Studies on the Sunlight Flavour of Beer

In order to establish the mechanism of occurrence of sunlight flavour of beer in continuation of the preceding paper, humulone, lupulone, and their analogues and degradation compounds, i.e., cohumulone, adhumulone, 5-acetyl-3-methylfilicinic acid including analogues, tetrahydrohumulone, hexahydrolupulone, humulinic acid and lupuloxinic acid, were tested for the occurrence of sunlight flavour of beer.

As a result, among the above-mentioned compounds, humulone, lupulone, cohumulone, adhumulone, tetrahydrohumulone and hexahydrolupulone were found to cause the sunlight flavour, but the other compounds did not cause typical sunlight flavour. This fact shows that some specific structural components seem requisite for the occurrence of sunlight flavour of beer.

It was also revealed that isomerization caused by boiling accelerates the occurrence of the sunlight flavour of beer. Finally, the result of the experiment conducted by the gas chromatographic procedure showed that any new component is not detected by the exposure of beer to sunlight but, two components somewhat increased.     

Bildung und weiterreaktion von acylvorstufen der hopfenbitterstoffe

Isobutyric acid is incorporated into desoxycohumulone, cohumulone and colupulone and isovaleric acid is incorporated into desoxyhumulone, humulone and lupulone. The compound 1-isobutyryl-3-(3,3-dimethylallyl)-phloroglucinol (CoX) can be considered as an intermediate for the first three components of the hop resins while isovaleryl-3-(3,3-dimethylallyl)-phloroglucinol (X) is an intermediate in the biosynthesis of the parent compounds.     

Biosynthesis of bitter acids in hops. A (13)C-NMR and (2)H-NMR study on the building blocks of humulone.

The biosynthesis of humulone, an antibacterial bitter acid from hops, was studied by isotope-incorporation experiments using (13)C-labelled glucose or (2)H(2)O. (13)C enrichments, (2)H enrichments and (13)C(13)C coupling patterns identify isovaleryl-CoA, malonyl-CoA and dimethylallyl pyrophosphate as precursors for humulone. Dimethylallyl pyrophosphate, which serves as a building block for the bitter acid, is generated via the deoxyxylulose pathway of terpenoid biosynthesis. The data confirm that a symmetrical intermediate is involved in humulone formation.     

Structural determination of the O-antigenic polysaccharide from the verocytotoxin-producing Escherichia coli O176

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O176 has been determined. Component analysis together with 1H and 13C NMR spectroscopy was employed to elucidate the structure. Inter-residue correlations were determined by 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [Formula: see text] Cross-peaks of low intensity from alpha-linked mannopyranosyl residues were present in the 1H, 1H TOCSY NMR spectra and further analysis of these showed that they originate from the terminal part of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O176 O-antigen is similar to those from E. coli O17 and O77, thereby explaining the reported cross-reactivities between the strains, and identical to that of Salmonella cerro (O:6, 14, 18).     

Structure and cross-reactivity of the O-antigen of Providencia stuartii O18 containing 3-acetamido-3,6-dideoxy-D-glucose

The O-polysaccharide (O-antigen) of Providencia stuartii O18 was obtained by mild acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [structure: see text] where Qui3NAc is 3-acetamido-3,6-dideoxyglucose. Anti-P. stuartii O18 serum cross-reacted with the O-antigen of Proteus genomospecies 4, which could be accounted for the marked structural similarities of the main chain.     

Structure and serological characterization of the O-antigen of Proteus mirabilis O18 with a phosphocholine-containing oligosaccharide phosphate repeating unit

A phosphorylated, choline-containing polysaccharide was obtained by O-deacylation of the lipopolysaccharide (LPS) of Proteus mirabilis O18 by treatment with aqueous 12% ammonia, whereas hydrolysis with dilute acetic acid resulted in depolymerisation of the polysaccharide chain by the glycosyl phosphate linkage. Treatment of the O-deacylated LPS with aqueous 48% hydrofluoric acid cleaved the glycosyl phosphate group but, unexpectedly, did not affect the choline phosphate group. The polysaccharide and the derived oligosaccharides were studied by NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HMQC and HMQC-TOSCY experiments, along with chemical methods, and the following structure of the pentasaccharide phosphate repeating unit was established: [carbohydrate structure in text] Where ChoP=Phosphocoline Immunochemical studies of the LPS, O-deacylated LPS and partially dephosphorylated pentasaccharide using rabbit polyclonal anti-P. mirabilis O18 serum showed the importance of the glycosyl phosphate group in manifesting the serological specificity of the O18-antigen.     

