Ionic currents influencing spontaneous firing and pacemaker frequency in dopamine neurons of the ventrolateral periaqueductal gray and dorsal raphe nucleus (vlPAG/DRN): A voltage-clamp and computational modelling study

Dopamine (DA) neurons of the ventrolateral periaqueductal gray (vlPAG) and dorsal raphe nucleus (DRN) fire spontaneous action potentials (APs) at slow, regular patterns in vitro but a detailed account of their intrinsic membrane properties responsible for spontaneous firing is currently lacking. To resolve this, we performed a voltage-clamp electrophysiological study in brain slices to describe their major ionic currents and then constructed a computer model and used simulations to understand the mechanisms behind autorhythmicity in silico. We found that vlPAG/DRN DA neurons exhibit a number of voltage-dependent currents activating in the subthreshold range including, a hyperpolarization-activated cation current (I_H), a transient, A-type, potassium current (I_A), a background, ‘persistent’ (I_NaP) sodium current and a transient, low voltage activated (LVA) calcium current (I_CaLVA). Brain slice pharmacology, in good agreement with computer simulations, showed that spontaneous firing occurred independently of I_H, I_A or calcium currents. In contrast, when blocking sodium currents, spontaneous firing ceased and a stable, non-oscillating membrane potential below AP threshold was attained. Using the DA neuron model we further show that calcium currents exhibit little activation (compared to sodium) during the interspike interval (ISI) repolarization while, any individual potassium current alone, whose blockade positively modulated AP firing frequency, is not required for spontaneous firing. Instead, blockade of a number of potassium currents simultaneously is necessary to eliminate autorhythmicity. Repolarization during ISI is mediated initially via the deactivation of the delayed rectifier potassium current, while a sodium background ‘persistent’ current is essentially indispensable for autorhythmicity by driving repolarization towards AP threshold.     

Giant congenital melanocytic nevus with vascular malformation and epidermal cysts associated with a somatic activating mutation in BRAF

Giant congenital melanocytic nevi may be symptomatically isolated or syndromic. Associations with capillary malformations are exceptional, and development of epidermal cysts has not been described. A 71‐year‐old patient with a giant congenital melanocytic nevus (CMN) of the lower back, buttocks, and thighs was asymptomatic except for unexpected hemorrhage during partial surgical excision years before. Blunt trauma at age 64 initiated recurrent, severe pain under the nevus; multiple large epidermal cysts then developed within it. Imaging and biopsy showed a large, non‐pulsatile venous malformation intermingled with the deep nevus. A low‐abundance, heterozygous BRAF c.1799T>A (p.V600E) mutation was present in both gluteal and occipital congenital nevi; additional mutations in NRAS, GNAQ, GNA11, HRAS, or PIK3CA were undetectable. This is the first demonstration of a recurrent BRAF mutation in multiple large congenital nevi from the same individual, confirming that this malformation can have multiple genetic origins. Early constitutive activation of BRAF can therefore cause unusual associations of giant nevi with vascular malformations, indicating that both pigment and endothelial cell physiology may be affected by mosaic RASopathies.     

Ralstonia eutropha H16 flagellation changes according to nutrient supply and state of poly(3-hydroxybutyrate) accumulation

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE), in combination with matrix-assisted laser desorption ionization-time of flight analysis, and the recently revealed genome sequence of Ralstonia eutropha H16 were employed to detect and identify proteins that are differentially expressed during different phases of poly(3-hydroxybutyric acid) (PHB) metabolism. For this, a modified protein extraction protocol applicable to PHB-harboring cells was developed to enable 2D PAGE-based proteome analysis of such cells. Subsequently, samples from (i) the exponential growth phase, (ii) the stationary growth phase permissive for PHB biosynthesis, and (iii) a phase permissive for PHB mobilization were analyzed. Among several proteins exhibiting quantitative changes during the time course of a cultivation experiment, flagellin, which is the main protein of bacterial flagella, was identified. Initial investigations that report on changes of flagellation for R. eutropha were done, but 2D PAGE and electron microscopic examinations of cells revealed clear evidence that R. eutropha exhibited further significant changes in flagellation depending on the life cycle, nutritional supply, and, in particular, PHB metabolism. The results of our study suggest that R. eutropha is strongly flagellated in the exponential growth phase and loses a certain number of flagella in transition to the stationary phase. In the stationary phase under conditions permissive for PHB biosynthesis, flagellation of cells admittedly stagnated. However, under conditions permissive for intracellular PHB mobilization after a nitrogen source was added to cells that are carbon deprived but with full PHB accumulation, flagella are lost. This might be due to a degradation of flagella; at least, the cells stopped flagellin synthesis while normal degradation continued. In contrast, under nutrient limitation or the loss of phasins, cells retained their flagella.     

