Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

Direct formation of axes in new plantlets of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Doty) Doty,as influenced by the use of AMPEP K<Superscript>+</Superscript>, spindle inhibitors,and plant growth hormones

In order to induce the direct formation of erect dark brown vegetative shoots of Kappahycus alvarezii, under micro-propagation conditions, the agricultural bio-stimulant “Acadian Marine Plant Extract Powder” (or AMPEP) K⁺ (a Canadian commercial extract of the brown seaweed Ascophyllum nodosum with enhanced levels of potassium) was used singly or in combination with colchicine or oryzalin at different concentrations plus (synthetic) plant growth regulators (i.e., PGR, IAA, and kinetin) over 45 days of incubation. Amongst the treatments tested, 5 mg L^?1 AMPEP K⁺ with PGR produced the longest direct axis shoots (9.6 ± 0.33 mm), followed by 0.1 mg L^?1 AMPEP K⁺ + 1.0 mg L^?1 oryzalin with PGR (8.7 ± 0.00 mm), 10 mg L^?1 AMPEP K⁺ + 0.5 mg L^?1 colchicine with PGR (8.6 ± 1.20 mm), and 0.5 mg AMPEP K⁺ only (5.3 ± 1.8 mm). The shortest, direct axis shoots measured were the following: 8.3 ± 0.33 mm with 0.1, 1 and 10 mg L^?1 AMPEP K⁺ with PGR, 6.0 ± 0.58 mm with 0.5 mg L^?1 AMPEP K⁺ + 0.1 mg L^?1 oryzalin with PGR, 5.3 ± 0.89 mm with 0.5 mg L^?1 AMPEP K⁺ + 0.1 mg L^?1 colchicine with PGR, and finally, 2.7 ± 0.33 mm with 0.1 mg L^?1 AMPEP K⁺ only. The percentage of direct shoots formed ranged from 93 to 100 % and 87 to 100 % in AMPEP K⁺ with PGR and AMPEP K⁺ only, respectively, while 62–100 % and 87–100 % were observed in AMPEP K⁺ + colchicine with PGR and AMPEP K⁺ + oryzalin with PGR, respectively. The use of AMPEP K⁺ with the PGR resulted in the longest and highest percentage formation of direct axis shoots. These observations, taken together, suggested the efficacy of AMPEP K⁺ used as a protocol to induce direct, erect shoots for the micro-propagation of K. alvarezii plantlets under laboratory conditions.     

The ZO-1 protein Polychaetoid as an upstream regulator of the Hippo pathway in Drosophila

The generation of a diversity of photoreceptor (PR) subtypes with different spectral sensitivities is essential for color vision in animals. In the Drosophila eye, the Hippo pathway has been implicated in blue- and green-sensitive PR subtype fate specification. Specifically, Hippo pathway activation promotes green-sensitive PR fate at the expense of blue-sensitive PRs. Here, using a sensitized triple heterozygote-based genetic screening approach, we report the identification of the single Drosophila zonula occludens-1 (ZO-1) protein Polychaetoid (Pyd) as a new regulator of the Hippo pathway during the blue- and green-sensitive PR subtype binary fate choice. We demonstrate that Pyd acts upstream of the core components and the upstream regulator Pez in the Hippo pathway. Furthermore, We found that Pyd represses the activity of Su(dx), a E3 ligase that negatively regulates Pez and can physically interact with Pyd, during PR subtype fate specification. Together, our results identify a new mechanism underlying the Hippo signaling pathway in post-mitotic neuronal fate specification.     

Production,characteristics and applications of phytase from a rhizosphere isolated <Emphasis Type="Italic">Enterobacter</Emphasis> sp. ACSS

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40–80 °C) and pH (2.0–6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m of the pure phytase were 0.21 mM, 131.58 nmol mg^?1 s^?1, 1.64 × 10³ s^?1, and 7.81 × 10⁶ M^?1 s^?1, respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca⁺², Mg⁺² and Mn⁺², but inhibited by Zn⁺², Cu⁺², Fe⁺², Pb⁺², Ba⁺² and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.     

