Sample records for n-3-4-hydroxyphenylpropionyl pro-hyp-gly-ala-gly-oh reduces

  1. Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing Cyclic Dipeptides with High Oral Bioavailability: Efficient Formation of Cyclo(X-Hyp) from X-Hyp-Gly-Type Tripeptides by Heating.

    PubMed

    Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2017-11-01

    Cyclic dipeptides (2,5-diketopiperazines) are present in a variety of foods and are reported to demonstrate antioxidant, antidepressant, and other beneficial effects. We recently developed a novel collagen hydrolysate characterized by a high content of X-hydroxyproline (Hyp)-Gly-type tripeptides using ginger protease. In the present study, we found that, through heating, X-Hyp-Gly can be easily converted into Hyp-containing cyclic dipeptides. After heating for 3 h at 85 °C and pH 4.8, Ala-Hyp-Gly was almost completely cyclized to cyclo(Ala-Hyp), in contrast to a slight cyclization of Ala-Hyp. The contents of cyclo(Ala-Hyp) and cyclo(Leu-Hyp) reached 0.5-1% (w/w) each in the ginger-degraded collagen hydrolysate under the heating conditions. Oral administration experiments using mice revealed that cyclo(Ala-Hyp) and cyclo(Leu-Hyp) were absorbed into the blood at markedly higher efficiencies compared to collagenous oligopeptides, including Pro-Hyp. The high productivity and oral bioavailability of the collagen-specific cyclic dipeptides suggest significant health benefits of the heat-treated ginger-degraded collagen hydrolysate.

  2. Efficient Absorption of X-Hydroxyproline (Hyp)-Gly after Oral Administration of a Novel Gelatin Hydrolysate Prepared Using Ginger Protease.

    PubMed

    Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2016-04-13

    Recent studies have reported that oral intake of gelatin hydrolysate has various beneficial effects, such as reduction of joint pain and lowering of blood sugar levels. In this study, we produced a novel gelatin hydrolysate using a cysteine-type ginger protease having unique substrate specificity with preferential peptide cleavage with Pro at the P2 position. Substantial amounts of X-hydroxyproline (Hyp)-Gly-type tripeptides were generated up to 2.5% (w/w) concomitantly with Gly-Pro-Y-type tripeptides (5%; w/w) using ginger powder. The in vivo absorption of the ginger-degraded gelatin hydrolysate was estimated using mice. The plasma levels of collagen-derived oligopeptides, especially X-Hyp-Gly, were significantly high (e.g., 2.3-fold for Glu-Hyp-Gly, p < 0.05) compared with those of the control gelatin hydrolysate, which was prepared using gastrointestinal proteases and did not contain detectable X-Hyp-Gly. This study demonstrated that orally administered X-Hyp-Gly was effectively absorbed into the blood, probably due to the high protease resistance of this type of tripeptide.

  3. Encapsulation of rat bone marrow stromal cells using a poly-ion complex gel of chitosan and succinylated poly(Pro-Hyp-Gly).

    PubMed

    Kusumastuti, Yuni; Shibasaki, Yoshiaki; Hirohara, Shiho; Kobayashi, Mime; Terada, Kayo; Ando, Tsuyoshi; Tanihara, Masao

    2017-03-01

    Encapsulation of stem cells into a three-dimensional (3D) scaffold is necessary to achieve tissue regeneration. Prefabricated 3D scaffolds, such as fibres or porous sponges, have limitations regarding homogeneous cell distribution. Hydrogels that can encapsulate cells such as animal-derived collagen gels need adjustment of the pH and/or temperature upon cell mixing. In this report, we fabricated a poly-ion complex (PIC) hydrogel of chitosan and succinylated poly(Pro-Hyp-Gly) and assessed its effect on cell viability after encapsulation of rat bone marrow stromal cells. PIC hydrogels were obtained successfully with a concentration of each precursor as low as 3.0-3.8 mg/ml. The maximum gelation and swelling ratios were achieved with an equal molar ratio (1:1) of anionic and cationic groups. Using chitosan acetate as a cationic precursor produced a PIC hydrogel with both a significantly greater gelation ratio and a better swelling ratio than chitosan chloride. Ammonium succinylated poly(Pro-Hyp-Gly) as an anionic precursor gave similar gelation and swelling ratios to those of sodium succinylated poly(Pro-Hyp-Gly). Cell encapsulation was also achieved successfully by mixing rat bone marrow stromal cells with the PIC hydrogel simultaneously during its formation. The PIC hydrogel was maintained in the culture medium for 7 days at 37°C and the encapsulated cells survived and proliferated in it. Although it is necessary to improve its functionality, this PIC hydrogel has the potential to act as a 3D scaffold for cell encapsulation and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Experimental verification of force fields for molecular dynamics simulations using Gly-Pro-Gly-Gly.

    PubMed

    Aliev, Abil E; Courtier-Murias, Denis

    2010-09-30

    β(PR) ⇆ β(S) ⇆ β(P) ⇆ α, where backbone secondary structures α(L) and α are associated with helices and turns, β(P) and β(PR) correspond to the left- and right-handed polyproline II structures and β(S) denotes the fully stretched backbone conformation. Compared to the force field dependence, less significant, but noteworthy, variations in the populations of the peptide backbone conformations were observed. For different solvent models considered, a correlation was noted between the number of torsional transitions in GPGG and the water self-diffusion coefficient on using TIP3P, TIP4P, and TIP5P models. In addition to MD results, we also report DFT derived Karplus relationships for Gly and Pro residues using B972 and B3LYP functionals.

  5. Effects of Pro-Gly-Pro tripeptide on the dopamine system.

    PubMed

    Meshavkin, V K; Batishcheva, E Yu; Kost, N V; Sokolov, O Yu; Trufanova, A V; Samonina, G E

    2011-08-01

    Tripeptide Pro-Gly-Pro interacted with dopamine receptors in vitro and reduced behavioral manifestations of apomorphine-induced hyperfunction of the dopamine system in verticalization, stereotypy, and yawning tests. Presumably, the behavioral effects of Pro-Gly-Pro tripeptide were mediated through post- and presynaptic D(2)and D(3)receptors.

  6. [Effects of Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly peptides on hormonal activity and thyroid morphology in hypophysectomized mature and old birds].

    PubMed

    Kuznik, B I; Pateiuk, A V; Rusaeva, N S; Baranchugova, L M; Obydenko, V I

    2011-01-01

    The aim of the paper was to investigate effects of Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly peptides which were designed and synthesized on the basis of amino acid study of the hypophyseal anterior and posterior lobe peptides on the thyroid morphology and hormonal activity in mature chicken and old birds. Hypophysectomy was established to produce atrophic changes in the thyroid gland and development of secondary hypothyrosis. Administration of Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly tetrapeptides significantly prevented these impairments by increasing the levels of the thyroid-stimulating hormone (TSH) as well as T3 and T4. Restoration of the thyroid functions and morphology was registered to be greater in one-year-old chicken as compared to five-year-old ones.

  7. Potent inhibition of endopeptidase 24.16 and endopeptidase 24.15 by the phosphonamide peptide N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid.

    PubMed Central

    Barelli, H; Dive, V; Yiotakis, A; Vincent, J P; Checler, F

    1992-01-01

    A phosphonamide peptide, N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid, previously shown to block Clostridium histolyticum collagenases, was examined as a putative inhibitor of endopeptidase 24.16 and endopeptidase 24.15. Hydrolysis of two endopeptidase 24.16 substrates, i.e. 3-carboxy-7-methoxycoumarin (Mcc)-Pro-Leu-Gly-Pro-D-Lys-dinitrophenyl (Dnp) and neurotensin, were completely and dose-dependently inhibited by the phosphonamide inhibitor with KI values of 0.3 and 0.9 nM respectively. In addition, the phosphonamide peptide inhibited the hydrolysis of benzoyl (Bz)-Gly-Ala-Ala-Phe-(pAB) p-aminobenzoate and neurotensin by endopeptidase 24.15 with about a 10-fold lower potency (KI values of 5 and 7.5 nM respectively). The selectivity of this inhibitor towards several exo- and endo-peptidases belonging to the zinc-containing metallopeptidase family established that a 1 microM concentration of this inhibitor was unable to affect leucine aminopeptidase, carboxypeptidase A, angiotensin-converting enzyme and endopeptidase 24.11. The present paper therefore reports on the first hydrophilic highly potent endopeptidase 24.16 inhibitor and describes the most potent inhibitory agent directed towards endopeptidase 24.15 developed to date. These tools should allow one to assess the contribution of endopeptidase 24.16 and endopeptidase 24.15 to the physiological inactivation of neurotensin as well as other neuropeptides. PMID:1332678

  8. Folding and conformational consequences of glycine to alanine replacements at different positions in a collagen model peptide.

    PubMed

    Bhate, Manjiri; Wang, Xin; Baum, Jean; Brodsky, Barbara

    2002-05-21

    The collagen model peptide T1-892 includes a C-terminal nucleation domain, (Gly-Pro-Hyp)(4), and an N-terminal (Gly-X-Y)(6) sequence taken from type I collagen. In osteogenesis imperfecta (OI) and other collagen diseases, single base mutations often convert one Gly to a larger residue, and T1-892 homologues modeling such mutations were synthesized with Gly to Ala substitutions in either the (Gly-Pro-Hyp)(4) domain, Gly25Ala, or the (Gly-X-Y)(6) domain, Gly10Ala. CD and NMR studies show the Gly10Ala peptide forms a normal triple-helix at the C-terminal end and propagates from the C- to the N-terminus until the Gly --> Ala substitution is encountered. At this point, triple-helix folding is terminated and cannot be reinitiated, leaving a nonhelical N-terminus. A decreased thermal stability is observed as a result of the shorter length of the triple-helix. In contrast, introduction of the Gly to Ala replacement at position 25, in the nucleation domain, shifts the monomer/trimer equilibrium toward the monomer form. The increased monomer and lower trimer populations are reflected in the dramatic decrease in triple-helix content and stability. Unlike the Ala replacement at position 10, the Ala substitution in the (Gly-Pro-Hyp)(4) region can still be incorporated into a triple-helix, but at a greatly decreased rate of folding, since the original efficient nucleation site is no longer operative. The specific consequences of Gly to Ala replacements in two distinctive sequences in this triple-helical peptide may help clarify the variability in OI clinical severity resulting from mutations at different sites along type I collagen chains.

  9. Synthesis, biological evaluation, and automated docking of constrained analogues of the opioid peptide H-Dmt-D-Ala-Phe-Gly-NH₂ using the 4- or 5-methyl substituted 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one scaffold.

    PubMed

    De Wachter, Rien; de Graaf, Chris; Keresztes, Atilla; Vandormael, Bart; Ballet, Steven; Tóth, Géza; Rognan, Didier; Tourwé, Dirk

    2011-10-13

    The Phe(3) residue of the N-terminal tetrapeptide of dermorphin (H-Dmt-d-Ala-Phe-Gly-NH(2)) was conformationally constrained using 4- or 5-methyl-substituted 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) stereoisomeric scaffolds. Several of the synthesized peptides were determined to be high affinity agonists for the μ opioid receptor (OPRM) with selectivity over the δ opioid receptor (OPRD). Interesting effects of the Aba configuration on ligand binding affinity were observed. H-Dmt-d-Ala-erythro-(4S,5S)-5-Me-Aba-Gly-NH(2)9 and H-Dmt-threo-(4R,5S)-5-Me-Aba-Gly-NH(2)12 exhibited subnanomolar affinity for OPRM, while they possess an opposite absolute configuration at position 4 of the Aba ring. However, in the 4-methyl substituted analogues, H-Dmt-d-Ala-(4R)-Me-Aba-Gly-NH(2)14 was significantly more potent than the (4S)-derivative 13. These unexpected results were rationalized using the binding poses predicted by molecular docking simulations. Interestingly, H-Dmt-d-Ala-(4R)-Me-Aba-Gly-NH(2)14 is proposed to bind in a different mode compared with the other analogues. Moreover, in contrast to Ac-4-Me-Aba-NH-Me, which adopts a β-turn in solution and in the crystal structure, the binding mode of this analogue suggests an alternative receptor-bound conformation.

  10. Infrared Spectroscopy of Mobility-Selected H+-Gly-Pro-Gly-Gly (GPGG)

    NASA Astrophysics Data System (ADS)

    Masson, Antoine; Kamrath, Michael Z.; Perez, Marta A. S.; Glover, Matthew S.; Rothlisberger, U.; Clemmer, David E.; Rizzo, Thomas R.

    2015-09-01

    We report the first results from a new instrument capable of acquiring infrared spectra of mobility-selected ions. This demonstration involves using ion mobility to first separate the protonated peptide Gly-Pro-Gly-Gly (GPGG) into two conformational families with collisional cross-sections of 93.8 and 96.8 Å2. After separation, each family is independently analyzed by acquiring the infrared predissociation spectrum of the H2-tagged molecules. The ion mobility and spectroscopic data combined with density functional theory (DFT) based molecular dynamics simulations confirm the presence of one major conformer per family, which arises from cis/ trans isomerization about the proline residue. We induce isomerization between the two conformers by using collisional activation in the drift tube and monitor the evolution of the ion distribution with ion mobility and infrared spectroscopy. While the cis-proline species is the preferred gas-phase structure, its relative population is smaller than that of the trans-proline species in the initial ion mobility drift distribution. This suggests that a portion of the trans-proline ion population is kinetically trapped as a higher energy conformer and may retain structural elements from solution.

  11. Inhibitory effect of collagen-derived tripeptides on dipeptidylpeptidase-IV activity.

    PubMed

    Hatanaka, Tadashi; Kawakami, Kayoko; Uraji, Misugi

    2014-12-01

    The collagen tripeptide fragments Gly-Ala-Hyp, Gly-Pro-Ala and Gly-Pro-Hyp were generated by hydrolyzing collagen from pig-skin, cattle-skin, fish-scales and chicken-feet, respectively, with Streptomyces collagenase. Collagenase treatment increased the concentration of tripeptides in the hydrolysates by 13-15% (w/w). Of the three peptides, Gly-Pro-Hyp was a true peptidic inhibitor of dipeptidylpeptidase-IV (DPP-IV), because DPP-IV could not hydrolyze the bond between Pro-Hyp. This tripeptide was a moderately competitive inhibitor (Ki=4.5 mM) of DPP-IV, and its level in the collagen hydrolysates could be greatly increased (4-9% [w/w]) using Streptomyces collagenase.

  12. Effect of peptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly on the morphology of the thymus in hypophysectomized young and old birds.

    PubMed

    Pateyk, A V; Baranchugova, L M; Rusaeva, N S; Obydenko, V I; Kuznik, B I

    2013-03-01

    Investigations were carried out on chicks of different age. It was found that the most pronounced changes in the morphology of the thymus occurred after neonatal hypophysectomy. These changes are least pronounced in old chicks. Peptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly synthesized on the basis of amino acid composition of peptide complexes of the anterior and posterior pituitary lobes administered to hypophysectomized birds regardless of age promoted recovery of the morphological structures of the thymus. The anterior pituitary peptide (Lys-Glu-Asp-Gly) had more pronounced effect on the recovery of thymic structure than posterior pituitary peptide (Ala-Glu-Asp-Gly).

  13. Pressure dependence of side chain 13C chemical shifts in model peptides Ac-Gly-Gly-Xxx-Ala-NH2.

    PubMed

    Beck Erlach, Markus; Koehler, Joerg; Crusca, Edson; Munte, Claudia E; Kainosho, Masatsune; Kremer, Werner; Kalbitzer, Hans Robert

    2017-10-01

    For evaluating the pressure responses of folded as well as intrinsically unfolded proteins detectable by NMR spectroscopy the availability of data from well-defined model systems is indispensable. In this work we report the pressure dependence of 13 C chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH 2 (Xxx, one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of a number of nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The size of the polynomial pressure coefficients B 1 and B 2 is dependent on the type of atom and amino acid studied. For H N , N and C α the first order pressure coefficient B 1 is also correlated to the chemical shift at atmospheric pressure. The first and second order pressure coefficients of a given type of carbon atom show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure also are weakly correlated. The downfield shifts of the methyl resonances suggest that gauche conformers of the side chains are not preferred with pressure. The valine and leucine methyl groups in the model peptides were assigned using stereospecifically 13 C enriched amino acids with the pro-R carbons downfield shifted relative to the pro-S carbons.

  14. Pharmacology and expression analysis of glycine transporter GlyT1 with [3H]-(N-[3-(4'-fluorophenyl)-3-(4'phenylphenoxy)propyl])sarcosine.

    PubMed

    Mallorga, Pierre J; Williams, Jacinta B; Jacobson, Marlene; Marques, Rosemary; Chaudhary, Ashok; Conn, P Jeffrey; Pettibone, Douglas J; Sur, Cyrille

    2003-10-01

    In the central nervous system, re-uptake of the neurotransmitter glycine is mediated by two different glycine transporters, GlyT1 and GlyT2. GlyT2 is found in brainstem and spinal cord, whereas GlyT1 is expressed in rat forebrain regions where it is responsible for most glycine transport activity. Initially, GlyT1 and GlyT2 were pharmacologically differentiated by sarcosine, a weak selective inhibitor of GlyT1. The recently described selective and potent GlyT1 antagonist, NFPS/ALX-5407 provided an important additional tool to further characterize GlyT1 pharmacology. In the present study, we have radiolabeled the racemic form of NFPS (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (also known as ALX-5407) to investigate its interaction with GlyT1, as well as define GlyT1 expression in the rat central nervous system. Kinetic studies indicated that [3H]NFPS binds rapidly to rat forebrain membranes and dissociates with a t(1/2) of 28 +/- 5 min. [3H]NFPS labeled a saturable population of sites in rat forebrain with a Kd of 7.1+/-1.3 nM and a B(max) of 3.14 +/- 0.26 pmol/mg protein. Bound [3H]NFPS was fully and potently displaced by unlabeled NFPS, whereas glycine and sarcosine were weak, Na+-dependent inhibitors with IC50 of 1,008 and 190 microM, respectively. Additional saturation experiments indicated that glycine and sarcosine were non-competitive antagonists of [3H]NFPS binding. Functional studies revealed that NFPS was a non-competitive inhibitor of [3H]glycine uptake and does not interact with Na+ and Cl- binding sites of GlyT1. Overall, this work shows that [3H]NFPS is a valuable tool in studying GlyT1 expression and pharmacology and that NFPS interacts with GlyT1 at a site different from the transporter translocation and ion binding sites.

  15. Reactivity of Ala-Gly dipeptide with β-turn secondary structure

    NASA Astrophysics Data System (ADS)

    Yu, Craig P.; Gerlei, Klára Z.; Rágyanszki, Anita; Jensen, Svend J. Knak; Viskolcz, Béla; Csizmadia, Imre G.

    2018-01-01

    The conformational space of β-turns of Ala-Gly dipeptide is analyzed theoretically using quantum mechanical methods. A number of potential minima are obtained and characterized. The potential energy surface suggests that β-turn conformers are susceptible to rapid radical formation, which leads to potential L and D epimerization. The calculated thermodynamics show that the radical mediated epimerization is possible and that the estimated barrier height for hydrogen abstraction on the Cα is the lowest for the Gly residue.

  16. Changes in composition and content of food-derived peptide in human blood after daily ingestion of collagen hydrolysate for 4 weeks.

    PubMed

    Shigemura, Yasutaka; Suzuki, Asahi; Kurokawa, Mihoko; Sato, Yoshio; Sato, Kenji

    2018-03-01

    Daily ingestion of collagen hydrolysate for a long period improves skin and joint conditions. It has been speculated that the beneficial effects are exerted by food-derived hydroxyproline (Hyp) peptides, which are detected in human blood after single ingestions. In the present study, to investigate the effect of long-term ingestion of collagen hydrolysate on Hyp peptides profile in blood, the concentrations of Hyp-peptides in human blood before and after daily ingestion for a long period were examined. Hyp-peptides increased to a maximum level at 1 h after ingestion and reverted to their initial levels within 24 h during experimental period. Pro-Gly and Hyp-peptides such as Pro-Hyp-Gly, Pro-Hyp, Ile-Hyp, Leu-Hyp, Hyp-Gly, Glu-Hyp and Ala-Hyp were identified in the blood after ingestion of collagen hydrolysate at 4.5 g day -1 for 4 weeks. For the whole period, Pro-Hyp was the leading compound. The compositional rate of Hyp-Gly showed a tendency to increase, while that of Pro-Hyp tended to decrease after daily ingestion. The present results indicate that daily ingestion of collagen hydrolysate for a long period can change compositional rate of Hyp peptides in human blood. This fact suggests that long-term ingestion of collagen hydrolysate might change exo- or endo-type protease activity in the digestive tract, which may consequently promote beneficial effects. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  17. Effect of tetrapeptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly on the structure and function of the thyroid gland in neonatally hypophysectomized chickens.

    PubMed

    Kuznik, B I; Pateyuk, A V; Rusaeva, N S

    2008-01-01

    Tetrapeptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly were synthesized on the basis of amino acid composition of pituitary cytomedins. Administration of these tetrapeptides to hypophysectomized chickens for 40 days was followed by an increase in the concentrations of thyrotropic hormone and thyroid hormones and recovery of thyroid gland structure.

  18. A C69-family cysteine dipeptidase from Lactobacillus farciminis JCM1097 possesses strong Gly-Pro hydrolytic activity.

    PubMed

    Sakamoto, Takuma; Otokawa, Takuya; Kono, Ryosuke; Shigeri, Yasushi; Watanabe, Kunihiko

    2013-11-01

    Dipeptide Gly-Pro, a hard-to-degrade and collagenous peptide, is thought to be hydrolysed by prolidases that can work on various X-Pro dipeptides. Here, we found an entirely different type of dipeptidase from Lactobacillus farciminis JCM1097 that cleaves Gly-Pro far more efficiently and with higher specificity than prolidases, and then investigated its properties by use of a recombinant enzyme. Although L. farciminis dipeptidase was expressed in the form of an inclusion body in Escherichia coli at 37 °C, it was smoothly over-expressed in a soluble form at a lower temperature. The maximal Gly-Pro hydrolytic activity was attained in E. coli at 30 °C. In contrast to prolidases that are metallopeptidases showing the modest or marginal activity toward Gly-Pro, this L. farciminis dipeptidase belongs to the cysteine peptidase family C69. Lactobacillus farciminis dipeptidase occurs in cytoplasm and utilizes the side chain of an amino-terminal cysteine residue to perform the nucleophilic attack on the target amide bond between Gly-Pro after processing eight amino acid residues at the N-terminus. Furthermore, L. farciminis dipeptidase is potent enough to synthesize Gly-Pro from Gly and Pro by a reverse reaction. These novel properties could be revealed by virtue of the success in preparing recombinant enzymes in higher yield and in a stable form.

  19. Effects of peptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly on hormonal activity and structure of the thyroid gland in hypophysectomized young chickens and old hens.

    PubMed

    Kuznik, B I; Pateyuk, A V; Rusaeva, N S; Baranchugova, L M; Obydenko, V I

    2011-02-01

    Hypophysectomy in 5-days chickens and old hens was followed by hormonal disturbances and structural changes in the thyroid gland. Administration of peptides Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly synthesized on the basis the amino acid composition of extracts from the anterior and posterior lobes of the pituitary gland, respectively, to hypophysectomized birds for 40 days significantly reduced the degree of these changes. The normalizing effect of synthetic peptides on the concentration of thyrotrophic hormone and thyroid hormones in old hens was less pronounced than in chickens.

  20. [Peptide Ala-Glu-Asp-Gly and interferon gamma: their role in immune response during aging].

    PubMed

    Lin'kova, N S; Kuznik, B I; Khavinson, V Kh

    2012-01-01

    The decrease of lymphocyte interferon gamma expression during aging is one of the main mechanisms leading to the immunodeficiency state in the elderly. Cell penetrating geroprotective peptide Ala-Glu-Asp-Gly has the capability to activate the proliferation of lymphocytes in thymus during its aging. The nucleotide sequence which is complementary contacted with peptide Ala-Glu-Asp-Gly was found in promoter region of interferon gamma gene. Thus, the immune protection of this peptide can be explained by its activation of the interferon gamma production in T-cells.

  1. Differential requirements for Gli2 and Gli3 in the regional specification of the mouse hypothalamus

    PubMed Central

    Haddad-Tóvolli, Roberta; Paul, Fabian A.; Zhang, Yuanfeng; Zhou, Xunlei; Theil, Thomas; Puelles, Luis; Blaess, Sandra; Alvarez-Bolado, Gonzalo

    2015-01-01

    Secreted protein Sonic hedgehog (Shh) ventralizes the neural tube by modulating the crucial balance between activating and repressing functions (GliA, GliR) of transcription factors Gli2 and Gli3. This balance—the Shh-Gli code—is species- and context-dependent and has been elucidated for the mouse spinal cord. The hypothalamus, a forebrain region regulating vital functions like homeostasis and hormone secretion, shows dynamic and intricate Shh expression as well as complex regional differentiation. Here we asked if particular combinations of Gli2 and Gli3 and of GliA and GliR functions contribute to the variety of hypothalamic regions, i.e., we wanted to approach the question of a possible hypothalamic version of the Shh-Gli code. Based on mouse mutant analysis, we show that: (1) hypothalamic regional heterogeneity is based in part on differentially stringent requirements for Gli2 or Gli3; (2) another source of diversity are differential requirements for Shh of neural vs. non-neural origin; (3) the medial progenitor domain known to depend on Gli2 for its development generates several essential hypothalamic nuclei plus the pituitary and median eminence; (4) the suppression of Gli3R by neural and non-neural Shh is essential for hypothalamic specification. Finally, we have mapped our results on a recent model which considers the hypothalamus as a transverse region with alar and basal portions. Our data confirm the model and are explained by it. PMID:25859185

  2. Frequency and Clinicopathological Characteristics of Presenilin 1 Gly206Ala Mutation in Puerto Rican Hispanics with Dementia

    PubMed Central

    Arnold, Steven E.; Vega, Irving E.; Karlawish, Jason H.; WoIk, David A.; Nunez, Jessica; Negron, Mirna; Xie, Sharon X.; Wang, Li-San; Dubroff, Jacob G.; McCarty-Wood, Elisabeth; Trojanowski, John Q.; Van Deerlin, Vivianna

    2012-01-01

    The frequency and clinical and pathological characteristics associated with the Gly206Ala presenilin 1 (PSEN1) mutation in Puerto Rican and non-Puerto Rican Hispanics were evaluated at the University of Pennsylvania’s Alzheimer’s Disease Center. DNAs from all cohort subjects were genotyped for the Gly206Ala PSEN1 mutation. Carriers and non-carriers with neurodegenerative disease dementias were compared for demographic, clinical, psychometric, and biomarker variables. Nineteen (12.6%) of 151 unrelated subjects with dementia were discovered to carry the PSEN1 Gly 206Ala mutation. Microsatellite marker genotyping determined a common ancestral haplotype for all carriers. Carriers were all of Puerto Rican heritage with significantly younger age of onset, but otherwise were clinically and neuropsychologically comparable to those of non-carriers with AD. Three subjects had extensive topographic and biochemical biomarker assessments that were also typical of non-carriers with AD. Neuropathological examination in one subject revealed severe, widespread plaque and tangle pathology without other meaningful disease lesions. The PSEN1 Gly206Ala mutation is notably frequent in unrelated Puerto Rican immigrants with dementia in Philadelphia. Considered together with the increased prevalence and mortality of AD reported in Puerto Rico, these high rates may reflect hereditary risk concentrated in the island which warrants further study. PMID:23114514

  3. Diastereoselective DNA Cleavage Recognition by Ni(II)•Gly-Gly-His Derived Metallopeptides

    PubMed Central

    Fang, Ya-Yin; Claussen, Craig A.; Lipkowitz, Kenny B.; Long, Eric C.

