The human bitter taste receptor, hTAS2R16, discriminates slight differences in the configuration of disaccharides

Sweetness and bitterness are key determinants of food acceptance and rejection, respectively. Sugars, such as sucrose and fructose, are generally recognized as sweet. However, not all sugars are sweet, and even anomers may have quite different tastes. For example, gentiobiose is bitter, whereas its anomer, isomaltose, is sweet. Despite this unique sensory character, the molecular basis of the bitterness of gentiobiose remains to be clarified. In this study, we used calcium imaging analysis of human embryonic kidney 293T cells that heterologously expressed human taste receptors to demonstrate that gentiobiose activated hTAS2R16, a bitter taste receptor, but not hT1R2/hT1R3, a sweet taste receptor. In contrast, isomaltose activated hT1R2/hT1R3. As a result, these anomers elicit different taste sensations. Mutational analysis of hTAS2R16 also indicated that gentiobiose and β-d-glucopyranosides, such as salicin share a common binding site of hTAS2R16.     

Gentiobiose: a novel oligosaccharin in ripening tomato fruit

Two neutral disaccharides, gentiobiose [beta- D-Glc p-(1-->6)- D-Glc] and nigerose [alpha- D-Glc p-(1-->3)- D-Glc], were detected in tomato ( Lycopersicon esculentum Mill.) pericarp and locule. Gentiobiose was present in the locule of green fruit and ripe fruit at 0.88 and 5.8 micro mol (kg fresh weight)(-1), respectively. When vacuum-infiltrated into green tomato fruit, exogenous gentiobiose (50 or 200 micro g per fruit) hastened the initiation of ripening (as judged by colour change) by 1-3 days relative to fruit that were infiltrated with glucose or isomaltose. Nigerose plus gentiobiose was particularly effective, but nigerose alone had no significant effect. The endogenous disaccharides were found to be present in the apoplastic fluid of the fruit, compatible with a proposed intercellular signalling role. The origin and metabolic fate of the disaccharides were investigated. Phenolic esters of these disaccharides were not detectable in tomato fruit and it is therefore unlikely that the free disaccharides were formed from a pool of such esters. An alternative possible biosynthetic origin, via transglycosylation, is discussed. When [(14)C]gentiobiose was vacuum-infiltrated into unripe or ripe fruit, the disaccharide remained intact for at least 1 h but was largely degraded within 24 h. The results suggest that gentiobiose is a new, naturally occurring oligosaccharin with a rapid turnover rate.     

Transglycosylation reaction and raw starch hydrolysis by novel carbohydrolase from<Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

A novel carbohydrolase, which is a DXAMase, containing both dextranase and amylase equivalent activities, was purified fromLipomyces starkeyi KSM22. The purified DXAMase was also found to hydrolyze cellobiose, gentiobiose, trehalose and melezitose, while disproportionation reactions were exhibited with various di-and tri-saccharides, such as maltose, isomaltose, gentiobiose, kojibiose, sophorose, panose, maltotriose, and isomaltotriose with various kinds of oligosaccharides produced as acceptor reaction products. Furthermore, the purified DXAMase hydrolyzed raw waxy rice starch and produced maltodextrin to the extent of 50% as a glucose equivalent.     

Effects of carbohydrate-structure changes on induced shifts in differential isotope-shift 13C-N.M.R.

Deuterium-induced, ¹³C-isotope shifts are shown to vary considerably from the initially predicted values calculated for ordinary pyranose and furanose sugars, when minor structural changes are introduced into the carbohydrate ring. Both substitution of C-OH groups or reduction of C-OH to CH₂ permitted the evaluation of γ effects of OD without the contribution of β-OD-induced shifting. The observed γ-shift values for these modified structures were twice as large as those previously noted. This difference is most probably due to favored salvation. Substitution of OH at C-6 led to the predicted loss of differential isotope-shift (d.i.s.) at C-6 because of its isolation from all β and γ OD groups. The ³¹P resonances of d-glucose 6-phosphate show downfield deuterium shifts. Based on d.i.s. values, new ¹³C-shift assignments are proposed for isomaltose and 2-amino-2-deoxy-α-d-glucose. A study of acidic carbohydrates has demonstrated that isotope shifts are somewhat larger for sp²-hybridized carbon atoms whose OH groups are acidic. Relaxation times for sp² carbon atoms isolated from dipolar interaction with protons were very long in D₂O relative to their relaxation time in the H₂O environment.     