Binding of more than one retinoid to visual opsins

Visual opsins bind 11-cis retinal at an orthosteric site to form rhodopsins but increasing evidence suggests that at least some are capable of binding an additional retinoid(s) at a separate, allosteric site(s). Microspectrophotometric measurements on isolated, dark-adapted, salamander photoreceptors indicated that the truncated retinal analog, β-ionone, partitioned into the membranes of green-sensitive rods; however, in blue-sensitive rod outer segments, there was an enhanced uptake of four or more β-ionones per rhodopsin. X-ray crystallography revealed binding of one β-ionone to bovine green-sensitive rod rhodopsin. Cocrystallization only succeeded with extremely high concentrations of β-ionone and binding did not alter the structure of rhodopsin from the inactive state. Salamander green-sensitive rod rhodopsin is also expected to bind β-ionone at sufficiently high concentrations because the binding site is present on its surface. Therefore, both blue- and green-sensitive rod rhodopsins have at least one allosteric binding site for retinoid, but β-ionone binds to the latter type of rhodopsin with low affinity and low efficacy.     

Optimization of heterologous expression of the phytase (PPHY) of Pichia anomala in P. pastoris and its applicability in fractionating allergenic glycinin from soy protein

The phytase (PPHY) of Pichia anomala has the requisite properties of thermostability and acidstability, broad substrate spectrum, and protease insensitivity, which make it a suitable candidate as a feed and food additive. The 1,389-bp PPHY gene was amplified from P. anomala genomic DNA, cloned in pPICZαA, and expressed extracellularly in P. pastoris X33. Three copies of PPHY have been detected integrated into the chromosomal DNA of the recombinant P. pastoris. The size exclusion chromatography followed by electrophoresis of the pure rPPHY confirmed that this is a homohexameric glycoprotein of ~420 kDa with a 24.3 % portion as N-linked glycans. The temperature and pH optima of rPPHY are 60 °C and 4.0, similar to the endogenous enzyme. The kinetic characteristics K _m, V _max, K _cat, and K _cat/K _m of rPPHY are 0.2 ± 0.03 mM, 78.2 ± 1.43 nmol mg^?1 s^?1, 65,655 ± 10.92 s^?1, and 328.3 ± 3.12 μM^?1 s^?1, respectively. The optimization of medium components led to a 21.8-fold improvement in rPPHY production over the endogenous yeast. The rPPHY titer attained in shake flasks could also be sustained in the laboratory fermenter. The rPPHY accounts for 57.1 % of the total secreted protein into the medium. The enzyme has been found useful in fractionating allergenic protein glycinin from soya protein besides dephytinization.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

Kinetics of chloride transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

The kinetics of the light-driven Cl^? uptake pump of Synechococcus R-2 (PCC 7942) were investigated. The kinetics of Cl^? uptake were measured in BG-11 medium (pH_o, 7·5; [K⁺]_o, 0·35 mol m^?3; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 mol m^?3) or modified media based on the above. Net³⁶Cl^? fluxes (?Cl^?_o,i) followed Michaelis-Menten kinetics and were stimulated by Na⁺ [18 mol m^?3 Na⁺ BG-11 ?Cl^?_max= 3·29±0·60 (49) nmol m^?2 s^?1 versus Na⁺-free BG-11 ?Cl^?_max= 1·02±0·13 (54) nmol m^?2 s^?1] but the Km was not significantly different in the presence or absence of Na⁺ at pH_o 10; the Km was lower, but not affected by the presence or absence of Na⁺ [Km = 22·3±3·54 (20) mmol m^?3]. Na⁺ is a non-competitive activator of net ?Cl^?_o,i. High [K⁺]_o (18 mol m^?3) did not stimulate net ?Cl^?_o,i or change the Km in Na⁺-free medium. High [K⁺]_o (18 mol m^?3) added to Na⁺ BG-11 medium decreased net ?Cl^?_o,i [18 mol m^?3K⁺ BG-11; ?Cl^?_max= 2·50±0·32 (20) nmol m^?2 s^?1 versus BG-11 medium; ?Cl^?_max= 3·35±0·56 (20) nmol m^?2 s^?1] but did not affect the Km 55·8±8·100 (40) mmol m^?3]. Na⁺-stimulation of net ?Cl^?_o,i followed Michaelis-Menten kinetics up to 2–5 mol m^?3 [Na⁺]_o but higher concentrations were inhibitory. The Km for Na⁺-stimulation of net ?Cl^?_o,i [K_1/2(Na⁺)] was different at 47 mmol m^?3 [Cl^?]_o (K_1/2[Na⁺] = 123±27 (37) mmol m^?3]. Li⁺ was only about one-third as effective as Na⁺ in stimulating Cl^? uptake but the activation constant was similar [K_1/2(Li⁺) = 88±46 (16) mmol m^?3]. Br^? was a competitive inhibitor of Cl^? uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na⁺. The overall Ki was 297±23 (45) mmol m^?3. The discrimination ratio of Cl^? over Br^? (δCl^?/δBr^?) was 6·38±0·92 (df = 147). Synechococcus has a single Na⁺-stimulated Cl^? pump because the Km of the Cl^? transporter and its discrimination between Cl^? and Br^? are not significantly different in the presence and absence of Na⁺. The Cl^? pump is probably driven by ATP.     

