A high-temperature water vapor equilibration method to determine non-exchangeable hydrogen isotope ratios of sugar, starch and cellulose.

The analysis of the non-exchangeable hydrogen isotope ratio (δ2 Hne ) in carbohydrates is mostly limited to the structural component cellulose, while simple high-throughput methods for δ2 Hne values of non-structural carbohydrates (NSC) such as sugar and starch do not yet exist. Here, we tested if the hot vapor equilibration method originally developed for cellulose is applicable for NSC, verified by comparison with the traditional nitration method. We set up a detailed analytical protocol and applied the method to plant extracts of leaves from species with different photosynthetic pathways (i.e., C3 , C4 and CAM). δ2 Hne of commercial sugars and starch from different classes and sources, ranging from -157.8 to +6.4‰, were reproducibly analysed with precision between 0.2‰ and 7.7‰. Mean δ2 Hne values of sugar are lowest in C3 (-92.0‰), intermediate in C4 (-32.5‰) and highest in CAM plants (6.0‰), with NSC being 2 H-depleted compared to cellulose and sugar being generally more 2 H-enriched than starch. Our results suggest that our method can be used in future studies to disentangle 2 H-fractionation processes, for improving mechanistic δ2 Hne models for leaf and tree-ring cellulose and for further development of δ2 Hne in plant carbohydrates as a potential proxy for climate, hydrology, plant metabolism and physiology.

Revisiting the Language of Glycoscience: Readers, Writers and Erasers in Carbohydrate Biochemistry.

The roles of carbohydrates in nature are many and varied. However, the lack of template encoding in glycoscience distances carbohydrate structure, and hence function, from gene sequence. This challenging situation is compounded by descriptors of carbohydrate structure and function that have tended to emphasise their complexity. Herein, we suggest that revising the language of glycoscience could make interdisciplinary discourse more accessible to all interested parties.

Presence of carbohydrate binding modules in extracellular region of class C G-protein coupled receptors (C GPCR): An in silico investigation on sweet taste receptor.

Sweet taste receptor (STR) is a C GPCR family member and a suggested drug target for metabolic disorders such as diabetes. Detailed characteristics of the molecule as well as its ligand interactions mode are yet considerably unclear due to experimental study limitations of transmembrane proteins. An in silico study was designed to find the putative carbohydrate binding sites on STR. To this end, α-D-glucose and its α-1,4-oligomers (degree of polymerization up to 14) were chosen as probes and docked into an ensemble of different conformations of the extracellular region of STR monomers (T1R2 and T1R3), using AutoDock Vina. Ensembles had been sampled from an MD simulation experiment. Best poses were further energy-minimized in the presence of water molecules with Amber14 forcefield. For each monomer, four distinct binding regions consisting of one or two binding pockets could be distinguished. These regions were further investigated with regard to hydrophobicity and hydrophilicity of the residues, as well as residue compositions and non-covalent interactions with ligands. Popular binding regions showed similar characteristics to carbohydrate binding modules (CBM). Observation of several conserved or semi-conserved residues in these binding regions suggests a possibility to extrapolate the results to other C GPCR family members. In conclusion, presence of CBM in STR and, by extrapolation, in other C GPCR family members is suggested, similar to previously proposed sites in gut fungal C GPCRs, through transcriptome analyses. STR modes of interaction with carbohydrates are also discussed and characteristics of non-covalent interactions in C GPCR family are highlighted.

Discoveries of the structures of sialic acid and CMP-sialic acid (1957-1960): a letter from Saul Roseman.

Sialic acids have a special place in vertebrate glycobiology, where they constitute the dominant terminal saccharides on many cell surface glycans. From early studies that identified sialoglycans as receptors for important pathogens and toxins to more recent discoveries of sialic acid-binding proteins essential for immune system (and other) functions in humans, sialic acids and sialoglycans have become cornerstones in understanding vertebrate glycobiology and pathology. During a remarkable 3-year period in the late 1950s, a newly minted postdoctoral fellow (Donald G. Comb) and his young mentor (Saul Roseman) made a surprising series of discoveries that put sialic acid research on sound chemical and biochemical footing. A detailed personal letter written by Dr. Roseman that describes this period of intense sialic acid discovery, complete with inserted figures, was given to one of us (Y.C.L.) several years later. The text and figures of this letter provide a look back at the enthusiasm, rigor and serendipity that led to their important findings through the eyes of one of the key figures in sialic acid research.

doGlycans-Tools for Preparing Carbohydrate Structures for Atomistic Simulations of Glycoproteins, Glycolipids, and Carbohydrate Polymers for GROMACS.

