Surface-induced synthesis of new sulfonolipids in the gliding bacterium Cytophaga johnsonae

Many simple gliding bacteria contain significant quantities of phosphate-free, sulfur-containing lipids (sulfonolipids; N-acylamino-3-hydroxyisoheptadecane-1-sulfonic acids, or N-acyl capnines) that recently were shown to function in the ability of Cytophaga johnsonae to migrate over solid surfaces. Reported here is the synthesis, by surface-grown Cytophaga johnsonae cells, of two additional sulfonolipids not present in cells grown in liquid media. These newly characterized sulfonolipids are more polar than the N-acylcapnines characteristic of liquid grown cells. Acid methanolysis of the sulfonolipids revealed that the aminosulfonate capnine was common to all, thus indicating that the chemical differences in the compounds resided in their N-fatty acyl groups, and not in the aminosulfonate moiety. Instead of the non-hydroxy and 3-hydroxy fatty acyl moieties present in sulfonolipids of liquid-grown cells, one new sulfonolipid contained a 2-hydroxy, branched C₁₅ fatty acid, while the other contained a 2,3-dihydroxy, isobranched C₁₆ fatty acid, as indicated by gas chromatographic and mass spectrometric analyses. Although the structure of sulfonolipids thus varies between surface- and liquid-grown cells, no difference was found between the total quantity of sulfonolipids present under either of these conditions. The surface-dependent synthesis of these more polar N-acyl-aminosulfonates ceased immediately when surface-grown populations were suspended in broth. The ability of Cytophaga johnsonae to synthesize these compounds in response to a solid surface may be significant in relation to the organism's ability to migrate over such surfaces; it is one of few instances where a physical interaction of the cell surface has been shown to influence the molecular composition of a prokaryote.Abbreviations LTY tryptone yeast extract medium - TLC thin layer chromatography - FAME fatty acid methyl ester - ECL equivalent chain length - T _r retention time - TMS trimethylsilyl - TFA trifluoroacetyl     

Purification of the N-acylaminosulfonates,unusual lipids of gliding bacteria

A group of unusual sulfonolipids that are major components of the cell envelope has been found in gliding bacteria of the genus Cytophaga and closely related genera. One of the lipids, capnine, has been previously isolated and shown to be 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid. Capnine accumulates in large quantities in some Capnocytophaga spp., but in other organisms the predominant sulfonolipids are compounds that are less polar than capnine and lack the free amino group of that compound. A method is described for the purification of these less polar lipids employing chromatography on hydroxylapatite and DEAE-cellulose in organic solvents. The sulfonolipids have been isolated in high yield and essentially pure from two Cytophaga spp., one Flexibacter sp. and one Capnocytophaga sp. Preliminary characterization of the lipids of infrared absorption spectroscopy, thin layer chromatography and other methods has shown them to be N-acylaminosulfonates, the aminosulfonate moiety of which is closely related (if not identical) to capnine.     

SL2-like spliced leader RNAs in the basal nematode Prionchulus punctatus: New insight into the evolution of nematode SL2 RNAs

Spliced-leader (SL) trans-splicing has been found in all molecularly characterized nematode species to date, and it is likely to be a nematode synapomorphy. Most information regarding SL trans-splicing has come from the study of nematodes from a single monophyletic group, the Rhabditida, all of which employ SL RNAs that are identical to, or variants of, the SL1 RNA first characterized in Caenorhabditis elegans. In contrast, the more distantly related Trichinella spiralis, belonging to the subclass Dorylaimia, utilizes a distinct set of SL RNAs that display considerable sequence diversity. To investigate whether this is true of other members of the Dorylaimia, we have characterized SL RNAs from Prionchulus punctatus. Surprisingly, this revealed the presence of a set of SLs that show clear sequence similarity to the SL2 family of spliced leaders, which have previously only been found within the rhabditine group (which includes C. elegans). Expression of one of the P. punctatus SL RNAs in C. elegans reveals that it can compete specifically with the endogenous C. elegans SL2 spliced leaders, being spliced to the pre-mRNAs derived from downstream genes in operons, but does not compete with the SL1 spliced leaders. This discovery raises the possibility that SL2-like spliced leaders were present in the last common ancestor of the nematode phylum.     

