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Bombyx batryticatus is a well-known animal in traditional Chinese medicine. The aim of the research was to reveal the quality formation mechanism of B. batryticatus and to screen out the characteristic component used for the quality control. The anticonvulsant effects of B. batryticatus with a stiff time of one, five, and nine days (D1, D5 and D9, respectively) and healthy silkworm of the same developmental stage (SW) were determined by animal experiment. The dynamic changes in chemical composition were analyzed using UPLC-Q-TOF-MS-based metabolomics. D5 and D9 B. batryticatus exhibited significant anticonvulsant effects ( p < 0.05 and p < 0.01, respectively). Accordingly, principal component analysis (PCA) and partial least squares discrimination analysis (PLS-DA) indicated that the chemical composition of D5 and D9 B. batryticatus changed significantly. The different metabolites mainly consisted of primary metabolites such as lipids and amino acids and secondary metabolites such as flavonoids, beauvericin, and glycolipids. Interestingly, the relative abundance of quercetin-7- O -β-d-4- O -methylglucoside, the characteristic component of B. batryticatus , increased with stiff time and was promised to be used as an index component of quality control. The results expand our understanding of the quality formation mechanism of B. batryticatus . In addition, it highlights the potential of UPLC-Q-TOF-MS-based metabolomics for the quality control purpose of TCMs.

4-(3,4-Dihydroxybenzoyloxymethyl)phenyl-O-β-d-glucopyranoside (DBPG), a polyphenolic glycoside, isolated from Origanum vulgare has shown 1,1-diphenyl-2-picrylhydrazyl (DPPH(•))-scavenging capacity in previous work. This study demonstrated that DBPG exhibits antioxidant activity by a series of DPPH(•), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS(•+)), and superoxide anion radical (O(2)(•-)) radical-scavenging assays. The inhibition of lipid peroxidation (LP) by DBPG exceeded that by l-ascorbic acid (AA) in a liposome model system. Adding DBPG to mouse liver and brain tissue inhibited the formation of thiobarbituric acid reactive substances (TBARS) to a greater extent than did trolox. In the oxygen stress test, BNLCL2 and HaCaT cells pretreated with DBPG showed increased activities of glutathione peroxidase (GPx), perhaps as a result of reduction of the production of reactive oxygen species (ROS). These findings proved that DBPG had antioxidant activities and a cytoprotective effect in hepatocytes and keratinocytes, suggesting that DBPG may be a useful food and cosmetic additive.

Mycobacteria produce two unusual polymethylated polysaccharides, the 6-O-methylglucosyl-containing lipopolysaccharides (MGLP) and the 3-O-methylmannose polysaccharides, which have been shown to regulate fatty acid biosynthesis in vitro. A cluster of genes dedicated to the synthesis of MGLP was identified in Mycobacterium tuberculosis and Mycobacterium smegmatis. Overexpression of the putative glycosyltransferase gene Rv3032 in M. smegmatis greatly stimulated MGLP production, whereas the targeted disruption of Rv3032 in M. tuberculosis and that of the putative methyltransferase gene MSMEG2349 in M. smegmatis resulted in a dramatic reduction in the amounts of MGLP synthesized and in the accumulation of precursors of these molecules. Disruption of Rv3032 also led to a significant decrease in the glycogen content of the tubercle bacillus, indicating that the product of this gene is likely to be involved in the elongation of more than one alpha-(1-->4)-glucan in this bacterium. Results thus suggest that Rv3032 encodes the alpha-(1-->4)-glucosyltransferase responsible for the elongation of MGLP, whereas MSMEG2349 encodes the O-methyltransferase required for the 6-O-methylation of these compounds.

Conventional relaxation parameters (T1(-1), T2(-1), and NOE), obtained at different temperatures and magnetic fields, are reported for the hydroxymethyl (C6) carbon in methyl-beta-D-glucopyranoside in a D2O/DMSO cryosolvent. These data are interpreted with the Lipari-Szabo model. In addition, two-field measurements of longitudinal and spin-locked relaxation rates related to the cross-correlated carbon-proton dipole-dipole interactions for the same carbon are reported. The complete data set consisting the conventional and cross-correlated relaxation parameters is interpreted using a new "hybrid" approach, in which the Lipari-Szabo model for the auto-correlated spectral densities is combined with the two-site jump model for the cross-correlated spectral densities, with the global correlation time as a common parameter. The two-site jump rates thus obtained are in reasonable agreement with the ultrasonic relaxation measurements, and have reasonable temperature dependence.

