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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.

Turkish Cypriot Literature has appeared to develop for the last fifthy years and come out as an independent literature. As the Turkish Cypriot intellectuals go through a struggle of existance, they could not establish a strong contact with the World and European Literature; besides, they have tried to follow ancient and current World and Eurepean writers from time to time. Dante Alieghieri who is an Italian poet of 13 th-14th century, is one of those writers. One might think that Shakespeare or some other British writers might have affected the Turkish Cypriot writers as the Cypriot Turks have lived under the British colonial administration for a long time. However, when a number of approximate 170 poem books that are published from 1920s through current day are investigated, it is seen that Turkish Cypriot poets and writers refered to Dante by various means, and used similar motives inspiring from ”Inferno“ chapter of Divina Commedia. It is also identical in Turkish Literature. Rauf Raif Denktaş, Semih Sait Umar, Pembe Marmara, Aydın Adamoğlu, Fikret Demirağ, Özbil Bedri, Zeki Ali, Özker Yaşın, Hatice İntaç, Feriha Altıok, Taner Baybars are some of those Turkish Cypriot writers and poets who are inspired from Dante. Kıbrıs Türk Edebiyatı son elli yılda bir gelişme göstermekte ve müstakil bir edebiyat olarak ele gelmeye başlamaktadır. Kıbrıs-Türk aydınları bir varoluş mücadelesi içinden geçtikleri için Avrupa ve Dünya edebiyatlarıyla büyük bir bağ kuramamışlar; bununla birlikte zaman zaman Avrupa ve Dünya yazarlarını takibe çalışmışlardır. Bunlardan biri de 13-14.yy İtalyan şâiri Dante Alighieri’dir. Kıbrıs Türkleri uzun süre İngiliz sömürge yönetimi altında kalmaları dolayısıyla öncelikle insanın aklına Shakespeare ve diğer İngiliz yazarlarının Kıbrıs Türk edebiyatçılarına etki edebileceği gibi bir fikir gelebilir. Ancak 1920’lerden günümüze Kıbrıs’ta çıkan 170’e yakın şiir kitabı incelendiğinde Kıbrıs-Türk şâir ve yazarlarının çeşitli vesilelerle Dante’ye göndermelerde bulundukları, onun İlahi Komedya’sının “Cehennem” kısmından etkilenerek eserlerine benzer motifler yerleştirdikleri görülmektedir. Bu durum Türkiye Edebiyatında da böyledir. Kıbrıs-Türk Edebiyatında Dante’den etkilenen, ilham alan yazarlar ve şâirler arasında Rauf Raif Denktaş, Semih Sait Umar, Pembe Marmara, Aydın Adamoğlu, Fikret Demirağ, Özbil Bedri, Zeki Ali, Özker Yaşın, Hatice İntaç, Feriha Altıok, Taner Baybars, Altay Burağan gibi isimler yer almaktadır.

Summary Background: Renin-angiotensin-aldosterone system (RAS) may be activated during atrial fibrillation (AF). It is unclear whether RAS inhibition may facilitate cardioversion from AF and may prevent acute recurrence of AF (ARAF). We thus investigated the effect of pretreatment with RAS blockers on cardioversion success and ARAF in patients with AF scheduled for elective cardioversion. Methods: This observational study included 356 patients with AF undergoing elective pharmacological or electrical cardioversion. Of these patients, 135 were not included based on exclusion criteria and the remaining 221 patients were divided into RAS group (n = 116, 69 male) or non-RAS group (n = 105, 58 male) based on precardioversion use of any RAS blocker. Results: Hypertension, coronary heart disease and heart failure were more frequent in the RAS group. Cardioversion from AF was more successful in the RAS group than in the non-RAS group (%92 vs. %82, p = 0.026). The rate of ARAF was lower in RAS group compared with that in non-RAS group (17% vs. 31%, p = 0.026). In multivariate analysis, pretreatment with RAS blockers in addition to shock number and enlarged left atrium, independently predicted ARAF (OR: 0.33, 95% CI: 0.15–0.75, p = 0.008). Independent predictors of cardioversion success were shock number and left atrial dilatation, but not use of RAS blocker. Conclusion: Precardioversion use of RAS blockers may reduce ARAF following successful cardioversion of AF, but did not improve electrical cardioversion.

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.

The polysaccharide (VTP) from the gum exudate of Vochysia tucanorum gave aqueous solutions of low viscosity. VTP contained two components, which on freeze–thawing, gave rise to soluble (S-VTP) and precipitated (P-VTP) fractions, with Mw 350,000 and 520,000 g/mol, and dn/dc 0.134 and 0.139, respectively. Acid hydrolysis of S-VTP provided Ara, Xyl, Man, Gal, Glc in a 43:1:5:18:2 molar ratio (GC–MS) with 31% uronic acid, whereas the values for P-VTP were 51:1:5:16:2 and 25%, respectively. Methylation analysis of S-VTP revealed 11 neutral, partially O-methylated alditol acetates, mainly from nonreducing end- (31%) and 5-O-substituted Araf (9%), 3-O- (16%) and 3,4-di-O-substituted Galp (9%), and 2,3-di-O-substituted Manp units (10%): P-VTP was similar, containing the same structures, consistent with their almost identical 13C NMR spectra. Partial hydrolysis of P-VTP removed side chains giving rise to the main chain (PH4h) of -β- d -GlcpA-(1 → 2)-α- d -Manp-(1 → 4)- repeating groups. A controlled Smith degradation of S-VTP gave a polysaccharide (S1-SVTP), shown by methylation analysis to contain α- l -Araf-(1 → 3)-β- d -Galp-, α- l -Araf-(1 → 3)-β- d -Galp-(1 → 3)-β- d -Galp-, α- l -Araf-(1 → 3)[α- l -Araf-(1 → 4]-β- d -Galp-, and α- l -Araf-(1 → 2)-α- d -Manp- groups, with the Araf region probably containing some additional, oxidized 5-O-substituted L-Araf units. This agreed with the structure of a polysaccharide (PH30), formed by milder hydrolysis of P-VTP, sufficient to only remove Araf groups. For a more reliable characterization and quantification of S-VTP structures, it was subjected to two carbodiimide reductions and methylation analysis of the product (CR2-SVTP) showed the former GlcpA units to be 3,4-di-O-substituted. Minute amounts of a (1 → 4)-linked β- d -xylan were present in the gum, along with free reducing oligosaccharides. ESI-MS–MS, 13C NMR, and methylation analysis showed Ara-Gal-Man side-chain sequences similar to those of the glycoglucuronomannan. Mainly present was Ara4-Hex2, whose daughter ions were Ara3-Hex2, and Ara2. The oligosaccharide mixture contained nonreducing end-, 2-O- and 3-O-substituted Araf, internal 3-O- and 3,4-di-O-substituted internal Galp, and 2-O-substituted Manp reducing end-units. The glycoglucuronomannan of Vochysia lehmannii gum, although having the same main-chain, contained less side-chains.

