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The arabinogalactan of Corynebacterianeae is a critical heteropolysaccharide that tethers outer membrane mycolic acids to peptidoglycan thus forming the characteristic cell wall core of these prokaryotes. An essential α-(1→5)-arabinosyltransferase, AftA, is responsible for the transfer of the first arabinofuranosyl (Araf) unit of the arabinan domain to the galactan backbone of arabinogalactan, but the number and precise position at which Araf residue(s) is/are added in mycobacteria remain ill-defined. Using membrane preparations from Mycobacterium smegmatis overexpressing aftA, farnesyl-phospho-arabinose as an Araf donor, and a series of synthetic galactan acceptors of various lengths, we here show that a single priming arabinosyl residue substitutes the C-5 position of a precisely positioned internal 6-linked galactofuranosyl residue of the galactan acceptors, irrespective of their length. This unexpected result suggests that, like the structurally related mycobacterial lipoarabinomannans, the arabinogalactan of mycobacteria may in fact harbor a single arabinan chain.

Drug addiction is a chronic relapsing brain disease. Alterations of glucose uptake and metabolism are found in the brain of drug addicts. Insulin mediates brain glucose metabolism and its abnormality could induce brain injury and cognitive impairment. Here, we established a rat model of phenobarbital addiction by 90 days of dose escalation and evaluated addiction-related symptoms. We also performed 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) to detect glucose uptake in the brain and proteomic analysis of the function of the differentially expressed (DE) proteins via bioinformatics in brain tissues by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) on days 60 and 90 of phenobarbital or 0.5% carboxymethyl cellulose sodium (CMC-Na) (vehicle) administration. The results showed that phenobarbital-addictive rats developed severe withdrawal symptoms after abstinence and glucose uptake was significantly increased in the brain. Proteomics analysis showed that numerous DE proteins were enriched after phenobarbital administration, among which CALM1, ARAF, and Cbl proteins (related to the insulin signaling pathway) were significantly downregulated on day 60 but not day 90. However, SLC27A3 and NF-κB1 proteins (related to insulin resistance) were significantly upregulated on day 90 (data are available via ProteomeXchange with identifier PXD021101). Our data indicate that the insulin signaling pathway and insulin resistance may play a role in the development of phenobarbital addiction and brain injury, so the findings may have important clinical implications.

Two neutral polysaccharides (PSNP-1, 104.7 kDa; PSNP-2, 24.5 kDa) were isolated from the pulp of Pyrus sinkiangensis Yu. by using the combined techniques of ion-exchange and gel permeation chromatography. According to the IR, NMR spectra, monosaccharide composition, and methylation analyses, PSNP-1 was mainly composed of glucose and xylose residues, which form a typical xyloglucan. PSNP-2 contained an arabinan region composed of 1,5-linked Araf residues, a xyloglucan region that was mainly composed of t-, 1,2-linked Xylp, and 1,4-, 1,4,6-linked Glcp residues. PSNP-1 and PSNP-2 could stimulate the cell viability, NO release, and cytokine secretion (IL-6 and TNF-α) of RAW264.7 macrophages at the adosage of 250 μg/mL. It was suggested that PSNP-1 and PSNP-2 may increase macrophage-mediated immunostimulatory activity.

Multiple rice GT61 members were demonstrated to be xylan arabinosyltransferases (XATs) mediating 3-O-arabinosylation of xylan and the functions of XATs and xylan 2-O-xylosyltransferases were shown to be conserved in grass species. Xylan is the major hemicellulose in the cell walls of grass species and it is typified by having arabinofuranosyl (Araf) substitutions. In this report, we demonstrated that four previously uncharacterized, Golgi-localized glycosyltransferases residing in clade A or B of the rice GT61 family were able to mediate 3-O-arabinosylation of xylan when heterologously expressed in the Arabidopsis gux1/2/3 triple mutant. Biochemical characterization of their recombinant proteins established that they were xylan arabinosyltransferases (XATs) capable of transferring Araf residues onto xylohexaose acceptors, and thus they were named OsXAT4, OsXAT5, OsXAT6 and OsXAT7. OsXAT5 and the previously identified OsXAT2 were shown to be able to arabinosylate xylooligomers with a degree of polymerization of as low as 3. Furthermore, a number of XAT homologs from maize, sorghum, Brachypodium and switchgrass were found to exhibit activities catalyzing Araf transfer onto xylohexaose, indicating that they are XATs involved in xylan arabinosylation in these grass species. Moreover, we revealed that homologs of another GT61 member, xylan 2-O-xylosyltransferase (XYXT1), from these grass species could mediate 2-O-xylosylation of xylan when expressed in the Arabidopsis gux1/2/3 mutant. Together, our findings indicate that multiple OsXATs are involved in 3-O-arabinosylation of xylan and the functions of XATs and XYXTs are conserved in grass species.

Background/aim Prostate cancer (PCa) is the second-most commonly occurring cancer among men, worldwide. Although the mechanisms associated with the progression of castration-resistant prostate cancer (CRPC) have been widely studied, the mechanism associated with more distant metastases from the bone remains unknown. This study aimed to characterize potential pathogenic kinases associated with highly metastatic PCa, that may regulate phosphorylation in extensively involved and diverse signaling pathways that are associated with the development of various cancers. Materials and methods A mass spectrometry (MS)-based comparative phosphoproteome strategy was utilized to identify differentially expressed kinases between the highly aggressive PCa cell-lines PC-3 and PC-3M. Results Among 2,968 phosphorylation sites in PCa cells, 151 differently expressed phosphoproteins were identified. Seven motifs: -SP-, -SxxE-, -PxS-, -PxSP-, -SxxK-, -SPxK-, and -SxxxxxP- were found to be highly expressed in PC-3M cells. Based on these motifs, the kinases p21-activated kinase (PAK)2, Ste20-like kinase (SLK), mammalian Ste20-like kinase (MST)4, mitogen-activated kinase kinase (MAP2K)2, and A-Raf proto-oncogene serine/threonine kinase (ARAF) were up-regulated in PC-3M cells. Conclusion PAK2, SLK, MST4, MAP2K2, and ARAF are kinases that are potentially associated with the progression of increased migration in PC-3M cells and may represent molecule regulators or drug targets for highly metastatic PCa therapy.

Azadirachta indica leaf is used by Indian population for the healing of various diseases including viral infection. Herein, we analyzed the antiherpetic (HSV-1) activity of two polysaccharides (P1 and P2) isolated from the leaf of A. indica and their chemically sulfated derivatives (P1S and P2S). The molecular weights of P1S and P2S are 41 and 11 kDa, respectively. Sulfate groups are located at positions C3 of the Araf and C6 of both Galp and Glcp residues of the most active polysaccharide (P1S). These compounds were not cytotoxic in HEp-2 cells, up to 1000 μg/mL. Both P1S and P2S exhibited antiviral activity when used simultaneously to HSV-1, with 50% inhibitory concentration/selectivity index, respectively, of 31.1 μg/mL/>51.4 and 80.5 μg/mL/>19.8. P1S showed better inhibitory effect (91.8%) compared to P1 (50%), P2 (71.1%) and P2S (70%) at 200 μg/mL. Synthesis of viral protein showed a dose–dependent response and the nucleic acid synthesis was inhibited up to 25 μg/mL, by P1 and P1S and up to 50 μg/mL, by P2 and P2S. The antiviral effect is probably due to the interference of polysaccharides at the early stages of HSV-1 replication, including adsorption. Further studies are under way to get insight into the mechanism of action of the substances.