An amperometric hydrogen peroxide sensor based on immobilization of hemoglobin in poly(o-aminophenol) film at iron-cobalt hexacyanoferrate-modified gold electrode

A series of hybrid iron-cobalt hexacyanoferrate (FeCoHCF) films were electrodeposited on gold electrodes from solutions containing 6mM Fe(CN)(6)(3-) with different concentrations of Co(2+) and Fe(3+). FeCoHCF films deposited from solutions with different molar ratios of iron were studied by cyclic voltammetry, and their solid states were characterized by Fourier transform infrared spectroscopy. The kind of FeCoHCF film that deposited from a solution with a molar ratio of iron of 0.4 showed the largest response current to H(2)O(2) and was characterized by energy-dispersive X-ray spectroscopy. Therefore, the optimized FeCoHCF film was combined with nonconducting poly(o-aminophenol) (POAP) film that entrapped the hemoglobin (Hb) to construct hydrogen peroxide biosensor. The response current of the Hb/POAP/FeCoHCF/Au electrode (29.8 nA) was nearly 40 and was 1.5 times that of the Hb/POAP/Au (0.7 nA) and POAP/FeCoHCF/Au (20 nA) electrodes, respectively. The Michaelis-Menten constant of Hb in the Hb/POAP/FeCoHCF/Au film was 9.31 mM. These results show that the immobilized Hb in the Hb/POAP/FeCoHCF/Au film exhibits higher catalytic activity and larger response current to H(2)O(2) by the mediation of FeCoHCF. In addition, effects of applied potential, solution pH, and electroactive interferent on the response current of the Hb/POAP/FeCoHCF/Au electrode were investigated in detail.     

Effects of long-term storage on the stability of OpMNPV DNA contained in TM Biocontrol-1

Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) DNA was extracted from samples representing 10 lots of TM Biocontrol-1 stored at -10 degrees C for 5-15 years and digested with the restriction enzymes BglII, PstI, and SalI. DNA from the OpMNPV virus strain (MEM-75-STANDARD) used to produce the TM Biocontrol-1 lots was also extracted and digested. No restriction fragment length polymorphisms were observed in any of the samples and there was no evidence of DNA degradation. This indicates that long-term cold storage of TM Biocontrol-1 had no adverse effect on the quality of the OpMNPV DNA. In addition to the expected >23 kb OpMNPV DNA, extracts from lots 4a, 5b, and 6 contained 10 additional nucleic acid segments, ranging in size from 0.9 to 4.2 kb. The electrophoretic profile of these segments was characteristic of O. pseudotsugata cypovirus (OpCPV). RNase A/DNase I treatment showed that the nucleic acid contaminants were composed of RNA, suggesting that lots 4a, 5b, and 6 contained OpCPV as well as OpMNPV. Bioassay results have shown that there is a decrease in efficacy of stored TM-biocontrol-1, but this did not appear to be directly correlated with the length of time in storage.     

The O18 antigens (lipopolysaccharides) of Escherichia coli. Structural characterization of the O18A, O18A1, O18B and O18B1-specific polysaccharides.

The O-specific polysaccharide moieties (PS) of the O18A, O18A1, O18B, and O18B1 antigens (lipopolysaccharides, LPS) consist of L-rhamnose (Rha), N-acetyl-D-glucosamine, D-galactose, and D-glucose in different molar ratios. By using chemical fragmentation, methylation, as well as one- and two-dimensional NMR spectroscopy, the structures of these polysaccharides were found to be [formula: see text] In O18A-PS and O18A1-PS x = 2, whereas in O18B-PS and in O18B11-PS x = 3. In all four polysaccharides alpha-D-Galp (residue D) is substituted at O-3. This substituent L (residue E) is beta-D-GlcpNAc-(1 in O18A-PS and O18A1-PS and it is alpha-D-Glcp-(1 in O18B-PS and O18B1-PS. Whereas there is no further substituent on the main chain of the O18A and O18B polysaccharides, in O18A1-PS and O18B1-PS the alpha-D-GlcpNAc residue A is substituted with alpha-Glcp-(1 (residue F), which is linked to O-6 in O18A1-PS and to O-4 in O18B1-PS. These results show that the O18 antigen comprises a group of four related LPS (O18A and O18B, with their glucosylated forms O18A1 and O18B1). The results are discussed with respect to epitope definition and biochemical implications.     

