Glucosinolate structures in evolution

By 2000, around 106 natural glucosinolates (GSLs) were probably documented. In the past decade, 26 additional natural GSL structures have been elucidated and documented. Hence, the total number of documented GSLs from nature by 2011 can be estimated to around 132. A considerable number of additional suggested structures are concluded not to be sufficiently documented. In many cases, NMR spectroscopy would have provided the missing structural information. Of the GSLs documented in the past decade, several are of previously unexpected structures and occur at considerable levels. Most originate from just four species: Barbarea vulgaris, Arabidopsis thaliana, Eruca sativa and Isatis tinctoria. Acyl derivatives of known GSLs comprised 15 of the 26 newly documented structures, while the remaining exhibited new substitution patterns or chain length, or contained a mercapto group or related thio-functionality.GSL identification methods are reviewed, and the importance of using authentic references and structure-sensitive detection methods such as MS and NMR is stressed, especially when species with relatively unknown chemistry are analyzed. An example of qualitative GSL analysis is presented with experimental details (group separation and HPLC of both intact and desulfated GSLs, detection and structure determination by UV, MS, NMR and susceptibility to myrosinase) with emphasis on the use of NMR for structure elucidation of even minor GSLs and GSL hydrolysis products. The example includes identification of a novel GSL, (R)-2-hydroxy-2-(3-hydroxyphenyl)ethylglucosinolate.Recent investigations of GSL evolution, based on investigations of species with well established phylogeny, are reviewed. From the relatively few such investigations, it is already clear that GSL profiles are regularly subject to evolution. This result is compatible with natural selection for specific GSL side chains. The probable existence of structure-specific GSL catabolism in intact plants suggests that biochemical evolution of GSLs has more complex implications than the mere liberation of a different hydrolysis product upon tissue disruption.     

Quantitative microanalysis of oligosaccharides by high-performance liquid chromatography

The rapid separation and quantitative determination of per-O-benzoyl oligosaccharides were obtained using high performance, liquid chromatography. Oligosaccharides were completely O-benzoylated without concomitant N-benzoylation of acetamidodeoxyhexoses. Benzoylation prior to analysis allowed a quantitative determination of picomolar amounts because the absorbance at 230 nm of these derivatives is directly proportional to the number of benzoyl groups present. Separation by normal and reversed-phase chromatography was demonstrated, and the best resolution was obtained on an Ultrasphere octyl column. Excellent separations of oligosaccharides containing up to 10 sugar residues present in mannosidosis urine and of malto-oligosaccharides containing up to 15 sugar residues present in Karo syrup were achieved within an analysis time of 30 min. Anomers of maltose, maltotriose, and maltotetraose were separated; for this reason, reduction of complex samples prior to analysis is advisable. The effect of linkage configuration on retention time was tested with reduced, α-linked di- and tri-glucopyranosides. The presence of an acetamidodeoxyhexose residue in an oligosaccharide significantly reduced its retention time, whereas branching had the opposite effect. A linear response was obtained for the injection of 1–600 pmol of raffinose, and the detection limit was 0.5 pmol. Derivatization and analysis of raffinose was shown to yield reproducible results within the range 0.01–1 μmol, and, with special precautions to minimize losses, as little as 100 pmol could be analyzed successfully.     