A neutral polysaccharide separated from Dendrobium nobile Lindl was designated as DNPE6(4). It was structurally characterized using a combination of spectral and chemical analysis. Its average molecule weight was 99.2 kDa. The monosaccharide composition was Araf, Glcp, Galp, and Manp in a molar ratio of 2.5:0.9:0.3:0.8. Their linkage types were →1)-L-Araf-(3→, →1)-D-Glcp-(4→, →1)-D-Galp-(3→, →1)-D-Galp-(6→, →1)-D-Manp-(3, 6→, and T-D-Manp. The polysaccharide was found to have anti-TMV and anti-CMV activities for the first time in vivo. Notably, DNPE6(4) exhibited excellent protective activity against TMV. Furthermore, several proteins related to calcium signaling pathway and pathogen related proteins were up-regulated, and we also found expression levels of EDS1, ICS1, and PR1 involved in SA pathway up-regulated after DNPE6(4) treatment. In addition, some defense enzymes increased in the same condition. All these findings revealed DNPE6(4) was an elicitor to stimulate calcium signaling pathway to enhance the tobacco defense against TMV. This study therefore revealed that DNPE6(4) was a promising antiviral agent for future study.

Three polysaccharides were isolated from the roots of Codonopsis pilosula by ion-exchange and gel-filtration chromatography. They were named RCNP, RCAP-1, and RCAP-2, and had apparent molecular weights of 1.14 × 104, 5.09 × 104, and 2.58 × 105, respectively. Their structures were characterized by HPGPC, chemical derivative analysis, GC–MS and NMR analyses. Results showed that RCNP contained arabinan and arabinogalactan regions. The arabinan region had a main chain comprising (1 → 5)-linked Araf residues, and the side chains branched at the O-3 position by the single Araf residues. The arabinogalactan region comprised alternating (1 → 4)-, (1 → 6)- or (1 → 3)-linked Galp along with small amounts of branches mainly at the O-3 position of the (1 → 6)-linked Galp or O-6 position of the (1 → 3)-linked Galp residues by terminally linked Araf residues. RCAP-1 and RCAP-2 were highly methyl-esterified pectin-type polysaccharides with long homogalacturonan regions interrupted by a short rhamnogalacturonan I (RG-I) region. The side chains of the RG-I region consisted of (1 → 2)-linked Rha residues attached to the position O-4 of rhamnose. Their degrees of methyl-esterification were approximately 60.6% and 68.1%, respectively. Bioactivity tests showed that RCAP-1 and RCAP-2 exerted a significant immunostimulatory effect based on NO production from RAW264.7 macrophages. These results suggested that these two pectin-type polysaccharides were potential immunostimulation agents.

Phellinus baumii is used to treat inflammatory bowel disease (IBD) and gastroenteritis. In this study, a 46 kDa heteropolysaccharide SHPS-1 was isolated from fruiting bodies of P. baumii. SHPS-1 consisted of arabinose, mannose, glucose, and galactose at a molar ratio of 2.2:15.7:49.3:32.8. SHPS-1 had a backbone containing 1,3-linked β-D-Glcp and 1,6-linked α-D-Galp residues, and Araf, Manp and Galp units were attached as oligosaccharidic side chains to the backbone at C-6 of some glucopyranoses. SHPS-1 decreased phosphorylation level of STAT-1 and expression levels of STAT-1 targeted genes such as iNOS and TNF-α in lipopolysaccharide-stimulated macrophage RAW 264.7 cells. Furthermore, SHPS-1 promoted the expression of IL-10 and macrophage mannose receptor CD 206, markers of tissue repairing macrophages. SHPS-1 alleviated ulcerative colitis in mice by decreasing pro-inflammatory genes and increasing anti-inflammatory and tissue repairing genes. Collectively, SHPS-1 polysaccharide from P. baumii had anti-inflammatory activity and can potentially treat IBD.

International audience; The GH-51 α-l-arabinofuranosidase from Thermobacillus xylanilyticus (TxAbf) possesses versatile catalytic properties, displaying not only the ability to hydrolyze glycosidic linkages but also to synthesize furanobiosides in α-l-Araf and β-d-Galf series. Herein, mutants are investigated to evaluate their ability to perform self-condensation, assessing both yield improvements and changes in regioselectivity. Overall yields of oligo-α-l-arabino- and oligo-β-d-galactofuranosides were increased up to 4.8-fold compared to the wild-type enzyme. In depth characterization revealed that the mutants exhibit increased transfer rates and thus a hydrolysis/self-condensation ratio in favor of synthesis. The consequence of the substitution N216W is the creation of an additional binding subsite that provides the basis for an alternative acceptor substrate binding mode. As a result, mutants bearing N216W synthesize not only (1,2)-linked furanobiosides, but also (1,3)- and even (1,5)-linked furanobiosides. Since the self-condensation is under kinetic control, the yield of homo-disaccharides was maximized using higher substrate concentrations. In this way, the mutant R69H-N216W produced oligo-β-d-galactofuranosides in > 70% yield. Overall, this study further demonstrates the potential usefulness of TxAbf mutants for glycosynthesis and shows how these might be used to synthesize biologically-relevant glycoconjugates.

A water-soluble polysaccharide (LCP-05) was isolated from the flowers of Leucosceptrum canum Smith. LCP-05 was an acidic polysaccharide with a molecular weight of approximately 8.9 kDa. Monosaccharide composition analysis indicated that LCP-05 was composed of Man, Rha, GlcA, GalA, Glc, Gal and Ara in a molar ratio of 0.83:1.68:0.33:2.15:1.00:1.45:1.22. The framework of LCP-05 was speculated to be a branched rhamnogalacturonan with the backbone consisting of α-1,2,4-linked Rhap and α-1,4-linked GalAp, and bearing branches at the O-4 position of the Rha residues. The side chains are terminated primarily with the Araf and Glcp residues. LCP-05 was found to be able to significantly induce the production of NO, IL-6, and TNF-α in RAW 264.7 cells, and to induce RAW 264.7 cell's suppressive effect on both cell growth and cell migration of 4 T1 mammary breast cancer cells.

RAF (rapidly accelerated fibrosarcoma) Ser/Thr kinases (ARAF, BRAF, and CRAF) link the RAS (rat sarcoma) protein family with the MAPK (mitogen-activated protein kinase) pathway and control cell growth, differentiation, development, aging, and tumorigenesis. Their activity is specifically modulated by protein–protein interactions, post-translational modifications, and conformational changes in specific spatiotemporal patterns via various upstream regulators, including the kinases, phosphatase, GTPases, and scaffold and modulator proteins. Dephosphorylation of Ser-259 (CRAF numbering) and dissociation of 14-3-3 release the RAF regulatory domains RAS-binding domain and cysteine-rich domain for interaction with RAS-GTP and membrane lipids. This, in turn, results in RAF phosphorylation at Ser-621 and 14-3-3 reassociation, followed by its dimerization and ultimately substrate binding and phosphorylation. This review focuses on structural understanding of how distinct binding partners trigger a cascade of molecular events that induces RAF kinase activation.

