Phenazine Methosulphate Research Articles

Photosynthetic efficiency and compositional alterations in microalgae Chlorella vulgaris in response to changes in the pH condition

Present investigation on the effect of varying extracellular pH conditions (pH 6.5–10.5) on growth, accumulation of cell constituents and photosynthetic performance of microalga Chlorella vulgaris revealed that slightly alkaline pH (7.5–8.5) was the preferred pH range for growth, synthesis of protein and carbohydrate. Whereas total organic carbon (TOC) and lipid content in the microalga gradually increased with pH ranging ranging from 6.5 to 10.5. The FTIR results on total lipid (1740 cm− 1), lipid/carbohydrate (1740/1650 cm− 1) and lipid/protein (1740/1040 cm− 1) ratio in the cells grown at pH 6.5, 8.5 and 10.5 supported the above observation on the estimated amount of macromolecules. The chlorophyll fluorescence induction kinetics (OJIP), exhibiting various photosynthetic parameters, showed initial improvement in the photosynthetic efficiency between pH 6.5–8.5, followed by pH dependent declining pattern. Addition of HCO3− (20 mM) showed little effect on the photosynthetic electron transport. The energy dependent quenching parameters of chlorophyll fluorescence like ABS/RC, NPQ and qE, usually associated with the energy state of the membrane, registered an increasing trend with rising pH (pH 6.5–10.5). Use of energy inhibitors DNP, CCCP and electron acceptors/donors (methyl viologen and phenazine methosulphate) on ABS/RC, NPQ and qE suggested that pH dependent regulation of photosynthetic performance in C. vulgaris was tightly coupled with proton gradient of the membrane and that was reversed by the energy uncouplers like DNP and CCCP or electron acceptor like MV.

Phenazine methosulphate-treated red blood cells activate NF-κB and upregulate endothelial ICAM-1 expression

Although enhanced Red Blood Cell (RBC) - Endothelial Cell (EC) interaction, as well as RBC induced EC activation, have been extensively studied in several RBC-linked pathologies, the specific individual effects of oxidatively modified RBC on EC activation has not yet been documented. However, increasing evidence in both experimental and clinical studies suggests that oxidatively modified RBC could be considered potential pathogenic determinants in several acute and chronic diseases displaying systemic oxidative stress. Therefore, the present study aimed to explore the specific effects of oxidized RBC interaction with endothelial cells on intracellular signaling pathways that promote EC activation. RBC were exposed to oxidative stress induced by phenazine methosulphate (PMS). It is shown that the interaction of oxidatively modified RBC with cultured human umbilical vein endothelial cells (HUVEC) results in: a) EC activation as indicated by the increased surface expression of intercellular adhesion molecule −1 (ICAM-1); b) the activation of transcription factor NF-κB, an indicator of cellular oxidant stress. These results emphasize the specific contribution of oxidatively modified RBC interaction to EC activation and their possible pathological role in vascular diseases and oxidative stress.

To determine the prevalence of glucose-6-phosphate dehydrogenase deficiency using a novel water-soluble tetrazolium-8 formazan method' for neonatal screening in region of Himachal Pradesh, India

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most significant enzyme defect in India with an incidence ranging from 2% to 27.9% in different communities. Prolonged neonatal jaundice and haemolytic crisis are known to occur in children with G6PD deficiency. Hence, screening of a population for G6PD deficiency is paramount. A Novel water soluble tetrazolium-8 (WST-8) Formazan Method has been used in this study for in-field mass-screening of G6PD in the region of Himachal Pradesh, India. Materials and Methods: In this prospective study, 5652 neonates were screened to assay G6PD activity using WST8/1-methoxy phenazine methosulphate method within the first 48 h of life. Orange colour at the end of the procedure indicated normal G6PD activity while pink or colourless appearance indicated G6PD deficiency. Results: After the screening of 5652 neonates, the prevalence of G6PD deficiency was 12.4%. 45 newborns (6%) had a severe G6PD deficiency. Males were more affected than females (70:30). Furthermore, males had higher prevalence of deficiency than females (64% [n = 29] and 16% [n = 16]). Conclusions: G6PD deficiency assessment by the method used for population screening in the study was easy to do and quite simple. Following this, the high prevalence of this deficiency was noted in Himachal Pradesh. This study highlights the need to do neonatal screening of G6PD deficiency in population so that untowards complications like haemolytic crisis, complications due to neonatal jaundice can be avoided.