Carbohydrates constitute a structurally and functionally diverse group of biological molecules and macromolecules. In cells they are involved in, e.g., energy storage, signaling, and cell-cell recognition. All of these phenomena take place in atomistic scales, thus atomistic simulation would be the method of choice to explore how carbohydrates function. However, the progress in the field is limited by the lack of appropriate tools for preparing carbohydrate structures and related topology files for the simulation models. Here we present tools that fill this gap. Applications where the tools discussed in this paper are particularly useful include, among others, the preparation of structures for glycolipids, nanocellulose, and glycans linked to glycoproteins. The molecular structures and simulation files generated by the tools are compatible with GROMACS.

Bimodal concentration-dependent reactivity pattern of a glycosyl donor: Is the solution structure involved?

Changes in concentration (0.001-0.1 M) of an arabinofuranosyl donor (1) have been shown to modulate the temperature T at which activation of 1 occurs (from -23 °C to +7 °C), the reaction time (from 1.5 h to 3 days) and the yield of the disaccharide formed (from 14% to 82%). At concentrations exceeding 0.01 M, these parameters, as well as the specific optical rotation of the solution of 1, virtually do not depend on concentration suggesting formation of reacting species (supramers) of glycosyl donor with similar structures, hence reactivities, but considerably different from those formed in more dilute solutions. The found critical concentration (0.01 M) separates two concentration ranges of reaction solutions corresponding to two types of solution structure that are featured by the presence of fundamentally different supramers of glycosyl donor, which have distinct chemical properties. These results allow a fresh look at the problems of reactivity of chemical compounds and selectivity of the reactions in which they participate.

AuCl3-AgOTf promoted O-glycosylation using anomeric sulfoxides as glycosyl donors at room temperature.

Activation of sulfoxide as glycosyl donors using AuCl3/AgOTf reagent system has been described. Under optimal reaction conditions, both armed and disarmed glycosyl sulfoxide donors were found to react with a range of primary, secondary, and tertiary alcohol acceptors, and sugar derived glycosyl acceptors to afford the corresponding glycosides in moderate to good yields with predictable selectivity. The reactions are quick (20-60 min), facile at room temperature and the reactions conditions tolerate acid sensitive groups.

Preparation of drug-in-cyclodextrin-in-liposomes at a large scale using a membrane contactor: Application to trans-anethole.

The present study aimed to prepare liposomes loaded with cyclodextrin/drug inclusion complexes at a pilot scale based on the ethanol injection technique. Anethole (ANE), a major component of anise and fennel essential oils, was used as a model of a volatile and highly hydrophobic drug. Membrane contactor (600mL) and a pilot plant (3L) were used for liposome production. The liposome preparations obtained were characterized for size, polydispersity index, zeta potential, morphology, stability and ANE release rate. All experimental set-ups were shown to be appropriate for the preparation of small, multilamellar vesicles with narrow size distribution and good stability at 4°C. The drug release study showed that only a small amount of ANE was released from liposome formulations after 21days of storage at 4°C. The loading rate of ANE was higher when ethanol was evaporated directly on the pilot plant compared to a rotary evaporation.

Cellulose oligomers production and separation for the synthesis of new fully bio-based amphiphilic compounds.

