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  Subjects -> BIOLOGY (Total: 2575 journals)
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BIOCHEMISTRY (190 journals)                  1 2     

AAPS PharmSciTech     Hybrid Journal   (Followers: 6)
Acetic Acid Bacteria     Open Access   (Followers: 1)
ACS Chemical Biology     Full-text available via subscription   (Followers: 230)
ACS Chemical Neuroscience     Full-text available via subscription   (Followers: 13)
Acta Crystallographica Section D : Biological Crystallography     Hybrid Journal   (Followers: 8)
Acta Crystallographica Section F: Structural Biology Communications     Hybrid Journal   (Followers: 5)
Advances and Applications in Bioinformatics and Chemistry     Open Access   (Followers: 7)
Advances in Biological Chemistry     Open Access   (Followers: 5)
Advances in Carbohydrate Chemistry and Biochemistry     Full-text available via subscription   (Followers: 5)
Advances in Plant Biochemistry and Molecular Biology     Full-text available via subscription   (Followers: 6)
Advances in Protein Chemistry and Structural Biology     Full-text available via subscription   (Followers: 9)
African Journal of Biochemistry Research     Open Access  
African Journal of Chemical Education     Open Access   (Followers: 1)
Alkaloids: Chemical and Biological Perspectives     Full-text available via subscription   (Followers: 4)
American Journal of Biochemistry     Open Access   (Followers: 6)
American Journal of Biochemistry and Biotechnology     Open Access   (Followers: 124)
American Journal of Biochemistry and Molecular Biology     Open Access   (Followers: 11)
Amino Acids     Hybrid Journal   (Followers: 7)
Analytical Biochemistry     Hybrid Journal   (Followers: 157)
Annals of Clinical Biochemistry     Hybrid Journal   (Followers: 1)
Annual Review of Biochemistry     Full-text available via subscription   (Followers: 28)
Annual Review of Chemical and Biomolecular Engineering     Full-text available via subscription   (Followers: 9)
Applied Biochemistry and Biotechnology     Hybrid Journal   (Followers: 18)
Applied Biochemistry and Microbiology     Hybrid Journal   (Followers: 7)
Applied Organometallic Chemistry     Hybrid Journal   (Followers: 4)
Archives of Biochemistry and Biophysics     Hybrid Journal   (Followers: 9)
Archives of Insect Biochemistry and Physiology     Hybrid Journal   (Followers: 1)
Archives Of Physiology And Biochemistry     Hybrid Journal   (Followers: 1)
Asian Journal of Biochemistry     Open Access   (Followers: 1)
Bangladesh Journal of Medical Biochemistry     Open Access   (Followers: 2)
BBA Clinical     Open Access  
BBR : Biochemistry and Biotechnology Reports     Open Access   (Followers: 4)
Biochemical and Biophysical Research Communications     Hybrid Journal   (Followers: 13)
Biochemical and Molecular Medicine     Full-text available via subscription   (Followers: 2)
Biochemical Engineering Journal     Hybrid Journal   (Followers: 8)
Biochemical Genetics     Hybrid Journal   (Followers: 2)
Biochemical Journal     Full-text available via subscription   (Followers: 14)
Biochemical Pharmacology     Hybrid Journal   (Followers: 6)
Biochemical Society Transactions     Full-text available via subscription   (Followers: 2)
Biochemical Systematics and Ecology     Hybrid Journal   (Followers: 3)
Biochemistry     Full-text available via subscription   (Followers: 163)
Biochemistry (Moscow)     Hybrid Journal   (Followers: 3)
Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology     Hybrid Journal   (Followers: 4)
Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry     Hybrid Journal   (Followers: 4)
Biochemistry and Cell Biology     Full-text available via subscription   (Followers: 8)
Biochemistry and Molecular Biology Education     Hybrid Journal   (Followers: 3)
Biochemistry and Molecular Biology of Fishes     Full-text available via subscription   (Followers: 1)
Biochemistry Research International     Open Access   (Followers: 4)
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids     Hybrid Journal   (Followers: 3)
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease     Hybrid Journal   (Followers: 16)
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research     Hybrid Journal   (Followers: 5)
Biochimie     Hybrid Journal   (Followers: 4)
Bioconjugate Chemistry     Full-text available via subscription   (Followers: 14)
BioDrugs     Full-text available via subscription   (Followers: 7)
Bioelectrochemistry     Hybrid Journal   (Followers: 3)
Biofuels     Hybrid Journal   (Followers: 7)
Biogeochemistry     Hybrid Journal   (Followers: 5)
BioInorganic Reaction Mechanisms     Full-text available via subscription   (Followers: 1)
Biokemistri     Open Access  
Biological Chemistry     Partially Free   (Followers: 11)
Biomedicines     Open Access  
BioMolecular Concepts     Full-text available via subscription   (Followers: 2)
Bioscience, Biotechnology, and Biochemistry     Hybrid Journal   (Followers: 6)
Biosimilars     Open Access   (Followers: 1)
Biotechnology and Applied Biochemistry     Hybrid Journal   (Followers: 18)
BMC Biochemistry     Open Access   (Followers: 8)
BMC Chemical Biology     Open Access   (Followers: 4)
Carbohydrate Polymers     Hybrid Journal   (Followers: 8)
Cell Biochemistry and Biophysics     Hybrid Journal   (Followers: 6)
Cell Biochemistry and Function     Hybrid Journal   (Followers: 3)
Cellular Physiology and Biochemistry     Open Access   (Followers: 3)
Central European Journal of Chemistry     Hybrid Journal   (Followers: 5)
ChemBioChem     Hybrid Journal   (Followers: 2)
Chemical Biology & Drug Design     Hybrid Journal   (Followers: 23)
Chemical Engineering Journal     Hybrid Journal   (Followers: 16)
Chemical Senses     Hybrid Journal   (Followers: 1)
Chemical Speciation and Bioavailability     Full-text available via subscription   (Followers: 1)
Chemico-Biological Interactions     Hybrid Journal   (Followers: 2)
Chemistry & Biodiversity     Hybrid Journal   (Followers: 5)
Chemistry & Biology     Full-text available via subscription   (Followers: 16)
Chemistry and Ecology     Hybrid Journal   (Followers: 1)
Clinical Biochemist Reviews     Full-text available via subscription   (Followers: 1)
Clinical Biochemistry     Hybrid Journal   (Followers: 3)
Clinical Chemistry and Laboratory Medicine     Full-text available via subscription   (Followers: 3)
Clinical Lipidology     Full-text available via subscription  
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology     Hybrid Journal   (Followers: 5)
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology     Hybrid Journal   (Followers: 2)
Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology     Hybrid Journal   (Followers: 4)
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics     Hybrid Journal   (Followers: 3)
Comprehensive Biochemistry     Full-text available via subscription   (Followers: 1)
Computational Biology and Chemistry     Hybrid Journal   (Followers: 8)
Critical Reviews in Biochemistry and Molecular Biology     Hybrid Journal   (Followers: 2)
Current Chemical Biology     Hybrid Journal   (Followers: 2)
Current Opinion in Chemical Biology     Hybrid Journal   (Followers: 13)
Current Opinion in Lipidology     Hybrid Journal   (Followers: 2)
DNA Barcodes     Open Access  
Doklady Biochemistry and Biophysics     Hybrid Journal   (Followers: 2)
Doklady Chemistry     Hybrid Journal  
Egyptian Journal of Biochemistry and Molecular Biology     Full-text available via subscription  
FEBS Letters     Hybrid Journal   (Followers: 24)

        1 2     

Journal Cover Archives of Biochemistry and Biophysics
   [11 followers]  Follow    
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
     ISSN (Print) 0003-9861 - ISSN (Online) 1096-0384
     Published by Elsevier Homepage  [2563 journals]   [SJR: 1.131]   [H-I: 115]
  • Titin-mediated control of cardiac myofibrillar function
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Laurin M. Hanft , Marion L. Greaser , Kerry S. McDonald
      According to the Frank–Starling relationship, ventricular pressure or stroke volume increases with end-diastolic volume. This is regulated, in large part, by the sarcomere length (SL) dependent changes in cardiac myofibrillar force, loaded shortening, and power. Consistent with this, both cardiac myofibrillar force and absolute power fall at shorter SL. However, when Ca2+ activated force levels are matched between short and long SL (by increasing the activator [Ca2+]), short SL actually yields faster loaded shortening and greater peak normalized power output (PNPO). A potential mechanism for faster loaded shortening at short SL is that, at short SL, titin becomes less taut, which increases the flexibility of the cross-bridges, a process that may be mediated by titin’s interactions with thick filament proteins. We propose a more slackened titin yields greater myosin head radial and azimuthal mobility and these flexible cross-bridges are more likely to maintain thin filament activation, which would allow more force-generating cross-bridges to work against a fixed load resulting in faster loaded shortening. We tested this idea by measuring SL-dependence of power at matched forces in rat skinned cardiac myocytes containing either N2B titin or a longer, more compliant N2BA titin. We predicted that, in N2BA titin containing cardiac myocytes, power-load curves would not be shifted upward at short SL compared to long SL (when force is matched). Consistent with this, peak normalized power was actually less at short SL versus long SL (at matched force) in N2BA-containing myocytes (N2BA titin: ΔPNPO (Short SL peak power minus long SL peak power)=−0.057±0.049 (n =5) versus N2B titin: ΔPNPO=+0.012±0.012 (n =5). These findings support a model whereby SL per se controls mechanical properties of cross-bridges and this process is mediated by titin. This myofibrillar mechanism may help sustain ventricular power during periods of low preloads, and perhaps a breakdown of this mechanism is involved in impaired function of failing hearts.


      PubDate: 2014-06-07T15:27:45Z
       
  • Sucrose increases the activation energy barrier for actin–myosin
           strong binding
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Del R. Jackson Jr. , Milad Webb , Travis J. Stewart , Travis Phillips , Michael Carter , Christine R. Cremo , Josh E. Baker
      To determine the mechanism by which sucrose slows in vitro actin sliding velocities, V, we used stopped flow kinetics and a single molecule binding assay, SiMBA. We observed that in the absence of ATP, sucrose (880mM) slowed the rate of actin–myosin (A–M) strong binding by 71±8% with a smaller inhibitory effect observed on spontaneous rigor dissociation (21±3%). Similarly, in the presence of ATP, sucrose slowed strong binding associated with Pi release by 85±9% with a smaller inhibitory effect on ATP-induced A–M dissociation, k T (39±2%). Sucrose had no noticeable effect on any other step in the ATPase reaction. In SiMBA, sucrose had a relatively small effect on the diffusion coefficient for actin fragments (25±2%), and with stopped flow we showed that sucrose increased the activation energy barrier for A–M strong binding by 37±3%, indicating that sucrose inhibits the rate of A–M strong binding by slowing bond formation more than diffusional searching. The inhibitory effects of sucrose on the rate of A–M rigor binding (71%) are comparable in magnitude to sucrose’s effects on both V (79±33% decrease) and maximal actin-activated ATPase, k cat, (81±16% decrease), indicating that the rate of A–M strong bond formation significantly influences both k cat and V.