Oxidative stress and phosphatidylserine exposure in red cells from patients with sickle cell anaemia.

SummaryPhosphatidylserine (PS) exposure increases as red cells age, and is an important signal for the removal of senescent cells from the circulation. PS exposure is elevated in red cells from sickle cell anaemia (SCA) patients and is thought to enhance haemolysis and vaso‐occlusion. Although precise conditions leading to its externalisation are unclear, high intracellular Ca2+ has been implicated. Red cells from SCA patients are also exposed to an increased oxidative challenge, and we postulated that this stimulates PS exposure, through increased Ca2+ levels. We tested four different ways of generating oxidative stress: hypoxanthine and xanthine oxidase, phenazine methosulphate, nitrite and tert‐butyl hydroperoxide, together with thiol modification with N‐ethylmaleimide (NEM), dithiothreitol and hypochlorous acid (HOCl), in red cells permeabilised to Ca2+ using bromo‐A23187. Unexpectedly, our findings showed that the four oxidants significantly reduced Ca2+‐induced PS exposure (by 40–60%) with no appreciable effect on Ca2+ affinity. By contrast, NEM markedly increased PS exposure (by about 400%) and slightly but significantly increased the affinity for Ca2+. Dithiothreitol modestly reduced PS exposure (by 25%) and HOCl had no effect. These findings emphasise the importance of thiol modification for PS exposure in sickle cells but suggest that increased oxidant stress alone is not important.

Identification and characterization of a new three-component nicotinic acid hydroxylase NahAB1 B2 from Pusillimonas sp. strain T2.

Nicotinic acid (NA) serves as a model system for the degradation of N-heterocyclic aromatic compounds and the microbial degradation mechanisms are diverse. This is the first time that a three-component hydroxylase has been identified. This study provides a novel insight into the NA degradation by bacteria.

In vitro and In vivo hepatoprotective studies on methanolic extract of aerial parts of Ludwigia hyssopifolia G. Don Exell

Background: Ludwigia hyssopifolia of family Onagraceae is traditionally used in the treatment of jaundice. There were no reports on the plant for both in vivo and in vitro hepatoprotective studies. Objective: The current study was aimed to evaluate the methanolic extract of aerial parts of L. hyssopifolia (LHME) for its in vitro and in vivo hepatoprotective activity against ethanol-induced oxidative damage in HepG2 cell lines and Wistar rats, respectively, and in vivo hepatoprotective activity against paracetamol and antihepatotoxic activity against D-galactosamine in rats. Materials and Methods: The antioxidant potential of the extract was investigated by employing different in vitro methods. The in vitro hepatoprotective activity of the extract was assessed by estimating cell supernatant for aspartate aminotransferase (AST), Alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) while the in vivo hepatoprotective activity of the extract was assessed on the basis of improvement in the altered level of various serum biochemical parameters and in the changes occurred in the histology of liver of the rats. Results: Among the three test doses of LHME, 100 μg/kg and 200 mg/kg were found to be the effective doses in in vitro and in vivo hepatoprotective methods, respectively. Conclusion: The extract, LHME, exhibited significant (P < 0.01) hepatoprotective activity in both in vitro and in vivo models, which may be attributed to its antioxidant property revealed in both in vitro and in vivo studies. Abbreviation used: DPPH: 1,1 Diphenyl-1-picryl hydrazyl; ALT: Alanine transaminase; ALB: Albumin; ALP: Alkaline phosphatase; AST: Aspartate transaminase; CHOL: Cholesterol; DB: Direct bilirubin; FBS: Fetal bovine serum; GAE: Gallic acid equivalents; GGT: Gamma glutamyl transferase; GLU: Glucose; LDH: Lactate dehydrogenase; MEM: Modified Eagle's Medium; NBT: Nitro blue tetrazolium; PMS: Phenazine methosulphate; RE: Rutin equivalents; LHME: The methanolic extract of Ludwigia hyssopifolia; TBA: Thiobarbituric acid; TB: Total bilirubin; TP: Total protein; TCA: Trichloro acetic acid.

