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Many species of the gastropod genus Philine have been named from northeastern Asia but scanty descriptions based predominantly on shells make it difficult to determine which are valid. This, plus the sporadic anatomical and genetic information available for many of these species has led to what may be described as an un-integrated taxonomy. In this situation, it is generally preferable to postpone dissection of rare and unusual specimens until relevant diagnostic characters can be established in broader studies. Micro-CT scanning and DNA sequencing were used to examine such a specimen collected recently from deep waters off northeastern Taiwan. Micro-CT examination of the morphology of the internal shell and gizzard plates suggested that, among named species, the sequenced specimen is most similar to P. otukai. It cannot, however, be definitively referred to P. otukai as that species lacks adequate anatomical description or known DNA sequences. Phylogenetic analyses of newly collected DNA sequences show the specimen to be most closely related to, but distinct from the northern Atlantic Ocean and Mediterranean species, Philine quadripartita. The sequences also confirm genetically that five or more species of Philine occur in northeast Asia, including at least three subject to considerable taxonomic uncertainty.

Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are the foremost causative agents of malaria. Due to the development of resistance to current antimalarial medications, new drugs for this parasitic disease need to be discovered. The activity of hypoxanthine-guanine-[xanthine]-phosphoribosyltransferase, HG[X]PRT, is reported to be essential for the growth of both of these parasites, making it an excellent target for antimalarial drug discovery. Here, we have used rational structure-based methods to design an inhibitor, [3R,4R]-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine, of PvHGPRT and PfHGXPRT that has Ki values of 8 and 7 nM, respectively, for these two enzymes. The crystal structure of PvHGPRT in complex with this compound has been determined to 2.85 A resolution. The corresponding complex with human HGPRT was also obtained to allow a direct comparison of the binding modes of this compound with the two enzymes. The tetra-(ethyl l-phenylalanine) tetraamide ...

Bruising of the mesocarp in avocado fruit is an important postharvest issue for the industry. Proton magnetic resonance imaging (H-1-MRI) was used as a non-destructive tool to monitor bruise expression over time in avocado cv. Hass fruit. H-1-MRI clearly identified fruit morphological features and bruised mesocarp tissue. The pixel intensity value of T-2 weighted spin echo H-1-MRI images of avocado fruit pericarp changed over time with fruit softening. Bruised mesocarp tissue in impacted fruit appeared relatively hyperintense (brighter) in T-2 weighted H-1-MRI images. For firm ripe fruit impacted from 25 cm drop height (0.38 J +/- 0.004) and for firm ripe fruit impacted from 50 cm drop height (0.81 J +/- 0.011), hyper-intensity in the mesocarp beneath the impact point was evident immediately after impact. However, visible symptoms of bruising in the form of flesh browning did not appear in parallel serial destructive assessments until after day 1 following impact on day 0. The brown, bruised mesocarp volume in ripe fruit increased progressively over the assessment period of 3 days. This trend was evident in destructive assessments as well as in H-1-MRI images. In hard green mature fruit impacted from 100 cm drop height (1.68 J +/- 0.020), contrast between mesocarp tissue beneath the impact site and surrounding sound mesocarp was evident in T-2 weighted H-1-MRI images from day 0. However, no bruise symptoms were evident as flesh browning upon serial destructive assessments of fruit over the 3 days assessment period. The average pixel intensity values at the impact site in T-2 weighted H-1-MRI images for both firm ripe and hard green mature fruit decreased over the period of assessment. In contrast, the pixel intensities in the T-2 weighted H-1-MRI images of diseased flesh increased over time. (C) 2014 Elsevier B.V. All rights reserved.

Principal component analysis of 1H NMR spectra of dichloromethane extracts taken from grapevine leaves reveals that phylloxera infestation of the root system causes metabolic changes in the leaves of infested grapevines, both in the field and in the glasshouse. A number of potential markers of phylloxera infestation were detected but their presence is transient and varies with the stage of the growing season. The changes in the metabolic profile caused by phylloxera infestation more closely resemble those caused by nitrogen deficiency than those induced by water stress. A reduction in the ratio of linoleic acid to linolenic acid in the triglyceride component of the leaf extract may provide an indicator of phylloxera infestation.

The malarial parasite Plasmodium falciparum depends on the purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) to convert purine bases from the host to nucleotides needed for DNA and RNA synthesis. An approach to developing antimalarial drugs is to use HGXPRT to convert introduced purine base analogs to nucleotides that are toxic to the parasite. This strategy requires that these compounds be good substrates for the parasite enzyme but poor substrates for the human counterpart, HGPRT. Bases with a chlorine atom in the 6-position or a nitrogen in the 8-position exhibited strong discrimination between P. falciparum HGXPRT and human HGPRT. The k(cat)/K(m) values for the Plasmodium enzyme using 6-chloroguanine and 8-azaguanine as substrates were 50 - 80-fold and 336-fold higher than for the human enzyme, respectively. These and other bases were effective in inhibiting the growth of the parasite in vitro, giving IC(50) values as low as 1 microM.