The crystal structure of CHIR-AB1: a primordial avian classical Fc receptor

CHIR-AB1 is a newly identified avian immunoglobulin (Ig) receptor that includes both activating and inhibitory motifs and was therefore classified as a potentially bifunctional receptor. Recently, CHIR-AB1 was shown to bind the Fc region of chicken IgY and to induce calcium mobilization via association with the common γ-chain, a subunit that transmits signals upon ligation of many different immunoreceptors. Here we describe the 1.8-Å-resolution crystal structure of the CHIR-AB1 ectodomain. The receptor ectodomain consists of a single C2-type Ig domain resembling the Ig-like domains found in mammalian Fc receptors such as FcγRs and FcαRI. Unlike these receptors and other monomeric Ig superfamily members, CHIR-AB1 crystallized as a 2-fold symmetrical homodimer that bears no resemblance to variable or constant region dimers in an antibody. Analytical ultracentrifugation demonstrated that CHIR-AB1 exists as a mixture of monomers and dimers in solution, and equilibrium gel filtration revealed a 2:1 receptor/ligand binding stoichiometry. Measurement of the 1:1 CHIR-AB1/IgY interaction affinity indicates a relatively low affinity complex, but a 2:1 CHIR-AB1/IgY interaction allows an increase in apparent affinity due to avidity effects when the receptor is tethered to a surface. Taken together, these results add to the structural understanding of Fc receptors and their functional mechanisms.     

Evaporation effects as reflected in freshwaters and ostracod calcite from modern environments in Central and Northeast Yakutia (East Siberia,Russia)

Taxonomical and geochemical investigations on freshwater ostracods from 15 waters in Central and Northeast (NE) Yakutia have been undertaken in order to estimate their potential usefulness in palaeoenvironmental reconstructions based on regional fossil records. Higher variability in environmental factors such as pH, electrical conductivity, and ionic content was observed in thermokarst-affected lakes in Central Yakutia than in NE Yakutia lakes. Species diversity of freshwater ostracods reached up to eight taxa per lake, mostly dominated by Candona weltneri Hartwig 1899, in Central Yakutia, whereas in NE Yakutian waters the diversity was lower and Candona muelleri jakutica Pietrzeniuk 1977 or Fabaeformiscandona inaequivalvis (Sars 1898) had highest frequencies. Coupled analyses of stable isotopes (δ¹⁸O, δ¹³C) and element ratios (Sr/Ca, Mg/Ca) were performed on both host waters and ostracod calcite, aiming to estimate the modern relationships. Correlations between host waters and ostracod calcite of single species were found for δ¹⁸O, δ¹³C and Sr/Ca and Mg/Ca ratios. The relationships between δ¹⁸O, Mg/Ca and Sr/Ca ratios and electrical conductivity (salinity) as an expression of solute concentrations in the waters mainly controlled by evaporation are more complicated but evident, and may be useful in future interpretation of geochemical data from fossil Siberian ostracods. Handling editor: K. Martens     

Isolated poly(3-hydroxybutyrate) (PHB) granules are complex bacterial organelles catalyzing formation of PHB from acetyl coenzyme A (CoA) and degradation of PHB to acetyl-CoA

Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from ¹⁴C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD⁺, indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD⁺/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.     

Interaction of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> non-O1/non-O139 with Copepods,Cladocerans and Competing Bacteria in the Large Alkaline Lake Neusiedler See,Austria