      PubDate: 2014-06-07T15:27:45Z
       
  • The structural dynamics of α-tropomyosin on F-actin shape the overlap
           complex between adjacent tropomyosin molecules
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): William Lehman , Xiaochuan (Edward) Li , Marek Orzechowski , Stefan Fischer
      Coiled-coil tropomyosin, localized on actin filaments in virtually all eukaryotic cells, serves as a gatekeeper regulating access of the motor protein myosin and other actin-binding proteins onto the thin filament surface. Tropomyosin’s modular pseudo-repeating pattern of approximately 39 amino acid residues is designed to allow binding of the coiled-coil to successive actin subunits along thin filaments. Even though different tropomyosin isoforms contain varying numbers of repeat modules, the pseudo-repeat length, in all cases, matches that of a single actin subunit. Thus, the seven pseudo-repeats of 42nm long muscle tropomyosin bind to seven successive actin subunits along thin filaments, while simultaneously bending into a super-helical conformation that is preshaped to the actin filament helix. In order to form a continuous cable on thin filaments that is free of gaps, adjacent tropomyosin molecules polymerize head-to-tail by means of a short (∼9 residue) overlap. Several laboratories have engineered peptides to mimic the N- and C-terminal tropomyosin association and to characterize the overlap structure. All overlapping domains examined show a compact N-terminal coiled-coil inserting into a partially opened C-terminal partner, where the opposing coiled-coils at the overlap junction face each other at up to ∼90° twist angles. Here, Molecular Dynamics (MD) simulations were carried out to determine constraints on the formation of the tropomyosin overlap complex and to assess the amount of twisting exhibited by full-length tropomyosin when bound to actin. With the exception of the last 20–40 C- and N-terminal residues, we find that the average tropomyosin structure closely resembles a “canonical” model proposed in the classic work of McLachlan and Stewart, displaying perfectly symmetrical supercoil geometry matching the F-actin helix with an integral number of coiled-coil turns, a coiled-coil helical pitch of 137Å, a superhelical pitch of 770Å, and no localized pseudo-rotation. Over the middle 70% of tropomyosin, the average twisting of the coiled-coil deviates only by 10° from the canonical model and the torsional freedom is very small (std. dev. of 7°). This small degree of twisting cannot yield the orthogonal N- and C-termini configuration observed experimentally. In marked contrast, considerable coiled-coil unfolding, splaying and twisting at N- and C-terminal ends is observed, providing the conformational plasticity needed for head-to-tail nexus formation.
      Graphical abstract image

      PubDate: 2014-06-07T15:27:45Z
       
  • Clusters of bound Ca2+ initiate contraction in fast skeletal muscle
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Philip W. Brandt , Corrado Poggesi
      Ca2+-binding to troponin C ultimately controls force in muscle leading to the expectation that the two curves, pCa/force and pCa/Ca2+ binding, will coincide. Using an improved fluorescence apparatus to measure Ca2+-binding, we confirm a displacement between the position and shape of the pCa/Ca2+-binding and pCa/force curves. This displacement may be part of a mechanism that reduces the noise inherent in the control process. There must always be some Ca2+-binding events even at 10 or 100nM, well below threshold for muscle contraction. To minimize the response to such random binding events we suggest that clusters of adjacent Ca2+-binding sites must be filled before contraction is initiated. Clusters promote the reconfiguration of the thin filament to the “On” state; this simultaneously increases thin filaments’ affinity for myosin heads and of troponin C for Ca2+ producing the highly cooperative pCa/force curve. The cluster requirement displaces the Ca2+-binding from the force curve as observed. The thin filament conformational changes and the accompanying affinity increases introduce a discontinuity in the pCa/Ca2+-binding curve. The curve, therefore, is most appropriately fit by two separate Hill equations, a simple non-cooperative one (midpoint, pK 1, n 1 ∼1) for the foot and a second cooperative one (pK 2, n 2 ∼2.5) for the upper part. With this fit pK 2 is larger than pK 1 as our argument requires, in contrast to fitting to the sum of two Hill equations. It also expresses the idea that there may be three states of the thin filament.


      PubDate: 2014-06-07T15:27:45Z
       
  • Influence of a constitutive increase in myofilament Ca2+-sensitivity on
           Ca2+-fluxes and contraction of mouse heart ventricular myocytes
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Jose L. Puglisi , Paul H. Goldspink , Aldrin V. Gomes , Megan S. Utter , Donald M. Bers , R. John Solaro
      Chronic increases in myofilament Ca2+-sensitivity in the heart are known to alter gene expression potentially modifying Ca2+-homeostasis and inducing arrhythmias. We tested age-dependent effects of a chronic increase in myofilament Ca2+-sensitivity on induction of altered alter gene expression and activity of Ca2+ transport systems in cardiac myocytes. Our approach was to determine the relative contributions of the major mechanisms responsible for restoring Ca2+ to basal levels in field stimulated ventricular myocytes. Comparisons were made from ventricular myocytes isolated from non-transgenic (NTG) controls and transgenic mice expressing the fetal, slow skeletal troponin I (TG-ssTnI) in place of cardiac TnI (cTnI). Replacement of cTnI by ssTnI induces an increase in myofilament Ca2+-sensitivity. Comparisons included myocytes from relatively young (5–7months) and older mice (11–13months). Employing application of caffeine in normal Tyrode and in 0Na+ 0Ca2+ solution, we were able to dissect the contribution of the sarcoplasmic reticulum Ca2+ pump (SR Ca2+-ATPase), the Na+/Ca2+ exchanger (NCX), and “slow mechanisms” representing the activity of the sarcolemmal Ca2+ pump and the mitochondrial Ca2+ uniporter. The relative contribution of the SR Ca2+-ATPase to restoration of basal Ca2+ levels in younger TG-ssTnI myocytes was lower than in NTG (81.12±2.8% vs 92.70±1.02%), but the same in the older myocytes. Younger and older NTG myocytes demonstrated similar contributions from the SR Ca2+-ATPase and NCX to restoration of basal Ca2+. However, the slow mechanisms for Ca2+ removal were increased in the older NTG (3.4±0.3%) vs the younger NTG myocytes (1.4±0.1%). Compared to NTG, younger TG-ssTnI myocytes demonstrated a significantly bigger contribution of the NCX (16±2.7% in TG vs 6.9±0.9% in NTG) and slow mechanisms (3.3±0.4% in TG vs 1.4±0.1% in NTG). In older TG-ssTnI myocytes the contributions were not significantly different from NTG (NCX: 4.9±0.6% in TG vs 5.5±0.7% in NTG; slow mechanisms: 2.5±0.3% in TG vs 3.4±0.3% in NTG). Our data indicate that constitutive increases in myofilament Ca2+-sensitivity alter the relative significance of the NCX transport system involved in Ca2+-homeostasis only in a younger group of mice. This modification may be of significance in early changes in altered gene expression and electrical stability hearts with increased myofilament Ca-sensitivity.


      PubDate: 2014-06-07T15:27:45Z
       
  • Conformation of the critical pH sensitive region of troponin depends upon
           a single residue in troponin I
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Ian M. Robertson , Sandra E. Pineda-Sanabria , Peter C. Holmes , Brian D. Sykes
      The calcium sensitivity of cardiac and skeletal muscle is reduced during cytosolic acidosis, and this inhibition is more pronounced in cardiac muscle. Replacing cardiac troponin I with skeletal troponin I reduces the pH sensitivity of cardiac muscle. This diminished pH sensitivity depends on a single amino acid difference in troponin I: an alanine in cardiac and a histidine in skeletal. Studies suggested that when this histidine is protonated, it forms an electrostatic interaction with glutamate 19 on the surface of cardiac troponin C. Structures of the skeletal and cardiac troponin complexes show very different conformations for the region of troponin I surrounding this residue. In this study, we determined the structure of skeletal troponin I bound to cardiac troponin C. Skeletal troponin I is found to bind to cardiac troponin C with histidine 130 in close proximity to glutamate 19. This conformation is homologous to the crystal structure of the skeletal troponin complex; but different than in the cardiac complex. We show that an A162H variant of cardiac troponin I adopts a conformation similar to the skeletal structure. The implications of these structural differences in the context of cardiac muscle regulation are discussed.
      Graphical abstract image

      PubDate: 2014-06-07T15:27:45Z
       
  • Allosteric effects of cardiac troponin TNT1 mutations on actomyosin
           binding: A novel pathogenic mechanism for hypertrophic cardiomyopathy
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Rachel K. Moore , Salwa Abdullah , Jil C. Tardiff
      The majority of hypertrophic cardiomyopathy mutations in (cTnT) occur within the alpha-helical tropomyosin binding TNT1 domain. A highly charged region at the C-terminal end of TNT1 unwinds to create a flexible “hinge”. While this region has not been structurally resolved, it likely acts as an extended linker between the two cTnT functional domains. Mutations in this region cause phenotypically diverse and often severe forms of HCM. Mechanistic insight, however, has been limited by the lack of structural information. To overcome this limitation, we evaluated the effects of cTnT 160–163 mutations using regulated in vitro motility (R-IVM) assays and transgenic mouse models. R-IVM revealed that cTnT mutations Δ160E, E163R and E163K disrupted weak electrostatic actomyosin binding. Reducing the ionic strength or decreasing Brownian motion rescued function. This is the first observation of HCM-linked mutations in cTnT disrupting weak interactions between the thin filament and myosin. To evaluate the in vivo effects of altering weak actomyosin binding we generated transgenic mice expressing Δ160E and E163R mutant cTnT and observed severe cardiac remodeling and profound myofilament disarray. The functional changes observed in vitro may contribute to the structural impairment seen in vivo by destabilizing myofilament structure and acting as a constant pathophysiologic stress.