    2008-01-01

    Site-selective DNA cleavage by diastereoisomers of Ni(II)•Gly-Gly-His-derived metallopeptides was investigated through high-resolution gel analyses and molecular dynamics simulations. Ni(II)•L-Arg-Gly-His and Ni(II)•D-Arg-Gly-His (and their respective Lys analogues) targeted A/T-rich regions; however, the L-isomers consistently modified a sub-set of available nucleotides within a given minor groove site while the D-isomers differed in both their sites of preference and ability to target individual nucleotides within some sites. In comparison, Ni(II)•L-Pro-Gly-His and Ni(II)•D-Pro-Gly-His were unable to exhibit a similar diastereoselectivity. Simulations of the above systems, along with Ni(II)•Gly-Gly-His, indicated that the stereochemistry of the amino-terminal amino acid produces either an isohelical metallopeptide that associates stably at individual DNA sites (L-Arg or L-Lys) or, with D-Arg and D-Lys, a non-complementary metallopeptide structure that cannot fully employ its side chain nor amino-terminal amine as a positional stabilizing moiety. In contrast, amino-terminal Pro-containing metallopeptides of either stereochemistry, lacking an extended side chain directed toward the minor groove, did not exhibit a similar diastereoselectivity. While the identity and stereochemistry of amino acids located in the amino-terminal peptide position influenced DNA cleavage, metallopeptide diastereoisomers containing L- and D-Arg (or Lys) within the second peptide position did not exhibit diastereoselective DNA cleavage patterns; simulations indicated that a positively-charged amino acid in this location alters the interaction of the metallopeptide equatorial plane and the minor groove leading to an interaction similar to Ni(II)•Gly-Gly-His. PMID:16522100

  4. ON THE RELATIONSHIPS OF SUBSTRATE ORIENTATION, HYDROGEN ABSTRACTION AND PRODUCT STEREOCHEMISTRY IN SINGLE AND DOUBLE DIOXYGENATIONS BY SOYBEAN LIPOXYGENASE-1 AND ITS ALA542GLY MUTANT*

    PubMed Central

    Coffa, Gianguido; Imber, Ann N.; Maguire, Brendan C.; Laxmikanthan, Gurunathan; Schneider, Claus; Gaffney, Betty J.; Brash, Alan R.

    2005-01-01

    Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-3H- and 11S-3H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective proS hydrogen abstraction from C-11 of the substrate (97–99% pro-S selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoyl-phosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S,15S- and 8S,15S-dihydroperoxides, a new product was formed and identified by HPLC, UV, GC-MS and NMR as 9R , 1 5 S -dihydroperoxy-eicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration “partner” of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site. PMID:16157595

  5. Pressure dependence of backbone chemical shifts in the model peptides Ac-Gly-Gly-Xxx-Ala-NH2.

    PubMed

    Erlach, Markus Beck; Koehler, Joerg; Crusca, Edson; Kremer, Werner; Munte, Claudia E; Kalbitzer, Hans Robert

    2016-06-01

    For a better understanding of nuclear magnetic resonance (NMR) detected pressure responses of folded as well as unstructured proteins the availability of data from well-defined model systems are indispensable. In this work we report the pressure dependence of chemical shifts of the backbone atoms (1)H(α), (13)C(α) and (13)C' in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH2 (Xxx one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of these nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The polynomial pressure coefficients B 1 and B 2 are dependent on the type of amino acid studied. The coefficients of a given nucleus show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure are also weakly correlated.

  6. Comparison of Allergenicity at Gly m 4 and Gly m Bd 30K of Soybean after Genetic Modification.

    PubMed

    Tsai, Jaw-Ji; Chang, Ching-Yun; Liao, En-Chih

    2017-02-15

    Despite rapid growth of genetically modified (GM) crops, effective evaluations of genetic modification on allergenicity are still lacking. Gly m Bd 30K is cross-reactive with cow's milk protein casein, Gly m 4, and with birch pollen allergen Bet v 1. Here we compared the allergenicity between GM and non-GM soybeans with respect to the foci Gly m 4 and Gly m Bd 30K. Recombinant allergens of Gly m Bd 30K and Gly m 4 were generated and polyclonal antibodies raised to identify these two allergenic components in soybeans. GM soybean was first PCR-confirmed using 35S promoter. A total of 20 soybeans (half GM, half non-GM) obtained from a food market were used to assess their allergenicity based on IgE-binding and histamine release. The concentrations of Gly m Bd 30K and Gly m 4 in soybeans were then determined. Most soybean-allergic patients (9 of 10) showed IgE-positive reactions to the allergen of 30 kDa in molecular weight. That allergen turned out to be Glycine max Gly m Bd 30K based on LC-MS/MS analyses. Gly m Bd 30K is therefore the major allergen in the soybean. An increase in the transcription of both the Gly m 4 (stress-induced protein SAM22) and Gly m Bd 28K (soybean allergen precursor) was found after genetic modification. The protein concentrations of Gly m 4 and Gly m Bd 30K were not statistically significant different between non-GM and GM soybeans. There were also no statistical significances between them in the tests of IgE binding and histamine release. In conclusion, soybeans showed similar concentrations of Gly m Bd 30K and Gly m 4 regardless of genetic modification or absence thereof. The allergenicity of both Gly m Bd 30K and Gly m 4 was therefore not altered after genetic modification. Patients showing hypersensitivity to soybeans and who had pre-existing allergy to birch pollen and cow's milk casein might not further increase their allergic reactions following exposures to the GM soybeans.

  7. Orchestration of Structural, Stereoelectronic, and Hydrogen-Bonding Effects in Stabilizing Triplexes from Engineered Chimeric Collagen Peptides (Pro(X)-Pro(Y)-Gly)6 Incorporating 4(R/S)-Aminoproline.

    PubMed

    Umashankara, Muddegowda; Sonar, Mahesh V; Bansode, Nitin D; Ganesh, Krishna N

    2015-09-04

    Collagens are an important family of structural proteins found in the extracellular matrix with triple helix as the characteristic structural motif. The collagen triplex is made of three left-handed polyproline II (PPII) helices with each PPII strand consisting of repetitive units of the tripeptide motif X-Y-Gly, where the amino acids X and Y are most commonly proline (Pro) and 4R-hydroxyproline (Hyp), respectively. A C4-endo pucker at X-site and C4-exo pucker at Y-site have been proposed to be the key for formation of triplex, and the nature of pucker is dependent on both the electronegativity and stereochemistry of the substituent. The present manuscript describes a new class of collagen analogues-chimeric cationic collagens-wherein both X- and Y-sites in collagen triad are simultaneously substituted by a combination of 4(R/S)-(OH/NH2/NH3(+)/NHCHO)-prolyl units and triplex stabilities measured at different pHs and in EG:H2O. Based on the results a model has been proposed with the premise that any factors which specifically favor the ring puckers of C4-endo at X-site and C4-exo at Y-site stabilize the PPII conformation and hence the derived triplexes. The pH-dependent triplex stability uniquely observed with ionizable 4-amino substituent on proline enables one to define the critical combination of factors C4-(exo/endo), intraresidue H-bonding, stereoelectronic (R/S) and n → π* interactions in dictating the triplex strength. The ionizable NH2 substituent at C4 in R/S configuration is thus a versatile probe for delineating the triplex stabilizing factors and the results have potential for designing of collagen analogues with customized properties for material and biological applications.

  8. Identification of the gene transcription repressor domain of Gli3.

    PubMed

    Tsanev, Robert; Tiigimägi, Piret; Michelson, Piret; Metsis, Madis; Østerlund, Torben; Kogerman, Priit

    2009-01-05

    Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N-terminal half. We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally. Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3. Unlike other mechanisms that inhibit Gli induced gene transcription, the repressor domain identified here does not utilize Histone deacetylases (HDACs) to achieve repression, as confirmed by HDAC inhibition studies and pull-down assays. This distinguishes the identified domain from other regulatory parts with negative influence on transcription.

  9. Glycine transporters GlyT1 and GlyT2 are differentially modulated by glycogen synthase kinase 3β.

    PubMed

    Jiménez, Esperanza; Núñez, Enrique; Ibáñez, Ignacio; Zafra, Francisco; Aragón, Carmen; Giménez, Cecilio

    2015-02-01

    Inhibitory glycinergic neurotransmission is terminated by the specific glycine transporters GlyT1 and GlyT2 which actively reuptake glycine from the synaptic cleft. GlyT1 is associated with both glycinergic and glutamatergic pathways, and is the main regulator of the glycine levels in the synapses. GlyT2 is the main supplier of glycine for vesicle refilling, a process that is vital to preserve the quantal glycine content in synaptic vesicles. Therefore, to control glycinergic neurotransmission efficiently, GlyT1 and GlyT2 activity must be regulated by diverse neuronal and glial signaling pathways. In this work, we have investigated the possible functional modulation of GlyT1 and GlyT2 by glycogen synthase kinase 3 (GSK3β). This kinase is involved in mood stabilization, neurodegeneration and plasticity at excitatory and inhibitory synapses. The co-expression of GSK3β with GlyT1 or GlyT2 in COS-7 cells and Xenopus laevis oocytes, leads to inhibition and stimulation of GlyT1 and GlyT2 activities, respectively, with a decrease of GlyT1, and an increase in GlyT2 levels at the plasma membrane. The specificity of these changes is supported by the antagonism exerted by a catalytically inactive form of the kinase and through inhibitors of GSK3β such as lithium chloride and TDZD-8. GSK3β also increases the incorporation of 32Pi into GlyT1 and decreases that of GlyT2. The pharmacological inhibition of the endogenous GSK3β in neuron cultures of brainstem and spinal cord leads to an opposite modulation of GlyT1 and GlyT2.Our results suggest that GSK3β is important for stabilizing and/or controlling the expression of functional GlyTs on the neural cell surface. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Gli function is essential for motor neuron induction in zebrafish.

    PubMed

    Vanderlaan, Gary; Tyurina, Oksana V; Karlstrom, Rolf O; Chandrasekhar, Anand

    2005-06-15

    The Gli family of zinc-finger transcription factors mediates Hedgehog (Hh) signaling in all vertebrates. However, their roles in ventral neural tube patterning, in particular motor neuron induction, appear to have diverged across species. For instance, cranial motor neurons are essentially lost in zebrafish detour (gli1(-)) mutants, whereas motor neuron development is unaffected in mouse single gli and some double gli knockouts. Interestingly, the expression of some Hh-regulated genes (ptc1, net1a, gli1) is mostly unaffected in the detour mutant hindbrain, suggesting that other Gli transcriptional activators may be involved. To better define the roles of the zebrafish gli genes in motor neuron induction and in Hh-regulated gene expression, we examined these processes in you-too (yot) mutants, which encode dominant repressor forms of Gli2 (Gli2(DR)), and following morpholino-mediated knockdown of gli1, gli2, and gli3 function. Motor neuron induction at all axial levels was reduced in yot (gli2(DR)) mutant embryos. In addition, Hh target gene expression at all axial levels except in rhombomere 4 was also reduced, suggesting an interference with the function of other Glis. Indeed, morpholino-mediated knockdown of Gli2(DR) protein in yot mutants led to a suppression of the defective motor neuron phenotype. However, gli2 knockdown in wild-type embryos generated no discernable motor neuron phenotype, while gli3 knockdown reduced motor neuron induction in the hindbrain and spinal cord. Significantly, gli2 or gli3 knockdown in detour (gli1(-)) mutants revealed roles for Gli2 and Gli3 activator functions in ptc1 expression and spinal motor neuron induction. Similarly, gli1 or gli3 knockdown in yot (gli2(DR)) mutants resulted in severe or complete loss of motor neurons, and of ptc1 and net1a expression, in the hindbrain and spinal cord. In addition, gli1 expression was greatly reduced in yot mutants following gli3, but not gli1, knockdown, suggesting that Gli3 activator

  11. Surface Chirality of Gly-Pro Dipeptide Adsorbed on a Cu(110) Surface.

    PubMed

    Cruguel, Hervé; Méthivier, Christophe; Pradier, Claire-Marie; Humblot, Vincent

    2015-07-01

    The adsorption of chiral Gly-Pro dipeptide on Cu(110) has been characterized by combining in situ polarization modulation infrared reflection absorption spectroscopy (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS). The chemical state of the dipeptide, and its anchoring points and adsorption geometry, were determined at various coverage values. Gly-Pro molecules are present on Cu(110) in their anionic form (NH2 /COO(-)) and adsorb under a 3-point binding via both oxygen atoms of the carboxylate group and via the nitrogen atom of the amine group. Low-energy electron diffraction (LEED) and scanning tunneling microscopy (STM) have shown the presence of an extended 2D chiral array, sustained via intermolecular H-bonds interactions. Furthermore, due to the particular shape of the molecule, only one homochiral domain is formed, creating thus a truly chiral surface. © 2015 Wiley Periodicals, Inc.

  12. Antithrombotic Protective Effects of Arg-Pro-Gly-Pro Peptide during Emotional Stress Provoked by Forced Swimming Test in Rats.

    PubMed

    Grigor'eva, M E; Lyapina, L A

    2017-01-01

    Blood coagulation was enhanced and all factors (total, enzyme, and non-enzyme) of the fibrinolytic system were suppressed in rats in 60 min after forced swimming test. Argininecontaining tetrapeptide glyproline Arg-Pro-Gly-Pro administered prior to this test activated fibrinolysis and prevented hypercoagulation. Administration of this peptide in 5 min after swimming test also enhanced anticoagulant, fibrinolytic, and antithrombotic activity of the blood. Therefore, glyproline Arg-Pro-Gly-Pro exerted both preventive and curative effects on the hemostasis system and prevented enhancement of blood coagulation provoked by emotional stress modeled by forced swimming test.

  13. Collagen Gly missense mutations: Effect of residue identity on collagen structure and integrin binding.

    PubMed

    Qiu, Yimin; Mekkat, Arya; Yu, Hongtao; Yigit, Sezin; Hamaia, Samir; Farndale, Richard W; Kaplan, David L; Lin, Yu-Shan; Brodsky, Barbara

    2018-05-11

    Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa) n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502 GFPGER 507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI. Copyright © 2018. Published by Elsevier Inc.

  14. The Structure of the Elusive Simplest Dipeptide Gly-Gly.

    PubMed

    Cabezas, Carlos; Varela, Marcelino; Alonso, José L

    2017-06-01

    Among the hundreds of peptide compounds for which conformations have been determined by using different spectroscopic techniques, the structure of the simplest dipeptide glycylglycine (Gly-Gly) is conspicuously absent. Herein, for the first time, solid samples of Gly-Gly have been vaporized by laser ablation and three different structures have been revealed in a supersonic expansion by Fourier transform microwave spectroscopy. The intramolecular hydrogen bonding interactions that stabilize the observed forms have been established based on the 14 N nuclear quadrupole hyperfine structure. We have illustrated how conformer interconversion distorts the equilibrium conformational distribution, giving rise to missing conformers in the conformational landscape. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Normal exon copy number of the GLI2 and GLI3 genes in patients with esophageal atresia.

    PubMed

    Bednarczyk, D; Smigiel, R; Patkowski, D; Laczmanska, I; Lebioda, A; Laczmanski, L; Sasiadek, M M

    2013-01-01

    Esophageal atresia (EA) is a congenital developmental defect of the alimentary tract concerning the interruption of the esophagus with or without connection to the trachea. The incidence of EA is 1 in 3000-3500 of live-born infants, and occurs in both isolated and syndromic (in combination with abnormalities in other organ systems) forms. The molecular mechanisms underlying the development of EA are poorly understood. Knockout studies in mice indicate that genes like Sonic hedgehog, Gli2, and Gli3 play a role in the etiology of EA. These facts led us to hypothesize that Sonic hedgehog-GLI gene rearrangements are associated with EA in humans. To test this hypothesis, we screened patients with isolated and syndromic EA for GLI2 and/or GLI3 microrearrangements using methods to estimate the copy number (Multiplex Ligation-dependent Probe Amplification, real-time polymerase chain reaction). To our best knowledge this is the first study assessing copy number of GLI2 and GLI3 genes in patients with EA. © 2013 Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.

  16. Surface-enhanced Raman spectrum of Gly-Gly adsorbed on the silver colloidal surface

    NASA Astrophysics Data System (ADS)

    Xiaojuan, Yuan; Huaimin, Gu; Jiwei, Wu

    2010-08-01

    Raman and SERS spectra of homodipeptide Gly-Gly and Gly were recorded and compared in this paper, and band assignment for the functional groups contained in these molecules was analyzed in detail. Time-dependent and pH-dependent SERS spectra of Gly-Gly molecule adsorbed on nano-colloidal silver surface were also studied. The time-dependent SERS spectra of Gly-Gly are characterized by the increase in intensity of bands primarily representing the vibrational signatures emanating from the amino and amide moiety of Gly-Gly molecule. It is found that the adsorption style of Gly-Gly on the silver colloid changes as time goes on; at 5 min after adding the sample to the silver colloid, Gly-Gly adsorbs on silver surface firstly through the carboxylate, amino and amide groups, and then the carboxylate group is far away from the silver surface at 10 min to 3 days. The SERS variation of Gly-Gly with the change of pH suggests that the adsorption style is pH-dependent, the different adsorption behavior of the Gly-Gly occurs on silver surface at different pH values.

  17. Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2.

    PubMed

    Subramanian, Nandhitha; Scopelitti, Amanda J; Carland, Jane E; Ryan, Renae M; O'Mara, Megan L; Vandenberg, Robert J

    2016-01-01

    The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1' site. We hypothesized that the third Na+ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na+ placed at the location consistent with the Na1' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.

  18. Lipid solvation effects contribute to the affinity of Gly-xxx-Gly motif-mediated helix-helix interactions.

    PubMed

    Johnson, Rachel M; Rath, Arianna; Melnyk, Roman A; Deber, Charles M

    2006-07-18

    Interactions between transmembrane helices are mediated by the concave Gly-xxx-Gly motif surface. Whether Gly residues per se are sufficient for selection of this motif has not been established. Here, we used the in vivo TOXCAT assay to measure the relative affinities of all 18 combinations of Gly, Ala, and Ser "small-xxx-small" mutations in glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP) homodimers. Affinity values were compared with the accessibility to a methylene-sized probe of the total surface area of each helix monomer as a measure of solvation by membrane components. A strong inverse correlation was found between nonpolar-group lipid accessibility and dimer affinity (R = 0.75 for GpA, p = 0.013, and R = 0.81 for MCP, p = 0.004), suggesting that lipid as a poor membrane protein solvent, conceptually analogous to water in soluble protein folding, can contribute to dimer stability and help to define helix-helix interfaces.

  19. DNA-fiber EPR investigation of the influence of amino-terminal residue stereochemistry on the DNA binding orientation of Cu(II)•Gly-Gly-His-derived metallopeptides

    PubMed Central

    Hamada, Hirokazu; Abe, Yuko; Nagane, Ryoichi; Fang, Ya-Yin; Lewis, Mark A.; Long, Eric C.; Chikira, Makoto

    2007-01-01

    DNA fiber EPR was used to investigate the DNA binding stabilities and orientations of Cu(II)•Gly-Gly-His-derived metallopeptides containing d- vs. l-amino acid substitutions in the first peptide position. This examination included studies of Cu(II)•d-Arg-Gly-His and Cu(II)•d-Lys-Gly-His for comparison to metallopeptides containing l-Arg/Lys substitutions, and also the diastereoisomeric pairs Cu(II)•d/l-Pro-Gly-His and Cu(II)•d/l-Pro-Lys-His. Results indicated that l-Arg/Lys to d-Arg/Lys substitutions considerably randomized the orientation of the metallopeptides on DNA whereas the replacement of l-Pro by d-Pro in Cu(II)•l-Pro-Gly-His caused a decrease in randomness. The difference in the extent of randomness of d- vs. l-Pro-Gly-His complexes was diminished through the substitution of Gly for Lys in the middle peptide position, supporting the notion that the ε-amino group of Lys triggered further randomization, likely through hydrogen bonding or electrostatic interactions that disrupt binding of the metallopeptide equatorial plane and the DNA. The relationship between the stereochemistry of amino acid residues and the binding and reaction of M(II)•Xaa-Xaa’-His metallopeptides with DNA are also discussed. PMID:17706784

  20. Sall4-Gli3 system in early limb progenitors is essential for the development of limb skeletal elements.

    PubMed

    Akiyama, Ryutaro; Kawakami, Hiroko; Wong, Julia; Oishi, Isao; Nishinakamura, Ryuichi; Kawakami, Yasuhiko

    2015-04-21

    Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed. Here we show that the zinc finger factors Sall4 and Gli3 cooperate for proper development of the anterior-proximal skeletal elements and also function upstream of Shh-dependent posterior skeletal element development. Conditional inactivation of Sall4 in the mesoderm before limb outgrowth caused severe defects in the anterior-proximal skeletal elements in the hindlimb. We found that Gli3 expression is reduced in Sall4 mutant hindlimbs, but not in forelimbs. This reduction caused posteriorization of nascent hindlimb buds, which is correlated with a loss of anterior digits. In proximal development, Sall4 integrates Gli3 and the Plzf-Hox system, in addition to proliferative expansion of cells in the mesenchymal core of nascent hindlimb buds. Whereas forelimbs developed normally in Sall4 mutants, further genetic analysis identified that the Sall4-Gli3 system is a common regulator of the early limb progenitor cells in both forelimbs and hindlimbs. The Sall4-Gli3 system also functions upstream of the Shh-expressing ZPA and the Fgf8-expressing AER in fore- and hindlimbs. Therefore, our study identified a critical role of the Sall4-Gli3 system at the early steps of limb development for proper development of the appendicular skeletal elements.

  1. Novel sodium channel inhibitor from Conus geographus: purification, structure, and pharmacological properties

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yanagawa, Y.; Abe, T.; Satake, M.

    1988-08-23

    A novel toxin, tentatively named conotoxin GS (CGS), has been isolated form a marine snail, Conus geographus. CGS was found to exist as a single polypeptide chain, consisting of 34 amino acid residues, cross-linked by three disulfide bonds. Its amino acid sequence was shown to be Ala-Cys-Ser-Gly-Arg-Gly-Ser-Arg-Cys-Hyp-Hyp-Gln-Cys-Cys-Met-Gly-Leu-Arg-Cys-Gly-Arg-Gly-Asn-Pro-Gln-Lys-Cys-Ile-Gly-Ala-His-Gla-Asp-Val. In competition experiments, CGS inhibited the bindings of (/sup 3/H)Lys-tetrodotoxin ((/sup 3/H)Lys-TTX) and (/sup 3/H)propionylconotoxin GIIIA to Electrophorus electricus electroplax membranes, with K/sub i/ values of 34 nM and 24 nM, respectively. The toxin inhibited the binding of (/sup 3/H)Lys-TTX (1 nM) to rat skeletal muscle homogenates with an IC/sub 50/ value ofmore » 880 nM but showed very little effect on this binding to the rat brain P/sub 2/ fraction at 10 ..mu..M. These binding studies indicate that CGS belongs to the same group of Na channel inhibitors as TTX, STX (saxitoxin), and ..mu..-conotoxins. Although CGS, like the ..mu..-conotoxins, is a pharmacological probe for distinguishing between neuronal and muscle Na channel subtypes, the homology in the sequences of CGS and ..mu..-conotoxins is very limited.« less

  2. Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2

    PubMed Central

    Subramanian, Nandhitha; Scopelitti, Amanda J.; Carland, Jane E.; Ryan, Renae M.; O’Mara, Megan L.; Vandenberg, Robert J.

    2016-01-01

    The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1' site. We hypothesized that the third Na+ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na+ placed at the location consistent with the Na1' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10. PMID:27337045

  3. [Effects of hypophyseal Lys-Glu-Asp-Gly and Ala-Glu-Asp-Gly synthetic peptides on immunity, hemostasis, morphology and functions of the thyroid gland in neonatally hypophysectomized chicken and one-year-old birds].

    PubMed

    Kuznik, B I; Pateiuk, A V; Rusaeva, N S; Baranchugova, L M; Obydenko, V I

    2010-01-01

    Neonatal hypophysectomy in chicken produces enlarged follicles of the thyroid gland, accumulation of colloids, impressed follicular epithelium, increased nucleus-cytoplasm ratio in thyrocytes, atrophied inter-follicular epithelium, depressed immunity, development of hypercoagulation and depressed fibrinolysis. When hypophysectomy is performed in one-year-old birds the impairments developing in thyroid morphology, immunity and hemostasis are less pronounced. Peptides of the anterior (Lys-Glu-Asp-Gly) and posterior (Ala-Glu-Asp-Gly) thyroid lobes injected to hypophysectomized birds prevent atrophic changes of the thyroid gland, normalize immune and hemostatic parameters.

