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Archives of Biochemistry and Biophysics

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ISSN (Print) 0003-9861 - ISSN (Online) 1096-0384
Published by Elsevier

[2556 journals]

2-Arachidonoylglycerol is a substrate for butyrylcholinesterase: a potential mechanism for extracellular endocannabinoid regulation
- Abstract: Publication date: Available online 17 May 2013
  Source:Archives of Biochemistry and Biophysics
  Author(s): Jason Barricklow , Matthew Blatnik
  2-Arachidonoylglycerol (2-AG) is a component of the endocannabinoid receptor pathway and is primarily hydrolyzed by monoacylglycerol lipase (MAGL) in vivo. We found that the non-specific serine esterase, butyrylcholinesterase (BChE), can hydrolyze 2-AG with reasonable affinity and may present a new compensatory mechanism for endocannabinoid regulation. In vitro hydrolysis reactions of 2-AG with equine BChE were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) positive/negative electrospray ionization (ESI+/-) to measure the formation of arachidonic acid (AA) and the loss of 2-AG over time (min). The resulting Michaelis-Menten approximations reveal that BChE has affinity towards 2-AG in phosphate buffer at neutral pH (7.4). The calculated Vmax, Km and kcat were 12.1 nmol s-1, 57.5 μM, and 0.073 s-1, respectively, which produced a diffusion-controlled rate of association (kcat/Km) of 1.3 x 103 M-1 s-1. Human BChE 2-AG hydrolysis was measured by immunoprecipitating BChE from fresh plasma and monitoring 2-AG loss and AA formation over time. These findings show that BChE can hydrolyze 2-AG and which may be evidence of a more specific role for BChE in endocannabinoid regulation.
  
  PubDate: 2013-05-20T21:25:25Z
Solute transport across the articular surface of injured cartilage
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Hooi Chuan Chin , Mohammad Moeini , Thomas M. Quinn
  Solute transport through extracellular matrix (ECM) is important to physiology and contrast agent-based clinical imaging of articular cartilage. Mechanical injury is likely to have important effects on solute transport since it involves alteration of ECM structure. Therefore it is of interest to characterize effects of mechanical injury on solute transport in cartilage. Using cartilage explants injured by an established mechanical compression protocol, effective partition coefficients and diffusivities of solutes for transport across the articular surface were measured. A range of fluorescent solutes (fluorescein isothiocyanate, 4 and 40kDa dextrans, insulin, and chondroitin sulfate) and an X-ray contrast agent (sodium iodide) were used. Mechanical injury was associated with a significant increase in effective diffusivity versus uninjured explants for all solutes studied. On the other hand, mechanical injury had no effects on effective partition coefficients for most solutes tested, except for 40kDa dextran and chondroitin sulfate where small but significant changes in effective partition coefficient were observed in injured explants. Findings highlight enhanced diffusive transport across the articular surface of injured cartilage, which may have important implications for injury and repair situations. Results also support development of non-equilibrium methods for identification of focal cartilage lesions by contrast agent-based clinical imaging.
  Graphical abstract
  
  PubDate: 2013-05-20T21:25:25Z
MADD/DENN/Rab3GEP functions as a guanine nucleotide exchange factor for Rab27 during granule exocytosis of rat parotid acinar cells
- Abstract: Publication date: Available online 20 May 2013
  Source:Archives of Biochemistry and Biophysics
  Author(s): Akane Imai , Morié Ishida , Mitsunori Fukuda , Tomoko Nashida , Hiromi Shimomura
  We previously reported that the small GTPase Rab27 and its effectors regulate isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Although activation of Rab27 by a specific guanine nucleotide exchange factor (GEF) is thought to be required for amylase release, its activation mechanism is poorly understood, because GEF for Rab27 has not been reported in parotid acinar cells. In the present study, we investigated the possible involvement of MADD/DENN/Rab3GEP, which was recently described as a Rab27-GEF in melanocytes, in amylase release from rat parotid acinar cells. Reverse transcription-PCR analyses indicated that mRNA of DENND family members, including MADD, was expressed in parotid acinar cells. MADD protein was also expressed in the cytosolic fraction of parotid acinar cells. Incubation of an antibody against the C-terminal 150 amino acids of MADD (anti-MADD-C antibody) with streptolysin O-permeabilized parotid acinar cells caused not only inhibition of IPR-induced amylase release but also reduction in the amount of GTP-Rab27. Our findings indicated that MADD functions as a GEF for Rab27 in parotid acinar cells and that its GEF activity for Rab27, which is GDP/GTP cycling, is required for IPR-induced amylase release.
  
  PubDate: 2013-05-20T21:25:25Z
The nemaline myopathy-causing E117K mutation in β-tropomyosin reduces thin filament activation
- Abstract: Publication date: Available online 17 May 2013
  Source:Archives of Biochemistry and Biophysics
  Author(s): Olga E. Karpicheva , Paul Robinson , Adam Piers , Yurii S. Borovikov , Charles S. Redwood
  The effect of the nemaline myopathy-causing E117K mutation in β-tropomyosin (TM) on the structure and function of this regulatory protein was studied. The E117K mutant was found to have indistinguishable actin affinity compared with wild-type (WT) and similar secondary structure as measured by circular dichroism. However the E117K mutation significantly lowered maximum activation of actomyosin ATPase. To explain the molecular mechanism of impaired ATPase activation, WT and E117K TMs were covalently labeled at Cys-36 with 5-Iodoacetimido-fluorescein and incorporated into ghost muscle fibers. The changes in the position and flexibility of tropomyosin strands on the thin filaments were observed at simulation of weak and strong binding states of actomyosin at high or low Ca2+ by polarized fluorescence techniques. The E117K mutation was found to shift the tropomyosin strands towards the closed position and restrict the tropomyosin displacement during the transformation of actomyosin from weak to strong binding state thus leading to a reduction in thin filament activation.
  
  PubDate: 2013-05-20T21:25:25Z
Inhibition of SAPK/JNK leads to enhanced IL-1-induced IL-6 synthesis in osteoblasts
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Akira Kondo , Takanobu Otsuka , Rie Matsushima-Nishiwaki , Gen Kuroyanagi , Jun Mizutani , Ikuo Wada , Osamu Kozawa , Haruhiko Tokuda
  Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) 1 Abbreviations used: Stress-activated protein kinase/c-Jun N-terminal kinase, SAPK/JNK; Interleukin 1, IL-1; Interleukin 6, IL-6; AMP-activated protein kinase, AMPK; Mitogen-activated protein, MAP; Inhibitor of κB, IκB; Nuclear factor-κB, NF-κB; Vascular endothelial growth factor, VEGF; Enzyme-linked immunosorbent assay, ELISA; Normal Human Osteoblasts, NHOst; Glyceraldehyde-3-phosphate dehydrogenase, GAPDH; α-minimum essential medium, α-MEM; Fetal calf serum, FCS; Sodium dodecyl sulfate, SDS; Polyacrylamide gel electrophoresis, PAGE. 1 which belongs to the MAP kinase superfamily regulates many cellular events. We previously reported that interleukin 1 (IL-1) stimulates the synthesis of interleukin 6 (IL-6) through activation of ERK and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that AMP-activated protein kinase (AMPK) negatively regulates the IL-1-induced IL-6 synthesis through IκB/NF-κB pathway. In the present study, we investigated the role of SAPK/JNK in the IL-1-stimulated IL-6 synthesis in these cells. IL-1 induced the phosphorylation of SAPK/JNK. SP600125, an inhibitor of SAPK/JNK, increased the release and the mRNA expression levels of IL-6 induced by IL-1. IL-1-stimulated IL-6 release was significantly up-regulated in SAPK/JNK-knocked down cells. SP600125 remarkably suppressed the IL-1-induced phosphorylation of both IκB and NF-κB, whereas SP600125 failed to affect the IL-1-induced phosphorylation of AMPK, STAT3 or Src. Compound C, an AMPK inhibitor, attenuated the IL-1-induced phosphorylation of SAPK/JNK. SP600125 enhanced IL-1-stimulated IL-6 release also in normal human osteoblasts. These results strongly suggest that SAPK/JNK negatively regulates IL-1-stimulated IL-6 synthesis and acts at the point between AMPK and IκB/NF-κB in osteoblasts.
  
  PubDate: 2013-05-17T03:33:39Z
Human airway trypsin-like protease induces mucin5AC hypersecretion via a protease-activated receptor 2-mediated pathway in human airway epithelial cells
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Chunyi Liu , Qi Li , Xiangdong Zhou , Victor P. Kolosov , Juliy M. Perelman
  Mucus hypersecretion is a common feature in chronic airway diseases, and serine proteases play a critical role in this process. However, the mechanisms by which serine proteases induce mucin5AC hypersecretion have not been fully explored. In this study, we characterized human airway trypsin-like protease (HAT), a serine protease that is found in the mucoid sputum of patients with chronic airway diseases and is an agonist of protease-activated receptor 2 (PAR2)-induced cellular responses in human bronchial epithelial cells (16HBE). We also investigated the potential involvement of PAR2 in this process. We found that both HAT and PAR2-AP enhance the exocytosis of mucin5AC protein, whereas HAT, but not PAR2-AP, enhances the expression of mucin5AC mRNA. PAR2 is expressed at a much higher level in the cells than the other three PARs. Transfection with an siRNA against the PAR2 receptor or Gαq/11 protein or pretreatment with the Gαq/11 protein inhibitor YM-254890, the PLC inhibitor U73122 or the intracellular Ca2+ chelator BAPTA-AM all effectively attenuated the HAT-induced cellular responses. Taken together, these results indicate that HAT can stimulate mucin5AC hypersecretion through a PAR2-mediated signaling pathway in 16HBE cells. Thus, PAR2 could represent a novel therapeutic target for chronic airway diseases with mucus hypersecretion.
  Highlights ► MUC5AC is the major secreted mucin in inflammatory airway diseases. ► HAT is isolated from the sputum of patients with chronic airway diseases. ► HAT is a serine protease and an agonist of the PAR2 receptor. ► HAT can induce MUC5AC hypersecretion in human airway epithelial cells. ► The PAR2-mediated pathway is involved in HAT-induced MUC5AC hypersecretion.
  