A new NMR experiment which allows a measurement of the chemical shift anisotropy (CSA) tensor under magic angle spinning (MAS) is described. This correlates a fast MAS spectrum in the omega2 dimension with a sideband pattern in omega1 in which the intensities mimic those for a sample spinning at a fraction of the rate omega r/N, and these sidebands result from an amplification by a factor N of the modulation caused by the CSA. Standard methods can be used to extract the principal tensor components from the omega1 sideband patterns, and the nature of the experiment is such that the use of a large number of t1 increments can be avoided without compromising the resolution of different chemical sites. The new experiment is useful for accurately measuring narrow shift anisotropies.

The specific mycobacterial methyl polysaccharides 3- O -methyl mannose polysaccharide (MMP) and the 6- O -methyl glucose lipopolysaccharides (MGLPs) were shown to modulate the fatty acid biosynthesis by the mycobacterial fatty acid synthetase I (FAS I). This activity is attributed to their fatty acid complexing properties allowing the release of the neo synthesized fatty acyl chain from the enzyme and probably their transport in the cell. To elucidate, at a molecular level, the mechanism of this unusual kind of polysaccharide-lipid biological interaction, we first analyzed, by mass spectrometry and proton nuclear magnetic resonance (1H NMR) spectroscopy, the structure of the polysaccharidic backbone (MGPs) of the MGLPs from Mycobacterium bovis BCG. This work reveals that this strain produces a new kind of MGP containing an unusual monosaccharide never described in the mycobacterial genus: a 2- N -acetyl-2,6-dideoxy-beta-glucopyranosyl. In addition,1H NMR data afforded evidence for the revision of three glycosidic linkages described previously. These modifications affect mainly the reducing end tetrasaccharide and have great consequences on the previously proposed molecular model of the MGP.

Mycobacterial methylglucose lipopolysaccharides (MGLPs) play an important regulatory role in the biosynthesis of long-chain fatty acids by forming complexes with neosynthesized acyl-CoA fatty acid derivatives. The MGLPs from Mycobacterium smegmatis were purified by high-performance anion-exchange chromatography and characterized by LSIMS and CE/ESI-MS. We investigated their interaction with palmitoyl-CoA using capillary zone electrophoresis with both direct and indirect UV detection. In the latter mode, the signal of the UV-transparent MGLPs decreased upon addition of increasing amounts of palmitoyl-CoA; while using direct UV detection, the addition of palmitoyl-CoA to the MGLPs revealed characteristic profiles. The major peak was assigned to the noncovalent MGLP/palmitoyl-CoA complex on the basis of its electrophoretic mobility. The abundance of the complex was found to increase until the MGLP/palmitoyl-CoA molar ratio reached a 1/1 stoichiometry. The existence of and the stoichiometry of this complex were assessed by CE/ESI mass spectrum analysis, showing pseudomolecular ions of the MGLP/palmitoyl-CoA complex. These results confirm that CE/ESI-MS is a powerful tool to characterize noncovalent molecular association.

The addition of various concentrations of monensin (1,5, and 10 micrograms/mL) to the culture medium inhibits the fungal growth and perturbs exopolysaccharides secretion, provoking a decrease of production in Botrytis cinerea and an increase in Sclerotium rolfsii. The ionophore induces also modifications in both polymer composition and structure. New monomers were observed in the two species and a decreased branching rate for Sclerotium rolfsii. These modifications show that monensin affects the enzymes responsible for normal wall synthesis and therefore vesicular traffic.

The intracellular volume of distribution (V) of 3-O-methylglucose (3OM), as calculated from mean artery-to-vein transit times (t)in the rabbit kidney, has been proposed as measure of cytoplasmic volume in renal cortex. This hypothesis requires that the non-concentrative uptake of the sugar be flow-dependent rather than diffusion-limited, and that total cell H2O (VHOH) exceed V3OM by a volume presumably reflecting that of 3OM-impermeable organelles. Diffusion-dependence of the calculated V3OM could be excluded by the finding that lengthening of t does not alter 3OM uptake. Determination of VHOH in kidney is complicated by presence of the luminal fluid compartment. VHOH and V3OM were therefore compared in leucocytes freshly prepared from rat blood; V3OM was found to equal 40% of VHOH. There is no evidence to suggest concentrative 3OM uptake by these cells; 3OM-impermeable organelles therefore occupy 60% of VHOH. These findings support the equation of V3OM with the cytoplasmic volume. As volume control is a basic function of intact cells, convenient determination of VHOH and V3OM on small numbers of readily available nucleated cells may prove useful in detecting early cytotoxic effects in vivo or in vitro.