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.

The action of three glycoside hydrolase family GH51 and one GH54 α-arabinofuranosidases (ABFs) was studied on polymeric arabinoxylan and arabinoxylooligosaccharides. The substrates covered all possible arabinofuranosyl (Araf) substituents, i.e., terminal and internal α(1→2) and α(1→3)-linked Araf monosubstitutions and disubstitutions. The GH51 ABFs removed nearly all mono- and disubstitutions from terminal non-reducing end xylopyranosyl (Xylp) residues, showing dual ABF-m/d activity. From internal Xylp, primarily monosubstitutions were removed, except after treatment with GH51 Aspergillus niger ABF. It degraded slowly internal disubstitutions as well, showing versatility in substrate specificity within GH51. GH54 Trichoderma reesei ABF core protein also presented dual ABF-m/d activity, slowly degrading Araf disubstitutions from both terminal and internal positions. Surprisingly, regioselectivity of the T. reesei ABF changed from α(1→3)-linked Araf on terminal Xylp to α(1→2)-linked Araf on internal Xylp on both mono- and disubstitutions. In conclusion, systematic analysis of natural substrates revealed interesting new details on the action T. reesei ABF and showed that the dual ABF-m/d activity could be more prevalent than previously thought, especially within GH51 ABFs.

Dear editor We have read with great interest the paper by Tang and Chen1 published in the most recent issue of the International Journal of Nanomedicine, in which the authors describe the protocol by which scientists constructed the ideal BRAF (V600E)-modeled structure through homology modeling and introduced the method of structure-based docking or virtual screening from a large compound database. They concluded that BRAF (V600E) has a quite prominent structural or conformational variation when compared to the wild-type BRAF protein by matrix of root mean square fluctuation and principal component analysis. On the basis of structure-based virtual screening, ligand-based quantitative structure activity relationship models, and molecular dynamics simulation, we recommend aknadicine and 16beta-hydroxy-19s-vindolinine N-oxide as potent compounds for developing novel inhibitors in the future. The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene is mutated in 40%–60% of melanomas, the most common being the V600E mutation, which leads to activation of the mitogen activated protein kinase (MAPK) pathway.2 BRAF is a member of the RAF family of serine/threonine protein kinases. This family consists of three kinases, ie, ARAF, CRAF (RAF-1), and BRAF, of which the latter has the highest basal kinase. BRAF functions to regulate the MAPK/ERK pathway, which is conserved in all eukaryotes. The RAS/RAF/MEK/ERK pathway acts as a signal transducer between the extracellular environment and the nucleus. Extracellular signals such as hormones, cytokines, and various growth factors interact with their receptors to activate the small G-proteins of the RAS family. BRAF-mutated tumors have a poor response to traditional chemotherapy and a poor prognosis in melanoma, thyroid, and colon cancers.3–5 Targeted therapies are of great interest for these types of cancer, and elucidation of the structure and functions of BRAF kinase is the subject of much ongoing research. The approach of targeting oncogenic kinases has been successful in the treatment of cancers with activating mutations in the kinase gene that drives their progression. It is likely that our evolving understanding of BRAF genetics and signaling will allow further personalization of cancer therapy with the goal of improving clinical responses. We are confident that the results reported Tang and Chen1 have brought progress to the field, but targeting a malignant cell with a monotherapy protocol is expected to fail and thus lead to clinical relapse of the disease. Thus, combination therapy is likely to be the most effective management plan for the treatment of BRAF-mutated tumors. Many BRAF-specific inhibitors display a cytostatic response inducing senescence and are susceptible to acquired resistance. Therefore, combination with traditional chemotherapeutic agents seems to be more effective than either treatment alone.

Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal, monocyte-derived dendritic cells or macrophages in Langerhans cell histiocytosis (LCH) and non-Langerhans cell histiocytosis (non-LCH), respectively. The discovery of BRAFV600E mutations in approximately 50% of these patients provided the first molecular therapeutic target in histiocytosis. However, recurrent driving mutations in the majority of patients with BRAFV600E–wild-type non-LCH are unknown, and recurrent cooperating mutations in non-MAP kinase pathways are undefined for the histiocytic neoplasms. Through combined whole-exome and transcriptome sequencing, we identified recurrent kinase fusions involving BRAF, ALK, and NTRK1, as well as recurrent, activating MAP2K1 and ARAF mutations in patients with BRAFV600E–wild-type non-LCH. In addition to MAP kinase pathway lesions, recurrently altered genes involving diverse cellular pathways were identified. Treatment of patients with MAP2K1- and ARAF-mutated non-LCH using MEK and RAF inhibitors, respectively, resulted in clinical efficacy, demonstrating the importance of detecting and targeting diverse kinase alterations in these disorders. Significance: We provide the first description of kinase fusions in systemic histiocytic neoplasms and activating ARAF and MAP2K1 mutations in non-Langerhans histiocytic neoplasms. Refractory patients with MAP2K1- and ARAF-mutant histiocytoses had clinical responses to MEK inhibition and sorafenib, respectively, highlighting the importance of comprehensive genomic analysis of these disorders. Cancer Discov; 6(2); 154–65. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 109

112th MPEG meeting, Warsaw, PL, June 2015 - ISO/IEC JTC 1/SC 29/WG 11 N15327

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.