Induction of prophage and mutagenic effects by alkyl alkylaminosulfonates, ethyl aminosulfonate and alkyl methanesulfonates

Prophage induction and mutation by alkylaminosulfonates, ethyl aminosulfonate and alkyl methanesulfonates were examined comparatively. Prophage induction was carried out with a lysozyme lysis technique on the lysogenic strain Micrococcus lysodeikticus 53-40 (N5). The sulfonic ester derivatives show a slight lysogenic induction. At higher concentrations their toxicity seems to mask phage detection. Only methyl isopropylaminosulfonate and ethyl aminosulfonate exhibit no or negligible toxic effects, and with these compounds at higher concentrations a strong prophage induction is found. Alkyl sulfonate derivatives induce mutations in the tester strain of Salmonella typhimurium TA1535. Methyl methylaminosulfonate and ethyl N-methyl-N-2-chloroethyl aminosulfonate show a mutagenicity comparable to that of the well-known methyl methanesulfonate or ethyl methanesulfonate. With ethyl aminosulfonate, however, which does not show inactivation, no significant mutagenic effect was observed. DNA alterations were found in the polymerase-deficient strain E. coli P3478. The results of prophage induction and mutagenicity are compared and discussed.     

mmBCFA C17iso ensures endoplasmic reticulum integrity for lipid droplet growth

In eukaryote cells, lipid droplets (LDs) are key intracellular organelles that dynamically regulate cellular energy homeostasis. LDs originate from the ER and continuously contact the ER during their growth. How the ER affects LD growth is largely unknown. Here, we show that RNAi knockdown of acs-1, encoding an acyl-CoA synthetase required for the biosynthesis of monomethyl branched-chain fatty acids C15iso and C17iso, remarkably prevented LD growth in Caenorhabditis elegans. Dietary C17iso, or complex lipids with C17iso including phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol, could fully restore the LD growth in the acs-1RNAi worms. Mechanistically, C17iso may incorporate into phospholipids to ensure the membrane integrity of the ER so as to maintain the function of ER-resident enzymes such as SCD/stearoyl-CoA desaturase and DGAT2/diacylglycerol acyltransferase for appropriate lipid synthesis and LD growth. Collectively, our work uncovers a unique fatty acid, C17iso, as the side chain of phospholipids for determining the ER homeostasis for LD growth in an intact organism, C. elegans.     

Sulfonolipids of gliding bacteria. Structure of the N-acylaminosulfonates

Earlier (Godchaux, W., and Leadbetter, E. R. (1980) J. Bacteriol. 144, 592-602; (1983) J. Bacteriol. 153, 1238-1246) we demonstrated that an unusual class of sulfonolipids are major components of the cell envelope of gliding bacteria of the genus Cytophaga and of closely related genera. One of these lipids, to which we have assigned the trivial name capnine, was purified and was shown to be 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid (which might also be named as 1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Though capnine accumulates as such in the cells of some Capnocytophaga spp., most organisms of the Cytophaga-like genera contain, instead, sulfonolipids that are less polar than capnine. These less polar lipids have been purified from a Capnocytophaga sp., a marine Cytophaga sp., Cytophaga johnsonae, and a Flexibacter sp. Acid methanolysis of the lipids yielded both aminosulfonates and a collection of fatty acid methyl esters. The infrared absorption spectra of the lipids indicated that the fatty acids were in amide (and not ester) linkage to the aminosulfonates. In every instance, analysis by mass spectrometry and other methods revealed that most, if not all, of the aminosulfonates obtained by methanolysis were structurally identical to capnine (though small amounts of variants of that compound may be present in some cases). The less polar sulfonolipids are, therefore, predominantly N-fatty acyl capnines, 1-deoxy-1-sulfonic acid analogs of ceramides. The fatty acid methyl esters obtained from the lipids were heterogeneous, but in all cases were rich in hydroxylated fatty acyl groups, which constituted 66 to 95% of the total.     

