Structure of the disodium salt of glucose 1-phosphate hydrate, 2Na+.C6H11O9P2-.3.5H2O

Mr= 367.2, monoclinic, C2, a = 8.429 (1),b= 10.184(2), c= 16.570(2)A, /~= 99.18 (1) °, U= 1404.2 A 3, z = 4, D m = 1.73, D x = 1.74 Mg m -3,Cu K~, 2 = 1.5418 A, g = 2.99 mm -1, F(000) = 764,T= 300K, final R for 1524 observed reflections is0.069. The endocyclic C-O bonds in the glucose ring are nearly equal with C(5)-O(5)= 1.445 (10) and C(1)-O(5)= 1.424(10). The pyranose sugar ring adopts a 4C 1 chair conformation. The conformation about the exocyclic C(5)-C(6) bond is gauche-gauche, in contrast to gauche-trans observed in the structure of the dipotassium salt of glucose 1-phosphate. The phosphate ester bond, P-O(1), is 1.641 (6)A, slightly longer than the 'high-energy' P-,.O bond in the monopotassium salt of phosphoenolpyruvate [1.612 (6)A]. Two sodium ions are six coordinated while the third has only five neighbours.

Temozolomide-modulated glioma proteome: Role of interleukin-1 receptor-associated kinase-4 (IRAK4) in chemosensitivity

The current treatment for glioblastoma includes temozolomide (TMZ) chemotherapy, yet the mechanism of action of TMZ is not thoroughly understood. Here, we investigated the TMZ-induced changes in the proteome of the glioma-derived cell line (U251) by 2D DIGE. We found 95 protein spots to be significantly altered in their expression after TMZ treatment. MS identified four upregulated spots: aspartyl tRNA synthetase glutathione synthetase, interleukin-1 receptor-associated kinase-4 (IRAK4), and breast carcinoma amplified sequence-1 and one downregulated spot: optineurin. TMZ-induced regulation of these five genes was validated by RT-qPCR andWestern blot analysis. RNAi-mediated knockdown of IRAK4, an important mediator of Toll-like receptors signaling and chemoresistance, rendered the glioma cells resistant to TMZ. High levels of IRAK4 induced upon TMZ treatment resulted in IRAK1 downregulation and inhibition of NFkB pathway. Endogenous IRAK4 protein, but not transcript levels in glioma cell lines, correlated with TMZ sensitivity. Thus, we have identified several TMZ-modulated proteins and discovered an important novel role for IRAK4 in determining TMZ sensitivity of glioma cells through its ability to inhibit Toll-like receptor signaling and NFkB pathway.

The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of beta-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.

Novel multimeric IL-1 receptor antagonist for the treatment of rheumatoid arthritis

Protein therapeutics targeting inflammatory mediators have shown great promise for the treatment of autoimmunities such as rheumatoid arthritis (RA). However, a significant challenge in this area has been their low in vivo stability and consequently their severely compromised therapeutic efficacy. One such therapeutic molecule IL-1 receptor antagonist (IL-1ra), used in the treatment of rheumatoid arthritis, has displayed only modest efficacy in human clinical trials owing to its short biological half-life. Herein, we report a novel approach to conglomerate individual protein entities into a drug depot by incorporation of an amyloidogenic motif Lys-Phe-Phe-Glu (KFFE) thereby dramatically improving their systemic persistence and in turn their therapeutic efficacy in a mice model of autoimmune arthritis. (C) 2014 Elsevier Ltd. All rights reserved.

Ac2PIM-responsive miR-150 and miR-143 Target Receptor-interacting Protein Kinase 2 and Transforming Growth Factor Beta-activated Kinase 1 to Suppress NOD2-induced Immunomodulators

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac2PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac2PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-alpha, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac2PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac2PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac2PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKC delta-MAPK pathway to suppress beta-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac2PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac2PIM and/or MDP.