      PubDate: 2014-06-07T15:27:45Z
       
  • In vitro rescue study of a malignant familial hypertrophic cardiomyopathy
           phenotype by pseudo-phosphorylation of myosin regulatory light chain
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Priya Muthu , Jingsheng Liang , William Schmidt , Jeffrey R. Moore , Danuta Szczesna-Cordary
      Pseudo-phosphorylation of cardiac myosin regulatory light chain (RLC) has never been examined as a rescue method to alleviate a cardiomyopathy phenotype brought about by a disease causing mutation in the myosin RLC. This study focuses on the aspartic acid to valine substitution (D166V) in the myosin RLC shown to be associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). The mutation has also been demonstrated to cause severe functional abnormalities in transgenic mice expressing D166V in the heart. To explore this novel rescue strategy, pseudo-phosphorylation of D166V was used to determine whether the D166V-induced detrimental phenotype could be brought back to the level of wild-type (WT) RLC. The S15D substitution at the phosphorylation site of RLC was inserted into the recombinant WT and D166V mutant to mimic constitutively phosphorylated RLC proteins. Non-phosphorylatable (S15A) constructs were used as controls. A multi-faceted approach was taken to determine the effect of pseudo-phosphorylation on the ability of myosin to generate force and motion. Using mutant reconstituted porcine cardiac muscle preparations, we showed an S15D-induced rescue of both the enzymatic and binding properties of D166V-myosin to actin. A significant increase in force production capacity was noted in the in vitro motility assays for S15D-D166V vs. D166V reconstituted myosin. A similar pseudo-phosphorylation induced effect was observed on the D166V-elicited abnormal Ca2+ sensitivity of force in porcine papillary muscle strips reconstituted with phosphomimic recombinant RLCs. Results from this study demonstrate a novel in vitro rescue strategy that could be utilized in vivo to ameliorate a malignant cardiomyopathic phenotype. We show for the first time that pseudo-RLC phosphorylation can reverse the majority of the mutation-induced phenotypes highlighting the importance of RLC phosphorylation in combating cardiac disease.


      PubDate: 2014-06-07T15:27:45Z
       
  • Ca2+-regulatory function of the inhibitory peptide region of cardiac
           troponin I is aided by the C-terminus of cardiac troponin T: Effects of
           familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C,
           alone and in combination, on filament sliding
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Nicolas M. Brunet , P. Bryant Chase , Goran Mihajlović , Brenda Schoffstall
      Investigations of cardiomyopathy mutations in Ca2+ regulatory proteins troponin and tropomyosin provide crucial information about cardiac disease mechanisms, and also provide insights into functional domains in the affected polypeptides. Hypertrophic cardiomyopathy-associated mutations TnI R145G, located within the inhibitory peptide (Ip) of human cardiac troponin I (hcTnI), and TnT R278C, located immediately C-terminal to the IT arm in human cardiac troponin T (hcTnT), share some remarkable features: structurally, biochemically, and pathologically. Using bioinformatics, we find compelling evidence that TnI and TnT, and more specifically the affected regions of hcTnI and hcTnT, may be related not just structurally but also evolutionarily. To test for functional interactions of these mutations on Ca2+-regulation, we generated and characterized Tn complexes containing either mutation alone, or both mutations simultaneously. The most important results from in vitro motility assays (varying [Ca2+], temperature or HMM density) show that the TnT mutant “rescued” some deleterious effects of the TnI mutant at high Ca2+, but exacerbated the loss of function, i.e., switching off the actomyosin interaction, at low Ca2+. Taken together, our experimental results suggest that the C-terminus of cTnT aids Ca2+-regulatory function of cTnI Ip within the troponin complex.
      Graphical abstract image

      PubDate: 2014-06-07T15:27:45Z
       
  • Biophysical and biomechanical properties of myofilament proteins
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Michael Regnier



      PubDate: 2014-06-07T15:27:45Z
       
  • Restrictive cardiomyopathy mutations demonstrate functions of the
           C-terminal end-segment of troponin I
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Shirin Akhter , Kenneth Bueltmann Jr. , Xupei Huang , J.-P. Jin
      The C-terminal end-segment of Troponin I (TnI) corresponding to the last 27–33 amino acids is the most conserved structure of TnI and interacts with tropomyosin in a Ca2+-regulated manner, suggesting a role in muscle relaxation. Mutations in the C-terminal end-segment of cardiac TnI cause restrictive cardiomyopathy. Here we demonstrate that mouse cardiac TnI containing R193H or R205H mutation have significantly conformational changes in the region interfacing with troponin T (TnT) and increased binding affinity for TnT. These restrictive cardiomyopathy mutations also exhibit increased binding affinity for troponin C at pCa 4. The effects of R193H mutation were more profound than that of R205H. Tertiary troponin complex was reconstituted using the TnI mutants and a mini TnT lacking tropomyosin-binding sites to examine the interaction between the C-terminal end-segment of TnI and tropomyosin. The results showed that, R193H, but not R205H, caused a moderate but statistically significant increase in the binding affinity for tropomyosin at pCa 9. Similar trend was observed at pCa 5.5 but not pCa 4. These results provide novel evidence for the function of the C-terminal end-segment of TnI, where mutations with conformational effects alter TnI’s interaction with other troponin subunits and tropomyosin to cause diastolic dysfunction.
      Graphical abstract image

      PubDate: 2014-06-07T15:27:45Z
       
  • Increased myocardial short-range forces in a rodent model of diabetes
           reflect elevated content of β myosin heavy chain
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Charles S. Chung , Mihail I. Mitov , Leigh Ann Callahan , Kenneth S. Campbell
      Diastolic dysfunction is a clinically significant problem for patients with diabetes and often reflects increased ventricular stiffness. Attached cross-bridges contribute to myocardial stiffness and produce short-range forces, but it is not yet known whether these forces are altered in diabetes. In this study, we tested the hypothesis that cross-bridge-based short-range forces are increased in the streptozotocin (STZ) induced rat model of type 1 diabetes. Chemically permeabilized myocardial preparations were obtained from 12week old rats that had been injected with STZ or vehicle 4weeks earlier, and activated in solutions with pCa (=−log10[Ca2+]) values ranging from 9.0 to 4.5. The short-range forces elicited by controlled length changes were ∼67% greater in the samples from the diabetic rats than in the control preparations. This change was mostly due to an increased elastic limit (the length change at the peak short-range force) as opposed to increased passive muscle stiffness. The STZ-induced increase in short-ranges forces is thus unlikely to reflect changes to titin and/or collagen filaments. Gel electrophoresis showed that STZ increased the relative expression of β myosin heavy chain. This molecular mechanism can explain the increased short-ranges forces observed in the diabetic tissue if β myosin molecules remain bound between the filaments for longer durations than α molecules during imposed movements. These results suggest that interventions that decrease myosin attachment times may be useful treatments for diastolic dysfunction associated with diabetes.


      PubDate: 2014-06-07T15:27:45Z
       
  • Effect of exercise training on post-translational and post-transcriptional
           regulation of titin stiffness in striated muscle of wild type and IG KO
           mice
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Carlos Hidalgo , Chandra Saripalli , Henk L. Granzier
      Exercise has beneficial effects on diastolic dysfunction but the underlying mechanisms are not well understood. Here we studied the effects of exercise on the elastic protein titin, an important determinant of diastolic stiffness, in both the left ventricle and the diaphragm. We used wild type mice and genetically engineered mice with HFpEF symptoms (IG KO mice), including diastolic dysfunction. In the diaphragm muscle, exercise increased the expression level of titin (increased titin:MHC ratio) which is expected to increase titin-based stiffness. This effect was absent in the LV. We also studied the constitutively expressed titin residues S11878 and S12022 that are known targets of CaMKIIδ and PKCα with increased phosphorylation resulting in an increase in titin-based passive stiffness. The phosphorylation level of S11878 was unchanged whereas S12022 responded to exercise with a reduction in the phosphorylation level in the LV and, interestingly, an increase in the diaphragm. These changes are expected to lower titin’s stiffness in the LV and increase stiffness in the diaphragm. We propose that these disparate effects reflect the unique physiological needs of the two tissue types and that both effects are beneficial.


      PubDate: 2014-06-07T15:27:45Z
       
  • The myofilament elasticity and its effect on kinetics of force generation
           by the myosin motor
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Gabriella Piazzesi , Mario Dolfi , Elisabetta Brunello , Luca Fusi , Massimo Reconditi , Pasquale Bianco , Marco Linari , Vincenzo Lombardi
      The half-sarcomere is the functional unit of striated muscle, in which, according to a “linear” mechanical model, myosin motors are parallel force generators with an average strain s acting between the opposing myosin and actin filaments that behave as a series elastic element with compliance C f. Thus the definition of the mechanism of force generation by myosin motors in muscle requires integration of the crystallographic model of the working stroke with the mechanical constraints provided by the organization of motors in the half-sarcomere. The relation between half-sarcomere compliance and force (C hs–T) during the development of isometric contraction deviates, at low forces, from that predicted by the linear model, indicating the presence of an elastic element in parallel with the myosin motors, which may influence the estimate of s. A working stroke model, kinetically constrained by the early phase of the isotonic velocity transient following a force step, predicts that the rate of quick force recovery following a length step is reduced to the observed value by a C f of 12.6nm/MPa. With this value of C f, the fit of C hs–T relation during the isometric force rise gives s =1.8–1.9nm, similar to the values estimated using the linear model.


      PubDate: 2014-06-07T15:27:45Z
       
  • Random myosin loss along thick-filaments increases myosin attachment time
           and the proportion of bound myosin heads to mitigate force decline in
           skeletal muscle
    • Abstract: Publication date: 15 June–1 July 2014
      Source:Archives of Biochemistry and Biophysics, Volumes 552–553
      Author(s): Bertrand C.W. Tanner , Mark McNabb , Bradley M. Palmer , Michael J. Toth , Mark S. Miller
      Diminished skeletal muscle performance with aging, disuse, and disease may be partially attributed to the loss of myofilament proteins. Several laboratories have found a disproportionate loss of myosin protein content relative to other myofilament proteins, but due to methodological limitations, the structural manifestation of this protein loss is unknown. To investigate how variations in myosin content affect ensemble cross-bridge behavior and force production we simulated muscle contraction in the half-sarcomere as myosin was removed either (i) uniformly, from the Z-line end of thick-filaments, or (ii) randomly, along the length of thick-filaments. Uniform myosin removal decreased force production, showing a slightly steeper force-to-myosin content relationship than the 1:1 relationship that would be expected from the loss of cross-bridges. Random myosin removal also decreased force production, but this decrease was less than observed with uniform myosin loss, largely due to increased myosin attachment time (ton ) and fractional cross-bridge binding with random myosin loss. These findings support our prior observations that prolonged ton may augment force production in single fibers with randomly reduced myosin content from chronic heart failure patients. These simulations also illustrate that the pattern of myosin loss along thick-filaments influences ensemble cross-bridge behavior and maintenance of force throughout the sarcomere.


