Endophytic fungi found in association with Smallanthus sonchifolius (Asteraceae) as resourceful producers of cytotoxic bioactive natural products

Smallanthus sonchifolius is a traditional Andean plant which has been cultured mainly in Brazil, Japan and New Zealand due to its medicinal properties. A study of the endophytic fungi associated to the plant was carried out in order to characterize new cytotoxic agents. Thirty two fungal strains were isolated and submitted to cultivation and extraction producing 186 extracts. Of these, 12% displayed moderate to high cytotoxic activities and were considered promising anticancer compound sources. The ethyl acetate fractions of Nigrospora sphaerica and Phoma betae liquid fermentations contained the synergistic compounds 8-hydroxy-6-methoxy-3-methylisocoumarin and (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one which are potential compounds for drug discovery. Another isolated compound, pimara-7,15-dien-3-beta-ol diterpene is being characterized for the first time through a detailed spectroscopic analysis including GC/MS, homo- and hetero-nuclear correlated NMR experiments (HMQC, HMBC, COSY and NOEdiff) along with its optical rotation.

Bioactive Chemical Constituents and Comparative Antimicrobial Activity of Callus Culture and Adult Plant Extracts from Alternanthera tenella

Crude extracts of a callus culture (two culture media) and adult plants (two collections) from Alternanthera tenella Colla (Amaranthaceae) were evaluated for their antibacterial and antifungal activity, in order to investigate the maintenance of antimicrobial activity of the extracts obtained from plants in vivo and in vitro. The antibacterial and antifungal activity was determined against thirty strains of microorganisms including Gram-positive and Gram-negative bacteria, yeasts and dermatophytes. Ethanolic and hexanic extracts of adult plants collected during the same period of the years 1997 and 2002 [Ribeirao Preto (SP), collections 1 and 2] and obtained from plant cell callus culture in two different hormonal media (AtT43 and AtT11) inhibited the growth of bacteria, yeasts and dermatophytes with inhibition halos between 6 and 20 mm. For the crude extracts of adult plants bioassay-guided fractionation, purification, and isolation were performed by chromatographic methods, and the structures of the isolated compounds were established by analysis of chemical and spectral evidences (UV, IR, NMR and ES-MS). Steroids, saponins and flavonoids (aglycones and C-glycosides) were isolated. The minimum inhibitory concentration (MIC) of the isolated compounds varied from 50 to 500 mu g/mL.

Molecular Sieve Silica (MSS) Membranes for Gas Separation and Reaction Processes

Weakly branched silica films formed by the two-step sol-gel process allow for the formation of high selectivity membranes for gas separation. 29Si NMR and gas permeation showed that reduced crosslinking leads to He/CH4 selectivity improvement from 300 to 1000. Applied in membrane reactor for cyclohexane conversion to benzene, conversions were achieved at 14 fold higher than a conventional reactor at 250°C. Hydrothermal stability studies showed that carbon templating of silica is required for hydrothermally stable membranes. From our work it was shown that with correct application of chemistry, practical membrane systems can be built to suit gas separation (e. g. hydrogen fuel) and reactor systems.

Hydrothermal Stability of Modified Silica Membranes for Gas Separation

A new class of hybrid molecular sieve silica (MSS) membranes is developed and tested against standard and organic templated membranes. The hybrid membrane is synthesized by the standard sol-gel process, integrating a template (methyltriethoxysilane - MTES) and a C6 surfactant (triethylhexylammonium bromide) into the silica film matrix. After hydro treatment under a relative humidity of 96% for 50h, the hybrid membrane shows no changes in its gas separation capabilities or energy of mobility. The structural characteristics and integrity of the hybrid membrane are retained due to a high concentration of organophilic functional groups and alkoxides observed using 29 Si NMR. In contrast, the structural integrity of the membranes prepared with non-templated films deteriorated during the hydro treatment due to a large percentage of silanol groups (Si-OH) which react with water. The hybrid membranes underwent a decrease in the H2/CO2 selectivity of only 1% whereas for the non-templated membrane a 21% decrease was observed. The transport mechanism of the hybrid membranes is activated as permeation increased with temperature. The activation energy for the permeation of H2 is positive while negative for CO2. The H2 permeation obtained was 3x 10 -8 mol.m -2 .s -1 .Pa -1 and permselectivities for H2/CO2 and H2/N2 varied between 1-7 and 31-34, respectively.

Fast Structure-Based Assignment of 15N HSQC Spectra of Selectively 15N-Labeled Paramagnetic Proteins

A novel strategy for fast NMR resonance assignment of N-15 HSQC spectra of proteins is presented. It requires the structure coordinates of the protein, a paramagnetic center, and one or more residue-selectively N-15-labeled samples. Comparison of sensitive undecoupled N-15 HSQC spectra recorded of paramagnetic and diamagnetic samples yields data for every cross-peak on pseudocontact shift, paramagnetic relaxation enhancement, cross-correlation between Curie-spin and dipole-dipole relaxation, and residual dipolar coupling. Comparison of these four different paramagnetic quantities with predictions from the three-dimensional structure simultaneously yields the resonance assignment and the anisotropy of the susceptibility tensor of the paramagnetic center. The method is demonstrated with the 30 kDa complex between the N-terminal domain of the epsilon subunit and the theta subunit of Escherichia Coll DNA polymerase III. The program PLATYPUS was developed to perform the assignment, provide a measure of reliability of the assignment, and determine the susceptibility tensor anisotropy.

Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus

The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, has been determined from 2D H-1 NMR data, Robustoxin is a polypeptide of 42 residues cross-linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1-15, 8-20, 14-31 and 16-42 (a 1-4/2-6/3-7/5-8 pattern), The structure consists of a small three-stranded, anti-parallel beta-sheet and a series of interlocking gamma-turns at the C-terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the omega-conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non-polar residues, Both of these structural features may play a role in its binding to the voltage-gated sodium channel. (C) 1997 Federation of European Biochemical Societies.

Solution structure and proposed binding mechanism of a novel potassium channel toxin kappa-conotoxin PVIIA

Background: kappa-PVIIA is a 27-residue polypeptide isolated from the venom of Conus purpurascens and is the first member of a new class of conotoxins that block potassium channels. By comparison to other ion channels of eukaryotic cell membranes, voltage-sensitive potassium channels are relatively simple and methodology has been developed for mapping their interactions with small-peptide toxins, PVIIA, therefore, is a valuable new probe of potassium channel structure. This study of the solution structure and mode of channel binding of PVIIA forms the basis for mapping the interacting residues at the conotoxin-ion channel interface. Results: The three-dimensional structure of PVIIA resembles the triple-stranded beta sheet/cystine-knot motif formed by a number of toxic and inhibitory peptides. Subtle structural differences, predominantly in loops 2 and 4, are observed between PVIIA and other conotoxins with similar structural frameworks, however. Electrophysiological binding data suggest that PVIIA blocks channel currents by binding in a voltage-sensitive manner to the external vestibule and occluding the pore, Comparison of the electrostatic surface of PVIIA with that of the well-characterised potassium channel blocker charybdotoxin suggests a likely binding orientation for PVIIA, Conclusions: Although the structure of PVIIA is considerably different to that of the alpha K scorpion toxins, it has a similar mechanism of channel blockade. On the basis of a comparison of the structures of PVIIA and charybdotoxin, we suggest that Lys19 of PVIIA is the residue which is responsible for physically occluding the pore of the potassium channel.

The crosslinking mechanism in gamma irradiation of polyarylsulfone: Evidence for Y-links

Measurements of molecular weights, soluble fractions and examination of NMR spectra of bisphenol-A polysulfone, after gamma irradiation in vacuum at 150 degrees C were used to elucidate the mechanism of crosslinking. Excellent agreement was obtained between G(S) and G(X) determined from measurements above and below the gel dose when a Y-linking mechanism was assumed, whereas poor agreement was obtained when an H-link mechanism was assumed, which is the mechanism normally believed to be responsible for crosslinking. New resonances were observed in the C-13 NMX spectra of the irradiated polymer which were consistent with the formation of Y-links involving phenylene units in the backbone chain. (C) 1998 John Wiley & Sons, Ltd.

Calcium is essential for the structural integrity of the cysteine-rich, ligand-binding repeat of the low-density lipoprotein receptor

Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. in the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional H-1 NMR spectral studies: only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.

Modification of MCM-41 by surface silylation with trimethylchlorosilane and adsorption study

Siliceous MCM-41 samples were modified by silylation using trimethylchlorosilane (TMCS). The surface coverage of functional groups was studied systematically in this work. The role of surface silanol groups during modification was evaluated using techniques of FTIR and Si-29 CP/MAS NMR. Adsorption of water and benzene on samples of various hydrophobicities was measured and compared. It was found that the maximum degree of surface attachments of trimethylsilyl (TMS) groups was about 85%, corresponding to the density of TMS groups of 1.9 per nm(2). The degree of silylation is found to linearly increase with increasing pre-outgassing temperature prior to silylation. A few types of silanol groups exist on MCM-41 surfaces, among which both free and geminal ones are responsible for active silylation. Results of water adsorption show that aluminosilicate MCM-41 materials are more or less hydrophilic, giving a type IV isotherm, similar to that of nitrogen adsorption, whereas siliceous MCM-41 are hydrophobic, exhibiting a type V adsorption isotherm. The fully silylated Si-MCM-41 samples are more hydrophobic giving a type III adsorption isotherm. Benzene adsorption on all MCM-41 samples shows type IV isotherms regardless of the surface chemistry. Capillary condensation occurs at a higher relative pressure for the silylated MCM-41 than that for the unsilylated sample, though the pore diameter was found reduced markedly by silylation. This is thought attributed to the diffusion constriction posed by the attached TMS groups. The results show that the surface chemistry plays an important role in water adsorption, whereas benzene adsorption is predominantly determined by the pore geometry of MCM-41.