Phenazine Methosulphate Modulating the Expression of Genes Involved in Yeast to Hyphal Form Signal Transduction in &amp;lt;i&amp;gt;Candida albicans&amp;lt;/i&amp;gt;

Candida albicans has ability to switch from yeast to hyphal form which is an important virulence factor. The objective of the research is to study the effect of Phenazine Methosulphate (PMS) on virulence factors and to study expression profile in yeast to hyphal form transition in C. albicans. Phenazine Methosulphate (PMS) acted as an inhibitor of yeast to hyphal form transition, adhesion and biofilm formation in C. albicans. RTPCR study demonstrated that PMS Modulate the expression of genes involved in Ras1-cAMP-Efg1 and Cek1-MAPK signal transduction pathways. Cell cycle of C. albicans was arrested at S phase on treatment of PMS. Hyphal suppressor genes like Tup1, Mig1 and Nrg1 were upregulated by PMS. Based on our data on expression of genes during yeast to hyphal form transition in presence and absence of PMS, we hypothesize that inhibition of hyphal formation may be due to the overexpression of negative regulators of hyphal growth. Targeting of hyphal specific genes involved in these pathways may be a promising strategy for anti-candida drug development.

Identification of Substrate Binding Site of Fungus Derived Flavin Adenine Dinucleotide Dependent Glucose Dehydrogenase

【Introduction】 The fungi derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (GDHs) are flavoproteins that catalyze the oxidation of first hydroxyl group of glucose and other sugar molecules, using FAD as the primary electron acceptor (systematic name; D-glucose:acceptor 1-oxidoreductase). Recently, fungi-derived FADGDHs have received attention as the enzymes for blood glucose monitoring in diabetes patients, especially for the use in the self-monitoring of blood glucose (SMBG), because of their oxygen insensitivity and relatively narrow substrate specificity, in particular their lack of activity toward maltose. However, unlike glucose oxidase, a gold standard enzyme for blood glucose monitoring, especially for the strictly narrow specificity toward glucose, fungi derived FADGDHs react with xylose as the substrate. We have previously reported on the characterization of Aspergillus flavus derived FADGDH (AfGDH) from genome database 1, and also the stabilization of AfGDH by introducing a disulfide bond 2. Recently we have reported on the first 3D structure of fungus derived FADGDH3. In order to construct an engineered enzyme meeting with the current increasing demands in the accuracy of sensors for SMBG, further advance in our understanding of the mechanism of substrate recognition of this enzyme is necessary.In this study, we report the site directed mutagenesis studies toward the residues expected to influence the binding of substrate, based on the structural analyses of AfGDH crystal structure (PDB ID：4ynu). 【Methods】The residues responsible for the substrate binding and/or recognition of AfGDH and Aspergillus niger derived GOx (AnGOx), were predicted based on the reported crystal structure (PDB ID:4ynu, 1cf3). According to this prediction, several mutant AfGDHs were designed and constructed by the sited directed mutagenesis. AfGDH wild type (WT) and mutants were prepared using recombinant Escherichia coli. The enzyme activities and substrate specificities were determined by measuring dye mediated dehydrogenase activity, spectrometrically, using phenazine methosulphate as the electron mediator and 2,6-dichlorophenolindophenol(DCIP) as the final electron acceptor, and by monitoring decrease in the absorbance of oxidized DCIP at 600nm. 【Results and Discussion】Several residues existing within the distance which may interact with glucono-δ-lactone by their side chain were observed from the ternary complex structure of AfGDH (4ynu). Assuming that this side chain of each residue may recognize and interact with glucose, AfGDH structure was compared with AnGOx. As results, residues, possibly recognize 1st, 2nd, 3rd or 4th hydroxyl group of glucose are conserved within AfGDH and AnGOx. Especially, two residues recognize 1st hydroxyl group of glucose are completely conserved. Mutational analyses resulted in the complete loss of the catalytic activity, thus suggesting that these residues are the catalytic residues. The residue interacting 4th hydroxyl group is conserved, and has been recognized that plays an important role in discrimination of monosaccharide from disaccharides, such as maltose. Interestingly, the introduction of mutation on this residue did not result in the significant change in the substrate specificity, especially toward maltose. The results suggested that the predicted residue may have a limited role in the determination of substrate specificity, and other residues may have more crucial roles. Significant difference in the substrate interaction was observed at the position where may recognize 6th hydroxyl group, which would be the reason of the AfGDH substrate specificity reacting with xylose. Further mutational analyses will be reported.(1) K. Mori, et al., Biotechnol Lett. 33(11) 2255-63(2011) (2) G. Sakai, et al., Biotechnol Lett. 37(5) 1091-99(2015) (3) H. Yoshida, et al., Sci. Rep. 5:13498 doi: 10.1038/srep13498(2015)