  4. Characterization of serine hydroxymethyltransferase GlyA as a potential source of D-alanine in Chlamydia pneumoniae

    PubMed Central

    De Benedetti, Stefania; Bühl, Henrike; Gaballah, Ahmed; Klöckner, Anna; Otten, Christian; Schneider, Tanja; Sahl, Hans-Georg; Henrichfreise, Beate

    2014-01-01

    For intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin. PMID:24616885

  5. Characterization of serine hydroxymethyltransferase GlyA as a potential source of D-alanine in Chlamydia pneumoniae.

    PubMed

    De Benedetti, Stefania; Bühl, Henrike; Gaballah, Ahmed; Klöckner, Anna; Otten, Christian; Schneider, Tanja; Sahl, Hans-Georg; Henrichfreise, Beate

    2014-01-01

    For intracellular Chlamydiaceae, there is no need to withstand osmotic challenges, and a functional cell wall has not been detected in these pathogens so far. Nevertheless, penicillin inhibits cell division in Chlamydiaceae resulting in enlarged aberrant bodies, a phenomenon known as chlamydial anomaly. D-alanine is a unique and essential component in the biosynthesis of bacterial cell walls. In free-living bacteria like Escherichia coli, penicillin-binding proteins such as monofunctional transpeptidases PBP2 and PBP3, the putative targets of penicillin in Chlamydiaceae, cross-link adjacent peptidoglycan strands via meso-diaminopimelic acid and D-Ala-D-Ala moieties of pentapeptide side chains. In the absence of genes coding for alanine racemase Alr and DadX homologs, the source of D-Ala and thus the presence of substrates for PBP2 and PBP3 activity in Chlamydiaceae has puzzled researchers for years. Interestingly, Chlamydiaceae genomes encode GlyA, a serine hydroxymethyltransferase that has been shown to exhibit slow racemization of D- and L-alanine as a side reaction in E. coli. We show that GlyA from Chlamydia pneumoniae can serve as a source of D-Ala. GlyA partially reversed the D-Ala auxotrophic phenotype of an E. coli racemase double mutant. Moreover, purified chlamydial GlyA had racemase activity on L-Ala in vitro and was inhibited by D-cycloserine, identifying GlyA, besides D-Ala ligase MurC/Ddl, as an additional target of this competitive inhibitor in Chlamydiaceae. Proof of D-Ala biosynthesis in Chlamydiaceae helps to clarify the structure of cell wall precursor lipid II and the role of chlamydial penicillin-binding proteins in the development of non-dividing aberrant chlamydial bodies and persistence in the presence of penicillin.

  6. Characterization of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands to thermolysin.

    PubMed

    Yasukawa, Kiyoshi; Kusano, Masayuki; Nakamura, Koji; Inouye, Kuniyo

    2006-04-01

    In this study, glycyl-D-phenylalanine (Gly-D-Phe), glycyl-L-leucine (Gly-L-Leu), and D-phenylalanine (D-Phe) were characterized for their abilities as affinity ligands to thermolysin. Each of the ligands was immobilized to the resin. The optimum pH for adsorption of thermolysin is 5.0-6.0 for each of the ligands. By the affinity column chromatography in which 2mg thermolysin was applied onto 4 ml volume of the resins at pH 5.5, the adsorption ratios based on casein hydrolysis activity were 100% for each of the ligands. However, the adsorption ratios of the resins containing Gly-L-Leu and D-Phe, unlike that of Gly-D-Phe, were progressively decreased with increasing the amounts of thermolysin applied to the column. Measurement of adsorption isotherms showed that the association constant to thermolysin at pH 5.5 of the resins containing Gly-D-Phe was (3.3+/-0.8)x10(5)M(-1), while those of Gly-L-Leu and D-Phe were approximately ten times less. This result is coincident with the observations of performances in affinity column chromatography. On the other hand, maximum thermolysin binding capacities were almost the same among the resins examined. These results indicate that Gly-D-Phe is more suitable than Gly-L-Leu and D-Phe as an affinity ligand for purification of thermolysin.

  7. New insights into genotype–phenotype correlation for GLI3 mutations

    PubMed Central

    Démurger, Florence; Ichkou, Amale; Mougou-Zerelli, Soumaya; Le Merrer, Martine; Goudefroye, Géraldine; Delezoide, Anne-Lise; Quélin, Chloé; Manouvrier, Sylvie; Baujat, Geneviève; Fradin, Mélanie; Pasquier, Laurent; Megarbané, André; Faivre, Laurence; Baumann, Clarisse; Nampoothiri, Sheela; Roume, Joëlle; Isidor, Bertrand; Lacombe, Didier; Delrue, Marie-Ange; Mercier, Sandra; Philip, Nicole; Schaefer, Elise; Holder, Muriel; Krause, Amanda; Laffargue, Fanny; Sinico, Martine; Amram, Daniel; André, Gwenaelle; Liquier, Alain; Rossi, Massimiliano; Amiel, Jeanne; Giuliano, Fabienne; Boute, Odile; Dieux-Coeslier, Anne; Jacquemont, Marie-Line; Afenjar, Alexandra; Van Maldergem, Lionel; Lackmy-Port-Lis, Marylin; Vincent- Delorme, Catherine; Chauvet, Marie-Liesse; Cormier-Daire, Valérie; Devisme, Louise; Geneviève, David; Munnich, Arnold; Viot, Géraldine; Raoul, Odile; Romana, Serge; Gonzales, Marie; Encha-Razavi, Ferechte; Odent, Sylvie; Vekemans, Michel; Attie-Bitach, Tania

    2015-01-01

    The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister–Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype–phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype–phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues. PMID:24736735

  8. New insights into genotype-phenotype correlation for GLI3 mutations.

    PubMed

    Démurger, Florence; Ichkou, Amale; Mougou-Zerelli, Soumaya; Le Merrer, Martine; Goudefroye, Géraldine; Delezoide, Anne-Lise; Quélin, Chloé; Manouvrier, Sylvie; Baujat, Geneviève; Fradin, Mélanie; Pasquier, Laurent; Megarbané, André; Faivre, Laurence; Baumann, Clarisse; Nampoothiri, Sheela; Roume, Joëlle; Isidor, Bertrand; Lacombe, Didier; Delrue, Marie-Ange; Mercier, Sandra; Philip, Nicole; Schaefer, Elise; Holder, Muriel; Krause, Amanda; Laffargue, Fanny; Sinico, Martine; Amram, Daniel; André, Gwenaelle; Liquier, Alain; Rossi, Massimiliano; Amiel, Jeanne; Giuliano, Fabienne; Boute, Odile; Dieux-Coeslier, Anne; Jacquemont, Marie-Line; Afenjar, Alexandra; Van Maldergem, Lionel; Lackmy-Port-Lis, Marylin; Vincent-Delorme, Catherine; Chauvet, Marie-Liesse; Cormier-Daire, Valérie; Devisme, Louise; Geneviève, David; Munnich, Arnold; Viot, Géraldine; Raoul, Odile; Romana, Serge; Gonzales, Marie; Encha-Razavi, Ferechte; Odent, Sylvie; Vekemans, Michel; Attie-Bitach, Tania

    2015-01-01

    The phenotypic spectrum of GLI3 mutations includes autosomal dominant Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS). PHS was first described as a lethal condition associating hypothalamic hamartoma, postaxial or central polydactyly, anal atresia and bifid epiglottis. Typical GCPS combines polysyndactyly of hands and feet and craniofacial features. Genotype-phenotype correlations have been found both for the location and the nature of GLI3 mutations, highlighting the bifunctional nature of GLI3 during development. Here we report on the molecular and clinical study of 76 cases from 55 families with either a GLI3 mutation (49 GCPS and 21 PHS), or a large deletion encompassing the GLI3 gene (6 GCPS cases). Most of mutations are novel and consistent with the previously reported genotype-phenotype correlation. Our results also show a correlation between the location of the mutation and abnormal corpus callosum observed in some patients with GCPS. Fetal PHS observations emphasize on the possible lethality of GLI3 mutations and extend the phenotypic spectrum of malformations such as agnathia and reductional limbs defects. GLI3 expression studied by in situ hybridization during human development confirms its early expression in target tissues.

  9. Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Seung Heon; Kang, Sukmo; Dong, Mi Sook

    2015-06-15

    Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E.more » coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. - Highlights: • Ala62 is located in α-helix A of the carcinogen-metabolizing enzyme CYP1A1. • Pro acts as an α-helix breaker. • A variant protein of CYP1A1, Ala62Pro, had lower heme content than the wild-type. • The variant of CYP1A1 had lower enzyme activities than the wild-type.« less

  10. The alpha-adducin Gly460Trp polymorphism and the antihypertensive effects of exercise among men with high blood pressure.

    PubMed

    Pescatello, Linda S; Blanchard, Bruce E; Tsongalis, Gregory J; Maresh, Carl M; O'Connell, Ann; Thompson, Paul D

    2007-09-01

    The alpha-adducin Gly460Trp polymorphism alters renal sodium transport and is associated with hypertension. Despite the immediate sodium- and volume-depleting effects of aerobic exercise, the influence of the alpha-adducin Gly460Trp polymorphism on PEH (postexercise hypotension) has not been studied. In the present study we examined the effects of the alpha-adducin Gly460Trp polymorphism on PEH among 48 men (42.6+/-1.6 years; mean+/-S.E.M.) with high BP (blood pressure; 144.0+/-1.7/84.7+/-1.1 mmHg). Subjects completed three experiments: non-exercise control and two cycle exercise sessions at 40% (light exercise) and 60% (moderate exercise) of maximal oxygen consumption. Subjects left the laboratory wearing an ambulatory BP monitor. PCR and restriction enzyme digestion determined the genotypes. No subjects had the Trp460Trp genotype due to the low frequency of 5% in the population. Repeated measure ANCOVA tested whether BP differed over time between experimental conditions and genotypes (Gly460Gly, n=36; Gly460Trp, n=12). Among Gly460Gly genotypes, SBP (systolic BP) was reduced by 5.2+/-1.4 mmHg after moderate exercise compared with non-exercise controls over 9 h (P<0.01). Among Gly460Trp genotypes, SBP was lowered by 7.8+/-2.3 mmHg; after light exercise compared with non-exercise controls over 9 h (P<0.05). The SBP reductions after light exercise (0.6+/-1.3 compared with 7.8+/-2.3 mmHg; P<0.05) but not moderate exercise (5.2+/-1.4 compared with 3.8+/-2.4 mmHg; P> or =0.05) differed between the Gly460Gly and Gly460Trp genotypes respectively. Men with Gly460Gly had a reduced SBP after moderate exercise, whereas men with Gly460Trp had a reduced SBP after light exercise. However, only the SBP reductions after light exercise differed between genotypes. Our findings indicate that the alpha-adducin Gly460Trp genotype may be useful in identifying men who have a reduced BP after lower intensity aerobic exercise.

  11. Gli3 Regulation of Myogenesis Is Necessary for Ischemia-Induced Angiogenesis

    PubMed Central

    Renault, Marie-Ange; Vandierdonck, Soizic; Chapouly, Candice; Yu, Yang; Qin, Gangjian; Metras, Alexandre; Couffinhal, Thierry; Losordo, Douglas W.; Yao, Qinyu; Reynaud, Annabel; Jaspard-Vinassa, Béatrice; Belloc, Isabelle; Desgranges, Claude; Gadeau, Alain-Pierre

    2015-01-01

    Rationale A better understanding of the mechanism underlying skeletal muscle repair is required to develop therapies that promote tissue regeneration in adults. Hedgehog signaling has been shown previously to be involved in myogenesis and angiogenesis: 2 crucial processes for muscle development and regeneration. Objective The objective of this study was to identify the role of the hedgehog transcription factor Gli3 in the crosstalk between angiogenesis and myogenesis in adults. Methods and Results Using conditional knockout mice, we found that Gli3 deficiency in endothelial cells did not affect ischemic muscle repair, whereas in myocytes, Gli3 deficiency resulted in severely delayed ischemia-induced myogenesis. Moreover, angiogenesis was also significantly impaired in HSA-CreERT2; Gli3Flox/Flox mice, demonstrating that impaired myogenesis indirectly affects ischemia-induced angiogenesis. The role of Gli3 in myocytes was then further investigated. We found that Gli3 promotes myoblast differentiation through myogenic factor 5 regulation. In addition, we found that Gli3 regulates several proangiogenic factors, including thymidine phosphorylase and angiopoietin-1 both in vitro and in vivo, which indirectly promote endothelial cell proliferation and arteriole formation. In addition, we found that Gli3 is upregulated in proliferating myoblasts by the cell cycle–associated transcription factor E2F1. Conclusions This study shows for the first time that Gli3-regulated postnatal myogenesis is necessary for muscle repair–associated angiogenesis. Most importantly, it implies that myogenesis drives angiogenesis in the setting of skeletal muscle repair and identifies Gli3 as a potential target for regenerative medicine. PMID:24044950

  12. Gli3 is a negative regulator of Tas1r3-expressing taste cells

    PubMed Central

    Jyotaki, Masafumi; Redding, Kevin; Jiang, Peihua

    2018-01-01

    Mouse taste receptor cells survive from 3–24 days, necessitating their regeneration throughout adulthood. In anterior tongue, sonic hedgehog (SHH), released by a subpopulation of basal taste cells, regulates transcription factors Gli2 and Gli3 in stem cells to control taste cell regeneration. Using single-cell RNA-Seq we found that Gli3 is highly expressed in Tas1r3-expressing taste receptor cells and Lgr5+ taste stem cells in posterior tongue. By PCR and immunohistochemistry we found that Gli3 was expressed in taste buds in all taste fields. Conditional knockout mice lacking Gli3 in the posterior tongue (Gli3CKO) had larger taste buds containing more taste cells than did control wild-type (Gli3WT) mice. In comparison to wild-type mice, Gli3CKO mice had more Lgr5+ and Tas1r3+ cells, but fewer type III cells. Similar changes were observed ex vivo in Gli3CKO taste organoids cultured from Lgr5+ taste stem cells. Further, the expression of several taste marker and Gli3 target genes was altered in Gli3CKO mice and/or organoids. Mirroring these changes, Gli3CKO mice had increased lick responses to sweet and umami stimuli, decreased lick responses to bitter and sour taste stimuli, and increased glossopharyngeal taste nerve responses to sweet and bitter compounds. Our results indicate that Gli3 is a suppressor of stem cell proliferation that affects the number and function of mature taste cells, especially Tas1r3+ cells, in adult posterior tongue. Our findings shed light on the role of the Shh pathway in adult taste cell regeneration and may help devise strategies for treating taste distortions from chemotherapy and aging. PMID:29415007

  13. Time-dependent, bidirectional, anti- and pro-spinal hyper-reflexia and muscle spasticity effect after chronic spinal glycine transporter 2 (GlyT2) oligonucleotide-induced downregulation.

    PubMed

    Kamizato, Kota; Marsala, Silvia; Navarro, Michael; Kakinohana, Manabu; Platoshyn, Oleksandr; Yoshizumi, Tetsuya; Lukacova, Nadezda; Wancewicz, Ed; Powers, Berit; Mazur, Curt; Marsala, Martin

    2018-07-01

    The loss of local spinal glycine-ergic tone has been postulated as one of the mechanisms contributing to the development of spinal injury-induced spasticity. In our present study using a model of spinal transection-induced muscle spasticity, we characterize the effect of spinally-targeted GlyT2 downregulation once initiated at chronic stages after induction of spasticity in rats. In animals with identified hyper-reflexia, the anti-spasticity effect was studied after intrathecal treatment with: i) glycine, ii) GlyT2 inhibitor (ALX 1393), and iii) GlyT2 antisense oligonucleotide (GlyT2-ASO). Administration of glycine and GlyT2 inhibitor led to significant suppression of spasticity lasting for a minimum of 45-60 min. Treatment with GlyT2-ASO led to progressive suppression of muscle spasticity seen at 2-3 weeks after treatment. Over the subsequent 4-12 weeks, however, the gradual appearance of profound spinal hyper-reflexia was seen. This was presented as spontaneous or slight-tactile stimulus-evoked muscle oscillations in the hind limbs (but not in upper limbs) with individual hyper-reflexive episodes lasting between 3 and 5 min. Chronic hyper-reflexia induced by GlyT2-ASO treatment was effectively blocked by intrathecal glycine. Immunofluorescence staining and Q-PCR analysis of the lumbar spinal cord region showed a significant (>90%) decrease in GlyT2 mRNA and GlyT2 protein. These data demonstrate that spinal GlyT2 downregulation provides only a time-limited therapeutic benefit and that subsequent loss of glycine vesicular synthesis resulting from chronic GlyT2 downregulation near completely eliminates the tonic glycine-ergic activity and is functionally expressed as profound spinal hyper-reflexia. These characteristics also suggest that chronic spinal GlyT2 silencing may be associated with pro-nociceptive activity. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Radiation chemical studies of Gly-Met-Gly in aqueous solution

    DOE PAGES

    Barata-Vallejo, Sebastian; Ferreri, Carla; Zhang, Tao; ...

    2016-10-25

    Important biological consequences are related to the reaction of HO radicals with methionine (Met). Several fundamental aspects remain to be defined when Met is an amino acid residue incorporated in the interior of peptides and proteins. The present study focuses on Gly-Met-Gly, the simplest peptide where Met is not a terminal residue. The reactions of HO with Gly-Met-Gly and its N-acetyl derivative were studied by pulse radiolysis technique. The transient absorption spectra were resolved into contributions from specific components of radical intermediates. Moreover, a detailed product analysis is provided for the first time for Met-containing peptides in radiolytic studies tomore » support the mechanistic proposal. By parallel radiolytical and electrochemical reactions and consequent product identification, the formation of sulfoxide attributed to the direct HO radical attack on the sulfide functionality of the Met residue could be excluded, with the in situ generated hydrogen peroxide responsible for this oxidation. LC–MS and high resolution MS/MS were powerful analytical tools to envisage the structures of five products, thus allowing to complete the mechanistic picture of the overall Met-containing peptide reactivity.« less

  15. Oleoyl-L-carnitine inhibits glycine transport by GlyT2

    PubMed Central

    Carland, JE; Mansfield, RE; Ryan, RM; Vandenberg, RJ

    2013-01-01

    Background and Purpose Concentrations of extracellular glycine in the CNS are regulated by two Na+/Cl–-dependent glycine transporters, GlyT1 and GlyT2. Selective inhibitors of GlyT1 have been developed for the treatment of schizophrenia, whilst selective inhibitors of GlyT2 are analgesic in animal models of pain. We have assessed a series of endogenous lipids as inhibitors of GlyT1 and GlyT2. Experimental Approach Human GlyT1 and GlyT2 were expressed in Xenopus laevis oocytes, and the inhibitory actions of a series of acylcarnitines on glycine transport were measured using electrophysiological techniques. Key Results Oleoyl-l-carnitine inhibited glycine transport by GlyT2, with an IC50 of 340 nM, which is 15-fold more potent than the previously identified lipid inhibitor N-arachidonyl-glycine. Oleoyl-l-carnitine had a slow onset of inhibition and a slow washout. Using a series of chimeric GlyT1/2 transporters and point mutant transporters, we have identified an isoleucine residue in extracellular loop 4 of GlyT2 that conferred differences in sensitivity to oleoyl-l-carnitine between GlyT2 and GlyT1. Conclusions and Implications Oleoyl-l-carnitine is a potent non-competitive inhibitor of GlyT2. Previously identified GlyT2 inhibitors show potential as analgesics and the identification of oleoyl-l-carnitine as a novel GlyT2 inhibitor may lead to new ways of treating pain. PMID:22978602

  16. Effects of polarizability on the structural and thermodynamics properties of [C{sub n}mim][Gly] ionic liquids (n = 1–4) using EEM/MM molecular dynamic simulations

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wu, Yang; Hu, Na; Yue, Lili

    2015-02-14

    An extended electronegativity equalization method/molecular mechanics (EEM/MM) model for ionic liquids is used to investigate the structures and properties of 1-alkyl-3-methylimidazolium glycine ionic liquids [C{sub n}mim][Gly] (n = 1–4) with alkyl substituents of different lengths. The EEM/MM model describes the electrostatic interactions of atoms and their changes in different ambient environments. This property is the most outstanding characteristic of the model. EEM parameters (i.e., valence electronegativities and valence hardness parameters) are calibrated using linear regression and least-squares methods, which can accurately predict the gas-phase properties of [C{sub n}mim]{sup +}, [Gly]{sup −}, and [C{sub n}mim][Gly] ion pairs. We utilize the EEM/MMmore » force field to systematically investigate the effects of polarizability on the accuracy of [C{sub n}mim][Gly] properties predicted through the molecular dynamic simulations. EEM/MM explicitly describes the atom-based polarizability of [C{sub n}mim][Gly]; thus, the densities, enthalpies of vaporization, self-diffusion coefficients, and conductivities of the [C{sub n}mim][Gly] are consistent with the experimental values. The calculated radial distribution functions provide a mechanistic understanding of the effects of polarizability on ionic aggregations in amino acid ionic liquids. The effects of alkyl chain length on the diffusion coefficient and conductivity are also discussed.« less

  17. An antibody reactive to the Gly63–Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding

    PubMed Central

    Lee, Yujean; Kim, Hyori; Chung, Junho

    2014-01-01

    The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63–Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63–Lys68 of NT-proBNP exhibits O-glycosylation-independent binding. PMID:25236766

  18. The 482Ser of PPARGC1A and 12Pro of PPARG2 Alleles Are Associated with Reduction of Metabolic Risk Factors Even Obesity in a Mexican-Mestizo Population.

    PubMed

    Vázquez-Del Mercado, Mónica; Guzmán-Ornelas, Milton-Omar; Corona Meraz, Fernanda-Isadora; Ríos-Ibarra, Clara-Patricia; Reyes-Serratos, Eduardo-Alejandro; Castro-Albarran, Jorge; Ruíz-Quezada, Sandra-Luz; Navarro-Hernández, Rosa-Elena

    2015-01-01

    The aim of this study was to investigate the relationship between functional polymorphisms Gly482Ser in PPARGC1A and Pro12Ala in PPARG2 with the presence of obesity and metabolic risk factors. We included 375 individuals characterized as Mexican-Mestizos and classified by the body mass index (BMI). Body dimensions and distribution of body fat were measured. The HOMA-IR and adiposity indexes were calculated. Adipokines and metabolic profile quantification were performed by ELISA and routine methods. Genetic polymorphisms were determined by polymerase chain reaction restriction fragment length polymorphism analysis. A difference between obese and nonobese subjects in polymorphism PPARGC1A distribution was observed. Among obese individuals, carriers of genotype 482Gly/Gly were observed to have decreased body fat, BMI, and body fat ratio versus 482Ser/Ser carriers and increased resistin and leptin levels in carriers Gly+ phenotype versus Gly- phenotype. Subjects with PPARG2 Ala- phenotype (genotype 12Pro/Pro) showed a decreased HOMA-IR index versus individuals with Ala+ phenotype (genotypes 12Pro/Ala plus 12Ala/Ala). We propose that, in obese Mexican-Mestizos, the combination of alleles 482Ser in PPARGC1A and 12Pro in PPARG2 represents a reduced metabolic risk profile, even when the adiposity indexes are increased.

  19. The hedgehog regulated oncogenes Gli1 and Gli2 block myoblast differentiation by inhibiting MyoD-mediated transcriptional activation

    PubMed Central

    Gerber, AN; Wilson, CW; Li, Y-J; Chuang, P-T

    2012-01-01

    The mechanism by which activation of the Hedgehog (Hh) pathway modulates differentiation and promotes oncogenesis in specific tissues is poorly understood. We therefore, analysed rhabdomyosarcomas from mice that were haploinsufficient for the Hh-binding protein, Hip1, or for the Hh receptor, Patched 1 (Ptch1). Transfection of the Hh-regulated transcription factor Gli1, which is expressed in a subset of mouse and human rhabdomyosarcomas, suppressed differentiation of myogenic rhabdomyosarcoma lines generated from Hip1+/− and Ptch1+/− mice. The closely related factor, Gli2, had similar effects. Gli1 and Gli2 inhibited myogenesis by repressing the capacity of MyoD to activate transcription. Deletion analysis of Gli1 indicated that multiple domains of Gli1 are required for efficient inhibition of MyoD. Gli1 reduced the ability of MyoD to heterodimerize with E12 and bind DNA, providing one mechanism whereby the Gli proteins modulate the activity of MyoD. This novel activity of Gli proteins provides new insights into how Hh signaling modulates terminal differentiation through inhibition of tissue-specific factors such as MyoD. This mechanism may contribute to the broad role of Hh signaling and the Gli proteins in differentiation decisions and cancer formation. PMID:16964293

  20. GlyQ-IQ: Glycomics Quintavariate-Informed Quantification with High-Performance Computing and GlycoGrid 4D Visualization

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kronewitter, Scott R.; Slysz, Gordon W.; Marginean, Ioan

    2014-05-31

    Dense LC-MS datasets have convoluted extracted ion chromatograms with multiple chromatographic peaks that cloud the differentiation between intact compounds with their overlapping isotopic distributions, peaks due to insource ion fragmentation, and noise. Making this differentiation is critical in glycomics datasets because chromatographic peaks correspond to different intact glycan structural isomers. The GlyQ-IQ software is targeted chromatography centric software designed for chromatogram and mass spectra data processing and subsequent glycan composition annotation. The targeted analysis approach offers several key advantages to LC-MS data processing and annotation over traditional algorithms. A priori information about the individual target’s elemental composition allows for exactmore » isotope profile modeling for improved feature detection and increased sensitivity by focusing chromatogram generation and peak fitting on the isotopic species in the distribution having the highest intensity and data quality. Glycan target annotation is corroborated by glycan family relationships and in source fragmentation detection. The GlyQ-IQ software is developed in this work (Part 1) and was used to profile N-glycan compositions from human serum LC-MS Datasets. The companion manuscript GlyQ-IQ Part 2 discusses developments in human serum N-glycan sample preparation, glycan isomer separation, and glycan electrospray ionization. A case study is presented to demonstrate how GlyQ-IQ identifies and removes confounding chromatographic peaks from high mannose glycan isomers from human blood serum. In addition, GlyQ-IQ was used to generate a broad N-glycan profile from a high resolution (100K/60K) nESI-LS-MS/MS dataset including CID and HCD fragmentation acquired on a Velos Pro Mass spectrometer. 101 glycan compositions and 353 isomer peaks were detected from a single sample. 99% of the GlyQ-IQ glycan-feature assignments passed manual validation and are backed with high

  1. Pharmacological characterization of the inhibitory activity of beta h-endorphin (beta h-EP), [Arg9,19,24,28,29]-beta h-EP, [Gln8,Gly31]-beta h-EP-Gly-Gly-NH2, in the neuroeffector junction of the mouse vas deferens.

    PubMed

    Valenzuela, R; Li, C H; Huidobro-Toro, J P

    1991-08-01

    The inhibitory opioid activities of beta h-endorphin (beta h-EP), its structurally related peptide analogues [Gln8,Gly31]-beta h-EP-Gly-Gly-NH2 (Gly-Gly-beta h-EP), [Arg9,19,24,28,29]-beta h-EP (Arg-beta h-EP) and methionine enkephalin have been examined in the electrically stimulated mouse vas deferens bioassay. All four peptides behaved as full agonists; methionine enkephalin was the most potent followed by Arg-beta h-EP, beta h-EP and Gly-Gly-beta h-EP. Neither Gly-Gly-beta h-EP nor Arg-beta h-EP antagonized the inhibitory action of beta h-EP or methionine enkephalin. An hour of tissue exposure to 30 nM beta-funaltrexamine followed by thorough washing, displaced to the right, in a parallel fashion, the concentration-response curves of beta h-EP and analogues. Whereas the displacement of the concentration response curves was 8 to 10-fold for beta h-EP and Arg-beta h-EP, it was only about 3-fold for Gly-Gly-beta h-EP and methionine enkephalin. Naltrindole was the most potent antagonist of methionine enkephalin with an apparent pA2 of 9.4; its potency as an antagonist of beta h-EP and related analogues was approximately one-tenth of this with pA2 values approximately 8.5. Norbinaltorphimine also antagonized the action of the opioid peptides with pA2 values close to 7.8.

