Action potentials in primary osteoblasts and in the MG-63 osteoblast-like cell line

Whole-cell patch-clamp analysis revealed a resting membrane potential of −60 mV in primary osteoblasts and in the MG-63 osteoblast-like cells. Depolarization-induced action potentials were characterized by duration of 60 ms, a minimal peak-to-peak distance of 180 ms, a threshold value of −20 mV and a repolarization between the spikes to −45 mV. Expressed channels were characterized by application of voltage pulses between −150 mV and 90 mV in 10 mV steps, from a holding potential of −40 mV. Voltages below −60 mV induced an inward current. Depolarizing voltages above −30 mV evoked two currents: (a) a fast activated and inactivated inward current at voltages between −30 and 30 mV, and (b) a delayed-activated outward current that was induced by voltages above −30 mV. Electrophysiological and pharmacological parameters indicated that hyperpolarization activated strongly rectifying K⁺ (K_ir) channels, whereas depolarization activated tetrodotoxin sensitive voltage gated Na⁺ (Na_v) channels as well as delayed, slowly activated, non-inactivating, and tetraethylammonium sensitive voltage gated K⁺ (K_v) channels. In addition, RT-PCR showed expression of Na_v1.3, Na_v1.4, Na_v1.5, Na_v1.6, Na_v1.7, and K_ir2.1, K_ir2.3, and K_ir2.4 as well as K_v2.1. We conclude that osteoblasts express channels that allow firing of action potentials.     

TEA-Sensitive Currents Contribute to Membrane Potential of Organ Surface Primo-node Cells in Rats

The primo-vascular (Bonghan) tissue has been identified in most tissues in the body, but its structure and functions are not yet well understood. We characterized electrophysiological properties of the cells of the primo-nodes (PN) on the surface of abdominal organs using a slice patch clamp technique. The most abundant were small round cells (~10 μm) without processes. These PN cells exhibited low resting membrane potential (−36 mV) and did not fire action potentials. On the basis of the current–voltage (I–V) relationships and kinetics of outward currents, the PN cells can be grouped into four types. Among these, type I cells were the majority (69%); they showed strong outward rectification in I–V relations. The outward current was activated rapidly and sustained without decay. Tetraethylammonium (TEA) dose-dependently blocked both outward and inward current (IC₅₀, 4.3 mM at ±60 mV). In current clamp conditions, TEA dose-dependently depolarized the membrane potential (18.5 mV at 30 mM) with increase in input resistance. The tail current following a depolarizing voltage step was reversed at −27 mV, and transient outward current like A-type K⁺ current was not expressed at holding potential of −80 mV. Taken together, the results demonstrate for the first time that the small round PN cells are heterogenous, and that, in type I cells, TEA-sensitive current with limited selectivity to K⁺ contributed to resting membrane potential of these cells.     

Responses from freshwater sediment during electricity generation using microbial fuel cells

In a two-electrode system, freshwater sediment was used as a fuel to examine the relationship between current generation and organic matter consumption with different types of electrode. Sediment microbial fuel cells using porous electrodes showed a superior performance in terms of generating current when compared with the use of non-porous electrodes. The maximum current densities with thicker and thin porous electrodes were 45.4 and 37.6 mA m⁻², respectively, whereas the value with non-porous electrodes was 13.9 mA m⁻². The amount of organic matter removed correlated with the current produced. The redox potential in the anode area under closed-circuit conditions was +246.3 ± 67.7 mV, while that under open-circuit conditions only reached −143.0 ± 7.18 mV. This suggests that an application of this system in organic-rich sediment could provide environmental benefits such as decreasing organic matter and prohibiting methane emission in conjunction with electricity production via an anaerobic oxidation process.     

Effect of of ATP on Neurons of the Rat Intact Nodose Ganglion

Under intracellular recording, we studied the effect of ATP on nerve cells of the rat intact nodose ganglion. The resting membrane potential of the examined neurons was, on average, –60.3 ± 1.4 mV (n = 84); among such units, 88% were classified as C cells. Local application of 2 mM ATP to the surface of the ganglion using a modified laminar flow system led to depolarization of neurons by 7.1 ± 0.9 mV, on average (n = 19). A blocker of P2X receptors, PPADS (100 μM), suppressed these depolarization responses, decreasing their amplitude, on average, to 16 ± 3% (n = 3) of the initial value. The obtained data indicate that an overwhelming majority of neurons of the intact nodose ganglion possess functional P2X receptors on their membranes. The absence of the corresponding responses in a considerable part of neurons of intact spinal ganglia [13-15] was, apparently, determined by the fact that P2X receptors in the course of the described experiments had enough time to desensitize before ATP reached the effective concentration.     