Structure of a colitose-containing O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O6

The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O6 and studied by sugar and methylation analysis, selective hydrolytic removal of 3,6-dideoxy-L-xylo-hexose (colitose, Col), (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY and H-detected (1)H,(13)C HSQC and HMBC experiments. The polysaccharide was found to have a branched pentasaccharide repeating unit with the following structure: [see text] Remarkably, the trisaccharide side chain of the O6-polysaccharide represents a colitose ('3-deoxy-L-fucose') analogue of the H type 1 (precursor) antigenic determinant.     

The biosynthetic origin of oxygen functions in phenylphenalenones of Anigozanthos preissii inferred from NMR- and HRMS-based isotopologue analysis

The biosynthetic origin of 9-phenylphenalenones and the sequence according to which their oxygen functionalities are introduced were studied using nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HRESIMS). ¹³C-labelled precursors were administered to root cultures of Anigozanthos preissii, which were simultaneously incubated in an atmosphere of ¹⁸O₂. Two major phenylphenalenones, anigorufone and hydroxyanigorufone, were isolated and analyzed by spectroscopic methods. Incorporation of ¹³C-labelled precursors from the culture medium and ¹⁸O from the atmosphere was detected. O-Methylation with ¹³C-diazomethane was used to attach ¹³C-labels to each hydroxyl and thereby dramatically enhance the sensitivity with which NMR spectroscopy can detect ¹⁸O by means of isotope-induced shifts of ¹³C signals. The isotopologue patterns inferred from NMR and HRESIMS analyses indicated that the hydroxyl group at C-2 of 9-phenylphenalenones had been introduced on the stage of a linear diarylheptanoid. The oxygen atoms of the carbonyl and lateral aryl ring originated from the hydroxyl group of the 4-coumaroyl moiety, which was incorporated as a unit.     

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E

A critical point in the V(1) sector and entire V(1)V(O) complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G(1-59)) of the V(1)V(O) ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of G(1-59) in the absence and presence of the N-terminal peptides E(1-18) and E(18-38) as well as the produced and purified C-terminal segment (E(39-233)) shows specific interactions only with the peptide fragment E(18-38). The binding of this peptide occurs via the residues M(1), V(2), S(3), and K(5) as well for V(22), S(23), K(24), A(25) and R(26) of G(1-59). The specific E(18-38)/G(1-59) binding has been confirmed by fluorescence correlation spectroscopy data. The E(18-38) peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E(18-38)/G(1-59) complex reveals the orientation of E(18-38) relative to G(1-59) via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.     

The stereochemical course of yeast hexokinase-catalysed phosphoryl transfer by using adenosine 5''[gamma(S)-16O,17O,18O]triphosphate as substrate.

Adenosine 5'[gamma(S)-16O, 17O, 18O]triphosphate has been synthesized and used to determine the stereochemical course of phosphoryl transfer catalysed by yeast hexokinase. The chirality at phosphorus of the D-glucose 6-[16O,17O,18O]phosphate formed was analysed, after cyclization and methylation, by 31P n.m.r. spectroscopy. The phosphoryl transfer was found to occur with inversion of configuration, with a stereoselectivity in excess of 94%. The simplest interpretation of this result is that the phosphoryl group is transferred between substrates in the enzyme-substrate ternary complex by an 'in line' mechanism.     

Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates

The transfer of 17O and/or 18O from (COOH-17O or -18O) enriched substrates to inorganic phosphate (Pi) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation. COOH-18O-labeled folate, methotrexate, and dihydropteroate, in addition to [17O]-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E. coli. Pi was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for 17O and 18O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy. In the reactions catalyzed by the E. coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate 18O-enriched carboxyl group to Pi occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions. Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes. The small amounts of Pi obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques. However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that 18O transfer had occurred.     

18-nor-Podocarpanes and podocarpanes from the Bark of Taiwania cryptomerioides

Seven nor- and podocarpane-type diterpenes were isolated from the bark of Taiwania cryptomerioides Hayata, including three 18-nor-podocarpanes: 18-nor-1beta,4alpha,14-trihydroxy-13-methoxy-8,11,13-podocarpatriene (1), 18-nor-1beta,4alpha,13,14-tetrahydroxy-8,11,13-podocarpatrien-7-one (2), 18-nor-1beta,4alpha,14-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (3), 1beta,14,19-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (4), 1beta,13,14,18-tetrahydroxy-8,11,13-podocarpatrien-7-one (5), 18-acetoxy-1beta,13,14-trihydroxy-8,11,13-podocarpatrien-7-one (6), and 1beta,14,18-trihydroxy-13-methoxy-8,11,13-podocarpatrien-7-one (7). Their structures were determined by application of 1D and 2D NMR spectroscopy and other techniques. Podocarpane-type diterpenes do not occur extensively in nature, and the presumed oxidative enzyme in this plant will be of interest to identify.     

