New NF-κB Inhibitory Steroids from the Marine Sponge Dysidea avara Collected from the South China Sea

Chemical investigation of the marine sponge Dysidea avara, collected from the South China Sea, yielded 13 steroids, including nine new ( 1 – 9 ) and four known ( 10 – 13 ) ones. The new structures were elucidated as (3S,14R)-3,14-dihydroxycholesta-5,8-dien-7-one ( 1 ), (22E,24R)-7α-ethoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol ( 2 ), 3β-hydroxy-7α-ethoxy-5α,6α-epoxy-8(14)-cholestene ( 3 ), 3β,5α-dihydroxy-6α-ethoxychofesta-7,9(11)-diene ( 4 ), 3β,5α-dihydroxy-6β-ethoxycholest-7-ene ( 5 ), (22E,24R)-24-ethoxy-3β,5α-dihydroxy-6β-ethoxyergosta-7,22-diene ( 6 ), (22E)-3β,5α-dihydroxy-6β-ethoxycholesta-7,22-diene ( 7 ), 24-ethoxy-3β,5α-dihydroxy-6β-ethoxycholest-7-ene ( 8 and 9 ), by extensive spectroscopic analyses, such as HR-ESI-MS, 1D and 2D NMR data. The absolute configuration of 1 was assigned by comparison the experimental ECD spectra with the calculated ones. Among the 13 metabolites, compounds 1 , 4 , 11 , 12 , and 13 showed NF-κB inhibitory activities in human HER-293 cells with IC₅₀ values of 6.4, 18.7, 8.1, 9.6, and 7.5 μM, respectively. Preliminary structure−activity relationship analysis unveiled that the conjugated ketones or unsaturated double bonds might be the functional groups for the five active steroids.     

Mode of formation of cholesta-5,7-dien-3β-ol from cholest-5-en-3β-ol by larvae of Calliphora erythrocephala

1. The conversion of cholest-5-en-3beta-ol (cholesterol) into cholesta-5,7-dien-3beta-ol by axenic Calliphora erythrocephala larvae was demonstrated. 2. The transformation is probably direct (Delta(5)-->Delta(5,7)) and does not involve a Delta(0) intermediate (Delta(5)-->Delta(0)-->Delta(7)--> Delta(5,7)). 3. Delta(7)-bond formation involves the stereospecific elimination of the 7beta hydrogen atom. 4. The relative amounts of free and esterified sterols were determined in larvae grown on cholesterol as sole sterol source and on 5alpha-cholestan-3beta-ol supplemented with minimal amounts of cholesterol. 5. The significance of the results is assessed in relation to the probable role of cholesta-5,7-dien-3beta-ol as an intermediate in the biosynthesis of ecdysones.     

Sterols of Xanthoria parietina: Evidence for two sterol ‘pools’ and the identification of a novel C,8 triene,ergosta-5,8,22-trien-3β-ol

Sterols extracted from Xanthoria parietina with organic solvents and released by saponification of the residual lichen tissue were analysed by GC-MS. The main components of the solvent-extractable sterols were two C₂₈ trienes and those of the more tightly bound sterols were ergost-5-en-3β-ol and two C₂₉ compounds. The structures of the C₂₈ compounds were shown to be ergosta-5,7,22-trien-3β-ol, Ia (ergosterol) and the previously unreported ergosta-5,8,22-trien-3β-ol, IIa, for which the name lichesterol is proposed. The main C₂₉ sterol was identified as (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol).     

Phylogenetic position of sponges from Chagytaĭ and Tore-Khol' lakes

Morphological and molecular genetic data for freshwater sponges from the lakes of Tuva Depression, Baikalospongia dzhegatajensis (Rezvo, 1936), forms Dzh05 and Dzh06, from Chagatai Lake, as well as forms TKhl and TKh2, from the Lake Tore-Khol, were obtained and examined. In the sponges examined, which on phylogenetic tree clustered together with the Ephydatia fluviatilis (Linneaus, 1758) sponge from the family Spongillidae, the ITS rDNA regions were sequenced. Comparison of highly variable interal spacer regions of the mitochondrial genome was performed using corresponding sequences of three sponges from the family Spongillidae (E. fluviatilis, E. muelleri and Spongilla lacustris), sponges from the Chagatai and Tore-Khol lakes (Dzh06 and TKh2) with an unknown status, and sponges from the Baikalian family Lubomirskiidae. Minimum genetic differences were observed between E. fluviatilis, Dzh06, and TKh2 (from 0.003 to 0.01% of nucleotide substitutions), while maximum differences were found between the species of Lubomirskiidae and Spongillidae (from 0.928 to 2.06%). The data obtained indicated that sponges from Chagatai and Tore-Khol lakes were most close to E. fluviatilis.     