Oscillation Kinetics of Post-illumination Increase in Chl Fluorescence in Cyanobacterium Synechocystis PCC 6803.

After termination of longer-illumination (more than 30 s), the wild type of Synechocystis PCC 6803 showed the oscillation kinetics of post-illumination increase in Chl fluorescence: a fast phase followed by one or two slow phases. Unlike the wild type, ndh-B defective mutant M55 did not show any post-illumination increase under the same conditions, indicating that not only the fast phase, but also the slow phases were related to the NDH-mediated cyclic electron flow around photosystem I (PS I) to plastoquinone (PQ). The fast phase was stimulated by dark incubation or in the presence of Calvin cycle inhibitor, iodoacetamide (IA) or cyclic photophosphorylation cofactor, phenazine methosulphate (PMS), implying the redox changes of PQ by electrons generated at PS I reduced side, probably NAD(P)H or ferredoxin (Fd). In contrast, the slow phases disappeared after dark starvation or in the presence of IA or PMS, and reappeared by longer re-illumination, suggesting that they are related to the redox changes of PQ by the electrons from the photoreductants produced in carbon assimilation process. Both the fast phase and slow phases were stimulated at high temperature and the slow phase was promoted by response to high concentration of NaCl. The mutant M55 without both phases could not survive under the stressed conditions.

L-Lactate-selective microbial sensor based on flavocytochrome b2-enriched yeast cells using recombinant and nanotechnology approaches

In the recent years, nanotechnology is the most developing branch due to a wide variety of potential applications in biomedical, biotechnological and agriculture fields. The binding nanoparticles with various biological molecules makes them attractive candidates for using in sensor technologies. The particularly actual is obtaining the bionanomembranes based on biocatalytic elements with improved sensing characteristics.The aim of this investigation is to study the properties of microbial L-lactate-selective sensor based on using the recombinant Hansenula polymorpha yeast cells overproducing flavocytochrome b2 (FC b2), as well as additionally enriched by the enzyme bound with gold nanoparticles (FC b2-nAu). Although, the high permeability of the living cells to nanoparticles is being intensively studied (mostly for delivery of drugs), the idea of using both recombinant technology and nanotechnology to increase the amount of the target enzyme in the biosensing layer is really novel.The FC b2-nAu-enriched living and permeabilized yeast cells were used for construction of a bioselective membrane of microbial L-lactate-selective amperometric biosensor. Phenazine methosulphate was served as a free defusing electron transfer mediator which provides effective electron transfer from the reduced enzyme to the electrode surface. It was shown that the output to L-lactate of FC b2-nAu-enriched permeabilized yeast cells is 2.5-fold higher when compared to the control cells.The obtained results confirm that additional enrichment of the recombinant yeast cell by the enzyme bound with nanoparticles improves the analytical parameters of microbial sensor.