Biosynthesis of a sulfonolipid in gliding bacteria

Gliding bacteria of the genus Cytophaga synthesize sulfonolipids (1,2) that contain capnine (1-deoxy-15-methylhexadecasphinganine-1-sulfonic acid). Studies of the incorporation of radiolabeled compounds by C. johnsonae show that cysteate is utilized preferentially to both cystine and inorganic sulfate as a precursor of capnine sulfur and to both cystine and serine as a precursor of carbons 1 and 2 of capnine. The results are consistent with a pathway in which capnine is formed by condensation of cysteate with a fatty acyl CoA. Cystine, added as the sole sulfur source in the presence of glucose, provides the sulfur but not the carbon for capnine. Hence, these cells form cysteate not by direct oxidation of cystine (or cysteine), but by transfer of its sulfur to a different carbon compound.     

Capnocytophaga spp. contain sulfonolipids that are novel in procaryotes.

A group of unusual sulfonolipids was found in bacteria of the genus Capnocytophaga. One of these lipids, to which we have assigned the trivial name capnine, was isolated in 98% pure form and was identified, by infrared absorption spectrometry, high-resolution mass spectrometry, and other methods, as 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid. Another lipid appears to be an N-acylated version of capnine; after acid hydrolysis, its sulfur was recovered in a form chromatographically indistinguishable from that of capnine. The new lipids are related structurally to sphingosine and the ceramides, respectively, but differ markedly from those compounds in important respects, notably the presence of the sulfonate group. Some Capnocytophaga strains accumulated mostly capnine, whereas others accumulated mostly N-acylcapnine. All seven strains examined were found to contain the new lipids, in amounts ranging from 7 to 16 mumol/g of cells (wet weight). The lipids were found in isolated cell envelopes, where they were present in amounts ranging up to 400 mg/g of envelope protein; they are, accordingly, major cell components.     

Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey

The present study was conducted to identify and characterize the thermophilic bacteria isolated from various hot springs in Turkey by using phenotypic and genotypic methods including fatty acid methyl ester and rep-PCR profilings, and 16S rRNA sequencing. The data of fatty acid analysis showed the presence of 17 different fatty acids in 15 bacterial strains examined in this study. Six fatty acids, 15:0 iso, 15:0 anteiso, 16:0, 16:0 iso, 17:0 iso, and 17:0 anteiso, were present in all strains. The bacterial strains were classified into three phenotypic groups based on fatty acid profiles which were confirmed by genotypic methods such as 16S rRNA sequence analysis and rep-PCR genomic fingerprint profiles. After evaluating several primer sets targeting the repetitive DNA elements of REP, ERIC, BOX and (GTG)₅, the (GTG)₅ and BOXA1R primers were found to be the most reliable technique for identification and taxonomic characterization of thermophilic bacteria in the genera of Geobacillus, Anoxybacillus and Bacillus spp. Therefore, rep-PCR fingerprinting using the (GTG)₅ and BOXA1R primers can be considered as a promising genotypic tool for the identification and characterization of thermophilic bacteria from species to strain level.     

Structural characterization of eight cyclic lipopeptides produced by Bacillus subtilis HSO121

Lipopeptides are amphiphilic compounds which contain both hydrophobic fatty acid moieties and amphiphilic peptide moieties. From the cell-free broth of Bacillus subtilis HSO121, eight cyclic lipopeptides were isolated by reversed-phase high performance liquid chromatography (RP-HPLC). The peptide part of each lipopeptide was elucidated according to electrospray ionization quadruple-time-of-flight mass spectrometry (ESI Q-TOF MS) and the fatty acid part was analyzed by electroionization gas chromatography/mass spectrometry (EI GC/MS). It showed that fractions 1-8 had molecular masses of 1007, 1021, 1021, 1035, 1035, 1035, 1063, and 1049, respectively. Analysis of hydrolyzed lipopeptides revealed that they had invariant amino acid compositions. The differences in molecular weights represent changes in the number of methylene groups and different types of branched chains in fatty acids. Peptide sequences of two of the eight lipopeptides appeared to be N-Asp-Leu-Leu-Val-Glu-Leu-Leu-C, which was different from previously reported lipopeptides. The remaining six had an identical peptide sequence of N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C. The fatty acid parts were found to be mixtures of iso C(12), iso C(13), anteiso C(13), iso C(14), n C(14), iso C(15), anteiso C(15), n C(15), anteiso C(16) and anteiso C(17) beta-hydroxy fatty acids. The structure of each lipopeptide was determined to be the beta-hydroxy fatty acid bonded to the peptide chain.     