A paragon of personalized medicine, the V600E gain-of-function mutation in the BRAF gene drives development of diverse types of human cancer, including 50% of malignant melanomas and 10% of colorectal cancers. Targeted BRAF inhibitors produce remarkable remission rates in patients suffering from otherwise incurable metastatic melanoma, but the development of drug resistance renders treatment responses transient. Further, nonmelanoma tumors such as colorectal cancer carrying the BRAF V600E mutation are generally intrinsically resistant to monotherapy with BRAF inhibitors. Finding a key to unlocking this intrinsic resistance is a crucial quest in extending the spectrum of tumors sensitive to BRAF inhibition. Now, Ahronian et al . take an important step toward overcoming colorectal-tumor resistance to BRAF inhibitors. The authors studied—both before treatment and after the development of resistance to BRAF inhibitors—three colorectal cancer patients who responded to BRAF inhibitors when administered in combination with other targeted therapeutic agents. A combination of whole-exome and RNA sequencing of tumors revealed that resistance development could be explained by the acquisition of new mutations after treatment. These mutations were diverse and included genomic amplification of BRAF and the KRAS oncogene and activating point mutations in the BRAF -signaling related ARAF and MEK genes. In spite of the diversity of resistance mechanisms, these mutations all occurred within the BRAF signaling pathway. Remarkably, the investigators unearthed an overarching Achilles’ heel of these diverse resistance mechanisms: In vitro inhibition of the ERK protein, a distal node within the BRAF signaling cascade, neutralized resistance to BRAF inhibitors in the case of every mutation. Availability of ERK inhibitors for clinical use lies on the horizon, as these are currently being tested in clinical trials. Although this study is small in size, it provides fundamental insights into the mechanisms underlying BRAF-inhibitor resistance in patients suffering from colorectal cancer. Further, Ahronian and colleagues provide the level of detail required to understand personalized medicine at the patient level. Tumors will need to be reevaluated, and treatments retailored, as a patient’s disease changes. L. G. Ahronian et al. , Clinical acquired resistance to RAF inhibitor combinations in BRAF-mutant colorectal cancer through MAPK pathway alterations. Cancer Discov . 5 , 358–367 (2015). [[Abstract]][1] [1]: http://cancerdiscovery.aacrjournals.org/content/early/2015/02/11/2159-8290.CD-14-1518.abstract?cited-by=yesl2159-8290.CD-14-1518v1

In the present study, structural characterization and antioxidant activity of a fraction (AAP-2A) of polysaccharides from angelica and astragalus (AAP) were investigated. Characteriztion assay showed that AAP-2A had molecular weight (Mw), root-mean square (RMS) radius and polydispersity index (Mw/Mn) of 2.252 × 10(3)kDa, 28.4 nm and 1.038, respectively. There were infrared characteristic absorption peaks of polysaccharides in FT-IR spectroscopy. AAP-2A was composed of rhamnose (Rha), galactose (Gal), arabinose (Ara) and glucose (Glc) with a molar ratio of 1:2.13:3.22:6.18 in GC analysis. Methylation analysis combined with NMR spectroscopic analysis demonstrated that a preliminary structure of AAP-2A was proposed as follows: 1,3-linked Rhap, 1,3-linked Galp, 1,3-linked Araf, 1,5-linked Araf, 1,3,5-linked Araf, 1,4-linked Glcp and 1,4,6-linked Glcp interspersed with terminal Glcp. AAP-2A exhibited a surface with a sheet-like appearance in scanning electron microscope and stronger antioxidant capacity compared with AAP.

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.

The MAPK signaling pathway plays a crucial role in tumorgenesis and cell proliferation in human papillary thyroid carcinoma (PTC). Ret/PTC rearrangements, RAS and BRAF mutations, the main non-overlapping genetic alterations all leading to MAPK cascade activation, are cumulatively identified in 60-80% of PTCs. In approximately one-fourth of the cases, oncogenic background potentially contributing to MAPK activation in PTC might be different. We therefore attempted to evaluate the mutational status of genes encoding other members of RAF family known to act upstream of MAPKs, ARAF and CRAF (RAF-1). In addition we also analyzed the MET gene that encodes hepatocyte growth factor/scatter factor receptor overexpressed in most of PTCs and a MAPK cascade contributor. In 129 Japanese patients with PTC, BRAF(T1799A) was detected in 65 cases (50.4%), and the remaining 64 tumor specimens were subjected to mutation analysis of kinase domains of ARAF, CRAF and MET genes, and hotspots of K- and N-RAS genes. No ARAF, CRAF, MET, K- and N-RAS mutations were revealed. Based on these observations, we concluded that despite the fact that ARAF, CRAF and MET are actively expressed, alterations of these genes are rare in PTC and unlikely to play a perceptible role in the molecular pathogenesis of this type of human malignancy.

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.

Colorectal cancer is a multi-step process characterized by a sequence of genetic alterations in cell growth regulatory genes, such as the adenomatous polyposis coli, KRAS, p53 and DCC genes. In the present study mutation analysis was performed with SSCA/direct sequencing of the hot-spot regions in exons 11 and 15 for the BRAF gene and exons 1-2 for the KRAS gene in 130 primary colorectal cancer tumors and correlated with clinico-pathological and mutational data. We also performed mutation analysis of the corresponding conserved regions in the ARAF and RAF-1 genes. Mutations in the BRAF and KRAS genes were found in 11.5 and 40% of the tumors, respectively. One germline exonic and nine germline intronic genetic variants were found in the ARAF and RAF-1 genes. All of the BRAF mutations were located in the kinase domain of the conserved region 3 in exon 15 of the BRAF gene. One novel somatic mutation was also identified in the BRAF gene. The majority of the BRAF mutations were found in colon compared with rectal tumors (P = 0.014). In agreement with others, a statistically significant correlation between BRAF mutations and microsatellite instability could be found. A negative correlation was also evident between mutations in the BRAF and KRAS genes, which supports earlier studies where somatic mutations in these genes are mutually exclusive. Collectively, our results provide support for the idea that activation of the MAP kinase pathway, especially via BRAF and KRAS mutations, is of critical importance for the development of colorectal cancer.

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.

Second RAF paralogue ( BRAF ) is a proto-oncogene that encodes a serine/threonine kinase that transduces regulatory signals through the Rat Sarcoma (RAS)/RAF/ mitogen-activated protein kinase extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. This pathway is hyperactivated in approximately 30% of human cancers.1 Activating mutations in the RAS oncogenes occur in 15%–30% of cancers.2 Kirsten murine sarcoma virus oncogene ( KRAS ) is an important and frequently mutated member of this group of oncogenes. Gain-of-function BRAF mutations results in aberrant activation of ERK signalling that is involved in several cancers such as thyroid papillary carcinoma, melanoma and colon carcinoma mainly but also as demonstrated in animal models in ovarian, skin, lung cancers and glioblastoma multiforme.3 ,4 There are three related RAF genes in mammals: first RAF paralogue ( ARAF ), BRAF and human homologue of v-RAF ( CRAF ) ( RAF-1 ). The BRAF gene, located at 7q34, contains 18 exons and encodes a serine–threonine kinase in the RAS/RAF/MAPK signalling pathway.5 All the main RAF proteins share three highly conserved regions: CR1, CR2 and CR3; however, BRAF has a number of structural differences from the other RAF proteins (ARAF and CRAF). CR1 contains the RAS binding domain and the cysteine-rich domain.6–10 CR2 is rich in serine and threonine and contains regulatory phosphorylation sites. CR3 contains the P-loop and the important kinase …

Plant cell walls are complex structural frameworks that affect cell shape and differentiation, and that protect the plant cell against environmental conditions (Scheller and Ulvskov, 2010). One of the major components of the wall is the hemicellulosic polysaccharide xylan, which is composed of a linear β-(1,4)-linked D-xylosyl (Xyl) backbone with various sidebranches. The latter include α-(1,2)-linked glucuronic acid (GlcAs), or α-(1,2)-linked 4-O-methyl-glucuronic acid residues (MeGlcA), and α-arabinofuranose (Araf) depending on tissues and species (Faik, 2010).