Human hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the synthesis of the purine nucleoside monophosphates, IMP and GMP, by the addition of a 6-oxopurine base, either hypoxanthine or guanine, to the 1-beta-position of 5-phospho-U-D-ribosyl-1-pyrophosphate (PRib-PP). The mechanism is sequential, with PRib-PP binding to the free enzyme prior to the base. After the covalent reaction, pyrophosphate is released followed by the nucleoside monophosphate. A number of snapshots of the structure of this enzyme along the reaction pathway have been captured. These include the structure in the presence of the inactive purine base analogue, 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and PRib-PP. Mg2+, and in complex with IMP or GMP. The third structure is that of the immucillinHP.Mg2+.PPi complex, a transition-state analogue. Here, the first crystal structure of free human HGPRT is reported to 1.9 angstrom resolution, showing that significant conformational changes have to occur for the substrate(s) to bind and for catalysis to proceed. Included in these changes are relative movement of subunits within the tetramer, rotation and extension of an active-site alpha-helix (D137-D153), reorientation of key active-site residues K68, D137 and K165, and the rearrangement of three active-site loops (100-128, 165-173 and 186-196). Toxoplasina gondii HGXPRT is the only other 6-oxopurine phosphoribosyltransferase structure solved in the absence of ligands. Comparison of this structure with human HGPRT reveals significant differences in the two active sites, including the structure of the flexible loop containing K68 (human) or K79 (T gondii). (c) 2005 Elsevier Ltd. All rights reserved.

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K(m) toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K(m) was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K(d) for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.

The sixth ligand-binding module of the low-density lipoprotein receptor contributes to the binding of apolipoprotein B100-containing lipoproteins. 1H NMR spectroscopy, DYANA and X-PLOR structure calculations were used to determine that this module has a well defined structure with a backbone conformation similar to other modules. Structures from calculations that simulated the presence of a calcium ion showed increased resolution without large increases in energy, increased deviations from idealised geometry or violations of experimental constraints. Investigation of the surface properties of this module indicates there are significant differences from the fifth module, which binds apolipoprotein E-containing lipoproteins in addition to apolipoprotein B100-containing lipoproteins.

Lepidozenal and isobicyclogermacrenal were isolated from the leaves of Eucalyptus dawsonii and a complete assignment of their 1H and 13C NMR spectra was carried out using 2D NMR methods. Copyright © 2007 John Wiley & Sons, Ltd.

The structures of haliclonacyclamines A (1) and B (2), and their methiodide salts (3) and (4), were investigated by 1D- and 2D-NMR experiments, notably DQFCOSY, HMBC, HMQC-HOHAHA, and HOHAHA. The relative stereochemistry and position of alkene substituents were determined by single crystal x-ray study at low temperature. The parent haliclonacyclamines show pronounced cytotoxic, antibacterial and antifungal activity.

R. J. Lewis, R. S. Norton, I. M. Brereton and C. D. Eccles . Ciguatoxin-2 is a diastereomer of ciguatoxin-3. Toxicon 31, 637–643, 1993.—Ciguatoxin-2, a major ciguatoxin present in the flesh and viscera of ciguateric fishes, has been shown by 1H nuclear magnetic resonance studies (2-dimensional homonuclear Hartman Hahn, nuclear Overhauser effect and decoupling difference experiments) to be a diastereomer of ciguatoxin-3, differing only in stereochemistry at carbon 52 (a quaternary carbon). This difference accounts for the significant changes in the chemical shift of resonances for protons in this region of ciguatoxin-2. Differences between ciguatoxin-1, -2 and -3 involve modifications at only one end of the ciguatoxins (ring M) and modest differences in potency, indicating that this ring contributes to, but is not critical for, high affinity binding of the ciguatoxins to voltage-dependent sodium channels. It is proposed that ciguatoxin-2 originates from a different precursor to the precursor (presumably gambiertoxin-4b) for ciguatoxin-1 and -3, and that both precursors are produced by a common biosynthetic pathway in Gambierdiscus toxicus .