Regulation of protoporphyrin IX biosynthesis by intraplastidic compartmentalization and adenosine triphosphate

Subplastidic preparations from cotyledons of cucumber (Cucumis sativus L.) were tested for their ability to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Envelope or thylakoid membranes failed to synthesize protoporphyrin IX from the substrate 5-aminolevulinic acid. Stromal preparations synthesized a very low amount of protoporphyrin IX. In a reconstitution experiment using stroma + envelope membranes, protoporphyrin IX synthesis from 5-aminolevulinic acid was enhanced by 660% over that of stroma alone. However, when thylakoids were added to the stroma + envelope mixture, protoporphyrin IX synthesis from 5-aminolevulinic acid was completely inhibited. In the reconstituted stroma + envelope membrane mixture, the reducing agent dithiothreitol enhanced the protoporphyrin IX-synthesizing ability and completely abolished the inhibition of protoporphyrin IX synthesis by thylakoids. This suggested that the oxidizing agents usually associated with the thylakoid membranes inhibited protoporphyrin IX biosynthesis and the inhibition was alleviated by the reducing power of dithiothreitol. This study exposes the weakness of in vitro reconstitution experiments in mimicking the in vivo-conditions. Addition of ATP stimulated protoporphyrin IX synthesis by 50% in the supernatant fraction of chloroplast lysate. This ATP-induced stimulation of protoporphyrin IX synthesis was due to the enhancement of the activities of uroporphyrinogen decarboxylase and protoporphyrinogen oxidase, involved in tetrapyrrole biosynthesis. The ATP-induced stimulation of porphyrinogen oxidase activity was an energy-dependent reaction. Received: 21 March 2000 / Accepted: 9 May 2000     

Catalyzing denitrification of <Emphasis Type="Italic">Paracoccus versutus</Emphasis> by immobilized 1,5-dichloroanthraquinone

The accelerating effect of non-dissolved redox mediator (1,5-dichloroanthraquinone) on the biological denitrification was investigated in this paper using 1,5-dichloroanthraquinone immobilized by calcium alginate (CA) and a heterotrophic denitrification bacterium of Paracoccus versutus (GU111570). The results suggested that the denitrification rate was enhanced 2.1 fold by 25 mmol l⁻¹ 1,5-dichloroanthraquinone of this study, and a positive correlation was found for the denitrification rate and 1,5-dichloroanthraquinone concentrations from 0 to 25 mmol l⁻¹. According to the change characteristic of NO₃ ⁻ and NO₂ ⁻ during the denitrification process, the tentative accelerating mechanism of the denitrification by redox mediators was put forward, and redox mediator might play the role of reduced cofactors like NADH, N(A)DH and SDH, or the similar ubiquinol/ubiquinone (Q/QH₂) role during the denitrification process.     

Single-channel properties of a stretch-sensitive chloride channel in the human mast cell line HMC-1

A stretch-activated (SA) Cl⁻ channel in the plasma membrane of the human mast cell line HMC-1 was identified in outside-out patch-clamp experiments. SA currents, induced by pressure applied to the pipette, exhibited voltage dependence with strong outward rectification (55.1 pS at +100 mV and an about tenfold lower conductance at −100 mV). The probability of the SA channel being open (P _o) also showed steep outward rectification and pressure dependence. The open-time distribution was fitted with three components with time constants of τ_1o = 755.1 ms, τ_2o = 166.4 ms, and τ_3o = 16.5 ms at +60 mV. The closed-time distribution also required three components with time constants of τ_1c = 661.6 ms, τ_2c = 253.2 ms, and τ_3c = 5.6 ms at +60 mV. Lowering extracellular Cl⁻ concentration reduced the conductance, shifted the reversal potential toward chloride reversal potential, and decreased the P _o at positive potentials. The SA Cl⁻ currents were reversibly blocked by the chloride channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) but not by (Z)-1-(p-dimethylaminoethoxyphenyl)-1,2-diphenyl-1-butene (tamoxifen). Furthermore, in HMC-1 cells swelling due to osmotic stress, DIDS could inhibit the increase in intracellular [Ca²⁺] and degranulation. We conclude that in the HMC-1 cell line, the SA outward currents are mediated by Cl⁻ influx. The SA Cl⁻ channel might contribute to mast cell degranulation caused by mechanical stimuli or accelerate membrane fusion during the degranulation process.     

