Non-genomic regulation of intermediate conductance potassium channels by aldosterone in human colonic crypt cells

BACKGROUND: Aldosterone has a rapid, non-genomic, inhibitory effect on macroscopic basolateral K+ conductance in the human colon, reducing its capacity for Cl− secretion. The molecular identity of the K+ channels constituting this aldosterone inhibitable K+ conductance is unclear. AIM: To characterise the K+ channel inhibited by aldosterone present in the basolateral membrane of human colonic crypt cells. METHODS: Crypts were isolated from biopsies of healthy sigmoid colon obtained during colonoscopy. The effect of aldosterone on basolateral K+ channels, and the possible involvement of Na+:H+ exchange, were studied by patch clamp techniques. Total RNA from isolated crypts was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific to intermediate conductance K+ channels (KCNN4) previously identified in other human tissues. RESULTS: In cell attached patches, 1 nmol/l aldosterone significantly decreased the activity of intermediate conductance (27 pS) K+ channels by 31%, 53%, and 54% after 1, 5 and 10, minutes, respectively. Increasing aldosterone concentration to 10 nmol/l produced a further 56% decrease in channel activity after five minutes. Aldosterone 1–10 nmol/l had no effect on channel activity in the presence of 20 µmol/l ethylisopropylamiloride, an inhibitor of Na+:H+ exchange. RT-PCR identified KCNN4 mRNA, which is likely to encode the 27 pS K+ channel inhibited by aldosterone. CONCLUSION: Intermediate conductance K+ channels (KCNN4) present in the basolateral membranes of human colonic crypt cells are a target for the non-genomic inhibitory effect of aldosterone, which involves stimulation of Na+:H+ exchange, thereby reducing the capacity of the colon for Cl− secretion

The use of tensiometers to control the irrigation of nursery stock in containers.

End of Project ReportThe use of digital tensiometers to control the irrigation of nursery stock in containers was studied over a three year period. Over this time the tensiometers performed satisfactorily and successfully automated the irrigation of the plants. The results indicate the feasibility of using them to control nursery stock irrigation under Irish conditions. An irrigation tension of 50 hPa to trigger an irrigation period resulted in larger plants than those grown under drier regimes with irrigation tensions of 100 and 200 hPa. Measurements of stomatal resistance indicated that the plants in the drier regimes were growing under greater moisture stress. The drier regimes reduced the number of irrigations and also the overall usage of water. They reduced plant size but did not impair plant appearance. It may be possible to use this approach in the future to control plant growth. There was no difference in performance between plants gown with ebb and flood irrigation and those irrigated via overhead spraylines. The ebb and flood system gave a considerable reduction in water use.European Union Structural Funds (EAGGF

Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina

The major photopigment of the cyanobacterium Acaryochloris marina is chlorophyll d , while its direct biosynthetic precursor, chlorophyll a , is also present in the cell. These pigments, along with the majority of chlorophylls utilized by oxygenic pho- totrophs, carry an ethyl group at the C-8 position of the molecule, having undergone reduction of a vinyl group during biosyn- thesis. Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB. The genome of Acaryochloris marina contains open reading frames (ORFs) encoding proteins displaying high sequence similarity to BciA or BciB, although they are annotated as genes involved in transcriptional control ( nmrA ) and methanogenesis ( frhB ), respectively. These genes were introduced into an 8-vinyl chlorophyll a -producing delta bciB strain of Synechocystis sp. strain PCC 6803, and both were shown to restore synthesis of the pigment with an ethyl group at C-8, demonstrating their activities as 8-vinyl reductases. We propose that nmrA and frhB be reassigned as bciA and bciB , respectively; transcript and proteomic analysis of Acaryochloris marina reveal that both bciA and bciB are expressed and their encoded proteins are present in the cell, possibly in order to ensure that all synthesized chlorophyll pigment carries an ethyl group at C-8. Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed

Proteorhodopsin overproduction enhances the long-term viability of Escherichia coli

Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli. Quantitative mass spectrometry shows that E. coli, exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ∼148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans

The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2- dependent insertion of a Mg2+ ion into protoporphyin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro , ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH. Quantitative analysis using microscale thermophoresis (MST) showed magnesium-dependent binding ( K d 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD-H interaction was investigated using chemical crosslinking coupled with mass spectrometry (XL-MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. Our work, showing the interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase, will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH

Unravelling Mycosphaerella: do you believe in genera?