Identification of Fatty Acids and Aliphatic Hydrocarbons in Sarcina lutea by Gas Chromatography and Combined Gas Chromatography-Mass Spectrometry

The composition and nature of the fatty acids and hydrocarbons of Sarcina lutea were elucidated by gas chromatography and by combined gas chromatography-mass spectrometry. The distribution of fatty acids found in S. lutea showed two families of pairs, or dyads, of saturated monocarboxylic acids (C12-C18) with and without methyl branching. These pairs of fatty acids showed a pattern of iso and anteiso structures for C13, C15, and C17, and iso and normal structures for C12, C14, and C16. Only the C18 showed unsaturation. The distribution of hydrocarbons in the range C22-C29 showed two families of tetrads of unsaturated aliphatic hydrocarbons all showing methyl branching. Each tetrad was composed of four isomers identified as two iso olefins and two anteiso olefins. The only difference between the tetrads pertaining to different families was found in the relative gas chromatographic retention times of the last two components of each group.     

Direct probing of RNA structures and RNA-protein interactions in the HIV-1 packaging signal by chemical modification and electrospray ionization fourier transform mass spectrometry

RNA hairpins of the HIV-1 packaging signal and their complexes with the nucleocapsid protein p7 (NC) were probed by solvent-accessibility reagents and electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The combination of dimethylsulfate, kethoxal, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMCT) offers the full range of information on base-pairing and solvent exposure concerning the four more abundant ribonucleotides. ESI-FTMS provides a universal method to achieve a direct and unambiguous characterization of alkylated structures, with no need for the different probe-specific procedures required by established methodologies based on gel electrophoresis. It enables us to streamline the optimization of the conditions for probe administration to minimize the incidence of probe-induced distortion of the structures under investigation. Nucleotides located in the single-stranded loops of hairpins SL2, SL3 and SL4 manifested different levels of protection, which were correlated directly to their conformation and structural surroundings. A common feature noted for all the hairpins was the limited susceptibility observed for the guanine base located at the 5'-end of each tetraloop, which assumes a stacked position upon the last base-pair of the double-stranded stems. The remaining loop bases were found to be clearly accessible by modifying reagents in free RNA, but were effectively protected in the NC-hairpin complexes. While this finding is consistent with the proven participation of SL2 and SL3 loops in interactions with NC, it contrasts with prior suggestions that tetraloop bases in SL4 might not be involved directly in NC binding. Alkylation was detected for stem nucleotides, which are not involved in the normal base-pairing and stacking typical of double-stranded structures, such as adenine 15 of the SL2 triple-base platform. Modification of the blunt ends of the double-stranded stems was found to be absent or extremely limited, due to the annealing stabilization introduced by the presence of G-C pairs at the end of the stems structures. Previously undetected alkylation of guanine 3 and guanine 13 in SL4 provides direct evidence of the destabilizing effects induced by the tandem G.U wobbles on the double-stranded structure of this stem, which is thought to be important for the hairpin's biological function.     

The Physcomitrium (Physcomitrella) patens PpKAI2L receptors for strigolactones and related compounds function via MAX2-dependent and -independent pathways