Magnesium availability regulates the development of root hairs in Arabidopsis thaliana (L.) Heynh.

Root hairs are reported to be plastic in response to nutrient supply, but relatively little is known about their development in response to magnesium (Mg) availability. Here, we showed that development of root hairs of Arabidopsis decreased progressively with increasing Mg supply, which was related to the initiation of new trichoblast files and likelihood of trichoblasts to form hairs. Tip-focused reactive oxygen species (ROS) and cytosolic Ca(2+) concentrations [(Ca(2+) )c] during elongation of root hairs were enhanced under low Mg but decreased under high Mg. Under low Mg, application of diphenylene iodonium (DPI) or BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] blocked the enhanced development of root hairs and the opposite was true when the plants under high Mg were treated with phenazine methosulphate (PMS), methyl viologen (MV) or CaCl2 . Furthermore, Mg availability did not alter root hair growth in rhd2-1 mutant that contains lower levels of ROS and cytosolic [Ca(2+) ]c. Transcriptome data and qPCR results revealed a greater fraction of morphogenetic H-genes, and cell wall organization genes were up-regulated by low Mg but down-regulated by high Mg. Our data suggest a profound effect of Mg supply on the development of root hairs in Arabidopsis, through the characterized Ca(2+) and ROS signals that modulate the elongation of root hairs and the expression of root-hair morphogenetic genes.

Flavonols and derivatives of gallic acid from young leaves of Toona sinensis (A. Juss.) Roemer and evaluation of their anti-oxidant capacity by chemical methods.

Background:Toona sinensis (A. Juss.) Roemer is an endemic species of Toona genus native to Asia. Its crude extract exhibits an effective anti-oxidant capacity against oxidative models, but the intrinsic substances responsible for this capacity in the extract remains unclear and is yet to be studied comprehensively.Objective:To investigate the chemical constituents of the young leaves of Toona sinensis and its anti-oxidant capacity.Materials and Methods:Silica gel column chromatography, preparative high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and mass spectrometry (MS) were used to isolate and characterize the chemical constituents. Four chemical-induced oxidative models including DPPH free-radical scavenging assay, phenazine methosulphate (PMS) nicotinamide adenine dinucleotide (NADH) PMS-NADH-NBT superoxide anion scavenging assay, FeCl3-K3Fe (CN)6 reducing power assay, and FeCl2-FerroZine metal chelation assay were applied in the present study for evaluating anti-oxidant capacity.Results:Five flavonols and three derivatives of gallic acid, including quercetrin, kaempferol-3-O-α-L-rhamopyranoside, astragalin, quercetin, kaempferol, methyl gallate, ethyl gallate, and 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucopyranose were isolated from the leaves. Results showed that these compounds exhibited various antioxidant properties, markedly either as the strong scavengers for superoxide and free radicals or as molecules that were reducing or metal chelating in nature.Conclusion:The findings suggested that the 8 compounds in the young leaves of T. sinensis that were isolated in our study were the active compounds responsible for its antioxidant activity. These compounds can be utilized as a potential health supplement, as an available source of natural antioxidants, and as an effective material in pharmaceutical applications.

β-Carotene as a factor in the reconstitution of cyclic phospho rylation in damaged chloroplast membranes

Phenazine methosulphate mediated cyclic phosphorylation suppressed by heptane extraction or galactolipase treatment of spinach chloroplasts is restored by β -carotene, in 100% and 50%, respectively. Xanthophylls are not able to reconstitute this reaction. β-Carotene replaces galactolipids in reactivation of galactolipase treated chloroplasts, indicating a nonspecific effect of lipids in photosystem I dependent reactions.

