   (Followers: 7)
Advances and Applications in Bioinformatics and Chemistry     Open Access   (Followers: 8)
Advances in Biological Chemistry     Open Access   (Followers: 6)
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: 13)
African Journal of Biochemistry Research     Open Access   (Followers: 1)
African Journal of Chemical Education     Open Access   (Followers: 2)
Alkaloids: Chemical and Biological Perspectives     Full-text available via subscription   (Followers: 3)
American Journal of Biochemistry     Open Access   (Followers: 6)
American Journal of Biochemistry and Biotechnology     Open Access   (Followers: 69)
American Journal of Biochemistry and Molecular Biology     Open Access   (Followers: 12)
American Journal of Polymer Science     Open Access   (Followers: 20)
Amino Acids     Hybrid Journal   (Followers: 8)
Analytical Biochemistry     Hybrid Journal   (Followers: 144)
Annals of Clinical Biochemistry     Hybrid Journal   (Followers: 8)
Annual Review of Biochemistry     Full-text available via subscription   (Followers: 49)
Annual Review of Chemical and Biomolecular Engineering     Full-text available via subscription   (Followers: 8)
Applied Biochemistry and Biotechnology     Hybrid Journal   (Followers: 43)
Applied Biochemistry and Microbiology     Hybrid Journal   (Followers: 15)
Applied Organometallic Chemistry     Hybrid Journal   (Followers: 5)
Archives of Biochemistry and Biophysics     Hybrid Journal   (Followers: 21)
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: 4)
Biocatalysis     Open Access  
Biochemical and Biophysical Research Communications     Hybrid Journal   (Followers: 19)
Biochemical and Molecular Medicine     Full-text available via subscription   (Followers: 4)
Biochemical Compounds     Open Access  
Biochemical Engineering Journal     Hybrid Journal   (Followers: 12)
Biochemical Genetics     Hybrid Journal   (Followers: 3)
Biochemical Journal     Full-text available via subscription   (Followers: 26)
Biochemical Pharmacology     Hybrid Journal   (Followers: 8)
Biochemical Society Transactions     Full-text available via subscription   (Followers: 3)
Biochemical Systematics and Ecology     Hybrid Journal   (Followers: 3)
Biochemistry     Full-text available via subscription   (Followers: 207)
Biochemistry & Pharmacology : Open Access     Open Access   (Followers: 2)
Biochemistry & Physiology : Open Access     Open Access  
Biochemistry (Moscow)     Hybrid Journal   (Followers: 4)
Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology     Hybrid Journal   (Followers: 3)
Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry     Hybrid Journal   (Followers: 3)
Biochemistry and Biophysics Reports     Open Access  
Biochemistry and Cell Biology     Full-text available via subscription   (Followers: 15)
Biochemistry and Molecular Biology Education     Hybrid Journal   (Followers: 5)
Biochemistry and Molecular Biology of Fishes     Full-text available via subscription   (Followers: 1)
Biochemistry Research International     Open Access   (Followers: 4)
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids     Hybrid Journal   (Followers: 8)
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease     Hybrid Journal   (Followers: 16)
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research     Hybrid Journal   (Followers: 7)
Biochimie     Hybrid Journal   (Followers: 6)
Biochimie Open     Open Access  
Bioconjugate Chemistry     Full-text available via subscription   (Followers: 30)
BioDrugs     Full-text available via subscription   (Followers: 8)
Bioelectrochemistry     Hybrid Journal   (Followers: 1)
Biofuels     Hybrid Journal   (Followers: 10)
Biogeochemistry     Hybrid Journal   (Followers: 10)
BioInorganic Reaction Mechanisms     Hybrid Journal   (Followers: 1)
Biokemistri     Open Access  
Biological Chemistry     Partially Free   (Followers: 27)
Biomaterials Research     Open Access   (Followers: 2)
Biomedicines     Open Access   (Followers: 1)
BioMolecular Concepts     Hybrid Journal   (Followers: 2)
Bioscience, Biotechnology, and Biochemistry     Hybrid Journal   (Followers: 25)
Biosimilars     Open Access   (Followers: 1)
Biotechnology and Applied Biochemistry     Hybrid Journal   (Followers: 45)
Bit├ícora Digital     Open Access  
BMC Biochemistry     Open Access   (Followers: 13)
Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca : Food Science and Technology     Open Access  
Carbohydrate Polymers     Hybrid Journal   (Followers: 7)
Cell Biochemistry and Biophysics     Hybrid Journal   (Followers: 5)
Cell Biochemistry and Function     Hybrid Journal   (Followers: 4)
ChemBioChem     Hybrid Journal   (Followers: 6)
Chemical and Biological Technologies for Agriculture     Open Access  
Chemical Biology & Drug Design     Hybrid Journal   (Followers: 22)
Chemical Engineering Journal     Hybrid Journal   (Followers: 21)
Chemical Senses     Hybrid Journal   (Followers: 1)
Chemical Speciation and Bioavailability     Open Access   (Followers: 1)
Chemico-Biological Interactions     Hybrid Journal   (Followers: 3)
Chemistry & Biodiversity     Hybrid Journal   (Followers: 5)
Chemistry & Biology     Full-text available via subscription   (Followers: 30)
Chemistry and Ecology     Hybrid Journal  
ChemTexts     Hybrid Journal  
Clinica Chimica Acta     Hybrid Journal   (Followers: 33)
Clinical Biochemist Reviews     Full-text available via subscription   (Followers: 1)
Clinical Biochemistry     Hybrid Journal   (Followers: 20)
Clinical Chemistry and Laboratory Medicine     Hybrid Journal   (Followers: 61)
Clinical Lipidology     Full-text available via subscription  
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology     Hybrid Journal   (Followers: 4)
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: 7)
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics     Hybrid Journal   (Followers: 2)
Comprehensive Biochemistry     Full-text available via subscription   (Followers: 1)
Computational Biology and Chemistry     Hybrid Journal   (Followers: 10)
Critical Reviews in Biochemistry and Molecular Biology     Hybrid Journal   (Followers: 4)
Current Chemical Biology     Hybrid Journal   (Followers: 2)
Current Opinion in Chemical Biology     Hybrid Journal   (Followers: 21)
Current Opinion in Lipidology     Hybrid Journal   (Followers: 5)
DNA Barcodes     Open Access  
Doklady Biochemistry and Biophysics     Hybrid Journal   (Followers: 1)
Doklady Chemistry     Hybrid Journal  
Egyptian Journal of Biochemistry and Molecular Biology     Full-text available via subscription  
FABICIB     Open Access  
FEBS Letters     Hybrid Journal   (Followers: 58)
FEBS Open Bio     Open Access   (Followers: 3)
Fish Physiology and Biochemistry     Hybrid Journal   (Followers: 4)
Food & Function     Full-text available via subscription   (Followers: 5)
Foundations of Modern Biochemistry     Full-text available via subscription  
Free Radicals and Antioxidants     Full-text available via subscription   (Followers: 3)
Frontiers in Molecular Biosciences     Open Access   (Followers: 2)
Frontiers in Natural Product Chemistry     Hybrid Journal  
Global Biogeochemical Cycles     Full-text available via subscription   (Followers: 8)
Green Chemistry     Full-text available via subscription   (Followers: 9)
Histochemistry and Cell Biology     Hybrid Journal   (Followers: 4)
Indian Journal of Biochemistry and Biophysics (IJBB)     Open Access   (Followers: 3)
Indian Journal of Clinical Biochemistry     Hybrid Journal   (Followers: 1)
Indonesian Biomedical Journal     Open Access  
Insect Biochemistry and Molecular Biology     Hybrid Journal   (Followers: 3)
International Journal of Biochemistry & Cell Biology     Hybrid Journal   (Followers: 6)
International Journal of Biochemistry and Biophysics     Open Access  
International Journal of Biological Chemistry     Open Access   (Followers: 4)
International Journal of Biomedical Nanoscience and Nanotechnology     Hybrid Journal   (Followers: 5)
International Journal of Food Contamination     Open Access  
International Journal of Plant Physiology and Biochemistry     Open Access  
International Journal of Plant Research     Open Access   (Followers: 3)
International Journal of Secondary Metabolite     Open Access   (Followers: 1)
Invertebrate Immunity     Open Access   (Followers: 1)
JBIC Journal of Biological Inorganic Chemistry     Hybrid Journal   (Followers: 4)
Journal of Microbial & Biochemical Technology     Open Access   (Followers: 1)
Journal of Applied Biology & Biotechnology     Open Access   (Followers: 1)
Journal of Bioactive and Compatible Polymers     Hybrid Journal   (Followers: 2)
Journal of Biochemistry     Hybrid Journal   (Followers: 41)
Journal of Biological Chemistry     Full-text available via subscription   (Followers: 170)
Journal of Biomaterials Science, Polymer Edition     Hybrid Journal   (Followers: 9)
Journal of Carbohydrate Chemistry     Hybrid Journal   (Followers: 7)
Journal of Cellular Biochemistry     Hybrid Journal   (Followers: 5)
Journal of Chemical Biology     Hybrid Journal   (Followers: 1)
Journal of Chemical Neuroanatomy     Hybrid Journal  
Journal of Clinical Lipidology     Hybrid Journal   (Followers: 1)
Journal of Comparative Physiology B : Biochemical, Systemic, and Environmental Physiology     Hybrid Journal   (Followers: 3)
Journal of Drug Discovery and Therapeutics     Open Access   (Followers: 1)
Journal of Enzyme Inhibition and Medicinal Chemistry     Hybrid Journal   (Followers: 4)
Journal of Evolutionary Biochemistry and Physiology     Hybrid Journal  
Journal of Food and Drug Analysis     Open Access  
Journal of Forensic Toxicology and Pharmacology     Hybrid Journal   (Followers: 3)
Journal of Inborn Errors of Metabolism and Screening     Open Access  
Journal of Inorganic Biochemistry     Hybrid Journal   (Followers: 3)
Journal of Investigational Biochemistry     Open Access   (Followers: 2)
Journal of Medical and Biomedical Sciences     Open Access  
Journal of Medical Biochemistry     Open Access   (Followers: 5)
Journal of Medicine and Biomedical Research     Open Access   (Followers: 1)
Journal of Molecular Biochemistry     Open Access   (Followers: 2)
Journal of Neurochemistry     Hybrid Journal   (Followers: 2)
Journal of Nutritional Biochemistry     Hybrid Journal   (Followers: 6)
Journal of Pediatric Biochemistry     Hybrid Journal   (Followers: 1)
Journal of Peptide Science     Hybrid Journal   (Followers: 24)
Journal of Photochemistry and Photobiology B: Biology     Hybrid Journal   (Followers: 3)
Journal of Physiobiochemical Metabolism     Hybrid Journal   (Followers: 1)
Journal of Physiology and Biochemistry     Hybrid Journal   (Followers: 3)
Journal of Plant Biochemistry and Biotechnology     Hybrid Journal   (Followers: 5)
Journal of Steroid Biochemistry and Molecular Biology     Hybrid Journal   (Followers: 2)
Journal of Virology & Antiviral Research     Hybrid Journal   (Followers: 3)
Journal of Wood Chemistry and Technology     Hybrid Journal   (Followers: 6)
La Rivista Italiana della Medicina di Laboratorio - Italian Journal of Laboratory Medicine     Hybrid Journal  
Marine Chemistry     Hybrid Journal   (Followers: 6)
Methods in Enzymology     Full-text available via subscription   (Followers: 12)
Molecular and Biochemical Parasitology     Hybrid Journal   (Followers: 2)
Molecular and Cellular Biochemistry     Hybrid Journal   (Followers: 3)
Molecular Informatics     Hybrid Journal   (Followers: 4)
Molecular inhibitors in targeted therapy     Open Access  
Moscow University Chemistry Bulletin     Hybrid Journal   (Followers: 1)
Mycology : An International Journal on Fungal Biology     Hybrid Journal   (Followers: 4)
Natural Products and Bioprospecting     Open Access   (Followers: 3)
Nature Chemical Biology     Full-text available via subscription   (Followers: 65)
Nature Communications     Open Access   (Followers: 100)
Novelty in Biomedicine     Open Access  
Ocean Acidification     Open Access   (Followers: 1)
Organic & Biomolecular Chemistry     Full-text available via subscription   (Followers: 80)
Parasitology Open     Open Access  
Peptidomics     Open Access  
Pesticide Biochemistry and Physiology     Hybrid Journal   (Followers: 4)
Pharmaceutical Bioprocessing     Full-text available via subscription   (Followers: 1)
Pharmacognosy Magazine     Open Access   (Followers: 2)
Pharmacognosy Research     Open Access   (Followers: 3)
Pharmacognosy Reviews     Open Access   (Followers: 1)
Phytochemistry Reviews     Hybrid Journal  
Plant Physiology and Biochemistry     Hybrid Journal   (Followers: 7)
Plasma Chemistry and Plasma Processing     Hybrid Journal   (Followers: 2)
Polymer Journal     Hybrid Journal   (Followers: 1)
Preparative Biochemistry and Biotechnology     Hybrid Journal   (Followers: 4)