  2. Possible Release of an ArgGlyArgProGln Pentapeptide with Innate Immunity Properties from Acidic Proline-Rich Proteins by Proteolytic Activity in Commensal Streptococcus and Actinomyces Species

    PubMed Central

    Li, Tong; Bratt, Per; Jonsson, Andreas P.; Ryberg, Mats; Johansson, Ingegerd; Griffiths, William J.; Bergman, Tomas; Strömberg, Nicklas

    2000-01-01

    This study suggests degradation of salivary acidic proline-rich proteins (PRPs) into potential innate-immunity-like peptides by oral Streptococcus and Actinomyces species. PRP degradation paralleled cleavage of Pro-containing substrates. PRP degradation by S. gordonii strain SK12 instantly released a Pyr1-Pro104Pro105 and a Gly111-Pro149Gln150 peptide together with a presumed Arg106Gly107Arg108Pro109Gln110 pentapeptide. The synthetic Arg106Gly107Arg108Pro109Gln110 peptide desorbed bound bacteria and counteracted sucrose-induced decrease of dental plaque pH in vitro. PMID:10948176

  3. Abrogation of Gli3 expression suppresses the growth of colon cancer cells via activation of p53

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kang, Han Na; Oh, Sang Cheul; Kim, Jun Suk

    2012-03-10

    p53, the major human tumor suppressor, appears to be related to sonic hedgehog (Shh)-Gli-mediated tumorigenesis. However, the role of p53 in tumor progression by the Shh-Gli signaling pathway is poorly understood. Herein we investigated the critical regulation of Gli3-p53 in tumorigenesis of colon cancer cells and the molecular mechanisms underlying these effects. RT-PCR analysis indicated that the mRNA level of Shh and Gli3 in colon tumor tissues was significantly higher than corresponding normal tissues (P < 0.001). The inhibition of Gli3 by treatment with Gli3 siRNA resulted in a clear decrease in cell proliferation and enhanced the level of expressionmore » of p53 proteins compared to treatment with control siRNA. The half-life of p53 was dramatically increased by treatment with Gli3 siRNA. In addition, treatment with MG132 blocked MDM2-mediated p53 ubiquitination and degradation, and led to accumulation of p53 in Gli3 siRNA-overexpressing cells. Importantly, ectopic expression of p53 siRNA reduced the ability of Gli3 siRNA to suppress proliferation of those cells compared with the cells treated with Gli3 siRNA alone. Moreover, Gli3 siRNA sensitized colon cancer cells to treatment with anti-cancer agents (5-FU and bevacizumab). Taken together, our studies demonstrate that loss of Gli3 signaling leads to disruption of the MDM2-p53 interaction and strongly potentiate p53-dependent cell growth inhibition in colon cancer cells, indicating a basis for the rational use of Gli3 antagonists as a novel treatment option for colon cancer.« less

  4. Geroprotective effect of ala-glu-asp-gly peptide in male rats exposed to different illumination regimens.

    PubMed

    Vinogradova, I A; Bukalev, A V; Zabezhinski, M A; Semenchenko, A V; Khavinson, V Kh; Anisimov, V N

    2008-04-01

    Exposure of male rats to permanent or natural illumination of North-Western Russia accelerated their death in comparison with animals exposed to standard (12 h) light. Permanent illumination promoted the development of spontaneous tumors in comparison with the standard photoregimen. Injection of epithalone (synthetic Ala-Glu-Asp-Gly peptide; subcutaneously 0.1 microg/rat 5 times a week from the age of 4 months until natural death) virtually did not change the mean lifespan of male rats, but was associated with a significant (p<0.05) normalization of population aging rate and hence, time of mortality rate doubling in groups exposed to natural or constant illumination. Epithalone injected to rats exposed to any photoregimen significantly inhibited the development of spontaneous tumors, primarily testicular leydigomas and leukemias.

  5. Targeting GLI by GANT61 involves mechanisms dependent on inhibition of both transcription and DNA licensing.

    PubMed

    Zhang, Ruowen; Wu, Jiahui; Ferrandon, Sylvain; Glowacki, Katie J; Houghton, Janet A

    2016-12-06

    The GLI genes are transcription factors and in cancers are oncogenes, aberrantly and constitutively activated. GANT61, a specific GLI inhibitor, has induced extensive cytotoxicity in human models of colon cancer. The FOXM1 promoter was determined to be a transcriptional target of GLI1. In HT29 cells, inhibition of GLI1 binding at the GLI consensus sequence by GANT61 led to inhibited binding of Pol II, the pause-release factors DSIF, NELF and p-TEFb. The formation of R-loops (RNA:DNA hybrids, ssDNA), were reduced by GANT61 at the FOXM1 promoter. Pretreatment of HT29 cells with α-amanitin reduced GANT61-induced γH2AX foci. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication to be linked. By co-immunoprecipitation and confocal microscopy, GLI1 co-localized with the DNA licensing factors ORC4, CDT1, and MCM2. Significant co-localization of GLI1 and ORC4 was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. CDT1 was found to be a transcription target of GLI1. Overexpression of CDT1 in HT29 and SW480 cells reduced GANT61-induced cell death, gH2AX foci, and cleavage of caspase-3. Data demonstrate involvement of transcription and of DNA replication licensing factors by non-transcriptional and transcriptional mechanisms in the GLI-dependent mechanism of action of GANT61.

  6. The topical penta-peptide Gly-Pro-Ile-Gly-Ser increases the proportion of thick hair in Japanese men with androgenetic alopecia.

    PubMed

    Iwabuchi, Tokuro; Takeda, Shunsuke; Yamanishi, Haruyo; Ideta, Ritsuro; Ehama, Ritsuko; Tsuruda, Akinori; Shibata, Hideaki; Ito, Tomoko; Komatsu, Nobuyuki; Terai, Keiko; Oka, Syuichi

    2016-06-01

    A penta-peptide, Gly-Pro-Ile-Gly-Ser (GPIGS), promotes proliferation of mouse hair keratinocytes and accelerates hair growth in mice. This study focused on the ability of the peptide to promote human hair growth. We used a human hair keratinocyte proliferation assay and organ cultures of human hair follicle as in vitro systems. The lotions with and without the penta-peptide were administered to 22 Japanese men with androgenetic alopecia (AGA) for 4 months in a double-blind and randomized clinical study. The penta-peptide significantly stimulated the proliferation of human hair keratinocytes at a concentration of 2.3 μm (P < 0.01), and 5.0 μm of this peptide had a marked effect on hair shaft elongation in the organ culture (P < 0.05). The change in the proportion of thick hair (≥60 μm) compared to baseline in patients that received the peptide was significantly higher than in the placebo (P = 0.006). The change in the proportion of vellus hair (<40 μm) was also significantly lower in the peptide group than in the placebo (P = 0.029). The penta-peptide also significantly improved the appearance of baldness (P = 0.020) when blinded reviewers graded photographs of the participants according to a standardized baldness scale. No adverse dermatological effects due to treatment were noted during this clinical study. This penta-peptide promotes proliferation of human hair keratinocytes and hair shaft elongation of human hair follicles, in vitro. This peptide increases thick hair ratio in vivo, and this compound is useful for the improvement of AGA. © 2016 Wiley Periodicals, Inc.

  7. A novel hemoglobin variant found on the α1 chain: Hb KSVGH (HBA1: p.Lys57_Gly58insSerHisGlySerAlaGlnValLys).

    PubMed

    Wang, Mei-Chun; Tsai, Kuo-Wang; Chu, Chih-Hsun; Yu, Ming-Sun; Lam, Hing-Chung

    2015-01-01

    Glycosylated hemoglobin (Hb A1C) is a crucial indicator for the long-term control and the diagnosis of diabetes. However, the presence of hemoglobin (Hb) variants may affect the measured value of Hb A1C and result in an abnormal graph trend and inconsistency between the clinical blood sugar test and Hb A1C values. In this study, laboratory data of 41,267 patients with diabetes were collected. The Hb A1C levels and the graph results were examined. We identified 74 cases containing abnormal Hb A1C graph trends. The conducted blood cell counts and capillary Hb electrophoresis were used to analyze Hb variants. We also determined gene variation for the Hb variants by a sequence approach. Fifteen different types of Hb variants were identified in this study. Among these, we found a novel variant in which the α1 subunit of Hb showed an insertion of 24 nucleotides (nts) between the 56th and 57th residues. We named this novel variant Hb Kaohsiung Veterans General Hospital (Hb KSVGH) (HBA1: p.Lys57_Gly58insSerHisGlySerAlaGlnValLys).

  8. Penfluridol suppresses glioblastoma tumor growth by Akt-mediated inhibition of GLI1

    PubMed Central

    Ranjan, Alok; Srivastava, Sanjay K.

    2017-01-01

    Glioblastoma (GBM) is the most common brain tumor with poor survival rate. Our results show that penfluridol, an antipsychotic drug significantly reduced the survival of ten adult and pediatric glioblastoma cell lines with IC50 ranging 2–5 μM after 72 hours of treatment and induced apoptosis. Penfluridol treatment suppressed the phosphorylation of Akt at Ser473 and reduced the expression of GLI1, OCT4, Nanog and Sox2 in several glioblastoma cell lines in a concentration-dependent manner. Inhibiting Akt with LY294002 and siRNA, or inhibiting GLI1 using GANT61, cyclopamine, siRNA and CRISPR/Cas9 resulted in enhanced cell growth suppressive effects of penfluridol. On the other hand, overexpression of GLI1 significantly attenuated the effects of penfluridol. Our results further demonstrated that penfluridol treatment inhibited the growth of U87MG tumors by 65% and 72% in subcutaneous and intracranial in vivo glioblastoma tumor models respectively. Immunohistochemical and western blot analysis of tumors revealed reduced pAkt (Ser 473), GLI1, OCT4 and increase in caspase-3 cleavage and TUNEL staining, confirming in vitro findings. Taken together, our results indicate that overall glioblastoma tumor growth suppression by penfluridol was associated with Akt-mediated inhibition of GLI1. PMID:28380428

  9. Topical treatment of corneal alkali burns with Gly-thymosin β4 solutions and in situ hydrogels via inhibiting corneal neovascularization and improving corneal epidermal recovery in experimental rabbits.

    PubMed

    Zhang, Weili; Nie, Liya; Du, Lina; Chen, Wenyang; Wu, Zhihong; Jin, Yiguang

    2017-12-01

    Corneal alkali burns are a severe disease and commonly encountered in the emergent clinic. A rapid medical treatment for the burn is very important. Gly-thymosin β 4 (Gly-Tβ 4 ) is a biomimic derivative of natural thymosin β 4 . The aim of this study is to evaluate the corneal recovery effects of Gly-Tβ 4 topical therapy on alkali burns in rabbit corneas. Rabbit alkali burns were induced with NaOH-contained filter paper. Phosphate-buffered solutions at pH 7.0, Gly-Tβ 4 solutions, blank in situ hydrogels, and Gly-Tβ 4 in situ hydrogels were dropped on the burned corneas. The treatments were continued for 14 days. Conjunctiva hyperemia, corneal edema, intraeye extravasation, hemorrhaging, corneal neovascularization (CNV), and corneal opacity were observed. Corneal immunohistochemistry and histopathology were performed. Gly-Tβ 4 solutions led to a lower corneal burn index than the other regimens. Hydrogels may stimulate the burned corneas due to the direct contact of them, and prevent the rapid release of Gly-Tβ 4 . Gly-Tβ 4 significantly inhibited CNV according to the images of the corneas, CNV areas, and CD31 expression. Furthermore, Gly-Tβ 4 improved corneal epidermal recovery according to the histopathological result. Gly-Tβ 4 solutions are a promising formulation for topical treatment of corneal alkali burns. Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.

  10. Lack of mixed agonist-antagonist properties of [Gln8-Gly31]-beta h-EP-Gly-Gly-NH2 and [Arg9,19,24,28,29]-beta h-EP in the rat vas deferens neuroeffector junction: studies with naloxone, beta-funaltrexamine and ICI 174,864.

    PubMed

    Valenzuela, R; Li, C H; Huidobro-Toro, J P

    1989-02-01

    The 1-27 truncated fragment of beta h-endorphin (beta h-EP) as well as [Gln8,Gly31]-beta h-EP-Gly-Gly-NH2 or [Arg9,19,24,28,29]-beta h-EP exhibited opiate agonist activity in the rat vas deferens bioassay; the potency of these peptides was 3 to 6 times less than that of beta h-EP. None of these compounds exhibited any degree of antagonism towards the inhibitory action of beta h-EP. Naloxone antagonized and reversed the inhibitory action of beta h-EP and its analogues though with varying potencies. The apparent naloxone-pA2 value for beta h-EP was 8.94; that for [Gln8-Gly31]-beta h-EP-Gly-Gly-NH2 was 8.08 and that for [Arg9,19,24,28,29]-beta h-EP was 8.38. beta-Funaltrexamine (beta-FNA) potently antagonized the inhibitory action of beta h-EP following non-equilibrium kinetics. Tissue preincubation with 10 nM beta-FNA for 60 min followed by extensive washing caused a 10-fold increase in the beta h-EP IC50. However, 10 nM beta-FNA caused only a 1.2 increase in the IC50 of [Gln8,Gly31]-beta h-EP-Gly-Gly-NH2 and a 4.1-fold increase in the IC50 of [Arg9,19,24,28,29]-beta h-EP. In contrast, preincubation of the tissue with 3 microM ICI 174,864 did not modify the potency of beta h-EP or its structural analogues. However, a 60 min pretreatment with 10 microM beta-FNA followed by the addition of 3 microM ICI 174,864 revealed a further decrease in the potency of the opiopeptins compared with tissues exposed to beta-FNA alone or ICI 174,864 alone. In conclusion, the inhibitory action of these peptides is remarkably sensitive to beta-FNA antagonism; in addition the peptides act as pure opiate agonists in marked contrast with the agonist-antagonist properties described in the CNS.

  11. Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma

    DOE PAGES

    Yang, Yi -Lin; Ni, Jian; Hsu, Ping -Chih; ...

    2015-07-27

    Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressedmore » and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi-square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma.« less

  12. Structure and Dynamics of End-to-End Loop Formation of the Penta-Peptide Cys-Ala-Gly-Gln-Trp in Implicit Solvents

    DTIC Science & Technology

    2009-01-01

    implicit solvents on peptide structure and dynamics , we performed extensive molecular dynamics simulations on the penta-peptide Cys-Ala-Gly-Gln-Trp. Two...end-to-end distances and dihedral angles obtained from molecular dynamics simulations with implicit solvent models were in a good agreement with those...to maintain the temperature of the systems. Introduction Molecular dynamics (MD) simulation techniques are widely used to study structure and

  13. The 482Ser of PPARGC1A and 12Pro of PPARG2 Alleles Are Associated with Reduction of Metabolic Risk Factors Even Obesity in a Mexican-Mestizo Population

    PubMed Central

    Vázquez-Del Mercado, Mónica; Guzmán-Ornelas, Milton-Omar; Corona Meraz, Fernanda-Isadora; Ríos-Ibarra, Clara-Patricia; Reyes-Serratos, Eduardo-Alejandro; Castro-Albarran, Jorge; Ruíz-Quezada, Sandra-Luz; Navarro-Hernández, Rosa-Elena

    2015-01-01

    The aim of this study was to investigate the relationship between functional polymorphisms Gly482Ser in PPARGC1A and Pro12Ala in PPARG2 with the presence of obesity and metabolic risk factors. We included 375 individuals characterized as Mexican-Mestizos and classified by the body mass index (BMI). Body dimensions and distribution of body fat were measured. The HOMA-IR and adiposity indexes were calculated. Adipokines and metabolic profile quantification were performed by ELISA and routine methods. Genetic polymorphisms were determined by polymerase chain reaction restriction fragment length polymorphism analysis. A difference between obese and nonobese subjects in polymorphism PPARGC1A distribution was observed. Among obese individuals, carriers of genotype 482Gly/Gly were observed to have decreased body fat, BMI, and body fat ratio versus 482Ser/Ser carriers and increased resistin and leptin levels in carriers Gly+ phenotype versus Gly− phenotype. Subjects with PPARG2 Ala− phenotype (genotype 12Pro/Pro) showed a decreased HOMA-IR index versus individuals with Ala+ phenotype (genotypes 12Pro/Ala plus 12Ala/Ala). We propose that, in obese Mexican-Mestizos, the combination of alleles 482Ser in PPARGC1A and 12Pro in PPARG2 represents a reduced metabolic risk profile, even when the adiposity indexes are increased. PMID:26185753

  14. Allergen Chip Diagnosis for Soy-Allergic Patients: Gly m 4 as a Marker for Severe Food-Allergic Reactions to Soy

    PubMed Central

    Berneder, M.; Bublin, M.; Hoffmann-Sommergruber, K.; Hawranek, T.; Lang, R.

    2016-01-01

    Background Gly m 5 and Gly m 6 are known to induce severe reactions in soy-allergic patients. For birch pollen (BP)-allergic patients, the Bet v 1 homologous allergen Gly m 4 is also a potential trigger of generalized severe reactions upon soy consumption. Therefore, reliable component-resolved diagnosis of soy allergy is needed. Methods IgE reactivity from sera of 20 patients from a BP environment with reported soy allergy was assessed. Skin prick tests (SPT) with BP and soy drink were performed. Specific IgE for BP, soy, Bet v 1 and Gly m 4 was analyzed by ImmunoCAP. In addition, ISAC microarray profiling was performed. Results Nineteen of 20 patients were BP allergic (positive SPT and/or CAP results for BP extract and Bet v 1). Eighteen soy-allergic patients were tested positive with soy drink in SPT. Soy CAP results were negative in the majority of tests (15/20), whereas 19/20 sera had specific IgE to Gly m 4. In the microarray approach, 14/20 sera displayed Gly m 4-specific IgE, the additional 6 sera had IgE levels below 0.3 ISAC standardized units. The BP-negative serum had Gly m 5- and Gly m 6-specific IgE which correlated with positive soy ImmunoCAP. Conclusions Soy sensitization detected by SPT and Gly m 4 ImmunoCAP were in good qualitative agreement with ISAC results. Soy ImmunoCAP was only specific for Gly m 5 and Gly m 6 sensitization. Gly m 4 ImmunoCAP has a higher sensitivity than ImmunoCAP ISAC. In this patient cohort, Gly m 4 sensitization was linked to the development of severe and generalized allergic reactions upon soy consumption. PMID:23548307

  15. Analysis of PGC-1{alpha} variants Gly482Ser and Thr612Met concerning their PPAR{gamma}2-coactivation function

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nitz, Inke; Ewert, Agnes; Klapper, Maja

    2007-02-09

    Peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) is a cofactor involved in adaptive thermogenesis, fatty acid oxidation, and gluconeogenesis. Dysfunctions of this protein are likely to contribute to the development of obesity and the metabolic syndrome. This is in part but not definitely confirmed by results of population studies. The aim of this study was to investigate if common genetic variants rs8192678 (Gly482Ser) and rs3736265 (Thr612Met) in the PGC-1{alpha} gene lead to a functional consequence in cofactor activity using peroxisome proliferator-activated receptor-{gamma} 2 (PPAR{gamma}2) as interacting transcription factor. Reporter gene assays in HepG2 cells with wildtype and mutant proteins of both PGC1{alpha}more » and PPAR{gamma}2 (Pro12Ala, rs1801282) using the acyl-CoA-binding protein (ACBP) promoter showed no difference in coactivator activity. This is First study implicating that the Gly482Ser and Thr612Met polymorphisms in PGC-1{alpha} and Pro12Ala polymorphism in PPAR{gamma}2 do not affect the functional integrity of these proteins.« less

  16. Association between Toll-like receptor 4 Asp299Gly polymorphism and coronary heart disease susceptibility.

    PubMed

    Wu, B W; Zhu, J; Shi, H M; Jin, B; Wen, Z C

    2017-08-07

    Published data on the association between Toll-like receptor 4 (TLR4) Asp299Gly polymorphism and coronary heart disease (CHD) susceptibility are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. English-language studies were identified by searching PubMed and Embase databases (up to November 2016). All epidemiological studies were regarding Caucasians because no TLR4 Asp/Gly and Gly/Gly genotypes have been detected in Asians. A total of 20 case-control studies involving 14,416 cases and 10,764 controls were included in the meta-analysis. Overall, no significant associations were found between TLR4 Asp299Gly polymorphism and CHD susceptibility in the dominant model (OR=0.89; 95%CI=0.74 to 1.06; P=0.20) pooled in the meta-analysis. In the subgroup analysis by CHD, non-significant associations were found in cases compared to controls. When stratified by control source, no significantly decreased risk was found in the additive model or dominant model. The present meta-analysis suggests that the TLR4 Asp299Gly polymorphism was not associated with decreased CHD risk in Caucasians.

  17. Investigation of collision-induced dissociation products and structures of gas-phase [ M·GlyGlyHis-H]+ ( M = Fe, Ni, Cu, and Zn) complexes.

    PubMed

    Gannamani, Bharathi; Shin, Joong-Won

    2017-02-01

    Collision-induced dissociation is carried out for electrosprayed [Fe·GlyGlyHis-H] + , [Ni·GlyGlyHis-H] + , [Cu·GlyGlyHis-H] + , and [Zn·GlyGlyHis-H] + complexes. [Fe·GlyGlyHis-H] + , [Ni·GlyGlyHis-H] + , and [Zn·GlyGlyHis-H] + yield metal-bound peptide sequence ions and dehydrated ions as primary products, whereas [Cu·GlyGlyHis-H] + generates a more extensive series of metal-bound sequence ions and a product arising from the unusual loss of a formaldehyde moiety; dehydration is significantly suppressed for this complex. Density functional theory calculations show that the copper ion-deprotonated peptide binding energy is substantially higher than those in other complexes, suggesting that there is a correlation between ion-ligand binding energy and their fragmentation behavior.

  18. Recombinant Brucella abortus gene expressing immunogenic protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mayfield, J.E.; Tabatabai, L.B.

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  19. Glaucocalyxin A exerts anticancer effect on osteosarcoma by inhibiting GLI1 nuclear translocation via regulating PI3K/Akt pathway.

    PubMed

    Zhu, Jianwei; Sun, Yang; Lu, Ying; Jiang, Xiubo; Ma, Bo; Yu, Lisha; Zhang, Jie; Dong, Xiaochen; Zhang, Qi

    2018-06-13

    Osteosarcoma, the most common malignant bone tumor with recurring disease or lung metastases, has become one of the leading causes of death in humans. In the current study, we made an investigation on the anticancer effect of glaucocalyxin A, a bioactive ent-kauranoid diterpenoid isolated from Rabdosia japonica var., and unraveled the underlying mechanisms. Here, we found that Glaucocalyxin A inhibited the cell viability of numerous osteosarcoma cells. Our results showed that Glaucocalyxin A exerted the pro-apoptotic effect on human osteosarcoma cells, MG-63 and HOS cells. Glaucocalyxin A induced apoptosis by mitochondrial apoptotic pathway through several steps including increasing the Bax/Bcl-2 ratio, triggering the intracellular reactive oxygen species (ROS) generation, reducing mitochondrial membrane potential (MMP), and inducing cleavage of caspase-9 and caspase-3. We demonstrated that Glaucocalyxin A induced apoptosis via inhibiting Five-zinc finger Glis 1 (GLI1) activation by overexpression and knockdown of GLI1 in vitro. We also found that Glaucocalyxin A inhibited GLI1 activation via regulating phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway. We further confirmed our findings by using PI3K activator and inhibitor to verify the inhibitory effect of Glaucocalyxin A on PI3K/Akt/GLI1 pathway. Moreover, our in vivo study revealed that glaucocalyxin A possessed a remarkable antitumor effect with no toxicity in the xenograft model inoculated with HOS tumor through the same mechanisms as in vitro. In conclusion, our results suggested that Glaucocalyxin A induced apoptosis in osteosarcoma by inhibiting nuclear translocation of GLI1 via regulating PI3K/Akt signaling pathway. Thus, Glaucocalyxin A might be a potential candidate for human osteosarcoma in the future.

  20. Msx genes are important apoptosis effectors downstream of the Shh/Gli3 pathway in the limb.

    PubMed

    Lallemand, Yvan; Bensoussan, Vardina; Cloment, Cécile Saint; Robert, Benoît

    2009-07-15

    In tetrapods, the anteroposterior (AP) patterning of the limb is under the control of the antagonistic activities of the secreted factor Sonic hedgehog (Shh) and Gli3R, the truncated repressor form of the transcription factor Gli3. In this report, we show that Msx1 and Msx2 are targets and downstream effectors of Gli3R. Consequently, in Shh null mutants, Msx genes are overexpressed and, furthermore, partially responsible for the limb phenotype. This is exemplified by the fact that reducing Msx activity in Shh mutants partially restores a normal limb development. Finally, we show that the main action of the Msx genes, in both normal and Shh(-/-) limb development, is to control cell death in the mesenchyme. We propose that, in the limb, Msx genes act downstream of the Shh/Gli3 pathway by transducing BMP signaling and that, in the absence of Shh signaling, their deregulation contributes to the extensive apoptosis that impairs limb development.

  1. Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa

    NASA Astrophysics Data System (ADS)

    Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

    2015-10-01

    A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms.

  2. Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa

    PubMed Central

    Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

    2015-01-01

    A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms. PMID:26423356

  3. Synergistic algicidal effect and mechanism of two diketopiperazines produced by Chryseobacterium sp. strain GLY-1106 on the harmful bloom-forming Microcystis aeruginosa.

    PubMed

    Guo, Xingliang; Liu, Xianglong; Pan, Jianliang; Yang, Hong

    2015-10-01

    A potent algicidal bacterium isolated from Lake Taihu, Chryseobacterium sp. strain GLY-1106, produces two algicidal compounds: 1106-A (cyclo(4-OH-Pro-Leu)) and 1106-B (cyclo(Pro-Leu)). Both diketopiperazines showed strong algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. Interestingly, these two algicidal compounds functioned synergistically. Compared with individual treatment, combined treatment with cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) significantly enhanced algicidal activity, accelerated the increase in intracellular reactive oxygen species (ROS) levels in M. aeruginosa, and further decreased the activities of antioxidases, effective quantum yield and maximal electron transport rate of M. aeruginosa. The results also showed that the algicidal characteristics of cyclo(4-OH-Pro-Leu) are distinct from those of cyclo(Pro-Leu). Cyclo(4-OH-Pro-Leu) mainly interrupted the flux of electron transport in the cyanobacterial photosynthetic system, whereas cyclo(Pro-Leu) mainly inhibited the activity of cyanobacterial intracellular antioxidases. A possible algicidal mechanism for the synergism between cyclo(4-OH-Pro-Leu) and cyclo(Pro-Leu) is proposed, which is in accordance with their distinct algicidal characteristics in individual and combined treatment. These findings suggest that synergism between algicidal compounds might be used as an effective strategy for the future control of Microcystis blooms.