Caribbean ciguatoxins (C-CTXs) are responsible for the widespread occurrence of ciguatera in the Caribbean Sea. The structure and configuration of C-CTX-1 (1), the major ciguatoxin isolated from the horse-eye jack (Caranx latus), has been determined from DQF-COSY, E-COSY, TOCSY, NOESY, POESY, ge-HSQC. and HMQC experiments performed at 750 MHz and 500 MHz on a 0.13 pmol sample. C-CTX-1 ([M + H](+) m/z 1141.6 Da, molecular formula C62H92O19) has a ciguatoxin/breveroxin ladder structure comprising 14 trans-fused, ether-linked rings (7/6/6/7/8/9/7/6/8/6/7/6/7/6) assembled fi um 6 protonated fragments. The relative stereochemistry and ring configuration of 1 was determined from an analysis of coupling constant and NOE data. Like ciguatoxins in the Pacific Ocean (P-CTX), C-CTX-1 possesses a flexible nine-membered ring which may be a conserved feature among ciguatoxins. However, C-CTX-1 has a longer contiguous carbon backbone (57 vs 55 carbons for P-CTX-1), one extra ring, and a hemiketal in ring N but no spiroketal as found in P-CTX. C-CTX-1 possesses a primary hydroxyl which may allow selective derivatization. A minor analogue, C-CTX-2, was also isolated from fish and assigned the structure 56 epi-C-CTX-1 (2). since it slowly rearranged to C-CTX-1 in solution. Given the structural similarities between Caribbean and Pacific ciguatoxins, we propose that C-CTX-1 and C-CTX-2 arise from a Caribbean strain of the benthic dinoflagellate, Gambierdiscus toxicus.

The malarial parasite, Plasmodium vivax (Pv), causes a serious infectious disease found primarily in Asia and the Americas. For protozoan parasites, 6-oxopurine phosphoribosyltransferases (PRTases) provide the only metabolic pathway to synthesize the purine nucleoside monophosphates essential for DNA/RNA production. We have purified the recombinant Pv 6-oxopurine (PRTase) and compared its properties with the human and Pf enzymes. The Pv enzyme uses hypoxanthine and guanine with similar catalytic efficiency to the Pf enzyme but xanthine is not a substrate, hence we identify this enzyme as PvHGPRT. Mass spectrometry suggests that PvHGPRT contains bound magnesium ions that are removed by EDTA resulting in loss of activity. However, the addition of Mg(2+) restores activity. Acyclic nucleoside phosphonates (ANPs) are good inhibitors of PvHGPRT having K(i) values as low as 3 microM. These compounds can form the basis for the design of new drugs aimed at combating malaria caused by Pv. ispartof: Molecular and Biochemical Parasitology vol:173 issue:2 pages:165-9 ispartof: location:Netherlands status: published

Different folivorous marsupials select their food from different subgenera of Eucalyptus, but the choices cannot be explained by known antifeedants, such as formylated phloroglucinol compounds or tannins, or by nutritional quality. Eucalypts contain a wide variety of plant secondary metabolites so it is difficult to use traditional methods to identify the chemicals that determine food selection. Therefore, we used a metabolomic approach in which we employed (1)H nuclear magnetic resonance spectroscopy to compare chemical structures of representatives from the two subgenera and to identify chemicals that consistently differ between them. We found that dichloromethane extracts of leaves from most species in the subgenus Eucalyptus differ from those in Symphyomyrtus by the presence of free flavanones, having no substitution in Ring B. Although flavanoids are known to deter feeding by certain insects, their effects on marsupials have not been established and must be tested with controlled feeding studies.

A cyclitol galactoside was isolated from the seeds of Vigna angularis and shown to be identical with galactosylononitol. However, detailed 2D NMR data and methylation analysis resulted in the revision of the structure of galactosylononitol to O-alpha-D-galactopyranosyl-(1-->3)-4-O-methyl-D-myo-inositol (1). Thus, compound 1 represents the only naturally occuring methylated derivative of galactinol.

A digalactosyl ononitol was isolated from seeds of adzuki bean (Vigna angularis [Willd.] Ohwi et Ohasi). Analysis of hydrolysis products and NMR spectroscopy established its structure as O-alpha-D-galactopyranosyl-(1-->6)-O-alpha-D-galactopyranosyl-(1-->3)-O-methyl- D-myo-inositol. (C) 2003 Elsevier Ltd. All rights reserved.

A novel furanoterpene (2), iso-dehydrodendrolasin, has been isolated from the tropical marine sponge Euryspongia deliculata and characterized by 2D NMR.