Kinetic Modeling for Simultaneous Organic Carbon Oxidation,Nitrification, and Denitrification of Abattoir Wastewater in Sequencing Batch Reactor

A laboratory-scale study was conducted in a 20.0-L sequencing batch reactor (SBR) to explore the feasibility of simultaneous removal of organic carbon and nitrogen from abattoir wastewater. The reactor was operated under three different combinations of aerobic-anoxic sequence, viz., (4+4), (5+3), and (5+4) h of total react period, with influent soluble chemical oxygen demand (SCOD) and ammonia nitrogen (NH₄⁺-N) level of 2200 ± 50 and 125 ± 5 mg L^?1, respectively. In (5+4) h cycle, a maximum 90.27% of ammonia reduction corresponding to initial NH₄⁺-N value of 122.25 mg L^?1 and 91.36% of organic carbon removal corresponding to initial SCOD value of 2215.25 mg L^?1 have been achieved, respectively. The biokinetic parameters such as yield coefficient (Y), endogenous decay constant (k_d), and half-velocity constant (K_s) were also determined to improve the design and operation of package effluent treatment plants comprising SBR units. The specific denitrification rate (q_DN) during anoxic condition was estimated as 6.135 mg N/g mixed liquor volatile suspended solid (MLVSS)·h on 4-h average contact period. The value of Y, k_d and K_s for carbon oxidation and nitrification were found to be in the range of 0.6225–0.6952 mg VSS/mg SCOD, 0.0481–0.0588 day^?1, and 306.56–320.51 mg L^?1, and 0.2461–0.2541 mg VSS/mg NH₄⁺-N, 0.0324–0.0565 day^?1, and 38.28–50.08 mg L^?1, respectively, for different combinations of react periods.     

Dioptrics of the facet lenses in the dorsal rim area of the cricket Gryllus bimaculatus

1. The optics of the corneal facet lenses from the dorsal rim area (DRA) and from the dorso-lateral areas (DA) of the compound eye of the cricket Gryllus bimaculatus were studied.
2. The DRA of the cricket eye contains quite normally shaped facet lenses. The diameter of the facet lens in the DA is 2-fold larger compared to that in the DRA. The radius of curvature of the front surface is distinctly less in the DA facet lenses, as the surface of the facet lenses in the DRA are virtually flat.
3. The averaged axial refractive index of the facet lenses of Gryllus bimaculatus, measured by interference microscopy, was 1.496 ± 0.008 (n = 42) in the DRA and 1.469 ± 0.004 (n = 39) in the DA. The geometrical thickness of the lenses was calculated to be 77 ± 3 μm (n = 42) in the DRA and 56 ± 1 μm (n = 39) in the DA.
4. Analysis of the diffraction pattern obtained with a point light source revealed distinct focusing properties of both the DRA and the DA facet lenses; striking Airy-like diffraction patterns were obtained in both cases.
5. Focal distances measured directly at the backfocal plane were 40 ± 8 μm (n = 84) in the DRA of all the animals studied, and 60–90 μm (n = 62) in DA depending on the animal. Analysis of the diffraction of the point light source yielded very similar focal distances: 40 ± 5 μm (n = 10) in DRA and 81 ± 8 μm (n = 11) in DA. In the DRA, focal distance of the facet lenses was smaller than the cone length, 58 ± 3 μm (n = 9) while in the DA the focal distance matched the effective cone length, 71 ± 5 μm (n = 16).

     

The Equilibria of Lipid–K<Superscript>+</Superscript> Ions in Monolayer at the Air/Water Interface

The effect of K⁺ ion interaction with monolayers of phosphatidylcholine (lecithin, PC) or cholesterol (Ch) was investigated at the air/water interface. We present surface tension measurements of lipid monolayers obtained using a Langmuir method as a function of K⁺ ion concentration. Measurements were carried out at 22°C using a Teflon trough and a Nima 9000 tensiometer. Interactions between lecithin and K⁺ ions or Ch and K⁺ ions result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants and area occupied by one molecule of lipid–K⁺ ion complex (LK⁺). The stability constants for lecithin–K⁺ ion (PCK⁺) complex, \( K_{{{\text{PCK}}^{ + } }} = { 3}. 2 6\times 10^{ 2} {\text{dm}}^{ 3} \,{\text{mol}}^{ - 1} \), and for cholesterol–K⁺ ion (ChK⁺) complex, \( K_{{{\text{ChK}}^{ + } }} = { 1}.00 \times 10^{ 3} {\text{dm}}^{ 3} \,{\text{mol}}^{ - 1} \), were calculated by inserting the experimental data. The value of area occupied by one PCK⁺ complex is 60 Å² molecule^?1, while the area occupied by one ChK⁺ complex is 40.9 Å² molecule^?1. The complex formation energy (Gibbs free energy) values for the PCK⁺ and ChK⁺ complexes are ?14.18 ± 0.71 and ?16.92 ± 0.85 kJ mol^?1, respectively.     