Vacuum-ultraviolet circular dichroism study of saccharides by synchrotron radiation spectrophotometry

Vacuum-ultraviolet circular dichroism (VUVCD) spectra of five monosaccharides (D-glucose, D-mannose, D-galactose, D-xylose, and D-lyxose) and five disaccharides (maltose, isomaltose, cellobiose, gentiobiose, and lactose) were measured to 160 nm using a synchrotron-radiation VUVCD spectrophotometer in aqueous solution under high vacuum at 25 degrees C. Most of the saccharides show a positive peak with some shoulders at around 170 nm, except for D-galactose and lactose, which show two distinct negative peaks at around 165 and 177 nm. These spectra are influenced by such structural factors as alpha and beta anomers at C-1, axial and equatorial hydroxyl groups at C-2 and C-4, trans (T) and gauche (G) conformations of the hydroxymethyl group at C-5, and the type of glycosidic linkage. Deconvolution of the VUVCD spectra of D-glucose, D-mannose, and D-galactose into six independent Gaussian components for alpha-GG, alpha-GT, alpha-TG, beta-GG, beta-GT, and beta-TG conformations suggests that the alpha anomer has red-shifted spectra relative to the beta anomer, and that GG and GT conformations have positive and negative circular dichroism signs, respectively, while the sign for TG conformation is anomer dependent. These speculations from the deconvolution analyses are also supported by the VUVCD spectra of disaccharides. These results give new insight into the equilibrium conformations of saccharides, demonstrating the usefulness of synchrotron-radiation VUVCD spectroscopy.     

Germacranolides from Viguiera microphylla

Three new germacranolides, including two heliangolides (niveusin C-2′,3′-epoxide and 1,2-dehydroniveusin C-2′,3′-epoxide) and a germacrolide (3β-hydroxy-8β-epoxyangeloyloxycostunolide-1β,10α-epoxide) were isolated from Viguiera microphylla. Their structures were established by spectroscopic analyses, including extensive ¹H NMR and ¹³C NMR decoupling experiments and chemical transformations. X-ray diffraction analysis confirmed the structure of niveusin C-2′,3′-epoxide.     

Purification and Some Properties of β-Glucosidase from Streptomyces sp

β-Glucosidases I, II, and III were isolated from the culture filtrate of a Streptomyces sp. by ammonium sulfate fractionation, hydroxylapatite column chromatography, filtration on Bio-Gel P-100, and DE-52 column chromatography. β-Glucosidase III had a single active band on disc-gel electrophoresis. Its optimum pH and temperature for activity were 6.0 and 60°C, respectively. The isoelectric point and molecular weight of the enzyme were pH 4.5 and 45,000, respectively. From an experiment using ¹⁴C-labeled glucose, gentiobiose seemed to be formed from laminaribiose as isomaltose is formed from maltose by fungal α-glucosidase. The enzyme showed transglucosylation and produced gentiobiose from β-gluco-disaccharides and 4-O-β-d-glucopyranosyl-d-manno-pyranose (epicellobiose). The enzyme acted on phenolic β-d-glucosides to produce unknown transfer products.     

Oxidation and degradation of maltose and isomaltose by persulfate

Maltose and isomaltose were oxidized and degraded by ammonium persulfate in 66.7 mmol aqueous phosphate buffer (pH 7.1). Product and e.s.r. studies suggested that radicals formed by C-H abstraction at C-1 and C-4 were predominant for oxidation of maltose, whereas C-H abstraction of isomaltose proceeded preferentially at C-5 of the reducing glucose group.     

Thermodynamics of hydrolysis of disaccharides. Cellobiose, gentiobiose, isomaltose, and maltose