  PubDate: 2013-05-17T03:33:39Z
Release of an ∼55kDa fragment containing the actin-binding domain of β-spectrin by caspase-8 during FND-induced apoptosis depends on the presence of protein 4.1
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Monika Toporkiewicz , Michał Grzybek , Justyna Meissner , Izabela Michalczyk , Patrycja M. Dubielecka , Justyna Korycka , Ewa Seweryn , Aleksander F. Sikorski
  Analyses of the status of the membrane spectrin-based skeleton during fludarabine/mitoxantrone/dexamethasone-induced (FND-induced) apoptosis revealed proteolytic degradation of β-spectrin, with the prevalent appearance of a specific fragment with a molecular weight of ∼55kDa, containing the actin-binding domain (ABD). Appearance of this fragment was dependent on induction of apoptosis. In silico proteolysis of spectrin identified caspase-8 as a candidate protease responsible for the generation of this ∼55kDa ABD-containing fragment. Analyses of spectrin and procaspase-8 localization during early apoptosis indicated temporary (<30–120min) submembranous colocalization of both proteins. Proteolytic release of the N-terminal ∼55kDa fragment of purified spectrin by recombinant caspase-8 does not occur in normal cells, but does occur in isolated membrane, such as red blood cell ghosts, or in vitro in the presence of apoptotic cell extracts. Surprisingly, proteolysis of purified spectrin by recombinant caspase-8 resulted in the generation of the ∼55kDa fragment only in the presence of purified protein 4.1. This suggests that only the appropriate spatial arrangement of the spectrin-based membrane skeleton or the appropriate conformational state of spectrin, which are both known to be induced by 4.1, can sensitize β-spectrin to cleavage by caspase-8 at the N-terminal ABD-containing region.
  
  PubDate: 2013-05-13T02:03:56Z
Corrigendum to: “Regulation of mouse Cyp24a1 expression via promoter-proximal and downstream-distal enhancers highlights new concepts of 1,25-dihydroxyvitamin D3 action” [Arch. Biochem. Biophys. 2012;523:2–8]
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): J. Wesley Pike , Mark B. Meyer
  
  PubDate: 2013-05-13T02:03:56Z
Deciphering the kinetic mechanisms controlling selected plant ADP-glucose pyrophosphorylases
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Susan K. Boehlein , Janine R. Shaw , Seon K. Hwang , Jon D. Stewart , L. Curtis Hannah
  ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes. With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last. 3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms. By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes. Redox state does not affect k cat of the two potato isoforms. Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex. This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme. Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis.
  
  PubDate: 2013-05-13T02:03:56Z
Phosphorylation accelerates geldanamycin-induced Akt degradation
- Abstract: Publication date: Available online 10 May 2013
  Source:Archives of Biochemistry and Biophysics
  Author(s): Chih-Hao Su , Keng-Hsin Lan , Chung-Pin Li , Yee Chao , Han-Chieh Lin , Shou-Dong Lee , Wei-Ping Lee
  Hsp90 (Heat shock protein-90) is a cellular buffer against erroneous gene products and also plays an essential role in facilitating proper folding, maturation, and activity of its client proteins. The phosphatidylinositol-3 kinase (PI-3K)–Akt pathway transduces a survival signal involved in tumor development. The kinase activity of Akt depends on its association with Hsp90. Hsp90 inhibition causes Akt degradation, but the mechanism remains unclear. Several reports showed that the Hsp90 inhibitor geldanamycin (GA) induces Thr308 and Ser473 phosphorylations of Akt, however, it is still unknown about the significance of GA-induced Akt activation in degradation of the kinase. We treated Hela cells with GA to observe Akt degradation and found that Ly294002 delayed Akt degradation. Mutation of Thr308 or Ser473 also caused delayed Akt ubiquitination and degradation. Inhibition of Akt dephosphorylation enhanced GA-mediated Akt degradation. In this report, we show that GA-mediated transient activation of Akt accelerates its association with the E3 ligase CHIP (C-terminal Hsp70-interacting protein)-mediated ubiquitination and subsequent proteasome degradation.
  
  PubDate: 2013-05-13T02:03:56Z
Transplatin enhances effect of cisplatin on both single DNA molecules and live tumor cells
- Abstract: Publication date: Available online 8 May 2013
  Source:Archives of Biochemistry and Biophysics
  Author(s): Yu-Ru Liu , Chao Ji , Hong-Yan Zhang , Shuo-Xing Dou , Ping Xie , Wei-Chi Wang , Peng-Ye Wang
  Cisplatin is the main platinum antitumor drug applied in clinical settings. However, its trans isomer, transplatin, is known to have an ineffective antitumor activity. Despite intensive studies in this field, the structural and biophysical properties of DNA molecules reacting with these two platinum complexes have not been fully elucidated. In the present study, we observed that transplatin made efficient cross-linking of DNA in the vicinity of cisplatin adducts. High-resolution atomic force microscopy studies revealed that the transplatin-induced cross-linkings of nucleotides flanking cisplatin adducts were characterized by kinked-loop structures with rod-like shapes of nanometer scales (∼10-60 nm). The results were further confirmed by denaturing gel electrophoresis and single-molecule experiment using magnetic tweezers. In vivo studies revealed that transplatin and cisplatin co-treatment could induce a considerable amount of kinked loops with smaller sizes (∼15 nm) in cellular DNA. Furthermore, compared with cisplatin treatment alone, the co-treatment resulted in enhanced cytotoxicity, increased amount of interstrand cross-links, and cell lesions more reluctant to cellular repair system. The results of the present study provide a new clue for understanding the stepwise reactions of DNA with platinum drugs and might serve as a basis for the development of a new antitumor strategy.
  
  PubDate: 2013-05-09T02:04:04Z
Structural and functional consequences of cardiac troponin C L57Q and I61Q Ca2+-desensitizing variants
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Dan Wang , Michelle E. McCully , Zhaoxiong Luo , Jonathan McMichael , An-Yue Tu , Valerie Daggett , Michael Regnier
  Two cTnC variants, L57Q and I61Q, both of which are located on helix C within the N domain of cTnC, were originally reported in the skeletal muscle system [Tikunova, Davis, J. Biol. Chem. 279 (2004) 35341–35352], as the analogous L58Q and I62Q sTnC, and demonstrated a decreased Ca2+ binding affinity. Here, we provide detailed characterization of structure–function relationships for these two cTnC variants, to determine if they behave differently in the cardiac system and as a framework for determining similarities and differences with other cTnC mutations that have been associated with DCM. We have used an integrative approach to study the structure and function of these cTnC variants both in solution and in silico, to understand how the L57Q and I61Q mutations influence Ca2+ binding at site II, the subsequent effects on the interaction with cTnI, and the structural changes which are associated with these changes. Steady-state and stopped flow fluorescence spectroscopy confirmed that a decrease in Ca2+ affinity for recombinant cTnC and cTn complexes containing the L57Q or I61Q variants. The L57Q variant was intermediate between WT and I61Q cTnC and also did not significantly alter cTnC–cTnI interaction in the absence of Ca2+, but did decrease the interaction in the presence of Ca2+. In contrast, I61Q decreased the cTnC–cTnI interaction in both the absence and presence of Ca2+. This difference in the absence of Ca2+ suggests a greater structural change in cNTnC may occur with the I61Q mutation than the L57Q mutation. MD simulations revealed that the decreased Ca2+ binding induced by I61Q may result from destabilization of the Ca2+ binding site through interruption of intra-molecular interactions when residue 61 forms new hydrogen bonds with G70 on the Ca2+ binding loop. The experimentally observed interruption of the cTnC–cTnI interaction caused by L57Q or I61Q is due to the disruption of key hydrophobic interactions between helices B and C in cNTnC. This study provides a molecular basis of how single mutations in the C helix of cTnC can reduce Ca2+ binding affinity and cTnC–cTnI interaction, which may provide useful insights for a better understanding of cardiomyopathies and future gene-based therapies.
  Highlights ► Study of L57Q and I61Q cTnC using solution and in silico techniques is presented. ► Solution studies indicate both variants decrease the Ca2+ binding affinity to cTnC. ► Both variants reduced cTnI affinity to cTnC in Ca2+ saturated states. ► MD simulation shows disruption between helices B/C by both cTnC variants. ► cTnC (I61Q) formed hydrogen bonds with the residues on the Ca2+ binding loop.
  