Microfluidic analysis of cellular deformability of normal and oxidatively damaged red blood cells

Microfluidic analysis of blood has potential clinical value for determining normal and abnormal erythrocyte deformability. To determine if a microfluidic device could reliably measure intra- and inter-personal variations of normal and oxidized human red blood cell (RBC), venous blood samples were collected from repeat donors over time. RBC deformability was defined by the cortical tension (pN/µm), as determined from the threshold pressure required to deform RBC through 2-2.5 μm funnel-shaped constrictions. Oxidized RBC were prepared by treatment with phenazine methosulphate (PMS; 50 µM). Analysis of the control and oxidized RBC demonstrated that the microfluidic device could clearly differentiate between normal and mildly oxidized (20.13 ± 1.47 versus 27.51 ± 3.64 pN/µm) RBC. In vivo murine studies further established that the PMS-mediated loss of deformability correlated with premature clearance. Deformability variation within an individual over three independent samplings (over 21 days) demonstrated minimal changes in the mean pN/µm. Moreover, inter-individual variation in mean control RBC deformability was similarly small (range: 19.37-21.40 pN/µm). In contrast, PMS-oxidized cells demonstrated a greater inter-individual range (range: 25.97-29.90 pN/µm) reflecting the differential oxidant sensitivity of an individual's RBC. Importantly, similar deformability profiles (mean and distribution width; 20.49 ± 1.67 pN/µm) were obtained from whole blood via finger prick sampling. These studies demonstrated that a low cost microfluidic device could be used to reproducibly discriminate between normal and oxidized RBC. Advanced microfluidic devices could be of clinical value in analyzing populations for hemoglobinopathies or in evaluating donor RBC products post-storage to assess transfusion suitability.

Alr0882 encoding a hypothetical protein of Anabaena PCC7120 protects Escherichia coli from nutrient starvation and abiotic stresses

This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (∆uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ∆uspA E. coli strain. The IPTG induced expression of a 56.6kDa GST fusion protein was visualized on SDS–PAGE and attested by immunoblotting. E. coli ∆uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl2, H2O2, UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl2 demonstrated about 22.6–51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria.

In vitro effect of calcium dobesilate on oxidative/inflammatory stress in human varicose veins

To determine whether calcium dobesilate can act in chronic venous insufficiency by similar antioxidant, anti-inflammatory mechanisms as in diabetic retinopathy. Calcium dobesilate was tested in vitro for its protective action against oxidative/inflammatory stress in human varicose veins. Varicose greater saphenous veins were obtained from 14 patients (11 men, 3 women) aged 53-65 years. Oxidative stress was induced exogenously in the vein segments, with the phenazine methosulphate (PMS)/NADH couple. Total antioxidant status (TAS) and malondialdehyde (MDA) contents were used as markers of oxidative stress. Calcium dobesilate significantly prevented oxidative disturbances in the micromolar range. PMS/NADH-dependent TAS decrease was fully prevented with IC(50) = 11.4 ± 2.3 µmol/L (n = 6 veins), whereas MDA increase was fully prevented with IC(50) = (102 ± -3) µmol/L (n = 6 veins). Calcium dobesilate acted quali- and quantitatively like rutin, the reference compound. Comparison with pharmacokinetic data suggests that calcium dobesilate can act at therapeutic concentrations. Calcium dobesilate protected human varicose veins against oxidative stress in vitro at levels that correspond to therapeutic concentrations. Further studies are required to investigate whether a similar action is found in varicose veins from patients orally treated with calcium dobesilate.

Antifungal mechanisms by which a novel Pseudomonas aeruginosa phenazine toxin kills Candida albicans in biofilms