      PubDate: 2014-06-07T15:27:45Z
       
  • Role of the disaggregase ClpB in processing of proteins aggregated as
           inclusion bodies
    • Abstract: Publication date: Available online 2 June 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Kamila Zblewska , Joanna Krajewska , Michal Zolkiewski , Sabina Kędzierska-Mieszkowska
      Overproduction of heterologous proteins in bacterial systems often results in the formation of insoluble inclusion bodies (IBs), which is a major impediment in biochemical research and biotechnology. In principle, the activity of molecular chaperones could be employed to gain control over the IB formation and to improve the recombinant protein yields, but the potential of each of the major bacterial chaperones (DnaK/J, GroEL/ES, and ClpB) to process IBs has not been fully established yet. We investigated the formation of inclusion bodies (IBs) of two aggregation-prone proteins, VP1LAC and VP1GFP, overproduced in E scherichia coli in the presence and absence of the chaperone ClpB. We found that both ClpB isoforms, ClpB95 and ClpB80 accumulated in E. coli cells during the production of IBs. The amount of IB proteins increased in the absence of ClpB. ClpB supported the resolubilization and reactivation of the aggregated VP1LAC and VP1GFP in E. coli cells. The IB disaggregation was optimal in the presence of both ClpB95 and ClpB80. Our results indicate an essential role of ClpB in controlling protein aggregation and inclusion body formation in bacteria.


      PubDate: 2014-06-07T15:27:45Z
       
  • Activated protease receptor-2 induces GATA6 expression to promote survival
           in irradiated colon cancer cells
    • Abstract: Publication date: Available online 1 June 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Wen-song Cai , Fei Shen , Jiang-lin Li , Zhe Feng , Ying-cheng Wang , Huan-qing Xiao , Bo Xu
      Background and aims The resistance to irradiation is common and a great drawback in the treatment of cancer with radiotherapy; the underlying mechanism is unclear. GATA binding protein 6 (GATA6) is associated with the pathogenesis of cancer. This study aims to investigate the role of GATA6 on compromising irradiation effect on HT55 and HT29 cells, 2 colorectal cancer cell lines. Methods Human colon cancer cell lines, HT55 and HT29 cells, were treated with irradiation in the culture. Apoptosis of HT55 and HT29 cells was determined by flow cytometry. The expression of PAR2 and GATA6 in HT55 and HT29 cells was analyzed by real time RT-PCR and Western blotting. The gene silence and gene over expression were employed to observe the effect of GATA6 on p53 expression in HT55 and HT29 cells. Results The results showed that HT55 and HT29 cells expressed protease-activated receptor-2 (PAR2). Irradiation induced 38.6% HT55 cell and 33.8% HT29 cell apoptosis, which reduced to 4.2% and 5.6%, respectively after activation of PAR2. Exposure to irradiation increased the expression of GATA6; the latter played a critical role in suppression of p53 expression in HT55 and HT29 cells. Inhibition of GATA6 significantly increased the effect of irradiation on HT55 and HT29 cells. Conclusions Activation of PAR2 compromises the effect of irradiation on inducing colorectal cancer cell apoptosis, which can be prevented by inhibition of GATA6 expression.


      PubDate: 2014-06-07T15:27:45Z
       
  • Regulation of energy metabolism by the skeleton: Osteocalcin and beyond
    • Abstract: Publication date: Available online 1 June 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Mathieu Ferron , Julie Lacombe
      The skeleton has recently emerged as an endocrine organ implicated in the regulation of glucose and energy metabolism. This function of bone is mediated, at least in part, by osteocalcin, an osteoblast-derived protein acting as a hormone stimulating insulin sensitivity, insulin secretion and energy expenditure. Osteocalcin secretion and bioactivity is in turn regulated by several hormonal cues including insulin, leptin, the sympathetic nervous system and glucocorticoids. Recent findings support the notion that osteocalcin functions and regulations are conserved between mice and humans. Moreover, studies in mice suggest that osteocalcin could represent a viable therapeutic approach for the treatment of obesity and insulin resistance. In this review, we summarize the current knowledge on osteocalcin functions, its various modes of action and the mechanisms implicated in the control of this hormone.


      PubDate: 2014-06-07T15:27:45Z
       
  • Effects of the melanin precursor 5,6-dihydroxy-indole-2-carboxylic acid
           (DHICA) on DNA damage and repair in the presence of reactive oxygen
           species
    • Abstract: Publication date: Available online 1 June 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Maria Carolina Pellosi , Andréia Akemi Suzukawa , Alexsandra Cristina Scalfo , Paolo Di Mascio , Carolina Parga Martins Pereira , Nadja Cristhina de Souza Pinto , Daniela de Luna Martins , Glaucia Regina Martinez
      Eumelanin is a heterogeneous polymer composed of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI). Studies have shown that DHICA promotes single strand breaks in plasmid DNA exposed to ultraviolet B radiation (UVB, 313nm) and in DNA from human keratinocytes exposed to ultraviolet A radiation (UVA, 340–400nm). Singlet molecular oxygen (1O2) is the main reactive species formed by UVA radiation on the skin. In this context, we now report that DHICA can cause single strand breaks in plasmid DNA even in the absence of light radiation. Interestingly, when DHICA was pre-oxidized by 1O2, it lost this harmful capacity. It was also found that DHICA could interact with DNA, disturbing Fpg activity and decreasing its recognition of lesions by ∼50%. Additionally, the free nucleoside deoxyguanosine (dGuo) was used to evaluate whether DHICA would interfere with the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and spiroiminodihydantoin (dSp) by 1O2 or with the formation of 8-oxodGuo by hydroxyl radical ( OH). We observed that when dGuo was oxidized by 1O2 in the presence of DHICA, 8-oxodGuo formation was increased. However, when dGuo was oxidized by OH in the presence of DHICA, 8-oxodGuo levels were lower than in the absence of the precursor. Overall, our data reveal an important role for this eumelanin precursor in both the promotion and the protection of DNA damage and imply that it can impair DNA repair.


      PubDate: 2014-06-07T15:27:45Z
       
  • Specific localization of quercetin-3-O-glucuronide in human brain
    • Abstract: Publication date: Available online 1 June 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Akari Ishisaka , Rie Mukai , Junji Terao , Noriyuki Shibata , Yoshichika Kawai
      In recent years, many papers have suggested that dietary flavonoids may exert beneficial effects in the brain tissue for the protection of neurons against oxidative stress and inflammation. However, the bioavailability of flavonoids across the blood–brain barrier and the localization in the brain remain controversial. Thus, we examined the localization of quercetin-3-O-glucuronide (Q3GA), a major phase-II metabolite of quercetin, in the human brain tissues with or without cerebral infarction by immunohistochemical staining using anti-Q3GA antibody. A significant immunoreactivity was observed in the epithelial cells of the choroid plexus, which constitute the structural basis of the blood–cerebrospinal fluid (CSF) barrier, and in the foamy macrophages of recent infarcts. The cellular accumulation of Q3GA was also reproduced in vitro in macrophage-like RAW264, microglial MG6, and brain capillary endothelial RBEC1. It is of interest that a common feature of these cell lines is the deconjugation of Q3GA, resulting in the cellular accumulation of non-conjugated quercetin and the methylated forms. We then examined the anti-inflammatory activity of Q3GA and the deconjugated forms in the lipopolysaccharide-stimulated macrophage cells and revealed that the deconjugated forms (quercetin and a methylated form isorhamnetin), but not Q3GA itself, exhibited inhibitory effects on the inflammatory responses through attenuation of the c-Jun N-terminal kinase pathway. These results suggested that a quercetin glucuronide can pass through the blood–brain barrier, perhaps the CSF barrier, accumulate in specific types of cells, such as macrophages, and act as anti-inflammatory agents in the brain through deconjugation into the bioactive non-conjugated forms.


      PubDate: 2014-06-07T15:27:45Z
       
  • Different involvement of extracellular calcium in two modes of cell death
           induced by nanosecond pulsed electric fields
    • Abstract: Publication date: Available online 1 June 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Keiko Morotomi-Yano , Hidenori Akiyama , Ken-ichi Yano
      Exposure of cultured cells to nanosecond pulsed electric fields (nsPEFs) induces various cellular responses, including the influx of extracellular Ca2+ and cell death. Recently, nsPEFs have been regarded as a novel means of cancer therapy, but their molecular mechanism of action remains to be fully elucidated. Here, we demonstrate the involvement of extracellular Ca2+ in nsPEF-induced cell death. Extracellular Ca2+ was essential for necrosis and consequent poly(ADP-ribose) (PAR) formation in HeLa S3 cells. Treatment with a Ca2+ ionophore enhanced necrosis as well as PAR formation in nsPEF-exposed HeLa S3 cells. In the absence of extracellular Ca2+, HeLa S3 cells were less susceptible to nsPEFs and exhibited apoptotic proteolysis of caspase 3 and PARP-1. HeLa S3 cells retained the ability to undergo apoptosis even after nsPEF exposure but instead underwent necrosis, suggesting that necrosis is the preferential mode of cell death. In K562 and HEK293 cells, exposure to nsPEFs resulted in the formation of necrosis-associated PAR, whereas Jurkat cells exclusively underwent apoptosis independently of extracellular Ca2+. These observations demonstrate that the mode of cell death induced by nsPEFs is cell-type dependent and that extracellular Ca2+ is a critical factor for nsPEF-induced necrosis.


