Ergosta-5,23-dien-3β-ol and ergosta-7,23-dien-3β-ol,two new sterols from Zea mays etiolated coleoptiles

Ergosta-5,23-dien-3β-ol and ergosta-7,23-dien-3β-ol were identified for the first time in maize etiolated coleoptiles. They represent more than 11 % of the total 4-desmethyl sterol fraction. It is suggested that they could play some role in the biosynthesis of 24-methyl sterols of this material.     

Experimental Evidence for the Existence of a Stable Half-Barrel Subdomain in the (β/α)8-Barrel Fold

The (β/α)₈-barrel is one of the most common folds functioning as enzymes. The emergence of two (β/α)₈-barrel enzymes involved in histidine biosynthesis, each of which has a twofold symmetric structure, has been proposed to be a consequence of tandem duplication and fusion of a (β/α)₄-half-barrel. However, little evidence has been found for the existence of an ancestral half-barrel in the evolution of other (β/α)₈-barrel proteins. In order to detect remnants of an ancestral half-barrel in the (β/α)₈-barrel structure of Escherichia coli N-(5′-phosphoribosyl)anthranilate isomerase, we engineered three potential half-barrel units, (β/α)_1-4, (β/α)_3-6, and (β/α)_5-8. Among these three arrangements, only (β/α)_3-6 is stable; it exists in equilibrium between monomeric and dimeric forms. Thus, the central segment of N-(5′-phosphoribosyl)anthranilate isomerase from E. coli can serve as a half-barrel precursor. A tandem duplication of (β/α)_3-6 yielded predominantly monomeric structures that were quite stable. This result exemplified that the structural characteristics of noncovalently assembled half-barrels could be improved by duplication and fusion. Moreover, our results may provide information regarding the local structural units that encompass interactions important for the early folding events of this ubiquitous protein conformation.     

Identification of (24S)-24-methylcholesta-5,22-dien-3β-ol as the major sterol of a marine cryptophyte and a marine prymnesiophyte

The major sterol in the two unicellular marine algae Cryptomonas sp. and Isochrysis galbana has been identified as (24S)-24-methylcholesta-5,22-dien-3β-ol (epibrassicasterol). The production of a sterol with the 24α-configuration by cryptophycean and prymnesiophycean algae and by diatoms contrasts with the situation in many other algae which produce sterols with the 24β-configuration.     

A Comparison of the Membrane Binding Properties of C1B Domains of PKCγ, PKCδ, and PKC?

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical γ, and the novel δ and ?), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bγ and C1B? exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1B? being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1B? domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.     

The conversion of (24S)-24-ethylcholesta-5,22,25-trien-3β-ol into poriferasterol,both in vivo and with a cell-free homogenate of the alga Trebouxia sp.

[6-³H₁] (24S)-24-Ethylcholesta-5,22,25-trien-3β-ol added to the growth medium of a culture of Trebouxia sp. 213/3 was efficiently taken-up by the cells and converted into (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol) which is one of the major sterols of this alga. A cell-free homogenate was obtained from Trebouxia which catalysed the NADPH-dependent reduction of [6-³H₁] (24S)-24-ethylcholesta-5,22,25-trien-3β-ol to yield poriferasterol. The δ²⁵-sterol reductase was found to be mainly localized in the microsomal fraction of the homogenate.     

Substoichiometric inhibition of Aβ1-40 aggregation by a tandem Aβ40-1-Gly8-1-40 peptide

Aβ peptides aggregate to form insoluble and neurotoxic fibrils associated with Alzheimer’s disease. Inhibition of the aggregation has been the subject of numerous studies. Here we describe a novel, substoichiometric inhibitor of Aβ_1-40 fibrillization as a tandem dimeric construct consisting of Aβ_40-1 (reverse sequence) linked to Aβ_1-40 via an eight residue glycine linker. At molar ratios of the tandem peptide to Aβ_1-40 of 1:10 to 1:25 inhibition of fibrillization, as measured by ThioflavinT, was observed. We postulate that the tandem construct binds to a fibrillar intermediate but the reverse sequence delays or prevents further monomer association.     

Synthesis, X-ray crystal structure and magnetic study of the 1D {[Cu2(N,N,N′,N′-tetramethyl-ethylenediamine)2(μ-1,5-dca)2(dca)2]}n complex

Reaction of Cu(NO₃)₂ · 3H₂O, N,N,N′,N′-tetramethyl-ethylenediamine (L) and sodium dicyanamide (Nadca) in aqueous medium yields a complex the {[Cu₂L₂(μ-1,5-dca)₂(dca)₂]}_n complex, 1. Single crystal X-ray analysis reveals that complex 1 has a 1D infinite chain structure in which copper(II) ions are bridged by single dicyanamide anions in an end-to-end fashion. The coordination environment around copper(II) is distorted square pyramidal. Two among the four coordination sites of the basal plane are occupied by the nitrogen atoms of the diamine and two remaining sites are occupied by the terminal nitrogen atom of a bridging and of a monodentate dca anions. The fifth coordination site (apical) is occupied by a nitrogen atom from a bridging dca anion of an adjacent CuL(dca)₂ moiety, yielding the [Cu₂L₂(μ-1,5-dca)₂(dca)₂] dinuclear unit. Dimeric units are connected to each other by single μ-1,5-dicyanamido group to form infinite 1D chains which propagate parallel to the crystallographic c-axis. The variable temperature magnetic susceptibility measurements evidenced weakly antiferromagnetic interactions (J = −0.26 cm⁻¹) in {[Cu₂L₂(μ-1,5-dca)₂(dca)₂]}_n, 1.     

