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Different elongation pathways in the biosynthesis of acyl groups of trichome exudate sugar esters from various solanaceous plants.

Two common pathways are known for elongation of aliphatic acids via acetate in biological organisms: the fatty acid synthase (FAS) and the alpha-ketoacid elongation (alphaKAE) pathways. The alphaKAE route is utilized in many biosynthetic pathways, including the tricarboxylic acid cycle, leucine biosynthesis, and in formation of coenzyme B, glucosinolates, alpha-ketoadipate, sugar-ester acyl acids, short-chain alcohols of yeast and Clostridium species, 2-amino-4-methylhex-4-enoic acid, and l-gamma-phenyl butyrine. In the FAS route, both carbons from acetyl-acyl carrier protein are retained per elongation cycle, while in the alphaKAE route only one carbon from acetyl-coenzyme A is retained. Available evidence indicates that different members of the family Solanaceae may use one or the other of these elongation mechanisms in the synthesis of acyl groups of trichome-exuded sugar esters. In both, precursors for elongation are derived from branched-chain amino acid metabolism. Here we compared radiolabeling patterns in sugar-ester acyl groups from trichomes (the specific tissue in which sugar esters are synthesized) of the tobaccos, Nicotiana benthamiana, N. gossei, N. glutinosa, of Petunia x hybrida cv. Falcon Red & White, and Datura metel, and epidermal peels of Lycopsersicon pennellii after their synthesis from [2-(14)C]-, [1-(14)C]- and [1,2-(14)C]acetate. Recovered acyl acids were purified and then degraded to determine label distribution between the carboxyl termini and the remainder of the molecules. Six- and 20-h incubations were studied, and membrane fatty acids were monitored as internal controls for FAS-mediated elongation. Results are consistent with participation of alphaKAE in synthesis of sugar-ester acyl groups of tobaccos and petunia, but apparently FAS is utilized in the formation of these groups in L. pennellii and D. metel.

Fatty Acid Synthases↗

A novel tumor-associated mucin of gastrointestinal carcinoma.

PURPOSE: To identify a new tumor-associated antigen, a monoclonal antibody, SC142, was produced by immunizing mice with a stomach cancer cell line. The tumor specificity of mAb SC142 was studied by immunohistochemical staining, and the biochemical characteristics of this new gastrointestinal tumor-associated antigen were also studied. METHODS: The expression of SC142-reactive antigen was investigated in various cancers by immunohistochemical staining. The SC142-reactive antigen was characterized by immunoblotting, sodium metaperiodate treatment assay, O-glycanase digestion assay, and lectin binding assay. RESULTS: The SC142-reactive antigen was highly expressed in 78% of gastric cancers (29/37) and 87% of colon cancers (27/31). No normal colon or stomach tissues remote from the tumor were positive for the antigen. The antibody also reacted with other tumors of epithelial origin such as lung squamous cell cancer (2/4), breast ductal cancer (2/20), bladder transitional cell carcinoma (4/6), and uterine cancer (3/16). Western blot analysis of the antigen revealed glycoprotein(s) which migrated as a smear ranging from the origin of the gel to about the 80 kDa region. The reactivity of this antigen with SC142 was reduced by sodium metaperiodate treatment or O-glycanase digestion, but not by N-glycanase, suggesting that the epitope is an O-glycan. In lectin-binding assay, this antigen reacted only with wheat germ agglutinin but not with Ricinus communis agglutinin, Datura stramonium agglutinin, and Sambucus nigra agglutinin. CONCLUSIONS: Our findings indicate that the antigen defined by SC142 is a tumor-associated antigen that could differentiate the gastrointestinal cancer cells from the normal cells. Therefore, SC142 may become a valuable tool for the immunohistochemical diagnosis and tumor immunoscintigraphy of the gastrointestinal cancer patients.

Animals↗

Composition of the sugar moiety of Tamm-Horsfall protein in patients with urinary diseases.