A pectic polysaccharide (CHIP3) was fractionated from the natural cupule of Castanea henryi. It contained mannose (10.70%), rhamnose (8.70%), galacturonic acid (38.21%), galactose (13.75%) and arabinose (28.63%) with a molecular weight of 2.44 × 104 g/mol by multi-laser light scattering. The structure was elucidated by using FT-IR spectroscopy, methylation analysis and NMR analysis. Results showed that the backbone of CHIP3 consisted of 1, 4-α-linked d -GalpA residues containing the non-methyl-esterified carboxyl groups, interspersed with a few 1,2-α- l -Rhap units. Its side chains were attached by two branches to O-4 of Rhap with 1,4-β-linked d -Galp units and 1,5-α- l -linked Araf units bearing 3,5-substituted α- l -linked Araf residues as branching points. AFM data revealed it existed as a flexible chain in 0.1 M NaNO3 aqueous solution. Furthermore, CHIP3 was demonstrated to have notable antioxidant activity of FRAP, ABTS + radical scavenging and reducing power. Cytotoxicity assay showed it displayed inhibitory activity against HepG2 cells with IC50 values of 242.6 μg/mL.

Stevia rebaudiana, a sweetener with medicinal functions, has attracted extensive attention due to its application in food and pharmaceutical fields. However, a few studies were performed to explore polysaccharides in this plant. Herein, SRP70-1 was derived from S. rebaudiana. Structural analysis (monosaccharide composition analysis, high-performance liquid chromatography-multi-angle light scattering detection, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy) revealed that SRP70-1 was composed of mannose, glucose, galactose, and arabinose at the molar ratio of 1.35:1.00:3.23:3.47, with an absolute molecular weight of 7698 Da. SRP70-1 was found to contain → 5)-α- l -Araf-(1→, →2,3,5)-α- l -Araf-(1→, →4)-β- l -Arap-(1→, →4)-β- d -Galp-(1→, →6)-β- d -Galp-(1→, →4)-β- d -Manp-(1→, →6)-β- d -Manp-(1→, and terminal α- l -Araf, β- d -Galp, and β- d -Glcp residues. Cell experiments showed that SRP70-1 could significantly promote phagocytosis and increase the release of nitric oxide and cytokines including IL-1β, IL-6, and TNF-α. Further zebrafish experiments confirmed the immunological enhancement effects of SRP70-1. This study revealed that SRP70-1 may be useful for the development of functional foods.

The RAF-MAPK signaling pathway regulates several very diverse cellular processes such as proliferation, differentiation, apoptosis, and transformation. While the canonical function of RAF kinases within the MAPK pathway is the activation of MEK, our group could demonstrate an important crosstalk between RAF signaling and the pro-apoptotic mammalian sterile 20-like kinase (MST2) tumor suppressor pathway in several cancer entities, including head and neck, colon, and breast. Here, the RAF kinases CRAF and ARAF sequester and inhibit the pro-apoptotic kinase MST2 independently of their own kinase activity. In our recent study, we showed that the ARAF-MST2 complex is regulated by subcellular compartmentalization during epithelial differentiation. Proliferating cells of the basal cell layer in squamous epithelia and tumor cells express ARAF at the mitochondria thus allowing for efficient sequestration of MST2. In contrast, non-malignant squamous epithelia have ARAF localized at the plasma membrane, where the control of MST2-mediated apoptosis is compromised. This re-distribution is regulated by the scaffold protein kinase suppressor of Ras 2 (KSR2). Here, we summarize how spatial and temporal regulation of RAF signaling complexes affect cellular signaling and functions.

RAF kinases (ARAF, BRAF, and CRAF) are highly conserved enzymes that trigger the RAF-MEK1/2-ERK1/2 (MAPK) pathway upon activation of RAS. Despite enormous clinical interest, relatively little is known on the role of RAFs in mediating immune responses. Here, we investigated the role of RAF kinases and MEK1/2 in dendritic cells (DCs), the central regulators of T cell-mediated antitumor immune responses and the adaptive immune system. We demonstrate that RAF kinases are active and stabilized at their protein levels during DC differentiation. Inhibition of RAF kinases but not MEK1/2 impaired the activation of DCs in both mice and human. As expected, DCs treated with RAF inhibitors show defects in activating T cells. Further, RAF and MEK1/2 kinases are directly required for the activation and proliferation of CD4+T cells. Our observations suggest that RAF and MEK1/2 have independent roles in regulating DC function that has important implications for administering RAF–MAPK inhibitors in the clinics.

This study presented the first purification and characterization of a hepatoprotective polysaccharide (PNPS-0.5 M) from the residue of Panax notoginseng (Burk.) F.H. Chen. This polysaccharide included a backbone of (4 → 1)-linked GalA and branches of (1→)-linked Araf, (1→)-linked Rhap, and (5 → 1)-linked Araf and had an extremely high molecular weight (2600 kDa). We investigated the hepatoprotective effects of PNPS-0.5 M on mice with alcoholic liver damage (ALD). After administration of PNPS-0.5 M, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), and hepatic malondialdehyde (MDA) were reduced to normal. In contrast, hepatic levels of alcohol dehydrogenase (ADH) and the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were elevated to normal. Further investigations indicated that PNPS-0.5 M activated Nrf2 signaling as a protective mechanism against Cyp2e1 toxicity in ALD mice. Meanwhile, it strengthened the ADH pathway and suppressed the CAT pathway of alcohol metabolism to prevent peroxide accumulation, thereby ameliorating ALD. In the present study, we describe a novel acidic polysaccharide from P. notoginseng with hepatoprotective activity that facilitates the development and utilization of P. notoginseng resources.

A water soluble polysaccharide CP-III was extracted and purified from Cyclocarya paliurus. CP-III is identified as a novel pectin-like polysaccharide with molecular weight (Mw) of 72.7 kDa. The structural features of CP-III were characterized by methylation and nuclear magnetic resonance (NMR) spectroscopy. Its depolymerized fragments were analyzed by hydrophilic interaction chromatography-Fourier transform mass spectrometry (HILIC-FTMS). The main chain of CP-III is composed of →4)GalAp(α1 → and →2)Rhap(α1 → 4)GalAp(α1→, repeatedly. The residue of →4)Galp(β1 → and →5)Araf(α1 → alternately exists on the O-4 of partial →2)Rhap(α1 → residues as side chains. On the O-3 of sectional →5)Araf(α1 → residues is a secondary branch assembled by →3)Araf(α1→. Moreover, on the non-reducing terminus of →4)Galp(β1 → occasionally have an →5)Araf(α1 → chain. Surprisingly, a sub-branch constructed by →6)Hexp(β1 → with a galacturonate or methyl galacturonate exists on the O-3 of certain →4)Galp(β1 → residues in the non-reducing terminus. In addition, a terminal Xyl is located on the O-3 of fractional →4)GalAp(β1 → residue. The highly branched polysaccharide CP-III with high water solubility can be used as food supplement and medicinal carrier in the future.

Selective BRafV600E inhibitors with DFG-in conformation have been proven effective against a subset of melanoma. However, representative inhibitor vemurafenib rapidly acquires resistance in the BRafWT cells through a CRaf or BRafWT dependent manner. Simultaneous targeting of all subtypes of Raf proteins offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Herein, we describe the design and characterization of a series of compounds I-01-I-22, based on a pyrimidine scaffold with DFG-out conformation as Pan-Raf inhibitors. Among them, I-15 binds to all Raf protomers with IC50 values of 12.6 nM (BRafV600E), 30.1 nM (ARaf), 19.7 nM (BRafWT) and 17.5 nM (CRaf) and demonstrates cellular activity against BRafWT phenotypic melanoma and BRafV600E phenotypic colorectal cancer cells. The western blot results for the P-Erk inhibition in human melanoma SK-Mel-2 cell line showed that I-15 inhibited the proliferation of the SK-Mel-2 cell line at concentrations as low as 400 nM, without paradoxical activation of Erk as vemurafenib, which supported that I-15 may become a good candidate compound to overcome the resistance of melanoma induced by vemurafenib. I-15 also has a favorable pharmacokinetic profile in rats. Rational design, synthesis, SAR, lead selection and evaluation of the key compounds studied are described.