Cellulose oligomers are water-soluble, on the contrary to cellulose, which greatly increase their application range. In this study, cellulose oligomers were obtained from the acidic hydrolysis of cellulose with phosphoric acid. The global yield in water-soluble oligomers was around 23% with polymerization degree (DP) ranging from 1 to 12. The cellulose oligomers DP distribution was successfully reduced by differential solubilisation in methanol as one of the goals of this work was to avoid the use of a time-consuming full chromatographic separation. The methanol-soluble oligomers were mainly low DP (≤3). The oligomers of higher molar mass, composed of 42% of cellotetraose and 36% of cellopentaose, were then functionalized and coupled with stearic acid through azide-alkyne click chemistry to obtain amphiphilic compounds. The self-assembly of these new bio-based compounds was finally investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM) and their critical micellar concentration (CMC) was found to be in the same range as alkylmaltosides and alkylglucosides.

A proposal for teaching undergraduate chemistry students carbohydrate biochemistry by problem-based learning activities.

This article presents a problem-based learning (PBL) approach to teaching elementary biochemistry to undergraduate students. The activity was based on "the foods we eat." It was used to engage students' curiosity and to initiate learning about a subject that could be used by the future teachers in the high school. The experimental activities (8-12 hours) were related to the questions: (i) what does the Benedict's Reagent detect? and (ii) What is determined by glucose oxidase (GOD)? We also ask the students to compare the results with those obtained with the Lugol reagent, which detects starch. Usually, students inferred that the Benedict reagent detects reducing sugars, while GOD could be used to detect glucose. However, in GOD assay, an open question was left, because the results could be due to contamination of the sugars (particularly galactose) with glucose. Though not stressed, GOD does not oxidize the carbohydrates tested and all the positive results are due to contamination. The activities presented here can be easily done in the high school, because they are simple and non-expensive. Furthermore, in the case of Benedict reaction, it is possible to follow the reduction of Cu (II) "macroscopically" by following the formation of the brick-orange precipitate. The concrete observation of a chemical reaction can motivate and facilitate students understanding about chemistry of life.

Variability of cell wall polysaccharides composition and hemicellulose enzymatic profile in an apple progeny.

The genetic variability of apple cell walls polysaccharides chemical composition and structure was assessed in a progeny of 141 individuals harvested over 2 years. The variability of the hemicelluloses oligosaccharides released by glucanase was analyzed by MALDI-TOF MS. The genetic contribution was distinguished from harvest year as well as from parental crossing patterns and scab resistance selection. Results showed that harvest year had a major impact on cell wall polysaccharide composition and structure. Within each harvest, genetic effect impact more significantly cell wall polysaccharide chemistry than does reciprocal crossing or early scab selection. Uronic acids, glucose, galactose and xylose contents as well as some glucomannan and xyloglucan structures have a high heritability. This first cell wall chemotyping of an apple progeny opens the way for future searches of genetic markers for the chemical variability of cell wall polysaccharides.

An evaluation of peptide-bond isosteres.

Peptide-bond isosteres can enable a deep interrogation of the structure and function of a peptide or protein by amplifying or attenuating particular chemical properties. In this Minireview, the electronic, structural, and conformational attributes of four such isosteres-thioamides, esters, alkenes, and fluoroalkenes-are examined in detail. In particular, the ability of these isosteres to partake in noncovalent interactions is compared with that of the peptide bond. The consequential perturbations provide a useful tool for chemical biologists to reveal new structure-function relationships, and to endow peptides and proteins with desirable attributes.

Trehalose has a protective effect on human brain-type creatine kinase during thermal denaturation.

We investigated the effects of trehalose on thermal inactivation and aggregation of human brain-type creatine kinase (hBBCK) in this study. In the presence of 1.0 M trehalose, the midpoint temperature of thermal inactivation (T (m)) of hBBCK increased by 4.6 °C, and the activation energy (E (a)) for thermal inactivation increased from 29.7 to 41.1 kJ mol(-1). Intrinsic fluorescence spectra also showed an increase in the apparent transition temperature (T (1/2)) of hBBCK from 43.0 °C to 46.5 °C, 47.7 °C, and 49.9 °C in 0, 0.6, 0.8, and 1.2 M trehalose, respectively. In addition, trehalose significantly blocked the aggregation of hBBCK during thermal denaturation. Our results indicate that trehalose has potential applications as a thermal stabilizer and may aid in the folding of other enzymes in addition to hBBCK.