The postmodern novel, that arises after the sixties in world literature, after the eighties in Turkish literature, vary from traditional novel concept in every respect. Characteristics of this trend are seen in most of the authors of the last period of Turkish literature. Here, the effective is that post-modern novel reflects value judgments and perceptions of the changing world more successfully. In today's literary works’ having more than one semantic layer takes the reader different readings and shows that he has the right to an equal voice with the author. Thus, the author presents his/her reader the opportunity and he/she fades from the scene and the reader complete the background to the text. Elif Şafak’s Araf is a narrative which reflects postmodern novel conception. The outstanding features in this study have been analyzed under some headings and postmodern conception of narrative has been pointed out. However, on the one hand the postmodern novel features have been focused on; on the other hand elements of a classic novel such as the plot, time, place, characters have been evaluated with a postmodern approach. In addition, on the occasion of some names, which are often emphasized in the novel, a number of evaluations have been done and the meaning of these names, added into the novel, has been focused on. Araf has been fictionalised with a complex plot in order to make the reader think. The time, as usual in postmodern texts, is scattered and fragmented. The place in narrative is sometimes used functionally even though it is identified with the reader and dominates them. On the contrary to giving importance to idealized and noncontradictory person in modern novel, the importance is given to the subject, who has paradoxes and can change according to circumstances. Intertextuality and image form the key features of the novel., Dünya edebiyatında altmışlardan sonra, Türk edebiyatında ise, seksenlerden sonra ortaya çıkan postmodern roman, hemen her açıdan geleneksel roman anlayışından farklılık gösterir. Türk edebiyatının son dönem yazarlarının çoğunda bu akımın özellikleri görülür. Bunda değişen dünyanın değer yargılarını ve algılarını postmodern romanın daha başarılı yansıttığı inancı etkilidir. Günümüz edebiyat eserinde birden fazla anlamsal katmanın olması, okuyucuyu farklı okumalara götürerek onun da anlatıyı kurgulama açısından yazarla eşit söz hakkına sahip olduğunu gösterir. Böylece yazar okuruna imkânı sunup aradan çekilir ve metnin arka plânını okuyucu tamamlar. Elif Şafak’ın Araf’ı, postmodern roman anlayışını yansıtan bir anlatıdır. Bu çalışmada öne çıkan özellikler, belli başlıklar altında incelenmiş ve anlatıdaki postmodern anlayışa dikkat çekilmiştir. Ancak romandaki postmodern özellikler üzerinde durulurken, bir yandan da olay örgüsü, zaman, mekân, şahıs kadrosu gibi klasik romanın unsurları, postmodern bir yaklaşımla değerlendirilmeye çalışılmıştır. Ayrıca romanda sık sık vurgulanan “isim”ler vesilesiyle birtakım değerlendirmeler yapılmış, bu isimlerin romana kattığı anlam üzerinde durulmuştur. Araf, okuyucuyu düşünmeye yöneltmek amacıyla karmaşık bir olay örgüsüyle kurgulanmıştır. Postmodern metinlerde olduğu gibi zaman, parçalı ve dağınıktır. Anlatıda mekân, genellikle bireyle özdeşleşip onu tahakkümü altına almasa da yer yer işlevsel olarak kullanılır. Ayrıca modern romanda idealize edilmiş, çelişiksiz bireye verilen önemin aksine, postmodern romanda paradoksları olan, duruma göre değişebilen özneye önem verilir. Metinlerarasılık ve imge romanın öne çıkan özelliklerini oluştururlar.

111th MPEG meeting, Genève, CH, February 2015 - ISO/IEC JTC 1/SC 29/WG 11 N15065

111th MPEG meeting, Genève, CH, February 2015 - ISO/IEC JTC 1/SC 29/WG 11 N15065

Intrahepatic cholangiocarcinoma (iCCA) is a fatal bile duct cancer with dismal prognosis and limited therapeutic options. By performing RNA- and exome-sequencing analyses, we report a novel fusion event, FGFR2-PPHLN1 (16%), and damaging mutations in the ARAF oncogene (11%). Here we demonstrate that the chromosomal translocation t(10;12)(q26;q12) leading to FGFR2-PPHLN1 fusion possesses transforming and oncogenic activity, which is successfully inhibited by a selective FGFR2 inhibitor in vitro. Among the ARAF mutations, N217I and G322S lead to activation of the pathway and N217I shows oncogenic potential in vitro. Screening of a cohort of 107 iCCA patients reveals that FGFR2 fusions represent the most recurrent targetable alteration (45%, 17/107), while they are rarely present in other primary liver tumours (0/100 of hepatocellular carcinoma (HCC); 1/21 of mixed iCCA-HCC). Taken together, around 70% of iCCA patients harbour at least one actionable molecular alteration (FGFR2 fusions, IDH1/2, ARAF, KRAS, BRAF and FGF19) that is amenable for therapeutic targeting.

113th MPEG meeting, Geneva, CH - October 2015- ISO/IEC JTC 1/SC 29/WG 11 N15621

Extracellular phosphate plays a key role in growth plate maturation by inducing Erk1/2 (Mapk3/1) phosphorylation, leading to hypertrophic chondrocyte apoptosis. The Raf kinases induce Mek1/2 (Map2k1/2) and Erk1/2 phosphorylation; however, a role for Raf kinases in endochondral bone formation has not been identified. Ablation of both A-Raf (Araf) and B-Raf (Braf) in chondrocytes does not alter growth plate maturation. Because c-Raf (Raf1) phosphorylation is increased by extracellular phosphate and c-Raf is the predominant isoform expressed in hypertrophic chondrocytes, chondrocyte-specific c-Raf knockout mice (c-Raf(f/f);ColII-Cre(+)) were generated to define a role for c-Raf in growth plate maturation. In vivo studies demonstrated that loss of c-Raf in chondrocytes leads to expansion of the hypertrophic layer of the growth plate, with decreased phospho-Erk1/2 immunoreactivity and impaired hypertrophic chondrocyte apoptosis. However, cultured hypertrophic chondrocytes from these mice did not exhibit impairment of phosphate-induced Erk1/2 phosphorylation. Studies performed to reconcile the discrepancy between the in vitro and in vivo hypertrophic chondrocyte phenotypes revealed normal chondrocyte differentiation in c-Raf(f/f);ColII-Cre(+) mice and lack of compensatory increase in the expression of A-Raf and B-Raf. However, VEGF (Vegfa) immunoreactivity in the hypertrophic chondrocytes of c-Raf(f/f);ColII-Cre(+) mice was significantly reduced, associated with increased ubiquitylation of VEGF protein. Thus, c-Raf plays an important role in growth plate maturation by regulating vascular invasion, which is crucial for replacement of terminally differentiated hypertrophic chondrocytes by bone.