      PubDate: 2014-06-07T15:27:45Z
       
  • Ammonia-induced energy disorders interfere with bilirubin metabolism in
           hepatocytes
    • Abstract: Publication date: Available online 27 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Qiongye Wang , Yanfang Wang , Zujiang Yu , Duolu Li , Bin jia[a] Jiangjing Li , Kelei Guan , Yubing Zhou , Yanling Chen , Quancheng Kan
      Hyperammonemia and jaundice are the most common clinical symptoms of hepatic failure. Decreasing the level of ammonia in the blood is often accompanied by a reduction in bilirubin in patients with hepatic failure. Previous studies have shown that hyperammonemia can cause bilirubin metabolism disorders, however it is unclear exactly how hyperammonemia interferes with bilirubin metabolism in hepatocytes. The purpose of the current study was to determine the mechanism or mechanisms by which hyperammonemia interferes with bilirubin metabolism in hepatocytes. Cell viability and apoptosis were analyzed in primary hepatocytes that had been exposed to ammonium chloride. Mitochondrial morphology and permeability were observed and analyzed, intermediates of the tricarboxylic acid (TCA)1 cycle were determined and changes in the expression of enzymes related to bilirubin metabolism were analyzed after ammonia exposure. Hyperammonemia inhibited cell growth, induced apoptosis, damaged the mitochondria and hindered the TCA cycle in hepatocytes. This led to a reduction in energy synthesis, eventually affecting the expression of enzymes related to bilirubin metabolism, which then caused further problems with bilirubin metabolism. These effects were significant, but could be reversed with the addition of adenosine triphosphate (ATP). This study demonstrates that ammonia can cause problems with bilirubin metabolism by interfering with energy synthesis.


      PubDate: 2014-06-01T14:28:35Z
       
  • Regulation of cortical and trabecular bone mass by communication between
           osteoblasts, osteocytes and osteoclasts
    • Abstract: Publication date: Available online 26 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Natalie A Sims , Christina Vrahnas
      The size and strength of bone is determined by two fundamental processes. One process, bone remodelling, renews the skeleton throughout life. In this process existing bone is resorbed by osteoclasts and replaced, in the same location, by osteoblasts. The other process is bone modelling, where bone formation and resorption occur at different sites so that the shape of bone is changed. Recent data suggests that both remodelling and modelling are controlled by signals between the cells that carry out these two processes. Osteoclasts both resorb bone, and provide inhibitory and stimulatory signals, including cardiotrophin-1 and sphingosine-1-kinase, to the osteoblast lineage thereby regulating their differentiation and activity on both trabecular and cortical surfaces. In addition, the osteoblast lineage, including osteoblast progenitors, matrix-producing osteoblasts, bone lining cells, and matrix-embedded osteocytes, produce both inhibitory and stimulatory factors that stimulate osteoclast differentiation. We will discuss the roles of osteoblast- and osteocyte-derived RANKL, and paracrine, autocrine and endocrine factors, such as ephrinB2, the IL-6 / gp130 family of cytokines, parathyroid hormone, and its related peptide, PTHrP. These factors not only stimulate RANKL production, but also stimulate osteoblast differentiation and activity. This review will focus on recent data, generated from pharmacological and genetic studies of mouse models and what these data reveal about these pathways at different stages of osteoblast differentiation and their impact on both bone remodelling and modelling in trabecular and cortical bone.


      PubDate: 2014-06-01T14:28:35Z
       
  • Polyphenols and mitochondria: An update on their increasingly emerging
           ROS-scavenging independent actions
    • Abstract: Publication date: Available online 26 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Cristian Sandoval-Acuña , Jorge Ferreira , Hernán Speisky
      Polyphenols, ubiquitously present in fruits and vegetables, have been traditionally viewed as antioxidant molecules. Such contention emerged, mainly from their well established in vitro ability to scavenge free radicals and other reactive oxygen species (ROS). During the last decade, however, increasing evidence has emerged supporting the ability of certain polyphenols to also exert numerous ROS-scavenging independent actions. Although the latter can comprise the whole cell, particular attention has been placed on the ability of polyphenols to act, whether favorably or not, on a myriad of mitochondrial processes. Thus, some particular polyphenols are now recognized as molecules capable of modulating pathways that define mitochondrial biogenesis (i.e., inducing sirtuins), mitochondrial membrane potential (i.e., mitochondrial permeability transition pore opening and uncoupling effects), mitochondrial electron transport chain and ATP synthesis (i.e., modulating complexes I to V activity), intra-mitochondrial oxidative status (i.e., inhibiting/inducing ROS formation/removal enzymes), and ultimately mitochondrially-triggered cell death (i.e., modulating intrinsic-apoptosis). The present review describes recent evidence on the ability of some polyphenols to modulate each of the formerly mentioned pathways, and discusses on how, by acting on such mitochondrial processes, polyphenols may afford protection against those mitochondrial damaging events that appear to be key in the cellular toxicity induced by various xenobiotics as well as that seen during the development of several ROS-related diseases.


      PubDate: 2014-06-01T14:28:35Z
       
  • Rutin inhibits UVB radiation-induced expression of COX-2 and iNOS in
           hairless mouse skin: p38 MAP kinase and JNK as potential targets
    • Abstract: Publication date: Available online 26 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Ki-Seok Choi , Joydeb Kumar Kundu , Kyung-Soo Chun , Hye-Kyung Na , Young-Joon Surh
      Exposure to excessive ultraviolet B (UVB) radiation, a complete environmental carcinogen, induces oxidative and inflammatory skin damage, thereby increasing the risk of skin carcinogenesis. The antioxidant and anti-inflammatory activities of a wide variety of plant polyphenols have been reported. Rutin (3-rhamnosyl-glucosylquercetin), a polyphenol present in many edible plants, possesses diverse pharmacological properties including antioxidant, anti-inflammatory, antimutagenic and anticancer activities. The present study was aimed to investigate the effects of rutin on UVB-induced inflammation in mouse skin in vivo. Topical application of rutin onto the dorsal skin of female HR-1 hairless mice 30 min prior to UVB irradiation diminished epidermal hyperplasia and the levels of proteins modified by 4-hydroxynonenal, which is a biochemical hallmark of lipid peroxidation. Topical application of rutin also significantly inhibited UVB-induced expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), two representative inflammatory enzymes, in hairless mouse skin. Rutin inhibited the DNA binding of activator protein-1 (AP-1) and phosphorylation of signal transducer and activator of transcription-3 (STAT3) in mouse skin exposed to UVB. Moreover, rutin attenuated UVB-induced phosphorylation of p38 mitogen-activated protein (MAP) kinase and c-Jun-N-terminal kinase (JNK). Pharmacological inhibition of p38 MAP kinase and JNK decreased UVB-induced expression of COX-2 in mouse skin. Taken together, these findings suggest that rutin exerts anti-inflammatory effects in UVB-irradiated mouse skin by inhibiting expression of COX-2 and iNOS, which is attributable to its suppression of p38 MAP kinase and JNK signaling responsible for AP-1 activation.
      Graphical abstract image Highlights

      PubDate: 2014-06-01T14:28:35Z
       
  • Possible allostery and oligomerization of recombinant plastidial
           sn-glycerol-3-phosphate acyltransferase
    • Abstract: Publication date: Available online 16 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Xue Chen , Robin Hryniuk , Crystal Snyder , Martin Truksa , Jian Zhang , Saleh Shah , Randall J. Weselake
      Plastidial acyl-acyl carrier protein:sn-glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the acyl-acyl carrier protein-dependent sn-1 acylation of sn-glycerol 3-phosphate (G3P) to produce lysophosphatic acid. Functional recombinant Erysimum asperum GPAT (EaGPAT), devoid of transit peptide, was produced in yeast. Analysis of the dependence of EaGPAT activity on increasing G3P concentration resulted in a hyperbolic response. EaGPAT exhibited a preference for 18-carbon unsaturated acyl-CoAs. Assays with concentrations of oleoyl-CoA up to 90μM revealed an exponential response to increasing concentrations of acyl donor, and the introduction of increasing concentrations of unlabeled linoleoyl-CoA into the standard reaction mixture resulted in increased incorporation of radiolabeled oleoyl moieties into lysophosphatidic acid. Collectively, the kinetic results suggest that acyl-CoA may act as both substrate and allosteric effector. EaGPAT was also shown to oligomerize to form higher molecular mass multimers, with the monomer and trimer being the predominant forms of the enzyme. Since most allosteric enzyme exhibit quaternary structure, the self-associating properties of EaGPAT are consistent with those of an allosteric enzyme. These results could have important regulatory implications when plastidial GPAT is introduced into a cytoplasmic environment where acyl-CoA is the acyl donor supporting cytoplasmic glycerolipid assembly.


      PubDate: 2014-05-25T16:15:16Z
       
  • The chalcones cardamonin and flavokawain B inhibit the differentiation of
           preadipocytes to adipocytes by activating ERK
    • Abstract: Publication date: Available online 17 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Tianshun Zhang , Norio Yamamoto , Yoko Yamashita , Hitoshi Ashida
      Aim We searched for polyphenols capable of inhibiting the lipid accumulation in 3T3-L1 cells, and investigated the mechanisms of two effective chalcones cardamonin and flavokawain B on differentiation of preadipocytes. Method and results We treated 3T3-L1 cells with a panel of 46 polyphenols and measured intracellular lipid accumulation by Sudan II staining. Four of them, including cardamonin and flavokawain B, inhibited lipid accumulation. In the further study, cardamonin and flavokawain B inhibited lipid accumulation by downregulating the expression of CCAAT/enhancer binding protein (C/EBP)-β, C/EBPα, and peroxisome proliferator-activated receptor-γ (PPARγ) at both mRNA and protein levels. Cardamonin and flavokawain B also increased phosphorylation of extracellular signal-regulated kinase (ERK) in the early phase of adipocyte differentiation. PD98059, an ERK inhibitor, restored C/EBPβ, PPARγ expression and intracellular lipid accumulation in adipocytes. Moreover, cardamonin and flavokawain B also modulated the secretion of C-reactive protein, dipeptidyl peptidase IV, interleukin-6, tumor necrosis factor-α and fibroblast growth factor-21 in mature adipocytes. Conclusions These results indicate that ERK activation and consequent downregulation of adipocyte-specific transcription factors are involved in the inhibitory effects of the chalcones cardamonin and flavokawain B on adipocyte differentiation. Moreover, cardamonin and flavokawain B are able to modulate secretion of adipokines in mature adipocytes.
      Graphical abstract image Highlights

      PubDate: 2014-05-25T16:15:16Z
       
  • Mechanisms of reduced flavin transfer in the two-component
           flavin-dependent monooxygenases
    • Abstract: Publication date: Available online 20 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Jeerus Sucharitakul , Ruchanok Tinikul , Pimchai Chaiyen
      Two-component flavin-dependent enzymes are abundant in nature and are involved in a wide variety of biological reactions. These enzymes consist of a reductase which generates a reduced flavin and a monooxygenase that utilizes the reduced flavin as a substrate for monooxygenation. As reduced flavin is unstable and can be oxidized by oxygen, these enzymes must have a means to efficiently coordinate the transfer of the reduced flavin such that auto-oxidation can be minimized. Various types of experiments and methodologies have been used to probe the mode of reduced flavin transfer. Results from many systems have indicated that the transfer can be achieved by free diffusion and that the presence of one component has no influence on the kinetics of the other component. Contradicting results indicating that the transfer of the reduced flavin may be achieved via protein–protein mediation also exist. Regardless of the mode of reduced flavin transfer, these enzymes have a means to control their overall kinetics such that the reaction rate is slow when the demand for oxygenation is not high.