  PubDate: 2013-05-05T02:05:02Z
Structural and kinetic effects of hypertrophic cardiomyopathy related mutations R146G/Q and R163W on the regulatory switching activity of rat cardiac troponin I
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Zhiqun Zhou , Daniel Rieck , King-Lun Li , Yexin Ouyang , Wen-Ji Dong
  Mutations in cardiac troponin I (cTnI) that cause hypertrophic cardiomyopathy (HCM) have been reported to change the contractility of cardiac myofilaments, but the underlying molecular mechanism remains elusive. In this study, Förster resonance energy transfer (FRET) was used to investigate the specific structural and kinetic effects that HCM related rat cTnI mutations R146G/Q and R163W exert on Ca2+ and myosin S1 dependent conformational transitions in rat cTn structure. Ca2+-induced changes in interactions between cTnC and cTnI were individually monitored in reconstituted thin filaments using steady state and time resolved FRET, and kinetics were determined using stopped flow. R146G/Q and R163W all changed the FRET distances between cTnC and cTnI in unique and various ways. However, kinetic rates of conformational transitions induced by Ca2+-dissociation were universally slowed when R146G/Q and R163W were present. Interestingly, the kinetic rates of changes in the inhibitory region of cTnI were always slower than that of the regulatory region, suggesting that the fly casting mechanism that normally underlies deactivation is preserved in spite of mutation. In situ rat myocardial fiber studies also revealed that FRET distance changes indicating mutation specific disruption of the cTnIIR−actin interaction were consistent with increased passive tension.
  Graphical abstract Highlights ► FRET study of effects of HCM-related cTnI mutations in reconstituted thin filaments. ► R146G/Q and R163W each uniquely impact interactions between cTnI and cTnC or actin. ► Fly casting mechanism underlying deactivation preserved in spite of mutation. ► R146G/Q and R163W cause TF to enter pre relax like state in the absence of S1-ADP. ► In situ effects on force−Ca2+ relationship of myocardial fibers match in vitro.
  
  PubDate: 2013-05-05T02:05:02Z
Asymmetric myosin binding to the thin filament as revealed by a fluorescent nanocircuit
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Pilar G. Coffee Castro-Zena , Douglas D. Root
  The interplay between myosin, actin, and striated muscle regulatory proteins involves complex cooperative interactions that propagate along the thin filament. A repeating unit of the tropomyosin dimer, troponin heterotrimer, and the actin protofilament heptamer is sometimes assumed to be able to bind myosin at any of its seven actins when activated even though the regulatory proteins are asymmetrically positioned along this repeating unit. Analysis of the impact of this asymmetry on actin and myosin interactions by sensitized emission luminescence resonance energy transfer spectroscopy and a unique fluorescent nanocircuit design reveals that the troponin affects the structure and function of myosin heads bound nearby in a different manner than myosin heads bound further away from the troponin. To test this hypothesis, a fluorescent nanocircuit reported the position of the myosin lever arm only when the myosin was bound adjacent to the troponin, or in controls, only when the myosin was bound distant from the troponin. Confirming the hypothesis, the myosin lever arm is predominantly in the pre powerstroke orientation when bound near troponin, but is predominantly in the post powerstroke orientation when bound distant from troponin. These data are consistent with the hypothesis that troponin is responsible for the formation of myosin binding target zones along the thin filament.
  Graphical abstract Highlights ► A fluorescent nanocircuit detects S1 lever arm orientations on the thin filament. ► Myosin S1 binds in different conformations along the thin filament. ► Troponin defines target zones for myosin binding along the thin filament. ► We provide evidence supporting the occurrence of a troponin bridge with myosin.
  
  PubDate: 2013-05-05T02:05:02Z
Myofilament and cytoskeleton proteins: Fine machineries of biological movements
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): J.-P. Jin
  
  PubDate: 2013-05-05T02:05:02Z
Length-dependent effects on cardiac contractile dynamics are different in cardiac muscle containing α- or β-myosin heavy chain
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Steven J. Ford , Murali Chandra
  Actomyosin crossbridges (XBs) are the fundamental source of force generation and pressure development in the myocardium. Faster kinetics are imparted on XBs comprised of the fast, α-myosin heavy chain (MHC) isoform, whereas slower kinetics are imparted on XBs comprised of the slow, β-MHC isoform. Other factors, such as sarcomere length (SL), influence XB formation, presumably acting through allosteric effects on the kinetics that regulate the XB cycle. We sought to determine whether the slower XB kinetics of β-MHC were more sensitive to such length-dependent effects than those of α-MHC. We studied the SL effects on mechanical properties of demembranated muscle fibers from normal and propylthiouracil-treated mouse hearts, which expressed predominantly α-MHC or β-MHC, respectively. Interestingly, XB detachment kinetics were more length-sensitive in β-MHC fibers, as estimated by tension cost and XB detachment rate constant (c), and as inferred by k tr. The nonlinearity in force responses to various-amplitude step-like changes in muscle length was more pronounced in β-MHC fibers. This phenomenon is attributed to a greater cooperative/allosteric mechanism in β-MHC fibers, as estimated by model parameter γ. These data suggest a mechanism whereby greater cooperative/allosteric effects impart an enhanced length-sensitivity of XB cycling kinetics in fibers containing the slower cycling β-MHC.
  Highlights ► Propylthiouracil-treated adult mice expressed cardiac β-myosin heavy chain (MHC). ► We studied length dependence of crossbridge dynamics of α- and β-MHC cardiac muscle. ► No length dependence of XB detachment was found in control, α-MHC cardiac muscle. ► XB detachment kinetics were significantly length dependent in β-MHC cardiac muscle. ► Cooperative/allosteric effects on XB recruitment dynamics were increased by β-MHC.
  
  PubDate: 2013-05-05T02:05:02Z
The human flavoproteome
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Wolf-Dieter Lienhart , Venugopal Gudipati , Peter Macheroux
  Vitamin B2 (riboflavin) is an essential dietary compound used for the enzymatic biosynthesis of FMN and FAD. The human genome contains 90 genes encoding for flavin-dependent proteins, six for riboflavin uptake and transformation into the active coenzymes FMN and FAD as well as two for the reduction to the dihydroflavin form. Flavoproteins utilize either FMN (16%) or FAD (84%) while five human flavoenzymes have a requirement for both FMN and FAD. The majority of flavin-dependent enzymes catalyze oxidation–reduction processes in primary metabolic pathways such as the citric acid cycle, β-oxidation and degradation of amino acids. Ten flavoproteins occur as isozymes and assume special functions in the human organism. Two thirds of flavin-dependent proteins are associated with disorders caused by allelic variants affecting protein function. Flavin-dependent proteins also play an important role in the biosynthesis of other essential cofactors and hormones such as coenzyme A, coenzyme Q, heme, pyridoxal 5′-phosphate, steroids and thyroxine. Moreover, they are important for the regulation of folate metabolites by using tetrahydrofolate as cosubstrate in choline degradation, reduction of N-5.10-methylenetetrahydrofolate to N-5-methyltetrahydrofolate and maintenance of the catalytically competent form of methionine synthase. These flavoenzymes are discussed in detail to highlight their role in health and disease.
  Graphical abstract Highlights ► 89 genes encoding flavoproteins were identified in the human genome. ► Two thirds of human flavoproteins are linked to human diseases. ► Flavoenzymes are essential for the biosynthesis of other coenzymes and hormones. ► Flavoenzymes play a critical role in folate and cobalamin metabolism.
  
  PubDate: 2013-05-05T02:05:02Z
Myofilament incorporation and contractile function after gene transfer of cardiac troponin I Ser43/45Ala
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Sarah E. Lang , Dustin A. Robinson , Helen C. Wu , Todd J. Herron , Philip A. Wahr , Margaret V. Westfall
  Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70–80% of endogenous cTnI replaced within 4days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca2+ sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca2+ transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response.
  Highlights ► Gene transfer of cTnISer43/45Ala is expressed and incorporated into myocyte sarcomeres. ► Myofilament contractile function is altered by cTnISer43/45Ala. ► Functional changes are not due to alterations in Ca2+ transients or phosphorylation. ► The response to the PKC agonist ET is changed by cTnISer43/45Ala.
  
  PubDate: 2013-05-05T02:05:02Z
Sexually dimorphic myofilament function and cardiac troponin I phosphospecies distribution in hypertrophic cardiomyopathy mice
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Laurel A.K. McKee , Hao Chen , Jessica A. Regan , Samantha M. Behunin , Jeffery W. Walker , John S. Walker , John P. Konhilas
  The pathological progression of hypertrophic cardiomyopathy (HCM) is sexually dimorphic such that male HCM mice develop phenotypic indicators of cardiac disease well before female HCM mice. Here, we hypothesized that alterations in myofilament function underlies, in part, this sex dimorphism in HCM disease development. Firstly, 10–12month female HCM (harboring a mutant [R403Q] myosin heavy chain) mice presented with proportionately larger hearts than male HCM mice. Next, we determined Ca2+-sensitive tension development in demembranated cardiac trabeculae excised from 10–12month female and male HCM mice. Whereas HCM did not impact Ca2+-sensitive tension development in male trabeculae, female HCM trabeculae were more sensitive to Ca2+ than wild-type (WT) counterparts and both WT and HCM males. We hypothesized that the underlying cause of this sex difference in Ca2+-sensitive tension development was due to changes in Ca2+ handling and sarcomeric proteins, including expression of SR Ca2+ ATPase (2a) (SERCA2a), β-myosin heavy chain (β-MyHC) and post-translational modifications of myofilament proteins. Female HCM hearts showed an elevation of SERCA2a and β-MyHC protein whereas male HCM hearts showed a similar elevation of β-MyHC protein but a reduced level of cardiac troponin T (cTnT) phosphorylation. We also measured the distribution of cardiac troponin I (cTnI) phosphospecies using phosphate-affinity SDS–PAGE. The distribution of cTnI phosphospecies depended on sex and HCM. In conclusion, female and male HCM mice display sex dimorphic myofilament function that is accompanied by a sex- and HCM-dependent distribution of sarcomeric proteins and cTnI phosphospecies.
  Highlights ► 10–12Month HCM females develop proportionately larger hearts than HCM males. ► HCM females display alterations in Ca2+-sensitivity over female WT; HCM males do not. ► The pattern of cTnI post-translational modification depends on sex and HCM genotype.
  