Objectives Aspergillus fumigatus is the most common agent of invasive aspergillosis (IA). In recent years, resistance to triazoles, the mainstay of IA therapy, has emerged in different countries worldwide. IA caused by azole-resistant A. fumigatus (ARAF) shows an exceedingly high mortality. In this study, IA due to ARAF isolates in HSCT recipients in Germany was investigated. Methods The epidemiology of azole resistance in IA was analysed in two German haematology departments. Between 2012 and 2013, 762 patients received HSCT in Essen (n = 388) and Cologne (n = 374). Susceptibility testing of A. fumigatus isolates was performed by Etest, followed by EUCAST broth microdilution testing if elevated MICs were recorded. In all ARAF isolates the cyp51A gene was sequenced and the genotype was determined by microsatellite typing using nine short tandem repeats. Results In total, A. fumigatus was recovered from 27 HSCT recipients. Eight patients had azole-resistant IA after HSCT, and seven of the cases were fatal (88%). All except one patient received antifungal prophylaxis (in five cases triazoles). TR34/L98H was the most common mutation (n = 5), followed by TR46/Y121F/T289A (n = 2). In one resistant isolate no cyp51A mutation was detected. Genotyping revealed genetic diversity within the German ARAF isolates and no clustering with resistant isolates from the Netherlands, India and France. Conclusions This report highlights the emergence of azole-resistant IA with TR34/L98H and TR46/Y121F/T289A mutations in HSCT patients in Germany and underscores the need for systematic antifungal susceptibility testing of A. fumigatus.

mAb CS-35 is representative of a large group of antibodies with similar binding specificities that were generated against the Mycobacterium leprae lipopolysaccharide, lipoarabinomannan (LAM), and which cross-reacted extensively with LAMs from Mycobacterium tuberculosis and other mycobacteria. That this antibody also cross-reacts with the arabinogalactan (AG) of the mycobacterial cell wall, suggesting that it recognizes a common arabinofuranosyl (Araf)-containing sequence in AG and LAM, is demonstrated. The antibody reacted more avidly with 'AraLAM' (LAM with naked Araf termini) compared to 'ManLAM' (in which many Araf termini are capped with mannose residues) and mycolylarabinogalactan-peptidoglycan complex (in which the terminal Araf units are substituted with mycolic acids). Neither did the antibody bind to AG from emb knock-out mutants deficient in the branched hexa-Araf termini of AG. These results indicate that the terminal Araf residues of mycobacterial arabinan are essential for binding. Competitive ELISA using synthetic oligosaccharides showed that the branched hexa-Araf methyl glycoside [beta-D-Araf-(1--2)-alpha-D-Araf-(1-)(2)-(3 and 5)-alpha-D-Araf-(1--5)-alpha-D-Araf-OCH(3)] was the best competitor among those tested. The related linear methyl glycoside, beta-D-Araf-(1--2)-alpha-D-Araf-(1--5)-alpha-D-Araf-(1--5)-alpha-D-Araf-OCH(3), representing one linear segment of the branched hexa-Araf, was less effective and the other linear tetrasaccharide, beta-D-Araf-(1--2)-alpha-D-Araf-(1--3)-alpha-D-Araf-(1--5)-alpha-D-Araf-OCH(3), was ineffective. The combined results suggest that the minimal epitope recognized by antibody CS-35 encompasses the beta-D-Araf-(1--2)-alpha-D-Araf-(1--5)-alpha-D-Araf-(1--5)-alpha-D-Araf within the branched hexa-Araf motif of mycobacterial arabinans, whether present in LAM or AG.

Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas. We present an expression-based variant-impact phenotyping (eVIP) method that uses gene expression changes to distinguish impactful from neutral somatic mutations. eVIP identified 69% of mutations analyzed as impactful and 31% as functionally neutral. A subset of the impactful mutations induces xenograft tumor formation in mice and/or confers resistance to cellular EGFR inhibition. Among these impactful variants are rare somatic, clinically actionable variants including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and multiple BRAF variants, demonstrating that rare mutations can be functionally important in cancer.

Activating mutations in the KRAS oncogene occur in about 40% of colorectal cancers (CRCs) and over 90% of pancreatic ductal adenocarcinomas (PDACs). Since development of small molecules capable of inhibiting KRAS directly has proven difficult, alternative strategies have instead focused on inhibiting downstream effector pathways, such as the MAPK pathway. However, inhibition of the MAPK pathway alone with MEK inhibitors, such as selumetinib and trametinib, produces only cytostatic effects and is insufficient to kill KRAS-mutant cancer cells. We hypothesized that inhibition of an additional kinase during MEK inhibitor treatment could improve response. We performed a kinase-targeting shRNA screen to find kinases whose knockdown would cooperate with trametinib in KRAS-mutant CRC and PDAC cell lines. The kinases found in this screen represent potential therapeutic targets to inhibit in combination with MEK. Interestingly, despite using a very high concentration of trametinib in the screen to enrich for hits outside of the MAPK pathway, the most highly ranked kinases in the screen were members of the MAPK pathway, including ARAF, BRAF, CRAF, and MEK1. This suggests that even at high concentration, trametinib produces suboptimal MAPK inhibition. Indeed, we found that while MEK inhibitors produce robust inhibition of MAPK signaling initially, pathway reactivation was observed by 48-96 hours despite regular replenishment of drug. This feedback reactivation was accompanied by marked increases in active CRAF and phosphorylated MEK. In fact, experimental approaches that artificially increased upstream signaling flux through the MAPK pathway led to a >10-fold reduction in the ability of MEK inhibitors to inhibit the MAPK pathway. Remarkably, despite triggering the same degree of adaptive upstream MAPK signaling as seen with MEK inhibitor, we found that ERK inhibitors were able to maintain MAPK pathway suppression. Importantly, these differences in MAPK pathway suppression amount to differences in cell viability. Over four weeks, ERK inhibitor treatment reduces the outgrowth of KRAS-mutant cell lines compared to those treated with MEK inhibitors. Additionally, as these inhibitors are not used as monotherapies, replacement of trametinib with an ERK inhibitor in therapeutically relevant combination treatments improved cell responses over four weeks. Despite the feedback reactivation of the MAPK pathway, we find that ERK inhibitors are less sensitive to this signaling than MEK inhibitors, and can effectively maintain suppression of MAPK signaling. The findings of our screen demonstrate that MAPK pathway targeting is key to successful treatment of KRAS-mutant cancers, and that ERK inhibition provides greater opportunity for inactivating MAPK. Further exploration into the mechanisms of pathway feedback will be necessary to developing valuable clinical combinations for KRAS-mutant cancers. Citation Format: Leanne G. Ahronian, Sandra Misale, Jason T. Godfrey, Koki Nishimura, Lifeng Chen, Jeffrey A. Engelman, Ryan B. Corcoran. Adaptive feedback reactivates MAPK signaling in KRAS-mutant cancers with inhibition of MEK, but not ERK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 101. doi:10.1158/1538-7445.AM2017-101