Mannitol, 3-O-methylglucose and lactulose administered orally are used to investigate small intestinal absorption pathways and mucosal integrity. Current methods of analysis include thin-layer chromatography, gas chromatography (GC) and enzymatic analysis, which require separate estimation of mono- and disaccharides and for GC, prior derivatization. We describe a high-pressure anion-exchange chromatographic method coupled with pulsed electrochemical detection allowing simultaneous measurement of all three sugars and its clinical application in monitoring intestinal damage in human immunodeficiency virus (HIV) infection. Sample preparation is simple and fast. All sugars are resolved within 10 min. Mean recovery is 93.3% for all sugars and the overall relative standard deviation is 4.2%. Intestinal permeability (lactulose/mannitol ratio) rises with disease progression to AIDS, indicating mucosal damage. The greatest increase in permeability is associated with chronic diarrhoea. The method is an ideal non-invasive test to assess gut mucosal damage in HIV infection.

Alkaline pH has been reported to cause release of Ca2+ from skeletal muscle sarcoplasmic reticulum (SR). Elevation of sarcoplasmic Ca2+ concentration is thought to stimulate glucose transport in skeletal muscle. In this context, we examined the effect of alkaline pH (extracellular pH of 8.6) on 3-O-methylglucose transport in skeletal muscle. Incubation of rat epitrochlearis muscles at pH 8.6 for 45 min resulted in an approx. 3-fold increase in glucose transport activity, which was not affected by reducing Ca2+ concentration in the incubation medium and essentially completely blocked by 25 microM dantrolene, an inhibitor of SR Ca2+ release. In addition to stimulating glucose transport by itself, alkaline pH may partially inhibit the stimulation of sugar transport by insulin hypoxia and contractions, as the combined effect of alkaline pH and the maximal effect of insulin, contractions, or hypoxia on glucose transport are not different from the maximal effects of insulin, hypoxia, or contractions alone. The maximal effects of insulin and contractions, and of insulin and hypoxia, on glucose transport are normally additive in muscle. Alkaline pH completely prevented this additivity. In summary, our results show that alkaline pH stimulates glucose transport activity in skeletal muscle and provide evidence suggesting that this effect is mediated by Ca2+. They further show that alkaline pH blocks the additivity of the maximal effects of insulin and contractions or hypoxia suggesting that alkaline pH may partially inhibit the stimulation of glucose transport by insulin, contraction and hypoxia.

The aqueous glucose concentration of rat milk is about 0.29mM, apparently unmodified by prolonged storage in the gland. Infused 3-0-methylglucose reaches a milk concentration similar to its measured mammary intracellular concentration. Milk glucose may therefore reflect its intracellular concentration in the mammary secretory cell.

Biosynthesis of sulfated saccharides that are linked to asparagine residues in the cell surface glycoprotein of Halobacterium halobium via a glucose residue involves sulfated dolichyl-monophosphoryl oligosaccharide intermediates (Lechner, J., Wieland, F., and Sumper, M. (1985) J. Biol. Chem. 260, 860-866). During isolation and characterization of these lipid oligosaccharides we detected a group of related compounds containing additional unidentified sugar residues. Here we report that: 1) the unknown sugar residues were 3-O-methylglucose, linked peripherally to the lipid-saccharide intermediates; 2) the 3-O-methylglucose residues in the oligosaccharides occur only at the lipid-linked level but are absent at the protein-linked level; 3) cell surface glycoprotein biosynthesis in Halobacteria in vivo is drastically depressed when S-adenosylmethionine-dependent methylation is inhibited, indicating that methylation is an obligatory step during glycoprotein synthesis. We propose a mechanism for the transport of lipid oligosaccharides through the cell membrane, involving an intermediate stage in which the saccharide moieties are transiently modified with 3-O-methylglucose.

A glucan of high molecular weight isolated from stale sugar-cane, and previously shown to have a marked effect on sucrose crystallisation processes-1, is a relatively linear dextran. Approximately 96-97% of its D-glucose residues are involved in (1 yields 6)-alpha-D linkages and constitute the linear backbone of the polymer. The remaining 3-4% of D-glucose residues form branch-points by (1 yields 3)-alpha-D linkages. The periodate-oxidation technique, which has been extensively used by other workers to determine dextran structure, gave erroneous results when applied to the dextran from stale sugar-cane.

The capsular type-specific antigen of Haemophilus influenzae, type b, has been reported to have a unit structure composed of D-ribose and phosphate. Recently, the presence of ribitol was found in preparations of type b capsular antigen. Our analytical results show equimolar proportions of ribose, ribitol, and phosphate. Periodate oxidation studies, paper chromatography of acidic and alkaline hydrolysates, and NMR spectral data indicate the structure of the capular antigen of H. influenzae b, strain Eagan to be a polyribosylribitol phosphate polymer.