Dialkyl (3-Aryl- 1,2,4-oxadiazol-5-yl)phosphonates: Synthesis and Thermal Behavior-Evidence for Monomeric Alkyl Metaphosphate

Dialkyl (3-aryl-l,2,4-oxadiazol-5-yl)phosphonate6sa -h have been obtained by 1,3-dipolar cycloaddition of arenenitrile oxides 5a-f to dialkyl phosphorocyanidates (4a and 4b) in yields ranging between 30% and 58%. A standardized method for obtaining cyanidates 4a and 4b has been established. The diethyl thiophosphorocyanidate (4c) is less reactive than 4a and 4b, only the 3-(4'-nitrophenyl) derivative 6i being obtainable. While the IR and NMFt spectra of 6a-i were unexceptional, their UV spectra showed evidence of conjugative interaction in high degrees between the phosphonate and heterocyclic moieties as well as a varying conjugative interaction between the heterocyclic and aryl moieties. The oxadiazoles 6a-h are thermally labile and yield trialkyl phosphates 7 as the only identifiable products. A mechanism based on the intermediacy of monomeric alkyl metaphosphate 11 in the formation of trialkyl phosphate was postulated, and supportive evidence in the form of trapping the metaphosphate with acetophenone has been obtained.

Paracrine action of sFLT-1 secreted by stably-transfected Ehrlich ascites tumor cells and therapy using sFLT-1 inhibits ascites tumor growth in vivo

Background Vascular endothelial growth factor (VEGF) is known to play a major role in angiogenesis. A soluble form of Flt-1, a VEGF receptor, is potentially useful as an antagonist of VEGF, and accumulating evidence suggests the applicability of sFlt-1 in tumor suppression. In the present study, we have developed and tested strategies targeted specifically to VEGF for the treatment of ascites formation.Methods As an initial strategy, we produced recombinant sFLT-1 in the baculovirus expression system and used it as a trap to sequester VEGF in the murine ascites carcinoma model. The effect of the treatment on the weight of the animal, cell number, ascites volume and proliferating endothelial cells was studied. The second strategy involved, producing Ehrlich ascites tumor (EAT) cells stably transfected with vectors carrying cDNA encoding truncated form of Flt-1 and using these cells to inhibit ascites tumors in a nude mouse model. Results The sFLT-1 produced by the baculovirus system showed potent antiangiogenic activity as assessed by rat cornea and tube formation assay. sFLT-1 treatment resulted in reduced peritoneal angiogenesis with a concomitant decrease in tumor cell number, volume of ascites, amount of free VEGF and the number of invasive tumor cells as assayed by CD31 staining. EAT cells stably transfected with truncated form of Flt-1 also effectively reduced the tumor burden in nude mice transplanted with these cells, and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Conclusions The inhibition of peritoneal angiogenesis and tumor growth by sequestering VEGF with either sFlt-1 gene expression by recombinant EAT cells or by direct sFLT-1 protein therapy is shown to comprise a potential therapy. Copyright (C) 2009 John Wiley & Sons, Ltd.

NMR relaxation study of disorder in the mixed system BP x GPI(1-x) and the low temperature transition from classical to quantum rotation

1H NMR spin-lattice relaxation time (T1) studies have been carried out in the temperature range 100 K to 4 K, at two Larmor frequencies 11.4 and 23.3 MHz, in the mixed system of betaine phosphate and glycine phosphite (BPxGPI(1-x)), to study the effects of disorder on the proton group dynamics. Analysis of T1 data indicates the presence of a number of inequivalent methyl groups and a gradual transition from classical reorientations to quantum tunneling rotations. At lower temperatures, microstructural disorder in the local environments of the methyl groups, result in a distribution in the activation energy (Ea) and the torsional energy gap (E01). For certain values of x, the magnetisation recovery shows biexponential behaviour at lower temperatures.

Hantzsch 1,4-dihydropyridine esters and analogs: candidates for generating reproducible one-dimensional packing motifs

Examination of the symmetric Hantzsch 1,4-dihydropyridine ester derivatives of the prototypical nifedipine molecule indicates the tendency of this class of molecule to form a common packing motif. Crystal structure analysis of 2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic diesters and analogs reveals that they form extended chains, characterized as the C(6) packing motif, via intermolecular (amine) N-H...O=C (C3,C5 carbonyl) hydrogen bonds. In addition, all the prepared derivatives also satisfy the basic structural requirements for their high binding efficiency to the receptor. The reproducible C(6) packing motif observed among these compounds has a use in the design of solid-state materials.