110th MPEG meeting, Strasbourg, France - October 2014 - ISO/IEC JTC 1/SC 29/WG 11 N14812

The RAF family of kinases mediates RAS signaling, and RAF inhibitors can be effective for treating tumors with BRAF V600E mutant protein. However, RAF inhibitors paradoxically accelerate metastasis in RAS -mutant tumors and become ineffective in BRAF V600E tumors because of reactivation of downstream mitogen-activated protein kinase (MAPK) signaling. We found that the RAF isoform ARAF has an obligatory role in promoting MAPK activity and cell migration in a cell type–dependent manner. Knocking down ARAF prevented the activation of MAPK kinase 1 (MEK1) and extracellular signal–regulated kinase 1 and 2 (ERK1/2) and decreased the number of protrusions from tumor cell spheroids in three-dimensional culture that were induced by BRAF V600E -specific or BRAF/CRAF inhibitors (GDC-0879 and sorafenib, respectively). RAF inhibitors induced the homodimerization of ARAF and the heterodimerization of BRAF with CRAF and the scaffolding protein KSR1. In a purified protein solution, recombinant proteins of the three RAF isoforms competed for binding to MEK1. In cells in culture, overexpressing mutants of ARAF that could not homodimerize impaired the interaction between ARAF and endogenous MEK1 and thus prevented the subsequent activation of MEK1 and ERK1/2. Our findings reveal a new role for ARAF in directly activating the MAPK cascade and promoting tumor cell invasion and suggest a new therapeutic target for RAS- and RAF-mediated cancers.

This Podcast features an interview with Juliane Mooz and Krishnaraj Rajalingam, authors of a Research Article that appears in the 5 August 2014 issue of Science Signaling , about the cellular functions of the kinase ARAF. RAF proteins are serine-threonine kinases that mediate signaling through the mitogen-activated protein kinase (MAPK) pathway, and aberrant RAF activity can transform normal cells into cancerous cells. There are three RAFs in mammals: ARAF, BRAF, and CRAF. The most studied of these is BRAF, mutations in which are associated with various cancers. Whereas the cellular functions of BRAF and CRAF have been extensively studied, not much is known about ARAF. Mooz et al . found that ARAF was required for the MAPK pathway activation and cell migration triggered by treating cancer cells with BRAF inhibitors.

Item does not contain fulltext OBJECTIVES: Azole resistance in Aspergillus fumigatus isolates has been increasingly reported with variable prevalence worldwide and is challenging the effective management of aspergillosis. Here we report the coexistence of both TR(3)(4)/L98H and TR(4)(6)/Y121F/T289A resistance mechanisms in azole-resistant A. fumigatus (ARAF) isolates originating from Tanzania, Africa. METHODS: A total of 30 soil and woody debris samples from the surroundings of Kilimanjaro Christian Medical Centre, Moshi, Tanzania, were processed for detection of ARAF isolates and were investigated for susceptibility to itraconazole, voriconazole, posaconazole and isavuconazole. All ARAF isolates were subjected to a real-time PCR assay for detection of mutations and were genotyped by microsatellite typing. RESULTS: Of the 30 samples, 29 yielded 108 A. fumigatus isolates. Overall, 15 ARAF isolates were obtained, which included 4 ARAF harbouring the TR(4)(6)/Y121F/T289A mutation and 11 isolates carrying TR(3)(4)/L98H. All four TR(4)(6)/Y121F/T289A A. fumigatus isolates showed high MICs of voriconazole (>16 mg/L) and isavuconazole (8 mg/L). In contrast, the 11 TR(3)(4)/L98H A. fumigatus isolates were pan-azole resistant. The isolates were cross-resistant to azole fungicides. Notably, 20% of environmental samples harboured ARAF and the TR(4)(6)/Y121F/T289A resistance mechanism was found in 5.5% of the soil samples, where it coexisted with TR(3)(4)/L98H. The Tanzanian TR(4)(6)/Y121F/T289A strains had a genotype identical to Dutch clinical TR(4)(6)/Y121F/T289A isolates. CONCLUSIONS: The present study reports the isolation of resistant A. fumigatus strains harbouring the TR(4)(6)/Y121F/T289A mutation from Africa. Recovery of TR(4)(6)/Y121F/T289A from the environment is worrisome and we must strive for effective surveillance of clinical and environmental sources to detect azole resistance in A. fumigatus.

109th MPEG meeting, Sapporo, JP - July 2014 - ISO/IEC JTC 1/SC 29/WG 11 N14537

Exudate gums, because of their beneficial properties, have been significant items of international trade in various industries for centuries. This manuscript sets out to gain insight into the fine structural details of an arabinogalactan protein (AGP) of gum ghatti (Anogeissus latifolia gum). The presence of a highly branched 554 kDa AGP having 1,6-linked Galp, 1,2-linked Manp, 1,3-linked Araf and 1,4-linked GlcpA main chain, substituted at O-4,6 of 1,2-linked Manp, and O-3/O-3,4 of 1,6-linked Galp residues by Araf, Arap and Galp units was revealed by chemical, chromatographic, ESMS, and NMR analyses. In particular, ESMS analysis of per acetylated oligomeric fragments derived from AGP by Smith degradation followed by acetylation was described as a commanding tool for providing critical structural information on a spectrum of glycerol tagged oligosaccharides. In addition, formation of an electrostatically driven complex between the isolated AGP and bovine serum albumin resulting in changes in the microenvironment around the tryptophan residues of BSA was established. A moderate radical scavenging activity comparable with those of standard antioxidants was observed from the AGP fraction (∼94% at 1 mg/mL) that could be valuable in foods or pharmaceutical products as alternatives to synthetic antioxidants.