Sterols from black sea invertebrates—I. Sterols from Scyphozoa and Anthozoa (Coelenterata)

• 1.1. Rhizostoma pulmo, Aurelia aurita and Actinia equina, the most widespread representatives of Coelenterata in Black Sea have been analysed and the occurrence of 20 sterols has been found.
• 2.2. Dinosterol and demethyldinosterol as well as a number of short side chain sterols have been found in Scyphozoa for the first time.
• 3.3. The occurrence of coprostanol in marine invertebrates has been shown for the first time.
• 4.4. Five groups of sterol esters were found, containing fatty acids with different polarity.

     

Purification and separation of the 20S immunoproteasome from the constitutive proteasome and identification of the subunits by LC–MS

The proteasome is a multicatalytic protease complex present in all eukaryotic cells, which plays a critical role in regulating essential cellular processes. During the immune response to pathogens, stimulation by γ interferon induces the production of a special form of proteasome, the immunoproteasome. Inappropriate increase of proteosomal activity has been linked to inflammatory and autoimmune diseases. Selective inhibition of the immunoproteasome specific LMP7 subunit was shown to block inflammatory cytokine secretion in human PBMC, thus making the immunoproteasome an interesting target to fight autoimmune diseases. This paper describes a method for purification and separation of the 20S immunoproteasomes from the constitutive proteasome, which is ubiquitously present in all cells, based on hydrophobic interaction chromatography. The purified immunoproteasome showed several bands, between 20–30 kDa, when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The purified proteasome complexes had a molecular mass of approximately 700 kDa as estimated by gel filtration. Identification of the catalytic subunits in the immunoproteasomes was performed in Western blot with antibodies directed specifically against either the constitutive or the immunoproteasome subunits. The purified immunoproteasome possessed all three protease activities associated with the proteasome complex. LC/MS analysis confirmed the presence of the three immunoproteasome catalytic subunits in the purified immunoproteasome.     

Bio-assay guided isolation and identification of α-glucosidase inhibitors from the leaves of Aquilaria sinensis

Eight α-glucosidase inhibitors including four new compounds were isolated from the 70% aqueous ethanolic extract of leaves of Aquilaria sinensis (Lour.) Gilg by activity-directed fractionation and purification processes. The ethanolic extract was first separated into petroleum ether, ethyl acetate, n-butanol and water soluble fractions and screened for inhibitory activity against α-glucosidase. Further activity-directed investigation lead to the isolation of four new compounds with moderate inhibitory activity, viz, aquilarisinin (1), aquilarisin (2), hypolaetin 5-O-β-D-glucuronopyranoside (3) and aquilarixanthone (4) from the n-butanol fraction, and four known compounds showing potent activity including mangiferin (5), iriflophenone 2-O-α-L-rhamnopyranoside (6), iriflophenone 3-C-β-D-glucoside (7) and iriflophenone 3,5-C-β-D-diglucopyranoside (8) from the most potent ethyl acetate fraction. The structures of these compounds were determined by extensive spectroscopic analyses, including IR, UV, ESIMS, HRESIMS, 1D and 2D NMR.     

Evaluation of the Vitek-MS™ system in the identification of Candida isolates from bloodstream infections

Background

Matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) represents a revolution in the identification of microorganisms of clinical interest. Many studies have confirmed the accuracy and fastness of this tool with routine strains.

Crystal Structure and Putative Mechanism of 3-Methylitaconate-Δ-isomerase from Eubacterium barkeri

3-Methylitaconate-Δ-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K_m but > 1000-fold lower k_cat. The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 Å. The asymmetric unit of the P2₁2₁2₁ unit cell with parameters a = 53.1 Å, b = 142.3 Å, and c = 228.4 Å contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an α/β-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Δ-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as thiolate) to deprotonate C-3 and (as thiol) to donate a proton to the methylene carbon atom of the resulting allylic carbanion. Interestingly, the active site of isopentenyl diphosphate isomerase type I also contains a cysteine that cooperates with glutamate rather than lysine. It has been proposed that the initial step in this enzyme is a protonation generating a tertiary carbocation intermediate.     