Nitrile-specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana

Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30–45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinase-catalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and properties of AtNSPs and related proteins and the biological implications.Brassicaceae plants such as oilseed rape (Brassica napus), turnip (Brassica rapa), and white mustard (Sinapis alba) as well as the model plant Arabidopsis thaliana contain a group of secondary metabolites known as glucosinolates (GSLs)² (1, 2). These are β-thioglucoside N-hydroxysulfates with a sulfur-linked β-d-glucopyranose moiety and a variable side chain that is derived from one of eight amino acids or their methylene group-elongated derivatives. Aliphatic GSLs are derived from alanine, leucine, isoleucine, valine, or predominantly methionine. Tyrosine or phenylalanine give aromatic GSLs, and tryptophan-derived GSLs are called indolic GSLs (for review, see Ref. 3). Although more than 120 different GSLs have been identified in total so far, individual plant species usually contain only a few GSLs (2). Quantitative and qualitative differences of GSL profiles are also observed within a species, such as, for example, for different A. thaliana ecotypes (4–6). In addition, GSL composition varies among organs and during the life cycle of plants (7, 8) and is affected by external factors (9).Intact GSLs are mostly considered to be biologically inactive. Most GSL degradation products have toxic effects on insect, fungal, and bacterial pests, serve as attractants for specialist insects, or may have beneficial health effects for humans (10–15). The enzymatic degradation of GSLs (Fig. 1A), which occurs massively upon tissue damage, is catalyzed by plant thioglucosidases called myrosinases (EC 3.2.1.147; glycoside hydrolase family 1). Depending on several factors (e.g. GSL structure, proteins, cofactors, pH) myrosinase-catalyzed hydrolysis of GSLs can lead to a variety of products (Fig. 1B; for review, see Refs. 16 and 17). Of these, isothiocyanates are the most common as their formation only requires myrosinase activity. Thiocyanates on the other hand are only produced from a very limited number of GSLs, and their formation necessitates the presence of a thiocyanate-forming factor in addition to myrosinase (18). A thiocyanate-forming protein (TFP) has recently been identified in Lepidium sativum (19). Alkenyl GSLs, a subgroup of aliphatic GSLs containing a terminal unsaturation in their side chain, can lead to the production of epithionitriles through the cooperative action of myrosinase and a protein called epithiospecifier protein (ESP (20)) in a ferrous ion-dependent way (21–23). Both TFP and ESP contain a series of Kelch repeats (19). Kelch repeats are involved in protein-protein interactions, and Kelch repeat-containing proteins are involved in a number of diverse biological processes (24). In addition to isothiocyanates, nitriles are the major group of GSL hydrolysis products. Although ESP and TFP activities can generate nitriles (19, 21, 25, 26), indications for an ESP-independent nitrile-specifier activity exist. The GSL hydrolysis profile of A. thaliana roots, an organ that does not show ESP expression or activity (27), reveals predominantly the presence of nitriles (28). In addition, leaf tissue of A. thaliana ecotypes supposedly devoid of ESP activity produces a certain amount of nitriles upon autolysis (21). Under acidic buffer conditions, a non-enzymatic production of nitriles from GSLs is observed (Ref. 29 and references therein). Increasing Fe²⁺ concentrations have also been shown to favor nitrile formation over isothiocyanate formation from a number of GSLs in the presence of myrosinase and absence of ESP (21, 22). Therefore, a non-enzymatic origin of this nitrile production cannot be excluded, although the presence of a nitrile-specifier protein is a tempting alternative. Although ESP is able to generate nitriles, it has also been shown that the conversion rates of GSLs to nitriles are lower than those of GSLs to epithionitriles for ESP (21, 22).Open in a separate windowFIGURE 1.Simplified scheme of enzymatic GSL hydrolysis (A) and structures and names of GSLs and their hydrolysis products that are mentioned in the article. (B). A, myrosinase acts on GSLs to form an unstable aglycone intermediate that can rearrange spontaneously to form an isothiocyanate. Hydrolysis can be diverted from this default route under certain conditions (e.g. the presence of NSPs, ferrous ions, or at pH < 5) to give the corresponding nitrile. ESP is responsible for the formation of epithionitriles from alkenyl GSLs in a ferrous ion-dependent mechanism. B, the general structure of GSLs, indicating the variable side chain as R, is given as well as the three major classes of hydrolysis products (i.e. isothiocyanates, nitriles, and epithionitriles). The listed GSLs are the ones mentioned in this article and are arranged according to the class of GSLs they belong to and with an increase in chain length or complexity. The names of the respective hydrolysis products are given for a better understanding of the present article, and not all were encountered during our studies.A nitrile-specifier protein (NSP) that is able to redirect the hydrolysis of GSLs toward nitriles has been cloned from the larvae of the butterfly Pieris rapae (30). This protein does not, however, exhibit sequence similarity to plant ESP, and a corresponding plant nitrile-specifier protein has not yet been identified. We report here the identification of a group of six A. thaliana genes with some sequence similarity to A. thaliana ESP, providing evidence for a new family of nitrile-specifier proteins and a more detailed characterization of one member that possesses nitrile-specifier activity in vitro, when applied exogenously to plant tissue and after ectopic expression in the two A. thaliana ecotypes Col-0 and C24. Despite its sequence homology to A. thaliana epithiospecifier protein (AtESP), it does not possess epithiospecifier activity under similar conditions. Therefore, we propose to designate this protein as A. thaliana nitrile-specifier protein 2 (AtNSP2). Although the biological roles of AtNSP2 and related proteins are not yet known, their specificities and distinctive expression patterns indicate the presence of a fine-tuned mechanism for GSL degradation controlling the outcome of an array of biologically active molecules.     