        1 2     

Journal Cover Archives of Biochemistry and Biophysics
  [SJR: 1.602]   [H-I: 124]   [21 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0003-9861 - ISSN (Online) 1096-0384
   Published by Elsevier Homepage  [2970 journals]
  • Ascorbic acid prevents acetaminophen-induced hepatotoxicity in mice by
           ameliorating glutathione recovery and autophagy
    • Abstract: Publication date: 15 August 2016
      Source:Archives of Biochemistry and Biophysics, Volume 604
      Author(s): Toshihiro Kurahashi, Jaeyong Lee, Atsunori Nabeshima, Takujiro Homma, Eun Sil Kang, Yuka Saito, Sohsuke Yamada, Toshiyuki Nakayama, Ken-ichi Yamada, Satoshi Miyata, Junichi Fujii
      Aldehyde reductase (AKR1A) plays a role in the biosynthesis of ascorbic acid (AsA), and AKR1A-deficient mice produce about 10–15% of the AsA that is produced by wild-type mice. We found that acetaminophen (AAP) hepatotoxicity was aggravated in AKR1A-deficient mice. The pre-administration of AsA in the drinking water markedly ameliorated the AAP hepatotoxicity in the AKR1A-deficient mice. Treatment of the mice with AAP decreased both glutathione and AsA levels in the liver in the early phase after AAP administration, and an AsA deficiency delayed the recovery of the glutathione content in the healing phase. While in cysteine supply systems; a neutral amino acid transporter ASCT1, a cystine transporter xCT, enzymes for the transsulfuration pathway, and autophagy markers, were all elevated in the liver as the result of the AAP treatment, the AsA deficiency suppressed their induction. Thus, AsA appeared to exert a protective effect against AAP hepatotoxicity by ameliorating the supply of cysteine that is available for glutathione synthesis as a whole. Because some drugs produce reactive oxygen species, resulting in the consumption of glutathione during the metabolic process, the intake of sufficient amounts of AsA would be beneficial for protecting against the hepatic damage caused by such drugs.

      PubDate: 2016-06-18T18:12:55Z
  • (−)-Rhazinilam and the diphenylpyridazinone NSC 613241: Two
           compounds inducing the formation of morphologically similar tubulin
           spirals but binding apparently to two distinct sites on tubulin
    • Abstract: Publication date: 15 August 2016
      Source:Archives of Biochemistry and Biophysics, Volume 604
      Author(s): Ruoli Bai, Ernest Hamel
      The most potent microtubule assembly inhibitor of newer diphenylpyridazinone derivatives examined was NSC 613241. Because NSC 613241 and (−)-rhazinilam also induce the formation of similar 2-filament spirals, these aberrant reactions were compared. Spiral formation with both compounds was enhanced by GTP and inhibited by GDP and by 15 other inhibitors of microtubule assembly. Similarly, microtubule assembly induced by paclitaxel or laulimalide is enhanced by GTP and inhibited by GDP and assembly inhibitors, but neither [3H]NSC 613241 nor [3H](−)-rhazinilam bound to microtubules or inhibited the binding of [3H]paclitaxel or [3H]peloruside A to microtubules. Differences in the pitch of aberrant polymers were found: NSC 613241-induced and (−)-rhazinilam-induced spirals had average repeats of 85 and 79–80 nm, respectively. We found no binding of [3H]NSC 613241 or [3H](−)-rhazinilam to αβ-tubulin dimer, but both compounds were incorporated into the polymers they induced in substoichiometric reactions, with as little as 0.1–0.2 mol compound/mol of tubulin, and no cross-inhibition by NSC 613241 or (−)-rhazinilam into spirals occurred. Under reaction conditions where neither compound induced spiral formation, both compounds together synergistically induced substantial spiral formation. We conclude that (−)-rhazinilam and NSC 613241 bind to different sites on tubulin that differ from binding sites for other antitubulin agents.

      PubDate: 2016-06-18T18:12:55Z
  • In-vitro, SDH5-dependent flavinylation of immobilized human respiratory
           complex II flavoprotein
    • Abstract: Publication date: 15 August 2016
      Source:Archives of Biochemistry and Biophysics, Volume 604
      Author(s): Lala Zafreen, Nancy Walker-Kopp, Li-Shar Huang, Edward Berry
      Mitochondrial Complex II (Succinate: ubiquinone oxidoreductase) has a covalently bound FAD cofactor in its largest subunit (SDHA), which accepts electrons from oxidation of succinate during catalysis. The mechanism of flavin attachment, and factors involved, have not been fully elucidated. The recent report of an assembly factor SDH5 (SDHAF2, SDHE) required for flavinylation (Hao et al., 2009 Science 325, 1139–1142) raises the prospect of achieving flavinylation in a completely defined system, which would facilitate elucidation of the precise role played by SDH5 and other factors. At this time that goal has not been achieved, and the actual function of SDH5 is still unknown. We have developed a procedure for in-vitro flavinylation of recombinant human apo-SDHA, immobilized on Ni-IMAC resin by a His tag, in a chemically defined medium. In this system flavinylation has a pH optimum of 6.5 and is completely dependent on added SDH5. The results suggest that FAD interacts noncovalently with SDHA in the absence of SDH5. This system will be useful in understanding the process of flavinylation of SDHA and the role of SDH5 in this process.

      PubDate: 2016-06-18T18:12:55Z
  • Toluidine blue O is a potent inhibitor of human cholinesterases
    • Abstract: Publication date: 15 August 2016
      Source:Archives of Biochemistry and Biophysics, Volume 604
      Author(s): Kevser Biberoglu, Melike Yuksel Tek, Seyhan Turk Ghasemi, Ozden Tacal
      In this study, the inhibitory effects of three phenothiazines [toluidine blue O (TBO), thionine (TH) and methylene violet (MV)] were tested on human plasma butyrylcholinesterase (BChE) and their inhibitory mechanisms were studied in detail. MV acted as a linear mixed type inhibitor of human BChE with Ki = 0.66 ± 0.06 μM and α = 13.6 ± 3.5. TBO and TH caused nonlinear inhibition of human BChE, compatible to double occupancy. Ki values estimated by nonlinear regression analysis for TBO and TH were 0.008 ± 0.003 μM and 2.1 ± 0.42 μM, respectively. The inhibitory potential of TBO was also tested on human erythrocyte AChE. TBO acted as a linear mixed type inhibitor of human AChE with Ki = 0.041 ± 0.005 μM and α = 1.6 ± 0.007. Using four site-directed BChE mutants, the role of peripheral anionic site residues of human BChE was also investigated in the binding of TBO to BChE. The peripheral anionic site mutants of BChE caused 16–69-fold increase in Ki value of TBO, compared to recombinant wild-type, suggesting that peripheral anionic site residues are involved in the binding of TBO to human BChE. In conclusion, TBO which is a potent inhibitor of human cholinesterases, may be a potential drug candidate for the treatment of Alzheimer’s disease.

