Involvement of GSK-3β in TWEAK-mediated NF-κB activation

AbstractGlycogen synthase kinase-3β (GSK-3β) is a key component of several signaling pathways. We found that a short variant of `TNF-like weak inducer of apoptosis' (shortTWEAK) formed a complex with GSK-3β in a yeast two-hybrid system. We demonstrate that shortTWEAK and GSK-3β colocalize in the nucleus of human neuroblastoma cells. We also show that TWEAK is internalized in different cell lines and that it translocates to the nucleus. This event causes the degradation of IκBα, the nuclear translocation of both GSK-3β and p65, and the induction of NF-κB-driven gene expression. We demonstrate that the induction of IL-8 expression by TWEAK can be counteracted by LiCl. Taken together, these data suggest that GSK-3β plays an important role in the signal transduction pathway between TWEAK and NF-κB

A CD3-Specific Antibody Reduces Cytokine Production and Alters Phosphoprotein Profiles in Intestinal Tissues From Patients With Inflammatory Bowel Disease

NOTICE: this is the author’s version of a work that was accepted for publication in Gastroenterology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in GASTROENTEROLOGY, 10.1053/j.gastro.2014.03.04

Synthesis of high-affinity, hydrophobic monosaccharide derivatives and study of their interaction with concanavalin A, the pea, the lentil, and fava bean lectins

Concanavalin A (Con A) and agglutinins from the pea (PSA), lentil (LCH), and fava bean (VFA) constitute a group of -mannose/-glucose binding legume lectins. In addition to their sugar binding specificity, these lectins also contain sites that bind hydrophobic ligands. The present study explores a class of nonpolar binding sites reportedly present adjacent to the carbohydrate binding site in PSA, LCH, and VFA. A series of 2-O- and 3-O-substituted nitrobenzoyl and nitrobenzyl derivatives of methyl [alpha]--glucopyranoside and methyl [alpha]--mannopyranoside were synthesized. Evaluation of their binding to Con A, PSA, LCH, and VFA was carried out by the technique of hapten inhibition of precipitation reaction. The hapten inhibition assay results reveal that the presence of a methyl or methylene group at the O-2 or O-3 position of the sugar is essential for hydrophobic interaction with PSA, LCH, and VFA. The substitution of methyl by nitrobenzyl leads to enhanced binding (1.7-16.7 times for the 2-O-substituted compounds and 7.9-40.5 times for the 3-O-substituted compounds) with the m-nitrobenzyl group contributing to maximum binding. A hydrophobic interaction is also involved between Con A and 2-O-nitrobenzyl derivatives, resulting in enhanced binding, but the corresponding 3-O-isomers bind poorly due probably to steric reasons. These results may be rationalized on the basis of the recently published X-ray data of Con A and VFA. The nitrobenzyl derivatives, after transformation to their azido analogs, have potential applications in the photoaffinity labeling of these lectins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29695/1/0000027.pd

Sinteza i farmakološko ispitivanje novih 4-(3-etilfenil)-1-supstituiranih 4H-[1,2,4]triazolo[4,3-a]kinazolin-5-ona kao nove klase H1-antihistaminika

A series of novel 4-(3-ethylphenyl)-1-substituted-4H-[1,2,4]triazolo[4,3-a]quinazolin-5-ones (4a-j) were synthesized by the cyclization of 3-(3-ethylphenyl)-2-hydrazino-3H-quinazolin-4-one (3) with various one-carbon donors. The starting material, compound 3, was synthesized from 3-ethyl aniline by a new innovative route with improved yield. When tested for their in vivo H1-antihistaminic activity on conscious guinea pigs, all test compounds protected the animals from histamine induced bronchospasm significantly. Compound 4-(3-ethylphenyl)-1-methyl-4H-[1,2,4]triazolo[4,3-a]quinazolin-5-one (4b) emerged as the most active compound of the series and it is more potent (74.6 % protection) compared to the reference standard chlorpheniramine maleate (71 % protection). Compound 4b shows negligible sedation (10 %) compared to chlorpheniramine maleate (30 %). Therefore compound 4b can serve as the leading compound for further development of a new class of H1-antihistamines.Ciklizacijom 3-(3-etilfenil)-2-hidrazino-3H-kinazolin-4-ona (3) s različitim donorima jednog C atoma sintetizirana je serija novih 4-(3-etilfenil)-1-supstituiranih 4H-[1,2,4]triazolo[4,3-a]kinazolin-5-ona (4a-j). Početni spoj 3 pripravljen je iz 3-etil anilina na novi, inovativni način, s poboljšanim iskorištenjem. U testovima in vivo na zamorcima, svi testirani spojevi pokazali su značajno zaštitno djelovanje protiv bronhospazma induciranog histaminom. Spoj 4-(3-etilfenil)-1-metil-4H-[1,2,4]triazolo[4,3-a]kinazolin-5-on (4b) najaktivniji je među testiranim spojevima (zaštita 74.6 %) i jači od referentnog standarda klorfeniramin maleata (zaštita 71 %). Spoj 4b pokazuje zanemarivu sedaciju (10 %) u usporedbi s klorfeniramin maleatom (30 %). Stoga spoj 4b može biti vodeći spoj za daljnji razvoj nove klase H1-antihistaminika