A procedure for the rapid, quantitative N-acetylation of amino sugar methyl glycosides.

A rapid and quantitative procedure is described for the re-N-acetylation of amino sugar methyl glycosides prior to their analysis by gas-liquid chromatography. Two equivalents of pyridine are added to acidic methanolysates containing amino sugars, serving both to neutralize the acid and to act as a catalyst for the subsequent N-acetylation reaction with acetic anhydride. The N-acetylation is quantitative and complete within 10 min at ambient temperature. Excess acetic anhydride is destroyed by solvolysis with the methanolic solvent. The procedure has been used effectively for methanolysates containing 0.01–2.0 mg/ml glucosamine. The procedures utilizing ion-exchange columns and insoluble salts are thus circumvented and all reaction byproducts are volatile. The procedure is therefore ideally suited for the simultaneous workup of numerous samples for analytical procedures such as gas-liquid chromatography.     

Isoferuloyl derivatives of five seed glucosinolates in the crucifer genus Barbarea

Five acylated glucosinolates (GSLs) were isolated as desulfated derivatives after enzymatic desulfation of anionic metabolites from seeds of two chemotypes of Barbareavulgaris, and their structures were elucidated by a combination of spectroscopic methods and HPLC analysis of products of enzymatic de-acylation. The acyl group was in all cases found to be a trans isoferuloyl group at the 6′-position of the thioglucose moiety. The GSL moieties of the native metabolites were found to be one Trp derived; indol-3-ylmethylGSL, as well as four homoPhe derived; phenethylGSL, (S)-2-hydroxy-2-phenylethylGSL, (R)-2-hydroxy-2-phenylethylGSL, and (R)-2-hydroxy-2-(4-hydroxyphenyl)ethylGSL. GSL analysis of B. vulgaris seed extracts by the commonly employed ‘desulfoGSL’ method (based on binding to anion exchange columns, enzymatic desulfation, elution and HPLC) was optimized for 6′-isoferuloyl derivatives of GSLs. From peak areas before and after de-acylation of the isolated desulfoGSL, the response factor of the 6′-isoferuloyl derivative of (S)-2-hydroxy-2-phenylethylGSL was estimated to be 0.37 (relative to 1.00 for sinigrin), allowing us to estimate the level in B. vulgaris to 3 μmol/g dry wt. in mature seeds and less than 0.1 μmol/g dry wt. in seedlings and floral parts of the insect resistant G-type of B. vulgaris var. arcuata. HPLC analysis of intact GSLs in crude extracts and after group separation did not reveal additional derivatives, but confirmed the existence of the deduced intact GSLs. A taxonomic screen showed that most (14/17) B. vulgaris accessions (with the exception of three accessions of var. vulgaris) contained relatively high levels of 6′-isoferuloyl GSLs. The profiles of 6′-isoferuloylated GSLs matched the profiles of non-acylated GSLs in the same seed accessions, suggesting a low side chain specificity of the isoferuloylation mechanism. A minor peak tentatively identified as a dimethoxycinnamoyl derivative of (S)-2-hydroxy-2-phenylethylGSL was detected by HPLC-MS of one accession, suggesting that GSLs with other acyl groups may occur at low levels. A single analyzed B. plantaginae accession contained relatively high levels of 6′-isoferuloylated phenethylGSL and (S)-2-hydroxy-2-phenylethylGSL. Five other tested Barbarea species (B. australis, B. bracteosa, B. intermedia, B. stricta, B. verna) also contained isoferuloylated GSLs, albeit at lower levels than in B. vulgaris and B. plantaginae, suggesting that seed GSL acylation is a general character of the Barbarea genus and possibly also of related genera including Arabidopsis.     