IAA-induced hyperpolarization of the membrane potential in isolated cells fromMimosa pulvinus

Upon treatment with 10⁻⁴ M IAA the membrane potential of an isolated cell from the main pulvinus, ofMimosa pudica L. depolarized by about 6 mV in 2–5 min, but later it gradually hyperpolarized by about 30 mV. The membrane potential of a motor cell in the main pulvinar tissue hyperpolarized by about 80 mV 1 hr after application of 10⁻⁴ M IAA.     

Single-Channel Analysis of a Delayed Rectifier K+ Channel in Xenopus Myelinated Nerve

Single-channel properties of a delayed rectifier voltage-gated K⁺ channel (I-type) were investigated in peripheral myelinated axons from Xenopus laevis. Channels activated between −60 and −40 mV with a potential of half-maximal activation, E₅₀, at −47.5 mV. Averaged single-channel currents activated with a time delay at all membrane potentials tested. Time to half-maximal activation decreased from 80 to 1.6 msec between −60 and +40 mV. The channel inactivated monoexponentially with a time constant of 10.9 sec at −40 mV. The time constant of deactivation was 126 msec at −80 mV and 16.9 msec at −110 mV. In symmetrical 105 mm K⁺, the single-channel conductance (γ) was 22 and 13 pS at negative and positive membrane potentials, respectively, at 13–15°C. In Na⁺-rich solution with 2.5 mm extracellular K⁺γ was 7 pS and the reversal potential was negative to −80 mV, indicating a high selectivity for K⁺ over Na⁺. γ depended on extracellular K⁺ concentration (K _D = 19.6 mm) and temperature (Q ₁₀= 1.45). External tetraethylammonium (TEA) reduced the apparent single-channel current amplitude at all potentials tested with a half-maximal inhibiting concentration (IC₅₀) of 0.6 mm. Open probability of the channel, but not single-channel current amplitude was decreased by extracellular dendrotoxin (DTX, IC₅₀= 6.8 nm) and mast cell degranulating peptide (MCDP, IC₅₀= 41.9 nm). In Ringer solution the membrane potential of macroscopic I-channel patches was about −65 mV and depolarized under TEA and DTX. It is concluded that besides their activation during action potentials, I-channels may also stabilize the resting membrane potential. Received: 2 June 1995/Revised: 13 October 1995     

Electron self-exchange and self-amplified posttranslational modification in the hemoglobins from <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 and <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539–552, 2011). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 10²–10³ M⁻¹ s⁻¹ and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment.     

Controlling the oxidoreduction potential of the culture of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> leads to an earlier initiation of solventogenesis,thus increasing solvent productivity

Fermentative production of solvents (acetone, butanol, and ethanol) by Clostridium acetobutylicum is generally a biphasic process consisting of acidogenesis and solventogenesis. We report that the biphasic metabolism of C. acetobutylicum could be changed by oxidoreduction potential (ORP) regulation. When using air to control the ORP of the fermentation broth at −290 mV, an earlier initiation of solventogenesis was achieved. Solvent production reached 25.6 g l⁻¹ (2.8 g acetone l⁻¹, 16.8 g butanol l⁻¹, 6.0 g ethanol l⁻¹), a 35% increase compared with the ORP uncontrolled process. Metabolic flux analysis revealed that there was a general increase of the central carbon flux in the first 24 h of fermentation when ORP was controlled at −290 mV, compared with the control. Specifically, the solvent ratio (acetone:butanol:ethanol) was changed from 25:64:11 to 11:66:23 at ORP level of −290 mV, which might have resulted from the rigidity at acetyl-CoA node and the flexibility at acetoacetyl-CoA and butyryl-CoA nodes in response to ORP regulation.     