Pseudomonas aeruginosa produces several phenazines including the recently described 5-methyl-phenazine-1-carboxylic acid (5MPCA), which exhibits a novel antibiotic activity towards pathogenic fungi such as Candida albicans. Here we characterize the unique antifungal mechanisms of 5MPCA using its analogue phenazine methosulphate (PMS). Like 5MPCA, PMS induced fungal red pigmentation and killing. Mass spectrometry analyses demonstrated that PMS can be covalently modified by amino acids, a process that yields red derivatives. Furthermore, soluble proteins from C. albicans grown with either PMS or P. aeruginosa were also red and demonstrated absorbance and fluorescence spectra similar to that of PMS covalently linked to either amino acids or proteins in vitro, suggesting that 5MPCA modification by protein amine groups occurs in vivo. The red-pigmented C. albicans soluble proteins were reduced by NADH and spontaneously oxidized by oxygen, a reaction that likely generates reactive oxygen species (ROS). Additional evidence indicated that ROS generation precedes 5MPCA-induced fungal death. Reducing conditions greatly enhanced PMS uptake by C. albicans and killing. Since 5MPCA was more toxic than other phenazines that are not modified, such as pyocyanin, we propose that the covalent binding of 5MPCA promotes its accumulation in target cells and contributes to its antifungal activity in mixed-species biofilms.

Purification and Characterization of Membrane-Associated Hydrogenase from the Deep-Sea EpsilonproteobacteriumHydrogenimonas thermophila

Membrane-associated hydrogenase was purified from the chemolithoautotrophic epsilonproteobacterium Hydrogenimonas thermophila at 152-fold purity. The hydrogenase was found to be localized in the periplasmic space, and was easily solubilized with 0.1% Triton X-100 treatment. Hydrogen oxidation activity was 1,365 micromol H(2)/min/mg of protein at 80 degrees C at pH 9.0, with phenazine methosulphate as the electron acceptor. Hydrogen production activity was 900 micromol H(2)/min/mg of protein at 80 degrees C and pH 6.0, with reduced methyl viologen as the electron donor. The hydrogenase from this organism showed higher oxygen tolerance than those from other microorganisms showing hydrogen oxidation activity. The structural genes of this hydrogenase, which contains N-terminal amino acid sequences from both small and large subunits of purified hydrogenase, were successfully elucidated. The hydrogenase from H. thermophila was found to be phylogenetically related with H(2) uptake hydrogenases from pathogenic Epsilonproteobacteria.

Enzyme separation and isozyme heterogeneity analysis using non-denaturing two-dimensional electrophoresis

Carboxylesterase and sorbitol dehydrogenase are separated by non-denaturing two-dimensional electrophoresis (2-DE) of isoelectric focusing separation using 5% carrier ampholyte (pH 6–8) and 1.25% carrier ampholyte (pH 3–10) and size separation. Furthermore, activities of sorbitol, malate and lactate dehydrogenases are sequentially examined when the enzymes are separated by 2-DE and are sequentially reacted to sorbitol, malic and lactic acid, respectively, in the presence of nicotinamide adenine dinucleotide, nitro blue tetrazolium and phenazine methosulphate. Several kinds of enzymes including lactate dehydrogenize isozymes can be simultaneously separated using 2-DE. Furthermore, the binding differences between lactate dehydrogenase isozymes and concanavalin A (con A) can be examined using a combination of 2-DE and non-denaturing stacking gel electrophoresis. The results of this study indicate that non-denaturing 2-DE can be applied to both enzyme separation and isozyme heterogeneity analysis.

Effect of yeast pretreatment on the characteristics of yeast-modified electrodes as mediated amperometric biosensors for lactic acid

Carbon paste electrode modified with baker' and wine yeast Saccharomyces cerevisiae (a source of flavocytochrome b2) were investigated as amperometric biosensors for l-lactic acid. Before immobilization on the electrode surface, yeast cells were pretreated with various electrolytes, alcohols and weak organic acids. Electrode responses to l-lactic acid were tested in the presence of various mediators (potassium ferricyanide, phenazine methosulfate, 2,6-dichlorophenolindophenol sodium salt hydrate, 1,2-naphthoquinone-4-sulfonic acid sodium salt). The highest (144±7 nA per 0.2 mM l-lactic acid) and the most stable responses were obtained after yeast pretreatment with 30% ethanol using potassium ferricyanide as a mediator. Different electrode sensitivities with mediator phenazine methosulphate probably reflected diverse changes in yeast membrane (and/or cell wall).