Many fungal genera have been defined based on single characters considered to be informative at the generic level. In addition, many unrelated taxa have been aggregated in genera because they shared apparently similar morphological characters arising from adaptation to similar niches and convergent evolution. This problem is aptly illustrated in Mycosphaerella. In its broadest definition, this genus of mainly leaf infecting fungi incorporates more than 30 form genera that share similar phenotypic characters mostly associated with structures produced on plant tissue or in culture. DNA sequence data derived from the LSU gene in the present study distinguish several clades and families in what has hitherto been considered to represent the Mycosphaerellaceae. In some cases, these clades represent recognisable monophyletic lineages linked to well circumscribed anamorphs. This association is complicated, however, by the fact that morphologically similar form genera are scattered throughout the order (Capnodiales), and for some species more than one morph is expressed depending on cultural conditions and media employed for cultivation. The present study shows that Mycosphaerella s.s. should best be limited to taxa with Ramularia anamorphs, with other well defined clades in the Mycosphaerellaceae representing Cercospora, Cercosporella, Dothistroma, Lecanosticta, Phaeophleospora, Polythrincium, Pseudocercospora, Ramulispora, Septoria and Sonderhenia. The genus Teratosphaeria accommodates taxa with Kirramyces anamorphs, while other clades supported in the Teratosphaeriaceae include Baudoinea, Capnobotryella, Devriesia, Penidiella, Phaeothecoidea, Readeriella, Staninwardia and Stenella. The genus Schizothyrium with Zygophiala anamorphs is supported as belonging to the Schizothyriaceae, while Dissoconium and Ramichloridium appear to represent a distinct family. Several clades remain unresolved due to limited sampling. Mycosphaerella, which has hitherto been used as a term of convenience to describe ascomycetes with solitary ascomata, bitunicate asci and 1-septate ascospores, represents numerous genera and several families yet to be defined in future studies

Decision Problems for Nash Equilibria in Stochastic Games

We analyse the computational complexity of finding Nash equilibria in stochastic multiplayer games with $\omega$-regular objectives. While the existence of an equilibrium whose payoff falls into a certain interval may be undecidable, we single out several decidable restrictions of the problem. First, restricting the search space to stationary, or pure stationary, equilibria results in problems that are typically contained in PSPACE and NP, respectively. Second, we show that the existence of an equilibrium with a binary payoff (i.e. an equilibrium where each player either wins or loses with probability 1) is decidable. We also establish that the existence of a Nash equilibrium with a certain binary payoff entails the existence of an equilibrium with the same payoff in pure, finite-state strategies.Comment: 22 pages, revised versio

High-precision determination of the critical exponents for the lambda-transition of 4He by improved high-temperature expansion

We determine the critical exponents for the XY universality class in three dimensions, which is expected to describe the $\lambda$-transition in ${}^4$He. They are obtained from the analysis of high-temperature series computed for a two-component $\lambda\phi^4$ model. The parameter $\lambda$ is fixed such that the leading corrections to scaling vanish. We obtain $\nu = 0.67166(55)$, $\gamma = 1.3179(11)$, $\alpha=-0.0150(17)$. These estimates improve previous theoretical determinations and agree with the more precise experimental results for liquid Helium.Comment: 8 pages, revte

Synthesis of poly(stearyl methacrylate)-poly(2-hydroxypropyl methacrylate) diblock copolymer nanoparticles via RAFT dispersion polymerization of 2-hydroxypropyl methacrylate in mineral oil

Poly(stearyl methacrylate)-poly(2-hydroxypropyl methacrylate) (PSMA-PHPMA) diblock copolymer nanoparticles are synthesized via reversible addition–fragmentation chain transfer (RAFT) dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA) in mineral oil at 90 °C. The relatively short PSMA precursor (mean degree of polymerization = 9) remains soluble in mineral oil, whereas the growing PHPMA block quickly becomes insoluble, resulting in polymerization-induced self-assembly (PISA). Relatively high HPMA monomer conversions (≥98%) were achieved within 70 min as confirmed by in situ1H NMR spectroscopy studies, while gel permeation chromatography (GPC) analyses indicated high blocking efficiencies and relatively narrow molecular weight distributions (Mw/Mn ≤ 1.37) for all PISA syntheses. Depending on the precise synthesis conditions, this PISA formulation can produce diblock copolymer spheres, worms or vesicles; a pseudo-phase diagram has been constructed to enable reproducible targeting of each pure phase. Thus this is a rare example of the use of a commercially available polar monomer for PISA syntheses in non-polar media that offers access to the full range of copolymer morphologies. The resulting nanoparticles were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), oscillatory rheology and small-angle X-ray scattering (SAXS). Interestingly, PSMA9-PHPMA70 worms undergo an unusual (partial) worm-to-vesicle transition at elevated temperature. Finally, PSMA9-PHPMA50 spheres were evaluated as putative Pickering emulsifiers. Using lower water volume fractions produced water-in-oil (w/o) emulsions after high shear homogenization, as expected. However, using higher water volume fractions, shear rates or copolymer concentrations favored the formation of w/o/w Pickering double emulsions

How the O2-dependent Mg-protoporphyrin monomethyl ester cyclase forms the fifth ring of chlorophylls

Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, producing protochlorophyllide a and contributing substantially to the absorption properties of chlorophylls and bacteriochlorophylls. The O2-dependent cyclase is found in both oxygenic phototrophs and some purple bacteria. We overproduced the simplest form of the cyclase, AcsF, from Rubrivivax gelatinosus, in Escherichia coli. In biochemical assays the di-iron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP+ reductase, or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain. Kinetic analyses yielded a turnover number of 0.9 min−1, a Michaelis–Menten constant of 7.0 µM for MgPME and a dissociation constant for MgPME of 0.16 µM. Mass spectrometry identified 131-hydroxy-MgPME and 131-keto-MgPME as cyclase reaction intermediates, revealing the steps that form the isocyclic ring and completing the work originated by Sam Granick in 1950