Análise da expressão de carboidratos no estroma do carcinoma ductal invasivo da mama e sua correlação com dados clínicos e histopatológicos / Carbohydrates expression analysis in stroma of breast invasive ductal carcinoma and its correlation with clinical and histopathological datas

Objetivos: Analisar o perfil de carboidratos no estroma do carcinoma ductal invasivo, usando histoquímica com lectinas, e correlacionar os achados com dados clínicos e histopatológicos. Métodos: Estudo retrospectivo baseado na análise dos casos de 30 pacientes diagnosticados com carcinoma ductal invasivo durante o período de Maio a Outubro de 2008, As mostras tumorais foram submetidas a histoquímica com lectinas, na qual foram utilizadas as lectinas conjugadas à peroxidase: PNA-Per, 25 mg/mL, UEA-I-Per, 40 mg/mL e Con A-Per, 40 mg/mL específicas para D-galactose, L-fucose e glicose/manose, respectivamente. Posteriormente, os achados histoquímicos foram relacionados com os dados clínicos e histopatológicos (idade, invasão linfonodal, status para p53, tamanho tumoral e variante histológica) das pacientes. Resultados: Na histoquímica com lectinas, a Con A reconheceu a matriz extracelular em 53,33% dos casos, enquanto a PNA e a UEA-I reconheceram 30 e 40%. Já o endotélio de vasos sanguíneos foi mais reconhecido pela UEA-I (40% dos casos) que a Con A (10% dos casos). Biópsias de pacientes cuja matriz extracelular foi reconhecida pela Con A foram positivas também para p53 (p = 0,029), e todos os casos positivos para p53 (p = 0,041) tiveram os respectivos endotélios vasculares reconhecidos por essa lectina. O reconhecimento do estroma pela UEA-I em relação à variante histológica pouco diferenciada mostrou-se significativo (p = 0,034) em relação às demais variáveis analisadas. Conclusões: Os resultados indicaram o potencial das lectinas como sondas eficientes e os glicoconjugados de estroma como biomarcadores ou fonte de informações da biologia tumoral do carcinoma ductal invasivo.

Objectives: To analyze the carbohydrates profile in the stroma of invasive ductal carcinoma, using lectin histochemistry, and correlate the findings with clinical and histopathological data. Methods: Retrospective study based on analysis of 30 cases from patients previously diagnosed with invasive ductal carcinoma during the period from May to October 2008. Tumour samples were subjected to the lectin histochemistry technique, in which the peroxidase-conjugated lectins were used: PNA-Per, 25 mg/mL, UEA-I-Per, 40 mg/mL e Con A-Per, 40 mg/mL specifics for D-galactose, L-fucose and glucose/mannose respectively. Subsequently the histochemical findings were correlated with clinical and histopathological data (age, lymph node invasion, status to p53, tumor size and histological variant) of the patients. Results: In lectin histochemistry, the Con A stained the extracellular matriz of 53.33% of cases, while the PNA and UEA-I recognized 30 and 40% of the ECM, respectively. The blood vessels endothelium was most recognized by the lectin UEA-I (40%) than Con A (10%). Biopsies from patients with ECM stained by Con A were also positive for p53 (p = 0.029), and all positive cases to p53 (p = 0.041) had the respective endothelium recognized by the same lectin. The ECM staining by the UEA-I in relation to poorly differentiated histological variant showed a significant difference (p = 0.034) between the variants analyzed. Conclusions: The results indicated the potential of lectins as efficient probres and stromal glycoconjugates as biomarker or source of information in the diagnosis of invasive ductal carcinoma.