      PubDate: 2016-06-18T18:12:55Z
  • Crystallographic studies on protein misfolding: Domain swapping and
           amyloid formation in the SH3 domain
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): Ana Cámara-Artigas
      Oligomerization by 3D domain swapping is found in a variety of proteins of diverse size, fold and function. In the early 1960s this phenomenon was postulated for the oligomers of ribonuclease A, but it was not until the 1990s that X-ray diffraction provided the first experimental evidence of this special manner of oligomerization. Nowadays, structural information has allowed the identification of these swapped oligomers in over one hundred proteins. Although the functional relevance of this phenomenon is not clear, this alternative folding of protomers into intertwined oligomers has been related to amyloid formation. Studies on proteins that develop 3D domain swapping might provide some clues on the early stages of amyloid formation. The SH3 domain is a small modular domain that has been used as a model to study the basis of protein folding. Among SH3 domains, the c-Src-SH3 domain emerges as a helpful model to study 3D domain swapping and amyloid formation.
      Graphical abstract image

      PubDate: 2016-06-13T12:23:21Z
  • Transfer RNA: From pioneering crystallographic studies to contemporary
           tRNA biology
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): Pablo Fernández-Millán, Cédric Schelcher, Joseph Chihade, Benoît Masquida, Philippe Giegé, Claude Sauter
      Transfer RNAs (tRNAs) play a key role in protein synthesis as adaptor molecules between messenger RNA and protein sequences on the ribosome. Their discovery in the early sixties provoked a worldwide infatuation with the study of their architecture and their function in the decoding of genetic information. tRNAs are also emblematic molecules in crystallography: the determination of the first tRNA crystal structures represented a milestone in structural biology and tRNAs were for a long period the sole source of information on RNA folding, architecture, and post-transcriptional modifications. Crystallographic data on tRNAs in complex with aminoacyl-tRNA synthetases (aaRSs) also provided the first insight into protein:RNA interactions. Beyond the translation process and the history of structural investigations on tRNA, this review also illustrates the renewal of tRNA biology with the discovery of a growing number of tRNA partners in the cell, the involvement of tRNAs in a variety of regulatory and metabolic pathways, and emerging applications in biotechnology and synthetic biology.
      Graphical abstract image

      PubDate: 2016-06-13T12:23:21Z
  • Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): Giovanna Di Nardo, Valentina Dell'Angelo, Gianluca Catucci, Sheila J. Sadeghi, Gianfranco Gilardi
      This paper reports the structure of the double mutant Asp251Gly/Gln307His (named A2) generated by random mutagenesis, able to produce 4′-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide from diclofenac, ibuprofen and tolbutamide, respectively. The 3D structure of the substrate-free mutant shows a conformation similar to the closed one found in the substrate-bound wild type enzyme, but with a higher degree of disorder in the region of the G-helix and F-G loop. This is due to the mutation Asp251Gly that breaks the salt bridge between Aps251 on I-helix and Lys224 on G-helix, allowing the G-helix to move away from I-helix and conferring a higher degree of flexibility to this element. This subtle structural change is accompanied by long-range structural rearrangements of the active site with the rotation of Phe87 and a reorganization of catalytically important water molecules. The impact of these structural features on thermal stability, reduction potential and electron transfer is investigated. The data demonstrate that a single mutation far from the active site triggers an increase in protein flexibility in a key region, shifting the conformational equilibrium toward the closed form that is ready to accept electrons and enter the P450 catalytic cycle as soon as a substrate is accepted.
      Graphical abstract image

      PubDate: 2016-06-13T12:23:21Z
  • Analysis of biostructural changes, dynamics, and interactions –
           Small-angle X-ray scattering to the rescue
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): Bente Vestergaard
      Solution small angle X-ray scattering from biological macromolecules (BioSAXS) plays an increasingly important role in biostructural research. The analysis of complex protein mixtures, dynamic equilibriums, intrinsic disorder and evolving structural processes is facilitated by SAXS data, either in stand-alone applications, or with SAXS taking a prominent role in hybrid biostructural analysis. This is not the least due to the significant advances in both hardware and software that have taken place in particular at the large-scale facilities. Here, recent developments and the future potential of BioSAXS are reviewed, exemplified by numerous examples of elegant applications to challenging systems.

      PubDate: 2016-06-13T12:23:21Z
  • SAD phasing: History, current impact and future opportunities
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): John P. Rose, Bi-Cheng Wang
      Single wavelength anomalous diffraction (SAD) can trace its beginnings to the early 1950s. Researchers at the time recognized that SAD offers some unique features that might be advantageous for crystallographic phasing, despite the fact that at that time recording accurate SAD data was problematic. In this review we will follow the trail from those early days, highlighting key advances in the field and interpreting them in terms on how they stimulated continued phasing development that produced the theoretical foundation for the routine macromolecular structure determination by SAD today. The technological advances over the past three decades in both hardware and software, which played a significant role in making SAD phasing a ‘first choice method’, will also be described.
      Graphical abstract image

      PubDate: 2016-06-13T12:23:21Z
  • Assessment of microcrystal quality by transmission electron microscopy for
           efficient serial femtosecond crystallography
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): Christopher O. Barnes, Elena G. Kovaleva, Xiaofeng Fu, Hilary P. Stevenson, Aaron S. Brewster, Daniel P. DePonte, Elizabeth L. Baxter, Aina E. Cohen, Guillermo Calero
      Serial femtosecond crystallography (SFX) employing high-intensity X-ray free-electron laser (XFEL) sources has enabled structural studies on microcrystalline protein samples at non-cryogenic temperatures. However, the identification and optimization of conditions that produce well diffracting microcrystals remains an experimental challenge. Here, we report parallel SFX and transmission electron microscopy (TEM) experiments using fragmented microcrystals of wild type (WT) homoprotocatechuate 2,3-dioxygenase (HPCD) and an active site variant (H200Q). Despite identical crystallization conditions and morphology, as well as similar crystal size and density, the indexing efficiency of the diffraction data collected using the H200Q variant sample was over 7-fold higher compared to the diffraction results obtained using the WT sample. TEM analysis revealed an abundance of protein aggregates, crystal conglomerates and a smaller population of highly ordered lattices in the WT sample as compared to the H200Q variant sample. While not reported herein, the 1.75 Å resolution structure of the H200Q variant was determined from ∼16 min of beam time, demonstrating the utility of TEM analysis in evaluating sample monodispersity and lattice quality, parameters critical to the efficiency of SFX experiments.

      PubDate: 2016-06-13T12:23:21Z
  • Neutron protein crystallography: A complementary tool for locating
           hydrogens in proteins
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): William B. O'Dell, Annette M. Bodenheimer, Flora Meilleur
      Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates hydrogen atom positions in a protein structure. The visibility of hydrogen and deuterium atoms arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction at resolutions typical of protein crystals. Neutrons have the additional benefit to structural biology of not inducing radiation damage in protein crystals. The same crystal could be measured multiple times for parametric studies. Here, we review the basic principles of neutron protein crystallography. The information that can be gained from a neutron structure is presented in balance with practical considerations. Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature. Available instruments for data collection and software for data processing and structure refinement are described along with technique-specific strategies including joint X-ray/neutron structure refinement. Examples are given to illustrate, ultimately, the unique scientific value of neutron protein crystal structures.

      PubDate: 2016-06-13T12:23:21Z
  • Computational crystallization
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): Irem Altan, Patrick Charbonneau, Edward H. Snell
      Crystallization is a key step in macromolecular structure determination by crystallography. While a robust theoretical treatment of the process is available, due to the complexity of the system, the experimental process is still largely one of trial and error. In this article, efforts in the field are discussed together with a theoretical underpinning using a solubility phase diagram. Prior knowledge has been used to develop tools that computationally predict the crystallization outcome and define mutational approaches that enhance the likelihood of crystallization. For the most part these tools are based on binary outcomes (crystal or no crystal), and the full information contained in an assembly of crystallization screening experiments is lost. The potential of this additional information is illustrated by examples where new biological knowledge can be obtained and where a target can be sub-categorized to predict which class of reagents provides the crystallization driving force. Computational analysis of crystallization requires complete and correctly formatted data. While massive crystallization screening efforts are under way, the data available from many of these studies are sparse. The potential for this data and the steps needed to realize this potential are discussed.
      Graphical abstract image

      PubDate: 2016-06-13T12:23:21Z
  • Current advances in synchrotron radiation instrumentation for MX
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): Robin L. Owen, Jordi Juanhuix, Martin Fuchs
      Following pioneering work 40 years ago, synchrotron beamlines dedicated to macromolecular crystallography (MX) have improved in almost every aspect as instrumentation has evolved. Beam sizes and crystal dimensions are now on the single micron scale while data can be collected from proteins with molecular weights over 10 MDa and from crystals with unit cell dimensions over 1000 Å. Furthermore it is possible to collect a complete data set in seconds, and obtain the resulting structure in minutes. The impact of MX synchrotron beamlines and their evolution is reflected in their scientific output, and MX is now the method of choice for a variety of aims from ligand binding to structure determination of membrane proteins, viruses and ribosomes, resulting in a much deeper understanding of the machinery of life. A main driving force of beamline evolution have been advances in almost every aspect of the instrumentation comprising a synchrotron beamline. In this review we aim to provide an overview of the current status of instrumentation at modern MX experiments. The most critical optical components are discussed, as are aspects of endstation design, sample delivery, visualisation and positioning, the sample environment, beam shaping, detectors and data acquisition and processing.

