Noradrenaline stimulates ATP release from DRG neurons by targeting β3 adrenoceptors as a factor of neuropathic pain

Noradrenaline (NA), released in association with sympathetic nerve sprouting into the dorsal root ganglion (DRG) after peripheral nerve injury, may enhance neuropathic pain. ATP serves as a pain mediator; however, NA‐regulated ATP mobilizations in the DRG is far from understanding. In the present study, we analyzed ATP mobilizations in acutely dissociated rat DRG neurons by recording single‐channel currents through P2X receptor channels as an ATP biosensor in an outside‐out patch‐clamp configuration and by monitoring real‐time enzymatic NADPH fluorescent imaging, and examined the role for β₃ adrenoceptors in allodynia using an in vivo rat model. We show here that NA stimulates ATP release from DRG neurons as mediated via β₃ adrenoceptors linked to G_s protein involving PKA activation, to cause allodynia. This represents a fresh regulatory pathway for neuropathic pain relevant to noradrenergic transmission in the DRG. J. Cell. Physiol. 224: 345–351, 2010. © 2010 Wiley‐Liss, Inc.     

Asymmetric Dimeric Structure of Ferredoxin-NAD(P) Oxidoreductase from the Green Sulfur Bacterium Chlorobaculum tepidum: Implications for Binding Ferredoxin and NADP

Ferredoxin-NAD(P)⁺ oxidoreductase (FNR) catalyzes the reduction of NAD(P)⁺ to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to plant-type FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR). Here, we report the crystal structure of C. tepidum FNR to 2.4 Å resolution, which reveals a unique structure-function relationship. C. tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically. One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form. We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge. The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd. Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by π-π stacking of Phe337 and the isoalloxazine ring of FAD. The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C. tepidum FNR.     

Arkadia complexes with clathrin adaptor AP2 and regulates EGF signalling

Arkadia is a positive regulator of transforming growth factor (TGF)-β signalling that induces ubiquitin-dependent degradation of several inhibitory proteins of TGF-β signalling through its C-terminal RING domain. We report here that, through yeast-two-hybrid screening for Arkadia-binding proteins, the μ2 subunit of clathrin-adaptor 2 (AP2) complex was identified as an interacting partner of Arkadia. Arkadia was located in both the nucleus and the cytosol in mammalian cells. The C-terminal YXXΦ-binding domain of the μ2 subunit associated with the N-terminal YALL motif of Arkadia. Arkadia ubiquitylated the μ2 subunit at Lys130. In addition, Arkadia interacted with the AP2 complex, and modified endocytosis of epidermal growth factor receptor (EGFR) induced by EGF. Arkadia thus appears to regulate EGF signalling by modulating endocytosis of EGFR through interaction with AP2 complex.     

Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate‐binding module (CBM)–cyan fluorescent protein (CFP)

In enzymatic saccharification of lignocellulosics, the access of the enzymes to exposed cellulose surfaces is a key initial step in triggering hydrolysis. However, knowledge of the structure–hydrolyzability relationship of the pretreated biomass is still limited. Here we used fluorescent‐labeled recombinant carbohydrate‐binding modules (CBMs) from Clostridium josui as specific markers for crystalline cellulose (CjCBM3) and non‐crystalline cellulose (CjCBM28) to analyze the complex surfaces of wood tissues pretreated with NaOH, NaOH–Na₂S (kraft pulping), hydrothermolysis, ball‐milling, and organosolvolysis. Japanese cedar wood, one of the most recalcitrant softwood species was selected for the analysis. The binding analysis clarified the linear dependency of the exposure of crystalline and non‐crystalline cellulose surfaces for enzymatic saccharification yield by the organosolv and kraft delignification processes. Ball‐milling for 5–30 min increased saccharification yield up to 77%, but adsorption by the CjCBM–cyan fluorescent proteins (CFPs) was below 5%. Adsorption of CjCBM–CFPs on the hydrothermolysis pulp were less than half of those for organosolvolysis pulp, in coincidence with low saccharification yields. For all the pretreated wood, crystallinity index was not directly correlated with the overall saccharification yield. Fluorescent microscopy revealed that CjCBM3–CFP and CjCBM28–CFP were site‐specifically adsorbed on external fibrous structures and ruptured or distorted fiber surfaces. The assay system with CBM–CFPs is a powerful measure to estimate the initiation sites of hydrolysis and saccharification yields from chemically delignified wood pulps. Biotechnol. Bioeng. 2010; 105: 499–508. © 2009 Wiley Periodicals, Inc.     

Micropatterning contractile C2C12 myotubes embedded in a fibrin gel

Contractile C₂C₁₂ myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C₂C₁₂ myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish. Biotechnol. Bioeng. 2010;105: 1161–1167. © 2009 Wiley Periodicals, Inc.     

Benzo[a]pyrene exposed to solar-simulated light inhibits apoptosis and augments carcinogenicity

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are widespread environmental pollutants and several lines of experimental evidence have suggested a role in carcinogenesis. PAHs in the environment are exposed to sunlight and photomodified PAHs have been detected in contaminated sediment and air particulate matter; however, the carcinogenicity of photomodified PAHs is not well understood. In this study, we found that solar-simulated light-irradiated BaP (LBaP) inhibited apoptosis, leading to cancer. LBaP suppressed apoptosis induced by cell detachment and serum depletion in a dose and light-irradiated time-dependent manner. The antiapoptotic effect was related to the production of reactive oxygen species from degraded BaP. The cells that survived apoptosis by LBaP treatment were transformed having the ability to form colonies in soft agar and tumors in nude mice. These capabilities were specific to LBaP, not BaP itself. The results suggested that the carcinogenicity of PAHs may be attributable not only to the genetic damage induced by their metabolites, but also to the antiapoptotic effects of oxidative products on exposure to sunlight.     

Detection of sequence-specific tyrosine nitration of manganese SOD and SERCA in cardiovascular disease and aging

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.