  4. Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells

    PubMed Central

    Faião-Flores, F; Alves-Fernandes, D K; Pennacchi, P C; Sandri, S; Vicente, A L S A; Scapulatempo-Neto, C; Vazquez, V L; Reis, R M; Chauhan, J; Goding, C R; Smalley, K S; Maria-Engler, S S

    2017-01-01

    BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib

  5. Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells.

    PubMed

    Faião-Flores, F; Alves-Fernandes, D K; Pennacchi, P C; Sandri, S; Vicente, A L S A; Scapulatempo-Neto, C; Vazquez, V L; Reis, R M; Chauhan, J; Goding, C R; Smalley, K S; Maria-Engler, S S

    2017-03-30

    BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib

  6. Synthetic peptide, Ala-Arg-Glu-Gly-Glu-Met, abolishes pro-proliferative and anti-apoptotic effects of high glucose in vascular smooth muscle cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cao, Xiaozhou; Lyu, Yi; Collaborative Innovation Centre for Modern Grain Circulation and Safety, Nanjing University of Finance and Economics, Nanjing 210023

    Apoptosis plays a critical role in normal vascular development and atherosclerosis. However, high glucose has been reported to generate a certain level of ROS that can inhibit vascular smooth muscle cell (VSMC) apoptosis, with the underlying mechanism remaining unclear. In this study, a synthetic peptide AREGEM (Ala-Arg-Glu-Gly-Glu-Met) exhibited antioxidative effects and was used to investigate its function in VSMCs during hyperglycaemia. MTT assay results demonstrated that AREGEM significantly attenuated high glucose-induced VSMCs proliferation. Flow cytometry displayed that high glucose levels inhibited cell apoptosis, whereas this effect was attenuated by pre-incubation with AREGEM. In addition, the 2′,7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probemore » assay further demonstrated that AREGEM reduced intracellular ROS accumulation in VSMCs. Furthermore, this peptide was able to prevent the decrease of caspase-3 activity and the increase of the ratio of Bcl-2/Bax protein in VSMCs exposed to high glucose. These findings demonstrated that AREGEM is able to abolish the effects of high glucose in VSMCs; therefore, this peptide can be a potential candidate to develop a novel strategy for curing diabetic related diseases. - Highlights: • A peptide, AREGEM, can reduce intracellular ROS accumulation in vascular smooth muscle cells (VSMCs). • AREGEM significantly inhibits high glucose-induced proliferation of VSMCs. • AREGEM attenuates the inhibitory effect of high glucose on VSMC Apoptosis and caspase-3 activity. • AREGEM decreases the ratio of Bcl-2/Bax protein in VSMCs exposed to high glucose.« less

  7. Arg16/Gly beta2-adrenergic receptor polymorphism alters the cardiac output response to isometric exercise.

    PubMed

    Eisenach, John H; Barnes, Sunni A; Pike, Tasha L; Sokolnicki, Lynn A; Masuki, Shizue; Dietz, Niki M; Rehfeldt, Kent H; Turner, Stephen T; Joyner, Michael J

    2005-11-01

    Normotensive adults homozygous for glycine (Gly) of the Arg16/Gly beta2-adrenergic-receptor polymorphism have 1) greater forearm beta2-receptor mediated vasodilation and 2) a higher heart rate (HR) response to isometric handgrip than arginine (Arg) homozygotes. To test the hypothesis that the higher HR response in Gly16 subjects serves to maintain the pressor response [increased cardiac output (CO)] in the setting of augmented peripheral vasodilation to endogenous catecholamines, we measured continuous HR (ECG), arterial pressure (Finapres), and CO (transthoracic echocardiography) during isometric, 40% submaximal handgrip to fatigue in healthy subjects homozygous for Gly (n = 30; mean age +/- SE: 30 +/- 1.2, 13 women) and Arg (n = 17, age 30 +/- 1.6, 11 women). Resting data were similar between groups. Handgrip produced similar increases in arterial pressure and venous norepinephrine and epinephrine concentrations; however, HR increased more in the Gly group (60.1 +/- 4.3% increase from baseline vs. 45.5 +/- 3.9%, P = 0.03), and this caused CO to be higher (Gly: 7.6 +/- 0.3 l/m vs. Arg: 6.5 +/- 0.3 l/m, P = 0.03), whereas the decrease in systemic vascular resistance in the Gly group did not reach significance (P = 0.09). We conclude that Gly16 homozygotes generate a higher CO to maintain the pressor response to handgrip. The influence of polymorphic variants in the beta2-adrenergic receptor gene on the cardiovascular response to sympathoexcitation may have important implications in the development of hypertension and heart failure.

  8. Specificity of the collagenolytic serine proteinase from the pancreas of the catfish (Parasilurus asotus).

    PubMed

    Yoshinaka, R; Sato, M; Yamashita, M; Itoko, M; Ikeda, S

    1987-01-01

    The collagenolytic serine proteinase from the pancreas of the catfish (Parasilus asotus) had a pH optimum of 7.5 for native, reconstituted calf skin collagen fibrils. The enzyme was most stable at pH 6-9. The enzyme hydrolyzed heat-denatured collagen (gelatin), casein, hemoglobin and elastin in addition to native collagen but not virtually Tos-Arg-OEe, Bz-Tyr-OEe and Suc-(Ala)3-NA. The enzyme cleaved Leu-Gly (or Gln-Gly), Gly-Ile and Ile-Ala bonds on DNP-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH2 and DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg.

  9. Ferrocene tripeptide Gly-Pro-Arg conjugates: synthesis and inhibitory effects on Alzheimer's Aβ(1-42) fibrillogenesis and Aβ-induced cytotoxicity in vitro.

    PubMed

    Zhou, Binbin; Li, Chun-Lan; Hao, Yuan-Qiang; Johnny, Muya Chabu; Liu, You-Nian; Li, Juan

    2013-01-15

    Alzheimer's disease (AD) is the most common cause of dementia, and currently there is no clinical treatment to cure it or to halt its progression. Aggregation and fibril formation of β-amyloid peptides (Aβ) are central events in the pathogenesis of AD. Many efforts have been spent on the development of effective inhibitors to prevent Aβ fibrillogenesis and cause disaggregation of preformed Aβ fibrils. In this study, the conjugates of ferrocene and Gly-Pro-Arg (GPR) tripeptide, Boc-Gly-Pro-Arg(NO(2))-Fca-OMe (4, GPR-Fca) and Fc-Gly-Pro-Arg-OMe (7, Fc-GPR) (Fc: ferrocene; Fca: ferrocene amino acid) were synthesized by HOBT/HBTU protocol in solution. These ferrocene GPR conjugates were employed to inhibit Aβ(1-42) fibrillogenesis and to disaggregate preformed Aβ fibrils. The inhibitory properties of ferrocene GPR conjugates on Aβ(1-42) fibrillogenesis were evaluated by thioflavin T (ThT) fluorescence assay, and confirmed by atomic force microscopy (AFM) analysis. The interaction between the ferrocene GPR conjugates and Aβ(1-42) was monitored by electrochemical means. Our results showed that both GPR and GPR-Fca can significantly inhibit the fibril formation of Aβ(1-42), and cause disaggregation of the preformed fibrils. As expected, GPR-Fca shows stronger inhibitory effect on Aβ(1-42) fibrillogenesis than that of its parent peptide GPR. In contrast, Fc-GPR shows no inhibitory effect on fibrillogenesis of Aβ(1-42). Furthermore, GPR-Fca demonstrates significantly protection against Aβ-induced cytotoxicity and exhibits high resistance to proteolysis and good lipophilicity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Gli1 protein participates in Hedgehog-mediated specification of osteoblast lineage during endochondral ossification.

    PubMed

    Hojo, Hironori; Ohba, Shinsuke; Yano, Fumiko; Saito, Taku; Ikeda, Toshiyuki; Nakajima, Keiji; Komiyama, Yuske; Nakagata, Naomi; Suzuki, Kentaro; Takato, Tsuyoshi; Kawaguchi, Hiroshi; Chung, Ung-il

    2012-05-18

    With regard to Hedgehog signaling in mammalian development, the majority of research has focused on Gli2 and Gli3 rather than Gli1. This is because Gli1(-/-) mice do not show any gross abnormalities in adulthood, and no detailed analyses of fetal Gli1(-/-) mice are available. In this study, we investigated the physiological role of Gli1 in osteogenesis. Histological analyses revealed that bone formation was impaired in Gli1(-/-) fetuses compared with WT fetuses. Gli1(-/-) perichondrial cells expressed neither runt-related transcription factor 2 (Runx2) nor osterix, master regulators of osteogenesis, in contrast to WT cells. In vitro analyses showed that overexpression of Gli1 up-regulated early osteogenesis-related genes in both WT and Runx2(-/-) perichondrial cells, and Gli1 activated transcription of those genes via its association with their 5'-regulatory regions, underlying the function of Gli1 in the perichondrium. Moreover, Gli1(-/-);Gli2(-/-) mice showed more severe phenotypes of impaired bone formation than either Gli1(-/-) or Gli2(-/-) mice, and osteoblast differentiation was impaired in Gli1(-/-);Gli3(-/-) perichondrial cells compared with Gli3(-/-) cells in vitro. These data suggest that Gli1 itself can induce early osteoblast differentiation, at least to some extent, in a Runx2-independent manner. It also plays a redundant role with Gli2 and is involved in the repressor function of Gli3 in osteogenesis. On the basis of these findings, we propose that upon Hedgehog input, Gli1 functions collectively with Gli2 and Gli3 in osteogenesis.

  11. Cis-regulatory underpinnings of human GLI3 expression in embryonic craniofacial structures and internal organs.

    PubMed

    Abbasi, Amir A; Minhas, Rashid; Schmidt, Ansgar; Koch, Sabine; Grzeschik, Karl-Heinz

    2013-10-01

    The zinc finger transcription factor Gli3 is an important mediator of Sonic hedgehog (Shh) signaling. During early embryonic development Gli3 participates in patterning and growth of the central nervous system, face, skeleton, limb, tooth and gut. Precise regulation of the temporal and spatial expression of Gli3 is crucial for the proper specification of these structures in mammals and other vertebrates. Previously we reported a set of human intronic cis-regulators controlling almost the entire known repertoire of endogenous Gli3 expression in mouse neural tube and limbs. However, the genetic underpinning of GLI3 expression in other embryonic domains such as craniofacial structures and internal organs remain elusive. Here we demonstrate in a transgenic mice assay the potential of a subset of human/fish conserved non-coding sequences (CNEs) residing within GLI3 intronic intervals to induce reporter gene expression at known regions of endogenous Gli3 transcription in embryonic domains other than central nervous system (CNS) and limbs. Highly specific reporter expression was observed in craniofacial structures, eye, gut, and genitourinary system. Moreover, the comparison of expression patterns directed by these intronic cis-acting regulatory elements in mouse and zebrafish embryos suggests that in accordance with sequence conservation, the target site specificity of a subset of these elements remains preserved among these two lineages. Taken together with our recent investigations, it is proposed here that during vertebrate evolution the Gli3 expression control acquired multiple, independently acting, intronic enhancers for spatiotemporal patterning of CNS, limbs, craniofacial structures and internal organs. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  12. Complementary Gli activity mediates early patterning of the mouse visual system.

    PubMed

    Furimsky, Marosh; Wallace, Valerie A

    2006-03-01

    The Sonic hedgehog (Shh) signaling pathway plays a key role in the development of the vertebrate central nervous system, including the eye. This pathway is mediated by the Gli transcription factors (Gli1, Gli2, and Gli3) that differentially activate and repress the expression of specific downstream target genes. In this study, we investigated the roles of the three vertebrate Glis in mediating midline Shh signaling in early ocular development. We examined the ocular phenotypes of Shh and Gli combination mutant mouse embryos and monitored proximodistal and dorsoventral patterning by the expression of specific eye development regulatory genes using in situ hybridization. We show that midline Shh signaling relieves the repressor activity of Gli3 adjacent to the midline and then promotes eye pattern formation through the nonredundant activities of all three Gli proteins. Gli3, in particular, is required to specify the dorsal optic stalk and to define the boundary between the optic stalk and the optic cup.

  13. Pre-synaptic glycine GlyT1 transporter--NMDA receptor interaction: relevance to NMDA autoreceptor activation in the presence of Mg2+ ions.

    PubMed

    Musante, Veronica; Summa, Maria; Cunha, Rodrigo A; Raiteri, Maurizio; Pittaluga, Anna

    2011-05-01

    Rat hippocampal glutamatergic terminals possess NMDA autoreceptors whose activation by low micromolar NMDA elicits glutamate exocytosis in the presence of physiological Mg(2+) (1.2 mM), the release of glutamate being significantly reduced when compared to that in Mg(2+)-free condition. Both glutamate and glycine were required to evoke glutamate exocytosis in 1.2 mM Mg(2+), while dizocilpine, cis-4-[phosphomethyl]-piperidine-2-carboxylic acid and 7-Cl-kynurenic acid prevented it, indicating that occupation of both agonist sites is needed for receptor activation. D-serine mimicked glycine but also inhibited the NMDA/glycine-induced release of [(3H]D-aspartate, thus behaving as a partial agonist. The NMDA/glycine-induced release in 1.2 mM Mg(2+) strictly depended on glycine uptake through the glycine transporter type 1 (GlyT1), because the GlyT1 blocker N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine hydrochloride, but not the GlyT2 blocker Org 25534, prevented it. Accordingly, [(3)H]glycine was taken up during superfusion, while lowering the external concentration of Na(+), the monovalent cation co-transported with glycine by GlyT1, abrogated the NMDA-induced effect. Western blot analysis of subsynaptic fractions confirms that GlyT1 and NMDA autoreceptors co-localize at the pre-synaptic level, where GluN3A subunits immunoreactivity was also recovered. It is proposed that GlyT1s coexist with NMDA autoreceptors on rat hippocampal glutamatergic terminals and that glycine taken up by GlyT1 may permit physiological activation of NMDA pre-synaptic autoreceptors. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  14. Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

    1986-05-01

    Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with (/sup 3/H)-NaBH/sub 4/, and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active sitemore » peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P.« less

  15. The Arg16/Gly beta2-adrenergic receptor polymorphism is associated with altered cardiovascular responses to isometric exercise.

    PubMed

    Eisenach, John H; McGuire, Antonio M; Schwingler, Rachel M; Turner, Stephen T; Joyner, Michael J

    2004-02-13

    A polymorphism in the gene encoding the beta(2)-adrenergic receptor (arginine or glycine at amino acid position 16) is associated with altered vasodilator responses to beta(2)-agonists, which may modulate the pressor response to endogenous catecholamines during stress. To test the hypothesis that the Arg16/Gly polymorphism is associated with differences in acute pressor responses to sympathoexcitation, we measured mean arterial pressure (MAP, Finapres) and heart rate (HR, ECG) during mental stress (MS), cold pressor test (CPT), and handgrip (HG) to fatigue in 31 healthy, nonobese, normotensive adults (mean age +/- SE: 31 +/- 1; 16 females). Subjects were homozygous for Gly16 (n = 16) or Arg16 (n = 15). Both groups had similar baseline MAP (Arg16, 86 +/- 3 mmHg; Gly16, 89 +/- 2 mmHg; P = 0.4) and HR (Arg16, 68 +/- 2 beats/min; Gly16, 65 +/- 3 beats/min; P = 0.3). For MS and CPT, MAP and HR did not differ between genotype groups. Handgrip also produced similar increases in MAP; however, the change in HR was greater in the Gly16 homozygotes (P(ANOVA) = 0.001, genotype-by-time interaction). During HG, peak HR at fatigue was 100 +/- 4 beats/min for Gly16 (54% increase from rest) vs. 93 +/- 3 beats/min for Arg16 (37% increase). We conclude that the cardiovascular responses to MS and CPT do not differ between Gly16 and Arg16 homozygotes. However, the greater HR response to exercise in the Gly16 homozygotes may serve to maintain the pressor response (increased cardiac output) in the face of augmented peripheral vasodilation (decreased total peripheral resistance) in this group.

  16. Abnormal Positioning of Diencephalic Cell Types in Neocortical Tissue in the Dorsal Telencephalon of Mice Lacking Functional Gli3

    PubMed Central

    Fotaki, Vassiliki; Yu, Tian; Zaki, Paulette A.; Mason, John O.; Price, David J.

    2008-01-01

    The transcription factor Gli3 (glioma-associated oncogene homolog) is essential for normal development of the mammalian forebrain. One extreme requirement for Gli3 is at the dorsomedial telencephalon, which does not form in Gli3Xt/Xt mutant mice lacking functional Gli3. In this study, we analyzed expression of Gli3 in the wild-type telencephalon and observed a highdorsal-to-lowventral gradient of Gli3 expression and predominance of the cleaved form of the Gli3 protein dorsally. This graded expression correlates with the severedorsal-to-mildventral telencephalic phenotype observed in Gli3Xt/Xt mice. We characterized the abnormal joining of the telencephalon to the diencephalon and defined the medial limit of the dorsal telencephalon in Gli3Xt/Xt mice early in corticogenesis. Based on this analysis, we concluded that some of the abnormal expression of ventral telencephalic markers previously described as being in the dorsal telencephalon is, in fact, expression in adjacent diencephalic tissue, which expresses many of the same genes that mark the ventral telencephalon. We observed occasional cells with diencephalic character in the Foxg1 (forkhead box)-expressing Gli3Xt/Xt telencephalon at embryonic day 10.5, a day after the anatomical subdivision of the forebrain vesicle. Large clusters of such cells appear in the Gli3Xt/Xt neocortical region at later ages, when the neocortex becomes highly disorganized, forming rosettes comprising mainly neural progenitors. We propose that Gli3 is indispensable for formation of an intact telencephalic-diencephalic boundary and for preventing the abnormal positioning of diencephalic cells in the dorsal telencephalon. PMID:16957084

  17. Light-induced Conversion of Trp to Gly and Gly Hydroperoxide in IgG1

    PubMed Central

    Haywood, Jessica; Mozziconacci, Olivier; Allegre, Kevin M.; Kerwin, Bruce A.; Schöneich, Christian

    2013-01-01

    The exposure of IgG1 in aqueous solution to light with λ = 254 nm or λ > 295 nm yields products consistent with Trp radical cation formation followed by αC-βC cleavage of the Trp side chain. The resulting glycyl radicals are either reduced to Gly, or add oxygen prior to reduction to Gly hydroperoxide. Photoirradiation at λ = 254 nm targets Trp at positions 191 (light chain), 309 and 377 (heavy chain) while photoirradiation at λ > 295 nm targets Trp at position 309 (heavy chain). Mechanistically, the formation of Trp radical cations likely proceeds via photo-induced electron- or hydrogen-transfer to disulfide bonds, yielding thiyl radicals and thiols, where thiols may serve as reductants for the intermediary glycyl or glycylperoxyl radicals. PMID:23363477

  18. Reactivity of cosmetic UV filters towards skin proteins: model studies with Boc-lysine, Boc-Gly-Phe-Gly-Lys-OH, BSA and gelatin.

    PubMed

    Stiefel, C; Schwack, W

    2014-12-01

    Organic UV filters are used as active ingredients in most sunscreens and also in a variety of daily care products. Their good (photo) stability is of special interest to guarantee protective function and to prevent interactions with the human skin. Due to the mostly electrophilic character of the UV filters, reactions with nucleophilic protein moieties like lysine side chains are conceivable. Prior studies showed that the UV filters octocrylene (OCR), butyl methoxydibenzoylmethane (BM-DBM), ethylhexyl salicylate (EHS), ethylhexyl methoxycinnamate (EHMC), benzophenone-3 (BP-3), ethylhexyl triazone (EHT) and dibenzoylmethane (DBM) were able to covalently bind to an HPTLC amino phase and the amino acid models ethanolamine and butylamine after slightly heating and/or radiation. Boc-protected lysine, the tetrapeptide Boc-Gly-Phe-Gly-Lys-OH, bovine serum albumin (BSA) and porcine gelatin were used as more complex models to determine the reactivity of the mentioned UV filters towards skin proteins under thermal or UV irradiation conditions. After gentle heating at 37°C, benzophenone imines were identified as reaction products of BP-3 and OCR with Boc-lysine and the tetrapeptide, whereas DBM and BM-DBM yielded enamines. For EHMC, a Michael-type reaction occurred, which resulted in addition of Boc-lysine or the tetrapeptide to the conjugated double bond. Ester aminolysis of EHS and EHT mainly afforded the corresponding amides. Reactions of the UV filters with BSA changed the UV spectrum of BSA, generally associated with an increase of the absorption strength in the UVA or UVB range. For all protein models, the UV filters showed an increasing reactivity in the order EHT < EHMC < EHS < BP-3 < OCR < DBM < BM-DBM. Especially the UV absorbers BM-DBM, OCR and BP-3, which are seen as common allergens or photoallergens, showed a high reactivity towards the different skin protein models. As the formation of protein adducts is recognized as important key element in the induction of

  19. A Novel Pathogenesis-Related Class 10 Protein Gly m 4l, Increases Resistance upon Phytophthora sojae Infection in Soybean (Glycine max [L.] Merr.)

    PubMed Central

    Fan, Sujie; Jiang, Liangyu; Wu, Junjiang; Dong, Lidong; Cheng, Qun; Xu, Pengfei; Zhang, Shuzhen

    2015-01-01

    Phytophthora root and stem rot of soybean, caused by Phytophthora sojae (P. sojae), is a destructive disease in many soybean planting regions worldwide. In a previous study, an expressed sequence tag (EST) homolog of the major allergen Pru ar 1 in apricot (Prunus armeniaca) was identified up-regulated in the highly resistant soybean ‘Suinong 10’ infected with P. sojae. Here, the full length of the EST was isolated using rapid amplification of cDNA ends (RACE). It showed the highest homolgy of 53.46% with Gly m 4 after comparison with the eight soybean allergen families reported and was named Gly m 4-like (Gly m 4l, GenBank accession no. HQ913577.1). The cDNA full length of Gly m 4l was 707 bp containing a 474 bp open reading frame encoding a polypeptide of 157 amino acids. Sequence analysis suggests that Gly m 4l contains a conserved ‘P-loop’ (phosphate-binding loop) motif at residues 47–55 aa and a Bet v 1 domain at residues 87–120 aa. The transcript abundance of Gly m 4l was significantly induced by P. sojae, salicylic acid (SA), NaCl, and also responded to methyl jasmonic acid (MeJA) and ethylene (ET). The recombinant Gly m 4l protein showed RNase activity and displayed directly antimicrobial activity that inhibited hyphal growth and reduced zoospore release in P. sojae. Further analyses showed that the RNase activity of the recombinant protein to degrading tRNA was significantly affected in the presence of zeatin. Over-expression of Gly m 4l in susceptible ‘Dongnong 50’ soybean showed enhanced resistance to P. sojae. These results indicated that Gly m 4l protein played an important role in the defense of soybean against P. sojae infection. PMID:26474489

  20. Effective Biotransformation of Ethyl 4-Chloro-3-Oxobutanoate into Ethyl (S)-4-Chloro-3-Hydroxybutanoate by Recombinant E. coli CCZU-T15 Whole Cells in [ChCl][Gly]-Water Media.

    PubMed

    Dai, Yong; Huan, Bin; Zhang, Hai-Sheng; He, Yu-Cai

    2017-04-01

    To increase the biocatalytic activity of Escherichia coli CCZU-T15 whole cells, choline chloride/glycerol ([ChCl][Gly]) was firstly used as biocompatible solvent for the effective biotransformation of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE]. Furthermore, L-glutamine (150 mM) was added into [ChCl][Gly]-water ([ChCl][Gly] 12.5 vol%, pH 6.5) media instead of NAD + for increasing the biocatalytic efficiency. To further improve the biosynthesis of (S)-CHBE (>99 % e.e.) by E. coli CCZU-T15 whole cells, Tween-80 (7.5 mM) was also added into this reaction media, and (S)-CHBE (>9 % e.e.) could be effectively synthesized from 2000 and 3000 mM COBE in the yields of 100 and 93.0 % by whole cells of recombinant E. coli CCZU-T15, respectively. TEM image indicated that the cell membrane was permeabilized and lost its integrity and when the cell was exposed to [ChCl][Gly]-water media with Tween-80. Clearly, this bioprocess has high potential for the effective biosynthesis of (S)-CHBE (>99 % e.e.).

  1. Aspirin inhibits the SHH/GLI1 signaling pathway and sensitizes malignant glioma cells to temozolomide therapy

    PubMed Central

    Li, Ziwei; Lin, Lin; Meng, Xiangqi; Han, Bo; Wang, Ruijia; Wu, Pengfei; Li, Jianlong; Cai, Jinquan; Jiang, Chuanlu

    2017-01-01

    Aberrant activation of sonic hedgehog (SHH)/glioma-associated oncogene homolog 1 (GLI1) pathway plays an important role in the tumorigenicity of malignant glioma cells and resistance to temozolomide (TMZ). Here we investigated the aspirin's antineoplastic molecular route by targeting SHH/GLI1 pathway and examined the feasibility of aspirin combined with TMZ therapy. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the activity of the SHH/GLI1 pathway was strongly inhibited by aspirin. Aspirin acted as the glioma growth-inhibitory and pro-apoptosis roles by inhibiting the SHH/GLI1 pathway and reprogramming the epithelial to mesenchymal transition (EMT). The immunofluorescence assay showed aspirin could prevent the nuclear translocation of GLI1 to inhibit its transcriptional regulation. The stable lentiviral overexpression of GLI1 reversed the DNA double strand breaks (DSBs) caused by the GANT61 and TMZ. Furthermore, aspirin combined with TMZ enhanced chemosensitivity and GLI1-induced chemoprotection was partly blocked by aspirin in vitro and in vivo. Collectively, aspirin has a therapeutic potential for SHH/GLI1 targeted therapy against glioma cells. Acquired activation of GLI1 protects glioma cells against TMZ therapy. Impairment of DNA DSBs repair activity might be involved in the route of aspirin-induced chemosensitivity. Combined aspirin with TMZ may be a promising strategy against malignant glioma. PMID:28446712

  2. Aspirin inhibits the SHH/GLI1 signaling pathway and sensitizes malignant glioma cells to temozolomide therapy.

    PubMed

    Ming, Jianguang; Sun, Bo; Li, Ziwei; Lin, Lin; Meng, Xiangqi; Han, Bo; Wang, Ruijia; Wu, Pengfei; Li, Jianlong; Cai, Jinquan; Jiang, Chuanlu

    2017-04-01

    Aberrant activation of sonic hedgehog (SHH)/glioma-associated oncogene homolog 1 (GLI1) pathway plays an important role in the tumorigenicity of malignant glioma cells and resistance to temozolomide (TMZ). Here we investigated the aspirin's antineoplastic molecular route by targeting SHH/GLI1 pathway and examined the feasibility of aspirin combined with TMZ therapy. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the activity of the SHH/GLI1 pathway was strongly inhibited by aspirin. Aspirin acted as the glioma growth-inhibitory and pro-apoptosis roles by inhibiting the SHH/GLI1 pathway and reprogramming the epithelial to mesenchymal transition (EMT). The immunofluorescence assay showed aspirin could prevent the nuclear translocation of GLI1 to inhibit its transcriptional regulation. The stable lentiviral overexpression of GLI1 reversed the DNA double strand breaks (DSBs) caused by the GANT61 and TMZ. Furthermore, aspirin combined with TMZ enhanced chemosensitivity and GLI1-induced chemoprotection was partly blocked by aspirin in vitro and in vivo . Collectively, aspirin has a therapeutic potential for SHH/GLI1 targeted therapy against glioma cells. Acquired activation of GLI1 protects glioma cells against TMZ therapy. Impairment of DNA DSBs repair activity might be involved in the route of aspirin-induced chemosensitivity. Combined aspirin with TMZ may be a promising strategy against malignant glioma.