The structure of the polysaccharide part of the LPS from Serratia marcescens serotype O19, including linkage region to the core and the residue at the non-reducing end

The structure of the LPS from Serratia marcescens serotype O19 was investigated. Deamination of the LPS released the O-chain polysaccharide together with a fragment of the core oligosaccharide. The following structure of the product was determined by NMR spectroscopy, mass spectrometry, and chemical methods: [carbohydrate structure: see text] The main polymer consists of a repeating disaccharide V-U and is present on average of 18 units per chain as estimated by integration of signals in the NMR spectra. The residue O corresponds to the primer, which initiates biosynthesis of the O-chain, and an oligomer of a disaccharide R-S is an insert between the primer and the main polymer. The polysaccharide has a beta-Kdo residue at the non-reducing end, a feature similar to that observed previously in the LPS from Klebsiella O12.     

A potent sphingomyelinase inhibitor from Cordyceps mycelia contributes its cytoprotective effect against oxidative stress in macrophages

A novel water-soluble polysaccharide fraction, CME-1, with a molecular mass of 27.6 kDa and containing mannose and galactose in a respective ratio of 4:6, was prepared from Cordyceps sinensis mycelia and identified by NMR and GC-MS. In the current study, we examined whether CME-1 has anti-inflammatory effects in RAW264.7 cells. The ability of CME-1 to inhibit H(2)O(2)-induced cell death in RAW264.7 cells was assessed by using an MTT assay and annexin V/propidium iodide double staining; we found that CME-1 protected cells against H(2)O(2)-induced injury. H(2)O(2)-induced intracellular oxidative stress and mitochondrial membrane depolarization were also diminished with CME-1 treatment. We evaluated the hydroxyl radical scavenging ability of CME-1 by using the DMPO-electron spin resonance technique, which indicated that CME-1 acts as an intracellular antioxidant in a concentration-dependent manner through a mechanism other than its scavenging activity. Activities of both neutral and acid sphingomyelinases (SMases) were assessed in vitro, and results showed that the CME-1 inhibited activities of both neutral and acid SMases in a concentration-dependent manner. CME-1 reduced H(2)O(2) treatment-elevated C16- and C18-ceramide levels measured by LC/MS/MS in RAW264.7 cells. Results suggest that CME-1 protects RAW264.7 cells against oxidative stress through inhibition of SMase activity and reduction of C16- and C18-ceramide levels.     

The stereochemical course of hydrolysis catalysed by snake venom 5''-nucleotide phosphodiesterase.

Adenosine 5'-(S)-[16O,17O,18O]phosphate was pyrophosphorylated by the combined action of adenylate kinase and pyruvate kinase. The isotopomers of adenosine 5'-[alpha-16O,17O,18O]triphosphate were hydrolysed by venom 5'-nucleotide phosphodiesterase (Crotalus adamanteus) in H2(17)O. Analysis by 31P nuclear magnetic resonance spectroscopy of the resulting adenosine 5'-[16O,17O,18O]phosphate, after cyclization and esterification, showed that the hydrolysis occurs with retention of configuration at phosphorus. The most likely explanation of this observation is that the enzymic hydrolysis involves a double displacement at phosphorus with a covalent nucleotidyl--enzyme intermediate on the reaction pathway.     

Structural investigation of a novel rhamnoglucogalactan isolated from the fruiting bodies of the fungus Hericium erinaceus

A new heteropolysaccharide (HEP-1) was isolated from the fruiting bodies of Hericium erinaceus. It was estimated to have a molecular weight of 1.8x10(4) da and showed [alpha](D)(20) +129 (c 0.295, H(2)O). HEP-1 is composed of rhamnose, galactose, and glucose in the ratio of 1.19:3.81:1.00. Its structural features were investigated using composition analysis, methylation analysis, partial hydrolysis, and IR and NMR spectroscopy. The results showed that HEP-1 has a (1-->6)-linked alpha-d-galactopyranosyl backbone with branches that are composed of rhamnose and glucose attached to O-2.