Synthesis and structural characterization of the molecular loop [cis-Mo2(N,N-di-p-anisylformamidinate)2]2(O2C-CHCCH-CO2)2

A new molecular loop composed of two quadruply bonded Mo₂(DAniF)₂ units (DAniF=N,N^′-di-p-anisylformamidinate) linked by two chiral allene-1,3-dicarboxylate anions has been prepared from the reaction of [cis-Mo₂(DAniF)₂(MeCN)₄](BF₄)₂ with the bis(tetraethylammonium) salt of allene-1,3-dicarboxylic acid. This compound, [cis-Mo₂(DAniF)₂]₂(O₂C-CHCCH-CO₂)₂ (1), has been characterized by X-ray crystallography and by ¹H NMR and UV-Vis spectroscopy. The molecule possesses a center of inversion and hence is meso. There is only weak electronic coupling between the two Mo₂ ⁴⁺ units as revealed by electrochemical measurements.     

The X-ray Crystal Structure of an Arthrobacter protophormiae Endo-β-N-Acetylglucosaminidase Reveals a (β/α)8 Catalytic Domain, Two Ancillary Domains and Active Site Residues Key for Transglycosylation Activity

Glycoside hydrolase family GH85 is a family of endo-β-N-acetylglucosaminidases that is responsible for the hydrolysis of β-1,4 linkage in the N,N-diacetylchitobiose core of N-linked glycans. The endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) is of particular interest, given its increasing use for the chemoenzymatic synthesis of bespoke N-glycans using N-glycan oxazolines as glycosyl donors. The E173Q variant of Endo-A is especially attractive for synthesis, as it is hydrolytically impaired but still able to catalyze N-glycan synthesis by transglycosylation using activated oxazoline donors. Here we present the three-dimensional structure of the A. protophormiae Endo-A E173Q variant, solved by multiple-wavelength anomalous scattering methods and refined at 1.8 Å resolution. The structure reveals that GH85 enzymes display a trimodular architecture in which a (β/α)₈ catalytic domain occurs with two ancillary β-sheet modules. The active centre is fully consistent with the known neighboring-group catalytic mechanism in which E173 acts as the catalytic acid/base for reaction via an oxazoline intermediate. Of note is the presence of an asparagine in the active centre, in a position likely to interact with the acetyl NH group that, in all other known families of glycosidase using this mechanism, is an aspartate or glutamate residue. The substrate-binding surface reveals an open topography, consistent with the ability to accept a large range of glycoprotein substrates and the ability to transglycosylate other acceptors. The three-dimensional structure of this important biocatalyst reveals that residues implicated in the enhancement of transglycosylation and synthetic capacity are proximal to the active centre, where they may act to favor binding of acceptor substrates.     

Synthesis and structure of the cyclic amido bismuth imide, {[(Me3Si)2N]Bi[μ-N(SiMe3)]}2, via loss of SiMe3 from a [N(SiMe3)2] ligand

The synthesis of Bi[N(SiMe₃)₂]₃ from BiCl₃ and KN(SiMe₃)₂ generates an unusual amido bismuth imide by-product, {[(Me₃Si)₂N]Bi[μ-N(SiMe₃)]}₂, evidently formed via SiN bond cleavage. X-ray diffraction shows that the complex contains a planar Bi₂N₂ ring with tetrahedral bismuth and trigonal planar imido nitrogen atoms.     

‘Twisted plywood’ structure and mineralization in the scales of a primitive living fish Amia calva

The basal plate of the scales of Amia calva is composed of regular double twisted plywood, as in Latimeria and Dipnoan scales. However, the progressive rotation of the fibrils direction is left-handed in Amia and right-handed in the ‘Sarcopterygians’. So, the similarity between these peculiar plywoods is probably the result of convergence. The basal plate of Amia scales is incompletely mineralized. There are numerous calcified ovoid corpuscles which look very like the Mandl's corpuscles of Teleost scales. The mineralization probably progresses essentially by the fusion of these corpuscles, as in Teleost scales, and would be inotropic rather than spheritic.     