      PubDate: 2016-06-13T12:23:21Z
  • Serial femtosecond crystallography: A revolution in structural biology
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): Jose M. Martin-Garcia, Chelsie E. Conrad, Jesse Coe, Shatabdi Roy-Chowdhury, Petra Fromme
      Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein.

      PubDate: 2016-06-13T12:23:21Z
  • Macromolecular crystallography: An old science with new perspectives
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): Ana Cámara-Artigas, José Antonio Gavira

      PubDate: 2016-06-13T12:23:21Z
  • Current trends in protein crystallization
    • Abstract: Publication date: 15 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 602
      Author(s): José A. Gavira
      Proteins belong to the most complex colloidal system in terms of their physicochemical properties, size and conformational-flexibility. This complexity contributes to their great sensitivity to any external change and dictate the uncertainty of crystallization. The need of 3D models to understand their functionality and interaction mechanisms with other neighbouring (macro)molecules has driven the tremendous effort put into the field of crystallography that has also permeated other fields trying to shed some light into reluctant-to-crystallize proteins. This review is aimed at revising protein crystallization from a regular-laboratory point of view. It is also devoted to highlight the latest developments and achievements to produce, identify and deliver high-quality protein crystals for XFEL, Micro-ED or neutron diffraction. The low likelihood of protein crystallization is rationalized by considering the intrinsic polypeptide nature (folded state, surface charge, etc) followed by a description of the standard crystallization methods (batch, vapour diffusion and counter-diffusion), including high throughput advances. Other methodologies aimed at determining protein features in solution (NMR, SAS, DLS) or to gather structural information from single particles such as Cryo-EM are also discussed. Finally, current approaches showing the convergence of different structural biology techniques and the cross-methodologies adaptation to tackle the most difficult problems, are presented. Synopsis Current advances in biomacromolecules crystallization, from nano crystals for XFEL and Micro-ED to large crystals for neutron diffraction, are covered with special emphasis in methodologies applicable at laboratory scale.
      Graphical abstract image

      PubDate: 2016-06-13T12:23:21Z
  • Role of SERCA and the sarcoplasmic reticulum calcium content on calcium
           waves propagation in rat ventricular myocytes
    • Abstract: Publication date: 15 August 2016
      Source:Archives of Biochemistry and Biophysics, Volume 604
      Author(s): Ayleen Salazar-Cantú, Perla Pérez-Treviño, Dolores Montalvo-Parra, Jaime Balderas-Villalobos, Norma L. Gómez-Víquez, Noemí García, Julio Altamirano
      In Ca2+-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca2+ level to trigger and sustain Ca2+ waves, that propagate at constant rate (ʋwave). High luminal Ca2+ sensitizes RyR2, thereby increasing Ca2+ sparks frequency, and the larger RyR2-mediated SR Ca2+ flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca2+ reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca2+ removal could instead impair RyR2 activation. We assessed whether rapid SR Ca2+ uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca2+ waves amplitude was largely unaffected. Furthermore, SR Ca2+ also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca2+ and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca2+ determines ʋwave, while rapid SERCA Ca2+ uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca2+.
      Graphical abstract image

      PubDate: 2016-06-13T12:23:21Z
  • Designing proteins to combat disease: Cardiac troponin C as an example
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Jonathan P. Davis, Vikram Shettigar, Svetlana B. Tikunova, Sean C. Little, Bin Liu, Jalal K. Siddiqui, Paul M.L. Janssen, Mark T. Ziolo, Shane D. Walton
      Throughout history, muscle research has led to numerous scientific breakthroughs that have brought insight to a more general understanding of all biological processes. Potentially one of the most influential discoveries was the role of the second messenger calcium and its myriad of handling and sensing systems that mechanistically control muscle contraction. In this review we will briefly discuss the significance of calcium as a universal second messenger along with some of the most common calcium binding motifs in proteins, focusing on the EF-hand. We will also describe some of our approaches to rationally design calcium binding proteins to palliate, or potentially even cure cardiovascular disease. Considering not all failing hearts have the same etiology, genetic background and co-morbidities, personalized therapies will need to be developed. We predict designer proteins will open doors for unprecedented personalized, and potentially, even generalized medicines as gene therapy or protein delivery techniques come to fruition.

      PubDate: 2016-06-08T12:06:57Z
  • Cardiac troponin structure-function and the influence of hypertrophic
           cardiomyopathy associated mutations on modulation of contractility
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Yuanhua Cheng, Michael Regnier
      Cardiac troponin (cTn) acts as a pivotal regulator of muscle contraction and relaxation and is composed of three distinct subunits (cTnC: a highly conserved Ca2+ binding subunit, cTnI: an actomyosin ATPase inhibitory subunit, and cTnT: a tropomyosin binding subunit). In this mini-review, we briefly summarize the structure-function relationship of cTn and its subunits, its modulation by PKA-mediated phosphorylation of cTnI, and what is known about how these properties are altered by hypertrophic cardiomyopathy (HCM) associated mutations of cTnI. This includes recent work using computational modeling approaches to understand the atomic-based structural level basis of disease-associated mutations. We propose a viewpoint that it is alteration of cTnC-cTnI interaction (rather than the Ca2+ binding properties of cTn) per se that disrupt the ability of PKA-mediated phosphorylation at cTnI Ser-23/24 to alter contraction and relaxation in at least some HCM-associated mutations. The combination of state of the art biophysical approaches can provide new insight on the structure-function mechanisms of contractile dysfunction resulting cTnI mutations and exciting new avenues for the diagnosis, prevention, and even treatment of heart diseases.
      Graphical abstract image

      PubDate: 2016-06-08T12:06:57Z
  • Dihydrocapsaicin suppresses proinflammatory cytokines expression by
           enhancing nuclear factor IA in a NF-κB-dependent manner
    • Abstract: Publication date: Available online 3 June 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Jing-Jing Zhao, Yan-Wei Hu, Chuan Huang, Xin Ma, Chun-Min Kang, Yuan Zhang, Feng-Xia Guo, Jing-Bo Lu, Jian-cheng Xiu, Yu-Rong Qiu, Yan-Hua Sha, Ji-Juan Gao, Yan-Chao Wang, Pan Li, Bang-Ming Xu, Lei Zheng, Qian Wang
      Background Atherosclerosis is a chronic inflammatory disease and represents the leading cause of morbidity and mortality throughout the world. Accumulating evidences have showed that Dihydrocapsaicin (DHC) has been found to exert multiple pharmacological and physiological effects. Nevertheless, the effects and possible mechanism of DHC on proinflammatory response remain largely unexplained. Methods and results We found that DHC markedly upregulated NFIA and suppressed NF-κB expression in THP-1 macrophages. Up-regulation of proinflammatory cytokines induced by LPS including TNF-α, IL-1β and IL-6 were markedly suppressed by DHC treatment. We also observed that protein level of NFIA was significantly increased while NF-κB and proinflammatory cytokines were decreased by DHC treatment in apoE−/− mice. Lentivirus-mediated overexpression of NFIA suppressed NF-κB and proinflammatory cytokines expression both in THP-1 macrophages and plaque tissues of apoE−/−mice. Moreover, treatment with lentivirus-mediated overexpression of NFIA made the down-regulation of DHC on NF-κB and proinflammatory cytokines expression notably accentuated in THP-1 macrophages and apoE−/− mice. In addition, treatment with siRNA targeting NF-κB accentuated the suppression of proinflammatory cytokines by lentivirus-mediated overexpression of NFIA. Conclusion These observations demonstrated that DHC can significantly decrease proinflammatory cytokines through enhancing NFIA and inhibiting NF-κB expression and thus DHC may be a promising candidate as an anti-inflammatory drug for atherosclerosis as well as other disorders.

      PubDate: 2016-06-08T12:06:57Z
  • The A31P missense mutation in cardiac myosin binding protein C alters
           protein structure but does not cause haploinsufficiency
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Sabine J. van Dijk, Kristina Bezold Kooiker, Stacy Mazzalupo, Yuanzhang Yang, Alla S. Kostyukova, Debbie J. Mustacich, Elaine R. Hoye, Joshua A. Stern, Mark D. Kittleson, Samantha P. Harris
      Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function.

      PubDate: 2016-06-08T12:06:57Z
  • Mutations in troponin T associated with Hypertrophic Cardiomyopathy
           increase Ca2+-sensitivity and suppress the modulation of Ca2+-sensitivity
           by troponin I phosphorylation
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Andrew E. Messer, Christopher R. Bayliss, Mohammed El-Mezgueldi, Charles S. Redwood, Douglas G. Ward, Man-Ching Leung, Maria Papadaki, Cristobal dos Remedios, Steven B. Marston
      We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca2+-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca2+-sensitivity when compared with donor heart troponin and the Ca2+-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca2+-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca2+-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca2+-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca2+-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca2+-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.