  PubDate: 2013-05-05T02:05:02Z
Tropomyosin Ser-283 pseudo-phosphorylation slows myofibril relaxation
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Benjamin R. Nixon , Bin Liu , Beatrice Scellini , Chiara Tesi , Nicoletta Piroddi , Ozgur Ogut , R. John Solaro , Mark T. Ziolo , Paul M.L. Janssen , Jonathan P. Davis , Corrado Poggesi , Brandon J. Biesiadecki
  Tropomyosin (Tm) is a central protein in the Ca2+ regulation of striated muscle. The αTm isoform undergoes phosphorylation at serine residue 283. While the biochemical and steady-state muscle function of muscle purified Tm phosphorylation have been explored, the effects of Tm phosphorylation on the dynamic properties of muscle contraction and relaxation are unknown. To investigate the kinetic regulatory role of αTm phosphorylation we expressed and purified native N-terminal acetylated Ser-283 wild-type, S283A phosphorylation null and S283D pseudo-phosphorylation Tm mutants in insect cells. Purified Tm’s regulate thin filaments similar to that reported for muscle purified Tm. Steady-state Ca2+ binding to troponin C (TnC) in reconstituted thin filaments did not differ between the 3 Tm’s, however disassociation of Ca2+ from filaments containing pseudo-phosphorylated Tm was slowed compared to wild-type Tm. Replacement of pseudo-phosphorylated Tm into myofibrils similarly prolonged the slow phase of relaxation and decreased the rate of the fast phase without altering activation kinetics. These data demonstrate that Tm pseudo-phosphorylation slows deactivation of the thin filament and muscle force relaxation dynamics in the absence of dynamic and steady-state effects on muscle activation. This supports a role for Tm as a key protein in the regulation of muscle relaxation dynamics.
  Highlights ► Sf9 insect cell expressed and purified αTm is N-terminal acetylated. ► Pseudo-phosphorylated αTm S283D regulates filaments like phosphorylated muscle αTm. ► αTm S283D does not alter steady-state Ca2+ binding to the thin filament. ► αTm S283D slows filament Ca2+ dissociation and prolongs myofibril force relaxation. ► Tm phosphorylation contributes to the modulation of muscle relaxation.
  
  PubDate: 2013-05-05T02:05:02Z
Post-translational modifications of myofilament proteins involved in length-dependent prolongation of relaxation in rabbit right ventricular myocardium
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Michelle M. Monasky , Domenico M. Taglieri , Alice K. Jacobson , Kaylan M. Haizlip , R. John Solaro , Paul M.L. Janssen
  The phosphorylation state of several cardiac myofilament proteins changes with the level of stretch in intact, twitch-contracting cardiac muscles. It remains unclear which kinases are involved in the length-dependent phosphorylation of these proteins. We set out to investigate which kinases are involved after a step-wise change in cardiac muscle length. We hypothesize that myofilament protein phosphorylation by PKCβII and PKA alters contractile kinetics during length-dependent activation. Right ventricular intact trabeculae were isolated from New Zealand White rabbit hearts and stimulated to contract at 1Hz. Twitch force recordings where taken at taut and optimal muscle lengths before and after administration of kinase inhibitors at 37°C. PKCβII inhibition significantly decreased time from stimulation to peak force (TTP), time from peak force to 50% relaxation (RT50), and 90% relaxation (RT90) at optimal muscle length. This led to a loss in the length-dependent increase of RT50 and RT90 in the presence of the PKCβII inhibitor, whereas the length-dependent increase in RT50 and RT90 was seen in the controls. PKA inhibition using H-89 significantly decreased TTP at both taut and optimal muscle lengths. Detection of Ser/Thr phosphorylation with ProQ-diamond staining indicates a role for PKCβII in the phosphorylation of tropomyosin and myosin light chain-2 (MLC2) and PKA for tropomyosin, troponin-I, MLC2, myosin binding protein-C, troponin-T (TnT) 3 and TnT4. Our data provide evidence for two signaling kinases acting upon myofilament proteins during length-dependent activation, and provide further insight for length-dependent myofilament function.
  
  PubDate: 2013-05-05T02:05:02Z
Protective effect of nicotinamide on high glucose/palmitate-induced glucolipotoxicity to INS-1 beta cells is attributed to its inhibitory activity to sirtuins
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Soo-Jin Lee , Sung-E. Choi , Ik-Rak Jung , Kwan-Woo Lee , Yup Kang
  This study was initiated to determine whether the protective effect of nicotinamide (NAM) on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death was due to its role as an anti-oxidant, nicotinamide dinucleotide (NAD+) precursor, or inhibitor of NAD+-consuming enzymes such as poly (ADP-ribose) polymerase (PARP) or sirtuins. All anti-oxidants tested were not protective against HG/PA-induced INS-1 cell death. Direct supplementation of NAD+ or indirect supplementation through NAD+ salvage or de novo pathway did not protect the death. Knockdown of the NAD+ salvage pathway enzymes such as nicotinamide phosphoribosyl transferase (NAMPT) or nicotinamide mononucleotide adenyltransferase (NMNAT) did not augment death. On the other hand, pharmacological inhibition or knockdown of PARP did not affect death. However, sirtinol as an inhibitor of NAD-dependant deacetylase or knockdown of SIRT3 or SIRT4 significantly reduced the HG/PA-induced death. These data suggest that protective effect of NAM on beta cell glucolipotoxicity is attributed to its inhibitory activity on sirtuins.
  
  PubDate: 2013-05-05T02:05:02Z
Ubiquitin C-terminal hydrolase-L5 is required for high glucose-induced transforming growth factor-β receptor I expression and hypertrophy in mesangial cells
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Yu-Min Ko , Chun-Ying Chang , Shean-Jaw Chiou , Fu-Jie Hsu , Jau-Shyang Huang , Yu-Lin Yang , Jinn-Yuh Guh , Lea-Yea Chuang
  Transforming growth factor-β (TGF-β) is pivotal in the pathogenesis of diabetic nephropathy. Type 1 TGF-β receptor (TGF-βR1) is degraded by Smad7-dependent ubiquitination–proteasomal pathway, which is deubiquitinated by ubiquitin C-terminal hydrolase-L5 (UCHL5). Therefore, we studied the role of UCHL5 in high glucose (27.8mM)-induced TGF-βR1 protein expression in mouse mesangial (MES13) cells. UCHL5 short hairpin RNA (shRNA) was used to knock down UCHL5 while LY294002 and the dominant-negative p85 were used to inhibit phosphatidylinositol-3-kinase (PI3K). We found that high glucose increased phospho-Akt, TGF-βR1 mRNA and protein expression. High glucose also increased UCHL5 protein expression, which was attenuated by LY294002, the dominant-negative p85 and the dominant-negative CREB. High glucose-induced TGF-βR1 protein expression and TGF-βR1 protein deubiquitination were attenuated by UCHL5 shRNA. Additionally, high glucose-induced p21WAF1, fibronectin protein expression and cell hypertrophy were attenuated by UCHL5 shRNA. However, high glucose-induced TGF-βR1 mRNA, p27kip1 protein expression and growth inhibition were not affected by UCHL5 shRNA. Finally, glomerular UCHL5 and TGF-βR1 protein expression were increased in streptozotocin-diabetic rats at 8weeks. We conclude that PI3K-dependent UCHL5 is required for high glucose-induced TGF-βR1 protein expression in mesangial cells. UCHL5 is also required for high glucose-induced TGF-βR1 protein deubiquitination, p21WAF1 and fibronectin protein expression and cell hypertrophy.
  
  PubDate: 2013-05-05T02:05:02Z
Regulation of phenylalanine hydroxylase: Conformational changes upon phosphorylation detected by H/D exchange and mass spectrometry
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Jun Li , Paul F. Fitzpatrick
  The enzyme phenylalanine hydroxylase catalyzes the hydroxylation of excess phenylalanine in the liver to tyrosine. The enzyme is regulated allosterically by phenylalanine and by phosphorylation of Ser16. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into any structural change upon phosphorylation. Peptides in both the catalytic and regulatory domains show increased deuterium incorporation into the phosphorylated protein. Deuterium is incorporated into fewer peptides than when the enzyme is activated by phenylalanine, and the incorporation is slower. This establishes that the conformational change upon phosphorylation of phenylalanine hydroxylase is different from and less extensive than that upon phenylalanine activation.
  