The thermodynamics of the enzymatic hydrolysis of cellobiose, gentiobiose, isomaltose, and maltose have been studied using both high pressure liquid chromatography and microcalorimetry. The hydrolysis reactions were carried out in aqueous sodium acetate buffer at a pH of 5.65 and over the temperature range of 286 to 316 K using the enzymes beta-glucosidase, isomaltase, and maltase. The thermodynamic parameters obtained for the hydrolysis reactions, disaccharide(aq) + H2O(liq) = 2 glucose(aq), at 298.15 K are: K greater than or equal to 155, delta G0 less than or equal to -12.5 kJ mol-1, and delta H0 = -2.43 +/- 0.31 kJ mol-1 for cellobiose; K = 17.9 +/- 0.7, delta G0 = -7.15 +/- 0.10 kJ mol-1 and delta H0 = 2.26 +/- 0.48 kJ mol-1 for gentiobiose; K = 17.25 +/- 0.7, delta G0 = -7.06 +/- 0.10 kJ mol-1, and delta H0 = 5.86 +/- 0.54 kJ mol-1 for isomaltose; and K greater than or equal to 513, delta G0 less than or equal to -15.5 kJ mol-1, and delta H0 = -4.02 +/- 0.15 kJ mol-1 for maltose. The standard state is the hypothetical ideal solution of unit molality. Due to enzymatic inhibition by glucose, it was not possible to obtain reliable values for the equilibrium constants for the hydrolysis of either cellobiose or maltose. The entropy changes for the hydrolysis reactions are in the range 32 to 43 J mol-1 K-1; the heat capacity changes are approximately equal to zero J mol-1 K-1. Additional pathways for calculating thermodynamic parameters for these hydrolysis reactions are discussed.     

Interaction of (1→4)- and (1→6)-linked disaccharides with the fenton reagent under physiological conditions

Reactions of (1→4)- and (1→6)-linked disaccharides, mainly of maltose and isomaltose, with the Fenton reagent under physiological conditions were studied. Chemical characterization of oxidation products was conducted by g.l.c. and g.l.c.-m.s. of their trimethylsilyl derivatives, and the results demonstrated that (1→6)-linked disaccharides are more reactive with the hydroxyl radical (·OH) generated by the Fenton reagent than (1→4)-linked disaccharides. About 35–40% of (1→6)-and 15–20% of (1→4)-linked disaccharides were oxidatively degraded to smaller molecules after incubation for 24 h. Of the four disaccharides examined, namely, maltose, isomaltose, cellobiose, and gentiobiose, the α-(1→6)-linked disaccharide isomaltose exhibited the highest reactivity, whereas the β-(1→4)-linked disaccharide cellobiose showed the lowest. These results suggest the existence of a relationship between the configuration of the glycosidic linkage and the reactivity with ·OH in aqueous solution.     

Similarity between 5'- and 3'-terminal nucleotide sequences and double-stranded RNA-derived sequences of eukaryotic mRNA

It has been reported recently that parts of the nucleotide sequences present in the 5′- and 3′-terminal regions of cytoplasmic mRNA are derived from double-stranded hairpin structures of heterogeneous nuclear RNA—a putative mRNA precursor (Naora, 1979). In order to explore the nature of double-stranded hairpin structures, using the sequencing data of human and rabbit globin mRNA and hen ovalbumin mRNA, we examined the following possibility: that certain regions of both the 5′- and 3′-terminal nucleotide sequences of mature mRNA were present in double-stranded hairpin structures covalently linked to both sides of the message sequence in the precursor mRNA molecule and that these double-stranded hairpin structures are similar to each other. The results support the above possibility by showing substantial similarity of nucleotide sequences between the 5′- and 3′-terminal regions of these mRNAs in terms of the formation of similar double-stranded hairpin structures.     

Cyclic glycolipids from glandular trichome exudates of Cerastium glomeratum

Fourteen cyclic glycolipids, named glomerasides A–N, have been isolated from the glandular trichome exudate of Cerastium glomeratum (Caryophyllaceae). Their structures were determined by spectroscopic analysis of the glycolipids, as well as by application of the Ohrui–Akasaka method to the fatty acid methyl esters derived from the glycolipids and GCMS studies of trimethylsilyl ether derivatives of the methyl esters. The various glomerasides have a glycosidic linkage between the anomeric hydroxy group of the glucose and the C-11, C-10 or C-9 positions of the docosanoyl moiety. They also contained an ester linkage between the C-6 hydroxy group of the glucose ring and the carboxyl group of the oxygenated fatty acid to form their macrocyclic structures. The glucose moiety was optionally acetylated and/or malonylated at the C-2 or C-3 hydroxy groups. Among these compounds, the 1,6′-cyclic ester of 11(R)-(2-O-acetyl-β-d-glucopyranosyloxy)docosanoic acid (glomeraside D) was the most abundant (25%).     