      PubDate: 2016-06-08T12:06:57Z
  • Gene expression patterns in transgenic mouse models of hypertrophic
           cardiomyopathy caused by mutations in myosin regulatory light chain
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Wenrui Huang, Katarzyna Kazmierczak, Zhiqun Zhou, Vanessa Aguiar-Pulido, Giri Narasimhan, Danuta Szczesna-Cordary
      Using microarray and bioinformatics, we examined the gene expression profiles in transgenic mouse hearts expressing mutations in the myosin regulatory light chain shown to cause hypertrophic cardiomyopathy (HCM). We focused on two malignant RLC-mutations, Arginine 58→Glutamine (R58Q) and Aspartic Acid 166 → Valine (D166V), and one benign, Lysine 104 → Glutamic Acid (K104E)-mutation. Datasets of differentially expressed genes for each of three mutants were compared to those observed in wild-type (WT) hearts. The changes in the mutant vs. WT samples were shown as fold-change (FC), with stringency FC ≥ 2. Based on the gene profiles, we have identified the major signaling pathways that underlie the R58Q-, D166V- and K104E-HCM phenotypes. The correlations between different genotypes were also studied using network-based algorithms. Genes with strong correlations were clustered into one group and the central gene networks were identified for each HCM mutant. The overall gene expression patterns in all mutants were distinct from the WT profiles. Both malignant mutations shared certain classes of genes that were up or downregulated, but most similarities were noted between D166V and K104E mice, with R58Q hearts showing a distinct gene expression pattern. Our data suggest that all three HCM mice lead to cardiomyopathy in a mutation-specific manner and thus develop HCM through diverse mechanisms.

      PubDate: 2016-06-08T12:06:57Z
  • Enhanced troponin I binding explains the functional changes produced by
           the hypertrophic cardiomyopathy mutation A8V of cardiac troponin C
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Henry G. Zot, Javier E. Hasbun, Clara A. Michell, Maicon Landim-Vieira, Jose R. Pinto
      Higher affinity for TnI explains how troponin C (TnC) carrying a causative hypertrophic cardiomyopathy mutation, TnCA8V, sensitizes muscle cells to Ca2+. Muscle fibers reconstituted with TnCA8V require ∼2.3-fold less [Ca2+] to achieve 50% maximum-tension compared to fibers reconstituted with wild-type TnC (TnCWT). Binding measurements rule out a significant change in N-terminus Ca2+-affinity of isolated TnCA8V, and TnCA8V binds the switch-peptide of troponin-I (TnIsp) ∼1.6-fold more strongly than TnCWT; thus we model the TnC-TnIsp interaction as competing with the TnI-actin interaction. Tension data are well-fit by a model constrained to conditions in which the affinity of TnCA8V for TnIsp is 1.5–1.7-fold higher than that of TnCWT at all [Ca2+]. Mean ATPase rates of reconstituted cardiac myofibrils is greater for TnCA8V than TnCWT at all [Ca2+], with statistically significant differences in the means at higher [Ca2+]. To probe TnC-TnI interaction in low Ca2+, displacement of bis-ANS from TnI was monitored as a function of TnC. Whereas Ca2+-TnCWT displaces significantly more bis-ANS than Mg2+-TnCWT, Ca2+-TnCA8V displaces probe equivalently to Mg2+-TnCA8V and Ca2+-TnCWT, consistent with stronger Ca2+-independent TnCA8V-TnIsp. A Matlab program for computing theoretical activation is reported. Our work suggests that contractility is constantly above normal in hearts made hypertrophic by TnCA8V.

      PubDate: 2016-06-08T12:06:57Z
  • The effect of cardiomyopathy mutation (R97L) in mouse cardiac troponin T
           on the muscle length-mediated recruitment of crossbridges is modified
           divergently by α- and β-myosin heavy chain
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Sampath K. Gollapudi, Murali Chandra
      Hypertrophic cardiomyopathy mutations in cardiac troponin T (TnT) lead to sudden cardiac death. Augmented myofilament Ca2+ sensitivity is a common feature in TnT mutants, but such observations fail to provide a rational explanation for severe cardiac phenotypes. To better understand the mutation-induced effect on the cardiac phenotype, it is imperative to determine the effects on dynamic contractile features such as the muscle length (ML)-mediated activation against α- and β-myosin heavy chain (MHC) isoforms. α- and β-MHC are not only differentially expressed in rodent and human hearts, but they also modify ML-mediated activation differently. Mouse analog of human TnTR94L (TnTR97L) or wild-type TnT was reconstituted into de-membranated muscle fibers from normal (α-MHC) and transgenic (β-MHC) mouse hearts. TnTR97L augmented myofilament Ca2+ sensitivity by a similar amount in α- and β-MHC fibers. However, TnTR97L augmented the negative impact of strained crossbridges on other crossbridges (γ) by 22% in α-MHC fibers, but attenuated γ by 21% in β-MHC fibers. TnTR97L decreased the magnitude of ML-mediated recruitment of crossbridges (E R) by 37% in α-MHC fibers, but increased E R by 35% in β-MHC fibers. We provide a mechanistic basis for the TnTR97L-induced effects in α- and β-MHC fibers and discuss the relevance to human hearts.

      PubDate: 2016-06-08T12:06:57Z
  • The functional significance of the last 5 residues of the C-terminus of
           cardiac troponin I
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Jennifer E. Gilda, Qian Xu, Margaret E. Martinez, Susan T. Nguyen, P. Bryant Chase, Aldrin V. Gomes
      The C-terminal region of cardiac troponin I (cTnI) is known to be important in cardiac function, as removal of the last 17 C-terminal residues of human cTnI has been associated with myocardial stunning. To investigate the C-terminal region of cTnI, three C-terminal deletion mutations in human cTnI were generated: Δ1 (deletion of residue 210), Δ3 (deletion of residues 208–210), and Δ5 (deletion of residues 206–210). Mammalian two-hybrid studies showed that the interactions between cTnI mutants and cardiac troponin C (cTnC) or cardiac troponin T (cTnT) were impaired in Δ3 and Δ5 mutants when compared to wild-type cTnI. Troponin complexes containing 2-[4′-(iodoacetamido) anilino] naphthalene-6-sulfonic acid (IAANS) labeled cTnC showed that the troponin complex containing cTnI Δ5 had a small increase in Ca2+ affinity (P < 0.05); while the cTnI Δ1- and Δ3 troponin complexes showed no difference in Ca2+ affinity when compared to wild-type troponin. In vitro motility assays showed that all truncation mutants had increased Ca2+ dependent motility relative to wild-type cTnI. These results suggest that the last 5 C-terminal residues of cTnI influence the binding of cTnI with cTnC and cTnT and affect the Ca2+ dependence of filament sliding, and demonstrate the importance of this region of cTnI.

      PubDate: 2016-06-08T12:06:57Z
  • Sarcomere length dependent effects on the interaction between cTnC and
           cTnI in skinned papillary muscle strips
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): King-Lun Li, Nazanin Bohlooli Ghashghaee, R. John Solaro, Wenji Dong
      Sarcomere length dependent activation (LDA) of myocardial force development is the cellular basis underlying the Frank-Starling law of the heart, but it is still elusive how the sarcomeres detect the length changes and convert them into altered activation of thin filament. In this study we investigated how the C-domain of cardiac troponin I (cTnI) functionally and structurally responds to the comprehensive effects of the Ca2+, crossbridge, and sarcomere length of chemically skinned myocardial preparations. Using our in situ technique which allows for simultaneous measurements of time-resolved FRET and mechanical force of the skinned myocardial preparations, we measured changes in the FRET distance between cTnI(167C) and cTnC(89C), labeled with FRET donor and acceptor, respectively, as a function of [Ca2+], crossbridge state and sarcomere length of the skinned muscle preparations. Our results show that [Ca2+], cross-bridge feedback and sarcomere length have different effects on the structural transition of the C-domain cTnI. In particular, the interplay between crossbridges and sarcomere length has significant impacts on the functional structural change of the C-domain of cTnI in the relaxed state. These novel observations suggest the importance of the C-domain of cTnI and the dynamic and complex interplay between various components of myofilament in the LDA mechanism.

      PubDate: 2016-06-08T12:06:57Z
  • Role of cardiac troponin I carboxy terminal mobile domain and linker
           sequence in regulating cardiac contraction
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Nancy L. Meyer, P. Bryant Chase
      Inhibition of striated muscle contraction at resting Ca2+ depends on the C-terminal half of troponin I (TnI) in thin filaments. Much focus has been on a short inhibitory peptide (Ip) sequence within TnI, but structural studies and identification of disease-associated mutations broadened emphasis to include a larger mobile domain (Md) sequence at the C-terminus of TnI. For Md to function effectively in muscle relaxation, tight mechanical coupling to troponin's core—and thus tropomyosin—is presumably needed. We generated recombinant, human cardiac troponins containing one of two TnI constructs: either an 8-amino acid linker between Md and the rest of troponin (cTnILink8), or an Md deletion (cTnI1-163). Motility assays revealed that Ca2+-sensitivity of reconstituted thin filament sliding was markedly increased with cTnILink8 (∼0.9 pCa unit leftward shift of speed-pCa relation compared to WT), and increased further when Md was missing entirely (∼1.4 pCa unit shift). Cardiac Tn's ability to turn off filament sliding at diastolic Ca2+ was mostly (61%), but not completely eliminated with cTnI1-163. TnI's Md is required for full inhibition of unloaded filament sliding, although other portions of troponin—presumably including Ip—are also necessary. We also confirm that TnI's Md is not responsible for superactivation of actomyosin cycling by troponin.
      Graphical abstract image

      PubDate: 2016-06-08T12:06:57Z
  • Insights into length-dependent regulation of cardiac cross-bridge cycling
           kinetics in human myocardium
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Nima Milani-Nejad, Jae-Hoon Chung, Benjamin D. Canan, Jonathan P. Davis, Vadim V. Fedorov, Robert S.D. Higgins, Ahmet Kilic, Peter J. Mohler, Paul M.L. Janssen
      Cross-bridge cycling kinetics play an essential role in the heart's ability to contract and relax. The rate of tension redevelopment (k tr) slows down as a muscle length is increased in intact human myocardium. We set out to determine the effect of rapid length step changes and protein kinase A (PKA) and protein kinase C-βII (PKC-βII) inhibitors on the k tr in ultra-thin non-failing and failing human right ventricular trabeculae. After stabilizing the muscle either at L90 (90% of optimal length) or at Lopt (optimal length), we rapidly changed the length to either Lopt or L90 and measured k tr. We report that length-dependent changes in k tr occur very rapidly (in the order of seconds or faster) in both non-failing and failing muscles and that the length at which a muscle had been stabilized prior to the length change does not significantly affect k tr. In addition, at L90 and at Lopt, PKA and PKC-βII inhibitors did not significantly change k tr. Our results reveal that length-dependent regulation of cross-bridge cycling kinetics predominantly occurs rapidly and involves the intrinsic properties of the myofilament rather than post-translational modifications that are known to occur in the cardiac muscle as a result of a change in muscle/sarcomere length.

