Molecular characterization of TRPA1 channel activation by cysteine-reactive inflammatory mediators

TRPA1 is a member of the transient receptor potential (TRP) cation channel family, and is predominantly expressed in nociceptive neurons of dorsal root ganglia (DRG) and trigeminal ganglia. Activation of TRPA1 by environmental irritants such as mustard oil, allicin and acrolein causes acute pain. However, the endogenous ligands that directly activate TRPA1 remain elusive in inflammation. Here, we show that a variety of inflammatory mediators (15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), nitric oxide (NO), hydrogen peroxide (H(2)O(2)), and proton (H(+))) activate human TRPA1 heterologously expressed in HEK cells. These inflammatory mediators induced robust Ca(2+) influx in a subset of mouse DRG neurons. The TRP channel blocker ruthenium red almost completely inhibited neuronal responses by 15d-PGJ(2) and NO, but partially suppressed responses to H(2)O(2) and H(+). Functional characterization of site-directed cysteine mutants of TRPA1 in combination with labeling experiments using biotinylated 15d-PGJ(2) demonstrated that modifications of cytoplasmic N-terminal cysteines (Cys421 and Cys621) were responsible for the activation of TRPA1 by 15d-PGJ(2). In TRPA1 responses to other cysteine-reactive inflammatory mediators, such as NO and H(2)O(2), the extent of impairment by respective cysteine mutations differed from those in TRPA1 responses to 15d-PGJ(2). Interestingly, the Cys421 mutation critically impaired the TRPA1 response to H(+) as well. Our findings suggest that TRPA1 channels are targeted by an array of inflammatory mediators to elicit inflammatory pain in the nervous system.     

Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera

A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.     

TNT biodegradation and production of dihydroxylamino-nitrotoluene by aerobic TNT degrader <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain TM15 in an anoxic environment

Anaerobic bacteria have been used to produce 2,4-dihydroxylamino-nitrotoluene (2,4DHANT), a reductive metabolite of 2,4,6-trinitrotoluene (TNT). Here, an aerobic TNT biodegrader Pseudomonas sp. strain TM15 produced 2,4DHANT as evidenced by the molecular ion with m/z of 199 identified from LC-TOFMS analyses. TNT biodegradation with a high cell concentration (10⁹ cells/ml) led to a significant accumulation of 2,4DHANT in the culture medium, as well as hydroxylamino-dinitrotoluenes (HADNTs), although these products were not accumulated when a low cell concentration was used; also, the accumulation of diamino-nitrotoluene and of an unidentified metabolite were observed in the culture medium with the high cell concentration (10¹⁰ cells/ml). 2,4DHANT overproduction was a function of the aeration speed since cultures with low aeration speeds (30 rpm) had a 19-fold higher DHANT productivity than those aerated with high speeds (180 rpm); this indicates that molecular oxygen was related to the formation of 2,4DHANT. The quantification of dissolved oxygen (DO) in the media demonstrated that the productivity of 2,4DHANT was increased at low DO values. Moreover, supplying oxygen to the culture media produced a remarkable decrease of 2,4DHANT accumulation; these results clearly indicate that high 2,4DHANT production was a consequence of the oxygen deficit in the culture medium. This finding is useful for understanding the TNT biodegradation (bioremediation technology) in an anoxic environment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Simulation of ATP metabolism in cardiac excitation–contraction coupling

To obtain insights into the mechanisms underlying the membrane excitation and contraction of cardiac myocytes, we developed a computer model of excitation–contraction coupling (Kyoto model: Jpn. J. Physiol. 53 (2003) 105). This model was further expanded by incorporating pivotal reactions of ATP metabolism; the model of mitochondrial oxidative phosphorylation by Korzeniewski and Zoladz (Biophys. Chem. 92 (2001) 17). The ATP-dependence of contraction, and creatine kinase and adenylate kinase were also incorporated. After minor modifications, the steady-state condition was well established for all the variables, including the membrane potential, contraction, and the ion and metabolite concentrations in sarcoplasmic reticulum, mitochondria and cytoplasm. Concentrations of major metabolites were close to the experimental data. Responses of the new model to anoxia were similar to experimental results of the P-31 NMR study in whole heart. This model serves as a prototype for developing a more comprehensive model of excitation–contraction–metabolism coupling.     