Neolignans and Sesquiterpenoid from Piper yunnanense

Two biphenyl-type neolignans with a rare dibenzofuran skeleton, including a new one piyunneolignan A ( 1 ) and a known one piperneolignan D ( 2 ), together with a new sesquiterpenoid piyunin A ( 3 ), were isolated from the leaves and twigs of Piper yunnanense. Their structures were established on the basis of comprehensive spectroscopic data analysis and electronic circular dichroism (ECD) calculation. Piyunneolignan A ( 1 ) featured a rare C-2−C-2′/C-3−O−C-3′ linkage. Compounds 1 – 3 were evaluated for their antimicrobial and cytotoxic activities against a panel of bacteria, fungi, and human cancer cell lines, respectively.     

Structural characterization of enzymatically synthesized dextran and oligosaccharides from Leuconostoc mesenteroides NRRL B-1426 dextransucrase

Leuconostoc mesenteroides NRRL B-1426 dextransucrase synthesized a high molecular mass dextran (>2 × 10⁶ Da) with ～85.5% α-(1→6) linear and ～14.5% α-(1→3) branched linkages. This high molecular mass dextran containing branched α-(1→3) linkages can be readily hydrolyzed for the production of enzyme-resistant isomalto-oligosaccharides. The acceptor specificity of dextransucrase for the transglycosylation reaction was studied using sixteen different acceptors. Among the sixteen acceptors used, isomaltose was found to be the best, having 89% efficiency followed by gentiobiose (64%), glucose (30%), cellobiose (25%), lactose (22.5%), melibiose (17%), and trehalose (2.3%) with reference to maltose, a known best acceptor. The β-linked disaccharide, gentiobiose, showed significant efficiency for oligosaccharide production that can be used as a potential prebiotic.     

我国两株登革2型病毒5′和3′端非编码区序列测定及二级结构分析

 为测定我国两株临床症状、乳鼠神经毒力不同的登革 2型病毒流行株 5′和 3′端非编码区序列 (untranslated region,UTR) ,分析二级结构差异与毒力变化的关系 ,分别从 D2 - 0 4、D2 - 44株感染的 C6/ 36细胞及鼠脑中提取总 RNA.以该 RNA为模板 ,利用 RACE法 ,分别扩增了 D2 - 0 4、D2 -44株的 5′和 3′末端 c DNA片段 .将其分别与 p GEM- T载体连接得到重组质粒 ,测定上述 c DNA插入片段的序列 .用 RNAdraw软件预测 D2 - 0 4、D2 - 44株 5′和 3′端非编码区的二级结构 .D2 - 0 4、D2 -44株 5′端和 3′端非编码区分别有 96和 454个核苷酸 .其中 5′非编码区 59位 C(D2 - 0 4 )→T(D2 -44 ) ,使 D2 - 44二级结构稳定性下降 ;3′端非编码区有 1 5个核苷酸不同 ,其中 T(355)→ A,T(32 6)→ G引起了所在位置二级结构自由能变化 ,且分别位于两个保守序列区 (conserved sequence,CS)CS1、CS2 A.这些位点变化可能与毒力有关 .     

Mechanism of Formation of Gentiobiose from Curdlan by Enzymes of Streptomyces sp.

The enzyme system (culture filtrate) from Streptomyces sp. W19-1 formed gentiobiose from curdlan (β-1,3-glucan). The mechanism of the formation of gentiobiose was investigated in this study.

Two kinds of enzymes, β-1,3-glucanase and β-glucosidase (transglucosidase), were isolated from the culture filtrate of the strain by hydroxylapatite column chromatography. The β-1,3-glucanase hydrolyzed curdlan to glucose and laminari-oligosaccharides, and the β-glucosidase formed gentiobiose by transglucosylation from the resultant laminari-oligosaccharides, especially laminaribiose. The two enzymes took part in the formation of gentiobiose from curdlan.     

Tritium incorporation in saccharinic acid production

d-Glucose, turanose, maltose, melibiose, isomaltose, and inulin were each degraded in aqueous barium tritioxide under nitrogen. The saccharinic acids subsequently isolated contained carbon-bound tritium. Maltose, similarly degraded in the presence of air, gave, in addition, O-d-glucosylaldonic acids which were not tritium-labelled. The application of these observations to the structural analysis of oligosaccharides is discussed.     