      PubDate: 2016-06-08T12:06:57Z
  • Effects of myosin light chain phosphorylation on length-dependent myosin
           kinetics in skinned rat myocardium
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Hannah C. Pulcastro, Peter O. Awinda, Jason J. Breithaupt, Bertrand C.W. Tanner
      Myosin force production is Ca2+-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca2+-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca2+-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 μm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 μm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles.

      PubDate: 2016-06-08T12:06:57Z
  • Functionally conservative substitutions at cardiac troponin I S43/45
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Sarah E. Lang, Tamara K. Stevenson, Dongyang Xu, Ryan O'Connell, Margaret V. Westfall
      A phospho-null Ala substitution at protein kinase C (PKC)-targeted cardiac troponin I (cTnI) S43/45 reduces myocyte and cardiac contractile function. The goal of the current study was to test whether cTnIS43/45N is an alternative, functionally conservative substitution in cardiac myocytes. Partial and more extensive endogenous cTnI replacement was similar at 2 and 4 days after gene transfer, respectively, for epitope-tagged cTnI and cTnIS43/45N. This replacement did not significantly change thin filament stoichiometry. In functional studies, there were no significant changes in the amplitude and/or rates of contractile shortening and re-lengthening after this partial (2 days) and extensive (4 days) replacement with cTnIS43/45N. The cTnIS43/45N substitution also was not associated with adaptive changes in the myocyte Ca2+ transient or in phosphorylation of the protein kinase A and C-targeted cTnIS23/24 site. These results provide evidence that cTnIS43/45N is a functionally conservative substitution, and may be appropriate for use as a phospho-null in rodent models designed for studies on PKC modulation of cardiac performance.

      PubDate: 2016-06-08T12:06:57Z
  • Molecule specific effects of PKA-mediated phosphorylation on rat isolated
           heart and cardiac myofibrillar function
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Laurin M. Hanft, Timothy D. Cornell, Colin A. McDonald, Michael J. Rovetto, Craig A. Emter, Kerry S. McDonald
      Increased cardiac myocyte contractility by the β-adrenergic system is an important mechanism to elevate cardiac output to meet hemodynamic demands and this process is depressed in failing hearts. While increased contractility involves augmented myoplasmic calcium transients, the myofilaments also adapt to boost the transduction of the calcium signal. Accordingly, ventricular contractility was found to be tightly correlated with PKA-mediated phosphorylation of two myofibrillar proteins, cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), implicating these two proteins as important transducers of hemodynamics to the cardiac sarcomere. Consistent with this, we have previously found that phosphorylation of myofilament proteins by PKA (a downstream signaling molecule of the beta-adrenergic system) increased force, slowed force development rates, sped loaded shortening, and increased power output in rat skinned cardiac myocyte preparations. Here, we sought to define molecule-specific mechanisms by which PKA-mediated phosphorylation regulates these contractile properties. Regarding cTnI, the incorporation of thin filaments with unphosphorylated cTnI decreased isometric force production and these changes were reversed by PKA-mediated phosphorylation in skinned cardiac myocytes. Further, incorporation of unphosphorylated cTnI sped rates of force development, which suggests less cooperative thin filament activation and reduced recruitment of non-cycling cross-bridges into the pool of cycling cross-bridges, a process that would tend to depress both myocyte force and power. Regarding MyBP-C, PKA treatment of slow-twitch skeletal muscle fibers caused phosphorylation of MyBP-C (but not slow skeletal TnI (ssTnI)) and yielded faster loaded shortening velocity and ∼30% increase in power output. These results add novel insight into the molecular specificity by which the β-adrenergic system regulates myofibrillar contractility and how attenuation of PKA-induced phosphorylation of cMyBP-C and cTnI may contribute to ventricular pump failure.

      PubDate: 2016-06-08T12:06:57Z
  • Liver Kinase B1 complex acts as a novel modifier of myofilament function
           and localizes to the Z-disk in cardiac myocytes
    • Abstract: Publication date: 1 July 2016
      Source:Archives of Biochemistry and Biophysics, Volume 601
      Author(s): Samantha M. Behunin, Marissa A. Lopez-Pier, Rachel M. Mayfield, Christiane A. Danilo, Yulia Lipovka, Camille Birch, Sarah Lehman, Jil C. Tardiff, Carol C. Gregorio, John P. Konhilas
      Contractile perturbations downstream of Ca2+ binding to troponin C, the so-called sarcomere-controlled mechanisms, represent the earliest indicators of energy remodeling in the diseased heart [1]. Central to cellular energy “sensing” is the adenosine monophosphate-activated kinase (AMPK) pathway, which is known to directly target myofilament proteins and alter contractility [2–6]. We previously showed that the upstream AMPK kinase, LKB1/MO25/STRAD, impacts myofilament function independently of AMPK [5]. Therefore, we hypothesized that the LKB1 complex associated with myofilament proteins and that alterations in energy signaling modulated targeting or localization of the LKB1 complex to the myofilament. Using an integrated strategy of myofilament mechanics, immunoblot analysis, co-immunoprecipitation, mass spectroscopy, and immunofluorescence, we showed that 1) LKB1 and MO25 associated with myofibrillar proteins, 2) cellular energy stress re-distributed AMPK/LKB1 complex proteins within the sarcomere, and 3) the LKB1 complex localized to the Z-Disk and interacted with cytoskeletal and energy-regulating proteins, including vinculin and ATP Synthase (Complex V). These data represent a novel role for LKB1 complex proteins in myofilament function and myocellular “energy” sensing in the heart.
      Graphical abstract image

      PubDate: 2016-06-08T12:06:57Z
  • IL-1β/NF-kb signaling promotes colorectal cancer cell growth through
           miR-181a/PTEN axis
    • Abstract: Publication date: Available online 3 June 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Pei Haiping, Tan Feng bo, Liu Li, Yu Nan hui, Zhu Hong
      To date, the role of miRNA in tumorigenesis has been largely reported. It was found that miR-181a may be involved in the tumorigenesis of colon cancer. The purpose of this study was to investigate the mechanism of miR-181a in colon cancer carcinogenesis. The expression levels of IL-1β, NF-κB (RelA), and miR-181a in colon cancer tissue were higher than that in normal control tissue when assessed by real-timePCR. In addition, it was found that IL-1β induced the expression of miR-181a. The expression of PTEN was regulated by IL-1β-stimulated miR-181a expression. In a PTEN reporter plasmid, miR-181a binding site mutations were introduced. By using a luciferase reporter assay, it was found that wild type reported activity was lower than that of the mutant registration system activity. Furthermore, a siRNA strategy was used to find that IL-1B regulates miR-181a expression via NF-κB and then regulates PTEN expression. Consequently, repression of PTEN by miR-181a promotes colon cancer cell proliferation. Taken together, our data support a critical role for NF-κB-dependent upregulation of miR-181a; this represents a new pathway for the repression of PTEN and the promotion of cell proliferation upon IL-1β induction.

      PubDate: 2016-06-03T11:40:22Z
  • The death enzyme CP14 is a unique papain-like cysteine proteinase with a
           pronounced S2 subsite selectivity
    • Abstract: Publication date: Available online 28 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Melanie Paireder, Ulrich Mehofer, Stefan Tholen, Andreas Porodko, Philipp Schähs, Daniel Maresch, Martin L. Biniossek, Renier A.L. van der Hoorn, Brigita Lenarcic, Marko Novinec, Oliver Schilling, Lukas Mach
      The cysteine protease CP14 has been identified as a central component of a molecular module regulating programmed cell death in plant embryos. CP14 belongs to a distinct subfamily of papain-like cysteine proteinases of which no representative has been characterized thoroughly to date. However, it has been proposed that CP14 is a cathepsin H-like protease. We have now produced recombinant Nicotiana benthamiana CP14 (NbCP14) lacking the C-terminal granulin domain. As typical for papain-like cysteine proteinases, NbCP14 undergoes rapid autocatalytic activation when incubated at low pH. The mature protease is capable of hydrolysing several synthetic endopeptidase substrates, but cathepsin H-like aminopeptidase activity could not be detected. NbCP14 displays a strong preference for aliphatic over aromatic amino acids in the specificity-determining P2 position. This subsite selectivity was also observed upon digestion of proteome-derived peptide libraries. Notably, the specificity profile of NbCP14 differs from that of aleurain-like protease, the N. benthamiana orthologue of cathepsin H. We conclude that CP14 is a papain-like cysteine proteinase with unusual enzymatic properties which may prove of central importance for the execution of programmed cell death during plant development.