The sugar moiety of Tamm-Horsfall protein (THP) is altered by pathological conditions. The aim of this study was to investigate the composition of THP glycans in urinary diseases. THP was isolated from the urine of patients with urinary tract infection (group A), glomerulonephritis or interstitial nephritis (group B), and Bartter's syndrome (BS) (group C). Monosaccharides, N-glycan profile, THP reactivity with specific lectins and some other proteins were analyzed. THP of patients from groups A, B, and C showed lower amounts of N-acetylgalactosamine (P<0.05, P<0.005, and P<0.05, respectively) than controls; this was reflected in lower reactivity with Phaseolus vulgaris lectin (P<0.005, P<0.05, and P<0.005). Reduced amounts of N-acetylglucosamine were noticed in groups A (P<0. 05) and B (P<0.05). In group A lower amounts of galactose and alpha2, 6-linked sialic acid, as determined by reactivity with Datura stramonium lectin (P<0.005) and Sambucus nigra lectin (P<0.005), were observed. In patients with BS there was a shift from tetrasialylated glycans towards less-sialylated chains. We found also that THP of all patients binds more strongly to IgG(1) (P<0.005, for all patient groups). Our results indicate that the urinary diseases examined affect the THP sugar moiety and the binding of THP to IgG(1).

Acetylglucosamine↗

The route of tomato spotted wilt virus inside the thrips body in relation to transmission efficiency.

The route of tomato spotted wilt virus (TSWV) in the body of its vectors, Frankliniella occidentalis and Thrips tabaci (Thysanoptera: Thripidae) was studied during their development. First instar larvae were allowed, immediately upon hatching, to acquire virus from mechanically infected Datura stramonium plants for 24 h. The rate of transmission by adults was determined in inoculation access feeding test on Emilia sonchifolia leaf disks. Thrips tissues were analysed for infection at 24 h intervals after the acquisition-access feeding period, and assayed by the whole-mount immuno-fluorescent staining technique. The virus was initially detected in the proximal midgut region in larvae of both species, and then in the second and third midgut regions, foregut, and salivary glands. Occasionally the first infections of the salivary glands were already detected in one-day-old second stage larvae. The intensity of the infection in the various organs of the thrips of each species was positively related to the transmission efficiency. In both thrips populations good agreement was found between the percentage of second instar larvae and adults with at least one infected salivary gland lobe and the percentage of transmitting adults. These results support the contention that the virus must reach the salivary glands before thrips pupation in order to be transmitted by old second instar larvae and adults.

Animals↗

Characterization and genetic diversity of potato yellow mosaic virus from the Caribbean.

The begomovirus Potato yellow mosaic virus (PYMV) is responsible of significant yield losses in tomato in Guadeloupe. Four field isolates from Guadeloupe were analyzed in term of their host range using three inoculation methods (mechanical, grafting and insect vector), sequences analysis of PCR fragments and phylogenetic analysis of an infectious clone, PYMV-[GP]. Capsicum annuum, Datura stramonium, Nicotiana benthamiana, N. tabacum 'Xanthi NC', Petunia hybrida, and Solanum tuberosum were found to be hosts. All isolates from Guadeloupe, Martinique, Puerto Rico and the Dominican Republic were closely related to PYMV-[GP]. Sequence identity between PYMV-[GP] and PYMV-Ve from Venezuela and PYMTV from Trinidad and Tobago clearly confirmed that it is a new strain of PYMV.

Caribbean Region↗

Further characterization of 'sweet potato virus 2': a distinct species of the genus Potyvirus.