      PubDate: 2014-05-25T16:15:16Z
       
  • Evolution of protein interactions: From interactomes to interfaces
    • Abstract: Publication date: Available online 20 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Jessica Andreani , Raphael Guerois
      Protein–protein interactions lie at the heart of most cellular processes. Many experimental and computational studies aim to deepen our understanding of these interactions and improve our capacity to predict them. In this respect, the evolutionary perspective is most interesting, since the preservation of structure and function puts constraints on the evolution of proteins and their interactions. However, uncovering these constraints remains a challenge, and the description and detection of evolutionary signals in protein–protein interactions is currently a very active field of research. Here, we review recent works dissecting the mechanisms of protein–protein interaction evolution and exploring how to use evolutionary information to predict interactions, both at the global level of the interactome and at the detailed level of protein–protein interfaces. We first present to what extent protein–protein interactions are found to be conserved within interactomes and which properties can influence their conservation. We then discuss the evolutionary and co-evolutionary pressures applied on protein–protein interfaces. Finally, we describe how the computational prediction of interfaces can benefit from evolutionary inputs.


      PubDate: 2014-05-25T16:15:16Z
       
  • Extracellular vesicles: Specialized bone messengers
    • Abstract: Publication date: Available online 22 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Jess Morhayim , Marta Baroncelli , Johannes P. van Leeuwen
      Mammalian cells actively secrete factors that contribute to shape their microenvironment. These factors either travel freely or they are enclosed within the lipid bilayer of extracellular vesicles (EVs), and regulate the function of neighboring and distant cells. EVs are secreted by a wide spectrum of cell types and are found in various biological fluids. They convey their message by mediating the horizontal transfer of bioactive molecules, such as proteins, mRNAs and miRNAs, between cells. Recent studies showed the vital roles of EVs in a wide range of physiological and pathophysiological processes. In this review, we highlight the recent developments in the newly emerging EV field, including their biogenesis, molecular content and function. Moreover, we discuss the role of EVs in bone biology and their promising applications in diagnosis, drug development and regenerative therapy.


      PubDate: 2014-05-25T16:15:16Z
       
  • Metabolomics-based search for therapeutic agents for non-alcoholic
           steatohepatitis
    • Abstract: Publication date: Available online 22 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Terashima Yoshihiko , Shin Nishiumi , Akihiro Minami , Yuuki Kawano , Namiko Hoshi , Takeshi Azuma , Masaru Yoshida
      Background Non-alcoholic fatty liver disease (NAFLD) is the commonest form of chronic liver disease in developed countries. Non-alcoholic steatohepatitis (NASH), which represents advanced stage NAFLD, is increasingly being recognized as a major cause of liver-related morbidity and mortality. However, no effective drugs against NASH have yet been developed. Therefore, we searched for candidate therapeutic agents based on the changes in levels of hepatic metabolites via gas chromatography mass spectrometry (GC/MS)-based metabolome analysis of livers from methionine-choline deficient (MCD) diet-fed mice, a mouse model of NASH. Methods The metabolites were extracted from the livers of the MCD diet-fed mice and then analyzed using GC/MS. Subsequently, the MCD diet-fed mice were supplemented with hypotaurine, and the therapeutic effects of hypotaurine against steatohepatitis were evaluated. Results Ninety-nine metabolites were detected in the livers of the MCD diet-induced steatohepatitis model mice. Among these metabolites, hypotaurine exhibited the greatest decrease in its concentration in the mice. Supplementation with 2 mmol/kgBW hypotaurine attenuated liver injuries and fat accumulation caused by the MCD diet-induced steatohepatitis. Furthermore, 10 mmol/kgBW hypotaurine supplementation ameliorated fibrosis and oxidative stress induced by the MCD diet. Conclusions The present metabolome analysis-based study demonstrated that hypotaurine is a novel candidate therapeutic agent for NASH.


      PubDate: 2014-05-25T16:15:16Z
       
  • Kinetic investigation of the rate-limiting step of manganese- and
           iron-lipoxygenases
    • Abstract: Publication date: Available online 21 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Anneli Wennman , Saeid Karkehabadi , Ernst H. Oliw
      Lipoxygenases (LOX) oxidize polyunsaturated fatty acids to hydroperoxides, which are generated by proton coupled electron transfer to the metal center with FeIIIOH- or MnIIIOH-. Hydrogen abstraction by FeIIIOH- of soybean LOX-1 (sLOX-1) is associated with a large deuterium kinetic isotope effect (D-KIE). Our goal was to compare the D-KIE and other kinetic parameters at different temperatures of sLOX-1 with 13R-LOX with catalytic manganese (13R-MnLOX). The reaction rate and the D-KIE of sLOX-1 with unlabeled and [11-2H2]18:2n-6 were almost temperature independent with an apparent D-KIE of ∼56 at 30˚C, which is in agreement with previous studies. In contrast, the reaction rate of 13R-MnLOX increased 7-fold with temperature (8 to 50˚C), and the apparent D-KIE decreased linearly from ∼38 at 8˚C to ∼20 at 50˚C. The kinetic lag phase of 13R-MnLOX was consistently extended at low temperatures. The Phe337Ile mutant of 13R-MnLOX, which catalyzes antarafacial hydrogen abstraction and oxygenation in analogy with sLOX-1, retained the large D-KIE and its temperature-dependent reaction rate. The kinetic differences between13R-MnLOX and sLOX-1 may be due to protein dynamics, hydrogen donor-acceptor distances, and to the metal ligands, which may not equalize the 0.7 V-gap between the redox potentials of the free metals.


      PubDate: 2014-05-25T16:15:16Z
       
  • Methotrexate inhibits the viability of human melanoma cell lines and
           enhances Fas/Fas-ligand expression, apoptosis and response to
           interferon-alpha: rationale for its use in combination therapy
    • Abstract: Publication date: Available online 23 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Minakshi Nihal , Jianqiang Wu , Gary S. Wood
      Melanoma, a highly aggressive form of cancer, is notoriously resistant to available therapies. Methotrexate (MTX), an antifolate, competitively inhibits DNA synthesis and is effective for several types of cancer. In cutaneous T-cell lymphoma (CTCL), MTX increases Fas death receptor by decreasing Fas promoter methylation by blocking the synthesis of SAM, the principal methyl donor for DNMTs, resulting in enhanced Fas-mediated apoptosis. The objective of this study was to explore the effects of MTX in human melanoma. MTX variably inhibits the survival of melanoma cells and induced apoptosis as evident by annexin V positivity and senescence associated β-galactosidase activity induction. Furthermore, MTX caused increased transcript and protein levels of extrinsic apoptotic pathway factors Fas and Fas-ligand, albeit at different levels in different cell lines. Our pyrosequencing studies showed that this increased expression of Fas was associated with Fas promoter demethylation. Overall, the ability of MTX to up-regulate Fas/FasL and enhance melanoma apoptosis through extrinsic as well as intrinsic pathways might make it a useful component of novel combination therapies designed to affect multiple melanoma targets simultaneously. In support of this concept, combination therapy with MTX and interferon-alpha (IFNα) induced significantly greater apoptosis in the aggressive A375 cell line than either agent alone.


      PubDate: 2014-05-25T16:15:16Z
       
  • Osteoblast and Osteocyte: Games without frontiers
    • Abstract: Publication date: Available online 14 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Mattia Capulli , Riccardo Paone , Nadia Rucci
      The portrait of osteoblasts and osteocytes has been subjected to a revision, since a large body of evidence are attributing these cells amazing roles both inside and outside the bone. The osteoblast, long confined to its bone building function, is actually a very eclectic cell, actively regulating osteoclast formation and function as well as hematopoietic stem cells homeostasis. It is also an endocrine cell, affecting energy metabolism, male fertility and cognition through the release of osteocalcin, a perfect definition-fitting hormone in its uncarboxylated state. As for the osteocytes, many evidence shows that they do not merely represent the final destination of the osteoblasts, but they are instead very active cells that, besides a mechanosensorial function, actively contribute to the bone remodelling by regulating bone formation and resorption. The regulation is exerted by the production of Sclerostin (SOST), which in turn inhibits osteoblast differentiation by blocking Wnt/beta-catenin pathway. At the same time, osteocytes influence bone resorption both indirectly, by producing RANKL, which stimulates osteoclastogenesis, and directly by means of a local osteolysis, which is observed especially under pathological conditions. The great versatility of both these cells reflects the complexity of the bone tissue, which has not only a structural role, but influences and is influenced by different organs, taking part in homeostatic and adaptive responses affecting the whole organism.