      PubDate: 2016-05-29T11:14:21Z
  • Simvastatin inhibits CD44 fragmentation in chondrocytes
    • Abstract: Publication date: Available online 28 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Kenya Terabe, Nobunori Takahashi, Toki Takemoto, Warren Knudson, Naoki Ishiguro, Kojima Toshihisa
      In human osteoarthritic chondrocytes, the hyaluronan receptor CD44 undergoes proteolytic cleavage at the cell surface. CD44 cleavage is thought to require transit of CD44 into cholesterol-rich lipid rafts. The purpose of this study was to investigate whether statins exert a protective effect on articular chondrocytes due to diminution of cholesterol. Three model systems of chondrocytes were examined including human HCS-2/8 chondrosarcoma cells, human osteoarthritic chondrocytes and normal bovine articular chondrocytes. Treatment with IL-1β + Oncostatin M resulted in a substantial increase in CD44 fragmentation in each of the three chondrocyte models. Pre-incubation with simvastatin prior to treatment with IL-1β + Oncostatin M decreased the level of CD44 fragmentation, decreased the proportion of CD44 that transits into the lipid raft fractions, decreased ADAM10 activity and diminished the interaction between CD44 and ADAM10. In HCS-2/8 cells and bovine articular chondrocytes, fragmentation of CD44 was blocked by the knockdown of ADAM10. Inhibition of CD44 fragmentation by simvastatin also resulted in improved retention of pericellular matrix. Addition of cholesterol and farnesyl-pyrophosphate reversed the protective effects of simvastatin. Thus, the addition of simvastatin exerts positive effects on chondrocytes including reduced CD44 fragmentation and enhanced the retention of pericellular matrix.

      PubDate: 2016-05-29T11:14:21Z
  • Dexamethasone rapidly inhibits glucose uptake via non-genomic mechanisms
           in contracting myotubes
    • Abstract: Publication date: Available online 28 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Hong Gong, Lei Liu, Chen-Xu Ni, Yi Zhang, Wen-Jun Su, Yong-Jie Lian, Wei Peng, Jun-Ping Zhang, Chun-Lei Jiang
      Glucocorticoids (GCs) are a class of steroid hormones that regulate multiple aspects of glucose homeostasis. In skeletal muscle, it is well established that prolonged GC excess inhibits glucose uptake and utilization through glucocorticoid receptor (GR)-mediated transcriptional changes. However, it remains obscure that whether the rapid non-genomic effects of GC on glucose uptake are involved in acute exercise stress. Therefore, we used electric pulse stimulation (EPS)-evoked contracting myotubes to determine whether the non-genomic actions of GC were involved and its underlying mechanism(s). Pretreatment with dexamethasone (Dex, 10 μM) significantly prevented contraction-stimulated glucose uptake and glucose transporter 4 (Glut4) translocation within 20 min in C2C12 myotubes. Neither GC nuclear receptor antagonist (RU486) nor protein synthesis inhibitor (cycloheximide, Chx) affected the rapid inhibition effects of Dex. AMPK and CaMKII-dependent signaling pathways were associated with the non-genomic effects of Dex. These results provide evidence that GC rapidly suppresses glucose uptake in contracting myotubes via GR-independent non-genomic mechanisms. AMPK and CaMKII-mediated Glut4 translocation may play a critical role in GC-induced rapid inhibition of glucose uptake.

      PubDate: 2016-05-29T11:14:21Z
  • Switching of actin-myosin motors by voltage-induced pH bias in vitro
    • Abstract: Publication date: Available online 20 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Kuniyuki Hatori, Takahiro Iwase, Reito Wada
      ATP-driven motor proteins, which function in cell motility and organelle transport, have potential applications as bio-inspired micro-devices; however, their control remains unsatisfactory. Here, we show rapid-velocity control of actin filaments interacting with myosin motors using voltage applied to Pt electrodes in an in vitro motility system, by which immediate increases and decreases in velocity were induced beside the cathode and anode, respectively. Indicator dye revealed pH changes after voltage application, and alternate voltage switching allowed actin filaments to cyclically alter their velocity in response to these changes. This principle provides a basis for on-demand control of not only motor proteins but also pH-sensitive events at a microscopic level.

      PubDate: 2016-05-24T10:51:59Z
  • Peroxynitrite-induced structural perturbations in human IgG: A
           physicochemical study
    • Abstract: Publication date: Available online 19 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Mir Yasir Arfat, Zarina Arif, Sumit Kumar Chaturvedi, Moinuddin, Khursheed Alam
      IgG is an important defence protein. To exhibit optimum function the molecule must maintain its native structure. Peroxynitrite is a potent oxidizing and nitrating agent produced in vivo under pathophysiological conditions. It can oxidize and/or nitrate various amino acids causing changes in the structure and function of proteins. Such proteins may be involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis. In the present work, peroxynitrite-induced structural changes in IgG have been studied by UV–visible, fluorescence, CD, FT-IR, DLS spectroscopy and DSC as well as by SDS–PAGE. Peroxynitrite-modified IgG exhibited hyperchromicity at 280 nm, quenching of tryptophan fluorescence, increase in ANS fluorescence, loss of β-sheet, shift in the positions of amide I and amide II bands, appearance of new peak in FT-IR, attachment of nitro residues and increase in melting temperature, compared to native IgG. Furthermore, peroxynitrite-modified IgG exhibited an additional peak at 420 nm, quenching in tyrosine fluorescence and enhancement in dityrosine fluorescence compared to native IgG. Generation of nitrotyrosine, dityrosine and nitrotryptophan was also observed in peroxynitrite-modified IgG. Gross structural changes in IgG caused by peroxynitrite and observed in vitro may favour autoantibodies induction in vivo under similar conditions.

      PubDate: 2016-05-24T10:51:59Z
  • Cross talk between MMP2-Spm-Cer-S1P and ERK1/2 in proliferation of
           pulmonary artery smooth muscle cells under angiotensin II stimulation
    • Abstract: Publication date: Available online 20 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Animesh Chowdhury, Jaganmay Sarkar, Pijush Kanti Pramanik, Tapati Chakraborti, Sajal Chakraborti
      The aim of the present study is to establish the mechanism associated with the proliferation of PASMCs under ANG II stimulation. The results showed that treatment of PASMCs with ANG II induces an increase in cell proliferation and 100 nM was the optimum concentration for maximum increase in proliferation of the cells. Pretreatment of the cells with AT1, but not AT2 receptor antagonist inhibited ANG II induced cell proliferation. Pretreatment with pharmacological and genetic inhibitors of sphingomyelinase (SMase) and sphingosine kinase (SPHK) prevented ANG II-induced cell proliferation. ANG II has also been shown to induce SMase activity, SPHK phosphorylation and S1P production. In addition, ANG II caused an increase in proMMP-2 expression and activation, ERK1/2 phosphorylation and NADPH oxidase activation. Upon inhibition of MMP-2, SMase activity and S1P level were curbed leading to inhibition of cell proliferation. SPHK was phosphorylated by ERK1/2 during ET-1 stimulation of the cells. ANG II-induced ERK1/2 phosphorylation and proMMP-2 expression and activation in the cells were abrogated upon inhibition of NADPH oxidase activity. Overall, NADPH oxidase plays an important role in proMMP-2 expression and activation and that MMP-2 mediated SMC proliferation occurs through the involvement of Spm-Cer-S1P signaling axis under ANG II stimulation of PASMCs.

      PubDate: 2016-05-24T10:51:59Z
  • Preparation of ribosomes for smFRET studies: A simplified approach
    • Abstract: Publication date: Available online 19 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Bassem Shebl, Drew E. Menke, Min Pennella, Raghav R. Poudyal, Donald H. Burke, Peter V. Cornish
      During the past decade, single-molecule studies of the ribosome have significantly advanced our understanding of protein synthesis. The broadest application of these methods has been towards the investigation of ribosome conformational dynamics using single-molecule Förster resonance energy transfer (smFRET). The recent advances in fluorescently labeled ribosomes and translation components have resulted in success of smFRET experiments. Various methods have been employed to target fluorescent dyes to specific locations within the ribosome. Primarily, these methods have involved additional steps including subunit dissociation and/or full reconstitution, which could result in ribosomes of reduced activity and translation efficiency. In addition, substantial time and effort are required to produce limited quantities of material. To enable rapid and large-scale production of highly active, fluorescently labeled ribosomes, we have developed a procedure that combines partial reconstitution with His-tag purification. This allows for a homogeneous single-step purification of mutant ribosomes and subsequent integration of labeled proteins. Ribosomes produced with this method are shown to be as active as ribosomes purified using classical methods. While we have focused on two labeling sites in this report, the method is generalizable and can in principle be extended to any non-essential ribosomal protein.

      PubDate: 2016-05-19T09:52:37Z
  • Approaches to the solution of coupled multiexponential transient-state
           rate kinetic equations: A critical review
    • Abstract: Publication date: Available online 9 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Harvey F. Fisher
      The transient-state kinetic approach has failed to reach its full potential despite its advantage over the steady-state approach in its ability to observe mechanistic events directly and in real time. This failure has been due in part to the lack of any rigorously derived and readily applicable body of theory corresponding to that which currently characterizes the steady-state approach. In order to clarify the causes of this discrepancy and to suggest a route to its solution we examine the capabilities and limitations of the various forms of transient-state kinetic approaches to the mathematical resolution of enzymatic reaction mechanisms currently available. We document a lack of validity inherent in their basic assumptions and suggest the need for a potentially more rigorous analytic approach.

      PubDate: 2016-05-14T09:45:05Z
  • Identification of the two-phase mechanism of arachidonic acid regulating
    • Abstract: Publication date: Available online 10 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Hironari Akasaka, Ke-He Ruan
      Through linking inducible cyclooxygenase (COX)-2 with microsomal prostaglandin E2 (PGE2) synthase-1 (mPGES-1), a Single-Chain Enzyme Complex (SCEC, COX-2-10aa-mPGES-1) was engineered to mimic a specific inflammatory PGE2 biosynthesis from omega-6 fatty acid, arachidonic acid (AA), by eliminating involvements of non-inducible COX-1 and other PGE2 synthases. Using the SCEC, we characterized coupling reactions between COX-2 and mPGES-1 at 1:1 ratio of inflammatory PGE2 production. AA demonstrated two phase activities to regulate inflammatory PGE2 production. In the first phase (<2 μM), AA was a COX-2 substrate and converted to increasing production of PGE2. In the second phase with a further increased AA level (2–10 μM), AA bound to mPGES-1 and inhibited the PGE2 production. The SCEC study was identical to the co-expression of COX-2 and mPGES-1. This was further confirmed by using mPGES-1 and PGH2 as a direct enzyme target and substrate, respectively. Furthermore, the carboxylic acid group of AA binding to R67 and R70 of mPGES-1 was identified by X-ray structure-based docking and mutagenesis. mPGES-1 mutants, R70A, R70K, R67A and R67K, lost 40–100% binding to [14C]-AA. To conclude, a cellular model, in which AA is involved in self-controlling initial initiating and later resolving inflammation by its two phase activities, was discussed.