Synthesis and pharmacology of the isomeric methylheptyl-Δ-tetrahydrocannabinols

The synthesis of the 3-heptyl, and the eleven isomeric 3-methylheptyl-Δ⁸-tetrahydrocannabinols (3–7, R and S methyl epimers, and 8) has been carried out. The synthetic approach entailed the synthesis of substituted resorcinols, which were subjected to acid catalyzed condensation with trans-para-menthadienol to provide the Δ⁸-THC analogue. The 1′-, 2′- and 3′-methylheptyl analogues (3–5) are considerably more potent than Δ⁸-THC. The 4′-, 5′- and 6′-methylheptyl isomers (6–8) are approximately equal in potency to Δ⁸-THC.     

Molecular cloning of rat liver 3α-hydroxysteroid dehydrogenase and identification of structurally related proteins from rat lung and kidney

3α-Hydroxysteroid dehydrogenase and related enzymes play important roles in the metabolism of endogenous compounds including androgens, corticosteroid, prostaglandins and bile acids, as well as drugs and xenobiotics such as benzo(a)pyrene. Complementary DNA clones encoding 3α-hydroxysteroid dehydrogenase were isolated from a rat liver cDNA lambda gt11 expression library using monoclonal antibodies as probes. A full-length cDNA clone of 1286 base pairs contained an open reading frame encoding a protein of 322 amino acids with an estimated M(w) of 37 kD. When expressed in E. coli, the encoded protein migrated to the same position on SDS-polycrylamide gels as the enzyme in rat liver cytosols. The protein expressed in bacteria was highly active in androsterone oxidation in the presence of NAD as cofactor and this activity was inhibited by indomethacin, a potent inhibitor of 3α-hydroxysteroid dehydrogenase. The predicted amino acid sequence of 3α-hydroxysteroid d dehydrogenase was related to sequences of several other aldo-keto reductases such as bovine prostaglandin F synthase, human chlordecone reductase, human aldose reductase, human aldehyde reductase and frog lens epsilon-crystallin, suggesting that these proteins belong to the same gene family. Recently, we have found that monoclonal antibodies against 3α-hydroxysteroid dehydrogenase also recognized multiple antigenically related proteins in rat lung, kidney and testis. Further screening of liver, lung and kidney cDNA libraries using these monoclonal antibodies as probes resulted in the isolation of additional five different cDNAs encoding proteins with high degree of structural homology to rat liver 3α-hydroxysteroid dehydrogenase.     

Specificity of the Yeast Rev3Δ Antimutator and Rev3 Dependency of the Mutator Resulting from a Defect (Rad1Δ) in Nucleotide Excision Repair

The yeast REV3 gene has been predicted to encode a DNA polymerase specializing in translesion synthesis. This polymerase likely participates in spontaneous mutagenesis, as rev3 mutants have an antimutator phenotype. Translesion synthesis also may be necessary for the mutator caused by a RAD1 (nucleotide excision repair) deletion (rad1Δ). To further examine the role of REV3 in spontaneous mutagenesis, we characterized SUP4-o mutations that arose spontaneously in strains having combinations of normal or mutant REV3 and RAD1 alleles. The largest fraction of the rev3Δ-dependent mutation rate decrease was observed for single base-pair substitutions and deletions, although the rates of all mutational classes detected in the RAD1 background were reduced by at least 30%. Interestingly, inactivation of REV3 was associated with a doubling of the number of sites at which the retrotransposon Ty inserted. rev3Δ also greatly diminished the magnitude of the rad1Δ mutator, but not to the rev3Δ antimutator level, implicating REV3-dependent and independent processes in the rad1Δ mutator effect. However, the specificity of the rev3Δ antimutator suggested that the same REV3-dependent processes gave rise to the majority of spontaneous mutations in the RAD1 and rad1Δ strains.     

Steroids from aquatic organisms—XII. Sterols from the antarctic sponge Homaxinella balfourensis (Ridley and Dendy)

• 1.1. The sterol composition of the sponge Homaxinella balfourensis (Ridley and Dendy) has been analysed and seven components detected.
• 2.2. These were separated by argentic column chromatography and studied by gas chromatography-mass spectrometry and by proton magnetic resonance spectroscopy.
• 3.3. It was established that the components were C₂₇, C₂₈ and C₂₉ fully saturated or side chain unsaturated stanols and colest-5-en-3β-ol as traces.