Purification and Regulatory Properties of Mung Bean (Vigna Radiata L.) Serine Hydroxymethyltransferase 1

Serine hydroxymethyltransferase, the first enzyme in the pathway for interconversion of C1 fragments, was purified to homogeneity for the first time from any plant source. The enzyme from 72-h mung bean (Vigna radiata L.) seedlings was isolated using Blue Sepharose CL-6B and folate-AH-Sepharose-4B affinity matrices and had the highest specific activity (1.33 micromoles of HCHO formed per minute per milligram protein) reported hitherto. The enzyme preparation was extremely stable in the presence of folate or L-serine. Pyridoxal 5'-phosphate, ethylenediaminetetraacetate and 2-mercaptoethanol prevented the inactivation of the enzyme during purification. The enzyme functioned optimally at pH 8.5 and had two temperature maxima at 35 and 55°C. The Km values for serine were 1.25 and 68 millimolar, corresponding to Vmax values of 1.8 and 5.4 micromoles of HCHO formed per minute per milligram protein, respectively. The K0.5 value for L-tetrahydrofolate (H4folate) was 0.98 millimolar. Glycine, the product of the reaction and D-cycloserine, a structural analog of D-alanine, were linear competitive inhibitors with respect to L-serine with Ki values of 2.30 and 2.02 millimolar, respectively. Dichloromethotrexate, a substrate analog of H4folate was a competitive inhibitor when H4folate was the varied substrate. Results presented in this paper suggested that pyridoxal 5'-phosphate may not be essential for catalysis.The sigmoid saturation pattern of H4folate (nH = 2.0), one of the substrates, the abolition of sigmoidicity by NADH, an allosteric positive effector (nH = 1.0) and the increase in sigmoidicity by NAD+ and adenine nucleotides, negative allosteric effectors (nH = 2.4) clearly established that this key enzyme in the folate metabolism was an allosteric protein. Further support for this conclusion were the observations that (a) serine saturation exhibited an intermediary plateau region; (b) partial inhibition by methotrexate, aminopterin, O-phosphoserine, DL-{alpha}-methylserine and DL-O-methylserine; (c) subunit nature of the enzyme; and (d) decrease in the nH value from 2.0 for H4folate to 1.5 in presence of L-serine. These results highlight the regulatory nature of mung bean serine hydroxymethyltransferase and its possible involvement in the modulation of the interconversion of folate coenzymes.

Indoleacetaldoxime hydro-lyase (4.2.1.29). III. Further studies on the nature and mode of action of the enzyme

Further purification of indoleacetaldoxime (IAOX) hydro-lyase from Gibberella fujikuroi by DEAE-cellulose chromatography is described. The purified enzyme was activated by dehydroascorbic acid (DHA), ascorbic acid (AA), and pyridoxal phosphate (PALP) and was inhibited by thiol compounds and thiol reagents including phenylthiocyanate. Ferrous ions but not ferric ions activated the purified enzyme. The enzyme was activated by dihydrofolic acid but inhibited by tetrahydrofolic acid. Phenylacetaldoxime, a competitive inhibitor, afforded partial protection of the enzyme from the action of N-ethylmaleimide suggesting the involvement of a thiol function at the active site or substrate-binding site. The inhibition of the enzyme by 2,3-dimercaptopropanol was reversed by DHA, PALP, or frozen storage. KCN inhibition of the enzyme was reversed by PALP. NaBH4 reduction of the purified enzyme in the presence of PALP gave an active enzyme which was further activated by PALP or DHA but not by ferrous ions. These results suggested a "structural" role for PALP in the activity of IAOX hydro-lyase. Dilute solutions of the purified enzyme, obtained during DEAE-cellulose chromatography and concentrated using sucrose, showed enhanced activity upon frozen storage and thawing. The increase in activity of the enzyme during certain culture conditions, the activation and inhibition of the enzyme by several unrelated compounds, and the effect of freezing indicate that IAOX hydro-lyase is probably a metabolically regulated enzyme with a structure composed of subunits.