An incompletely described potyvirus isolate from sweet potato in Taiwan, referred to as 'sweet potato virus 2' (SPV2), was further characterised. Electron microscopy revealed that SPV2 has filamentous particles of 850 nm in length and induces cytoplasmic cylindrical inclusions consisting of pinwheels and scrolls. The virus was mechanically transmitted to several species of the genera Chenopodium, Datura, Nicotiana, and Ipomoea. Two biotypes of Myzus persicae transmitted SPV2 in a non-persistent manner. Decoration titer experiments revealed a distant serological relationship between SPV2 and other potyviruses infecting sweet potato. The 3'-terminal 2006 nucleotides of the viral RNA were determined and shown to be a potyviral genome fragment comprising the coding region for the C-terminal half of the NIb protein, the entire coat protein cistron, and the 3' untranslated region (UTR). Comparison of the capsid protein and 3' UTR sequences of SPV2 with those of other potyviruses demonstrated that it is a distinct member of the genus Potyvirus (family Potyviridae). We propose that SPV2 is named Sweet potato virus Y.

Amino Acid Sequence↗

Daphne mosaic virus (DapMV), a new potyvirus from Daphne mezereum in the Czech Republic.

Daphne shrubs with light green rings and mosaic on leaves contained flexuous filamentous virions (696 x 13 nm) and cylindrical inclusions typical of the subdivision III of Edwardson's classification for inclusions induced by members of the family Potyviridae. Decoration tests using antisera to 67 potyviruses revealed distant serological relations among chilli veinal mottle virus, Colombian datura virus, papaya ringspot virus, tobacco vein mottling virus and yam mosaic virus. The 3' terminal region of the virus genome was amplified by RT-PCR using primers specific for cloned and sequenced members of the family Potyviridae. The most similar sequences in the GenBank were those of isolates of wild potato mosaic virus (WPMV) and yam mild mosaic virus (YMMV), originating from Peru and Guadeloupe, respectively. The new sequence had 63.2% and 61.9% nucleotide identity to WPMV and YMMV in the coat protein gene. The results suggest that the Czech isolate from daphne should be regarded as a new member of the genus Potyvirus. The name daphne mosaic virus (DapMV) is suggested for this virus.

Capsid Proteins↗

Analysis of sugar chain-binding specificity of tomato lectin using lectin blot: recognition of high mannose-type N-glycans produced by plants and yeast.

The sugar chain-binding specificity of tomato lectin (LEA) against glycoproteins was investigated qualitatively using lectin blot analysis. Glycoproteins containing tri- and tetra-antennary complex-type N-glycans were stained with LEA. Unexpectedly, glycoproteins containing high mannose-type N-glycans and a horseradish peroxidase were stained with LEA. LEA blot analysis of the glycoproteins accompanied by treatment with exoglycosidase revealed that the binding site of LEA for the complex-type N-glycans was the N-acetyllactosaminyl side chains, whereas the proximal chitobiose core appeared to be the binding site of LEA for high mannose-type N-glycans. Despite these results, the glycoproteins did not inhibit the hemagglutinating activity of LEA. Among the chitin-binding lectins compared, potato tuber lectin showed specificity similar to LEA on lectin blot analysis, while Datura stramonium lectin and wheat germ agglutinin (WGA) did not interact with glycoproteins containing high mannose-type N-glycans, except that RNase B was stained by WGA. Based on these observations, LEA blot analysis was applied to sugar chain analysis of tomato glycoproteins. The most abundant LEA-reactive glycoprotein was purified from the exocarp of ripe tomato fruits, and was identified as the tomato anionic peroxidase1 (TAP1). These results suggest that LEA interacts with glycoproteins produced by tomatoes, which participate in biological activities in tomato plants.

Amino Acid Sequence↗

Utility of pentose colorimetric assay for the purification of potato lectin, an arabinose-rich glycoprotein.

