Galactomannans for Entrapment of Gliomastix murorum Laccase and Their Use in Reactive Blue 2 Decolorization

In the present study, a novel laccase from ascomycete Gliomastix murorum was produced in agro-industrial wastes and entrapped in galactomannan beads for Reactive Blue 2 (Rb-2) decolorization. The maximum laccase production in agave bagasse-based medium occurred at 72 h (1798.6 UL−1). Entrapped laccase decolorized ˃80% of 0.5 mM Rb-2 in 2 h without the addition of redox mediator. Km for Rb-2 substrate was 1.42 mM, with a Vmax of 1.19 µmol min−1 for entrapped laccase. Galactomannan matrices produce stability to acid pH (2–5) and temperatures from 20–70 °C. Reusability assays showed that entrapped laccase could retain efficient Rb-2 decolorization of ˃80% six times. In general, galactomannan used for entrapment of laccase provides economic advantages in large-scale wastewater treatment due to its natural origin and efficient results.

The effects of extracellular ATP and its receptor antagonists on pig oocytes during in vitro maturation

SummaryWe measured the ATP concentrations in the porcine follicular fluid derived from three sizes of follicles (small: <3 mm, medium: 3–6 mm, large: >6 mm in diameter). Then, the effects of pre-treatment (100 μM each for 30 min before maturation) with antagonists for extracellular ATP receptor P2X or P2Y on the nuclear maturation rate of cumulus-cell-enclosed (COs) or -denuded oocytes (DOs) up to the preovulatory stage in the presence or absence of 20 nM ATP (a similar concentration to that of medium-sized follicle fluid) were investigated. The antagonists used were pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) or reactive blue 2 (RB2), for extracellular ATP receptor P2X and P2Y, respectively. In addition, the embryonic development rates of COs pre-treated with RB2 were also evaluated. It was found that when the follicular sizes increased, the ATP concentrations significantly decreased (P < 0.05). No differences were observed in the nuclear maturation rates among all COs, regardless of pre-treatment with (+) or without (–) PPADS and in the presence (+) or absence (–) of ATP during maturation. In contrast, the nuclear maturation rate of the COs, but not DOs, in the ATP(–) RB2(+) group was significantly lower (P < 0.05) than that of the ATP(–) RB2(–) and ATP(+)RB2(–) groups. The pronuclear formation and blastocyst formation rates by parthenogenetic activation in the ATP(–) RB2(+) and ATP(+) RB2(+) groups were significantly lower (P < 0.05) than those in the ATP(–) RB2(–) group. In conclusion, it is suggested that the nuclear maturation of porcine oocytes may be influenced by the ATP receptor P2Y present in the cumulus cells.

Effect of epithelium ATP release on cyclic pressure-induced airway mucus secretion

The cyclic mechanical effect of airflow during breathing creates the optimal airway hydration state. MUC (mucin) 5AC is an important component of the airway mucus. The formation of MUC5AC is related to ATP and intracellular calcium in the epithelial cells. In this study, we evaluated the effect of ATP release from intracellular calcium in epithelial cells on cyclic pressure-induced mucus secretion in the airway. 16HBE (human bronchial epithelial cells) were cultured in vitro on cyclically tilted cultured plates and divided into five groups: control, tilt, tilt and BAPTA–AM (1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid–acetoxymethyl ester), tilt and EGTA and tilt and RB-2 (reactive blue-2). The shear stress and compressive stress were induced by the surface tension of the liquid, atmospheric pressure and liquid gravity. Cell activity, MUC5AC mRNA expression level, MUC5AC protein expression level and ATP release and intracellular calcium changes were measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, RT–PCR (reverse transcription–PCR), HPLC and inverted fluorescence microscope, respectively. We detected that cyclic pressure significantly increased MUC5AC secretion and ATP release. The enhanced ATP release could be inhibited by both BAPTA–AM and RB-2, while EGTA did not have a suppressive effect. BAPTA–AM, EGTA and RB-2 did not obviously inhibit MUC5AC mRNA expression. Cyclic pressure did not induce MUC5AC secretion in the airway mucus epithelium via Ca2+-dependent ATP release, and nearly all Ca2+ was provided by stored intracellular Ca2+.