Complex formation of amphotericin B in sterol-containing membranes as evidenced by surface plasmon resonance

Amphotericin B (AmB) is a membrane-active antibiotic that increases the permeability of fungal membranes. Thus, the dynamic process of its interaction with membranes poses intriguing questions, which prompted us to elaborate a quick and reliable method for real-time observation of the drug's binding to phospholipid liposomes. We focused on surface plasmon resonance (SPR) and devised a new modification method of sensor chips, which led to a significant reduction in the level of nonspecific binding of the drug in a control lane. With this method in hand, we examined the affinity of AmB for various membrane preparations. As expected, AmB exhibited much higher affinity for sterol-containing palmitoyloleoylphosphatidylcholine membranes than those without sterol. The sensorgrams recorded under various conditions partly fitted theoretical curves, which were based on three interaction models. Among those, a two-state reaction model reproduced well the sensorgram of AmB binding to an ergosterol-containing membrane; in this model, two states of membrane-bound complexes, AB and AB*, are assumed, which correspond to a simple binding to the surface of the membrane (AB) and formation of another assembly in the membrane (AB*) such as an ion channel complex. Kinetic analysis demonstrated that the association constant in ergosterol-containing POPC liposomes is larger by 1 order of magnitude than that in the cholesterol-containing counterpart. These findings support the previous notion that ergosterol stabilizes the membrane-bound assembly of AmB.     

Immunohistochemical characterization of brush cells in the rat larynx

The immunohistochemical characteristics of brush cells in the laryngeal mucosa were examined using immunohistochemistry for various immunohistochemical cell markers including villin at the light and electron microscopic levels. Cells that were immunoreactive to villin were barrel-shaped with thick cytoplasmic processes extending toward the lumen of the laryngeal cavity. Immunoelectron microscopic observations revealed thick and short microvilli with long rootlets of microfilaments. Numerous small clear vesicles and small finger-like cytoplasmic processes were observed in the apical process and lateral membrane, respectively. Double immunofluorescence showed villin-immunoreactive cells were not immunoreactive for the markers of solitary chemosensory cells, GNAT3 and phospholipase C, β2-subunit (PLCβ2), or for that of neuroendocrine cells, synaptosome-associated protein 25kD. Furthermore, immunoreactivities for cytokeratin 18 (CK18) and doublecortin like-kinase 1 in the perinuclear cytoplasm of villin-immunoreactive cells. However, some CK18-immunoreactive cells were immunoreactive to GNAT3 but not to villin. Regarding sensory innervation, only a few intraepithelial nerve endings with P2X3, SP, or CGRP immunoreactivity attached to villin-immunoreactive cells. In the present study, brush cells in the rat laryngeal mucosa were classified by immunoreactivity for villin, and were independent of other non-ciliated epithelial cells such as solitary chemosensory cells and neuroendocrine cells.     

Multiple antiviral activities of the antimalarial and anti-hepatitis C drug candidates N-89 and N-251

The chemically synthesized endoperoxide compound N-89 and its derivative N-251 were shown to have potent antimalarial activity. We previously demonstrated that N-89 and N-251 potently inhibited the RNA replication of hepatitis C virus (HCV), which belongs to the Flaviviridae family. Since antimalarial and anti-HCV mechanisms have not been clarified, we were interested whether N-89 and N-251 possessed the activity against viruses other than HCV. In this study, we examined the effects of N-89 and N-251 on other flaviviruses (dengue virus and Japanese encephalitis virus) and hepatitis viruses (hepatitis B virus and hepatitis E virus). Our findings revealed that N-89 and N-251 moderately inhibited the RNA replication of Japanese encephalitis virus and hepatitis E virus, although we could not detect those anti-dengue virus activities. We also observed that N-89 and N-251 moderately inhibited the replication of hepatitis B virus at the step after viral translation. These results suggest the possibility that N-89 and N-251 act on some common host factor(s) that are necessary for viral replications, rather than the possibility that N-89 and N-251 directly act on the viral proteins except for HCV. We describe a new type of antiviral reagents, N-89 and N-251, which are applicable to multiple different viruses.