  3. Ectodermal Influx and Cell Hypertrophy Provide Early Growth for All Murine Mammary Rudiments, and Are Differentially Regulated among Them by Gli3

    PubMed Central

    Lee, May Yin; Racine, Victor; Jagadpramana, Peter; Sun, Li; Yu, Weimiao; Du, Tiehua; Spencer-Dene, Bradley; Rubin, Nicole; Le, Lendy; Ndiaye, Delphine; Bellusci, Saverio; Kratochwil, Klaus; Veltmaat, Jacqueline M.

    2011-01-01

    Mammary gland development starts in utero with one or several pairs of mammary rudiments (MRs) budding from the surface ectodermal component of the mammalian embryonic skin. Mice develop five pairs, numbered MR1 to MR5 from pectoral to inguinal position. We have previously shown that Gli3Xt-J/Xt-J mutant embryos, which lack the transcription factor Gli3, do not form MR3 and MR5. We show here that two days after the MRs emerge, Gli3Xt-J/Xt-J MR1 is 20% smaller, and Gli3Xt-J/Xt-J MR2 and MR4 are 50% smaller than their wild type (wt) counterparts. Moreover, while wt MRs sink into the underlying dermis, Gli3Xt-J/Xt-J MR4 and MR2 protrude outwardly, to different extents. To understand why each of these five pairs of functionally identical organs has its own, distinct response to the absence of Gli3, we determined which cellular mechanisms regulate growth of the individual MRs, and whether and how Gli3 regulates these mechanisms. We found a 5.5 to 10.7-fold lower cell proliferation rate in wt MRs compared to their adjacent surface ectoderm, indicating that MRs do not emerge or grow via locally enhanced cell proliferation. Cell-tracing experiments showed that surface ectodermal cells are recruited toward the positions where MRs emerge, and contribute to MR growth during at least two days. During the second day of MR development, peripheral cells within the MRs undergo hypertrophy, which also contributes to MR growth. Limited apoptotic cell death counterbalances MR growth. The relative contribution of each of these processes varies among the five MRs. Furthermore, each of these processes is impaired in the absence of Gli3, but to different extents in each MR. This differential involvement of Gli3 explains the variation in phenotype among Gli3Xt-J/Xt-J MRs, and may help to understand the variation in numbers and positions of mammary glands among mammals. PMID:22046263

  4. Synthesis, properties and crystal structure of (Gly) 2H 4SiW 12O 40·5.5H 2O

    NASA Astrophysics Data System (ADS)

    Lihua, Bi; Qizhuang, He; Qiong, Jia; Enbo, Wang

    2001-10-01

    A novel polyoxometalate containing Glycine (Gly), (Gly)2H4SiW12O40·5.5H2O (I), has been synthesized and characterized by single-crystal X-ray diffraction, elemental analyzes, IR spectrum, cyclic voltammograms and thermogravimetric analysis. The compound crystallizes in the monoclinic space group C2/C with a=40.362 (8) Å, b=12.478 (3) Å, c=19.879 (4) Å, β=96.22 (3)°, V=9953 (4) Å3, Z=8 and R1 (wR2)=0.0699 (0.1609). The crystal structure consists of [SiW12O40]4- units linked together with Gly molecules through hydrogen bonding. The electrochemical properties of I showed that the electrode reaction is surface-controlled. The compound has photosensitivity under irradiation of sunlight to result in charge transfer by oxidation of Gly and the reduction of SiW12O404-. We also found that the compound exhibited effectiveness in preventing cucumber mosaic virus (CMV).

  5. Role of GLI2 in hypopituitarism phenotype.

    PubMed

    Arnhold, Ivo J P; França, Marcela M; Carvalho, Luciani R; Mendonca, Berenice B; Jorge, Alexander A L

    2015-06-01

    GLI2 is a zinc-finger transcription factor involved in the Sonic Hedgehog pathway. Gli2 mutant mice have hypoplastic anterior and absent posterior pituitary glands. We reviewed the literature for patients with hypopituitarism and alterations in GLI2. Twenty-five patients (16 families) had heterozygous truncating mutations, and the phenotype frequently included GH deficiency, a small anterior pituitary lobe and an ectopic/undescended posterior pituitary lobe on magnetic resonance imaging and postaxial polydactyly. The inheritance pattern was autosomal dominant with incomplete penetrance and variable expressivity. The mutation was frequently inherited from an asymptomatic parent. Eleven patients had heterozygous non-synonymous GLI2 variants that were classified as variants of unknown significance, because they were either absent from or had a frequency lower than 0.001 in the databases. In these patients, the posterior pituitary was also ectopic, but none had polydactyly. A third group of variants found in patients with hypopituitarism were considered benign because their frequency was ≥ 0.001 in the databases. GLI2 is a large and polymorphic gene, and sequencing may identify variants whose interpretation may be difficult. Incomplete penetrance implies in the participation of other genetic and/or environmental factors. An interaction between Gli2 mutations and prenatal ethanol exposure has been demonstrated in mice dysmorphology. In conclusion, a relatively high frequency of GLI2 mutations and variants were identified in patients with congenital GH deficiency without other brain defects, and most of these patients presented with combined pituitary hormone deficiency and an ectopic posterior pituitary lobe. Future studies may clarify the relative role and frequency of GLI2 alterations in the aetiology of hypopituitarism. © 2015 Society for Endocrinology.

  6. Inhibitors of tripeptidyl peptidase II. 2. Generation of the first novel lead inhibitor of cholecystokinin-8-inactivating peptidase: a strategy for the design of peptidase inhibitors.

    PubMed

    Ganellin, C R; Bishop, P B; Bambal, R B; Chan, S M; Law, J K; Marabout, B; Luthra, P M; Moore, A N; Peschard, O; Bourgeat, P; Rose, C; Vargas, F; Schwartz, J C

    2000-02-24

    The cholecystokinin-8 (CCK-8)-inactivating peptidase is a serine peptidase which has been shown to be a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10). It cleaves the neurotransmitter CCK-8 sulfate at the Met-Gly bond to give Asp-Tyr(SO(3)H)-Met-OH + Gly-Trp-Met-Asp-Phe-NH(2). In seeking a reversible inhibitor of this peptidase, the enzymatic binding subsites were characterized using a fluorimetric assay based on the hydrolysis of the artificial substrate Ala-Ala-Phe-amidomethylcoumarin. A series of di- and tripeptides having various alkyl or aryl side chains was studied to determine the accessible volume for binding and to probe the potential for hydrophobic interactions. From this initial study the tripeptides Ile-Pro-Ile-OH (K(i) = 1 microM) and Ala-Pro-Ala-OH (K(i) = 3 microM) and dipeptide amide Val-Nvl-NHBu (K(i) = 3 microM) emerged as leads. Comparison of these structures led to the synthesis of Val-Pro-NHBu (K(i) = 0.57 microM) which served for later optimization in the design of butabindide, a potent reversible competitive and selective inhibitor of the CCK-8-inactivating peptidase. The strategy for this work is explicitly described since it illustrates a possible general approach for peptidase inhibitor design.

  7. Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation.

    PubMed

    Canettieri, Gianluca; Di Marcotullio, Lucia; Greco, Azzura; Coni, Sonia; Antonucci, Laura; Infante, Paola; Pietrosanti, Laura; De Smaele, Enrico; Ferretti, Elisabetta; Miele, Evelina; Pelloni, Marianna; De Simone, Giuseppina; Pedone, Emilia Maria; Gallinari, Paola; Giorgi, Alessandra; Steinkühler, Christian; Vitagliano, Luigi; Pedone, Carlo; Schinin, M Eugenià; Screpanti, Isabella; Gulino, Alberto

    2010-02-01

    Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling.

  8. Involvement of the strychnine-sensitive glycine receptor in the anxiolytic effects of GlyT1 inhibitors on maternal separation-induced ultrasonic vocalization in rat pups.

    PubMed

    Komatsu, Hiroko; Furuya, Yoshiaki; Sawada, Kohei; Asada, Takashi

    2015-01-05

    Several studies have shown that glycine transporter 1 (GlyT1) inhibitors have anxiolytic actions. There are two types of glycine receptor: the strychnine-sensitive glycine receptor (GlyA) and the strychnine-insensitive glycine receptor (GlyB); however, which receptor is the main contributor to the anxiolytic actions of GlyT1 inhibitors is yet to be determined. Here, we clarified which glycine receptor is the main contributor to the anxiolytic effects of GlyT1 inhibitors by using maternal separation-induced ultrasonic vocalization (USV) by rat pups as an index of anxiety. We confirmed that administration of the benzodiazepine diazepam or the selective serotonin reuptake inhibitor escitaloplam, which are both clinically proven anxiolytics, or the GlyT1 inhibitor SSR504734 (2-chloro-N-[(S)-phenyl[(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide), decreases USV in rat pups. In addition, we showed that another GlyT1 inhibitor, ALX5407 ((R)-N-[3-(4'-fluorophenyl)-3(4'-phenylphenoxy)propyl]sarcosine) also decreases USV in rat pups. SSR504734- or ALX5407-induced decreases in USV were dose-dependently reversed by administration of the GlyA antagonist strychnine, whereas the diazepam- or escitalopram-induced decreases in USV were not. Furthermore, GlyT1-induced decreases in USV were not reversed by administration of the GlyB antagonist L-687,414. Together, these results suggest that GlyA activation is the main contributor to the anxiolytic actions of GlyT1 inhibitors and that the anxiolytic actions of diazepam and escitalopram cannot be attributed to GlyA activation. Our findings provide new insights into the importance of the activation of GlyA in the anxiolytic effects of GlyT1 inhibitors. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Pirfenidone exerts antifibrotic effects through inhibition of GLI transcription factors.

    PubMed

    Didiasova, Miroslava; Singh, Rajeev; Wilhelm, Jochen; Kwapiszewska, Grazyna; Wujak, Lukasz; Zakrzewicz, Dariusz; Schaefer, Liliana; Markart, Philipp; Seeger, Werner; Lauth, Matthias; Wygrecka, Malgorzata

    2017-05-01

    Pirfenidone is an antifibrotic drug, recently approved for the treatment of patients with idiopathic pulmonary fibrosis (IPF). Although pirfenidone exhibits anti-inflammatory, antioxidant, and antifibrotic properties, the molecular mechanism underlying its protective effects remains unknown. Here, we link pirfenidone action with the regulation of the profibrotic hedgehog (Hh) signaling pathway. We demonstrate that pirfenidone selectively destabilizes the glioma-associated oncogene homolog (GLI)2 protein, the primary activator of Hh-mediated gene transcription. Consequently, pirfenidone decreases overall Hh pathway activity in patients with IPF and in patient-derived primary lung fibroblasts and leads to diminished levels of Hh target genes, such as GLI1, Hh receptor Patched-1, α-smooth muscle actin, and fibronectin, and to reduced cell migration and proliferation. Interestingly, Hh-triggered TGF-β1 expression potentiated Hh responsiveness of primary lung fibroblasts by elevating the available pool of glioma-associated oncogene homolog (GLI)1/GLI2, thus creating a vicious cycle of amplifying fibrotic processes. Because GLI transcription factors are not only crucial for Hh-mediated changes but are also required as mediators of TGF-β signaling, our findings suggest that pirfenidone exerts its clinically beneficial effects through dual Hh/TGF-β inhibition by targeting the GLI2 protein.-Didiasova, M., Singh, R., Wilhelm, J., Kwapiszewska, G., Wujak, L., Zakrzewicz, D., Schaefer, L., Markart, P., Seeger, W., Lauth, M., Wygrecka, M. Pirfenidone exerts antifibrotic effects through inhibition of GLI transcription factors. © FASEB.

  10. N-Glycan Structure Annotation of Glycopeptides Using a Linearized Glycan Structure Database (GlyDB)

    PubMed Central

    Ren, Jian Min; Rejtar, Tomas; Li, Lingyun; Karger, Barry L.

    2008-01-01

    While glycoproteins are abundant in nature, and changes in glycosylation occur in cancer and other diseases, glycoprotein characterization remains a challenge due to the structural complexity of the biopolymers. This paper presents a general strategy, termed GlyDB, for glycan structure annotation of N-linked glycopeptides from tandem mass spectra in the LC-MS analysis of proteolytic digests of glycoproteins. The GlyDB approach takes advantage of low-energy collision induced dissociation of N-linked glycopeptides that preferentially cleaves the glycosidic bonds while the peptide backbone remains intact. A theoretical glycan structure database derived from biosynthetic rules for N-linked glycans was constructed employing a novel representation of branched glycan structures consisting of multiple linear sequences. The commonly used peptide identification program, Sequest, could then be utilized to assign experimental tandem mass spectra to individual glycoforms. Analysis of synthetic glycopeptides and well-characterized glycoproteins demonstrate that the GlyDB approach can be a useful tool for annotation of glycan structures and for selection of a limited number of potential glycan structure candidates for targeted validation. PMID:17625816

  11. Retardation of ice crystallization by short peptides

    NASA Astrophysics Data System (ADS)

    Kim, Jun Soo; Yethiraj, Arun

    2009-03-01

    The effect of short peptides on the growth of ice crystals is studied using molecular dynamics simulations. The simulations focus on two sequences (Gly-Pro-Ala-Gly and Gly-Gly-Ala-Gly) that are found in collagen hydrolysate, a substance that is known to retard crystal growth. In the absence of peptides, the growth of ice crystal in the solution with the ice/water interface is observed in at a rate comparable to the experimental data. When peptides are present in the liquid phase, the crystal growth is retarded to a significant extent compared to the pure water. It is found that Gly-Pro-Ala-Gly is more effective (crystallization is up to 5 times slower than in its absence) than Gly-Gly-Ala-Gly (up to 3 times slower) implying that the role of the proline residue is important. The mechanism can be understood in the nature of binding of the peptides to the growing crystal.

  12. Affinity capillary electrophoresis and density functional theory study of noncovalent interactions of cyclic peptide [Gly6 ]-antamanide with small cations.

    PubMed

    Pangavhane, Sachin; Böhm, Stanislav; Makrlík, Emanuel; Ruzza, Paolo; Kašička, Václav

    2017-08-01

    ACE and density functional theory were employed to study the noncovalent interactions of cyclic decapeptide glycine-6-antamanide ([Gly 6 ]AA), synthetic derivative of native antamanide (AA) peptide from the deadly poisonous fungus Amanita phalloides, with small cations (Li + , Rb + , Cs + , NH 4 + , and Ca 2+ ) in methanol. The strength of these interactions was quantified by the apparent stability constants of the appropriate complexes determined by ACE. The stability constants were calculated using the nonlinear regression analysis of the dependence of the effective electrophoretic mobility of [Gly 6 ]AA on the concentration of the above ions in the BGE (methanolic solution of 20 mM chloroacetic acid, 10 mM Tris, pH MeOH 7.8, containing 0-70 mM concentrations of the above ions added in the form of chlorides). Prior to stability constant calculation, the effective mobilities measured at actual temperature inside the capillary and at variable ionic strength of the BGEs were corrected to the values corresponding to the reference temperature of 25°C and to the constant ionic strength of 10 mM. From the above ions, Rb + and Cs + cations interacted weakly with [Gly 6 ]AA but no interactions of [Gly 6 ]AA with univalent Li + and NH 4 + ions and divalent Ca 2+ ion were observed. The apparent stability constants of [Gly 6 ]AA-Rb + and [Gly 6 ]AA-Cs + complexes were found to be equal to 13 ± 4 and 22 ± 3 L/mol, respectively. The structural characteristics of these complexes, such as position of the Rb + and Cs + ions in the cavity of the [Gly 6 ]AA molecule and the interatomic distances within these complexes, were obtained by the density functional theory calculations. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease

    PubMed Central

    Tirnitz-Parker, Janina Elke Eleonore; Hamson, Elizabeth Jane; Warren, Alessandra; Maneck, Bharvi; Chen, Jinbiao; Patkunanathan, Bramilla; Boland, Jade; Cheng, Robert; Shackel, Nicholas Adam; Seth, Devanshi; Bowen, David Geoffrey; Martelotto, Luciano Gastón; Watkins, D. Neil; McCaughan, Geoffrey William

    2017-01-01

    Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (>99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMO-dependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness. PMID:28187190

  14. Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease.

    PubMed

    Grzelak, Candice Alexandra; Sigglekow, Nicholas David; Tirnitz-Parker, Janina Elke Eleonore; Hamson, Elizabeth Jane; Warren, Alessandra; Maneck, Bharvi; Chen, Jinbiao; Patkunanathan, Bramilla; Boland, Jade; Cheng, Robert; Shackel, Nicholas Adam; Seth, Devanshi; Bowen, David Geoffrey; Martelotto, Luciano Gastón; Watkins, D Neil; McCaughan, Geoffrey William

    2017-01-01

    Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (>99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMO-dependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness.

  15. Evaluation of GLI Reflectance and Vegetation Indices With MODIS Products

    DTIC Science & Technology

    2005-07-25

    collective picture of a warming world and other changes in the climate system . Vegetation over land surfaces contains carbon that is re- leased to atmosphere...irradiance based on Thuiller 2002 (Thuiller et al., 2003), Lsat[W/m2/str/µm] is GLI observed radiance, and θs[rad] is solor zenith angle. The GLI Project...longer wavelength than 2500nm, MODTRAN4.0 IR solor irradiance is used. GLI atmospheric correction for land is conducted for Rayleigh scattering and

  16. Identification of a "glycine-loop"-like coiled structure in the 34 AA Pro,Gly,Met repeat domain of the biomineral-associated protein, PM27.

    PubMed

    Wustman, Brandon A; Santos, Rudolpho; Zhang, Bo; Evans, John Spencer

    2002-12-05

    Fracture resistance in biomineralized structures has been linked to the presence of proteins, some of which possess sequences that are associated with elastic behavior. One such protein superfamily, the Pro,Gly-rich sea urchin intracrystalline spicule matrix proteins, form protein-protein supramolecular assemblies that modify the microstructure and fracture-resistant properties of the calcium carbonate mineral phase within embryonic sea urchin spicules and adult sea urchin spines. In this report, we detail the identification of a repetitive keratin-like "glycine-loop"- or coil-like structure within the 34-AA (AA: amino acid) N-terminal domain, (PGMG)(8)PG, of the spicule matrix protein, PM27. The identification of this repetitive structural motif was accomplished using two capped model peptides: a 9-AA sequence, GPGMGPGMG, and a 34-AA peptide representing the entire motif. Using CD, NMR spectrometry, and molecular dynamics simulated annealing/minimization simulations, we have determined that the 9-AA model peptide adopts a loop-like structure at pH 7.4. The structure of the 34-AA polypeptide resembles a coil structure consisting of repeating loop motifs that do not exhibit long-range ordering. Given that loop structures have been associated with protein elastic behavior and protein motion, it is plausible that the 34-AA Pro,Gly,Met repeat sequence motif in PM27 represents a putative elastic or mobile domain. Copyright 2002 Wiley Periodicals, Inc.

  17. Gly389Arg polymorphism of beta1-adrenergic receptor is associated with the cardiovascular response to metoprolol.

    PubMed

    Liu, Jie; Liu, Zhao-Qian; Tan, Zhi-Rong; Chen, Xiao-Ping; Wang, Lian-Sheng; Zhou, Gan; Zhou, Hong-Hao

    2003-10-01

    Our objectives were to determine whether the Gly389 polymorphism of the beta(1)-adrenergic receptor exhibits reduced responsiveness in vivo and to test the hypothesis that the Gly389Arg polymorphism affects the blood pressure and heart rate response to metoprolol. beta(1)-Adrenergic receptor genotype was determined by polymerase chain reaction-restriction fragment length polymorphism assay. Exercise-induced heart rate increases were compared to determine the functional significance in vivo in 8 healthy Chinese men homozygous for Gly389 and 8 homozygous for Arg389. All of the subjects were given 25, 50, or 75 mg of metoprolol every 8 hours; the dosages were given in a random order, and each dosage was given for 1 day. The degree of beta-blockade was measured as the reduction in resting and exercise heart rates and blood pressures. Plasma metoprolol concentrations were measured by the use of HPLC-fluorescence detection. Exercise led to a workload-dependent increase in heart rate. There were no differences in exercise-induced heart rate increases between Arg389 and Gly389 homozygotes. Oral metoprolol caused significant dose-dependent decreases in both resting and exercise heart rates in both groups. The reductions in the resting heart rate in 3 dosage levels of metoprolol were 6.3% +/- 0.8% versus 4.1% +/- 0.7%, 10.1% +/- 1.0% versus 6.2% +/- 1.1%, and 14.4% +/- 1.4% versus 10.9% +/- 1.3% in homozygous Arg389 subjects and Gly389 subjects, respectively (P =.008). We also found differences with respect to the exercise heart rate (8.9% +/- 0.5%, 14.0% +/- 0.9%, and 20.1% +/- 1.5% in Arg389 subjects and 6.6% +/- 0.7%, 11.7% +/- 1.0%, and 16.4% +/- 1.3% in Gly389 subjects; P =.017) and systolic pressure (5.9% +/- 0.7%, 9.2% +/- 1.0%, and 11.6% +/- 1.2% in Arg389 subjects and 4.6% +/- 0.5%, 6.0% +/- 0.8%, and 9.9% +/- 0.9% in Gly389 subjects; P =.011). However, the difference in the fall in diastolic pressure was not statistically significant (P =.442). The Arg389 variant

  18. Identification of novel non-coding RNA-based negative feedback regulating the expression of the oncogenic transcription factor GLI1.

    PubMed

    Villegas, Victoria E; Rahman, Mohammed Ferdous-Ur; Fernandez-Barrena, Maite G; Diao, Yumei; Liapi, Eleni; Sonkoly, Enikö; Ståhle, Mona; Pivarcsi, Andor; Annaratone, Laura; Sapino, Anna; Ramírez Clavijo, Sandra; Bürglin, Thomas R; Shimokawa, Takashi; Ramachandran, Saraswathi; Kapranov, Philipp; Fernandez-Zapico, Martin E; Zaphiropoulos, Peter G

    2014-07-01

    Non-coding RNAs are a complex class of nucleic acids, with growing evidence supporting regulatory roles in gene expression. Here we identify a non-coding RNA located head-to-head with the gene encoding the Glioma-associated oncogene 1 (GLI1), a transcriptional effector of multiple cancer-associated signaling pathways. The expression of this three-exon GLI1 antisense (GLI1AS) RNA in cancer cells was concordant with GLI1 levels. siRNAs knockdown of GLI1AS up-regulated GLI1 and increased cellular proliferation and tumor growth in a xenograft model system. Conversely, GLI1AS overexpression decreased the levels of GLI1, its target genes PTCH1 and PTCH2, and cellular proliferation. Additionally, we demonstrate that GLI1 knockdown reduced GLI1AS, while GLI1 overexpression increased GLI1AS, supporting the role of GLI1AS as a target gene of the GLI1 transcription factor. Activation of TGFβ and Hedgehog signaling, two known regulators of GLI1 expression, conferred a concordant up-regulation of GLI1 and GLI1AS in cancer cells. Finally, analysis of the mechanism underlying the interplay between GLI1 and GLI1AS indicates that the non-coding RNA elicits a local alteration of chromatin structure by increasing the silencing mark H3K27me3 and decreasing the recruitment of RNA polymerase II to this locus. Taken together, the data demonstrate the existence of a novel non-coding RNA-based negative feedback loop controlling GLI1 levels, thus expanding the repertoire of mechanisms regulating the expression of this oncogenic transcription factor. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. A Novel C2H2 Transcription Factor that Regulates gliA Expression Interdependently with GliZ in Aspergillus fumigatus

    PubMed Central

    Schoberle, Taylor J.; Nguyen-Coleman, C. Kim; Herold, Jennifer; Yang, Ally; Weirauch, Matt; Hughes, Timothy R.; McMurray, John S.; May, Gregory S.

    2014-01-01

    Secondary metabolites are produced by numerous organisms and can either be beneficial, benign, or harmful to humans. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Gliotoxin, which is produced by a variety of Aspergillus species, Trichoderma species, and Penicillium species, exhibits immunosuppressive properties and has therefore been the subject of research for many laboratories. There have been a few proteins shown to regulate the gliotoxin cluster, most notably GliZ, a Zn2Cys6 binuclear finger transcription factor that lies within the cluster, and LaeA, a putative methyltransferase that globally regulates secondary metabolism clusters within numerous fungal species. Using a high-copy inducer screen in A. fumigatus, our lab has identified a novel C2H2 transcription factor, which plays an important role in regulating the gliotoxin biosynthetic cluster. This transcription factor, named GipA, induces gliotoxin production when present in extra copies. Furthermore, loss of gipA reduces gliotoxin production significantly. Through protein binding microarray and mutagenesis, we have identified a DNA binding site recognized by GipA that is in extremely close proximity to a potential GliZ DNA binding site in the 5′ untranslated region of gliA, which encodes an efflux pump within the gliotoxin cluster. Not surprisingly, GliZ and GipA appear to work in an interdependent fashion to positively control gliA expression. PMID:24784729

  20. The LacI family protein GlyR3 co-regulates the celC operon and manB in Clostridium thermocellum

    DOE PAGES

    Choi, Jinlyung; Klingeman, Dawn M.; Brown, Steven D.; ...