Potato lectin (Solanum tuberosum agglutinin, STA) is an unusual glycoprotein containing approximately 50% carbohydrates by weight. Of the total carbohydrates, 92% is contributed by L: -arabinose, which are O-linked to hydroxyproline residues. The ferric chloride-orcinol assay (Bial's test), which is specific for pentoses has so far been used only for the determination of free pentoses in biological samples. However, this colorimetric assay has not been used for the detection of pentoses in bound form as it occurs in Solanaceae lectins (potato, tomato, and Datura lectins). Utilizing the pentose colorimetric assay for monitoring the presence of potato lectin, a simpler and shorter procedure for the purification of this lectin from potato tubers has been developed. The yield of potato lectin (1.73 mg per 100 g potato tuber) is twice compared to the yields reported in earlier procedures. Although potato lectin is well known for its specificity to free trimers and tetramers of N-acetyl-D: -glucosamine (GlcNAc), it possesses a similar specificity to the core (GlcNAc)(2) of N-linked glycoproteins. The utilization of the pentose assay in the purification of arabinose-rich lectins/agglutinins obviates the necessity for the use of agglutination assay in the various purification steps. The pentose assay appears to be a simple and convenient colorimetric assay for detecting any pentose-rich glycoprotein in plant extracts. The utility of the pentose assay appears to have a significant potential in the detection of hydroxyproline-rich glycoproteins (HRGPs), which are generally O-arabinosylated.

Arabinose↗

The tidemark of the chondro-osseous junction of the normal human knee joint.

The chondro-osseous junction includes the junction between calcified and non-calcified cartilage matrices often referred to as the tidemark. A detailed knowledge of the structure, function and pathophysiology of the chondro-osseous junction is essential for an understanding both of the normal elongation of bones and of the pathogenesis of osteoarthrosis. In this study the molecular anatomy of the tidemark was studied using histochemical techniques, including lectin histochemistry, on blocks of normal cartilage from human knee joints. The tidemark stained with H and E, picro-sirius red, toluidine blue, safranin O and methyl green, but not with alcian blue in the presence of magnesium chloride at 0.05 M or above. It stained with only four lectins, those from Datura stramonium, Maclura pomifera, Erythrina crystagalli and Helix pomatia, out of the 19 used. Therefore, it is rich in collagen and contains hyaluronan, but appears to lack the glycosaminoglycans of 'conventional' proteoglycans and it expresses a very limited and distinctive lectin staining glycoprofile, which is probably attributable to specific glycoproteins. In addition, the tidemark had a distinct microanatomical trilaminate appearance. From all of these results it is clear that this part of the chondro-osseous junctional region is chemically more complex and distinctive than has previously been described.

Adult↗

Method for the detection of glycopeptides at the picomole level in HPLC peptide maps.

Glycopeptide-containing fractions in HPLC peptide maps can be detected by a simple application of the microtiter plate-bound streptavidin-biotinylated glycopeptide-lectin method (M.-C. Shao, 1992, Anal. Biochem., 205, 77-82). To illustrate this application, the glycoproteins, ovalbumin and asialofetuin, reduced and S-alkylated with vinylpyridine, were digested with trypsin-L-1-p-tosylamino-2-phenylethylchloromethyl ketone and the tryptic peptides were fractionated by reverse-phase HPLC, monitoring for absorbance at 230 nm. Aliquots of the HPLC fractions (typically 0.2-0.5% of the total volume) were biotinylated and complexed with streptavidin in the wells of a microtiter plate, allowing the streptavidin-glycopeptide complex to adhere to the plate. Suitable lectins, such as concanavalin A, Datura stramonium agglutinin, and peanut agglutinin, all of which had been coupled to horse radish peroxidase, were added, and after thorough washing, only the wells containing streptavidin-bound glycopeptides retained the complementary lectin and gave a positive peroxidase reaction. Less than 1 pmol of glycopeptide can be detected. The demonstration that the glycopeptide detection could be inhibited either by addition of an excess of the appropriate sugar inhibitor to the different lectins or by digestion of the biotinylated glycopeptides with N-glycosidase F or O-glycosidase shows that the glycopeptide-lectin interaction is the basis for the reaction.