      PubDate: 2014-05-14T12:56:24Z
       
  • Protein kinase C restricts transport of carnitine by amino acid
           transporter ATB0,+ apically localized in the blood-brain barrier
    • Abstract: Publication date: Available online 10 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Katarzyna Michalec , Caroline Mysiorek , Mélanie Kuntz , Vincent Bérézowski , Andrzej A. Szczepankiewicz , Grzegorz M. Wilczyński , Roméo Cecchelli , Katarzyna A. Nałęcz
      Carnitine (3-hydroxy-4-trimethylammoniobutyrate) is necessary for transfer of fatty acids through the inner mitochondrial membrane. Carnitine, not synthesized in the brain, is delivered there through the strongly polarized blood-brain barrier (BBB). Expression and presence of two carnitine transporters – organic cation/carnitine transporter (OCTN2) and amino acid transporter B0,+ (ATB0,+) have been demonstrated previously in an in vitro model of the BBB. Due to potential protein kinase C (PKC) phosphorylation sites within ATB0,+ sequence, the present study verified effects of this kinase on transporter function and localization in the BBB. ATB0,+ can be regulated by estrogen receptor α and up-regulated in vitro, therefore its presence in vivo was verified with the transmission electron microscopy. The analyses of brain slices demonstrated ATB0,+ luminal localization in brain capillaries, confirmed by biotinylation experiments in an in vitro model of the BBB. Brain capillary endothelial cells were shown to control carnitine gradient. ATB0,+ was phosphorylated by PKC, what correlated with inhibition of carnitine transport. PKC activation did not change the amount of ATB0,+ present in the apical membrane of brain endothelial cells, but resulted in transporter exclusion from raft microdomains. ATB0,+ inactivation by a lateral movement in plasma membrane after transporter phosphorylation has been postulated.
      Graphical abstract image Highlights

      PubDate: 2014-05-14T12:56:24Z
       
  • Phosphorylation of protein kinase C sites Ser42/44 decreases
           Ca2+-sensitivity and blunts enhanced length-dependent activation in
           response to protein kinase A in human cardiomyocytes
    • Abstract: Publication date: Available online 9 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Paul J.M. Wijnker , Vasco Sequeira , E. Rosalie Witjas-Paalberends , D. Brian Foster , Cristobal G. dos Remedios , Anne M. Murphy , Ger J.M. Stienen , Jolanda van der Velden
      Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca2+-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca2+-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca2+-sensitivity at physiological Ca2+-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.


      PubDate: 2014-05-14T12:56:24Z
       
  • The p65 subunit of NF-κB involves in RIP140-mediated inflammatory and
           metabolic dysregulation in cardiomyocytes
    • Abstract: Publication date: Available online 10 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Luankun Zhang , Yanfang Chen , Zhongbao Yue , Yanhong He , Jian Zou , Shaorui Chen , Min Liu , Xi Chen , Zhiping Liu , Xueping Liu , Xiaojun Feng , Min Li , Peiqing Liu
      The transcription factor NF-κB regulates expression of many genes that are involved in inflammation, fatty acid and glucose metabolism, and plays a crucial role in cardiac pathological processes. RIP140 is a corepressor that down-regulates expression of genes involved in the cellular substrate uptake and mitochondrial β-oxidation. In addition to this, RIP140 also acts as a coactivator for p65-NF-κB, potentiating the secretion of proinflammatory cytokines in macrophages, but the effects in cardiomyocytes are still unknown. In this study, overexpression of RIP140 induced proinflammatory gene expression and cytokine release in neonatal rat cardiomyocytes, which could be reversed by P65-NF-κB inhibition. Furthermore, RIP140-mediated repression of metabolic-related genes, mitochondrial biogenesis and metabolic function were weakened after knocking down of p65-NF-κB. These findings suggest that p65-NF-κB plays an important role in RIP140-mediated proinflammatory response and energy metabolism in cardiomyocytes, and provide evidence for the crosstalk between proinflammatory processes and metabolic dysregulation in the heart.
      Graphical abstract image Highlights

      PubDate: 2014-05-14T12:56:24Z
       
  • Modulation of tumor cell stiffnessand migration by type IV collagenthrough
           direct activation of integrin signaling pathway
    • Abstract: Publication date: Available online 10 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Sheng-Yi Chen , Jo-Shi Lin , Bei-Chang Yang
      Excessive collagen deposition plays a critical rolein tumor progression and metastasis. To understand how type IV collagen affects mechanical stiffness andmigration,low-collagen-IV-expressing transfectantsof B16F10, U118MG, and Huh7(denoted shCol cells) were established by the lentiviral-mediated delivery of small interfering RNA againsttype IV-α1 collagen (Col4A1). Although having similar growth rates, shCol cells showed a flatter morphology compared to that of thecorresponding controls. Notably, knocking down theCol4A1geneconferred the cells withhigher levels of elasticityand lower motility.Exposure to blocking antibodies against human β1integrinor α2β1integrinor the pharmacologicalinhibition ofSrc and ERK activity by PP1 and U0126, respectively, effectively reducedcell motility and raisedcell stiffness. Reduced Src and ERK activitiesin shCol cells indicate theinvolvement of a collagen IV/integrin signaling pathway. The forced expression ofβ1integrinsignificantly stimulated Src and ERK phosphorylation, reduced cell stiffness, and acceleratedcell motility. In an experimental metastasis assay using C57BL/6 mice, B16F10 shColcells formed significantly fewer and smaller lung nodules,confirming the contribution of collagen to metastasis.In summary, theintegrin signaling pathway activated in a tumor environment with collagendeposition is responsible for low cell elasticity and high metastatic ability.


      PubDate: 2014-05-14T12:56:24Z
       
  • Redox equilibration after one-electron reduction of cytochrome c oxidase:
           radical formation and a possible hydrogen relay mechanism
    • Abstract: Publication date: Available online 5 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Damian Ashe , Trevor Alleyne , Michael Wilson , Dimitri Svistunenko , Peter Nicholls
      Kinetic studies using UV/visible and EPR spectroscopy were carried out to follow the distribution of electrons within beef heart cytochrome c oxidase (CcO), both active and cyanide-inhibited, following addition of reduced cytochrome c as electron donor. In the initial one-electron reduced state the electron is shared between three redox centres, heme a, CuA and a third site, probably CuB. Using a rapid freeze system and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) a protein radical was also detected. The EPR spectrum of the DMPO adduct of this radical was consistent with tyrosyl radical capture. This may be a feature of a charge relay mechanism involved in some part of the CcO electron transfer system from bound cytochrome c via CuA and heme a to the a 3CuB binuclear centre.


      PubDate: 2014-05-09T10:51:04Z
       
  • Delivery of Nitric Oxide to the Interior of Mammalian Cell by Carbon
           Nanotube: MD Simulation
    • Abstract: Publication date: Available online 4 May 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Przemysław Raczyński , Krzysztof Górny , Aleksander Dawid , Zygmunt Gburski
      Computer simulations have been performed to study the nanoindentationof phospholipid bilayer by the single-walled armchair carbon nanotube, filled with the nitric oxide molecules. The process has been simulated by means of molecular dynamics (MD) technique at physiological temperature T = 310 K with a constant pulling velocity of the nanotube. The force acting on the nanotube during membrane penetration has been calculated. We show that the indentation by carbon nanotube does not permanently destroy the membrane structure (self-sealing of the membrane occurs). The mobility of nitric oxide molecules during the membrane nanoindentation is discussed.


      PubDate: 2014-05-05T08:26:49Z
       
  • Screening of integrin-binding peptides in a laminin peptide library
           derived from the mouse laminin β chain short arm regions
    • Abstract: Publication date: Available online 28 April 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Fumihiko Katagiri , Masaharu Takagi , Minako Nakamura , Yoichiro Tanaka , Kentaro Hozumi , Yamato Kikkawa , Motoyoshi Nomizu
      Laminins, major components of basement membrane, consist of three different subunits, α, β, and γ chains, and so far, five α, three β, and three γ chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin α chain sequences (α1—α5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the β1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin β2 and β3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin β1 chain, 255-266), B67 (IPYSMEYEILIRY, mouse laminin β1 chain, 604-616), B2-105 (APNFWNFTSGRG, mouse laminin β2 chain, 1081-1092), and B3-19 (GHLTGGKVQLNL, mouse laminin β3 chain, 182-193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2-105, and B3-19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with α3, α6, and β1 integrins, and B34 and B3-19 interact with β1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2-64 (LPRAMDYDLLLRW, mouse laminin β2 chain, 618-630), the Glu8 residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide has potential for use as a molecular probe for integrins.


      PubDate: 2014-04-29T06:48:06Z
       
  • Membrane Permeability of the Human Pluripotent Stem Cells to Me2SO,
           Glycerol and 1,2-Propanediol
    • Abstract: Publication date: Available online 26 April 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Yanqing Xu , Liang Zhang , Jiandong Xu , Yuping Wei , Xia Xu
      Due to the unlimited capacity of self-renew and ability to differentiate into derivatives of three germ layers, the human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have a great potential in regenerative medicine. A major challenge we are facing during the long-term storage of human pluripotent stem cells with the conventional slow cooling rate is the low cell recovery rate after the cryopreservation which cannot meet the requirements for the future clinical applications. Evaluating the cell membrane permeability and the corresponding activation energy of hESCs and hiPSCs for water and different cryoprotective agents (CPA), including dimethyl sulfoxide (Me2SO), 1,2-propandiol and glycerol, is important for facilitating the development of cryopreservation protocol to enhance cell recovery after the cryopreservation. The osmotically inactive volume of hESCs and hiPSCs determined using the Boyle-van’t Hoff model was 0.32V0 and 0.42V0, respectively. The membrane permeability was assessed from the volume changes of cells exposed to Me2SO, 1,2-propanediol and glycerol at the temperatures ranging from 8°C to 30°C. These results showed the biophysical differences between hESCs and hiPSCs. Their activation energy for water and CPAs extrapolated from the Arrhenius relationship indicated that the water transport was probably not through the channel-mediated mechanism.


      PubDate: 2014-04-29T06:48:06Z
       
  • Effect of temperature, pH and heme ligands on the reduction of Cygb(Fec+)
           by ascorbate
    • Abstract: Publication date: Available online 26 April 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Jianjing Tong , Joseph R. Zweier , Rachael L. Huskey , Raed S. Ismail , Craig Hemann , Jay L. Zweier , Xiaoping Liu
      Cytoglobin (Cygb) plays a role in regulating vasodilation in response to changes in local oxygen concentration by altering the rate of nitric oxide (NO) metabolism. Because the reduction of Cygb(Fe3+) by a reductant is the control step for Cygb-mediated NO metabolism, we examined the effects of temperature, pH, and heme ligands on the Cygb(Fe3+) reduction by ascorbate (Asc) under anaerobic conditions. The standard enthalpy of Cygb(Fe3+) reduction by Asc was determined to be 42.4 ± 3.1 kJ/mol. The rate of Cygb(Fe3+) reduction increased ∼6% per °C when temperature varied from 35 °C to 40 °C. The yield and the rate of Cygb(Fe3+) reduction significantly increases with pH (2-3 times per pH unit), paralleling the formation of the Asc ion (A2-) and the increased stability of reduced state of heme iron at high pH values. Heme ligand cyanide (CN-) decreased the yield and the rate of Cygb(Fe3+) reduction, but ligands CO and NO allowed the process of Cygb(Fe3+) reduction to continue to completion. Critical information is provided for modeling and prediction of the process of Cygb-mediated NO metabolism in vessels in a range of temperature and pH values.