  PubDate: 2013-05-05T02:05:02Z
The impact of V30A mutation on transthyretin protein structural stability and cytotoxicity against neuroblastoma cells
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Feng Zhang , Cheng Hu , Yang Dong , Ming-shen Lin , Jingyao Liu , Xinmei Jiang , Yubin Ge , Yingjie Guo
  Single point mutations in the transthyretin (TTR) gene may cause a hereditary neurodegenerative disease termed familial amyloidotic polyneuropathy (FAP) due to accelerated deposition of amyloid fibrils, resulting in peripheral and autonomic nervous system dysfunction. Recently, we found a Chinese FAP family involving a TTR V30A mutation. To understand the pathogenic mechanisms of this V30A TTR, we investigated the effects of this mutation on TTR quaternary and tertiary structural stabilities and cytotoxicities against neuroblastoma cells along with the most common variant V30M TTR and the wild-type (WT) TTR. Our results showed that the V30A mutation impaired the thermodynamic and kinetic stabilities of the TTR protein by increasing the extent and rate of tetramer dissociation and unfolded monomer and amyloid fibril formation, even to a greater extent than the V30M mutation under several experimental conditions. Further, an obviously cytotoxic effect of the V30A TTR on the human neuroblastoma cell line, IMR-32, was observed. The V30A TTR induced apoptosis and autophagy concomitant with the accumulation of reactive oxygen species (ROS) and DNA double-strand breaks, reflected in the induction of phosphor-H2A.X. These results suggest that the V30A mutation in the TTR gene promotes the formation of unfolded monomers and amyloid fibrils, which potentially contribute to the increased neurotoxicity and the pathology associated with this FAP family.
  
  PubDate: 2013-05-05T02:05:02Z
Collagen degradation by tumor-associated trypsins
- Abstract: Publication date: 15 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 2
  Author(s): Lynn S. Mirigian , Elena Makareeva , Hannu Koistinen , Outi Itkonen , Timo Sorsa , Ulf-Håkan Stenman , Tuula Salo , Sergey Leikin
  In normal soft tissues, collagen is degraded primarily by collagenases from the matrix metalloproteinase family. Yet, collagenase-like activity of tumor-associated isoforms of other enzymes might be involved in cancer invasion as well. In the present study, we systematically examined collagen degradation by non-sulfated isoforms of trypsins, which were proposed to possess such an activity. We found that non-sulfated trypsin-1, -2, and -3 were able to cleave non-helical and unfolded regions of collagen chains but not the intact triple helix, similar to sulfated trypsins produced by the pancreas. Trypsin-2 sulfation did not affect the cleavage rate either. An apparent triple helix cleavage by tumor-associated trypsin-2 reported earlier likely occurred after triple helix unfolding during sample denaturation for gel electrophoresis. Nevertheless, tumor-associated trypsins might be important for releasing collagen from fibers through telopeptide cleavage as well as for degrading unfolded collagen chains, e.g. after initial cleavage and destabilization of triple helices by collagenases.
  
  PubDate: 2013-05-05T02:05:02Z
Calcium sensitivity and myofilament lattice structure in titin N2B KO mice
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Eun-Jeong Lee , Joshua Nedrud , Peter Schemmel , Michael Gotthardt , Thomas C. Irving , Henk L. Granzier
  The cellular basis of the Frank–Starling “Law of the Heart” is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information to altered calcium sensitivity has remained elusive. Here the effect of titin-based passive tension on the length-dependence of activation (LDA) was studied by measuring the tension–pCa relation in skinned mouse LV muscle at two sarcomere lengths (SLs). N2B KO myocardium, where the N2B spring element in titin is deleted and passive tension is elevated, was compared to WT myocardium. Myofilament lattice structure was studied with low-angle X-ray diffraction; the myofilament lattice spacing (d 1,0) was measured as well as the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I 1,1/I 1,0) as an estimate of the degree of association of myosin heads with the thin filaments. Experiments were carried out in skinned muscle in which the lattice spacing was reduced with Dextran-T500. Experiments with and without lattice compression were also carried out following PKA phosphorylation of the skinned muscle. Under all conditions that were tested, LDA was significantly larger in N2B KO myocardium compared to WT myocardium, with the largest differences following PKA phosphorylation. A positive correlation between passive tension and LDA was found that persisted when the myofilament lattice was compressed with Dextran and that was enhanced following PKA phosphorylation. Low-angle X-ray diffraction revealed a shift in mass from thin filaments to thick filaments as sarcomere length was increased. Furthermore, a positive correlation was obtained between myofilament lattice spacing and passive tension and the change in I 1,1/I 1,0 and passive tension and these provide possible explanations for how titin-based passive tension might regulate calcium sensitivity.
  Highlights ► We studied passive tension, length-dependent activation (LDA), and muscle structure. ► A positive correlation between titin-based passive tension and LDA was found. ► Passive tension and myofilament lattice spacing were positively correlated. ► A shift in mass from thin filaments to thick filaments occurred.
  
  PubDate: 2013-05-05T02:05:02Z
Prostate-apoptosis response-4 phosphorylation in vascular smooth muscle
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Justin A. MacDonald , Lori D. Moffat , Abdulhameed Al-Ghabkari , Cindy Sutherland , Michael P. Walsh
  The protein prostate-apoptosis response (Par)-4 has been implicated in the regulation of smooth muscle contraction, based largely on studies with the A7r5 cell line. A mechanism has been proposed whereby Par-4 binding to MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase, MLCP) blocks access of zipper-interacting protein kinase (ZIPK) to Thr697 and Thr855 of MYPT1, whose phosphorylation is associated with MLCP inhibition. Phosphorylation of Par-4 at Thr155 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition and contraction. We tested this “padlock” hypothesis in a well-characterized vascular smooth muscle system, the rat caudal artery. Par-4 was retained in Triton-skinned tissue, suggesting a tight association with the contractile machinery, and indeed Par-4 co-immunoprecipitated with MYPT1. Treatment of Triton-skinned tissue with the phosphatase inhibitor microcystin (MC) evoked phosphorylation of Par-4 at Thr155, but did not induce its dissociation from the contractile machinery. Furthermore, analysis of the time courses of MC-induced phosphorylation of MYPT1 and Par-4 revealed that MYPT1 phosphorylation at Thr697 or Thr855 preceded Par-4 phosphorylation. Par-4 phosphorylation was inhibited by the non-selective kinase inhibitor staurosporine, but not by inhibitors of ZIPK, Rho-associated kinase or protein kinase C. In addition, Par-4 phosphorylation did not occur upon addition of constitutively-active ZIPK to skinned tissue. We conclude that phosphorylation of Par-4 does not regulate contraction of this vascular smooth muscle tissue by inducing dissociation of Par-4 from MYPT1 to allow phosphorylation of MYPT1 and inhibition of MLCP.
  Highlights ► The prostate-apoptosis response (Par)-4 protein is implicated in smooth muscle contraction. ► Par-4 is tightly associated with the contractile machinery of demembranated rat caudal artery. ► Microcystin-evoked Par-4 phosphorylation does not precede MYPT1 phosphorylation. ► Findings do not support a ‘padlock’ mechanism of regulation of contraction by Par-4 in vascular smooth muscle tissue.
  
  PubDate: 2013-05-05T02:05:02Z
Intercalated disc protein, mXinα, suppresses p120-catenin-induced branching phenotype via its interactions with p120-catenin and cortactin
- Abstract: Publication date: 1 July 2013
  Source:Archives of Biochemistry and Biophysics, Volume 535, Issue 1
  Author(s): Qinchuan Wang , Te-Ling Lu , Eric Adams , Jenny Li-Chun Lin , Jim Jung-Ching Lin
  The Xin repeat-containing proteins, Xinα (Xirp1) and Xinβ (Xirp2), localize to the intercalated discs (ICDs) of mammalian hearts. Mouse Xinα (mXinα) directly interacts with β-catenin and actin filaments, potentially coupling the N-cadherin/β-catenin complexes to the underlying actin cytoskeleton and modulating ICD integrity and function. Supporting this possibility, mXinα-null hearts develop ICD structural defects and cardiomyopathy with conduction defects. However, the underlying mechanisms leading to these defects remain unclear. Here, we showed that mXinα also interacted with p120-catenin and cortactin. Different from the β-catenin binding domain, there existed multiple p120-catenin binding sites on mXinα, while only the extreme N-terminus of mXinα containing a SH3-binding motif could interact with cortactin. In mouse heart, a significant fraction of cortactin was co-localized with N-cadherin to ICDs, whereas in mXinα-null heart, this fraction of cortactin was drastically reduced. Therefore, mXinα may modulate ICD integrity and function through its interactions with catenins and cortactin. Analyses of the in vivo consequence of p120-catenin and mXinα interaction revealed that force-expressed mXinα or its fragments significantly suppressed the p120-catenin-induced branching phenotypes. It is known that p120-catenin directly regulates Rho GTPases, leading to the branching phenotype. Thus, mXinα may sequester the p120-catenin from inhibiting RhoA activity and/or from activating Rac1 activity.
  Highlights ► mXinα interacts with p120-catenin and suppresses p120-catenin-induced branching. ► Multiple p120-catenin-binding sites present on mXinα. ► N-terminus of mXinα containing a consensus SH3-binding motif binds to cortactin. ► mXinα links and modulates the adherens junctions to the underlying actin network.
  