Variation of glucosinolates on position orders of flower buds in turnip rape (Brassica rapa)

To glucosinolate (GSL) contents on flower buds depending on their position orders in turnip rape (Brassica rapa), three Japanese ‘Nabana’ cultivars such as cv. No. 21 (Brassica rapa, early type), cv. Husanohana (B. rapa, late type) and cv. Norin No. 20 (B. napus) were investigated using HPLC analysis. Ten GSLs including glucoraphanin, sinigrin, glucoalyssin, napoleiferin, gluconapin, 4-hydroxyglucobrassicin, glucobrassicanapin, glucobrassicin, and gluconasturtiin were detected. Differences in individual and total GSL contents were found between two plant varieties, which are also depending on various developmental stages. Among the GSLs, gluconapin (mean 23.11 μmole/g dry weight (DW) and glucobrassicanapin (mean 13.41 μmole/g DW) documented the most abundant compounds and contributed average 39 and 27% of the total GSLs, but indolyl and aromatic GSLs together accounted >10% of the total GSLs. The presence of significant quantities of gluconapin in the cultivars should be studied more extensively, since the GSL is mainly responsible for the bitter taste.     

Glucosinolates in Reseda lutea L.: Distribution in plant tissues during flowering time

Reseda lutea L. belongs to the Resedaceae family included in the order of Brassicales. R. lutea is a plant worthy of investigation on an ecological level for its ability to adapt to extreme environmental conditions and for its capacity to attract honeybees and wild pollinators. In the ancient pharmacotherapy it was also known for its healing properties. R. lutea glucosinolates (GSLs) were investigated by HPLC-UV considering their accumulation pattern and their quality profiles during flowering time. 3-Hydroxybenzyl GSL and 2-(α-L-rhamnopyranosyloxy)benzyl GSL were identified by NMR and HPLC-MS of the desulfo derivatives, while benzyl GSL, indol-3-ylmethyl GSL and traces of 2-phenylethyl GSL were identified by HPLC-UV comparison with authentic standards. Our data showed that the uncommon 2-(α-l-rhamnopyranosyloxy)benzyl GSL, until now identified as the main GSL in R. lutea, reached its highest content in the racemes during the full flowering stage, the most pollinator attractive phenological phase of the plant. The 2-(α-l-rhamnopyranosyloxy)benzyl GSL then decreased during late flowering, when the presence of 3-hydroxybenzyl GSL increased. This is the first report of 3-hydroxybenzyl GSL in R. lutea as well as of the full characterization by means of NMR and HPLC-APCI-MS of the desulfated derivative of 2-(α-l-rhamnopyranosyloxy)benzyl GSL. Finally, the identified R. lutea GSL profiles are discussed with reference to the actual knowledge on the Reseda genus GSLs. The results added new evidence to complete the characterization of the GSL profile of this species.     

Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography.

Reaction of ceramides containing nonhydroxy fatty acids with benzoyl chloride in pyridine at 70 degrees C for 1 hr resulted in N-benzoylation to form N,N-acyl,benzoyl derivatives; O-benzoylation also occurred. However with ceramides containing 2-hydroxy fatty acids and phytosphingosine only O-benzoylation occurred even on prolonged treatment. Only O-benzoylation occurred on reaction with benzoic an hydride. However, the benzoylation of ceramides with phytosphingosine could not be achieved with benzoic anhydride and this benzoylation was performed by reaction with benzoyl chloride at 70 degrees C for 4 hr. Because N,N-acyl,benzoyl derivatives of ceramides containing nonhydroxy fatty acids produced by treatment with benzoyl chloride overlap methyl benzoate on high-performance liquid chromatography, benzoic anhydride was preferable for benzoylation of ceramides with nonhydroxy and 2-hydroxy fatty acids. On the other hand, the reaction with benzoyl chloride at 70 degrees C for 4 hr was used for quantitation of benzoylated ceramides containing 2-hydroxy fatty acids and phytosphingosine. 3-(p-Phenylbenzoyl)estrone was used as an internal standard for both reactions and values for ceramides containing 2-hydroxy fatty acids obtained by the two reactions were in good agreement. This procedure was applied to measurement of the ceramide levels in the brain, liver, and kidney of rats during development. The levels of ceramides containing nonhydroxy and 2-hydroxy fatty acids in the brain, liver, and kidney increased to the adult levels and then remained unchanged. Ceramide with phytosphingosine was detected in the liver and kidney, where its concentration gradually increased with age, but it was not found in the brain. The composition of nonhydroxy fatty acids were also analyzed.     