Optimal conditions for isotachophoretic separation of carboxymethyl-D-glucoses formed by acidic depolymerization of carboxymethylcellulose were found. 6-O-carboxymethyl-D-glucose, 2-O-carboxymethyl-D-glucose and 3-O-carboxymethyl-D-glucose were identified and determined in the reaction mixture after carboxymethylcellulose hydrolysis. Relative reactivity of hydroxy groups in the glucopyranose unit of cellulose decreased in the following order: O(6)H greater than O(2)H much greater than O(3)H. This was found to be in agreement with the data published by other authors.

A reserve glucan of Megasphaera elsdenii was studied by methylation analysis before and after treatment with isoamylase. The results of this study indicate that the glucan is of the amylopectin-glycogen type.

Lipid molecular species compositions of chloroplast thylakoid membranes of mesophyll cells from Spinacia oleracea, Glycine max, Oryza sativa and Zea mays and of bundle sheath cells from Zea mays have been quantitatively determined. No significant difference in the lipid molecular species composition was found among the five membrane sources. The predominant molecular species of monogalactosyldiacylglycerol was the 1-linolenoyl parallel to 2-linolenoyl species. The 1-linolenoyl parallel to 2-linoenoyl and 1-palmitoyl parallel to 2-linolenoyl species were the major molecular species of digalactosyldiacylglycerol. 6-Sulfoquinovosyldiacylglycerol was mainly composed of the palmitoyl parallel to linolenoyl and palmitoyl parallel to lineolyl species. Almost all of the C-2 position of phosphatidylglycerol were esterified with the palmitoyl or delta 3-trans-hexadecenoyl residue. The molecular species compositions of phosphatidylcholine and phosphatidylinositol were basically similar to those of membranes in non-photosynthetic tissues.

The structure of the native dextran produced by Streptococcus sanguis ATCC 10558 was analyzed by g.l.c.-m.s. of the methylated alditol acetates derived from the polymer. The results indicate that the polymer contains D-glucosyl residues substituted at C-6 or C-3, or both, as well as unsubstituted D-glucosyl residues. These data aially purified dextransucrase on sucrose. The proportion of D-glucosyl residues substituted at C-3 is diminished in this case. It is concluded that several enzymes are involved in the dextran synthesis.

In order to study the structure of the methylglucose-containing polysaccharide (MGP) of Mycobacterium smegmatis by NMR spectroscopy, we have prepared the model compound O-alpha-D-glucopyranosyl-(1 leads to 2)-D-glyceric acid. This compound, which represents the aglycon-containing terminus of MGP, was made from leucorse [O-alpha-D-glucopyranosyl-(1 leads to 5)-D-fructopyranose] by successive treatment with sodium borohydride, lead tetraacetate, and hypobromite. The structure of O-alpha-D-glucopyranosy.-(1 leads to 2)-D-glyceric acid was confirmed by chemical and enzymic methods. 13C and 1H NMR spectra of this compound, together with spectra of several disaccharides, were obtained for future reference in the polysaccharide study. The nine resonances in the 13C spectrum were assigned by comparison with the spectrum of methyl alpha-D-glucopyranoside. Analysis of the 1H NMR spectrum showed that the two methylene protons on C-3 of the glycerate moiety were less equivalent in the sodium salt than in the acid. This may be attributable to hydrogen bonding between the carboxylate and the hydrogen atom of the glycerate 3-hydroxyl group.

Mycobacterial polymethyl polysaccharides, which bind long-chain fatty acids tightly [Ballou, C.E. (1981) Pure Appl. Chem. 53, 107-112], have been purified on a preparative scale by use of an affinity column packing consisting of (palmitoylamino)alkylsilyl silicate. The relatively large amount of material obtained in this way has allowed a study of the polysaccharide-lipid interactions at millimolar concentrations. The anomeric protons for all of the alpha 1----4-linked hexose units in the mycobacterial methylglucose polysaccharide occur in an envelope centered at delta 5.40, and, on titration with hexadecyltrimethylammonium bromide, the majority of these resonances move upfield to about delta 5.15. This shift is consistent with a change in the polysaccharide from a less ordered chain to one that has a significant proportion of helical conformation, and it is probable that the alkyl chain is included in the coiled portion of the polysaccharide in a manner analogous to the interaction of methylmannose polysaccharide with palmitic acid [Yabusaki, K. K., Cohen, R. E., & Ballou, C. E. (1979) J. Biol. Chem. 254, 7282-7286]. The native methylglucose lipopolysaccharide, which contains several short-chain acyl groups as well as an esterified octanoyl group, has an anomeric proton nuclear magnetic resonance spectrum similar to that of the methylglucose polysaccharide-hexadecyltrimethylammonium bromide complex. This suggests that the acylation stabilizes the polysaccharide chain in the same conformation it assumes when complexed to a long-chain lipid. Thus, acylation of the methylglucose polysaccharide could have an important role in regulating its shape and lipid-binding properties.