Histamine Induces ATP Release from Human Subcutaneous Fibroblasts, via Pannexin-1 Hemichannels, Leading to Ca2+ Mobilization and Cell Proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Activation of P2Y1 and P2Y2 nucleotide receptors by adenosine 5′-triphosphate analogues augmented nerve-mediated relaxation of human corpus cavernosum

Introduction: Adenosine 5′-triphosphate (ATP) is a ubiquitous cellularenergy source. We evaluated the effect of ATP and its analogueson nonadrenergic and noncholinergic relaxation in precontractedhuman corpus cavernosal smooth muscle (HCCSM).Methods: We obtained specimens of human corpus cavernosum(HCC) from patients undergoing penile prosthesis surgery (patientage 46–70 yr, n = 17) with prior approval from the local institutionalreview board. Isolated HCC strips were placed in organbaths containing Krebs solution and functional experiments wereconducted. Immunohistochemical localization studies were performedto establish the presence of purinergic P2X1, P2Y1 andP2Y2 receptors in HCC.Results: The amplitude of relaxation induced by electrical-fieldstimulation (EFS) on HCC was significantly increased after exposureto ATP (P2X and P2Y agonists), 2-MeSATP (P2Y1 agonist), and uridine5’ triphosphate (P2Y2 agonist), but not α,β-methylene ATP(P2X1 agonist). The P2X1 antagonist pyridoxal-5’-phosphate-6-azophenyl-2’, 4’-disulfonate, and the nonspecific P2Y antagonist,reactive blue 2, did not inhibit the potentiated response of EFS onHCC. Although immunoreactivity for both P2Y1 and P2Y2 receptorswas localized abundantly in HCC, there was only low-levelimmunostaining for the P2X1 receptor.Conclusion: These data demonstrate that nerve-mediated relaxation ofHCCSM strips precontracted with phenylephrine in organ bath preparationsis amplified by stimulating purinergic P2Y1 and P2Y2 receptors.Although nucleotides are important regulators of HCCSM tone, theseobservations suggest an independent purinergic relaxing mechanismin the HCC, separate from the better known nitrergic system.Introduction : L’adénosine 5’-triphosphate (ATP) est une sourced’énergie cellulaire générale. L’effet de l’ATP et de ses analoguessur le relâchement non adrénergique et non cholinergique du musclelisse précontracté du corps caverneux a été évalué.Méthode : Des échantillons de corps caverneux humains (CCH)ont été obtenus à partir de patients porteurs d’une prothèse pénienne(âgés de 46 à 70 ans, n = 17) avec l’approbation préalabledu comité d’éthique de l’établissement. Des bandes isolées deCCH ont été placées dans des bains organiques contenant unesolution de Krebs et des expériences fonctionnelles ont ensuite étéréalisées. On a eu recours à des tests de localisation immunohistochimiquepour déceler la présence des récepteurs purinergiquesP2X1, P2Y1 et P2Y2 dans les échantillons de CCH.Résultats : L’ampleur du relâchement produit par stimulation électriquedes échantillons de CCH a été significativement accrue aprèsexposition à l’ATP (agoniste des récepteurs P2X et P2Y),à la 2-MeSATP (agoniste du récepteur P2Y1) et à l’UTP (agonistedu récepteur P2Y2), mais pas à la α,β-MeATP (agoniste du récepteurP2X1). L’antagoniste du récepteur P2X1, le pyridoxal-5'-phosphate-6-azophényl-2’, 4’-disulfonate, et l’agoniste nonspécifique du récepteur P2Y, le bleu chimiquement réactif, n’ontpas inhibé la réponse potentialisée par stimulation électriquedes bandes de CCH. Même si une immunoréactivité des récep teursP2Y1 et P2Y2 a été grandement notée dans les bandes de CCH, onn’a obtenu qu’une faible immunocoloration pour le récepteur P2X1.Conclusion : Ces données montrent que le relâchement par voienerveuse des bandes de CCH précontractées par phényléphrinedans des bains organiques est amplifié par la stimulation des récepteurspurinergiques P2Y1 et P2Y2. Bien que les nucléotides constituentdes facteurs importants de régulation du tonus des CCH,ces observations portent à croire à l’existence d’un mécanismeindépendant de relâchement purinergique distinct du système nitrergiquemieux connu.

A simple and accurate analytical method for determination of three commercial dyes in different water systems using partial least squares regression

A simple analytical procedure is proposed for simultaneous determination of three common dyes (Basic Blue 9, Brilliant Blue E-4BA, and Reactive Blue 2) in natural waters without prior separation of the solutes. A popular chemometric method, partial least squares regression PLS-1, was effectively applied for spectral resolution of a highly overlapping system. At the best modeling conditions, mean recoveries and relative standard deviations (RSD) for dyes quantification by PLS-1 were found to be 102.1 (4.4), 95.7 (8.4), and 98.9 (6.2) for Basic Blue, Brilliant Blue, and Reactive Blue, respectively. The estimated limits of detection (LOD) were estimated using net-analyte signal concept and were 0.11, 0.52, 0.49 mg L−1 for Basic Blue, Brilliant Blue, and Reactive Blue, respectively. The quantitative determination of dyes spiked in real water samples was carried out successfully by PLS-1 with satisfactory recoveries for dyes (90–106%).