Synthesis, characterization and in vitro anti-cancer activity of N-(ferrocenyl)benzoyl tri- and tetrapeptide esters

N-ortho, N-meta and N-para-(ferrocenyl)benzoyl tri- and tetrapeptide esters (2-7) were prepared by coupling ortho, meta and para-ferrocenyl benzoic acids to the tri- and tetrapeptide ethyl esters of GlyGlyGly(OEt) and GlyGlyGlyGly(OEt) in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole. The compounds were characterized by a range of NMR spectroscopic techniques, mass spectrometry and cyclic voltammetry. The anti-proliferative effects of the ortho derivatives 2 and 5 were measured in vitro against H1299 lung cancer cells and both gave IC₅₀ values greater than 50 μM. Therefore, extending the length of the peptide chain had a negative effect on activity, relative to N-(ferrocenyl)benzoyl amino acid and dipeptide derivatives.     

Combined effect of Nitrogen,Phosphorus and Potassium fertilizers on the contents of glucosinolates in rocket salad (Eruca sativa Mill.)

Nitrogen (N), phosphorous (P) and potassium (K) are the most limiting factors in crop production. N often affects the amino acid composition of protein and in turn its nutritional quality. In Brassica plants, abundant supply of N fertilizer decreases the relative proportion of glucosinolates (GSLs), thus reducing the biological and medical values of the vegetables. Hence effort was made to evaluate the influence of different proportions of nutrient solutions containing N–P–K on the GSL profiles of rocket salad (Eruca sativa Mill.). Fifteen desulpho-(DS) GSLs were isolated and identified using liquid chromatography–mass spectrometry (LC/MS) analysis. Rocket salad plants supplied with lesser amount of N, P or higher concentrations of K showed a typical improvement in total GSL contents. In contrast, total GSL levels were less at higher N supply. Furthermore, with N concentrations above 5 mM and K concentrations less than 2.5 mM, the GSL amounts were on average 13.51 and 13.75 μmol/g dry weight (DW), respectively. Aliphatic GSLs predominated in all concentrations of NPK while indolyl GSLs made up marginally less amount of the total compositions. Five and 2 mM N and P possessed much higher levels of several types of aliphatic GSLs than other concentrations, including glucoerucin, glucoraphanin and dimeric 4-mercaptobutyl GSL. From this perspective, it is contended that supply of less N results in enhancing the metabolic pathway for the synthesis of GSLs in rocket salad.     

Structural studies on the neutral glycosphingolipids of Manduca sexta

Glycosphingolipids (GSLs) have been implicated as playing major roles in cellular interactions and control of cell proliferation in muticellular organisms. Moreover GSLs and other sphingolipids such as sphingomyelins, ceramides and sphingosines serve a variety of roles in signal transduction. Hence, identification of structures of GSLs in different biota will shed light in understanding their physiological role. During this study, the major glycosphingolipid component present in the extracts of stage-12 and stage-17/18 metamorphosing adults of Manduca sexta was identified as mactosyl ceramide. We report the isolation of several ceramide disaccharides, a ceramide trisaccharide and a ceramide tetrasaccharide. The GSL structures were confirmed by high-resolution mass spectrometry and tandem mass spectrometry. The identity of the monosaccharides was proved using exoglycosidases. The predominant sphingosine chain-length varied from C-14 (tetradecasphing-4-enine) to C-16 (hexadecasphing-4-enine) in these GSLs. Sphingosines of both chain lengths were accompanied by their doubly unsaturated counterparts tetradecasphinga-4,6-diene and hexadecasphinga-4,6-diene. It is also interesting to note the presence of tetradecasphinganine and hexadecasphinganine in minute amounts in the form of a GSL in the extracts of M. sexta. The varying degrees of unsaturation in the sphingosine moiety of GSLs in M. sexta may be biologically significant in insect metamorphosis. The ceramide trisaccharides and ceramide tetrasaccharide belong to the arthro-series, The observation of fucose in the M. sexta GSLs is the first report of the presence of fucose in an arthroseries GSL.     