In angiosperms, the α/β hydrolase DWARF14 (D14), along with the F-box protein MORE AXILLARY GROWTH2 (MAX2), perceives strigolactones (SL) to regulate developmental processes. The key SL biosynthetic enzyme CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) is present in the moss Physcomitrium patens, and PpCCD8-derived compounds regulate moss extension. The PpMAX2 homolog is not involved in the SL response, but 13 PpKAI2LIKE (PpKAI2L) genes homologous to the D14 ancestral paralog KARRIKIN INSENSITIVE2 (KAI2) encode candidate SL receptors. In Arabidopsis thaliana, AtKAI2 perceives karrikins and the elusive endogenous KAI2-Ligand (KL). Here, germination assays of the parasitic plant Phelipanche ramosa suggested that PpCCD8-derived compounds are likely noncanonical SLs. (+)-GR24 SL analog is a good mimic for PpCCD8-derived compounds in P. patens, while the effects of its enantiomer (−)-GR24, a KL mimic in angiosperms, are minimal. Interaction and binding assays of seven PpKAI2L proteins pointed to the stereoselectivity toward (−)-GR24 for a single clade of PpKAI2L (eu-KAI2). Enzyme assays highlighted the peculiar behavior of PpKAI2L-H. Phenotypic characterization of Ppkai2l mutants showed that eu-KAI2 genes are not involved in the perception of PpCCD8-derived compounds but act in a PpMAX2-dependent pathway. In contrast, mutations in PpKAI2L-G, and -J genes abolished the response to the (+)-GR24 enantiomer, suggesting that PpKAI2L-G, and -J proteins are receptors for moss SLs.

The study of moss PpKAI2L receptors for strigolactones and related compounds highlights MORE AXILLARY GROWTH2-dependent and -independent pathways for the perception of these compounds.     

Spontaneous rosette formation of rat thymus cells with guinea pig erythrocytes.

C57 black mice were immunised intraperitoneally with a DBA2 lymphoma (SL2). Fourteen days later spleen cells were prepared. These cells lysed the specific target (SL2) in vitro. Spleen cells were cultured for 24 hr at 37 ° C. Cell-free culture supernatants contained IgG and lysed SL2 cells either in the presence of a source of complement or in the presence of a monolayer of macrophages (a good source of antibody-dependent effectors). The cells producing cell-dependent antibody adhered to nylon wool and were unaffected by anti-theta serum. It was found that the production of antibody in vitro did not make a significant contribution to the observed cytolysis of SL2 by sensitised spleen cells. This effect was mediated by thymus-derived lymphocytes.     

New branching inhibitors and their potential as strigolactone mimics in rice

Strigolactones (SLs) are rhizosphere communication chemicals. Recent studies of highly branched mutants revealed that SL or its metabolites work as a phytohormone to inhibit shoot branching. When SLs are exogenously applied to the rice d10-1 mutant that has a highly branched phenotype caused by a defect in the SL biosynthesis gene (CCD8), they inhibit tiller bud outgrowth (branching in rice) of the mutant. We focused our attention on the SL function as a phytohormone and tried to find new chemicals mimicking the hormonal action of SL by screening chemicals that inhibit branching of rice d10-1 mutant. Fortunately, we found 5-(4-chlorophenoxy)-3-methylfuran-2(5H)-one (3a) as a new chemical possessing SL-like activity against the rice d10-1 mutant. Then, we prepared several derivatives of 3a (3b-3k) to examine their ability to inhibit shoot branching of rice d10-1. These derivatives were synthesized by a one-pot coupling reaction between phenols and halo butenolide to give 5-phenoxy 3-methylfuran-2(5H)-one (3) derivatives, which possess a common substructure with SLs. Some of the derivatives showed SL-like activity more potently than GR24, a typical SL derivative, in a rice assay. As SLs also show activity by inducing seed germination of root parasitic plants, the induction activity of these derivatives was also evaluated. Here we report the structure-activity relationships of these compounds.     

Exploring the relationship between bacterial genera and lipid metabolism in bovine rumen