Amino Acid Sequence↗

The use of streptavidin-biotinylglycans as a tool for characterization of oligosaccharide-binding specificity of lectin.

A new rapid and sensitive method for characterizing lectin specificity using streptavidin-biotinylglycans as a tool is presented. This assay is analogous to enzyme immunoassay and takes advantage of the strong, irreversible adsorption of streptavidin to the wells of the chambers of titer plates. A series of streptavidin-biotinylglycans was first coated on a microtiter plate, and then one of six lectins, concanavalin A, wheat germ agglutinin, Phaseolus vulgaris (red kidney bean) erythro-agglutinin, Lens culinaris (lentil) agglutinin, Datura stramoniun agglutinin, or Sambucus nigra (elderberry bark) agglutinin coupled to horseradish peroxidase, was added. After incubation and thorough washing, only the lectin bound to a complementary glycan remained and could be detected and quantified by the peroxidase reaction. It was established that the lectins retained their oligosaccharide-binding specificities after coupling to the peroxidase, that the binding was inhibited by addition of the corresponding sugar inhibitors, and that the color intensity produced by the enzyme reaction is proportional to the amount of lectin-peroxidase bound to biotinylglycan complexed with streptavidin immobilized on the plate. As an example, it was found that the peroxidase-D. stramoniun agglutinin conjugate strongly bound biotinylglycans, GlcNAc3-Man5-R, GalGlcNAc3Man5-R, and GlcNAc3-4Man3-R (R = GlcNAc2-[6-(biotinamido)hexanoyl]-Asn). As little as 10 pmol/ml of lectin was detected. With the growing availability of biotinylglycans, the method should represent a reliable and simple procedure for screening lectin-oligosaccharide recognition qualitatively and quantitatively.

Bacterial Proteins↗

Modulation of N-acetylglucosaminyltransferase III, IV and V activities and alteration of the surface oligosaccharide structure of a myeloma cell line by interleukin 6.

The activity of N-acetylglucosaminyltransferase (GnT) III, IV and V on a myeloma cell line, OPM-1, was examined after incubation with interleukin 6 (IL-6). While augmenting cell proliferation, IL-6 resulted in a decrease of GnT III activity and an increase of GnT IV and V activities. Consistent with this, OPM-1 cultured with IL-6 showed an increased affinity to Datura stramonium lectin, which recognizes asialo-tri- and asialo-tetraantenary N-linked oligosaccharides. These results indicate that IL-6 modulates glycosyltransferase activity and the oligosaccharide structure of target cells.

Cell Membrane↗

gamma-L-glutamyl-L-aspartate induces specific deficits in long-term memory and inhibits [3H]glutamate binding on hippocampal membranes.

gamma-L-Glutamyl-L-aspartic acid (gamma-LGLA) has been isolated from Datura stramonium; its structure has been determined and it was then synthesized. In male Swiss mice intraperitoneal administration of the natural peptide (125 nmol/kg) or of the synthetic peptide (25-2500 nmol/kg) 24 h after acquisition of a Y-maze avoidance task induced a dose-dependent deficit in retention performance 48 h later. gamma-LGLA had no effect on locomotor activity or emotional reactivity at the doses used. Separate or simultaneous administration of aspartate or glutamate (each at 250 nmol/kg) had no effect on learning retention, indicating that deficit induced by gamma-LGLA was specific to the peptide. gamma-LGLA impaired learning acquisition in a time-dependent manner when administered from 3 min to 24 h post-training, but had no effect when administered 3 days following acquisition. gamma-LGLA administered just after the training session did not affect retention performance during the first 3 h, but suppressed the retention improvement observed in control animals from 6 to 24 h after acquisition; this deficit was still evident 7 days after the treatment. gamma-LGLA partially inhibited L-[3H]glutamate binding on crude hippocampal or striatal membrane preparations; this inhibition was not observed on cerebellar membrane preparations. These results suggest a specific action of gamma-LGLA on excitatory amino acid systems which may be responsible for its effects on learning retention.