Traveling for the glycosphingolipid path

Our studies on glycosphingolipids (GSLs) were initiated through isolation and structural characterization of lacto-series type 1 and 2 GSLs, and globo-series GSLs. Lacto-series structures included histo-blood group ABH and I/i antigens. Our subsequent studies were focused on GSL changes associated with: (i) ontogenic development and differentiation; (ii) oncogenic transformation and tumor progression. Various novel types of GSLs such as extended globo-series, sialyl-Le^x (SLe^x), sialyl-dimeric-Le^x (SLe^x-Le^x), dimeric-Le^x (Le^x-Le^x), Le^y-on-Le^x, dimeric-Le^a (Le^a-Le^a), Le^b-on-Le^a, etc. were identified as tumor-associated antigens. These studies provide an essential basis for up- or down-regulation of key glycosyltransferase genes controlling development, differentiation, and oncogenesis. GSL structures established in our laboratory are summarized in Table 1, and structural changes of GSLs associated with ontogenesis and oncogenesis are summarized in Sections 2 and 3.Based on these results, we endeavored to find out the cell biological significance of GSL changes, focused on (i) cell adhesion, e.g., the compaction process of preimplantation embryo in which Le^x-to-Le^x, Gb4-to-GalGb4 or -nLc₄ play major roles; and (ii) modulation of signal transduction through interaction of growth factor receptor tyrosine kinase with ganglioside, e.g., EGF receptor tyrosine kinase with GM3. Recent trends of studies on i and ii lead to the concept that GSL clusters (microdomains) are organized with various signal transducer molecules to form glycosignaling domains (GSD). GSL-dependent adhesion occurs through clustered GSLs, and is coupled with activation of signal transducers (cSrc, Src family kinase, Rho A, etc.). Clustered GSLs involved in cell adhesion are recognized by GSLs on counterpart cells (carbohydrate-to-carbohydrate interaction), or by lectins (e.g., siglecs, selectins).Our major effort in utilization of GSLs in medical science has been for: (i) cancer diagnosis and treatment (vaccine development) based on tumor-associated GSLs and glycoepitopes; (ii) genetically defined phenotype for susceptibility to E. coli infection; (iii) clear identification of physiological E-selectin epitope (myeloglycan) expressed on neutrophils and myelocytes; (iv) characterization of sialyl poly-LacNAc epitopes recognized as male-specific antigens. Utilization of these GSLs or glycoepitopes in development of anti-adhesion approach to prevent tumor metastasis, infection, inflammation, or fertilization (i.e., contraceptive) is discussed. For each approach, development of mimetics of key GSLs or glycoepitopes is an important subject of future study.     

Influence of glycosphingolipids on cancer cell energy metabolism

A growing number of studies describe a connection between glycosphingolipids (GSLs) and glutamine metabolism, glucose metabolism and mitochondrial dysfunction in cancer cells. Since deregulated cell energy metabolism is one of cancer cells hallmarks, investigating this connection is an important step in the development of anti-cancer therapies. GSL species are often aberrantly regulated in human cancers. They cluster in signaling platforms in the plasma membrane and organelle membranes in so called glycosphingolipid enriched microdomains (GEMs), thereby regulating cell signaling pathways. The most important glutamine transporter for epithelial cells, alanine-serine-cysteine transporter 2 (ASCT2) locates in GEMs and is regulated by GEM composition. The accumulation of glucosylceramide and lactosylceramide in mitochondria associated ER membranes (MAMs) leads to increased oxidative phosphorylation. This increases mitochondrial reactive oxygen species (ROS) levels and influences mitochondrial dynamics. Here, we review current knowledge about deregulated GSL species in cancer, GSL influence on glutamine and glucose metabolism. In addition, the role of GSLs in MAMs, oxidative phosphorylation (OXPHOS) and mitochondrial dynamics with a special focus on mechanistic target of rapamycin (mTOR) signaling is discussed. mTOR seems to play a pivotal role in the connection between GSLs and glutamine metabolism as well as in mitochondrial signaling.     