A water-soluble polysaccharide extracted from the roots of Angelica sinensis (Oliv.) Diels, which is a traditional Chinese medicine herb, was fractioned and purified by Sephadex G-50 gel filtration chromatography. The structural characterization and antitumor activities of the purified polysaccharide fraction, named as ASP, were evaluated in the present study. ASP, which molecular weight was determined to be 80kDa by high-performance gel-permeation chromatography, is an acidic heteropolysaccharide consisting of glucuronic acid, glucose, arabinose and galactose in ratio of 1.00:1.70:1.85:5.02. It has a backbone composed of (1→3)-linked Galp, (1→6)-linked Galp and 2-OMe-(1→6)-linked Galp with three branches attached to O-3 of 2-OMe-(1→6)-linked Galp and terminated with GlcpA and Araf, and all of Araf and the majority of Glcp are distributed in branches. Moreover, all of GlcpA were presented as (1→)-linked GlcpA in branches. In in vitro antitumor assays, ASP displayed cytotoxicity against HepG2 cells (34.32±3.50% at the concentration of 1mg/mL) and MCF-7 cells (28.90±1.50% at the concentration of 1mg/mL) in a dose-dependent manner, and ASP also showed mild inhibitory activity against A549 cells.

In our search for bioactive polysaccharides as immunomodulatory agents, an arabinofuranan (GMP90-1) was purified and characterized from the rinds of Garcinia mangostana L. GMP90-1 (absolute molecular weight: 5.30 × 103 g/mol) was found to be composed of arabinose, galactose, and rhamnose. The backbone of GMP90-1 was determined as (1→5)-linked α- l -Araf, (1→2,3,5)-linked α- l -Araf, (1→3,5)-linked α- l -Araf, (1→6)-linked β- d -Galp, and (1→2)-linked α- l -Rhap. Conformational analysis revealed GMP90-1 to exist as a rigid rod structure in sodium chloride solution. To explore its potential as immunomodulatory agents, an in vitro cell screening was performed and GMP90-1 was found to significantly enhance the phagocytic uptake of neutral red and improve the secreted level of nitric oxide (NO), interleukin (IL)-6, IL-1β, and tumor necrosis factor-α (TNF-α) of macrophages. Furthermore, the cellular immunomodulatory activities were confirmed by the in vivo zebrafish experiment, which suggested that GMP90-1 with immunomodulatory effects could be considered as a potential immunomodulatory for immune diseases.

In this present study, the structure and immunomodulatory activity of a novel polysaccharide (WSRP-1b) from Kushui rose (Rosa setate x Rosa rugosa) waste were investigated. Structure characterization demonstrated that WSRP-1b had a weight-average molecular weight of 1.11 × 104 Da and consisted of glucose (42.6 %), mannose (21.4 %), arabinose (9.9 %), xylose (2.2 %), and galactose (23.9 %). Its backbone was composed of 1, 4-linked α-Glcp, 1, 4-linked β-Glcp, and 1, 4-linked β-Manp, with branches of 1, 4-linked α-Glcp and 1, 4-linked β-Manp substituted at C-6 by 1, 6-linked β-Galp. The branches mainly contained 1, 5-linked Araf, terminal arabinose and terminal glucose. Bioactivity assays showed that WSRP-1b had immunomodulatory activity by enhancing phagocytosis of macrophages, increasing production of ROS, NO, cytokines (IL-6, TNF-α), and activating NF-κB signaling pathway. These results suggested that it could be developed as a potential and safe immunomodulatory agent in fields of pharmacological or functional foods.

Type II arabinogalactan (AG) is a soluble prebiotic fiber stimulating the proliferation of bifidobacteria in the human gut. Larch AG, which is comprised of type II AG, is known to be utilized as an energy source for Bifidobacterium longum subsp. longum (B. longum). We have previously characterized GH43_24 exo-β-1,3-galactanase (Bl1,3Gal) for the degradation of type II AG main chains in B. longum JCM1217. In this study, we characterized GH30_5 exo-β-1,6-galactobiohydrolase (Bl1,6Gal) and GH43_22 α-L-arabinofuranosidase (BlArafA), which are degradative enzymes for type II AG side chains in cooperation with exo-β-1,3-galactanase. The recombinant exo-β-1,6-galactobiohydrolase specifically released β-1,6-galactobiose (β-1,6-Gal2) from the nonreducing terminal of β-1,6-galactooligosaccharides, and the recombinant α-L-arabinofuranosidase released arabinofuranose (Araf) from α-1,3-Araf-substituted β-1,6-galactooligosaccharides. β-1,6-Gal2 was additively released from larch AG by the combined use of type II AG degradative enzymes, including Bl1,3Gal, Bl1,6Gal, and BlArafA. The gene cluster encoding the type II AG degradative enzymes is conserved in all B. longum strains, but not in other bifidobacterial species. The degradative enzymes for type II AG side chains are thought to be important for the acquisition of type II AG in B. longum.

A new water-soluble polysaccharide, APS-1II, with a molecular weight of 42.1 kDa was isolated from the roots of Angelica sinensis (Oliv.) Diels. APS-1II consists of arabinose (Ara), glucose (Glc) and fucose (Fuc) with a molar ratio of 2.48:1.05:1.00. The backbone of APS-1II is composed of 1,3-α- l -Araf and 1,6-α- d -Glcp with the branches containing 1,5-α- l -Araf, 1,4-β- d -Glcp, T-β- d -Glcp, 1,3-α- l -Fucp and T-α- l -Fucp. APS-1II inhibited the proliferation of human leukemia K562 and mouse L1210 cells in vitro and markedly prolonged the life span of L1210-bearing mice in vivo. APS-1II also increased the numbers of leukocytes and lymphocytes in peripheral blood, and significantly decreased plasma tumor necrosis factor, interleukin-2 and interferon-γ levels in L1210-bearing mice. Moreover, APS-1II administration concentration-dependently promoted the proliferation of the splenocytes, enhanced phagocytic activity of peritoneal macrophages and cytotoxicity of natural killer cells. These results suggest that APS-1II could effectively inhibit leukemia and induce a protective immune response, and it may be used as a suitable candidate reagent for leukemia therapy.

Wheat germ is well known for as a rich source of nutrients. In this study, a novel water soluble wheat germ glycoprotein (WGPII), with an average molecular weight of 14.5 kDa, was extracted from commercial wheat germ. The protein segments occupy 70.38% (w/w) of WGPII, and are rich in glutamic acid. The sugar residues of WGPII are primarily composed of (1 → 5)-linked α- l -Araf, (1 → 3, 6)-linked-α- d -Manp, (1→)-α- d -Glcp, (1 → 6)-linked-α- d -Glcp, (1 → 6)-linked-α- d -Galp, (1 → 4)-linked-α- d -Xylp and (1 → 4,6)-linked-α- d -Glcp. The protein and polysaccharide parts are linked by O-linkage bonds. WGPII is highly stable in aqueous solution at low concentrations (0.1–1 mg/mL), and show high thermo stability.