    2017-06-24

    In this paper, we demonstrate that the GlyR3 protein mediates the regulation of manB. We first identify putative GlyR3 binding sites within or just upstream of the coding regions of manB and celT. Using an electrophoretic mobility shift assay (EMSA), we determined that a higher concentration of GlyR3 is required to effectively bind to the putative manB site in comparison to the celC site. Neither the putative celT site nor random DNA significantly binds GlyR3. While laminaribiose interfered with GlyR3 binding to the celC binding site, binding to the manB site was unaffected. In the presence of laminaribiose, in vivomore » transcription of the celC–glyR3–licA gene cluster increases, while manB expression is repressed, compared to in the absence of laminaribiose, consistent with the results from the EMSA. An in vitro transcription assay demonstrated that GlyR3 and laminaribiose interactions were responsible for the observed patters of in vivo transcription.« less

  1. The novel RAF1 mutation p.(Gly361Ala) located outside the kinase domain of the CR3 region in two patients with Noonan syndrome, including one with a rare brain tumor.

    PubMed

    Harms, Frederike L; Alawi, Malik; Amor, David J; Tan, Tiong Y; Cuturilo, Goran; Lissewski, Christina; Brinkmann, Julia; Schanze, Denny; Kutsche, Kerstin; Zenker, Martin

    2018-02-01

    Noonan syndrome is characterized by typical craniofacial dysmorphism, postnatal growth retardation, congenital heart defect, and learning difficulties and belongs to the RASopathies, a group of neurodevelopmental disorders caused by germline mutations in genes encoding components of the RAS-MAPK pathway. Mutations in the RAF1 gene are associated with Noonan syndrome, with a high prevalence of hypertrophic cardiomyopathy (HCM). RAF1 mutations cluster in exons encoding the conserved region 2 (CR2), the kinase activation segment of the CR3 domain, and the C-terminus. We present two boys with Noonan syndrome and the identical de novo RAF1 missense variant c.1082G>C/p.(Gly361Ala) affecting the CR3, but located outside the kinase activation segment. The p.(Gly361Ala) mutation has been identified as a RAF1 allele conferring resistance to RAF inhibitors. This amino acid change favors a RAF1 conformation that allows for enhanced RAF dimerization and increased intrinsic kinase activity. Both patients with Noonan syndrome showed typical craniofacial dysmorphism, macrocephaly, and short stature. One individual developed HCM and was diagnosed with a disseminated oligodendroglial-like leptomeningeal tumor (DOLT) of childhood at the age of 9 years. While there is a well-established association of NS with malignant tumors, especially childhood hemato-oncological diseases, brain tumors have rarely been reported in Noonan syndrome. Our data demonstrate that mutation scanning of the entire coding region of genes associated with Noonan syndrome is mandatory not to miss rare variants located outside the known mutational hotspots. © 2017 Wiley Periodicals, Inc.

  2. hPEPT1 is responsible for uptake and transport of Gly-Sar in the human bronchial airway epithelial cell-line Calu-3.

    PubMed

    Søndergaard, Helle Bach; Brodin, Birger; Nielsen, Carsten Uhd

    2008-06-01

    The purpose of this work was to investigate the apical uptake and transepithelial transport of Gly-Sar along with the expression of the di-/tripeptide transporters hPEPT1 and hPEPT2 in human Calu-3 bronchial epithelial cells. The apical Gly-Sar uptake rate in Calu-3 cells followed Michaelis-Menten kinetics with a Km value of 1.3 +/- 0.3 mM and a Vmax value of 0.60 +/- 0.06 nmol cm(-2) min(-1). Transepithelial apical to basolateral transport of 50 microM [3H]-labelled Gly-Sar across the Calu-3 cell monolayer was pH-dependent. The Gly-Sar flux was significantly reduced in the presence of delta-aminolevulinic acid (2.5 mM), cephalexin (25 mM), and captopril (25 mM; p < 0.05, n = 3). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed the presence of both hPEPT1 and hPEPT2 mRNA in the Calu-3 cells. These findings were confirmed in healthy human bronchial cDNA. Restriction-endonuclease analysis identified hPEPT2 in Calu-3 cells to be the hPEPT2*1 haplotype. Western blotting demonstrated expression of the hPEPT1 protein (approximately 80 kDa), and the immunolabel was mainly localized in the apical membrane as judged by immunolocalization studies using confocal laser scanning microscopy (CLSM). This work presents for the first time hPEPT1 and hPEPT2*1 expression in human Calu-3 cells. Surprisingly, the results indicate that Gly-Sar uptake and transport in Calu-3 cells are hPEPT1-mediated rather than hPEPT2-mediated.

  3. Direct and indirect requirements of Shh/Gli signaling in early pituitary development.

    PubMed

    Wang, Yiwei; Martin, James F; Bai, C Brian

    2010-12-15

    Induction of early pituitary progenitors is achieved through combined activities of signals from adjacent embryonic tissues. Previous studies have identified a requirement for oral ectoderm derived Sonic Hedgehog (Shh) in specification and/or proliferation of early pituitary progenitors, however how different Gli genes mediate Shh signaling to control pituitary progenitor development has not yet been determined. Here we show that Gli2, which encodes a major Gli activator, is required for proliferation of specific groups of pituitary progenitors but not for initial dorsoventral patterning. We further show that the action of Gli2 occurs prior to the closure of Rathke' pouch. Lastly, we show that Shh/Gli2 signaling controls the diencephalic expression of Bone morphogenetic protein 4 (Bmp4) and Fibroblast growth factor 8 (Fgf8), two genes that are known to play critical roles in patterning and growth of Rathke's pouch. Our results therefore suggest both cell-autonomous and non-cell-autonomous requirements for Gli2 in regulation of pituitary progenitor specification, proliferation and differentiation. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. The hedgehog/Gli signaling paradigm in prostate cancer

    PubMed Central

    Chen, Mengqian; Carkner, Richard; Buttyan, Ralph

    2011-01-01

    Hedgehog is a ligand-activated signaling pathway that regulates Gli-mediated transcription. Although most noted for its role as an embryonic morphogen, hyperactive hedgehog also causes human skin and brain malignancies. The hedgehog-related gene anomalies found in these tumors are rarely found in prostate cancer. Yet surveys of human prostate tumors show concordance of high expression of hedgehog ligands and Gli2 that correlate with the potential for metastasis and therapy-resistant behavior. Likewise, prostate cancer cell lines express hedgehog target genes, and their growth and survival is affected by hedgehog/Gli inhibitors. To date, the preponderance of data supports the idea that prostate tumors benefit from a paracrine hedgehog microenvironment similar to the developing prostate. Uncertainty remains as to whether hedgehog’s influence in prostate cancer also includes aspects of tumor cell autocrine-like signaling. The recent findings that Gli proteins interact with the androgen receptor and affect its transcriptional output have helped to identify a novel pathway through which hedgehog/Gli might affect prostate tumor behavior and raises questions as to whether hedgehog signaling in prostate cancer cells is suitably measured by the expression of Gli target genes alone. PMID:21776292

  5. Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

    PubMed

    Saito, M; Takenouchi, Y; Kunisaki, N; Kimura, S

    2001-05-01

    The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.

  6. The effect of glycosylation on plasma N-terminal proBNP-76 levels in patients with heart or renal failure.

    PubMed

    Nishikimi, Toshio; Ikeda, Masashi; Takeda, Yosuke; Ishimitsu, Toshihiko; Shibasaki, Ikuko; Fukuda, Hirotsugu; Kinoshita, Hideyuki; Nakagawa, Yasuaki; Kuwahara, Koichiro; Nakao, Kazuwa

    2012-01-01

    Pro-brain natriuretic peptide (proBNP)-108 and N-terminal proBNP-76 (NT-BNP) contain seven sites for O-linked oligosaccharide attachment. Currently, levels of glycosylated NT-BNP are probably underestimated because it is not recognised by one antibody in the sandwich assay system. The pathophysiological significance of cardiac and plasma levels of non-glycosylated (nonglyNT-BNP) and glycosylated NT-BNP (glyNT-BNP) in heart failure (HF) and chronic renal failure (CRF) was investigated. Plasma samples from 186 patients with HF and 76 patients with CRF on haemodialysis were studied, together with 11 atrial tissue samples. To measure nonglyNT-BNP and glyNT-BNP, samples were incubated with or without deglycosylating enzymes and NT-BNP was measured using Roche Elecsys proBNP I. The percentage glyNT-BNP was calculated as glyNT-BNP/(glyNT-BNP + nonglyNT-BNP). In HF, plasma BNP, nonglyNT-BNP and glyNT-BNP levels all increased with increasing disease severity (New York Heart Association class; p<0.0001), though the molar ratio remained constant (molar ratio, BNP:nonglyNT-BNP:glyNT-BNP = 1:2.4:9.6). Before haemodialysis for CRF, plasma BNP and nonglyNT-BNP were somewhat elevated, and glyNT-BNP was markedly increased (molar ratio, BNP:nonglyNT-BNP:glyNT-BNP = 1:8.5:82). After haemodialysis, plasma BNP, nonglyNT-BNP, atrial natriuretic protein and cGMP all declined (p<0.0001), but glyNT-BNP was unchanged. Notably, the percentage of glyNT-BNP was elevated before haemodialysis, and was further increased after haemodialysis (p<0.0001). Atrial tissue levels of BNP, nonglyNT-BNP and glyNT-BNP were similar. THE findings suggest that most endogenous plasma NT-BNP is glycosylated and therefore undetectable with the current assay system, and that the relative glycosylation level is increased by haemodialysis.

  7. Synthesis and Characterization of Novel Acyl-Glycine Inhibitors of GlyT2.

    PubMed

    Mostyn, Shannon N; Carland, Jane E; Shimmon, Susan; Ryan, Renae M; Rawling, Tristan; Vandenberg, Robert J

    2017-09-20

    It has been demonstrated previously that the endogenous compound N-arachidonyl-glycine inhibits the glycine transporter GlyT2, stimulates glycinergic neurotransmission, and provides analgesia in animal models of neuropathic and inflammatory pain. However, it is a relatively weak inhibitor with an IC 50 of 9 μM and is subject to oxidation via cyclooxygenase, limiting its therapeutic value. In this paper we describe the synthesis and testing of a novel series of monounsaturated C18 and C16 acyl-glycine molecules as inhibitors of the glycine transporter GlyT2. We demonstrate that they are up to 28 fold more potent that N-arachidonyl-glycine with no activity at the closely related GlyT1 transporter at concentrations up to 30 μM. This novel class of compounds show considerable promise as a first generation of GlyT2 transport inhibitors.

  8. Arg16Gly polymorphism of the beta2-adrenergic receptor is associated with differences in cardiovascular function at rest and during exercise in humans.

    PubMed

    Snyder, Eric M; Beck, Kenneth C; Dietz, Niki M; Eisenach, John H; Joyner, Michael J; Turner, Stephen T; Johnson, Bruce D

    2006-02-15

    In humans, subjects homozygous for arginine (ArgArg) at codon 16 of the beta2-adrenergic receptor (beta2AR) have been shown to have greater agonist-mediated desensitization than subjects homozygous for glycine (GlyGly). We sought to determine if this substitution differentially influenced cardiovascular function during short duration (9 min) low and high intensity exercise (40 and 75% of peak work). Healthy Caucasian ArgArg (n = 16), GlyGly (n = 31) and ArgGly (n = 17) subjects matched for age, sex and peak oxygen uptake were studied. There were no differences in adrenaline (ADR) at rest or with heavy exercise, but the ArgArg group had lower ADR with light exercise (P = 0.04). Resting heart rate (HR) was higher in ArgArg (P < 0.01), while cardiac output (Q), stroke volume (SV), and mean arterial pressure (MAP) were lower than the other groups (HR = 86+/-2, 78+/-2, 80+/-1 beats min(-1); Q = 5.7+/-0.81, 6.1+/-0.18, 6.7+/-0.22 l min(-1); SV = 68+/-3, 82+/-3, 89+/-4 ml beat(-1); MAP = 92+/-1, 103+/-2, 98+/-1 mmHg-- for ArgArg, ArgGly and GlyGly, respectively, means +/-s.e.m., P < 0.01), however, no differences were observed in systemic vascular resistance (SVR). With low intensity exercise and high intensity exercise the ArgArg group continued to have a lower , SV and MAP compared to the other groups (P < 0.05), with no differences observed in SVR. During recovery, the ArgArg subjects continued to have a lower MAP but there were no differences in HR, , or SVR. These data suggest that subjects homozygous for Arg at codon 16 of the beta2AR have reduced and MAP at rest that persist during exercise with no evidence for differential changes over the course of exercise despite large changes in catecholamines. This may suggest possible genotype-related differences in baseline receptor function or density which causes phenotypic differences at rest that are sustained during short-term exercise.

  9. Mass Spectrometry Identification of N-Chlorinated Dipeptides in Drinking Water.

    PubMed

    Huang, Guang; Jiang, Ping; Li, Xing-Fang

    2017-04-04

    We report the identification of N-chlorinated dipeptides as chlorination products in drinking water using complementary high-resolution quadrupole time-of-flight (QTOF) and quadrupole ion-trap mass spectrometry techniques. First, three model dipeptides, tyrosylglycine (Tyr-Gly), tyrosylalanine (Tyr-Ala), and phenylalanylglycine (Phe-Gly), reacted with sodium hypochlorite, and these reaction solutions were analyzed by QTOF. N-Cl-Tyr-Gly, N,N-di-Cl-Tyr-Gly, N-Cl-Phe-Gly, N,N-di-Cl-Phe-Gly, N-Cl-Tyr-Ala, and N,N-di-Cl-Tyr-Ala were identified as the major products based on accurate masses, 35 Cl/ 37 Cl isotopic patterns, and MS/MS spectra. These identified N-chlorinated dipeptides were synthesized and found to be stable in water over 10 days except N,N-di-Cl-Phe-Gly. To enable sensitive detection of N-chlorinated dipeptides in authentic water, we developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method with multiple reaction monitoring (MRM) mode. N-Cl-Tyr-Gly, N,N-di-Cl-Tyr-Gly, N-Cl-Phe-Gly, N-Cl-Tyr-Ala, and N,N-di-Cl-Tyr-Ala along with their corresponding dipeptides were detected in authentic tap water samples. The dipeptides were clearly detected in the raw water, but the N-chlorinated dipeptides were at background levels. These results suggest that the N-chlorinated dipeptides are produced by chlorination. This study has identified N-chlorinated dipeptides as new disinfection byproducts in drinking water. The strategy developed in this study can be used to identify chlorination products of other peptides in drinking water.

  10. Structure-activity relationship of linear peptide Bu-His-DPhe-Arg-Trp-Gly-NH(2) at the human melanocortin-1 and -4 receptors: histidine substitution.

    PubMed

    Cheung, Adrian Wai-Hing; Danho, Waleed; Swistok, Joseph; Qi, Lida; Kurylko, Grazyna; Rowan, Karen; Yeon, Mitch; Franco, Lucia; Chu, Xin-Jie; Chen, Li; Yagaloff, Keith

    2003-01-06

    Systematic substitution of His(6) residue using non-selective hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)) as the template led to the identification of Bu-Atc(6)(2-aminotetraline-2-carboxylic acid)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) which showed moderate selectivity towards hMC4R over hMC1R. Further SAR studies resulted in the discovery of Penta-5-BrAtc(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) and Penta-5-Me(2)NAtc(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) which are potent hMC4R agonists and are inactive in hMC1R, hMC3R and hMC5R agonist assays.

  11. Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice.

    PubMed

    Liu, Jessica Ai-Jia; Lai, Frank Pui-Ling; Gui, Hong-Sheng; Sham, Mai-Har; Tam, Paul Kwong-Hang; Garcia-Barcelo, Maria-Mercedes; Hui, Chi-Chung; Ngan, Elly Sau-Wai

    2015-12-01

    Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

  12. Superpotent [Dmt¹] dermorphin tetrapeptides containing the 4-aminotetrahydro-2-benzazepin-3-one scaffold with mixed μ/δ opioid receptor agonistic properties.

    PubMed

    Vandormael, Bart; Fourla, Danai-Dionysia; Gramowski-Voss, Alexandra; Kosson, Piotr; Weiss, Dieter G; Schröder, Olaf H-U; Lipkowski, Andrzej; Georgoussi, Zafiroula; Tourwé, Dirk

    2011-11-24

    Novel dermorphin tetrapeptides are described in which Tyr(1) is replaced by Dmt(1), where d-Ala(2) and Gly(4) are N-methylated, and where Phe(3)-Gly(4) residue is substituted by the constrained Aba(3)-Gly(4) peptidomimetic. Most of these peptidic ligands displayed binding affinities in the nanomolar range for both μ- and δ-opioid receptors but no detectable affinity for the κ-opioid receptor. Measurements of cAMP accumulation, phosphorylation of extracellular signal-regulated kinase (ERK1/2) in HEK293 cells stably expressing each of these receptors individually, and functional screening in primary neuronal cultures confirmed the potent agonistic properties of these peptides. The most potent ligand H-Dmt-NMe-d-Ala-Aba-Gly-NH(2) (BVD03) displayed mixed μ/δ opioid agonist properties with picomolar functional potencies. Functional electrophysiological in vitro assays using primary cortical and spinal cord networks showed that this analogue possessed electrophysiological similarity toward gabapentin and sufentanil, which makes it an interesting candidate for further study as an analgesic for neuropathic pain.

  13. Hedgehog-GLI signaling drives self-renewal and tumorigenicity of human melanoma-initiating cells.

    PubMed

    Santini, Roberta; Vinci, Maria C; Pandolfi, Silvia; Penachioni, Junia Y; Montagnani, Valentina; Olivito, Biagio; Gattai, Riccardo; Pimpinelli, Nicola; Gerlini, Gianni; Borgognoni, Lorenzo; Stecca, Barbara

    2012-09-01

    The question of whether cancer stem/tumor-initiating cells (CSC/TIC) exist in human melanomas has arisen in the last few years. Here, we have used nonadherent spheres and the aldehyde dehydrogenase (ALDH) enzymatic activity to enrich for CSC/TIC in a collection of human melanomas obtained from a broad spectrum of sites and stages. We find that melanomaspheres display extensive in vitro self-renewal ability and sustain tumor growth in vivo, generating human melanoma xenografts that recapitulate the phenotypic composition of the parental tumor. Melanomaspheres express high levels of Hedgehog (HH) pathway components and of embryonic pluripotent stem cell factors SOX2, NANOG, OCT4, and KLF4. We show that human melanomas contain a subset of cells expressing high ALDH activity (ALDH(high)), which is endowed with higher self-renewal and tumorigenic abilities than the ALDH(low) population. A good correlation between the number of ALDH(high) cells and sphere formation efficiency was observed. Notably, both pharmacological inhibition of HH signaling by the SMOOTHENED (SMO) antagonist cyclopamine and GLI antagonist GANT61 and stable expression of shRNA targeting either SMO or GLI1 result in a significant decrease in melanoma stem cell self-renewal in vitro and a reduction in the number of ALDH(high) melanoma stem cells. Finally, we show that interference with the HH-GLI pathway through lentiviral-mediated silencing of SMO and GLI1 drastically diminishes tumor initiation of ALDH(high) melanoma stem cells. In conclusion, our data indicate an essential role of the HH-GLI1 signaling in controlling self-renewal and tumor initiation of melanoma CSC/TIC. Targeting HH-GLI1 is thus predicted to reduce the melanoma stem cell compartment. Copyright © 2012 AlphaMed Press.

  14. The potential role of toll-like receptor 4 Asp299Gly polymorphism and its association with recurrent cystic echinococcosis in postoperative patients.

    PubMed

    Noori, Jafar; Spotin, Adel; Ahmadpour, Ehsan; Mahami-Oskouei, Mahmoud; Sadeghi-Bazargani, Homayoun; Kazemi, Tohid; Sakhinia, Ebrahim; Aghebati-Maleki, Leili; Shahrivar, Firooz

    2018-06-01

    The study of pathogenesis mechanisms of larval stages in the Taeniidae has recently focused on host genetic factors, particularly toll-like receptor (TLR) variations. However, the potential role of TLR4 polymorphism in hydatidosis has not yet been sufficiently elucidated in postoperative patients. In this case-control investigation, 80 patients from Iran, including 40 with acute hydatidosis (AH) and 40 with recurrent hydatidosis (RH), and 80 ethnically matched controls were evaluated from February 2015 to February 2017. Hydatidosis patients were confirmed using radiological, immunological, and histopathological examinations. Genotyping of Asp299Gly and Thr399Ile of TLR4 single-nucleotide polymorphisms was determined by restriction fragment length polymorphism, sequencing, and phylogenetic strategies. The heterozygous mutant-type TLR4 Asp299Gly genotype indicated a tendency to be associated with the occurrence of RH (P = 0.060) and conferred a 3-fold risk for susceptibility. There was no difference in genotype frequency of Asp299Gly between patients with AH and healthy controls (P = 0.42; OR, 1.82; 95% CI, 0.11-30.1%). Interestingly, a frequency of the G allele (12%: Gly) was observed to be a risk factor for susceptibility to RH patients (P = 0.050; OR, 7.08; 95% CI, 0.97-51.5%). A relative genetic variability of TLR4 Asp299Gly was found in RH patients (haplotype diversity: 0.700) compared to AH patients and healthy controls (Hd: 0.000). The Asp299Gly genotype was dominantly identified in patients with hepatic hydatid cysts. The TLR4 Thr399Ile codon was not detected except in a patient with a pulmonary hydatid cyst. The current findings enhance our knowledge regarding the TLR4 Asp299Gly polymorphism potentially leading to the development of RH, by skewing the immune system towards a Th2 response. Identification of the Asp299Gly codon may be a diagnostic hallmark in RH patients who have undergone unsuccessful postoperative intervention. However, further

  15. Dipeptidyl-peptidase IV inhibitory activity of peptides derived from tuna cooking juice hydrolysates.

    PubMed

    Huang, Shih-Li; Jao, Chia-Ling; Ho, Kit-Pan; Hsu, Kuo-Chiang

    2012-05-01

    The in vitro DPP-IV inhibitory activity of isolated peptides from of tuna cooking juice hydrolyzed by Protease XXIII (PR) and orientase (OR) was determined. The results showed that the peptide fractions with the molecular weight over 1,422 Da possessed the greatest DPP-IV inhibitory activity. The amino acid sequences of the three peptides isolated from PR and OR hydrolysates were identified by MALDI-TOF/TOF MS/MS, and they were Pro-Gly-Val-Gly-Gly-Pro-Leu-Gly-Pro-Ile-Gly-Pro-Cys-Tyr-Glu (1412.7 Da), Cys-Ala-Tyr-Gln-Trp-Gln-Arg-Pro-Val-Asp-Arg-Ile-Arg (1690.8 Da) and Pro-Ala-Cys-Gly-Gly-Phe-Try-Ile-Ser-Gly-Arg-Pro-Gly (1304.6 Da), while they showed the dose-dependent inhibition effect of DPP-IV with IC(50) values of 116.1, 78.0 and 96.4 μM, respectively. In vitro simulated gastrointestinal digestion retained or even improved the DPP-IV inhibitory activities of the three peptides. The results suggest that tuna cooking juice would be a good precursor of DPP-IV inhibitor, and the DPP-IV inhibitory peptides can successfully passed through the digestive tract. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Suppressor of Fused Chaperones Gli Proteins To Generate Transcriptional Responses to Sonic Hedgehog Signaling

    PubMed Central

    Zhang, Ziyu; Shen, Longyan; Law, Kelvin; Zhang, Zengdi; Liu, Xiaotong; Hua, Hu; Li, Sanen; Huang, Huijie; Yue, Shen; Hui, Chi-chung

    2016-01-01

    ABSTRACT Cellular responses to the graded Sonic Hedgehog (Shh) morphogenic signal are orchestrated by three Gli genes that give rise to both transcription activators and repressors. An essential downstream regulator of the pathway, encoded by the tumor suppressor gene Suppressor of fused (Sufu), plays critical roles in the production, trafficking, and function of Gli proteins, but the mechanism remains controversial. Here, we show that Sufu is upregulated in active Shh responding tissues and accompanies Gli activators translocating into and Gli repressors out of the nucleus. Trafficking of Sufu to the primary cilium, potentiated by Gli activators but not repressors, was found to be coupled to its nuclear import. We have identified a nuclear export signal (NES) motif of Sufu in juxtaposition to the protein kinase A (PKA) and glycogen synthase kinase 3 (GSK3) dual phosphorylation sites and show that Sufu binds the chromatin with both Gli1 and Gli3. Close comparison of neural tube development among individual Ptch1−/−, Sufu−/−, and Ptch1−/−; Sufu−/− double mutant embryos indicates that Sufu is critical for the maximal activation of Shh signaling essential to the specification of the most-ventral neurons. These data define Sufu as a novel class of molecular chaperone required for every aspect of Gli regulation and function. PMID:27849569

  17. The algicidal activity of Aeromonas sp. strain GLY-2107 against bloom-forming Microcystis aeruginosa is regulated by N-acyl homoserine lactone-mediated quorum sensing.

    PubMed

    Guo, Xingliang; Liu, Xianglong; Wu, Lishuang; Pan, Jianliang; Yang, Hong

    2016-11-01

    Cyanobacterial blooms have disrupted the efficient utilization of freshwater worldwide. A new freshwater bacterial strain with strong algicidal activity, GLY-2107, was isolated from Lake Taihu and identified as Aeromonas sp. It produced two algicidal compounds: 2107-A (3-benzyl-piperazine-2,5-dione) and 2107-B (3-methylindole). Both compounds exhibited potent algicidal activities against Microcystis aeruginosa, the dominant bloom-forming cyanobacterium in Lake Taihu. The EC 50 values (concentration for 50% maximal effect) of 3-benzyl-piperazine-2,5-dione and 3-methylindole were 4.72 and 1.10 μg ml -1 respectively. Based on a thin-layer chromatography biosensor assay and ultra-performance liquid chromatography-coupled high resolution-tandem mass spectrometry (UPLC-HRMS/MS), the N-acyl homoserine lactone (AHL) profile of strain GLY-2107 was identified as two short side-chain AHLs: N-butyryl-homoserine lactone (C4-HSL) and N-hexanoyl-homoserine lactone (C6-HSL). The production of the two algicidal compounds was controlled by AHL-mediated quorum sensing (QS), and C4-HSL was the key QS signal for the algicidal activity of the strain GLY-2107. Moreover, 3-methylindole was found to be positively regulated by C4-HSL-mediated QS, whereas 3-benzyl-piperazine-2,5-dione might be negatively controlled by C4-HSL-mediated QS. This study suggests that a QS-regulated algicidal system may have potential use for the development of a novel control strategy for harmful cyanobacterial blooms. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  18. Molecular Mechanism Underlying the Action of Substituted Pro-Gly Dipeptide Noopept.