      PubDate: 2014-04-29T06:48:06Z
       
  • Analysis of the Solution Structure of Thermosynechococcus elongatus
           Photosystem I in n-dodecyl-β-D-maltoside Using Small-Angle Neutron
           Scattering and Molecular Dynamics Simulation
    • Abstract: Publication date: Available online 24 April 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Rosemary Le , Brad Harris , Ifeyinwa J. Iwuchukwu , Barry D. Bruce , Xiaolin Cheng , Shuo Qian , William T. Heller , Hugh O’Neill , Paul Frymier
      Small-angle neutron scattering (SANS) and molecular dynamics (MD) simulation were used to investigate the structure of trimeric photosystem I (PSI) from Thermosynechococcus elongatus (T. elongatus) stabilized in n-dodecyl-β-D-maltoside (DDM) detergent solution. Scattering curves of detergent and protein-detergent complexes were measured at 18% D2O, the contrast match point for the detergent, and 100% D2O, allowing observation of the structures of protein/detergent complexes. It was determined that the maximum dimension of the PSI-DDM complex was consistent with the presence of a monolayer belt of detergent around the periphery of PSI. A dummy-atom reconstruction of the shape of the complex from the SANS data indicates that the detergent envelope has an irregular shape around the hydrophobic periphery of the PSI trimer rather than a uniform, toroidal belt around the complex. A 50 ns MD simulation model (a DDM ring surrounding the PSI complex with extra interstitial DDM) of the PSI-DDM complex was developed for comparison with the SANS data. The results suggest that DDM undergoes additional structuring around the membrane-spanning surface of the complex instead of a simple, relatively uniform belt, as is generally assumed for studies that use detergents to solubilize membrane proteins.
      Graphical abstract image Highlights

      PubDate: 2014-04-24T09:15:28Z
       
  • Mechanistic studies on the flavin-dependent N6-lysine monooxygenase MbsG
           reveal an unusual control for catalysis
    • Abstract: Publication date: Available online 24 April 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Reeder M. Robinson , Pedro J. Rodriguez , Pablo Sobrado
      The mechanism of Mycobacterium smegmatis G (MbsG), a flavin-dependent L-lysine monooxygenase, was investigated under steady-state and rapid reaction conditions using primary and solvent kinetic isotope effects, substrate analogs, pH and solvent viscosity effects as mechanistic probes. The results suggest that L-lysine binds before NAD(P)H, which leads to a decrease in the rate constant for flavin reduction. L-lysine binding has no effect on the rate of flavin oxidation, which occurs in a one-step process without the observation of a C4a-hydroperoxyflavin intermediate. Similar effects were determined with several substrate analogs. Flavin oxidation is pH independent while the k cat/K m and k red/K D pH profiles for NAD(P)H exhibit single pK a value of ∼6.0, with increasing activity as the pH decrease. At lower pH, the enzyme becomes more uncoupled, producing more hydrogen peroxide and superoxide. Hydride transfer is partially rate-limiting at neutral pH and becomes more rate-limiting at low pH. An inverse solvent viscosity effect on k cat/K m for NAD(P)H was observed at neutral pH whereas a normal solvent viscosity effect was observed at lower pH. Together, the results indicate a unique mechanism where a rate-limiting and pH-sensitive conformational change occurs in the reductive half-reaction, which affects the efficiency of lysine hydroxylation.
      Graphical abstract image Highlights

      PubDate: 2014-04-24T09:15:28Z
       
  • Acetylation of Acetyl-CoA Synthetase from Mycobacterium tuberculosis Leads
           to Specific Inactivation of the Adenylation Reaction
    • Abstract: Publication date: Available online 18 April 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Tahel Noy , Hua Xu , John S. Blanchard
      Acetyl-CoA synthetase (ACS) catalyzes the formation of AcCoA from acetate, ATP and Coenzyme A, allowing the organism to grow on acetate as the sole carbon source. ACS was the first enzyme in Mycobacterium tuberculosis shown to be regulated by posttranslational acetylation by the cAMP-dependent protein acetyltransferase. This modification results in the inactivation of the enzyme and can be reversed in the presence of NAD+ and a mycobacterial sirtuin-like deacetylase. In this study we characterize the kinetic mechanism of MtACS, where the overall reaction can be divided into two half-reactions: the acetyl-adenylate forming reaction and the thiol-ligation reaction. We also provide evidence for the existence of the acetyl-adenylate intermediate via 31P-NMR spectroscopy. Furthermore, we dissect the regulatory role of K617 acetylation and show that acetylation inhibits only the first, adenylation half-reaction while leaving the second half reaction unchanged. Finally, we demonstrate that the chemical mechanism of the enzyme relies on a conformational change which is controlled by the protonation state of aspartate 525. Together with our earlier results, this suggests a degree of regulation of enzyme activity that is appropriate for the role of the enzyme in central carbon metabolism.
      Graphical abstract image Highlights

      PubDate: 2014-04-19T08:42:40Z
       
  • Altered Interactions Between Cardiac Myosin Binding Protein-C and
           α-Cardiac Actin Variants Associated with Cardiomyopathies
    • Abstract: Publication date: Available online 13 April 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Melissa L. Chow , Justin F. Shaffer , Samantha P. Harris , John F. Dawson
      The two genes most commonly associated with mutations linked to hypertrophic or dilated cardiomyopathies are β-myosin and cardiac myosin binding protein-C (cMyBP-C). Both of these proteins interact with cardiac actin (ACTC). Currently there are 16 ACTC variants that have been found in patients with HCM or DCM. While some of these ACTC variants exhibit protein instability or polymerization-deficiencies that might contribute to the development of disease, other changes could cause changes in protein-protein interactions between sarcomere proteins and ACTC. To test the hypothesis that changes in ACTC disrupt interactions with cMyBP-C, we examined the interactions between seven ACTC variants and the N-terminal C0C2 fragment of cMyBP-C. We found there was a significant decrease in binding affinity (increase in Kd values) for the A331P and Y166C variants of ACTC. These results suggest that a change in the ability of cMyBP-C to bind actin filaments containing these ACTC protein variants might contribute to the development of disease. These results also provide clues regarding the binding site of the C0C2 fragment of cMyBP-C on F-actin.


      PubDate: 2014-04-15T07:39:12Z
       
  • FRET Study of the Structural and Kinetic Effects of PKC Phosphomimetic
           Cardiac Troponin T Mutants on Thin Filament Regulation
    • Abstract: Publication date: Available online 5 April 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): William Schlecht , Zhiqun Zhou , King-Lun Li , Daniel Rieck , Yexin Ouyang , Wen-Ji Dong
      FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca2+ and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca2+ and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca2+ sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca2+-sensitivty. The rate of Ca2+-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca2+-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca2+-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca2+-sensitivity and likely play an important role in cardiac regulation.


      PubDate: 2014-04-10T04:51:08Z
       
  • Regulation of alkaline ceramidase activity by the c-Src-mediated pathway
    • Abstract: Publication date: Available online 5 April 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Hirotsune Sasaki , Kaori Toyomura , Wataru Matsuzaki , Aya Okamoto , Naoto Yamaguchi , Hiroyuki Nakamura , Toshihiko Murayama
      Ceramidase hydrolyzes ceramide to fatty acids and sphingosine, and sphingosine is then converted to sphingosine-1-phosphate. Ceramide and sphingosine-1-phosphate act as signaling molecules. Although stimuli coupling to protein kinases-dependent systems have been shown to regulate ceramidase activity, the exact role of c-Src-mediated signal has not been elucidated. We examined the effects of the downregulation of c-Src activity and c-Src overexpression on ceramidase activity in cells. In A549, CHO, and HeLa cells labeled with a fluorescent ceramide, 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide), the downregulation of c-Src by c-Src-shRNA and pharmacological inhibitors including SU6656 decreased levels of NBD-caproic acid. The overexpression of c-Src increased NBD-caproic acid levels in CHO and HeLa cells. Similar results were obtained in Na3VO4-treated cells having higher NBD-caproic acid levels. The downregulation and overexpression of c-Src decreased and increased ceramidase activity, respectively, in the lysates of A549 cells at pH 8.8. The ceramidase sensitivity to substrates, pH, and Ca2+ suggest that the c-Src- and SU6656-sensitive ceramidase is alkaline ceramidase (ACER), possibly Ca2+-activated ACER2. Serum starvation increased both ceramidase activity at pH 8.8 and expression of ACER2. Our data suggest that c-Src-mediated signal positively regulates ACER activity in a Ca2+-independent manner.


      PubDate: 2014-04-10T04:51:08Z
       
  • The sole tryptophan of amicyanin enhances its thermal stability but does
           not influence the electronic properties of the type 1 copper site
    • Abstract: Publication date: Available online 1 April 2014
      Source:Archives of Biochemistry and Biophysics
      Author(s): Brian A. Dow , Narayanasami Sukumar , Jason O. Matos , Moonsung Choi , Alfons Schulte , Suren A. Tatulian , Victor L. Davidson
      The cupredoxin amicyanin possesses a single tryptophan residue, Trp45. Its fluorescence is quenched when copper is bound even though it separated by 10.1 Å. Mutation of Trp45 to Ala, Phe, Leu and Lys resulted in undetectable protein expression. A W45Y amicyanin variant was isolated. The W45Y mutation did not alter the spectroscopic properties or intrinsic redox potential of amicyanin, but increased the pKa value for the pH-dependent redox potential by 0.5 units. This is due to a hydrogen-bond involving the His95 copper ligand which is present in reduced W45Y amicyanin but not in native amicyanin. The W45Y mutation significantly decreased the thermal stability of amicyanin, as determined by changes in the visible absorbance of oxidized amicyanin and in the circular dichroism spectra for oxidized, reduced and apo forms of amicyanin. Comparison of the crystal structures suggests that the decreased stability of W45Y amicyanin may be attributed to the loss of a strong interior hydrogen bond between Trp45 and Tyr90 in native amicyanin which links two of the β-sheets that comprise the overall structure of amicyanin. Thus, Trp45 is critical for stabilizing the structure of amicyanin but it does not influence the electronic properties of the copper which quenches its fluorescence.
      Graphical abstract image Highlights

      PubDate: 2014-04-05T03:21:01Z
       
 
 
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