The water-soluble bioactive polysaccharides can contribute to the health benefits of Lycium barbarium fruit. However, the structure characteristics of these polysaccharides remain unclear yet. An important polysaccharide (LBPA) was isolated and purified from L. barbarium in this work. It was identified by chemical and spectroscopic methods as arabinogalactan with β-d-(1→6)-galactan as backbone, which was different to any reported polysaccharides from this species before. This arabinogalactan was comprised of Araf, Galp, GlcpA and Rhap with a molar ratio of 9.2:6.6:1.0:0.9. The side chains, including α-l-Araf-(1→, α-l-Araf-(1→5)-α-l-Araf-(1→, β-l-Araf-(1→5)-α-l-Araf-(1→ and α-l-Rhap-(1→4)-β-d-GlcpA-(1→6)-β-d-Galp-(1→, were linked to β-d-(1→6)-galactan at O-3. The putative structure was drawn as below. The molecular weight was determined to be 470,000g/mol by gel permeation chromatography.

Background: The full-length Araf (Araf-tv1) inhibits Nodal/Smad2-induced mesendodermal induction in zebrafish embryos. Results: Araf-tv2, a kinase-deficient isoform of zebrafish Araf, can inhibit developmental functions of Fgf/Ras signaling. Conclusion: Araf-tv1 and Araf-tv2 regulate distinct signaling pathways in zebrafish embryos. Significance: The zebrafish araf gene is expressed to produce different variants with distinct functions during embryogenesis., Raf kinases are important components of the Ras-Raf-Mek-Erk pathway and also cross-talk with other signaling pathways. Araf kinase has been demonstrated to inhibit TGF-β/Smad2 signaling by directly phosphorylating and accelerating degradation of activated Smad2. In this study, we show that the araf gene expresses in zebrafish embryos to produce a shorter transcript variant, araf-tv2, in addition to the full-length variant araf-tv1. araf-tv2 is predicted to encode a C-terminally truncated peptide without the kinase activity domain. Araf-tv2 can physically associate with Araf-tv1 but does not antagonize the inhibitory effect of Araf-tv1 on TGF-β/Smad2 signaling. Instead, Araf-tv2 interacts strongly with Kras and Nras, ultimately blocking MAPK activation by these Ras proteins. In zebrafish embryos, overexpression of araf-tv2 is sufficient to inhibit Fgf/Ras-promoted Erk activation, mesodermal induction, dorsal development, and neuroectodermal posteriorization. Therefore, different isoforms of Araf may participate in similar developmental processes but by regulating different signaling pathways.

The berries of Sambucus nigra have traditionally been used and are still used to treat respiratory illnesses such as cold and flu in Europe, Asia and America. The aim of this paper was to elucidate the structures and the immunomodulating properties of the pectic polymers from elderberries. All the purified fractions obtained from 50% ethanol, 50°C water and 100°C water extracts showed potent dose-dependent complement fixating activity and macrophage stimulating activity. The isolated fractions consisted of long homogalacturonan regions, in addition to arabinogalactan-I and arabinogalactan-II probably linked to a rhamnogalacturonan backbone. Reduced bioactivity was observed after reduction of Araf residues and 1→3,6 Gal by weak acid hydrolysis. The rhamnogalacturonan region in SnBe50-I-S3-I and SnBe50-I-S3-II showed higher activity compared to the native polymer, SnBe50-S3, after enzymatic treatment with endo-α-d-(1→4)-polygalacturonase. These results indicated that elderberries contained immunomodulating polysaccharides, where the ramified regions express the activities observed.

To the editor: Erdheim-Chester disease (ECD) is a myeloid neoplasm characterized by recurrent mutations in mitogen-activated protein kinase pathway genes, including BRAF , ARAF , N/KRAS , MAP2K1 , and PIK3CA mutations and fusions in ALK and NTRK1 .[1][1][⇓][2][⇓][3]-[4][4] Lesional ECD cells

Decoction of Psidium guajava leaves has been used as medication for chronic coughs and breathlessness for ages. Despite demonstration of antitussive activity, the specific molecule responsible for this remains unidentified. Herein, we report chemical profile and antitussive activity of its water extract (WE) and a polysaccharide (F1) present therein. This polysaccharide (F1), purified from WE by precipitation with ethanol and then through Cu(II)acetate, contains Ara, Gal, Rha, Glc and GalA residues, and has a molecular mass of 156 kDa. It comprises of terminal-, (1,5)- and (1,3,5)-linked Araf; (1,3)-, (1,6)- and (1,3,6)-linked Galp alongside (1,2)- and (1,2,4)-linked Rhap residues. Oligosaccharides indicating polysaccharide structure have been generated by Smith degradation and characterized. The WE fraction suppressed citric acid induced cough efforts in guinea pigs in the dose of 50 mg kg−1. Assessment of antitussive activity of fractions prepared from WE namely F1 (polysaccharide) and F2 (ethanol soluble fraction) revealed that polysaccharide is the active component. Remarkably, tested samples do not alter the specific airway smooth muscle reactivity in animals significantly. The simple extraction method, prominent activity and favorable reactions profile suggest that this macromolecule could be an antitussive drug candidate.

l-Arabinose is one of the main constituents of cell wall polysaccharides such as pectic rhamnogalacturonan I (RG-I), glucuronoarabinoxylans and other glycoproteins. It is found predominantly in the furanose form rather than in the thermodynamically more stable pyranose form. UDP-L-arabinofuranose (UDP-Araf), rather than UDP-L-arabinopyranose (UDP-Arap), is a sugar donor for the biosynthesis of arabinofuranosyl (Araf) residues. UDP-arabinopyranose mutases (UAMs) have been shown to interconvert UDP-Araf and UDP-Arap and are involved in the biosynthesis of polysaccharides including Araf. The UAM gene family has three members in Oryza sativa. Co-expression network in silico analysis showed that OsUAM3 expression was independent from OsUAM1 and OsUAM2 co-expression networks. OsUAM1 and OsUAM2 were expressed ubiquitously throughout plant development, but OsUAM3 was expressed primarily in reproductive tissue, particularly at the pollen cell wall formation developmental stage. OsUAM3 co-expression networks include pectin catabolic enzymes. To determine the function of OsUAMs in reproductive tissues, we analyzed RNA interference (RNAi)-knockdown transformants (OsUAM3-KD) specific for OsUAM3. OsUAM3-KD plants grew normally and showed abnormal phenotypes in reproductive tissues, especially in terms of the pollen cell wall and exine. In addition, we examined modifications of cell wall polysaccharides at the cellular level using antibodies against polysaccharides including Araf. Immunolocalization of arabinan using the LM6 antibody showed low levels of arabinan in OsUAM3-KD pollen grains. Our results suggest that the function of OsUAM3 is important for synthesis of arabinan side chains of RG-I and is required for reproductive developmental processes, especially the formation of the cell wall in pollen.

Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder characterized by lesions composed of pathological CD207+ dendritic cells with an inflammatory infiltrate. BRAFV600E remains the only recurrent mutation reported in LCH. In order to evaluate the spectrum of somatic mutations in LCH, whole exome sequencing was performed on matched LCH and normal tissue samples obtained from 41 patients. Lesions from other histiocytic disorders, juvenile xanthogranuloma, Erdheim-Chester disease, and Rosai-Dorfman disease were also evaluated. All of the lesions from histiocytic disorders were characterized by an extremely low overall rate of somatic mutations. Notably, 33% (7/21) of LCH cases with wild-type BRAF and none (0/20) with BRAFV600E harbored somatic mutations in MAP2K1 (6 in-frame deletions and 1 missense mutation) that induced extracellular signal-regulated kinase (ERK) phosphorylation in vitro. Single cases of somatic mutations of the mitogen-activated protein kinase (MAPK) pathway genes ARAF and ERBB3 were also detected. The ability of MAPK pathway inhibitors to suppress MAPK kinase and ERK phosphorylation in cell culture and primary tumor models was dependent on the specific LCH mutation. The findings of this study support a model in which ERK activation is a universal end point in LCH arising from pathological activation of upstream signaling proteins.

Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. In a previous article, we cloned and characterized the hypBA2 gene as a β-l-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel β-l-arabinofuranosidase that liberates l-arabinose from the l-arabinofuranose (Araf)-β1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-366 is a critical residue for catalytic activity. This report provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the GH family 127.

Bioactivity-guided fractionation of hot-water extract from the roots of Bupleurum chinense DC led to the isolation of a homogeneous polysaccharide BC-PS1 composed of Gal, GalA, Glc, Ara and Man in the ratio of 1.6:1.1:1.8:1.7:1.0, along with trace of Rha, Fuc and Xyl. The main linkages of the residues of BC-PS1 include terminal, 1,4-linked and 1,3-linked Galp (GalA), terminal and 1,4-linked Glcp, terminal and 1,5-linked Araf, 1,4,6-linked Manp. Bioassays revealed that the concentrations of BC-PS1 that resulted in 50% hemolytic inhibition on classical complement pathway (CH50) and alternative complement pathway (AP50) were 0.199±0.025 mg/mL and 0.371±0.017 mg/mL, respectively. Preliminary mechanism study indicated that BC-PS1 interacted with C1q, C2, C5 and C9.

The l -arabinose-inducible araBAD promoter (P BAD ) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where P BAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli , Spot 42 sRNA regulates P BAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF , a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of P BAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the P BAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the P BAD system and has implications for improving its usage for tunable gene expression.

Indexación: Scopus. In plants, L-Arabinose (Ara) is a key component of cell wall polymers, glycoproteins, as well as flavonoids, and signaling peptides. Whereas the majority of Ara found in plant glycans occurs as a furanose ring (Araf), the activated precursor has a pyranose ring configuration (UDP-Arap). The biosynthesis of UDP-Arap mainly occurs via the epimerization of UDP-xylose (UDP-Xyl) in the Golgi lumen. Given that the predominant Ara form found in plants is Araf, UDP-Arap must exit the Golgi to be interconverted into UDPAraf by UDP-Ara mutases that are located outside on the cytosolic surface of the Golgi. Subsequently, UDP-Araf must be transported back into the lumen. This step is vital because glycosyltransferases, the enzymes mediating the glycosylation reactions, are located within the Golgi lumen, and UDP-Arap, synthesized within the Golgi, is not their preferred substrate. Thus, the transport of UDP-Araf into the Golgi is a prerequisite. Although this step is critical for cell wall biosynthesis and the glycosylation of proteins and signaling peptides, the identification of these transporters has remained elusive. In this study, we present data demonstrating the identification and characterization of a family of Golgilocalized UDP-Araf transporters in Arabidopsis. The application of a proteoliposome-based transport assay revealed that four members of the nucleotide sugar transporter (NST) family can efficiently transport UDP-Araf in vitro. Subsequent analysis of mutant lines affected in the function of these NSTs confirmed their role as UDP-Araf transporters in vivo. http://www.pnas.org/content/114/16/4261.full

Ethnopharmacological relevance The spikes of Prunella vulgaris have long been used as a traditional Chinese medicine to treat various inflammation-related diseases. The aim of this study was to isolate and characterize homogenous polysaccharides from this herb and to evaluate their anticomplement activity. Materials and methods Anticomplement activity-guided fractionation of the hot water extract of P. vulgaris was performed by DEAE-cellulose and size-exclusion chromatography, yielding two homogeneous polysaccharides PW-PS1 and PW-PS2. The homogeneity, molecular weight, monosaccharide composition and linkage of the two polysaccharides were determined in addition to other chemical characterizations. The anticomplement activity of the polysaccharides was evaluated and expressed as 50% hemolytic inhibition concentration through the classical pathway (CH50 value) and alternative pathway (AP50 value). The preliminary mechanism for the complement activation cascade was also assessed. Results PW-PS1 and PW-PS2 were both branched acidic polysaccharides. PW-PS1 was composed of Ara, Xyl, and 4-methoxy-Glc A in a ratio of 1.0: 2.6: 0.8. The main linkages of the sugar residues of PW-PS1 included terminal β- d -Xylp, 1,4-linked β- d -Xylp, 1,3-linked α- d -Arap, 1,3,5-linked α- d -Arap, and terminal 4-methoxy-α- d -Glcp A. PW-PS2 was composed of Rha, Ara, Xyl, Gal, and Gal A in a ratio of 0.6: 1.0: 1.3: 1.8: 3.4. The main linkages between the sugar residues of PW-PS2 included terminal Araf, 1,4-linked β- d -Xylp, 1,3-linked α- d -Rhap, terminal α- d -Galp, and 1,4,6-linked α- d -Galp. PW-PS1 and PW-PS2 inhibited complement activation through both the classical and alternative pathways with CH50 values of 0.28 and 0.13 mg/mL, respectively, and AP50 values of 0.40 and 0.35 mg/mL, respectively. Preliminary mechanism studies using complement component-depleted sera showed that PW-PS1 acted on the C1q, C3, and C9 components and that PW-PS2 acted on the C1q, C2, C3, C5, and C9 components. Conclusion Our study suggested that PW-PS1 and PW-PS2 could be valuable for the treatment of diseases associated with the excessive activation of the complement system.

PLP-3 (Plantago asiatica L. polysaccharide 3) was isolated and purified from the seeds of Plantago asiatica L. Its structure characters were elucidated by monosaccharide composition analysis, partial acid hydrolysis and methylation analysis, combined with FT-IR, GC/MS, 1D and 2D NMR spectroscopy. PLP-3 was found to be arabinoxylan, containing glucuronic acid. It was consisted of β-1,4-linked Xylp backbone with short side chains attached to its O-2 (1,2,4-linked Xylp, 17.87%) or O-3 (1,3,4-linked Xylp, 24.24%) positions. The main terminal residues were T-linked Araf (8.13%), T-linked Xylp (15.58%) and T-linked GlcAp (13.22%). Small amounts of other residues were also found in PLP-3, such as 1,2,5-linked Araf, 1,2-linked Rhap, T-linked Glcp and T-linked Galp. A possible molecular structure was proposed.

A type I arabinogalactan (CPP) was isolated from the viscous gum exuded by Cereus peruvinus (Cactaceae). It contained arabinose, galactose, galacturonic acid, and rhamnose in a 15:66:6:13 molar ratio and had Mw 9 × 105 g mol−1. Methylation analysis and 13C NMR spectroscopy indicated that CPP is composed of a (1 → 4)-linked β- d -Galp main-chain with substituents of α- l -Araf at O-2, O-3 and O-6, which are in turn substituted at O-2, O-3, and O-2,3. These are probably linked to O-4 of some rhamnosyl units of a type I rhamnogalacturonan (RG1). The main chain is formed by repeating (1 → 4)-α- d -GalpA-(1 → 2)-α- l -Rhap groups. The methylation analysis suggested the presence of (1 → 3)-linked β-Galp structures, perhaps linked to the (1 → 4)-linked β- d -Galp backbone. CPP significantly inhibited ethanol-induced gastric lesions in rats at an ED50 of 49 mg kg−1, indicating that it has a gastroprotective effect. The gastric lesion inhibition by CPP suggests a potential use of this polysaccharide, or the crude plant extract, in phytotherapy.