The extracellular signal-regulated kinase (ERK) signaling pathway is activated in Langerhans cell histiocytosis (LCH) histiocytes, but only 60% of cases carry somatic activating mutations of BRAF. To identify other genetic causes of ERK pathway activation, we performed whole exome sequencing on purified LCH cells in 3 cases. One patient with wild-type BRAF alleles in his histiocytes had compound mutations in the kinase domain of ARAF. Unlike wild-type ARAF, this mutant was a highly active mitogen-activated protein kinase kinase in vitro and was capable of transforming mouse embryo fibroblasts. Mutant ARAF activity was inhibited by vemurafenib, a BRAF inhibitor, indicating the importance of fully evaluating ERK pathway abnormalities in selecting LCH patients for targeted inhibitor therapy.

The extracellular signal-regulated kinase (ERK) signaling pathway is activated in Langerhans cell histiocytosis (LCH) histiocytes, but only 60% of cases carry somatic activating mutations of BRAF. To identify other genetic causes of ERK pathway activation, we performed whole exome sequencing on purified LCH cells in 3 cases. One patient with wild-type BRAF alleles in his histiocytes had compound mutations in the kinase domain of ARAF. Unlike wild-type ARAF, this mutant was a highly active mitogen-activated protein kinase kinase in vitro and was capable of transforming mouse embryo fibroblasts. Mutant ARAF activity was inhibited by vemurafenib, a BRAF inhibitor, indicating the importance of fully evaluating ERK pathway abnormalities in selecting LCH patients for targeted inhibitor therapy.

Biomarker development takes two principal forms. In recent years, numerous large-scale genomic studies resulted in a deluge of data. Identifying the genetic alterations that drive and maintain tumorigenesis (“driver” mutations) from these data sets requires complex bioinformatics methods. Even after a candidate driver is identified, however, it may not be amenable to pharmacologic targeting. An alternative approach to biomarker identification is to begin in the clinic. A decade ago, after observing that a subset of patients with advanced lung cancer responded dramatically to epidermal growth factor receptor (EGFR) inhibitors, investigators sequenced the EGFR gene in biopsies of responding patients. From this work came the recognition that sensitizing EGFR mutations result in heightened kinase activity, oncogene addiction, and exquisite sensitivity to EGFR inhibitor therapy. Imielinski and colleagues used this second approach to determine the molecular underpinnings of outlier responses to sorafenib in a lung cancer clinical trial. Sorafenib, a multitargeted kinase inhibitor, has modest activity in lung cancer, and the Eastern Cooperative Oncology Group (ECOG) 2501 trial was no exception. Among the 306 evaluable patients, there were only nine responders, one of whom achieved ongoing disease control for 5 years. The investigators obtained biospecimens from this case and performed massively parallel DNA sequencing of tumor tissue and peripheral blood. Initially, this yielded 25,150 somatic mutations. These included 101 nonsynonymous mutations affecting the coding of 99 genes, 15 of which were consistently detected in RNA sequencing. Within this subset, based on pathway biology, the investigators surmised that the most likely candidate oncogenic driver was an ARAF S214C mutation. ARAF encodes a serine-threonine kinase in the Raf protein family. Similar to other Raf family members, ARAF transduces mitogen-activated protein kinase (MAPK) pathway signaling from Ras to MAPK kinase (MEK) and extracellular signal–regulated kinase (ERK) and is a sorafenib target. However, it had not been previously implicated in tumorigenesis. To investigate the broader importance of their findings, Imielinski and colleagues queried The Cancer Genome Atlas (TCGA) and identified three ARAF codon 214 somatic mutations and three mutations in the related gene RAF1 , representing ~1% of lung cancers. ARAF mutations were also identified in rare cases of colorectal cancer (2%), gastric cancer (1%), and melanoma (1%). The functional impact of ARAF mutations was demonstrated by ectopic expression of ARAF S214 variants in immortalized human airway epithelial cells, which increased soft agar colony formation and phosphoMEK. This transformation was inhibited by clinically achievable concentrations of sorafenib and the MEK inhibitor trametinib. ARAF mutations are not ready for targeting in clinical practice, but this initial work paves the way for confirmatory preclinical studies and testing of other clinical specimens. If ARAF mutations are ultimately validated as a predictive biomarker, the present work will serve as a key reminder that clinical observation of outlier patient responses remains a powerful and efficient approach to driver mutation discovery. M. Imielinski et al ., Oncogenic and sorafenib-sensitive ARAF mutations in lung adenocarcinoma. J. Clin. Invest. 10.1172/JCI72763 (2014). [[Full Text]][1] [1]: http://www.jci.org/articles/view/72763