Norwalk virus-like particles bind specifically to A,H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids

Noroviruses and norovirus virus-like particles (VLPs) exhibit strain specific patterns in their binding to ABH and Lewis histo-blood group antigens. In this study we demonstrate for the first time specific binding of Norwalk virus VLPs to type 1 and type 2 chain glycosphingolipids (GSLs) carrying ABH and Lewis antigens. N-succinimidyl-3-tributylstannyl benzoate (ATE) was precursor labeled with ¹²⁵I and then conjugated to VLPs. The ¹²⁵I-VLPs were used in GSL thin-layer chromatogram binding assays and displayed binding to H type 1, Lewis b, A type 1, A Lewis b GSLs but no binding to B type 1 or B Lewis b GSLs. For the type 2 chain GSLs the Norwalk VLPs bound to H type 2, Lewis y, A type 2 and A Lewis y. In addition, the VLPs bound to several complex GSLs from blood group O and A, but not from blood group B red blood cells.     

Dynamic modulation of the glycosphingolipid content in supported lipid bilayers by glycolipid transfer protein

Supported lipid bilayers (SLBs) are popular models of cell membranes. Owing to the importance of glycosphingolipids (GSLs) in modulating structure and function of membranes and membrane proteins, methods to tune the GSL content in SLBs would be desirable. Glycolipid transfer protein (GLTP) can selectively transfer GSLs between membrane compartments. Using the ganglioside GM1 as a model GSL, and two mass-sensitive and label-free characterization techniques—quartz crystal microbalance with dissipation monitoring and ellipsometry—we demonstrate that GLTP is an efficient and robust biochemical tool to dynamically modulate the GSL content of SLBs up to 10 mol % GM1, and to quantitatively control the GSL content in the bulk-facing SLB leaflet. By exploiting what we believe to be a novel tool, we provide evidence that GM1 distributes highly asymmetrically in silica-supported lipid bilayers, with ∼85% of the ganglioside being present in the bulk-facing membrane leaflet. We report also that the pentameric B-subunit of cholera toxin binds with close-to-maximal stoichiometry to GM1 in SLBs over a large range of GM1 concentrations. Furthermore, we quantify the liganding affinity of GLTP for GM1 in an SLB context to be 1.5 μM.     

9-(2-Deoxy-ß-D-xylofuranosyl)adenine and 1-(2-Deoxy-ß-D-xylofuranosyl)thymine: Phosphorylation and Stability

Abstract

Phosphorylation of 1-(2-deoxy-β-D-xylofuranosyl)thymine (1) or 9-(2-deoxy-β-D-xylofuranosyl)adenine (3) with phosphoryl chloride gives the cyclic 3′,5′-phosphates (2 and 4a) but not the 5′-monophosphates 8a or 8b. The latter are obtained by phosphorylation of the 3′-0-benzoylated 2′-deoxy-β-D-xylonucleosides (7a, b) and subsequent base-catalyzed removal of the benzoyl groups. Compound 3, as the parent dA, depurinates in acidic medium, a reaction which is facilitated in the case of the N⁶-benzoyl derivative 9b and reduced after the introduction of an amidine protecting group. N-Glycosylic bond hydrolysis of 2′-deoxy-β-D-xylofuranosyl nucleosides is enhanced by a factor of two compared to 2′-deoxy-β-D-ribofuranosyl nucleosides.     