The rumen is characterised by a complex microbial ecosystem, which is particularly active in lipid metabolism. Several studies demonstrated a role of diet and breed on bacterial community profile, with the effect on metabolic pathways. Despite the knowledge achieved on metabolism and the bacterial profile, little is known about the relationship between individual bacteria and metabolic pathways. Therefore, a multivariate approach was used to search for possible relationships between bacteria and products of several pathways. The correlation between rumen bacterial community composition and rumen lipid metabolism was assessed in 40 beef steers (20 Maremmana and 20 Aubrac) reared with the same system and fed the same diet. A canonical discriminant analysis combined with a canonical correlation analysis (CCA) was performed to explore this correlation. The variables showing a Pearson correlation higher than 0.6 as absolute value and significant were retained for CCA considering the relationship of bacterial composition with several metabolic pathways. The results indicated that some bacterial genera could have significant impacts on the presence of several fatty acids. However, the relationship between genera and fatty acid changes according to the breed, demonstrating that the metabolic pathways change according to the host genetic background, related to breed evolution, although there is also an intra-breed genetic background which should not be ignored. In Maremmana, Succiniclasticum and Rikenellaceae_RC9_gut_group showed a high positive correlation with dimethylacetals (DMAs) DMA_C13:0, DMA_C14:0, DMA_C14:0iso, DMA_C15:0, DMA_C15:0iso, and DMA_C18:0. Prevotellaceae_UCG-003 correlates with C18:3c9c12c15 and C18:1t11, while Fibrobacter and Succiniclasticum correlate with C18:2c9t11 and Lachnospiraceae_NK3A20_group correlates with C18:1c12. Prevotellaceae_UCG-003, Ruminococcaceae_UCG-010, and Oribacterium showed a positive correlation with C13:0iso, and C17:0. Conversely, in Aubrac, Treponema_2 and Rikenellaceae_RC9_gut_group correlated with DMA_C14:0iso, DMA_C16:0iso, DMA_C17:0iso, while Ruminococcaceae_UCG-010, Christensenellaceae_R-7_group and Ruminococcaceae_NK4A214_group correlated with DMA_C18:1t11, DMA_C14:0, DMA_C18:1c12. Acetitomaculum correlated with C18:2c9c12, C18:1c12, C18:1c13, C18:1t12 and Lachnospiraceae_NK3A20_group with C18:1t6-8 and C18:1t9. Saccharofermentas, Ruminococcaceae_UCG-010 and Rikenellaceae_RC9_gut_group correlated with C18:2c9t11 while, Prevotellaceae_UCG-001 and Ruminococcus_1 correlated with C14:0iso, C15:0, C15:0iso, C17:0. Saccharofermentans, Rikenellaceae_RC9_gut_group, Ruminococcaceae_NK4A214_group, and Ruminococcaceae_UCG-010 correlated with C13:1c12 and C16:0iso. These results lead to hypothesise a possible association between several metabolic pathways and one or a few bacterial genera. If these associations are confirmed by further investigations that verify the causality of a bacterial genus with a particular metabolic process, it will be possible to deepen the knowledge on the activity of the rumen population in lipid metabolism. This approach appears to be a promising tool for uncovering the correlation between bacterial genera and products of rumen lipid metabolism.     

Sulfonolipids are localized in the outer membrane of the gliding bacterium Cytophaga johnsonae

Earlier work in our laboratory demonstrated that gliding bacteria of the Cytophaga-Flexibacter group contain, in their cell envelopes, large quantities of unusual sulfonolipids (N-fatty acyl 2-amino-3-hydroxyisoheptadecane-1-sulfonic acids). Recently, it has been shown that these lipids are necessary for the gliding motility of C. johnsonae. As one approach to determining the role of the lipids in motility, methods have now been developed for separating the inner (cytoplasmic) and outer membranes of a strain (ATCC 43786) of this Gram-negative bacterium. Sulfonolipid is at least five times as abundant in the outer membrane as in the inner. The inner membrane has properties similar to those found for other Gram-negative bacteria; it has a buoyant density of 1.14 g/ml and is highly enriched in cytochromes and succinate dehydrogenase. The outer membrane (1.18 g/ml) is enriched in bound carbohydrate and sulfonolipid, but contains little or no 2-keto-3-deoxyoctonate (such as is found in the enterobacteria). The localization of the sulfonolipids in the outer membrane permits focus on the possible roles these unusual substances may play in gliding motility.Abbreviations used IM inner membrane - OM outer membrane - KDO 2-keto-3-deoxyoctonate - EDTA ethylenediaminetetraacetic acid - SDH succinate dehydrogenase     