Carbohydrate-binding specificity of the daffodil (Narcissus pseudonarcissus) and amaryllis (Hippeastrum hybr.) bulb lectins

The carbohydrate binding specificity of the daffodil (Narcissus pseudonarcissus; NPA) and amaryllis (Hippeastrum hybr.; HHA) lectins, isolated from extracts of their bulbs by affinity chromatography on immobilized mannose, was studied by quantitative precipitation, sugar hapten inhibition, and affinity chromatography on the immobilized lectins. These lectins gave strong precipitation reactions with several yeast mannans, but did not precipitate with [alpha]--glucans (e.g., dextrans and glycogen). Interestingly, both lectins reacted strongly with yeast galactomannans having multiple nonreducing terminal [alpha]--galactosyl groups, a synthetic linear [alpha]-1,6-mannan, and an [alpha]-1,3-mannan (DP = 30). Treatment of the linear [alpha]-1,3-mannan with periodate, resulting in oxidation of the terminal, nonreducing mannosyl group, did not reduce its reactivity with NPA or HHA. Taken together, these observations suggest that NPA and HHA react not only with terminal but also with internal [alpha]--mannosyl residues. Sugar hapten inhibition studies showed these lectins to possess the greatest specific activity for [alpha]--mannosyl units whereas -Glc and -GlcNAc did not inhibit either lectin precipitation system. Of the oligosaccharides tested, the best inhibitor of NPA interaction was [alpha]-1,6-linked mannotriose, which was twice as good an inhibitor as Man[alpha] 1,6Man[alpha]-O-Me and 10 times better than methyl [alpha]--mannoside. On the other hand, oligosaccharides containing either 1,3- or 1,6-linked mannosyl units were good inhibitors of the HHA-mannan precipitation system (6- to 20-fold more active than -Man). These results indicate that both lectins appear to possess an extended binding site(s) complementary to at least three 1,6-linked [alpha]-mannosyl units. Various glycosylasparagine glycopeptides which contain [alpha]-1,6-Man units were retarded on the immobilized NPA column. On the other hand, those containing either [alpha]-1,3- or [alpha]-1,6-mannosyl residues were retarded on the immobilized HHA column; Man5-GlcNAc2-Asn [containing two Man[alpha] 1,3 (Man[alpha] 1,6) units] bound to the HHA column. On the contrary, glycopeptides with hybrid type glycan chains were not retarded on either column. These two new lectins which differ in their fine sugar binding specificity from each other, and also from the snowdrop lectin, should prove to be useful probes for the detection and preliminary characterization of glycoconjugates on cell surfaces and in solution.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28546/1/0000345.pd

Inhibition of all-TRANS-retinoic acid metabolism by R116010 induces antitumour activity

All-trans-retinoic acid is a potent inhibitor of cell proliferation and inducer of differentiation. However, the clinical use of all-trans-retinoic acid in the treatment of cancer is significantly hampered by its toxicity and the prompt emergence of resistance, believed to be caused by increased all-trans-retinoic acid metabolism. Inhibitors of all-trans-retinoic acid metabolism may therefore prove valuable in the treatment of cancer. In this study, we characterize R116010 as a new anticancer drug that is a potent inhibitor of all-trans-retinoic acid metabolism. In vitro, R116010 potently inhibits all-trans-retinoic acid metabolism in intact T47D cells with an IC50-value of 8.7 nM. In addition, R116010 is a selective inhibitor as indicated by its inhibition profile for several other cytochrome P450-mediated reactions. In T47D cell proliferation assays, R116010 by itself has no effect on cell proliferation. However, in combination with all-trans-retinoic acid, R116010 enhances the all-trans-retinoic acid-mediated antiproliferative activity in a concentration-dependent manner. In vivo, the growth of murine oestrogen-independent TA3-Ha mammary tumours is significantly inhibited by R116010 at doses as low as 0.16 mg kg−1. In conclusion, R116010 is a highly potent and selective inhibitor of all-trans-retinoic acid metabolism, which is able to enhance the biological activity of all-trans-retinoic acid, thereby exhibiting antitumour activity. R116010 represents a novel and promising anticancer drug with an unique mechanism of action