      PubDate: 2016-05-14T09:45:05Z
  • Biochemical properties of Glu-SH3 as a family 13 glycoside hydrolase with
           remarkable substrate specificity for trehalose: Implications to
           sequence-based classification of CAZymes
    • Abstract: Publication date: Available online 10 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Kamran Khalili Ghadikolaei, Maral Shojaei, Armin Ghaderi, Farzaneh Hojjati, Kambiz Akbari Noghabi, Hossein Shahbani Zahiri
      A novel glycoside hydrolase from Exiguobacterium sp. SH3 was characterized. The enzyme, designated as Glu-SH3, was predicted by in silico analysis to have structural similarity with members of oligo-1,6-glucosidase and trehalose-6-phosphate hydrolase subfamilies in the GH-13 family of glycoside hydrolases. The gene was expressed in E. coli and the recombinant enzyme was purified as a His-tagged protein of about 60 kDa. The enzyme was shown to have remarkable substrate specificity for trehalose. The characteristic ability of Glu-SH3 to hydrolyze trehalose was ascertained by zymography, thin layer chromatography, and NMR spectroscopy. The maximum activity of Glu-SH3 was obtained at 35 °C and pH 7, but it was able to exhibit more than 90% of the activity within the pH range of 5-8. The V max and K m values were estimated to be 170 U and 4.5 mg ml−1, respectively. By comparison with trehalases, Glu-SH3 with K cat and K cat /K m values of 1552 s−1 and 119.4 mM−1 s−1 can be recognized as a very efficient trehalose-hydrolyzing glycosidase. Given the phylogeny and the substrate specificity of Glu-SH3, it may be assumed that the enzyme shares a common ancestor with oligo-1,6-glucosidases but have evolved distinctly to serve a physiological function in trehalose metabolism.

      PubDate: 2016-05-14T09:45:05Z
  • Nuclear localization of formyl-peptide receptor 2 in human cancer cells
    • Abstract: Publication date: Available online 10 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Fabio Cattaneo, Melania Parisi, Tiziana Fioretti, Daniela Sarnataro, Gabriella Esposito, Rosario Ammendola
      Current models of G protein-coupled receptors (GPCRs) signaling describe binding of external agonists to cell surface receptors which, in turn, trigger several biological responses. New paradigms indicate that GPCRs localize to and signal at the nucleus, thus regulating distinct signaling cascades. The formyl-peptide receptor FPR2 belongs to the GPCR super-family and is coupled to PTX-sensitive Gi proteins. We show by western blot analysis, immunofluorescence experiments and radioligand binding assays that FPR2 is expressed at nuclear level in CaLu-6 and AGS cells. Nuclear FPR2 is a functional receptor, since it participates in intra-nuclear signaling, as assessed by decreased G protein-FPR2 association and enhanced ERK2, c-Jun and c-Myc phosphorylation upon stimulation of intact nuclei with the FPR2 agonist, WKYMVm. We analyzed FPR2 sequence for the search of a nuclear localization sequence (NLS) and we found a stretch of basic aminoacids (227-KIHKK-231) in the third cytoplasmic loop of the receptor. We performed single (K230A) and multiple (H229A/K230A/K231A) mutagenesis of NLS. The constructs were individually overexpressed in HEK293 cells and immunofluorescence and western blot analysis showed that nuclear localization or translocation of FPR2 depends on the integrity of the H229 and K231 residues within the NLS.

      PubDate: 2016-05-14T09:45:05Z
  • Diastolic dysfunction and cardiac troponin I decrease in aging hearts
    • Abstract: Publication date: Available online 13 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): B. Pan, Z.W. Xu, Y. Xu, L.J. Liu, J. Zhu, X. Wang, C. Nan, Z. Zhang, W. Shen, X.P. Huang, J. Tian
      Cardiac tropnoin I (cTnI) plays a critical role in the regulation of diastolic function, and its low expression may result in cardiac diastolic dysfunction, which is the most common form of cardiovascular disorders in older adults. In this study, cTnI expression levels were determined in mice at various ages and cardiac function was measured and compared between young adult mice (3 and 10 months) and older mice (18 months). The data indicated that the cTnI levels reached a peak high in young adult hearts (3 months), but decreased in older hearts (18 months). Furthermore, the older hearts showed a significant diastolic dysfunction observed by P–V loop and echocardiography measurements. To further define the mechanism underlying the cTnI decrease in aging hearts, we tested DNA methylation and histone acetylation modifications of cTnI gene. We found that acetylation of histone near the promoter region of cTnI gene played an important role in regulation of cTnI expression in the heart at different ages. Our study indicates that epigenetic modification caused cTnI expression decrease is one of the possible causes that result in a reduced cTnI level and diastolic dysfunction in the older hearts.

      PubDate: 2016-05-14T09:45:05Z
  • Cold-inducible RNA binding protein regulates mucin expression induced by
           cold temperatures in human airway epithelial cells
    • Abstract: Publication date: Available online 13 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): DanHua Ran, LingXiu Chen, WenYao Xie, Qing Xu, Zhong Han, HuaPing Huang, XiangDong Zhou
      Mucus overproduction is an important manifestation of chronic airway inflammatory diseases, however, the mechanisms underlying the association between cold air and mucus overproduction remain unknown. We found that the expression of the cold-inducible RNA binding protein (CIRP) was increased in patients with chronic obstructive pulmonary disease (COPD). In the present study, we tested whether CIRP was involved in inflammatory factors and mucin5AC (MUC5AC) expression after cold stimulation and investigated the potential signaling pathways involved in this process. We found that CIRP was highly expressed in the bronchi of COPD patients. The expression of CIRP, interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were increased, and the CIRP was localized in cytoplasm after cold stimulation. MUC5AC mRNA and protein expression levels were elevated in a temperature- and time-dependent manner after cold stimulation and were associated with the phosphorylation of ERK and NF-κB, which reflected their activation. These responses were suppressed by knockdown of CIRP with a specific siRNA or the ERK and NF-κB inhibitors. These results demonstrated that CIRP was expressed in the bronchi of human COPD patients and was involved in inflammatory factors and MUC5AC expression after cold stimulation through the ERK and NF-κB pathways.

      PubDate: 2016-05-14T09:45:05Z
  • Myofilament Modulation of Contraction
    • Abstract: Publication date: Available online 6 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Brandon J. Biesiadecki

      PubDate: 2016-05-09T09:26:32Z
  • What is the concentration of hydrogen peroxide in blood and plasma'
    • Abstract: Publication date: Available online 9 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Henry Jay Forman, Angelito Bernardo, Kelvin J.A. Davies
      The concentration of hydrogen peroxide (H2O2) in blood and plasma is a measurement that has often been made, but the absolute values remain unsettled due the great variability of results actually published in the literature. As in every tissue, the concentration of H2O2 in blood and plasma is determined by the dynamics of its production versus its removal. The major sources of H2O2 in cells will only be briefly described as they are already well documented, The production of H2O2 in red blood cells will be described as it is less well known. But, the concentration of H2O2 within cells is more problematic. Intracellular H2O2 concentration has been estimated based on the kinetics of production and elimination, while its determination is technically difficult. Furthermore, compartmentalization and gradients result in its quantitation only as an average. The sources of extracellular H2O2, particularly in plasma, will also be described briefly. The major question addressed here however, is the actual concentration of H2O2 in plasma, which has been studied extensively, but still remains controversial.
      Graphical abstract image

      PubDate: 2016-05-09T09:26:32Z
  • Aggregation of intrinsically disordered fibrinogen as the influence of
           backbone conformation
    • Abstract: Publication date: Available online 3 May 2016
      Source:Archives of Biochemistry and Biophysics
      Author(s): Aabgeena Naeem, Sheraz Ahmad Bhat, Afshin Iram, Rizwan Hasan Khan
      Fib having intrinsically disordered αC domains is involved in coagulation cascade and thrombosis. Fib molecules forms prefibrillar oligomers at 30%, and associate in 40% and 50% TFE to proceed α to β transition, suggesting the formation of an intermolecular β-structure. AFM images confirmed the nature of Fib aggregates at 40% and 50% TFE to be prefibrillar and fibrillar respectively. These aggregates possess high thioflavin T fluorescence with a shifted Congo red absorbance. Kinetics of Fib aggregation data at 50% TFE supports nucleation-dependent polymerization mechanism. At 60 and 70% TFE, no aggregation was observed. The inhibition of protein aggregation appears due to weakening of the hydrophobic interactions that were initially stabilizing the intermolecular β-sheet structure in the protein aggregation. The loss of hydrophobic contacts seems to favor the formation of intra-molecular hydrogen bonds over intermolecular hydrogen bonds leads to helix formation. To conclude, protein aggregation is accompanied by the formation of β-sheet conformation, and induction of non-native helical segments in the protein inhibits aggregation. The discrepancy of the secondary structures on aggregation is proposed to stem from the disparity in the nature of the hydrogen bonds and packing of hydrophobic residues of the side chains in the β-sheet and α-helix conformation.
      Graphical abstract image

      PubDate: 2016-05-05T08:45:50Z
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