Simultaneous determination of dimethylamphetamine and its metabolites in rat hair by gas chromatography–mass spectrometry

In order to study the disposition of dimethylamphetamine (DMAP) and its metabolites, DMAP N-oxide, methamphetamine (MA) and amphetamine (AP), from plasma to hair in rats, a simultaneous determination method for these compounds in biological samples using gas chromatography–mass spectrometry with selected ion monitoring (GC–MS-SIM) was developed. As DMAP N-oxide partially degrades to DMAP and MA during GC–MS analysis, it was necessary to avoid conditions which co-extract the N-oxide in the sample preparation so as to assure no contribution of artifactual products from DMAP N-oxide in the detection of the other compounds. For confirmation of the satisfactory separation of DMAP N-oxide from the others, the internal standards used for quantification were labeled with different numbers of deuterium atoms. Determination of unchanged DMAP was performed without any derivatization, that of DMAP N-oxide was carried out after conversion into trifluoroacetyl-MA by reaction with trifluoroacetic anhydride, and MA and AP were quantified after trifluoroacetyl-derivatization.After intraperitoneal administration of DMAP HCl to pigmented hairy rats (5 mg kg⁻¹ day⁻¹, 10 days, n=3), concentrations of DMAP and its metabolites in urine, plasma and hair were measured by GC–MS-SIM. The area under the concentration versus time curves (AUCs) of DMAP, DMAP N-oxide, MA and AP in the plasma were 397.2±97.5, 279.7±68.3, 18.4±1.2 and 15.9±2.2 μg min ml⁻¹, while their concentrations in the hair newly grown for 4 weeks after administration were 4.82±0.67. 0.45±0.09, 3.25±0.36 and 0.89±0.05 ng mg⁻¹, respectively. This fact suggested that the incorporation tendency of DMAP N-oxide from plasma into hair was distinctly low in comparison with the other compounds.     

Homogeneous modification of cellulose with succinic anhydride in ionic liquid using 4-dimethylaminopyridine as a catalyst

Cellulose, extracted from sugarcane bagasse, was successfully succinylated in ionic liquid 1-buty-3-methylimidazolium (BMIMCl) using 4-dimethylaminopyridine (DMAP) as a catalyst. Parameters investigated included the mass ratio of DMAP/succinic anhydride in a range from 0% to 15%, reaction time (from 30 to 120 min), reaction temperature (from 60 to 110 °C). The succinylated cellulosic derivatives had a degree of substitution (DS) ranging from 0.24 to 2.34. It was found that the DS of succinylated cellulosic derivatives using DMAP as a catalyst was higher than that without any catalyst under the same reaction conditions. The products were characterized by FT-IR, solid-state CP/MAS ¹³C NMR, and thermal analysis. FT-IR and solid-state CP/MAS ¹³C NMR spectra showed that succinoylation occurred at C-6, C-2 and C-3 positions. The thermal stability of the succinylated cellulose decreased upon chemical modification.     

Novel bioresources for studies of Brassica oleracea: identification of a kale MYB transcription factor responsible for glucosinolate production

Plants belonging to the Brassicaceae family exhibit species‐specific profiles of glucosinolates (GSLs), a class of defence compounds against pathogens and insects. GSLs also exhibit various human health–promoting properties. Among them, glucoraphanin (aliphatic 4‐methylsulphinylbutyl GSL) has attracted the most attention because it hydrolyses to form a potent anticancer compound. Increased interest in developing commercial varieties of Brassicaceae crops with desirable GSL profiles has led to attempts to identify genes that are potentially valuable for controlling GSL biosynthesis. However, little attention has been focused on genes of kale (Brassica oleracea var. acephala). In this study, we established full‐length kale cDNA libraries containing 59 904 clones, which were used to generate an expressed sequence tag (EST) data set with 119 204 entries. The EST data set clarified genes related to the GSL biosynthesis pathway in kale. We specifically focused on BoMYB29, a homolog of Arabidopsis MYB29/PMG2/HAG3, not only to characterize its function but also to demonstrate its usability as a biological resource. BoMYB29 overexpression in wild‐type Arabidopsis enhanced the expression of aliphatic GSL biosynthetic genes and the accumulation of aliphatic GSLs. When expressed in the myb28myb29 mutant, which exhibited no detectable aliphatic GSLs, BoMYB29 restored the expression of biosynthetic genes and aliphatic GSL accumulation. Interestingly, the ratio of methylsulphinyl GSL content, including glucoraphanin, to that of methylthio GSLs was greatly increased, indicating the suitability of BoMYB29 as a regulator for increasing methylsulphinyl GSL content. Our results indicate that these biological resources can facilitate further identification of genes useful for modifications of GSL profiles and accumulation in kale.     

Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies

In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.