Carrageenans with complex substitution patterns from red algae of the genus Erythroclonium

Extracellular polysaccharides from three Erythroclonium spp. were shown, by a combination of compositional, linkage analyses, and Fourier transform infrared and C-13-nuclear magnetic resonance spectroscopy, to be highly substituted carrageenans with at least five types of repeating disaccharide units. These are the carrabiose 2,4'-disulfate of iota-carrageenan, carrabiose 2-sulfate of alpha-carrageenan, the 6'-O-methylated counterparts of each of these repeating units, and 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate. The polysaccharides also contain significant amounts of unsubstituted, 4-linked galactopyranose and small amounts of 4-linked 3-O-methylgalactopyranose and terminal glycosyl residues. The carrageenan preparations of the three species are similar, differing only in the proportions of some components. (C) 1998 Elsevier Science Ltd.

Ester prodrugs of a potent analgesic, morphine-6-sulfate: syntheses, spectroscopic and physicochemical properties

The aim of this work is to develop 3-acyl prodrugs of the potent analgesic morphine-6-sulfate (M6S). These are expected to have higher potency and/or exhibit longer duration of analgesic action than the parent compound. M6S and the prodrugs were synthesized, then purified either by recrystallization or by semi-preparative HPLC and the structures confirmed by mass spectrometry, IR spectrophotometry and by detailed 1- and 2-D NMR studies. The lipophilicities of the compounds were assessed by a combination of shake-flask, group contribution and HPLC retention methods. The octanol-buffer partition coefficient could only be obtained directly for 3-heptanoylmorphine-6-sulfate, using the shake-flask method. The partition coefficients (P) for the remaining prodrugs were estimated from known methylene group contributions. A good linear relationship between log P and the HPLC log capacity factors was demonstrated. Hydrolysis of the 3-acetyl prodrug, as a representative of the group, was found to occur relatively slowly in buffers (pH range 6.15-8.01), with a small buffer catalysis contribution. The rates of enzymatic hydrolysis of the 3-acyl group in 10% rat blood and in 10% rat brain homogenate were investigated. The prodrugs followed apparent first order hydrolysis kinetics, with a significantly faster hydrolysis rate found in 10% rat brain homogenate than in 10% rat blood for all compounds. (C) 1998 Elsevier Science B.V. All rights reserved.

Structure and distribution of N-glycans on the S-7-allele stylar self-incompatibility ribonuclease of Nicotiana alata

S-RNases are the stylar products of the self-incompatibility (S)-locus in solanaceous plants (including Nicotiana alata), and as such, are involved in the prevention of self-pollination. All cDNA sequences of S-RNase products of functional S-alleles contain potential N-glycosylation sites, with one site being conserved in all cases, suggesting that N-glycosylation is important in self-incompatibility. In this study, we report on the structure and localization of the N-glycans on the S-7-allele RNase of N, alata, A total of nine N-glycans, belonging to the high-mannose- and xylosylated hybrid-classes, were identified and characterized by a combination of electrospray-ionization mass-spectrometry (ESI-MS), H-1-NMR spectroscopy, and methylation analyses. The glycosylation pattern of individual glycosylation sites was determined by ESI-MS of the glycans released from isolated chymotryptic glycopeptides, All three N-glycosylation sites showed microheterogeneity and each had a unique complement of N-glycans, The N-glycosylation pattern of the S-7-RNase is significantly different to those of the S-1- and S-2-RNases.

Structure determination of the three disulfide bond isomers of alpha-conotoxin GI: A model for the role of disulfide bonds in structural stability

The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by H-1 NMR spectroscopy. alpha-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2-7;3-13), GI(2-13;3-7) and GI(2-3;7-13). The NMR structure for the native isomer GI(2-7;3-13) is of excellent quality, with a backbone pairwise RMSD of 0.16 Angstrom for a family of 35 structures, and comprises primarily a distorted 3(10),, helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2-7;3-13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in alpha-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2-7;3-13) < GI(2-13;3-7) < GI(2-3;7-13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2-3;7-13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms. A model for the binding of native GI(2-7;3-13) to the mammalian nAChR is proposed, with an alpha-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the alpha and delta subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2-7;3-13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity. (C) 1998 Academic Press Limited.

Stage-specific proteophosphoglycan from Leishmania mexicana amastigotes - Structural characterization of novel mono-, di-, and triphosphorylated phosphodiester-linked oligosaccharides

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as H-1 and P-31 NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase, However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glc beta(1-3)](1-2)Gal beta 1-4Man, Gal beta 1-3Gal beta 1-4Man, Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta 1-4Man) but carrying stage-specific modifications (R = Gal beta 1-, [Glc beta 1-3](1-2)Glc beta 1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glc beta 1-3Gal beta 1-4Man, PO4-6-Glc beta 1-3Glc beta 1-3Gal beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3 [PO4-6-Gal]beta 1-4Man, PO4-6-GaL beta 1-3Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6Gal beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, and PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man. These glycans are linked together by the conserved phosphodiester R-Man alpha 1-PO4-6-Gal-R or the novel phosphodiester R-Man alpha 1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Man alpha 1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.