  PubDate: 2013-05-05T02:05:02Z
Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase associated with X-linked neurodevelopmental disorders
- Abstract: Publication date: Available online 4 May 2013
  Source:Archives of Biochemistry and Biophysics
  Author(s): Mari Sekiguchi , Syouichi Katayama , Naoya Hatano , Yasushi Shigeri , Noriyuki Sueyoshi , Isamu Kameshita
  Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in brain and mutations of its gene are known to be associated with neurodevelopmental disorders such as X-linked West syndrome and Rett syndrome. However, the physiological substrates of CDKL5 that are directly linked to these neurodevelopmental disorders are currently unknown. In this study, we explored endogenous substrates for CDKL5 in mouse brain extracts fractionated by a liquid-phase isoelectric focusing. In conjunction with CDKL5 phosphorylation assay, this approach detected a protein band with an apparent molecular mass of 120 kDa that is remarkably phosphorylated by CDKL5. This 120-kDa protein was identified as amphiphysin 1 (Amph1) by LC-MS/MS analysis, and the site of phosphorylation by CDKL5 was determined to be Ser-293. The phosphorylation mimic mutants, Amph1(S293E) and Amph1(S293D), showed significantly reduced affinity for endophilin, a protein involved in synaptic vesicle endocytosis. Introduction of point mutations in the catalytic domain of CDKL5, which are disease-causing missense mutations found in Rett patients, resulted in the impairment of kinase activity toward Amph1. These results suggest that Amph1 is the cytoplasmic substrate for CDKL5 and that its phosphorylation may play crucial roles in the neuronal development.
  
  PubDate: 2013-05-05T02:05:02Z
From neuroepithelial cells to neurons: Changes in the physiological properties of neuroepithelial stem cells
- Abstract: Publication date: June 2013
  Source:Archives of Biochemistry and Biophysics, Volume 534, Issues 1–2
  Author(s): Masayuki Yamashita
  The central nervous system, which includes the spinal cord, retina, and brain, is derived from the neural tube. The neural tube is formed of a sheet of cells called the neuroepithelium. During embryonic development, neuroepithelial cells function as neural stem cells: they renew themselves while undergoing interkinetic nuclear movements along the apico-basal axis during the cell cycle, and they produce postmitotic cells that function as newborn neurons. Neuroepithelial cells exhibit a robust increase in nucleoplasmic [Ca2+] in response to G protein-coupled receptor activation during S-phase when the nucleus is located in the basal region of the cell. This Ca2+ rise is caused by the release of Ca2+ from intracellular Ca2+ stores, and the Ca2+ release in turn activates Ca2+ entry from the extracellular space, which is called capacitative (or store-operated) Ca2+ entry. The Ca2+ release and store-operated Ca2+ entry are essential for DNA synthesis during S-phase. The activity of this store-operated Ca2+ signaling system declines in parallel with the decreasing proliferative activity of neuroepithelial cells. When exiting the cell cycle, the cells lose the apical process where gap junctions are located. Following the loss of gap junction coupling, the postmitotic cells show a high input resistance, which allows them to be readily depolarized. The Ca2+ response to the excitatory neurotransmitter glutamate appears and develops during neuronal differentiation. The glutamate-induced Ca2+ rise increases transiently during natural cell death (apoptosis). The rise in Ca2+ levels mediated by voltage-gated Ca2+ channels also develops during neuronal differentiation. Thus, when neuroepithelial cells differentiate into neurons, a transition from a store-operated system to a voltage-operated system occurs in the main Ca2+ signaling system. This transition may reflect a change in the mode of intercellular communication from a stored Ca2+-dependent mode to a plasma membrane potential-dependent mode.
  Graphical abstract Highlights ► Neuroepithelial cells act as neural stem cells during embryonic development. ► The main calcium signaling system changes during neurogenesis. ► Gap junction uncoupling functions as a signal for neuronal differentiation.
  
  PubDate: 2013-04-27T03:31:53Z
Paracrine regulation of neural stem cells in the subependymal zone
- Abstract: Publication date: June 2013
  Source:Archives of Biochemistry and Biophysics, Volume 534, Issues 1–2
  Author(s): Eva Porlan , Ana Perez-Villalba , Ana C. Delgado , Sacri R. Ferrón
  Stem cells maintain their self-renewal and multipotency capacities through a self-organizing network of transcription factors and intracellular pathways activated by extracellular signaling from the microenvironment or “niche” in which they reside in vivo. In the adult mammalian brain new neurons continue to be generated throughout life of the organisms and this lifelong process of neurogenesis is supported by a reservoir of neural stem cells in the germinal regions. The discovery of adult neurogenesis in the mammalian brain has sparked great interest in defining the conditions that guide neural stem cell (NSC) maintenance and differentiation into the great variety of neuronal and glial subtypes. Here we review current knowledge regarding the paracrine regulation provided by the components of the niche and its function, focusing on the main germinal region of the adult central nervous system (CNS), the subependymal zone (SEZ).
  Graphical abstract Highlights ► Neural stem cells (NSCs) reside in a specialized “niche” that supports neurogenesis. ► Vasculature regulate NSCs function by secretion of molecules in the niche. ► The choroid plexus-CSF system produces factors that promote NSC maintenance. ► Astrocytes participate in the creation of the niche. ► Brain damage enhances proliferation of vasculature and NSCs.
  
  PubDate: 2013-04-27T03:31:53Z
The role of redox environment in neurogenic development
- Abstract: Publication date: June 2013
  Source:Archives of Biochemistry and Biophysics, Volume 534, Issues 1–2
  Author(s): E.A. Ostrakhovitch , O.A. Semenikhin
  The dynamic changes of cellular redox elements during neurogenesis allow the control of specific programs for selective lineage progression. There are many redox couples that influence the cellular redox state. The shift from a reduced to an oxidized state and vice versa may act as a cellular switch mechanism of stem cell mode of action from proliferation to differentiation. The redox homeostasis ensures proper functioning of redox-sensitive signaling pathways through oxidation/reduction of critical cysteine residues on proteins involved in signal transduction. This review presents the current knowledge on the relation between changes in the cellular redox environment and stem cell programming in the course of commitment to a restricted neural lineage, focusing on in vivo neurogenesis and in vitro neuronal differentiation. The first two sections outline the main systems that control the intracellular redox environment and make it more oxidative or reductive. The last section provides the background on redox-sensitive signaling pathways that regulate neurogenesis.
  Highlights ► Cellular redox state defines decisions of cell fate and proliferation. ► Cellular redox systems participate in regulation of neurogenesis. ► Redox-mediated signalling controls neuronal differentiation.
  
  PubDate: 2013-04-27T03:31:53Z
FoxOs in neural stem cell fate decision
- Abstract: Publication date: June 2013
  Source:Archives of Biochemistry and Biophysics, Volume 534, Issues 1–2
  Author(s): Seung-Hyun Ro , Debra Liu , Hyeonju Yeo , Ji-hye Paik
  Neural stem cells (NSCs) persist over the lifespan of mammals to give rise to committed progenitors and their differentiated cells in order to maintain the brain homeostasis. To this end, NSCs must be able to self-renew and otherwise maintain their quiescence. Suppression of aberrant proliferation or undesired differentiation is crucial to preclude either malignant growth or precocious depletion of NSCs. The PI3K-Akt-FoxO signaling pathway plays a central role in the regulation of multiple stem cells including one in the mammalian brain. In particular, members of FoxO family transcription factors are highly expressed in these stem cells. As an important downstream effector of growth, differentiation, and stress stimuli, mammalian FoxO transcription factor family controls cellular proliferation, oxidative stress response, homeostasis, and eventual maintenance of long-term repopulating potential. The review will focus on the current understanding of FoxO function in NSCs as well as discuss their biological activities that contribute to determining neural stem cell fate.
  Highlights ► FoxOs are necessary for maintenance of adult NSC. ► FoxOs regulate critical processes for NSC quiescence, homeostasis, stress resistance. ► Pathways both regulate or are regulated by FoxOs serve to maintain adult NSC.
  
  PubDate: 2013-04-27T03:31:53Z
Analysis of neural stem cell self-renewal and differentiation by transgenic RNAi in Drosophila
- Abstract: Publication date: June 2013
  Source:Archives of Biochemistry and Biophysics, Volume 534, Issues 1–2
  Author(s): Yanrui Jiang , Heinrich Reichert
  The fruit fly, Drosophila melanogaster, has proved to be a useful model organism for studying the biology of neural stem cells. Notably, significant progress has been made in identifying the molecular mechanisms that regulate the asymmetric cell divisions of the neural stem cell-like neuroblasts during brain development. Recently, the emerging technology of genome-wide transgenic RNA interference (RNAi), which makes it possible to analyze complicated developmental processes in a targeted, tissue-specific way, has been used for the analysis of gene function in Drosophila neuroblasts. Here, we review the key molecular mechanisms that regulate the asymmetric cell divisions of neuroblasts during brain development in Drosophila. We then summarize recent genome-wide transgenic RNAi screens in Drosophila and report on the identification of new regulators and gene networks that are required in balancing neuroblast self-renewal and differentiation.
  Highlights ► Neuroblasts are neural stem cells in the Drosophila brain. ► Cell fate determinants control self-renewal and differentiation of neuroblasts. ► Genome-wide transgenic RNAi identify new genes regulating neuroblasts proliferation. ► Drosophila neuroblasts are a new model to study cancer stem cells.
  