Amino Acids↗

Caracterisations immunochimiques de deux auxine oxydases extraites de tumeurs vegetables.

Two auxin oxidases with distinct antigenic specificities have been identified in extracts of plant tumor induced in Datura stramonium by Agrobacterium tumefaciens. Auxin oxidase and peroxidase activities are demonstrated for both enzymes. The adaptation of an immunochemical method allows the quantitative evaluation of both enzymes in different plant tissue extracts. The amount of these enzymes which is low in the organ of the healthy plant increases during tumorisation, healing and in vitro culture of healthy tissues.

Journal Article↗

Characterization of carbohydrate chains of C1-inhibitor and of desialylated C1-inhibitor.

Carbohydrate chains of C1-inhibitor were identified with a binding assay using different lectins. Lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA) that are specific for sialic acids bound to C1-inhibitor. Lectin from Datura stramonium (DSA) reacted also with the inhibitor indicating complex and hybrid sugar structures. C1-inhibitor was enzymatically desialylated and reexamined for lectin binding. SNA and MAA did not react anymore, but in addition to DSA, peanut agglutinin, which can bind to carbohydrate chains after sialic acids are removed, bound to desialylated C1-inhibitor. C1-inhibitor contains about 30 sialic acid residues per molecule. SDS-polyacrylamide gel electrophoresis showed that desialylated C1-inhibitor had a faster mobility than native C1-inhibitor. The N-terminal sequence of desialylated C1-inhibitor was the same as of native C1-inhibitor and no change in the inhibition of human plasma kallikrein was observed.

Blotting, Western↗

Characterization of human arylsulfatase A glycans.

Despite numerous studies on arylsulfatase A, the structure of the glycans present in each of its two subunits has not been determined. This is important because the carbohydrate component of human arylsulfatase A synthesized in tumor tissues and transformed cells has been shown to undergo apparent changes. This study elucidates some of their major features. Glycan chain analysis of native and deglycosylated arylsulfatase A as well as its subunits was performed with the use of a Glycan Differentiation Kit and lectin affinity chromatography. Each of the two subunits of arylsulfatase A from placenta, separated electrophoretically on polyacrylamide gel in reducing conditions, reacted with digoxigenin-labelled Galantus nivalis agglutinin and Aleuria aurantia agglutinin, while those from liver enzyme reacted with the former only. The subunits of both enzymes did not react with Sambucus nigra, Maakia amuriensis, Datura stramonium or Peanut agglutinin. Deglycosylation of arylsulfatase A with peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase F resulted in complete cleavage of its carbohydrate component from each subunit. Their molecular weights decreased by 3 kDa. Neuraminidase treatment of the enzyme from liver and placenta followed by isoelectrofocusing separation showed the presence of sialylated forms which constituted a small percentage of total enzyme activity. Placental arylsulfatase A became bound to Lens culinaris agglutinin agarose, while no interaction with Ricinus communis or Griffonia simplicifolia agglutinin agarose was observed. The study shows that both subunits of arylsulfatase A from human placenta possess two high mannose/hybrid type glycans as major structures, with at least one 6-O-L-fucose bound to the innermost N-acetylglucosamine on each.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Sequence↗

Immobilized lectin columns: useful tools for the fractionation and structural analysis of oligosaccharides.

Elucidation of the binding specificity of a concanavalin A-Sepharose column led to the possibility of the affinity chromatography of oligosaccharides and glycopeptides with the use of immobilized lectin columns. Subsequent addition of immobilized erythroagglutinating phytohaemagglutinin, Aleuria aurantia lectin, Datura stramonium agglutinin, Ricinus communis agglutinin and Allomyrina dichotoma agglutinin to the range of well characterized lectin columns has afforded a way to fractionate a mixture of N-linked oligosaccharides even to a single component.

Animals↗