Water, sodium, potassium, ATP, amino acids, and sugars are not uniformly distributed in Rana pipiens oocytes. Concentration differences exist between nucleus (germinal vesicle) and ooplasm and between animal and vegetal ooplasmic regions. The mechanisms responsible for these differences were investigated using intracellular reference-phase (iRP) analysis. The iRP is an artificial "organelle" that has the solvent properties of a dilute salt solution and is in diffusional equilibrium with water and solutes present in other cellular compartments. Ooplasm/iRP solute distributions show that ooplasm differs from ordinary aqueous solutions--exhibiting both solute exclusion and solute binding. Yolk platelets are an important cause of this behavior, largely because their proteins are present as hydrate crystals, which are rich in anionic sites and which interact intensely with associated water. Because of yolk's abundance, it obscures the solvent and binding properties of ooplasmic ground substance. The oocyte nucleus is yolk and organelle free and the nuclear envelope is readily permeable. Consequently, nucleus/iRP solute concentration differences reflect the binding and solvent properties of nuclear ground substance. Nucleoplasm binds approximately 19 meq of potassium. Furthermore, the monosaccharides, 3-O-methylglucose, L-glucose, and D-xylose, are selectively excluded, their nucleus/iRP concentration ratios averaging about 0.7; ratios for other solutes studied are unity. We interpret monosaccharide exclusion to mean that nuclear ground substance water is different in its "instantaneous" structure from ordinary saline water. Because of this difference, hydrogen bond interaction between nuclear water and certain sterically restricted solutes, of which ringed monosaccharides are examples, is reduced. Some implications of modified ground substance water and selective solute exclusion are discussed.

The four major sulfolipids in the non-photosynthetic marine diatom, Nitzschia alba, were isolated in pure form and their structures were established spectrometrically and by identification of their hydrolysis products as (a) 24-methylene cholesterol sulfate, (b) 1-deoxyceramide-1-sulfonate, (c) phosphatidyl sulfocholine (a sulfonium analogue of phosphatidylcholine) and (d) sulfoquinovosyl diglyceride. The major characteristic fatty acids of the sulfolipids were: for the deoxyceramide sulfonate, 16 : 0 (26%) and 16 : 1-delta3-trans (64%); for the sulfonium analogue, 14 : 0 (30%), 18 : 1 (12%), 18 : 2 (8%), 20 : 5 (27%) and 22 : 6 (4%); and for the sulfoquinovosyl diglyceride (two species, respectively), 14 : 0 (9%, 22%), 16 : 0 (16%, 28%), 18 : 1 (8%, 22%), 20 : 5 (42%, 23%) and 22 : 6 (14%, 2%). Traces of lyso-derivatives of sulfoquinovosyl diglyceride and phosphatidyl sulfocholine were also detected. The deoxyceramide sulfonate and the phosphatidyl sulfocholine represent novel membrane lipid components not previously detected in other organisms. They may however have a widespread distribution in marine diatoms and perhaps in marine organisms generally.

The low-Mr lipopolysaccharide produced by Nocardia otitidis-cavirum is composed of 6-O-methyl-D-glucose (11 mol), D-glucose (8 mol) and 3-O-methyl-D-glucose (1 mol). Glyceric acid was also found as a constituent. Methylation and periodate oxidation analyses suggested that the backbone was formed by (1----4)-linked glucose and O-methylglucose residues. Glucose (1 mol) and 6-O-methylglucose (1 mol) served as branching points. Acetic acid was the major acyl substituent esterifying glucose residues. The lipopolysaccharide was isolated from the cytoplasmic fraction. Several other Nocardia strains were examined; all possessed the same lipopolysaccharide in their extracts.

Four products derived from Staphylococcus aureus cultures were partially purified and tested for inhibitory activity against staphylococcal phage. Phage inhibition, a specific stable phenomenon, was concentration dependent. All inhibitory products contained carbohydrate and amino acids, the most active (phage 73 lysate product) having a high carbohydrate content. Galactose, glucosamine, five or six amino acids, and possibly 3-O-methylglucose and a uronic acid were found as components in all active preparations. However, the exact nature of the active material remains undetermined.