  PubDate: 2013-04-27T03:31:53Z
Nucleosome assembly proteins and their interacting proteins in neuronal differentiation
- Abstract: Publication date: June 2013
  Source:Archives of Biochemistry and Biophysics, Volume 534, Issues 1–2
  Author(s): Mikaël Attia , Christophe Rachez , Philip Avner , Ute Christine Rogner
  Neuronal differentiation from neural stem cells into mature neurons is guided by the concerted action of specific transcription factors that stepwise exercise their role in the context of defined chromatin states. Amongst the classes of proteins that influence chromatin compaction and modification are nucleosome assembly proteins (NAPs). Mammals possess several nucleosome assembly protein 1 like proteins (NAP1L) that show either ubiquitous or neuron-restricted expression. The latter group is presumably involved in the process of neuronal differentiation. Mammalian NAP1Ls can potentially form both homo- and hetero-dimers and octamers, in theory allowing thousands of different combinations to be formed. Detailed studies have been performed on several of the NAP1Ls that point to a range of molecular roles, including transcriptional regulation, nuclear import, and control of cell division. This article aims at summarizing current knowledge of the mammalian NAP1L family and its interactions.
  Highlights ► Mammals possess five nucleosome assembly protein 1 (NAP1) likes (NAP1Ls). ► The three intronless NAP1L genes show increased expression in neurons. ► NAP1Ls and their protein partners point to specific roles in neuronal differentiation. ► Their roles may include chromatin remodelling and modification and nuclear import.
  
  PubDate: 2013-04-27T03:31:53Z
The role of nuclear receptors in controlling the fine balance between proliferation and differentiation of neural stem cells
- Abstract: Publication date: June 2013
  Source:Archives of Biochemistry and Biophysics, Volume 534, Issues 1–2
  Author(s): Athanasios Stergiopoulos , Panagiotis K. Politis
  In the central nervous system (CNS) of vertebrates a large variety of cell types are specified from a pool of highly plastic neural stem/progenitor cells (NSCs) via a combined action of extrinsic morphogenetic cues and intrinsic transcriptional regulatory networks. Nuclear receptors and their ligands are key regulators of fate decisions in NSCs during development and adulthood, through their ability to control transcription of downstream genes. In the last few years considerable progress has been made towards the understanding of the actions of nuclear receptors in NSCs as well as elucidating the mechanistic basis for these actions. Here we summarize recent progress in the role of nuclear receptors in the biology of NSCs. These studies highlight the importance of this family of transcriptional regulators in CNS development and function in health and disease. Furthermore, they raise the intriguing possibility of using nuclear receptors as therapeutic targets for nervous system related diseases and traumas.
  Highlights ► We review recent progress in the role of nuclear receptors in neural stem cells. ► We focus on proliferation and differentiation decisions in health and disease. ► We summarize the action of specific receptors in neural development and function.
  
  PubDate: 2013-04-27T03:31:53Z
In the wake of neural progenitors
- Abstract: Publication date: June 2013
  Source:Archives of Biochemistry and Biophysics, Volume 534, Issues 1–2
  Author(s): Elena Ostrakhovitch
  
  PubDate: 2013-04-27T03:31:53Z
Pluripotent stem cells as a model to study oxygen metabolism in neurogenesis and neurodevelopmental disorders
- Abstract: Publication date: June 2013
  Source:Archives of Biochemistry and Biophysics, Volume 534, Issues 1–2
  Author(s): Bruna da Silveira Paulsen , Mariana Souza da Silveira , Antonio Galina , Stevens Kastrup Rehen
  Reactive oxygen species (ROS) and oxygen (O2) have been implicated in neurogenesis and self-renewal of neural progenitor cells (NPCs). On the other hand, oxidative unbalance, either by an impairment of antioxidant defenses or by an intensified production of ROS, is increasingly related to risk factors of neurodevelopmental disorders, such as schizophrenia. In this scenario, human induced pluripotent stem cells (hiPSCs) emerged as an interesting platform for the study of cellular and molecular aspects of this mental disorder, by complementing other experimental models, with exclusive advantages such as the recapitulation of brain development. Herein we discuss the role of O2/ROS signaling for neuronal differentiation and how its unbalance could be related to neurodevelopmental disorders, such as schizophrenia. Identifying the role of O2/ROS in neurogenesis as well as tackling oxidative stress and its disturbances in schizophrenic patients’ derived cells will provide an interesting opportunity for the study of neural stem cells differentiation and neurodevelopmental disorders.
  Highlights ► O2/ROS play a crucial role in neurogenesis and self-renewal of stem cells. ► Impaired O2/ROS is detrimental to neurogenesis and contribute to schizophrenia’s onset. ► iPSCs are useful for studying O2/ROS in neurogenesis, with benefits upon other models.
  
  PubDate: 2013-04-27T03:31:53Z
Functional Analysis and Expression of The Mono-Heme Containing Cytochrome C Subunit Of Alternative Complex Iii In Chloroflexus Aurantiacus
- Abstract: Publication date: Available online 12 April 2013
  Source:Archives of Biochemistry and Biophysics
  Author(s): Xinliu Gao , Erica Wunderlich Majumder , Yisheng Kang , Hai Yue , Robert E. Blankenship
  The filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus possesses an unusual electron transfer complex called Alternative Complex III instead of the cytochrome bc or bf type complex found in nearly all other known groups of phototrophs. Earlier work has confirmed that Alternative Complex III behaves as a menaquinol:auracyanin oxidoreductase in the photosynthetic electron transfer chain. In this work, we focus on elucidating the contribution of individual subunits to the overall function of Alternative Complex III. The monoheme subunit ActE has been expressed and characterized in E. coli. A partially dissociated Alternative Complex III missing subunit ActE and subunit ActG was obtained by treatment with the chaotropic agent KSCN, and was then reconstituted with the expressed ActE. Enzymatic activity of the partially dissociated Alternative Complex III was greatly reduced and was largely restored in the reconstituted complex. The redox potential of the heme in the recombinant ActE was +385 mV vs. NHE, similar to the highest potential heme in the intact complex. The results strongly suggest that the monoheme subunit, ActE, is the terminal electron carrier for Alternative Complex III.
  
  PubDate: 2013-04-15T03:30:37Z
MicroRNA-24 Inhibits Osteosarcoma Cell Proliferation Both In Vitro and In Vivo by Targeting LPAATβ
- Abstract: Publication date: Available online 8 April 2013
  Source:Archives of Biochemistry and Biophysics
  
  Lysophosphatidic Acid Acyltransferase β (LPAATβ) may be critically involved in osteosarcoma cell proliferation. However, the comprehensive mechanisms responsible for regulation of LPAATβ in osteosarcoma cells remain unclear. This study found that enhanced LPAATβ expression was correlated with osteosarcoma cell proliferation. MiR-24, targeted to LPAATβ, was down-regulated in osteosarcoma cells. Overexpression of miR-24 down-regulated LPAATβ expression in osteosarcoma cells. Specifically, overexpression of miR-24 inhibited osteosarcoma cell proliferation, however, such effect was blocked when LPAATβ activity was inhibited. In conclusion, our study indicates that miR-24 is reduced in osteosarcoma cells, contributing to up-regulation of LPAATβ and resultant osteosarcoma cell proliferation.
  
  PubDate: 2013-04-11T21:27:45Z
Crystal Structure of Arginase from Leishmania mexicana and Implications for the Inhibition of Polyamine Biosynthesis in Parasitic Infections
- Abstract: Publication date: Available online 9 April 2013
  Source:Archives of Biochemistry and Biophysics
  
  Arginase from parasitic protozoa belonging to the genus Leishmania is a potential drug target for the treatment of leishmaniasis because this binuclear manganese metalloenzyme catalyzes the first committed step in the biosynthesis of polyamines that enable cell growth and survival. The high resolution X-ray crystal structures of the unliganded form of Leishmania mexicana arginase (LmARG) and four inhibitor complexes are now reported. These complexes include the reactive substrate analogue 2(S)-amino-6-boronohexanoic acid (ABH) and the hydroxylated substrate analogue nor-Nω-hydroxy-L-arginine (nor-NOHA), which are the most potent arginase inhibitors known to date. Comparisons of the LmARG structure with that of the archetypal arginase, human arginase I, reveal that all residues important for substrate binding and catalysis are strictly conserved. However, three regions of tertiary structure differ between the parasitic enzyme and the human enzyme corresponding to the G62 – S71, L161 – C172, and I219 – V230 segments of LmARG. Additionally, variations are observed in salt link interactions that stabilize trimer assembly in LmARG. We also report biological studies in which we demonstrate that localization of LmARG to the glycosome, a unique subcellular organelle peculiar to Leishmania and related parasites, is essential for robust pathogenesis.
  
  PubDate: 2013-04-11T21:27:45Z
Protein kinase C-mediated ATP stimulation of Na+-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor
- Abstract: Available online 6 April 2013
  Publication year: 2013
  Source:Archives of Biochemistry and Biophysics
  
  ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10–6 M) or Ado (10–6 M) specifically stimulated Na+-ATPase activity without any changes in (Na++K+)-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na+-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na+-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling.
  