Using Redox Potential to Detect Microbial Activities During Clavulanic Acid Biosynthesis in <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>

Production of clavulanic acid (CA) by Streptomyces clavuligerus in a shake-flask culture increased from 92 to 180 mg l⁻¹ with an increased O₂ transfer efficiency (0.039 → 0.058 s⁻¹), which maintained the redox potential values above −250 mV. Compared with traditional measures, such as dissolved O₂ concentration and respiratory activity, the redox potential can easily be determined and correlates closely with CA production. It can therefore be used to monitor microbial activities during biosyntheses of secondary metabolites. Revisions requested 5 April 2005 and 19 July 2005; Revisions received 19 July 2005 and 9 September 2005     

Cl Anion-Dependent Mg-ATPase

We studied, in the rat brain, the synaptosomal and microsomal membrane fractions of Cl⁻ ion-activated, Mg²⁺-dependent ATPase, satisfying the necessary kinetic peculiarities of transport ATPases, by a novel method of kinetic analysis of the multisite enzyme systems: (1) the [Mg-ATP] complex constitutes the substrate of the enzymic reaction; (2) the V = f(Cl⁻) dependence-reflecting curve is bell-shaped; (3) substrate dependence, V = f(S), curves at a constant concentration of free ligands (Mg_f, ATP_f, Cl⁻); (4) as known from the literature, in the process of reaction a phosphorylated intermediate is formed (Gerencser, Crit Rev Biochem Mol Biol 31:303–337, 1996). We report on the Cl-ATPase molecular mechanism and its place in the “P-type ATPase” classification.     

A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q

Only two genome-wide association (GWA) screens have been published for melanoma (Nat Genet 47:920–925, 2009; Nat Genet 40:838–840, 2008). Using a unique approach, we performed a genome-wide association study in 156 related melanoma cases from 34 high-risk Utah pedigrees. Genome-wide association analysis was performed on nearly 500,000 markers; we compared cases to 2,150 genotypically matched samples from Illumina’s iControls database. We performed genome-wide association with EMMAX software, which is designed to account for population structure, including relatedness between cases. Three SNPs exceeded a genome-wide significance threshold of p < 5 × 10⁻⁸ on chromosome arm 10q25.1 (rs17119434, rs17119461, and rs17119490), where the most extreme p value was 7.21 × 10⁻¹². This study represents a new and unique approach to predisposition gene identification; and it is the first genome-wide association study performed in related cases in high-risk pedigrees. Our approach illustrates an example of using high-risk pedigrees for the identification of new melanoma predisposition variants.     

Purinergic signalling in rat GFSHR-17 granulosa cells: an <Emphasis Type="Italic">in vitro</Emphasis> model of granulosa cells in maturing follicles

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca²⁺-imaging and perforated patch-clamp. We observed a resting intracellular Ca²⁺-concentration ([Ca²⁺]_i) of 100 nM and a membrane potential of −40 mV. This was consistent with high K⁺− and Cl⁻ permeability and a high intracellular Cl⁻ concentration of 40 mM. Application of ATP for 5–15 s every 3 min induced repeated [Ca²⁺]_i increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP₃-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y₂ and P2Y₄ receptors and that the [Ca²⁺]_i increase was a prerequisite for hyperpolarization. Inhibitors of Ca²⁺-activated channels or K⁺ channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl⁻ channels hyperpolarized cells to −70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y₂ and P2Y₄ receptors in granulosa cells modulate Cl⁻ permeability by regulating Ca²⁺-release.     

Effect of headgroup on the dipole potential of phospholipid vesicles

The dipole potentials, ψ _d, of phospholipid vesicles composed of pure dimyristoylphosphatidylcholine (DMPC) or vesicles in which 50 mol% of the DMPC was substituted by dimyristoylphosphatidylserine (DMPS), dimyristoylphosphatidylglycerol (DMPG), dimyristoylethanolamine (DMPE), dimyristoylphosphatidic acid (DMPA) or monomyristoylphosphatidylcholine (MMPC) were measured via a fluorescent ratiometric method utilizing the probe di-8-ANEPPS. The PS and PG headgroups were found to cause only minor changes in ψ _d. PE caused an increase in ψ _d of 51 mV. This could be explained by a decrease in the dielectric constant of the glycerol backbone region as well as a movement of the P⁻–N⁺ dipole of the less bulky PE headgroup to a position more parallel to the membrane surface than in PC. The negatively charged PA headgroup increases ψ _d by 215 mV relative to PC alone. This indicates that the positive pole of the dipole predominantly responsible for the dipole potential is located at a position closer to the interior of the membrane than the phosphate group. The increase in the charge of the negative pole of the dipole by the phosphate group of PA increases the electrical potential drop across the lipid headgroup region. The incorporation of the single chain lipid MMPC into the membrane causes a decrease in ψ _d of 142 mV. This can be explained by a decrease in packing density within the membrane of carbonyl dipoles from the sn-2 chain of DMPC. The results presented should contribute to a better understanding of the electrical effect of lipid headgroups on the functioning of membrane proteins.     