Aclimatação de gengibre (Zingiber officinale Roscoe) e a relação com carboidratos endógenos / Acclimation of ginger (Zingiber officinale Roscoe) and its relation to endogenous carbohydrates

O objetivo deste trabalho foi determinar as variações dos teores de açúcares solúveis totais e de reserva nas diferentes fases de desenvolvimento de mudas de gengibre. Foram avaliados os parâmetros de sobrevivência, teores de açúcares solúveis totais e de reserva na fase de micropropagação, aclimatação e cultivo em campo. A sobrevivência das plantas no processo de aclimatação aos 60 dias foi de 100 por cento, enquanto que as plantas em condições de campo apresentaram um índice de sobrevivência de 80 por cento aos 60 dias de cultivo. Os teores médios de amido, no primeiro ano de cultivo, foram superiores nas plantas micropropagadas, tanto nas folhas (303,19±0,17 mgGlu gMF-1 mgGlu gMF-1) quanto nas raízes (3341,59±1,24 mgGlu gMF-1). Foram observados altos teores médios de amido nos rizomas (164,91±2,4 mgGlu gMF-1) e gemas (190,88±0,25 mgGlu gMF-1). Os teores médios de açúcares solúveis totais apresentaram um decréscimo nas plantas in vitro com valores médios de 86,56±0,55 mgGlug MF-1 e 94,26±0,40 mgGlu gMF-1 para folhas e raízes, respectivamente. Obteve-se um aumento nesses valores nas plantas aclimatadas em casa de vegetação, tanto nas folhas (168,22±0,77 mgGlu gMF-1) quanto nas raízes (189,68±0,70 mgGlu gMF-1), sendo superadas pelos teores médios de folhas e raízes das plantas cultivadas a campo, cujos valores foram 227,51±0,8 e 183,97±0,32 mgGlu gMF-1, respectivamente. No segundo ciclo de cultivo observou-se um aumento nos teores médios de amido nos rizomas, gemas e raízes, com valores de 210,87±0,85 mgGlu gMF-1 , 203,45±0,91 mgGlu gMF-1 e 201 ±0,69 mgGlu gMF-1 e um declínio nos teores médios nas folhas (41,55±0,88 mgGlu gMF-1) com uma queda gradativa até os 240 dias de cultivo. Porém, em relação à quantidade de açúcares solúveis totais as folhas apresentaram-se com teores médios altos no período inicial, com um declínio ao longo do desenvolvimento em casa de vegetação. O oposto foi verificado para as raízes (225,29±0,75 mgGlu gMF-1), rizoma (250,08±0,93 mgG...

The aim of this work was to determine the variations in total soluble sugar and starch levels during the different development stages of ginger seedlings. Survival parameters, as well as total soluble sugar and starch levels, were evaluated during micropropagation, acclimation and field cultivation phases. The survival of plants was 100 percent after 60 days of acclimation, whereas under field conditions such value was 80 percent after the same period. In the first year of cultivation, mean starch levels were higher in micropropagated plants, both in leaves (303.19 ± 0.17 mgGlu gFM-1) and in roots (3341.59 ± 1.24 mg Glu gFM-1). High mean starch levels were detected in rhizomes (164.91 ± 2.4 mg Glu gFM-1) and buds (190.88 ± 0.25 mgGlu gMF-1). The mean levels of total soluble sugars were lower in plants in vitro: 86.56 ± 0.55 mgGlu gFM-1 and 94.26 ± 0.40 mgGlu gFM-1 in leaves and roots, respectively. Higher values were obtained for plants acclimatized in greenhouse, both in leaves (168.22 ± 0.77 mgGlu gFM-1) and roots (189.68 ± 0.70 mgGlu gFM-1); these values were inferior to those detected in leaves and roots of plants grown in the field, which were 227.51 ± 0.8 and 183.97 ± 0.32 mgGlu gFM-1, respectively. In the second cultivation cycle, mean starch levels in rhizomes, buds and roots increased to 210.87 ± 0.85 mgGlu gFM-1, 203.45 ± 0.91 mgGlu gFM-1 and 201 ± 0.69 mgGlu gFM-1, respectively, whereas in leaves these values progressively decreased (41.55 ± 0.88 mgGlu gFM-1) until 240 days of cultivation. However, leaves presented high mean total soluble sugar levels in the initial period, with a posterior decline over the development in greenhouse. The opposite was observed in roots (225.29 ± 0.75 mgGlu gFM-1), rhizomes (250.08 ± 0.93 mgGlu gFM-1) and buds (225.75 ± 0.80 mgGlu gFM-1). Based on the high survival rate of acclimatized plants and the higher levels of total soluble sugars and starch in early stages of cultivation, acclimation is recommended to assure...