Anti-inflammatory and Immune-regulatory Effects of Subcutaneous Perillae Fructus Extract Injections on OVA-induced Asthma in Mice

Perillae fructus (perilla seed) is a traditional medicinal herb used to treat bronchial asthma in Oriental medical clinics. ST36 is one of the most widely used acupuncture points, particularly for immune system regulation. Injection of an herbal extract into an acupuncture point (herbal acupuncture) is a therapeutic technique combining both acupuncture and herbal treatment. Perillae fructus extract was injected subcutaneously (Perillae fructus herbal acupuncture; PF-HA) at acupoint ST36 of OVA-induced asthmatic mice. The lung weight, bronchoalveolar fluid (BALF) cell count, the number of CCR3+, CD11b+, CD4+ and CD3e+/CD69+ cells in the lung, and the level of IgE, IL-4, IL-5 and IL-13 in BALF and serum were then measured. RT-PCR was used to measure the mRNA expression of IL-4, IL-5, IL-13 and TNF-α in the lung. Lung sections were analyzed histologically. PF-HA significantly reduced lung weight, the number of inflammatory cells in the lung and BALF, the levels of IgE and Th2 cytokines in BALF and serum, mRNA expression of Th2 cytokines in the lung, and pathological changes in lung tissue. Our results suggest that PF-HA may have an anti-inflammatory and immune-regulatory effect on bronchial allergic asthma by restoring the Th1/Th2 imbalance in the immune system and suppressing eosinophilic inflammation in airways

Imaging Pulmonary NF-kappaB Activation and Therapeutic Effects of MLN120B and TDZD-8

NF-κB activation is a critical signaling event in the inflammatory response and has been implicated in a number of pathological lung diseases. To enable the assessment of NF-κB activity in the lungs, we transfected a luciferase based NF-κB reporter into the lungs of mice or into Raw264.7 cells in culture. The transfected mice showed specific luciferase expression in the pulmonary tissues. Using these mouse models, we studied the kinetics of NF-κB activation following exposure to lipopolysaccharide (LPS). The Raw264.7 cells expressed a dose-dependent increase in luciferase following exposure to LPS and the NF-κB reporter mice expressed luciferase in the lungs following LPS challenge, establishing that bioluminescence imaging provides adequate sensitivity for tracking the NF-κB activation pathway. Interventions affecting the NF-κB pathway are promising clinical therapeutics, thus we further examined the effect of IKK-2 inhibition by MLN120B and glycogen synthase kinase 3 beta inhibition by TDZD-8 on NF-κB activation. Pre-treatment with either MLN120B or TDZD-8 attenuated NF-κB activation in the pulmonary tissues, which was accompanied with suppression of pro-inflammatory chemokine MIP-1ß and induction of anti-inflammatory cytokine IL-10. In summary, we have established an imaging based approach for non-invasive and longitudinal assessment of NF-κB activation and regulation during acute lung injury. This approach will potentiate further studies on NF-κB regulation under various inflammatory conditions

Arabidopsis CPR5 Independently Regulates Seed Germination and Postgermination Arrest of Development through LOX Pathway and ABA Signaling

The phytohormone abscisic acid (ABA) and the lipoxygenases (LOXs) pathway play important roles in seed germination and seedling growth and development. Here, we reported on the functional characterization of Arabidopsis CPR5 in the ABA signaling and LOX pathways. The cpr5 mutant was hypersensitive to ABA in the seed germination, cotyledon greening and root growth, whereas transgenic plants overexpressing CPR5 were insensitive. Genetic analysis demonstrated that CPR5 gene may be located downstream of the ABI1 in the ABA signaling pathway. However, the cpr5 mutant showed an ABA independent drought-resistant phenotype. It was also found that the cpr5 mutant was hypersensitive to NDGA and NDGA treatment aggravated the ABA-induced delay in the seed germination and cotyledon greening. Taken together, these results suggest that the CPR5 plays a regulatory role in the regulation of seed germination and early seedling growth through ABA and LOX pathways independently