108th MPEG meeting, Valencia, ES - April 2014 - ISO/IEC JTC 1/SC 29/WG 11 N14310

Remarkable clinical activity with the RAF inhibitor PLX-4032 in melanomas containing mutant BRAF has recently been described in the New England Journal of Medicine by Flaherty et al. (2010). In a second manuscript published in Nature, the discovery and preclinical development of the drug is chronicled by the scientist at Plexxicon who led this effort, Gideon Bollag (Bollag et al., 2010). The initial preliminary report of the Phase 1 data revealing the activity of this drug was first presented at the Annual Meeting of the American Society of Clinical Oncology Annual Meeting in 2009, 7 years after the discovery of BRAF mutations in human cancer (Davies et al., 2002). In a disease in which the development of therapies against novel targets is often frustrating and slow, this is an amazing achievement, the origins of which can be traced back over 25 years. In 1983, Rapp et al. cloned v-raf as the transformative factor in a replication-defective type C murine virus (3611-MSV) but no mutations were detected in the homologous human RAF genes (ARAF, BRAF and CRAF) until 2002 (Rapp et al. 1983). At that time, Davies et al. (2002) at the Sanger identified BRAF as commonly mutated in human tumors. These mutations were particularly common in melanoma where they are found in upwards of 50% of patients but were also identified in approximately 10% of colorectal tumors and a third of papillary thyroid cancers. BRAF mutations are typically missense substitutions found clustered in two regions – the glycine rich loop in exon 11 and the activation segment within exon 15. The most common mutations disrupt an inactive conformation of the protein and lead to constitutive hyperactivation of the kinase (Wan et al., 2004). Notably, a single mutation within codon 600 (V600E) is found in over 90% of cases. RAF activation occurs in response to activation of RAS proteins by upstream growth factor-induced signals. Activated RAF in turn phosphorylates and activates MEK1 and 2 [mitogen-activated protein ⁄ extracellular signal-regulated kinase kinase (MAPKK)] which in turn phosphorylate and activate the extracellular signal-related kinases 1 and 2 (ERK1 ⁄2). The activation of this signaling cascade (the ‘classical’ MAP kinase pathway) regulates a number of effector kinases and transcription factors that drive key cellular processes including cell proliferation and survival. Phosphorylation of other RAF substrates may also be important in normal physiology and cellular transformation; however, this remains to be firmly established. Tumor cells with BRAF mutations depend on expression of BRAF for proliferation (Wellbrock et al. 2004). Furthermore, melanoma cell lines with the mutation are almost universally sensitive to inhibition of ERK signaling with MEK inhibitors (Solit et al., 2006). These findings suggested that inhibitors of RAF, MEK or ERK could have major clinical activity in patients whose tumors harbored activating BRAF mutations. Unfortunately, sorafenib (Nexavar, Bayer), the first RAF inhibitor to be tested clinically, lacked meaningful clinical activity in melanoma. This result suggested to some that mutant RAF did not play an important role in these tumors. In retrospect, this conclusion was unwarranted. Sorafenib is an unselective inhibitor of many kinases, and it inhibits tumors with mutant and wild-type RAF with equal potency. It is likely that the toxicity of sorafenib precluded administration of a high enough dose to inhibit ERK signaling adequately. In contrast to sorafenib, allosteric MEK inhibitors are exceptionally selective. These drugs potently inhibit ERK signaling in tumors. In patients, the major dose-limiting toxicity of these agents is skin rash. In clinical trials, the MEK inhibitor AZD6244 exhibited activity in melanoma, with a 13% partial response rate in tumors harboring a BRAF mutation. However, given the expectations from trials of oncoprotein-targeted therapy in other diseases, these results were disappointing. They suggested to many that BRAF ⁄ERK signaling was dispensible for maintenance of most melanomas, possibly because of the cellular heterogeneity and genetic complexity of the disease, as well as to postulated inherent resistance of melanoma stem cells. The two reports by Flaherty et al. (2010) and Bollag et al. (2010) dispel these concerns and validate the dependence of these tumors on BRAF. In the study by Flaherty et al., the authors report the Phase 1 results of the RAF inhibitor PLX4032 (also known as RG7204). PLX4032 is a potent nanomolar, ATP-competitive inhibitor of all three RAF isoforms as well as the V600E mutant form of BRAF. In this trial, a 77% response rate

An acidic heteroxylan, SSb2, was isolated from the alkaline extract of Sophora subprostrata by DEAE-Sephadex A-25 and Sephacryl S-300 chromatography, with an Mr of 4.4×105 and a specific optical rotation of −49.2° (c 0.5, H2O). Its structural features were elucidated by methylation analysis, partial acid hydrolysis, periodate oxidation and 13C NMR spectroscopy. The data obtained indicated that SSb2 has a backbone consisting of 1,4-linked xylopyranosyl residues with branches at O-2 or O-3, composed of l -Araf(1→5) l -Araf(1→, 4-O-Me- d -GlcA(1→ or d -GlcA(1→. SSb-2 exhibited an immunostimulating activity in vivo in pharmacological experiments.

Hot water extract (ALR-0) of rhizomes of Atractylodes lanceo DC. was fractionated into MeOH-soluble fraction (ALR-1), supernatant fraction of EtOH precipitation (ALR-3 + 4), and crude polysaccharide fraction (ALR-5). Among these fractions, only ALR-5 showed potent stimulating activity for proliferation of bone marrow cells mediated by Peyer's patch cells. ALR-5 gave three potently active carbohydrate-rich fractions (ALR-5IIa, 5IIb, and 5IIc) by anion-exchange chromatography on DEAE-Sepharose CL-6B, and three active polysaccharides (ALR-5IIa-1-1, ALR-5IIb-2-2, and ALR-5IIc-3-1) were further purified from the respective fractions. The order of activity was revealed to be ALR-5IIb-2-2 > or = ALR-5IIa-1-1 > ALR-5IIc-3-1, ALR-5IIa-1-1, 5IIb-2-2, and 5IIc-3-1 each was eluted as a single peak on HPLC and their molecular weights were estimated to be 74,000, 3,100, 16,000, respectively. ALR-5IIa-1-1 consisted mainly of Ara and Gal (molar ratio; 0.6: 1.0) in addition to a trace amount of uronic acid whereas ALR-5IIb-2-2 and ALR-5IIc-3-1 mainly comprised Ara, Gal, GlcA, and GalA (molar ratio; 0.2: 1.0: 0.2: 0.8, and 0.5: 1.0: 0.7: 1.5, respectively). Methylation analysis indicated that ALR-5IIa-1-1 consisted mainly of terminal Araf, 4- or 5-linked Ara, 3.4- or 3.5-branched Ara, and 3-linked, 4-linked, and 3,6-branched Gal. ALR-5IIb-2-2 and ALR-5IIc-3-1 were composed mainly of terminal Araf, 4- or 5-linked Ara, 4-linked Gal, 4-linked GalA, and terminal GlcA. In addition, ALR-5IIb-2-2 mainly comprised 4-linked Xyl whereas ALR-5IIc-3-1 consisted mainly of 2,4-branched Rha. Single radial gel diffusion indicated that ALR-5IIa-1-1 showed a strong reactivity with beta-glucosyl-Yariv antigen, whereas ALR-5IIb-2-2 and ALR-5IIc-3-1 did not show the reactivity with the antigen. Treatments of ALR-5IIa with NalO4, NaClO2 and pronase did not reduce the stimulating activity for Peyer's patch cells, however combination of exo-alpha-L-arabinofuranosidase and exo-beta-D-(1-->3)-galactanase digestions of ALR-5IIa-1-1 significantly decreased its activity.

International audience; Background: DsbC is a protein-disulfide isomerase present in the periplasm of Gram-negative bacteria. Results: We discovered that DsbC also regulates the redox state of the single cysteine residue of the l-arabinose-binding protein AraF. Conclusion: DsbC is involved in the protection of single cysteine residues against oxidative stress. Significance: This finding reveals a new link between oxidative stress protection and oxidative protein folding. We report a new function for Escherichia coli DsbC, a protein best known for disulfide bond isomerization in the periplasm. We found that DsbC regulates the redox state of the single cysteine of the l-arabinose-binding protein AraF. This cysteine, which can be oxidized to a sulfenic acid, mediates the formation of a disulfide-linked homodimer under oxidative stress conditions, preventing l-arabinose binding. DsbC, unlike the homologous protein DsbG, reduces the intermolecular disulfide, restoring AraF binding properties. Thus, our results reveal a new link between oxidative protein folding and the defense mechanisms against oxidative stress.