Vibrio cholerae is a human pathogen and natural inhabitant of aquatic environments. Serogroups O1/O139 have been associated with epidemic cholera, while non-O1/non-O139 serogroups usually cause human disease other than classical cholera. V. cholerae non-O1/non-O139 from the Neusiedler See, a large Central European lake, have caused ear and wound infections, including one case of fatal septicaemia. Recent investigations demonstrated rapid planktonic growth of V. cholerae non-O1/non-O139 and correlation with zooplankton biomass. The aim of this study was to elucidate the interaction of autochthonous V. cholerae with two dominant crustacean zooplankton species in the lake and investigate the influence of the natural bacterial community on this interaction. An existing data set was evaluated for statistical relationships between zooplankton species and V. cholerae and co-culture experiments were performed in the laboratory. A new fluorescence in situ hybridisation protocol was applied for quantification of V. cholerae non-O1/non-O139 cells, which significantly reduced analysis time. The experiments clearly demonstrated a significant relationship of autochthonous V. cholerae non-O1/non-O139 with cladocerans by promoting growth of V. cholerae non-O1/non-O139 in the water and on the surfaces of the cladocerans. In contrast, copepods had a negative effect on the growth of V. cholerae non-O1/non-O139 via competing bacteria from their surfaces. Thus, beside other known factors, biofilm formation by V. cholerae on crustacean zooplankton appears to be zooplankton taxon specific and may be controlled by the natural bacterial community.     

Nitrate uptake and carbon exudation – do plant roots stimulate or inhibit denitrification?

Plant and Soil - Plant growth affects soil moisture, mineral N and organic C availability in soil, all of which influence denitrification. With increasing plant growth, root exudation may stimulate...     

Acrylyl-Coenzyme A Reductase,an Enzyme Involved in the Assimilation of 3-Hydroxypropionate by Rhodobacter sphaeroides

The anoxygenic phototroph Rhodobacter sphaeroides uses 3-hydroxypropionate as a sole carbon source for growth. Previously, we showed that the gene (RSP_1434) known as acuI, which encodes a protein of the medium-chain dehydrogenase/reductase (MDR) superfamily, was involved in 3-hydroxypropionate assimilation via the reductive conversion to propionyl-coenzyme A (CoA). Based on these results, we speculated that acuI encoded acrylyl-CoA reductase. In this work, we characterize the in vitro enzyme activity of purified, recombinant AcuI using a coupled spectrophotometric assay. AcuI from R. sphaeroides catalyzes the NADPH-dependent acrylyl-CoA reduction to produce propionyl-CoA. Two other members of the MDR012 family within the MDR superfamily, the products of SPO_1914 from Ruegeria pomeroyi and yhdH from Escherichia coli, were shown to also be part of this new class of NADPH-dependent acrylyl-CoA reductases. The activities of the three enzymes were characterized by an extremely low K_m for acrylyl-CoA (<3 μM) and turnover numbers of 45 to 80 s⁻¹. These homodimeric enzymes were highly specific for NADPH (K_m = 18 to 33 μM), with catalytic efficiencies of more than 10-fold higher for NADPH than for NADH. The introduction of codon-optimized SPO_1914 or yhdH into a ΔacuI::kan mutant of R. sphaeroides on a plasmid complemented 3-hydroxypropionate-dependent growth. However, in their native hosts, SPO_1914 and yhdH are believed to function in the metabolism of substrates other than 3-hydroxypropionate, where acrylyl-CoA is an intermediate. Complementation of the ΔacuI::kan mutant phenotype by crotonyl-CoA carboxylase/reductase from R. sphaeroides was attributed to the fact that the enzyme also uses acrylyl-CoA as a substrate.     

Serratia odorifera: analysis of volatile emission and biological impact of volatile compounds on Arabidopsis thaliana

Bacteria emit a wealth of volatiles. The combination of coupled gas chromatography/mass spectrometry (GC/MS) and proton-transfer-reaction mass spectrometry (PTR-MS) analyses provided a most comprehensive profile of volatiles of the rhizobacterium Serratia odorifera 4Rx13. An array of compounds, highly dominated by sodorifen (approximately 50%), a bicyclic oligomethyl octadiene, could be detected. Other volatiles included components of the biogeochemical sulfur cycle such as dimethyl disulfide (DMDS), dimethyl trisulfide and methanethiol, terpenoids, 2-phenylethanol, and other aromatic compounds. The composition of the bouquet of S. odorifera did not change significantly during the different growth intervals. At the beginning of the stationary phase, 60 μg of volatiles per 24 h and 60 easily detectable components were released. Ammonia was also released by S. odorifera, while ethylene, nitric oxide (NO) and hydrogen cyanide (HCN) could not be detected. Dual culture assays proved that 20 μmol DMDS and 2.5 μmol ammonia, individually applied, represent the IC₅₀ concentrations that cause negative effects on Arabidopsis thaliana.