Lipids from the guinea pig Harderian gland: use of picolinyl and other pyridine-containing derivatives to investigate the structures of novel branched-chain fatty acids and glycerol ethers

The lipid extracted from guinea pig Harderian glands was hydrolysed and the constituents were examined as trimethylsilyl (TMS), (2H9)TMS, methyl ester/TMS, acetonide/TMS, nicotinate/TMS, picolinyl/TMS and nicotinylidene/TMS derivatives by capillary gas-liquid chromatography and gas chromatography/mass spectrometry. Over 70 compounds amounting to over 93% of the extract were identified. These consisted of 1-O-alkyl glycerols (glycerol ethers) with alkyl chains containing from 17 to 21 carbon atoms and fatty acids ranging from 14 to 26 carbon atoms. The alkyl chains in the glycerol ethers were straight, mono- and dimethyl-branched with the major site of branching being at C-14. All straight-chain acids from C14 to C26 were present, with the most abundant being n-24:0. Again mono- and dimethyl branched structures comprised the bulk of the remaining acids. Methyl groups tended to be towards the middle of the chain rather than in the more usual omega-1 (iso) and omega-2 (anteiso) positions, with C-14 again being a major site. The shorter-chain acids tended to have methyl groups closer to the acid group, with several of the short-chain compounds being substituted at C-2. Structural information on the acids was provided by the picolinyl derivatives and the sample provided an opportunity to evaluate these derivatives with branched acids other than the iso and anteiso compounds studied previously. They were found to be satisfactory for analysis of both mono- and dimethyl branched acids with the possible exception of compounds containing a methyl branch at C-4. However, in this case, structural information was provided by the methyl ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and biological activity of abscisic acid esters

16 ABA esters including 11 new compounds were prepared by two different esterification routes. All the structures of ABA esters were confirmed by ¹H NMR, ¹³C NMR and HRMS. Their biological activity and hydrolysis stability were investigated. Fortunately, there were 15 and 9 compounds which displayed much better or nearly the same inhibition activity for rice seedling growth and Arabidopsis thaliana seed germination compared to ABA, respectively. Especially, compounds 2d and 2g showed better biological activities than ABA in the three tests. Moreover, we found that chemical hydrolysis ability of the esters in vitro had little relationship to their biological activity.     

NMR structure of varkud satellite ribozyme stem-loop V in the presence of magnesium ions and localization of metal-binding sites

In the Neurospora VS ribozyme, magnesium ions facilitate formation of a loop-loop interaction between stem-loops I and V, which is important for recognition and activation of the stem-loop I substrate. Here, we present the high-resolution NMR structure of stem-loop V (SL5) in the presence of Mg(2+) (SL5(Mg)) and demonstrate that Mg(2+) induces a conformational change in which the SL5 loop adopts a compact structure with most characteristics of canonical U-turn structures. Divalent cation-binding sites were probed with Mn(2+)-induced paramagnetic line broadening and intermolecular NOEs to Co(NH(3))(6)(3+). Structural modeling of Mn(H(2)O)(6)(2+) in SL5(Mg) revealed four divalent cation-binding sites in the loop. Sites 1, 3, and 4 are located in the major groove near multiple phosphate groups, whereas site 2 is adjacent to N7 of G697 and N7 of A698 in the minor groove. Cation-binding sites equivalent to sites 1-3 in SL5 are present in other U-turn motifs, and these metal-binding sites may represent a common feature of the U-turn fold. Although magnesium ions affect the loop conformation, they do not significantly change the conformation of residues 697-699 involved in the proposed Watson-Crick base pairs with stem-loop I. In both the presence and the absence of Mg(2+), G697, A698, and C699 adopt an A-form structure that exposes their Watson-Crick faces, and this is compatible with their proposed interaction with stem-loop I. In SL5(Mg), however, U700 becomes exposed on the minor groove face of the loop in the proximity of the bases of G697, A698, and C699, suggesting that the Mg(2+)-bound conformation of stem-loop V allows additional contacts with stem-loop I. These studies improve our understanding of the role of Mg(2+) in U-turn structures and in substrate recognition by the VS ribozyme.