A water-soluble polysaccharide isolated from Cynomorium songaricum which is a traditional Chinese food was purified by Ultrahydrogel 500 gel filtration chromatography. The structural characterisation and hypoglycaemic activity of the purified polysaccharide fraction (CSPA) were evaluated. CSPA (Mw of 1.394 × 105 Da) was a heteropolysaccharide consisting of Ara, Glu and Gal. The conformation of CSPA was characterised as a compact chain of sphere-like structure in aqueous solution with a df value of 2.86. Linkage analysis and NMR (1D and 2D) revealed the presence of the following unit: → 3 ) - α - araf - ( 1 → 3 ) - α - d - glcp - ( 1 → 4 ) - α - d -Gal p A6Me- ( 1 → . Pharmaceutical experiments showed that the administration of CSPA could significantly decrease blood glucose levels, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, blood urea nitrogen, creatinine activities, and effectively increase serum insulin level and the content of liver glycogen in streptozotocin-induced diabetic rats. Histopathology studies showed that the reduced islet cells were restored to near normal upon treatment with CSPA. The results suggest CSPA could be considered as a potential candidate for developing a new anti-diabetic agent.

To characterize the macrophage stimulating polysaccharide in grape (Vitis labrusca) peels, the active crude polysaccharide (VL-3) has been fractionated from the hot-water extract of grape peels. A macrophage stimulating polysaccharide-rich fraction (VL-3IIb-1-1) was purified from VL-3 by 3 successive column chromatographies on DEAE-Sepharose CL-6B, Sepharose CL-6B, and Sephacryl S-300. VL-3IIb-1-1 was eluted as a single peak on high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 194 kDa. VL-3IIb-1-1 consisted mainly of Ara and Gal in addition to uronic acid (GalA+GlcA) (molar ratio 1.00:0.81:0.72). Methylation analysis indicated that VL-3IIb-1-1 consisted mainly of terminal Araf, 4- or 5-linked Ara, 2,4-branched Rha, 6- or 3,4- or 3,6-branched Gal, and 3,4,6-branched Glc. Single radial gel diffusion also indicated that VL-3IIb-1-1 showed an intermediate reactivity with β-glucosyl-Yariv antigen. In addition, oral administration of VL-3IIb enhanced the stimulatory responses of macrophage stimulating activity ex vivo. Therefore, VL-3IIb-1-1 purified from grape peels is suggested to be pectic polysaccharide with arabino-3,6-galactan, and it is assumed that VL-3IIb-1-1 plays an important role for expression of its activity.

Arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are potential drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis (MTB). Herein, we report the synthesis and acceptor/inhibitory activity of Araf alpha(1-->5) Araf disaccharides possessing deoxygenation at the reducing sugar of the disaccharide. Deoxygenation at either the C-2 or C-3 position of Araf was achieved via a free radical procedure using xanthate derivatives of the hydroxyl group. The alpha(1-->5)-linked disaccharides were produced by coupling n-octyl alpha-Araf 2-/3-deoxy, 2-fluoro glycosyl acceptors with an Araf thioglycosyl donor. The target disaccharides were tested in a cell free mycobacterial AraTs assay as well as an in vitro assay against MTB H(37)Ra and M. avium complex strains.

Glycosyltransferases (GTs) play a crucial role in mycobacterial cell wall biosynthesis and are necessary for the survival of mycobacteria. Hence, these enzymes are potential new drug targets for the treatment of tuberculosis (TB), especially multiple drug-resistant TB (MDR-TB). Herein, we report the efficient syntheses of Araf(α 1 → 5)Araf, Galf(β 1 → 5)Galf, and Galf(β 1 → 6)Galf disaccharides possessing a 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl) unit that were prepared as fluorescent disaccharide acceptors for arabinosyl- and galactosyl-transferases, respectively. Such analogs may offer advantages relative to radiolabeled acceptors or donors for studying the enzymes and for assay development and compound screening. Additionally, analogs possessing a 5-azidonaphthalene-1-sulfonamidoethyl unit were prepared as photoaffinity probes for their potential utility in studying active site labeling of the GTs (arabinosyl and galactosyl) in Mycobacterium tuberculosis (MTB). Beyond their preparation, initial biological testing and kinetic analysis of these disaccharides as acceptors toward glycosyltransferases are also presented.

An immunomodulating pectic polymer, GOA1, obtained from the aerial parts of the Malian medicinal plant Glinus oppositifolius (L.) Aug. DC. (Aizoaceae) has previously been reported to consist of arabinogalactans type I and II, probably linked to a rhamnogalacturonan backbone. To further elucidate the structure of the polymer GOA1, enzymatic degradation studies and weak acid hydrolysis were performed. Five different glycosidases were used, endo-alpha-D-(1--4)-polygalacturonase, exo-alpha-L-arabinofuranosidase, endo-alpha-L-(1--5)-arabinanase, endo-beta-D-(1--4)-galactanase and exo-beta-D-galactosidase. It appears that GOA1 may contain a structural moiety consisting of a 1,3-linked galactopyranosyl (Galp) main chain with 1,6-linked Galp side chains attached to position 6 of the main chain. The 1,6-linked Galp side chain may be branched in position 3 with arabinofuranosyl (Araf) side chains. A 1,4-linked Galp backbone which might carry side chains or glycosyl units attached to position 3 is also a structural element in the polymer. We further show that GOA1 induce proliferation of B cells and the secretion of IL-1beta by macrophages, in addition to a marked increase of mRNA for IFN-gamma in NK-cells. To elucidate structure-activity relations the native polymer and the digested fractions were tested for complement fixing activity and intestinal immune stimulating activity. The partial removal of Araf residues after enzymatic degradations did not affect the bioactivities, while the acid hydrolysed fraction showed reduced complement fixing activity. A decrease in Araf units, 1,3,6-linked Galp units and a partial hydrolysed rhamnogalacturonan backbone, in addition to a reduction in molecular weight are factors that might have contributed to reduced bioactivity.

Cell wall material from whole oat grains was sequentially extracted to study the structural characteristics of individual arabinoxylan (AX) populations. Araf was singly substituted at both O-3 (mainly) and O-2 positions of Xylp, and no disubstitution of Xylp with Araf residues was found in oat AXs. Both highly substituted and sparsely substituted segments were found in AXs in Ba(OH)2 extracts, whereas AXs in 1 and 6 M NaOH extracts were rarely branched and easily aggregated. Both O-2-linked GlcA and 4-O-MeGlcA residues were present in oat AXs. A series of AX oligomers with galactose as a substituent was detected for the first time in oats. The present study suggested that the distribution of Araf was contiguous in oat AXs, different from the homogeneous distribution of Araf in wheat and barley AXs, which might result in different fermentation patterns in humans and animals.