Chemical synthesis was used to prepare the HIV-1 protease specifically 13C-labelled in the catalytically essential Asp 25 in each monomer. The NMR chemical shift of the 13C-enriched homodimeric enzyme was measured in the presence of the inhibitor pepstatin, a mimic of the tetrahedral intermediate formed in enzyme catalysis. In this complex, the catalytic carboxyls do not titrate in the pH range where the enzyme is active; throughout the range pH 2.5-6.5, one Asp 25 side chain is protonated and the other deprotonated. By contrast, in the absence of inhibitor the two Asp side chains are chemically equivalent and both deprotonated at pH 6, the optimum for enzymatic activity. These direct observations of the chemical properties of the catalytic apparatus of the enzyme provide concrete information on which to base the design of improved HIV-1 protease inhibitors.

Two new polypropionates, caloundrin B (11), systematic name (1″R,2R,3″R,4R,5S,5″R,6S,-7″R,8″S,9″S,10″R)-2-(6′-ethyl-3′,5′-dimethyl-4′-oxopyran-2′-yl)-6-(5″-ethyl-7″-hydroxy-8″,9″,10″-trimethyl-2″,4″,6″-trioxatricyclo[3.3.1.13,7]dec-3″-yl)-5-hydroxy-4-methylheptan-3-one, and funiculatin A (12), systematic name (2ξ,1′S,4R,5′S,6′S)-2-(1′-ethyl-4′,6′,8′-trimethyl-2𔈀,9′-dioxabicyclo[3.3.1]nona-3′,7′-dien-3′-yl)-4,6-dimethylnon-6-en-3-one, have been isolated from Siphonaria zelandica and S. funiculata respectively. The structures of the new compounds were deduced by two-dimensional n.m.r. spectroscopy, particularly long-range 13C- 1H correlation spectroscopy (HMBC), by biosynthetic reasoning and by comparison with the known polypropionates denticulatin A (5), siphonarin B (8), muamvatin (9) and baconipyrone C (14). Information about the relative stereochemistry of the tricyclic ring system of (11) and the bicyclic ring system of (12) was deduced from coupling constant values, and by n.O.e. difference and NOESY experiments, and was confirmed by molecular modelling studies. The relative stereochemistry of the side chains and the absolute stereochemistry were inferred from biosynthetic comparison with the above known polypropionates, and by correlation of funiculatin A with denticulatin A (5) of known absolute stereochemistry. The stereochemistry at C10 of funiculatin A (position 2 of the non-6-en-3-one chain) could not be unambiguously determined. A third new polypropionate funiculatin B, epimeric with funiculatin A at C10, was isolated and partially characterized.

1H NMR studies of glycolysis in washed cell suspensions of Streptococcus faecalis indicated that intracellular lactate is not 1H NMR visible. Evidence for this was gained from time course studies of glycolysis at increasing concentrations of glucose. A close correlation existed between the relative increase in the lactate integral and the enzymatically determined extracellular lactate concentration [Lo]. When ionophores which cause the collapse of the positive intracellular/extracellular lactate gradient were added to cell suspensions following fermentation of 5, 10 and 50 mM glucose, the increase in the lactate integral was proportional to the respective increase in [Lo]. A more direct method for determining the origin of the lactate signal involved centrifugation of a cell suspension after fermentation of 50 mM glucose and measurement of lactate in the extracellular and intracellular fluid. 1H spectra of the cell suspension, supernatant and sonicated pellet revealed that the lactate observed in the cell suspension was equivalent to the lactate in the supernatant alone. The intracellular lactate contained in the pellet represented 42% of the total lactate, indicating that only 58% of lactate is detected by in vivo 1H MRS of S. faecalis. This result is in contrast with the high percentage (70-90%) of in vitro lactate which is detected by in vivo 1H MRS of mammalian brain tissue (Williams S. R. et al. Magn. Res. Med. 7, 425-431, 1988). This may be due to a higher proportion of extracellular lactate in mammalian tissue or differences in the intracellular environments of bacterial and mammalian cells.

An apparatus is described that facilitates the determination of incorporation levels of isotope labelled, gaseous precursors into volatile insect-derived metabolites. Atmospheres of varying gas compositions can be generated by evacuation of a working chamber followed by admission of the required levels of component gases, using a precision, digitised pressure read-out system. Insects such as fruit-flies are located initially in a small introduction chamber, from which migration can occur downwards into the working chamber. The level of incorporation of labelled precursors is continuously assayed by the Solid Phase Micro Extraction (SPME) technique and GC-MS analyses. Experiments with both Bactrocera species (fruit-flies) and a parasitoid wasp, Megarhyssa nortoni nortoni (Cresson) and oxygen-18 labelled dioxygen illustrate the utility of this system. The isotope effects of oxygen-18 on the carbon-13 NMR spectra of 1,7- dioxaspiro[5,5]undecane are also described.