     

Novel photoreception system in sponges? Unique transmission properties of the stalk spicules from the hexactinellid Hyalonemasieboldi

Sponges (phylum Porifera) of the classes Hexactinellida and Demospongiae possess a skeleton composed of siliceous spicules, which are synthesized enzymatically. The longest spicules are found among the Hexactinellida, with the stalk spicules (length: 30 cm; diameter: 300 microm) of Hyalonema sieboldi as prominent examples. These spicules are constructed around a central axial filament, which is formed by approximately 40 siliceous layers. The stratified spicules function as optical glass fibers with unique properties. If free-spaced coupled with a white light source (WLS), the entire fiber is illuminated. Special features of the light transmission: (i) only wavelengths between 615 and 1310 nm can pass through the fibers and (ii) light below wavelengths of 615 nm and above 1310 nm is completely cut-off. The transmission efficiency is around 60% (measured at 1080-1100 nm [length of the fiber: 5 cm]). The spicules acts as sharp high- and low-pass filters, suggesting that these silica-based fibers might be involved in a photoreception system. This assumption is supported by the finding that sponges are provided with a bioluminescent system. It is hypothesized that the spicules/siliceous fibers might be involved in a photoreception system in these animals.     

Bioactivity-guided identification of flavonoids with cholinesterase and β-amyloid peptide aggregation inhibitory effects from the seeds of Millettia pachycarpa

Millettia pachycarpa Benth, a widely used anthelminthic drug in folk, is rich in flavonoids with various bioactivities. This study aimed to identify active flavonoids with anti-Alzheimer’s disease (AD) effect from its seeds by a bioassay-guided isolation. A novel rotenoid with unusual oxidative ring-opening skeleton (10) and nine known flavonoids (1–9) were obtained, and their structures were elucidated by NMR and HR-ESIMS analysis. Among all isolates, 7 and 8 showed selective butyrylcholinesterase (BChE) inhibitory activities (IC₅₀?=?2.34 and 11.49?μM, respectively), while 3 was classified as a dual-action inhibitor against acetylcholinesterase (AChE) and BChE (IC_{50 AChE}?=?17.14?μM, IC_{50 BChE}?=?5.68?μM). Further kinetic study revealed that 3, 7, and 8 were mixed-type BChE inhibitors, but 3 was a competitive AChE inhibitor. Their strong binding affinities to BChE were confirmed by fluorescence quenching analysis. Additionally, 3 and 8 exhibited potent inhibitory effects against β-amyloid peptide aggregation. These results revealed M. pachycarpa could be a valuable source for anti-AD leads development, and compounds 3, 7 and 8 were worthy of further study as multifunctional or specific agents for AD treatment.     

Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata,identification and specificity of a branching β1→6 galactosyltransferase

Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-³H]-uridine 5diphosphogalactose the incorporation of labelled d-galactose into the polysaccharide can be traeed in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltrasferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient -(16) galactosyltransferase was detected when methyl 3-O-(-d-galactopyranosyl)--d-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by ¹H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked 13. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer d-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(-d-galactopyranosyl)-d-galactopyranose. but without any for methyl-3-O-(-d-galactopyranosyl)--d-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan.Abbreviation Gal galactose - glc gas-liquid chromatography - Gro glycerol - tlc thin layer chromatography     

A novel olefinic rearrangement. The enzymic conversion of cholesta-7,9-dien-3β-ol into cholesta-8,14-dien-3β-ol

1. [3alpha-(3)H]Cholesta-7,9-dien-3beta-ol is converted in high yield into cholesterol by a 10000g(av.) supernatant fraction of rat liver homogenate. 2. Incubation of cholesta-7,9-dien-3beta-ol with [4-(3)H]NADPH and rat liver microsomal fractions under anaerobic conditions resulted in (3)H being incorporated into the 14alpha-position of cholest-7-en-3beta-ol. 3. Under anaerobic conditions in the absence of NADPH cholesta-7,9-dien-3beta-ol was isomerized into cholesta-8,14-dien-3beta-ol by rat liver microsomal fractions.