Synthesis and Antileukemic Activity of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea Derivatives

Heterocyclic urea derivatives play an important role as anticancer agents because of their good inhibitory activity against receptor tyrosine kinases (RTKs), raf kinases, protein tyrosine kinases (PTKs), and NADH oxidase, which play critical roles in many aspects of tumorigenesis. Benzothiazole moiety constitutes an important scaffold of drugs, possessing several pharmacological functions, mainly the anticancer activity. Based on these interesting properties of benzothiazoles and urea moiety to obtain new biologically active agents, we synthesized a series of novel 1-((S)-2-amino-4,5,6.7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea derivatives and evaluated for their efficacy as antileukemic agents against two human leukemic cell lines (K562 and Reh). These compounds showed good and moderate cytotoxic effect to cancer cell lines tested. Compounds with electron-withdrawing chloro and fluoro substituents on phenyl ring showed good activity and compounds with electron-donating methoxy group showed moderate activity. Compound with electron-withdrawing dichloro substitution on phenyl ring of aryl urea showed good activity. Further, lactate dehydrogenase (LDH) assay, flow cytometric analysis of annexin V-FITC/propidium iodide (PI) double staining and DNA fragmentation studies showed that compound with dichloro substitution on phenyl ring of aryl urea can induce apoptosis.

Recognition of Polycyclic Aromatic Hydrocarbons and Their Derivatives by the 1,3-Dinaphthalimide Conjugate of Calix4]arene: Emission, Absorption, Crystal Structures, and Computational Studies

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants as well as well-known carcinogens. Therefore, it is important to develop an effective receptor for the detection and quantification of such molecules in solution. In view of this, a 1,3-dinaphthalimide derivative of calix4]arene (L) has been synthesized and characterized, and the structure has been established by single crystal XRD. In the crystal lattice, intermolecular arm-to-arm pi center dot center dot center dot pi overlap dominates and thus L becomes a promising receptor for providing interactions with the aromatic species in solution, which can be monitored by following the changes that occur in its fluorescence and absorption spectra. On the basis of the solution studies carried out with about 17 derivatives of the aromatic guest molecular systems, it may be concluded that the changes that occur in the fluorescence intensity seem to be proportional to the number of aromatic rings present and thus proportional to the extent of pi center dot center dot center dot pi interaction present between the naphthalimide moieties and the aromatic portion of the guest molecule. Though the nonaromatic portion of the guest species affects the fluorescence quenching, the trend is still based on the number of rings present in these. Four guest aldehydes are bound to L with K-ass of 2000-6000 M-1 and their minimum detection limit is in the range of 8-35 mu M. The crystal structure of a naphthaldehyde complex, L.2b, exhibits intermolecular arm-to-arm as well as arm-to-naphthaldehyde pi center dot center dot center dot pi interactions. Molecular dynamics studies of L carried out in the presence of aromatic aldehydes under vacuum as well as in acetonitrile resulted in exhibiting interactions observed in the solid state and hence the changes observed in the fluorescence and absorption spectra are attributable for such interactions. Complex formation has also been delineated through ESI MS studies. Thus L is a promising receptor that can recognize PAHs by providing spectral changes proportional to the aromatic conjugation of the guest and the extent of aromatic pi center dot center dot center dot pi interactions present between L and the guest.

On the importance of backbone loop and peptide flip in the Walker sequence in F-1-ATPase action

The Walker sequence, GXXXXGKT, present in all the six subunits of F-1-ATPase exists in a folded form, known as phosphate-binding loop (P-loop). Analysis of the Ramachandran angles showed only small RMS deviation between the nucleotide-bound and nucleotide-free forms. This indicated a good overlap of the backbone loops. The catalytic beta-subunits (chains D, E and F) showed significant changes in the Ramachandran angles and the side chain torsion angles, but not the structural alpha-subunits (chains A, B and C). Most striking among these are the changes associated with Val160 and Gly161 corresponding to a flip in the peptide unit between them when a nucleotide is bound (chains D or F compared to nucleotide-free chain E). The conformational analysis further revealed a hitherto unnoticed hydrogen bond between amide-N of the flipped Gly161 and terminal phosphate-O of the nucleotide. This assigns a role for this conserved amino acid, otherwise ignored, of making an unusual direct interaction between the peptide backbone of the enzyme protein and the incoming nucleotide substrate. Significance of this interaction is enhanced, as it is limited only to the catalytic subunits, and also likely to involve a mechanical rotation of bonds of the peptide unit. Hopefully this is part of the overall events that link the chemical hydrolysis of ATP with the mechanical rotation of this molecule, now famous as tiny molecular motor.