Plant cell walls constitute the bulk of the earth renewable source of energy and are a component in the diet of humans and herbivores. l-Arabinofuranosyl (Araf) residues are a quantifiably important constituent of these walls. Plants use uridine diphosphate (UDP)-l-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing polysaccharides, proteoglycans, and glycoproteins. However, little is known about the formation of UDP-Araf. We now describe the purification and partial characterization of a rice UDP-arabinopyranose mutase (UAM) that catalyzes the formation of UDP-Araf from UDP-arabinopyranose (UDP-Arap). The reaction is reversible and at thermodynamic equilibrium the pyranose form is favored over the furanose form (90 : 10). Three related proteins that are encoded by rice gene loci Os03g40270, Os04g56520, and Os07g41360 were identified from partial amino acid sequences of UAM. These proteins have >80% sequence identity with polypeptides that are reversibly glycosylated in the presence of UDP-sugars. The rice mutase and two functionally active recombinant mutases were shown to be reversibly glycosylated in the presence of UDP-Glc. The cofactor, flavin-adenine-dinucleotide (FAD), is required for the catalytic activity of UDP-galactose mutases of prokaryotes, fungi, and protozoa. The plant mutases, which do not require a cofactor, must therefore have a different catalytic mechanism. Putative UAM-encoding genes are present in the green algae Chlamydomonas reinhardtii, the moss Physcomitrella patens, the gymnosperm Pinus taeda (loblolly pine), and in numerous dicots and monocots, indicating that UAMs are widespread in green plants.

Arabinofuranosyltransferase activity was identified in Golgi membranes obtained from mung bean (Vigna radiata) hypocotyls. The enzyme transfers the arabinofuranosyl (Araf) residue from UDP-β-l-arabinofuranose to exogenous (1, 5)-linked α-l-arabino-oligosaccharides labeled at their reducing ends with 2-aminobenzamide. The transferred residue was shown, using 1H-nuclear magnetic resonance spectroscopy and α-l-arabinofuranosidase treatment, to be α-l-Araf and to be linked to O-5 of the nonreducing terminal Araf residue of the acceptor oligosaccharide. The enzyme was nonprocessive because only a single Araf residue was added to the acceptor molecule. Arabino-oligosaccharides with a degree of polymerization between 3 and 8 were acceptor substrates. The 2-aminobenzamide-labeled arabino-tetra- and pentasaccharides were the most effective acceptor substrates analyzed. The enzyme has a pH optimum between 6.5 and 7.0 and its activity is stimulated by Mn2+ and Co2+ ions. The apparent Km and Vmax values of the arabinofuranosyltransferase for UDP-arabinofuranose are 243 μm and 243 pmol min−1 mg protein−1, respectively. The highest enzyme activity was detected in the elongating regions of mung bean hypocotyls. The data show that UDP-arabinofuranose is the donor molecule for the generation of arabino-oligosaccharides composed of Araf residues.

This work describes the purification and characterization of enzymes that exhibit β-d-xylosidase activity in stem tissues of Arabidopsis. This is the first detailed investigation that concerns the characterization of catalytic properties and sequence identity of enzymes with β-d-xylosidase activities in a dicotyledonous plant. Three different enzymes, ARAf, XYL4, and XYL1 with apparent molecular masses of 75, 67, and 64 kD, respectively, were purified to homogeneity. ARAf was identified as a putative α-l-arabinofuranosidase, and XYL4 and XYL1 as putative β-d-xylosidases using matrix-assisted laser-desorption ionization time of flight. ARAf belongs to family 51 and XYL4 and XYL1 to family 3 of glycoside hydrolases. ARAf and XYL1 have highest specificity for p-nitrophenyl-α-l-arabinofuranoside and XYL4 for p-nitrophenyl-β-d-xylopyranoside and natural substrates such as xylobiose and xylotetraose. XYL4 was shown to release mainly d-Xyl from oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, and oligoarabinoxylans. ARAf and XYL1 can also release d-Xyl from these substrates but less efficiently than XYL4. Moreover, they can also release l-Ara from arabinoxylans and arabinan. Overall, the results indicate that XYL4 possesses enzymatic specificity characteristic for a β-d-xylosidase, while ARAf and XYL1 act as bifunctional α-l-arabinofuranosidase/β-d-xylosidases. Analysis of the activity of these three enzymes in stem tissues at different stages of development has shown that young stems possess the highest activities for all three enzymes in comparison to the activities of the enzymes present in stems at older stages of development. High enzyme activities are most likely related to the necessary modifications of cell wall structure occurring during plant growth.

The genus Tsukamurella is a member of the phylogenetic group nocardioform actinomycetes and is closely related to the genus Mycobacterium. The mycobacterial cell envelope contains lipoglycans, and of particular interest is lipoarabinomannan, one of the most potent mycobacterial immunomodulatory molecules. We have investigated the presence of lipoglycans in Tsukamurella paurometabola and report here the isolation and structural characterization of a new lipoarabinomannan variant, designated TpaLAM. Matrix-assisted laser desorption ionization-mass spectrometric analysis revealed that TpaLAM had an average molecular mass of 12.5 kDa and consequently was slightly smaller than Mycobacterium tuberculosis lipoarabinomannan. Using a range of chemical degradations, NMR experiments, capillary electrophoresis, and mass spectrometry analyses, TpaLAM revealed an original carbohydrate structure. Indeed, TpaLAM contained a mannosylphosphatidyl-myo-inositol (MPI) anchor glycosylated by a linear (alpha1-->6)-Manp mannan domain, which is further substituted by an (alpha1-->5)-Araf chain. Half of the Araf units are further substituted at the O-2 position by a Manp-(alpha1-->2)-Manp-(alpha1--> dimannoside motif. Altogether, TpaLAM appears to be the most elaborated non-mycobacterial LAM molecule identified to date. TpaLAM was found to induce the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha when tested with either human or murine monocyte/macrophage cell lines. This induction was completely abrogated in the presence of an anti-toll-like receptor-2 (TLR-2) antibody, suggesting that TLR-2 participates in the mediation of TNF-alpha production in response to TpaLAM. Moreover, we established that the lipomannan core of TpaLAM is the primary moiety responsible for the observed TNF-alpha-inducing activity. This conclusively demonstrates that a linear (alpha1-->6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity.

Structural features of the acidic, highly substituted glycanoxylan (LCP; 87% yield) from the gum exudate of the palm, Livistona chinensis, family Arecaceae, were determined. It had [alpha]D -30 degrees, Mw 1.9x10(5) and a polydispersity ratio Mw/Mn of approximately 1.0. Acid hydrolysis gave rise to Rha, Fuc, Ara, Xyl, and Gal, in a 1:6:46:44:3 molar ratio, and 12% of uronic acid was present. LCP had a highly branched structure with side-chains containing nonreducing end-units (% values are approximate) of Araf (15%), Fucp (4%), Xylp (7%), GlcpA, and 4-Me-GlcpA, and internal 2-O- (5%) and 3-O-substituted Araf (8%), and 2-O-substituted Xylp (14%) units. The (1-->4)-linked beta-Xylp main-chain units of LCP were substituted at O-3 (4%), O-2 (17%), and O-2,3 (16%). Partial acid hydrolysis gave 4-Me-alpha-GlcpA-(1-->2)-[beta-Xylp-(1-->4)](0-2)-Xyl, identified by showing that the uronic acids were single-unit side-chain substituents on O-2. Milder hydrolysis conditions removed from O-3 other side-chains containing Fucp and Araf nonreducing end-units and internal Arap, and 2-O- and 3-O-substituted Araf units. Carboxyl-reduced LCP contained 4-O-methylglucose and glucose in a 3.2:1 molar ratio, arising from GlcpA and 4-OMe-GlcpA nonreducing end-units, respectively. The gum contained small amounts of free alpha-Fucp-(1-->2)-Ara, which corresponds to structures in the polysaccharide. Free myo- and D- or L-chiro-inositol were present in a 9:1 ratio.