  PubDate: 2013-04-08T06:15:12Z
Insights into molecular interactions between the juxtamembrane and kinase subdomains of the Arabidopsis Crinkly-4 receptor-like kinase
- Abstract: Available online 6 April 2013
  Publication year: 2013
  Source:Archives of Biochemistry and Biophysics
  
  Arabidopsis CRINKLY4 (ACR4), a receptor-like kinase required for plant growth and development, possesses an extracellular ligand binding domain, a transmembrane helix, and an intracellular domain (ICD). The ICD contains the juxtamembrane (JMD) and the C-terminal (CTD) subdomains, which flank the core kinase domain (KD), with at least 16 autophosphorylation sites. Phosphorylation sites are often docking sites for the modification-dependent recruitment of interacting proteins that orchestrate many downstream signaling events. In this context, we have specifically probed the role of the two phosphorylation sites Ser475 and Thr478 in the JMD using mutagenesis and phage-peptide screening techniques. Thus, naïve and phosphorylated 15-mer peptides derived from the JMD were panned against a 21-amino acid random phage peptide library. The phosphorylated peptide preferentially recognized the consensus sequence LxSLL. This sequence harbors the LxxLL motif, a known protein-protein interaction motif that is also present in the N-terminal lobe of the KD. We demonstrate the binding of JMD peptides to the KD and also show through kinetic analyses of mutants that phosphorylation of Ser475 and Thr478 in the JMD is necessary for optimal substrate phosphorylation in vitro. Our experiments suggest that an intramolecular interaction can occur between the JM and the N-terminal lobe of the KD.
  Graphical abstract Highlights
  
  PubDate: 2013-04-08T06:15:12Z
Evaluation of UGT Protein Interactions in Human Hepatocytes: Effect of siRNA Down Regulation of UGT1A9 and UGT2B7 on Propofol Glucuronidation in Human Hepatocytes
- Abstract: Available online 4 April 2013
  Publication year: 2013
  Source:Archives of Biochemistry and Biophysics
  
  Previous experiments performed in recombinant systems have suggested that protein-protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein-protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein-protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.
  
  PubDate: 2013-04-08T06:15:12Z
Anti-inflammatory effects of propofol are mediated by apolipoprotein M in a hepatocyte nuclear factor-1α-dependent manner
- Abstract: Available online 13 March 2013
  Publication year: 2013
  Source:Archives of Biochemistry and Biophysics
  
  Propofol (2,6-diisopropylphenol) is probably the most widely used intravenous hypnotic agent in daily practice. However, its anti-inflammatory properties have seldom been addressed. In this study, we evaluated the anti-inflammatory activity and mechanisms of propofol on lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro and found that propofol markedly inhibited LPS-induced production of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and expression of inducible nitric oxide synthase (iNOS). At the same time, the expression of hepatocyte nuclear factor-1α (HNF-1α) and apolipoprotein M (APOM) was inhibited by treatment with LPS and LPS-induced down-regulation of H NF-1α expression and APOM expression could be compensated by propofol treatment. However, propofol could not compensate LPS-induced down-regulation of APOM expression by treatment with HNF-1α siRNA and the suppressive effect on LPS-induced pro-inflammatory cytokines production by propofol was significantly compensated by treatment with APOM siRNA. These results provide evidence that propofol may first up-regulate APOM expression by enhancing HNF-1α expression and then inhibit pro-inflammatory cytokine production in LPS-stimulated cells. Therefore, our study may be useful in understanding the critical effect of propofol in patients with systemic inflammatory response syndrome.
  
  PubDate: 2013-03-15T07:19:16Z
Neural stem cell survival factors
- Abstract: Available online 5 March 2013
  Publication year: 2013
  Source:Archives of Biochemistry and Biophysics
  
  Neural stem and progenitor cells (NSCs and NPs) give rise to the central nervous system (CNS) during embryonic development. NSCs and NPs differentiate into three main cell-types of the CNS; astrocytes, oligodendrocytes, and neurons. NSCs are present in the adult CNS and are important in maintenance and repair. Adult NSCs hold great promise for endogenous or self-repair of the CNS. Intriguingly, NSCs have been implicated as the cells that give rise to brain tumors. Thus, the balance between survival, growth and differentiation is a critical aspect of NSC biology, during development, in the adult, and in disease processes. In this review we survey what is known about survival factors that control both embryonic and adult NSCs. We discuss the neurosphere culture system as this is widely used to measure NSC activity and behavior in vitro and emphasize the importance of clonality. We define here NSC survival factors in their broadest sense to include any factor that influences survival and proliferation of NSC and NPs. NSC survival factors identified to date include; growth factors, morphogens, proteoglycans, cytokines, hormones and neurotransmitters Understanding NSC and NP interaction in response to these survival factors will provide insight to CNS development, disease and repair.
  Highlights ► Survival factors include any factor that influences the survival and proliferation of NSCs and NPs. ► During development five types of NSCs are found. ► Neural stem cell markers used in vitro and in vivo are discussed. ► The importance of clonal neurosphere assays for measuring NSCs frequency is demonstrated. ► Understanding the NSC survival factors will provide insight to CNS development, disease, and repair.
  
  PubDate: 2013-03-08T22:22:26Z
Differential expression of adenylate kinase 4 in the context of disparate stress response strategies of HEK293 and HepG2 cells
- Abstract: Available online 6 March 2013
  Publication year: 2013
  Source:Archives of Biochemistry and Biophysics
  
  Adenylate kinase isozyme 4 (AK4) belongs to a family of nucleotide monophosphate kinases involved in energy metabolism. Recently, AK4 was reported to play a role in protection from stress: In HEK293 cells, hypoxia increases AK4 expression but does not affect proliferation or viability, while RNA interference (RNAi) directed against AK4 inhibits proliferation and promotes death. By contrast, we show here that HepG2 cells showed much higher AK4 levels, which decreased under hypoxia along with markedly reduced cell proliferation and increased cell death. Nevertheless, RNAi directed against AK4 inhibited cell proliferation and caused death in both cell types, although cell cycle parameters were affected only in HepG2 cells. Hence reductions of AK4 levels were always associated with cell death. These results extend the notion of a stress-protective function of AK4 to a novel physiological context and show that AK4-mediated stress protection is not limited to one particular death scenario. Our data also allow the hypothesis that the different basal AK4 levels reflect different basal stress levels, causing alternative responses to additional stress.
  Graphical abstract Highlights ► HepG2 cells show a much higher basal AK4 expression than HEK293 cells. ► HEK293 cells increase, but HepG2 cells decrease, AK4 levels under hypoxia. ► HEK293 cells survive, but HepG2 cells die, under hypoxia. ► Under normoxia, RNAi directed against AK4 caused death in both cell types. ► Thus cell death was always observed when AK4 levels were decreased.
  
  PubDate: 2013-03-08T22:22:26Z
Ischemic brain injury: a consortium analysis of key factors involved in mesenchymal stem cell-mediated inflammatory reduction
- Abstract: Available online 4 March 2013
  Publication year: 2013
  Source:Archives of Biochemistry and Biophysics
  
  Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia.Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.
  Highlights ► IL-4 and Oncostatin M activate heterodimerisation of STAT3 and STAT1 pathways of human mesenchymal stem cells. ► hMSCs are capable of reducing STAT3 gene and Y705-phosphorylated STAT3 in microglial cells. ► Environmental influences must be considered in cellular therapy but may be reduced by inclusion of mesenchymal stem cells.
  
  PubDate: 2013-03-05T04:31:29Z
A system for reconstructing B cell antigen receptor signaling in the mouse myeloma J558L cell line
- Abstract: Available online 27 February 2013
  Publication year: 2013
  Source:Archives of Biochemistry and Biophysics
  
  B cell antigen receptor (BCR) signaling is positively and negatively regulated by various cell surface receptors such as CD19 and CD45. Functional analysis of these receptors has been performed using gene targeting technology, which is a valid approach to elucidate their functions. However, this type of analysis is restricted when multiple molecules are evaluated simultaneously. From a different perspective, synthetic biology provides a high degree of freedom for analyzing various molecules. Here we developed a system to reconstruct BCR signaling using the J558L myeloma cell line in combination with the protein-based Ca2+ indicator YC3.60. BCR-reconstituted J558L cells harboring YC3.60 (J558Lμv11 cells) permitted monitoring of Ca2+ mobilization. Reconstituting CD19 in J558Lμv11 cells resulted in detectable BCR-induced Ca2+ mobilization but with kinetics different from that of CD45-expressing cells. Furthermore, we evaluated the validity of the J558L system by proteomic analysis of tyrosine-phosphorylated proteins after antigen stimulation. Identification of more than 100 BCR-induced tyrosine-phosphorylated proteins in J558Lμv11 cells revealed a similarity to that observed in B cells, and a novel member, non-receptor protein tyrosine kinase Fer, was found. Thus, this reconstruction system using J558L cells appeared to be valid for comprehensively investigating BCR signaling.
  Highlights ► We developed a system to reconstitute BCR signaling in non-B cell line J558L. ► YC3.60 permits monitoring of BCR-mediated Ca2+ signaling. ► CD45 and CD19 differentially regulated Lyn and Akt activation and Ca2+ mobilization. ► We identified more than 100 tyrosine-phosphorylated proteins in BCR-reconstituted J558L cells. ► Fer was involved in BCR signaling.
  
  PubDate: 2013-03-01T22:24:43Z