Cation-permeable vacuolar ion channels in the moss Physcomitrella patens: a patch-clamp study

Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca²⁺ on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K⁺ (E _K), indicating K⁺ selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at ?80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na⁺ and K⁺ selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na⁺ influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca²⁺ revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca²⁺ activated SV channels, vacuolar Ca²⁺ inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca²⁺ was also determined. These channels were active even without Ca²⁺ (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca²⁺ on the cytoplasmic side. Apart from monovalent cations (K⁺ and Na⁺), SV channels were permeable to divalent cations (Ca²⁺ and Mg²⁺). Both monovalent and divalent cations passed through the channels in the same direction—from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.     

Swainsonine,a novel fungal metabolite: optimization of fermentative production and bioreactor operations using evolutionary programming

The optimization of bioreactor operations towards swainsonine production was performed using an artificial neural network coupled evolutionary program (EP)-based optimization algorithm fitted with experimental one-factor-at-a-time (OFAT) results. The effects of varying agitation (300–500 rpm) and aeration (0.5–2.0 vvm) rates for different incubation hours (72–108 h) were evaluated in bench top bioreactor. Prominent scale-up parameters, gassed power per unit volume (P _g/V _L, W/m³) and volumetric oxygen mass transfer coefficient (K _L a, s^?1) were correlated with optimized conditions. A maximum of 6.59 ± 0.10 μg/mL of swainsonine production was observed at 400 rpm-1.5 vvm at 84 h in OFAT experiments with corresponding P _g/V_L and K _L a values of 91.66 W/m³ and 341.48 × 10^?4 s^?1, respectively. The EP optimization algorithm predicted a maximum of 10.08 μg/mL of swainsonine at 325.47 rpm, 1.99 vvm and 80.75 h against the experimental production of 7.93 ± 0.52 μg/mL at constant K _L a (349.25 × 10^?4 s^?1) and significantly reduced P _g/V _L (33.33 W/m³) drawn by the impellers.     

M3 cholinoreceptors alter electrical activity of rat left atrium via suppression of L-type Ca<Superscript>2+</Superscript> current without affecting K<Superscript>+</Superscript> conductance

Electrophysiological effects produced by selective activation of M3 cholinoreceptors were studied in isolated left atrium preparations from rat using the standard sharp glass microelectrode technique. The stimulation of M3 receptors was obtained by application of muscarinic agonist pilocarpine (10^?5 M) in the presence of selective M2 antagonist methoctramine (10^?7 M). Stimulation of M3 receptors induced marked reduction of action potential duration by 14.4 ± 2.4% and 16.1 ± 2.5% of control duration measured at 50 and 90% of repolarization, respectively. This effect was completely abolished by selective M3 blocker 4-DAMP (10^?8 M). In isolated myocytes obtained from the rat left atrium, similar pharmacological stimulation of M3 receptors led to suppression of peak L-type calcium current by 13.9 ± 2.6% of control amplitude (measured at +10 mV), but failed to affect K⁺ currents I _to, I _Kur, and I _Kir. In the absence of M2 blocker methoctramine, pilocarpine (10^?5 M) produced stronger attenuation of I _CaL and induced an increase in I _Kir. This additive inward rectifier current could be abolished by highly selective blocker of K_ir3.1/3.4 channels tertiapin-Q (10^?6 M) and therefore was identified as I _KACh. Thus, in the rat atrial myocardium activation of M3 receptors leads to shortening of action potentials via suppression of I _CaL, but does not enhance the major potassium currents involved in repolarization. Joint stimulation of M2 and M3 receptors produces stronger action potential shortening due to M2-mediated activation of I _KACh.     

Reaction Kinetics of the Invertase from Yeast (S. Cerevisiae)

Invertase converts sucrose to glucose and fructose. The reaction mechanism for the formation of glucose and fructose was studied by stopped flow spectrophotometer and circular dichroism. The reaction mechanism follows biphasic mode with rate constants of k₁0.0053 s^?1?±?0.001 s^?1 and k₂ 0.030 s^?1?±?0.01 s^?1 for 25 mM concentration of sucrose. Far UV circular dichroic spectrum of invertase in presence of sucrose shows 18 % increase in β conformation as a function of time. Taken together, the invertase hydrolysis follows biphasic mode where it undergoes conformational changes followed by hydrolysis of the sucrose.     

