Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates

Background

The β-carbonic anhydrase (CA, EC 4.2.1.1) enzymes have been reported in a variety of organisms, but their existence in animals has been unclear. The purpose of the present study was to perform extensive sequence analysis to show that the β-CAs are present in invertebrates and to clone and characterize a member of this enzyme family from a representative model organism of the animal kingdom, e.g., Drosophila melanogaster.

Results

The novel β-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of β-CA sequences in 55 metazoan species. The corresponding recombinant enzyme was produced in Sf9 insect cells, purified, kinetically characterized, and its inhibition was investigated with a series of simple, inorganic anions. Holoenzyme molecular mass was defined by dynamic light scattering analysis and gel filtration, and the results suggested that the holoenzyme is a dimer. Double immunostaining confirmed predictions based on sequence analysis and localized DmBCA protein to mitochondria. The enzyme showed high CO₂ hydratase activity, with a k_cat of 9.5 × 10⁵ s^-1 and a k_cat/K_M of 1.1 × 10⁸ M^{- 1}s^{- 1}. DmBCA was appreciably inhibited by the clinically-used sulfonamide acetazolamide, with an inhibition constant of 49 nM. It was moderately inhibited by halides, pseudohalides, hydrogen sulfide, bisulfite and sulfate (K_I values of 0.67 - 1.36 mM) and more potently by sulfamide (K_I of 0.15 mM). Bicarbonate, nitrate, nitrite and phenylarsonic/boronic acids were much weaker inhibitors (K_Is of 26.9 - 43.7 mM).

Conclusions

The Drosophila β-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.     

X-Ray Crystal Structure of Phyllostin,a Metabolite Produced by <Emphasis Type="Italic">Phyllosticta cirsii</Emphasis>, a Potential Mycoherbicide of <Emphasis Type="Italic">Cirsium arvense</Emphasis>

Abstract  

The X-ray crystal structure of phyllostin (8-hydroxy-3-methyl-2-oxo-2,3,4a,5,8,8a-hexahydro-benzo[1,4]dioxine-6-carboxylic acid methyl ester) is reported. Phyllostin was recently and for the first time isolated, together with phytotoxin phyllostoxin and the phyllostictines A–D, from the culture filtrates of Phyllosticta cirsii, a fungal pathogen isolated from diseased Cirsium arvense leaves and evaluated as biocontrol agent of this noxious perennial weed. Phyllostin crystallizes in the monoclinic space group P2₁ with unit cell parameters: a = 8.371(2), b = 7.026(1), c = 9.918(1) ?, β = 92.013(8)°, Z = 2. The final refinement converged to R1 = 0.0530 for 1026 observed reflections having I > 2σ(I). In the molecule two trans joined six-membered rings are present. The cyclohexene ring adopts a half-chair conformation. The 2-oxo-1,4-dioxan ring adopts a distorted half-chair conformation. The relative stereochemistry of the four chiral centers turns out to be 3R,4aS,8R,8aS. In the crystal packing molecules form infinite chains through O–H···O intermolecular hydrogen bonds. Chains run along the crystallographic a axis forming layers of molecules stacking in the [0 1 0] direction.     

Freudenthal duality and generalized special geometry

Freudenthal duality, introduced in Borsten et al. (2009) [1] and defined as an anti-involution on the dyonic charge vector in d=4 space-time dimensions for those dualities admitting a quartic invariant, is proved to be a symmetry not only of the classical Bekenstein-Hawking entropy but also of the critical points of the black hole potential.Furthermore, Freudenthal duality is extended to any generalized special geometry, thus encompassing all N>2 supergravities, as well as N=2 generic special geometry, not necessarily having a coset space structure.     

Fractal regularity results on optimal irrigation patterns

In this paper the problem of the regularity, i.e. fractal behaviour, of the minima of the branched transport problem is addressed. We show that, under suitable conditions on the irrigated measure, the minima present a fractal regularity, that is on a given branch of length l the number of branches bifurcating from it whose length is comparable with ε   can be estimated both from above and below by l/ε$l/\epsilon$.     

Negatively buoyant plume dispersion: a comparison of simple mathematical models, hydraulic simulations and field data

Summary The ground-level concentration distribution for negatively buoyant gases of various densities, emitted from stacks with heights, diameters and exit velocities varying in the typical range of geothermal drilling activities, was studied by means of hydraulic simulations performed in neutral conditions at different flow speeds. These measurements, and a few others taken in the field in nearly neutral and unstable atmosphere during tests of CO₂ release from a geothermal well, were compared with the values expected from a Gaussian formula corrected for denser-than-air, tilted plumes. For calculating theoretical concentrations, three couples of horizontal and vertical plume standard deviations as functions of downwind distance from source were employed: those given by Singer-Smith and Briggs and a third one obtained by our group during a previous water channel experiment. Also the maximum ground-level concentration and its distance from the source were measured in each simulation and compared with the prediction of our Gaussian formula, as well as with a best-fit expression given by Meroney. The concentration pattern χ(x, y, 0) given by the corrected Gaussian formula proved to be in good agreement with experimental data, especially in association with Singer-Smith's or our sigmas. When only maximum g.l. concentrations are needed, also the simple formula suggested by Meroney can give a good approximation.