Ligand induced PH bond formation and a comparison of the reactivity of two isomers of the carbonyl hydride [Pt2(μ-PBu2)2(H)(CO)(PBu2H)]CF3SO3

The Pt₂ ^(II) isomeric terminal hydrides [(CO)(H)Pt(μ-PBu^t ₂)₂Pt(PBu^t ₂H)]CF₃SO₃ (1a), and [(CO)Pt(μ-PBu^t ₂)₂Pt(PBu^t ₂H)(H)]CF₃SO₃ (1b), react rapidly with 1 atm of carbon monoxide to give the same mixture of two isomers of the Pt₂ ^(I) dicarbonyl [Pt₂(μ-PBu^t ₂)(CO)₂(PBu^t ₂H)₂]CF₃SO₃ (3-Pt); the solid state structure of the isomer bearing the carbonyl ligands pseudo-trans to the bridging phosphide was solved by X-ray diffraction. A remarkable difference was instead found between the reactivity of 1a and 1b towards carbon disulfide or isoprene. In both cases 1b reacts slowly to afford [Pt₂(μ-PBu^t ₂)(μ,η²,η²-CS₂)(PBu^t ₂H)₂]CF₃SO₃ (4-Pt), and [Pt₂(μ-PBu^t ₂)(μ,η²,η²-isoprene) (PBu^t ₂H)₂]CF₃SO₃ (6-Pt), respectively. In the same experimental conditions, 1a is totally inert. A common mechanism, proceeding through the preassociation of the incoming ligand followed by the PH bond formation between one of the bridging P atoms and the hydride ligand, has been suggested for these reactions.     

1,3-Bis(α-aminoisopropyl)benzene, meta-C6H4(CMe2NH2)2: An N,N-bridging and N,C,N-cyclometalating ligand

Saponification of the bis(carbamic acid ester) 1,3-C₆H₄(CMe₂NHCO₂Me)₂ (1), made by the addition of methanol to commercial 1,3-C₆H₄(CMe₂NCO)₂, yielded the meta-phenylene-based bis(tertiary carbinamine) 1,3-C₆H₄(CMe₂NH₂)₂ (2). Dinuclear [{(η⁴-1,5-C₈H₁₂)RhCl}₂{μ-1,3-C₆H₄(CMe₂NH₂)₂}] (3) resulted from the action of 2 on [{(η⁴-1,5-C₈H₁₂)Rh(μ-Cl)}₂] in toluene. Combination of 2 with PdCl₂ or K₂[PdCl₄] gave the dipalladium macrocycle trans,trans-[{μ-1,3-C₆H₄(CMe₂NH₂)₂}₂(PdCl₂)₂] (4) along with cyclometalated [{2,6-C₆H₃(CMe₂NH₂)₂-κN,κC¹,κN′}PdCl] (5). Substitution of PEt₃ for the labile chlorido ligand of 5 afforded [{2,6-C₆H₃(CMe₂NH₂)₂-κN,κC¹, κN′}Pd(PEt₃)]Cl (6). The crystal structures of the following compounds were determined: bis(carbamic acid ester) 1, ligand 2 as its bis(trifluoroacetate) salt [1,3-C₆H₄(CMe₂NH₃)₂](O₂CCF₃)₂, 2 · (HAc_f)₂, complexes 3 and 6, as well as 1,3-C₆H₄(CMe₂OH)₂ (the diol analogue of 2).     

Ric-8A,a GEF,and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.     

Reduction chemistry of the mixed ligand metallocene [(C5Me5)(C8H8)U]2(μ-C8H8) with bipyridines

The U⁴⁺ cyclooctatetraenyl complex, [(C₅Me₅)(C₈H₈)U]₂(μ-C₈H₈), 1, reacts with two equiv of 4,4′-dimethyl-2,2′-bipyridine (Me₂bipy) and 2 equiv of 2,2′-bipyridine (bipy) to form 2 equiv of (η⁵-C₅Me₅)(η⁸-C₈H₈)U(Me₂bipy-κ²N,N′) and (η⁵-C₅Me₅)(η⁸-C₈H₈)U(bipy-κ²N,N′), respectively. X-ray crystallography, infrared spectroscopy, and density functional theory calculations indicate that the products are best described as U⁴⁺ complexes of bipyridyl radical anions. Hence, only one of the (C₈H₈)²⁻ ligands in 1 acts as a reductant and delivers 2 electrons per equiv of 1. Since the reduction potentials of uncomplexed (C₈H₈)²⁻, Me₂bipy, and bipy are −1.86, −2.15, and −2.10 V vs SCE, respectively, it is likely that prior coordination of the bipyridine reagents enhances the electron transfer.     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

The synthesis of 6,9-deepoxy-6,9-N-phenylimino-Δ6,8-prostaglandin I1 a novel inhibitor of leukotriene C and D synthesis

The pyrrole analog of prostacyclin, 6,9-deepoxy-6,9-N-phenylimino-Δ^6,8-prostaglandin I₁ was synthetized from PGF₂α methyl ester. This pyrroloprostacyclin (U-60, 257) and its methyl ester (U-56, 467) have been shown to inhibit leukotriene C/D biosynthesis and antagonized leukotriene C/D contractions in vitro. Antigen induced bronchopulmonary changes in monkeys and guinea pigs are inhibited by U-60, 257 in vivo.     

A 3D Structure Model of Integrin α4β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4β1, we began constructing a structure model for the integrin α4β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg²⁺ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/β7 ligand binding selectivity.