Crystal Structure and Computational Modeling of the Fab Fragment from a Protective Anti-Ricin Monoclonal Antibody

Background

Many antibody crystal structures have been solved. Structural modeling programs have been developed that utilize this information to predict 3-D structures of an antibody based upon its sequence. Because of the problem of self-reference, the accuracy and utility of these predictions can only be tested when a new structure has not yet been deposited in the Protein Data Bank.

Methods

We have solved the crystal structure of the Fab fragment of RAC18, a protective anti-ricin mAb, to 1.9 Å resolution. We have also modeled the Fv structure of RAC18 using publicly available Ab modeling tools Prediction of Immunoglobulin Structures (PIGS), RosettaAntibody, and Web Antibody Modeling (WAM). The model structures underwent energy minimization. We compared results to the crystal structure on the basis of root-mean-square deviation (RMSD), template modeling score (TM-score), Z-score, and MolProbity analysis.

Findings

The crystal structure showed a pocket formed mainly by AA residues in each of the heavy chain complementarity determining regions (CDRs). There were differences between the crystal structure and structures predicted by the modeling tools, particularly in the CDRs. There were also differences among the predicted models, although the differences were small and within experimental error. No one modeling program was clearly superior to the others. In some cases, choosing structures based only on sequence homology to the crystallized Ab yielded RMSDs comparable to the models.

Conclusions

Molecular modeling programs accurately predict the structure of most regions of antibody variable domains of RAC18. The hypervariable CDRs proved most difficult to model, particularly H chain CDR3. Because CDR3 is most often involved in contact with antigen, this defect must be considered when using models to identify potential contacts between antibody and antigen. Because this study represents only a single case, the results cannot be generalized. Rather they highlight the utility and limitations of modeling programs.     

Homoisoflavonoids from the medicinal plant Portulaca oleracea

Four homoisoflavonoids named portulacanones A−D, identified as 2′-hydroxy- 5,7-dimethoxy-3-benzyl-chroman-4-one, 2′-hydroxy-5,6,7-trimethoxy-3-benzyl-chroman-4-one, 5,2′-dihydroxy-6,7-dimethoxy-3-benzyl-chroman-4-one, and 5,2′-dihydroxy-7-methoxy-3-benzylidene-chroman-4-one, were isolated from aerial parts of the plant Portulaca oleracea along with nine other known metabolites. Their structures were established on the basis of extensive spectroscopic analyses. Portulacanones A−D is the first group of homoisoflavonoids so far reported from the family Portulacaceae. They represent a rare subclass of homoisoflavonoids in nature with a structural feature of a single hydroxyl group substituted at C-2′ rather than at C-4′ in ring B of the skeleton. Three homoisoflavonoids and the known compound 2,2′-dihydroxy-4′,6′-dimethoxychalcone selectively showed in vitro cytotoxic activities towards four human cancer cell lines. Especially 2,2′-dihydroxy-4′,6′-dimethoxychalcone showed cytotoxic activity against cell line SGC-7901 with an IC₅₀ value of 1.6 μg/ml, which was more potent than the reference compound mitomycin C (IC₅₀ 13.0 μg/ml).     

Three new isoflavonoids with antioxidant properties from Ptycholobium contortum (N.E.Br.) Brummitt (Leguminosae)

Two new pterocarpans, seputhecarpan A (1) and B (2) and a new isoflavanone seputheisoflavone (3) were isolated from the roots of Ptycholobium contortum. Seputhecarpan A and B were identified respectively as 9-hydroxylated and 9,16-dihydroxylated pterocarpans possessing a linear (C-2/C-3) isopropenyldihydrofuran substituent and seputheisoflavone as 5′-α,α-dimethylallyl-4′,5,7-trihydroxy-2′-methoxyisoflavanone. In addition, four known compounds, genistein, isoliquiritigenin, thonningiol and (−)-medicarpin were isolated. Their structures were elucidated using intensive 1D and 2D NMR spectroscopies, CD and MS data. The new compounds were assayed for their antimicrobial, α-glucosidase and antioxidant activities. If the antimicrobial and α-glucosidase inhibitory properties were weak, all the tested compounds exhibited antioxidant activities and seputhecarpan A (1) was most effective as an antioxidant with an EC50 value of 0.030 mg/ml.