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Riassunto Una serie di simulazioni in vasca senza stratificazione, condotte a varie velocità della vena idraulica, ha permesso di studiare la distribuzione di concentrazione al suolo dovuta a gas con varie densità maggiori di quella dell'aria, emessi da ciminiere con altezze, diametri e velocità di efflusso variabili entro intervalli tipici delle attività di perforazione geotermica. Queste misure, ed alcune altre ottenute in campo in condizioni di atmosfera circa neutra e instabile durante prove di rilascio di CO₂ da un pozzo geotermico, sono state confrontate con i valori attesi in base ad una formula gaussiana corretta per pennacchi inclinati. Nel calcolo delle concentrazioni teoriche si sono impiegate tre diverse coppie di espressioni per le deviazioni standard orizzontale e verticale del pennacchio: quelle date da Singer-Smith e Briggs ed una terza ottenuta dal nostro gruppo durante una precedente esperienza in canale idraulico. In ciascuna prova si è misurata anche la concentrazione massima al suolo e la sua distanza dalla sorgente; queste quantità sono state raffrontate con le previsioni della formula gaussiana e con una espressione empirica proposta da Meroney. Il campo di concentrazione χ(x, y, 0) dato dalla formula gaussiana corretta è risultato in buon accordo con le misure, specialmente se associato alle sigma di Singer-Smith o alle nostre. Quando sia sufficiente una valutazione della concentrazione massima, anche la semplice formula di Meroney può dare una buona approssimazione.

     

Quantitative analysis of thin samples by differential absorption imaging using a laser-plasma soft X-ray source

We consider the application of the differential absorption imaging technique in the soft X-ray region. The surface-density maps of test samples containing bromine, resulting from the application of the differential absorption imaging technique, are presented. Images of the samples were obtained in an X-ray monochromatic projection imaging scheme based on a spherically bent crystal using line emission close to the $L_2$ edge of bromine from a microplasma produced by focusing a nanosecond laser on the surface of an aluminum target.     

Competitive Gold‐Promoted Meyer–Schuster and oxy‐Cope Rearrangements of 3‐Acyloxy‐1,5‐enynes: Selective Catalysis for the Synthesis of (+)‐(S)‐γ‐Ionone and (−)‐(2S,6 R)‐cis‐γ‐Irone

We report a simple, highly stereoselective synthesis of (+)‐(S)‐γ‐ionone and (‐)‐(2S,6R)‐cis‐γ‐irone, two characteristic and precious odorants; the latter compound is a constituent of the essential oil obtained from iris rhizomes. Of general interest in this approach are the photoisomerization of an endo trisubstituted cyclohexene double bond to an exo vinyl group and the installation of the enone side chain through a [(NHC)Au^I]‐catalyzed Meyer–Schuster‐like rearrangement. This required a careful investigation of the mechanism of the gold‐catalyzed reaction and a judicious selection of reaction conditions. In fact, it was found that the Meyer–Schuster reaction may compete with the oxy‐Cope rearrangement. Gold‐based catalytic systems can promote either reaction selectively. In the present system, the mononuclear gold complex [Au(IPr)Cl], in combination with the silver salt AgSbF₆ in 100:1 butan‐2‐one/H₂O, proved to efficiently promote the Meyer–Schuster rearrangement of propargylic benzoates, whereas the digold catalyst [{Au(IPr)}₂(μ‐OH)][BF₄] in anhydrous dichloromethane selectively promoted the oxy‐Cope rearrangement of propargylic alcohols.     

The inorganic perspective of nerve growth factor: interactions of Cu2+ and Zn2+ with the N-terminus fragment of nerve growth factor encompassing the recognition domain of the TrkA receptor

There is a significant overlap between brain areas with Zn(2+) and Cu(2+) pathological dys-homeostasis and those in which the nerve growth factor (NGF) performs its biological role. The protein NGF is necessary for the development and maintenance of the sympathetic and sensory nervous systems. Its flexible N-terminal region has been shown to be a critical domain for TrkA receptor binding and activation. Computational analyses show that Zn(2+) and Cu(2+) form pentacoordinate complexes involving both the His4 and His8 residues of the N-terminal domain of one monomeric unit and the His84 and Asp105 residues of the other monomeric unit of the NGF active dimer. To date, neither experimental data on the coordination features have been reported, nor has one of the hypotheses according to which Zn(2+) and Cu(2+) may have different binding environments or the Ser1 α-amino group could be involved in coordination been supported. The peptide fragment, encompassing the 1-14 sequence of the human NGF amino-terminal domain (NGF(1-14)), blocked at the C terminus, was synthesised and its Cu(2+) and Zn(2+) complexes characterized by means of potentiometric and spectroscopic (UV/Vis, CD, NMR, and EPR) techniques. The N-terminus-acetylated form of NGF(1-14) was also investigated to evaluate the involvement of the Ser1 α-amino group in metal-ion coordination. Our results demonstrate that the amino group is the first anchoring site for Cu(2+) and is involved in Zn(2+) coordination at physiological pH. Finally, a synergic proliferative activity of both NGF(1-14) and the whole protein on SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). This effect was not observed after treatment with the N-acetylated peptide fragment, demonstrating a functional involvement of the N-terminal amino group in metal binding and peptide activity.     

Base promoted synthesis of activated cyclopropanes bearing homologated carbonyl groups via tandem Michael addition-intramolecular enolate trapping

A simple base promoted intramolecular Michael initiated ring closure reaction of γ-hydroxyenone derived diphenyl phosphinates with 1,3-indandione, enabled the synthesis of novel activated cyclopropanes with homologated carbonyl moiety in good yield. Promising levels of enantioselectivity are achieved when using cinchona derivatives as promoters.