Influence of submerged macrophytes,temperature, and nutrient loading on the development of redox potential around the sediment–water interface in lakes

Redox potential is a significant factor in aquatic systems to regulate the availability of nutrients and some metals. To assess the driving variables regulating redox potential, background parameters (dissolved oxygen, pH, temperature, chlorophyll-a, soluble reactive and total phosphorus content of water, coverage and height of submerged macrophytes) and redox potential profiles around the sediment–water interface (SWI) were measured in simulated shallow lake ecosystems. There were two nutrient regimes (enriched and non-enriched) and three temperature scenarios (unheated; +3.5°C; +5°C) installed in the experimental setups, which were constructed to study the effects of global climate change. Temperature did not have any detectable effect on redox potentials, and we presume that nutrient addition had only indirect positive effects through triggering phytoplankton dominance which causes macrophyte absence. When submerged macrophytes were present in high density (80–100% coverage), redox potentials at the SWI varied between 60–215 mV and the mean redox potential was 133 ± 34 mV (mean ± 1 SD). In contrast to this, when phytoplankton dominance was coupled to low macrophyte density (0–20% coverage), the range of redox potentials at the SWI was 160–290 mV and the mean redox potential was 218 ± 34 mV. The results revealed the primary importance of submersed macrophytes; macrophyte coverage determined alone the redox potential of the sediment–water interface by 81%. This study suggests that possible positive effects of macrophytes on redox potential can be suppressed by their negative effects in case of 80–100% coverage and total inhabitation of the water column.     

An extracellular haem-protein from Coriolus versicolor

A haem-containing protein has been isolated from the growth medium of Coriolus versicolor, a wood-rotting basidiomycete. The polypeptide was identified as a ‘peroxidase-type’ haem protein of MW 53 700, which appeared to be a glycoprotein and had a protoporphyrin IX prosthetic group with a mid-point redox potential of ?121 mV. It also bound carbon monoxide suggesting it may act as an oxidase, and liberated hydroxyl radicals from hydrogen peroxide as measured by its ability to release ethylene from methional.     

Combined action of redox potential and pH on heat resistance and growth recovery of sublethally heat-damaged Escherichia coli

The combined effect of redox potential (RP) (from −200 to 500 mV) and pH (from 5.0 to 7.0) on the heat resistance and growth recovery after heat treatment of Escherichia coli was tested. The effect of RP on heat resistance was very different depending on the pH. At pH 6.0, there was no significant difference, whereas at pH 5.0 and 7.0 maximum resistance was found in oxidizing conditions while it fell in reducing ones. In sublethally heat-damaged cells, low reducing and acid conditions allowed growth ability to be rapidly regained, but a decrease in the redox potential and pH brought about a longer lag phase and a slower exponential growth rate, and even led to growth failure (pH 5.0, ≤−100 mV). Received: 28 June 1999 / Received revision: 22 October 1999 / Accepted: 22 October 1999     

Desulforubrerythrin from <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, a novel multidomain protein

A novel multidomain metalloprotein from Campylobacter jejuni was overexpressed in Escherichia coli, purified, and extensively characterized. This protein is isolated as a homotetramer of 24-kDa monomers. According to the amino acid sequence, each monomer was predicted to contain three structural domains: an N-terminal desulforedoxin-like domain, followed by a four-helix bundle domain harboring a non-sulfur μ-oxo diiron center, and a rubredoxin-like domain at the C-terminus. The three predicted iron sites were shown to be present and were studied by a combination of UV–vis, EPR, and resonance Raman spectroscopies, which allowed the determination of the electronic and redox properties of each site. The protein contains two FeCys₄ centers with reduction potentials of +240 mV (desulforedoxin-like center) and +185 mV (rubredoxin-like center). These centers are in the high-spin configuration in the as-isolated ferric form. The protein further accommodates a μ-oxo-bridged diiron site with reduction potentials of +270 and +235 mV for the two sequential redox transitions. The protein is rapidly reoxidized by hydrogen peroxide and has a significant NADH-linked hydrogen peroxide reductase activity of 1.8 μmol H₂O₂ min⁻¹ mg⁻¹. Owing to its building blocks and its homology to the rubrerythrin family, the protein is named desulforubrerythrin. It represents a novel example of the large diversity of the organization of domains exhibited by this enzyme family.