Can uranium follow the iron-acquisition pathway? Interaction of uranyl-loaded transferrin with receptor 1

Transferrin receptor 1 (R_D) binds iron-loaded transferrin and allows its internalization in the cytoplasm. Human serum transferrin also forms complexes with metals other than iron, including uranium in the uranyl form (UO₂ ²⁺). Can the uranyl-saturated transferrin (TUr₂) follow the receptor-mediated iron-acquisition pathway? In cell-free assays, TUr₂ interacts with R_D in two different steps. The first is fast, direct rate constant, k ₁ = (5.2 ± 0.8) × 10⁶ M^?1 s^?1; reverse rate constant, k _?1 = 95 ± 5 s^?1; and dissociation constant K ₁ = 18 ± 6 μM. The second occurs in the 100-s range and leads to an increase in the stability of the protein–protein adduct, with an average overall dissociation constant K _d = 6 ± 2 μM. This kinetic analysis implies in the proposed in vitro model possible but weak competition between TUr₂ and the C-lobe of iron-loaded transferrin toward the interaction with R _D.     

Ecosystem respiration derived from 222Rn measurements on Rishiri Island, Japan

We evaluated the nighttime CO₂ flux (ecosystem respiration) on Rishiri Island, located at the northern tip of Hokkaido, Japan, from 2009 to 2011, by using the relationship between atmospheric ²²²Rn and CO₂ concentrations. The annual mean CO₂ flux was 1.8 μmol m^?2 s^?1, with a maximum monthly mean in July (4.6 ± 2.6 μmol m^?2 s^?1) and a broad minimum from December to March (0.33 ± 0.29 μmol m^?2 s^?1). The annual mean was comparable to fluxes at the JapanFlux sites in northern Japan. During the season of snow cover (mid-December to early April), the CO₂ flux was low (0.45 ± 0.43 μmol m^?2 s^?1). Total annual respiration was estimated at 679 ± 174 g cm^?2, about 8 % of which occurred during the season of snow cover.     

Voltage-sensitive chloride ion channels in Anopheles gambiae Sua-1B cells

In this study, we performed electrophysiological analysis of Anopheles gambiae Sua-1B cells having “neuron-like” morphologies using the patch clamp method. The recorded cells (n = 79) had processes resembling axons/dendrites, with 63 % unipolar, 22 % bipolar, and 15 % multipolar. While no inward currents were observed following step depolarizations (holding potential = ?80 mV), a slowly activating outward current was observed in 96 % of the cells, especially at depolarized potentials. The amplitude of the current was attenuated nearly 70 % by reducing extracellular Cl^? ion concentration, or by incubating with 100 μM DIDS, a known voltage-sensitive chloride channel blocker, suggesting that the current was mediated by chloride ions. No qualitative difference was found between recordings made with Cs⁺ ions in the intracellular pipette solution (inhibits K⁺ currents) and those made with normal physiological solution, indicating a deficiency of potassium channels. Additionally, recordings made with Ca²⁺-free extracellular bath solution eliminated the slowly activating outward current. A subset of cells (n = 3) lacked this current, but had outward currents with voltage-dependent properties similar to those of volume-regulated chloride channels. Taken together, our results suggest that the voltage-sensitive currents observed in the majority of Sua-1B cells are mediated primarily by chloride channels of the calcium-dependent subtype.     

Taurine Stimulation of Isolated Hamster Brain Na+, K+-ATPase: Activation Kinetics and Chemical Specificity

Epileptic foci are associated with locally reduced taurine (2-aminoethanesulfonic acid) concentration and Na⁺, K⁺-ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic “free” calcium activity, and neuronal excitability. Taurine may act in part by modulating Na⁺, K⁺-ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na⁺, K⁺-ATPase by taurine. Normal whole brain homogenate Na⁺, K⁺-ATPase activity is 5.1 ± 0.4 (4) μmol P_i± h^?1± mg^?1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane-bound Na⁺, K⁺-ATPase activity of 204.6 ± 5.8 (4) mol P_i± h^?1± mg^?1 Lowry protein. Taurine activates the Na⁺, K⁺-ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K_1/2= 39 mM taurine, activation maximum =+87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid > hypotaurine > no activation =β-alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na⁺, K⁺-ATPase. Its action is mediated by a membrane-bound protein.