    PubMed

    Vakhitova, Y V; Sadovnikov, S V; Borisevich, S S; Ostrovskaya, R U; A Gudasheva, T; Seredenin, S B

    2016-01-01

    This study was performed in order to reveal the effect of Noopept (ethyl ester of N-phenylacetyl-Lprolylglycine, GVS-111) on the DNA-binding activity of transcriptional factors (TF) in HEK293 cells transiently transfected with luciferase reporter constructs containing sequences for CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF-1. Noopept (10 μM) was shown to increase the DNA-binding activity of HIF-1 only, while lacking the ability to affect that of CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, and HSF1. Noopept provoked an additional increase in the DNA-binding activity of HIF-1 when applied in conditions of CoCl2-induced HIF- 1 stabilization. The degree of this HIF-positive effect of Noopept was shown to be concentration-dependent. Piracetam (1 mM) failed to affect significantly any of the TF under study. The results of molecular docking showed that Noopept (L-isomer), as well as its metabolite, L-isomer of N-phenyl-acetylprolyl, unlike its pharmacologically ineffective D-isomer, is able to bind to the active site of prolyl hydroxylase 2. Taking into account the important role of the genes activated by HIF-1 in the formation of an adaptive response to hypoxia, data on the ability of Noopept to provoke a selective increase in the DNA-binding activity of HIF-1 explain the wide spectrum of neurochemical and pharmacological effects of Noopept revealed before. The obtained data allow one to propose the HIF-positive effect as the primary mechanism of the activity of this Pro-Gly-containing dipeptide.

  19. Molecular Mechanism Underlying the Action of Substituted Pro-Gly Dipeptide Noopept

    PubMed Central

    Vakhitova, Y. V.; Sadovnikov, S. V.; Borisevich, S. S.; Ostrovskaya, R. U.; A.Gudasheva, T.; Seredenin, S. B.

    2016-01-01

    This study was performed in order to reveal the effect of Noopept (ethyl ester of N-phenylacetyl-Lprolylglycine, GVS-111) on the DNA-binding activity of transcriptional factors (TF) in HEK293 cells transiently transfected with luciferase reporter constructs containing sequences for CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF-1. Noopept (10 μM) was shown to increase the DNA-binding activity of HIF-1 only, while lacking the ability to affect that of CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, and HSF1. Noopept provoked an additional increase in the DNA-binding activity of HIF-1 when applied in conditions of CoCl2-induced HIF- 1 stabilization. The degree of this HIF-positive effect of Noopept was shown to be concentration-dependent. Piracetam (1 mM) failed to affect significantly any of the TF under study. The results of molecular docking showed that Noopept (L-isomer), as well as its metabolite, L-isomer of N-phenyl-acetylprolyl, unlike its pharmacologically ineffective D-isomer, is able to bind to the active site of prolyl hydroxylase 2. Taking into account the important role of the genes activated by HIF-1 in the formation of an adaptive response to hypoxia, data on the ability of Noopept to provoke a selective increase in the DNA-binding activity of HIF-1 explain the wide spectrum of neurochemical and pharmacological effects of Noopept revealed before. The obtained data allow one to propose the HIF-positive effect as the primary mechanism of the activity of this Pro-Gly-containing dipeptide. PMID:27099787

  20. GLI1 inhibitor GANT61 exhibits antitumor efficacy in T-cell lymphoma cells through down-regulation of p-STAT3 and SOCS3

    PubMed Central

    Geng, Lingyun; Lu, Kang; Li, Peipei; Li, Xinyu; Zhou, Xiangxiang; Li, Ying; Wang, Xin

    2017-01-01

    T-cell lymphomas are lymphoid malignancies with aggressive clinical course and poor prognosis. Increasing evidences suggest that deregulation of signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) is associated with the pathogenesis of T-cell lymphomas. The hedgehog (Hh)/glioma-associated oncogene-1 (GLI1) pathway, aberrantly activated in a number of tumors, has also been extensively studied. We found that protein expressions of GL11, p-STAT3, STAT3, and SOCS3 were up-regulated in T-cell lymphoma tissues and cell lines. Moreover, the protein expressions of p-STAT3 and SOCS3 were positively correlated with GLI1 in T-cell lymphomas. GLI1 inhibitor GANT61 and lentivirus-mediated siGLI1 exhibited inhibitory effects in the three T-cell lines (Jurkat, Karpass299 and Myla3676 cells). The protein expressions of p-STAT3 and SOCS3 were decreased accompanied with the inhibition of GLI1. These findings indicated that GANT61 is a promising agent against T-cell lymphoma and the antitumor activity might be partly mediated by down-regulating p-STAT3 and SOCS3. PMID:27275540

  1. GLI1 inhibition promotes epithelial-to-mesenchymal transition in pancreatic cancer cells

    PubMed Central

    Joost, Simon; Almada, Luciana L.; Rohnalter, Verena; Holz, Philipp S.; Vrabel, Anne M.; Fernandez-Barrena, Maite G.; McWilliams, Robert R.; Krause, Michael; Fernandez-Zapico, Martin E.; Lauth, Matthias

    2011-01-01

    The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility and synergized with TGFβ to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC. PMID:22086851

  2. PHEX Mimetic (SPR4-Peptide) Corrects and Improves HYP and Wild Type Mice Energy-Metabolism

    PubMed Central

    Zelenchuk, Lesya V.; Hedge, Anne-Marie; Rowe, Peter S. N.

    2014-01-01

    Context PHEX or DMP1 mutations cause hypophosphatemic-rickets and altered energy metabolism. PHEX binds to DMP1-ASARM-motif to form a complex with α5β3 integrin that suppresses FGF23 expression. ASARM-peptides increase FGF23 by disrupting the PHEX-DMP1-Integrin complex. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide/motif to study the DMP1-PHEX interaction and to assess SPR4 for the treatment of energy metabolism defects in HYP and potentially other bone-mineral disorders. Design Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle (VE) into wild-type mice (WT) and HYP-mice (PHEX mutation) for 4 weeks. Results SPR4 partially corrected HYP mice hypophosphatemia and increased serum 1.25(OH)2D3. Serum FGF23 remained high and PTH was unaffected. WT-SPR4 mice developed hypophosphatemia and hypercalcemia with increased PTH, FGF23 and 1.25(OH)2D3. SPR4 increased GAPDH HYP-bone expression 60× and corrected HYP-mice hyperglycemia and hypoinsulinemia. HYP-VE serum uric-acid (UA) levels were reduced and SPR4 infusion suppressed UA levels in WT-mice but not HYP-mice. SPR4 altered leptin, adiponectin, and sympathetic-tone and increased the fat mass/weight ratio for HYP and WT mice. Expression of perlipin-2 a gene involved in obesity was reduced in HYP-VE and WT-SPR4 mice but increased in HYP-SPR4 mice. Also, increased expression of two genes that inhibit insulin-signaling, ENPP1 and ESP, occurred with HYP-VE mice. In contrast, SPR4 reduced expression of both ENPP1 and ESP in WT mice and suppressed ENPP1 in HYP mice. Increased expression of FAM20C and sclerostin occurred with HYP-VE mice. SPR4 suppressed expression of FAM20C and sclerostin in HYP and WT mice. Conclusions ASARM peptides and motifs are physiological substrates for PHEX and modulate osteocyte PHEX-DMP1-α5β3-integrin interactions and thereby FGF23 expression. These interactions also provide a nexus that regulates bone and energy metabolism. SPR4 suppression of

  3. GLI1, a master regulator of the hallmark of pancreatic cancer.

    PubMed

    Kasai, Kenji

    2016-12-01

    Hedgehog signaling is highly conserved across species and governs proper embryonic development. Germline gene mutations that reduce this signaling activity cause a variety of developmental abnormalities such as holoprosencephaly, while those that enhance Hedgehog signaling activity induce a tumor-predisposition condition Nevoid basal cell carcinoma syndrome. Furthermore, dysregulated activation of Hedgehog signaling has been recognized in various sporadic malignancies, including pancreatic adenocarcinoma. Pancreatic adenocarcinoma develops through a multistep carcinogenesis starting with oncogenic mutation of the KRAS gene. During this process, precancerous or cancer cells secrete Hedgehog ligand proteins to promote characteristic desmoplastic stroma around the cells, which in turn activates the expression of the downstream transcription factor GLI1 inside the cells. The quantitative and spatiotemporal dysregulation of GLI1 subsequently leads to the expression of transcriptional target genes of GLI1 that govern the hallmark of malignant properties. Here, after a brief introductory outline, a perspective is offered of Hedgehog signaling with a special focus on the role of GLI1 in pancreatic carcinogenesis. © 2016 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

  4. Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells.

    PubMed

    Piirsoo, Alla; Kasak, Lagle; Kauts, Mari-Liis; Loog, Mart; Tints, Kairit; Uusen, Piia; Neuman, Toomas; Piirsoo, Marko

    2014-04-01

    Observations that Glioma-associated transcription factors Gli1 and Gli2 (Gli1/2), executers of the Sonic Hedgehog (Shh) signaling pathway and targets of the Transforming Growth Factor β (TGF-β) signaling axis, are involved in numerous developmental and pathological processes unveil them as attractive pharmaceutical targets. Unc-51-like serine/threonine kinase Ulk3 has been suggested to play kinase activity dependent and independent roles in the control of Gli proteins in the context of the Shh signaling pathway. This study aimed at investigating whether the mechanism of generation of Gli1/2 transcriptional activators has similarities regardless of the signaling cascade evoking their activation. We also elucidate further the role of Ulk3 kinase in regulation of Gli1/2 proteins and examine SU6668 as an inhibitor of Ulk3 catalytic activity and a compound targeting Gli1/2 proteins in different cell-based experimental models. Here we demonstrate that Ulk3 is required not only for maintenance of basal levels of Gli1/2 proteins but also for TGF-β or Shh dependent activation of endogenous Gli1/2 proteins in human adipose tissue derived multipotent stromal cells (ASCs) and mouse immortalized progenitor cells, respectively. We show that cultured ASCs possess the functional Shh signaling axis and differentiate towards osteoblasts in response to Shh. Also, we demonstrate that similarly to Ulk3 RNAi, SU6668 prevents de novo expression of Gli1/2 proteins and antagonizes the Gli-dependent activation of the gene expression programs induced by either Shh or TGF-β. Our data suggest SU6668 as an efficient inhibitor of Ulk3 kinase allowing manipulation of the Gli-dependent transcriptional outcome. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  5. The study on the relationship between IRS-1 Gly972Arg and IRS-2 Gly1057Asp polymorphisms and type 2 diabetes in the Kurdish ethnic group in West Iran.

    PubMed

    Haghani, Karimeh; Bakhtiyari, Salar

    2012-11-01

    An association between the IRS-1 Gly972Arg and IRS-2 Gly1057Asp polymorphisms and type 2 diabetes mellitus (T2DM) in different ethnic groups is controversial. We aimed to identify the association of these polymorphisms with T2DM in the Kurdish ethnic group of Iran. Study groups included 336 T2DM and 341 normoglycemic subjects. Genotyping was determined by polymerase chain reaction-restriction fragment length polymorphism. Genotypic and allelic frequencies were then evaluated. GR and RR genotypes of IRS-1 Gly972Arg variant gave a higher risk for T2DM (odds ratios [OR]=1.76 and OR=3.86, respectively). IRS-1 Gly972Arg polymorphism was found to be significantly associated with T2DM (OR=1.63) for the dominant model (GG vs. GR+RR). GD genotypes of the IRS-2 Gly1057Asp variant gave a higher risk for T2DM (OR=1.63). The dominant model analysis of the IRS-2 Gly1057Asp genotypes (GG vs. GD+DD) also showed an enhanced association with T2DM (OR=1.69). Among several combinations, GR/GD gave the highest risk for T2DM (OR=3.1). Other combinations were also significantly associated with T2DM, including, GR/GG (OR=1.86), RR/GG (OR=1.76), GG/GD (OR=1.83), and GG/DD (OR=2.35). HbA1c, serum triglyceride, and systolic blood pressure were higher in the control subjects with GR+RR genotypes compared with the GG genotype. Among the T2DM subjects, fasting plasma glucose was significantly lower in subjects with the GG genotype in relation to those with the GR+RR genotypes. Normoglycemic subjects carrying GD+DD genotypes of IRS-2 Gly1057Asp variation had a significantly higher fasting plasma glucose and total cholesterol, as compared with those with the GG genotype. Our findings revealed that IRS-1 Gly972Arg and IRS-2 Gly1057Asp polymorphisms are associated with T2DM in the Kurdish ethnic group.

  6. The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

    PubMed Central

    Gallagher, Lorna; Owens, Rebecca A.; Dolan, Stephen K.; O'Keeffe, Grainne; Schrettl, Markus; Kavanagh, Kevin; Jones, Gary W.

    2012-01-01

    The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H2O2 (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P = 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P < 0.001) and H2O2 (P < 0.01), unexpectedly, exogenous gliotoxin relieved H2O2-induced growth inhibition in a dose-dependent manner (0 to 10 μg/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P < 0.05) the growth of the ΔgliK26933 deletion mutant. The A. fumigatus ΔgliK26933 mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus ΔgliK26933 mutant than in those of the wild type (P = 0.0024 [fold difference, 24] and P = 0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus ΔgliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus ΔgliK26933 mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P = 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ΔgliK46645 mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus. PMID:22903976

  7. Large Conformational Changes of Insertion 3 in Human Glycyl-tRNA Synthetase (hGlyRS) during Catalysis

    DOE PAGES

    Deng, Xiangyu; Qin, Xiangjing; Chen, Lei; ...

    2016-01-21

    Glycyl-tRNA synthetase (GlyRS) is the enzyme that covalently links glycine to cognate tRNA for translation. It is of great interest because of its nonconserved quaternary structures, unique species-specific aminoacylation properties, and noncanonical functions in neurological diseases, but none of these is fully understood. We report two crystal structures of human GlyRS variants, in the free form and in complex with tRNA Gly respectively, and reveal new aspects of the glycylation mechanism. We discover that insertion 3 differs considerably in conformation in catalysis and that it acts like a "switch" and fully opens to allow tRNA to bind in a cross-subunitmore » fashion. The flexibility of the protein is supported by molecular dynamics simulation, as well as enzymatic activity assays. The biophysical and biochemical studies suggest that human GlyRS may utilize its flexibility for both the traditional function (regulate tRNA binding) and alternative functions (roles in diseases).« less

  8. Genetic polymorphisms in extracellular superoxide dismutase Leu53Leu, Arg213Gly, and Ala40Thr and susceptibility to type 2 diabetes mellitus.

    PubMed

    Yang, Y M; Xie, X R; Jin, A L

    2016-12-02

    The most common type of endocrine disease is type 2 diabetes mellitus (T2DM); genetic factors contribute to the development to T2DM. In this study, we investigated the role of the Leu53Leu, Arg213Gly, and Ala40Thr polymorphisms in extracellular superoxide dismutase (EC-SOD) gene in the development of T2DM in a Chinese population. DNA was extracted from peripheral blood samples obtained from 256 T2DM patients and 324 control subjects recruited from our hospital between January 2013 and March 2015. DNA was genotyped by polymerase chain reaction-restriction fragment length polymorphism. The obtained data was then statistically analyzed. The chi-square test revealed a statistically significant difference in the genotype frequencies of EC-SOD Ala40Thr (χ 2 = 13.26, P = 0.001) between the patients and controls. Unconditional regression analysis indicated that the GA and AA genotypes of EC-SOD Ala40Thr were associated with an increased risk of T2DM compared to the GG genotype {adjusted odds ratio (OR) [95% confidence interval (CI)] = 1.46 (1.01-2.11) and 2.67 (1.48-4.85), respectively}. In the dominant model, the GA+AA genotype of EC-SOD Ala40Thr was correlated with a higher risk of T2DM, in comparison with the GG genotype (OR = 1.64, 95%CI = 1.16-2.33). In the recessive model, AA of EC-SOD Ala40Thr showed a 2.19-fold higher risk of developing T2DM than the GG+GA genotype. In conclusion, people with the Ala40Thr polymorphism in EC-SOD are at a higher risk of developing T2DM; therefore, this may be utilized as a biomarker for early screening of T2DM in a Chinese population.

  9. Free energy landscapes of peptides by enhanced conformational sampling.

    PubMed

    Nakajima, N; Higo, J; Kidera, A; Nakamura, H

    2000-02-11

    The free energy landscapes of peptide conformations in water have been observed by the enhanced conformational sampling method, applying the selectively enhanced multicanonical molecular dynamics simulations. The conformations of the peptide dimers, -Gly-Gly-, -Gly-Ala-, -Gly-Ser-, -Ala-Gly-, -Asn-Gly-, -Pro-Gly-, -Pro-Ala-, and -Ala-Ala-, which were all blocked with N-terminal acetyl and C-terminal N-methyl groups, were individually sampled with the explicit TIP3P water molecules. From each simulation trajectory, we obtained the canonical ensemble at 300 K, from which the individual three-dimensional landscape was drawn by the potential of mean force using the three reaction coordinates: the backbone dihedral angle, psi, of the first amino acid, the backbone dihedral angle, phi, of the second amino acid, and the distance between the carbonyl oxygen of the N-terminal acetyl group and the C-terminal amide proton. The most stable state and several meta-stable states correspond to extended conformations and typical beta-turn conformations, and their free energy values were accounted for from the potentials of mean force at the states. In addition, the contributions from the intra-molecular energies of peptides and those from the hydration effects were analyzed. Consequently, the stable beta-turn conformations in the free energy landscape were consistent with the empirically preferred beta-turn types for each amino acid sequence. The thermodynamic values for the hydration effect were decomposed and they correlated well with the empirical values estimated from the solvent accessible surface area of each molecular conformation during the trajectories. The origin of the architecture of protein local fragments was analyzed from the viewpoint of the free energy and its decomposed factors. Copyright 2000 Academic Press.

  10. Characterization and receptor specific toxicity of two diphtheria toxin-related interleukin-3 fusion proteins DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3.

    PubMed

    Liger, D; vanderSpek, J C; Gaillard, C; Cansier, C; Murphy, J R; Leboulch, P; Gillet, D

    1997-04-07

    We have constructed two fusion proteins, DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3, in which the receptor-binding domain of diphtheria toxin is replaced by mouse interleukin-3 (IL-3). Cytotoxic activity of the fusion toxins was observed on three out of six cell lines assayed. This toxicity was mediated through binding to the IL-3 receptor as it was inhibited in a dose-dependent manner with murine IL-3 or anti-IL-3 neutralizing antibodies. DAB389-(Gly4Ser)2-mIL-3 was up to 5 times more toxic than DAB389-mIL-3, depending on the cell line (0.8 x 10(-10) M < IC50 < 3 x 10(-10) M). These proteins can be used for the detection of IL-3 receptors on mouse cells and should allow for the selective elimination of IL-3 receptor-positive pluripotent hematopoietic stem cells prior to bone marrow transplantation.

  11. Sodium caprate enables the blood pressure-lowering effect of Ile-Pro-Pro and Leu-Lys-Pro in spontaneously hypertensive rats by indirectly overcoming PepT1 inhibition.

    PubMed

    Gleeson, John P; Frías, Jesús M; Ryan, Sinéad M; Brayden, David J

    2018-04-20

    The tripeptides, Ile-Pro-Pro (IPP) and Leu-Lys-Pro (LKP), inhibit angiotensin-converting enzyme (ACE) resulting in lowered blood pressure. Our hypothesis was that the medium chain fatty acid permeation enhancer, sodium caprate (C 10 ), may prevent the decrease in permeability of the tripeptides when PepT1 is inhibited by glycyl-sarcosine (Gly-Sar), a situation that may occur in the presence of food hydrolysates. Using Caco-2 monolayers and isolated rat jejunal tissue, the apparent permeability coefficients (P app ) of [ 3 H]-IPP and [ 3 H]-LKP were assessed in the presence of Gly-Sar with and without C 10 . Gly-Sar decreased the P app of both tripeptides across monolayers and isolated jejunal tissue, but C 10 restored it. C 10 likely increased the paracellular permeability of the tripeptides, as indicated by immunofluorescence changes in tight junction proteins in Caco-2 monolayers accompanied by a concentration-dependent decrease in transepithelial electrical resistance (TEER). [ 3 H]-IPP and [ 3 H]-LKP were orally-gavaged to normal rats with Gly-Sar, C 10 , or with a mixture. Plasma levels of both peptides were reduced by Gly-Sar to less than half that of the levels detected in its absence, but were restored when C 10 was co-administered. In spontaneously hypertensive rats (SHRs), unlabelled IPP and LKP lowered blood pressure when delivered either by i.v. or oral routes. Oral gavage of Gly-Sar reduced the hypotensive action of peptides in SHRs, but the effect was restored in the presence of C 10 . In conclusion, there was a reduction in the hypotensive effects of IPP and LKP in SHRs when intestinal PepT1 was inhibited by Gly-Sar, but C 10 may circumvent this by enhancing paracellular permeability. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. The role of collagen charge clusters in the modulation of matrix metalloproteinase activity.

    PubMed

    Lauer, Janelle L; Bhowmick, Manishabrata; Tokmina-Roszyk, Dorota; Lin, Yan; Van Doren, Steven R; Fields, Gregg B

    2014-01-24

    Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-L-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23-P23' subsites of collagenous substrates.

  13. Kif3a Controls Murine Nephron Number Via GLI3 Repressor, Cell Survival, and Gene Expression in a Lineage-Specific Manner

    PubMed Central

    Chi, Lijun; Galtseva, Alevtina; Chen, Lin; Mo, Rong; Hui, Chi-chung; Rosenblum, Norman D.

    2013-01-01

    The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms. PMID:23762375

  14. Obesity is associated with the Arg389Gly ADRB1 but not with the Trp64Arg ADRB3 polymorphism in children from San Luis PotosÍ and León, México.

    PubMed

    Aradillas-Garc X Cd, Celia; Cruz, Miguel; Pérez-Luque, Elva; Garay-Sevilla, María E; Malacara, Juan M; R, Aduna; Peralta, Jesús; Burguete-García, Ana; Alegría-Torres, Jorge A

    2016-10-17

    This research was designed to analyze the possible associations of Arg389Gly ADRB1 and Trp64Arg ADRB3 polymorphisms in children with obesity. A cross-sectional study included 1,046 school-age Mexican participants (6-12 years old) from the cities of San Luis PotosÍ and León. Children were classified as non-obese or obese according to their body mass index (BMI) percentile; obese children had a BMI≥95th percentile for sex and age. Biochemical data were collected. Polymorphisms were detected using TaqMan qPCR assay. A logistic regression analysis was used to calculate the risk of obesity based on genotypes. Differences were found between groups where obese children had a significant increase in systolic and diastolic blood pressure, fasting plasma glucose, insulin, HOMA-IR, LDL-cholesterol, triglycerides, and lower HDL-cholesterol compared with the normal weight group (P<0.05). The distribution of allele frequency in the population was Arg= 87.4 and Gly= 12.6 (Hardy Weinberg equilibrium c 2 = 3.16 , P = 0.07 ); Trp= 81.5 and Arg= 18.5 (Hardy Weinberg equilibrium c 2 = 2.2, P = 0.14 ) for ADRB1 and ADRB3, respectively. Even though no different frequencies of Arg389Gly polymorphism between groups were found (P = 0.08), children carriers of one Gly389 ADRB1 allele had a risk for obesity of OR=1.40 (95%CI, 1.03-1.90, P = 0.03) after adjustment for age and gender. No other association was found for Trp64Arg ADRB3 polymorphism. Only the Arg389Gly ADRB1 polymorphism was associated with risk for obesity in Mexican children.

  15. Direct Interactions Between Gli3, Wnt8b, and Fgfs Underlie Patterning of the Dorsal Telencephalon.

    PubMed

    Hasenpusch-Theil, Kerstin; Watson, Julia A; Theil, Thomas

    2017-02-01

    A key step in the development of the cerebral cortex is a patterning process, which subdivides the telencephalon into several molecularly distinct domains and is critical for cortical arealization. This process is dependent on a complex network of interactions between signaling molecules of the Fgf and Wnt gene families and the Gli3 transcription factor gene, but a better knowledge of the molecular basis of the interplay between these factors is required to gain a deeper understanding of the genetic circuitry underlying telencephalic patterning. Using DNA-binding and reporter gene assays, we here investigate the possibility that Gli3 and these signaling molecules interact by directly regulating each other's expression. We show that Fgf signaling is required for Wnt8b enhancer activity in the cortical hem, whereas Wnt/β-catenin signaling represses Fgf17 forebrain enhancer activity. In contrast, Fgf and Wnt/β-catenin signaling cooperate to regulate Gli3 expression. Taken together, these findings indicate that mutual interactions between Gli3, Wnt8b, and Fgf17 are crucial elements of the balance between these factors thereby conferring robustness to the patterning process. Hence, our study provides a framework for understanding the genetic circuitry underlying telencephalic patterning and how defects in this process can affect the formation of cortical areas. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Role of toll-like receptor 4 Asp299Gly polymorphism in the development of cardiovascular diseases in HIV-infected patients.

    PubMed

    Tarancon-Diez, Laura; De Pablo-Bernal, Rebeca S; Jiménez, José L; Álvarez-Ríos, Ana I; Genebat, Miguel; Rosado-Sánchez, Isaac; Muñoz-Fernández, María-Ángeles; Ruiz-Mateos, Ezequiel; Leal, Manuel

    2018-05-15

    Cardiovascular diseases (CVDs) are one of the main causes of morbimortality in HIV-infected patients on suppressive antiretroviral therapy. The objective of this work was to evaluate the role of single nucleotide polymorphisms (SNPs) in lipopolysaccharide (LPS) Toll-like receptor 4 (TLR4) and CVDs occurrence in HIV-infected patients. Additionally, the functional consequences of carrying these SNPs were analyzed. The association of TLR4 SNPs, Asp299Gly/Thr399Ile with CVDs occurrence was analyzed using multivariate logistic regression models. Clinical, immunological, and traditional cardiovascular risk factors were used as covariates. The monocyte phenotype and response were assessed by multiparametric flow cytometry comparing carriers with noncarriers of this SNP. Asp299Gly SNP, assayed in 253 HIV-infected patients, was independently associated with the occurrence of CVDs after adjusting for CD4 T-cell nadir, HCV-coinfection, bacterial pneumonia, diabetes mellitus, and traditional cardiovascular risk factors [odds ratio (confidence interval 95%) = 3.672 (1.061-12.712), P = 0.04). Carriers of Asp299Gly SNP showed higher percentage of patrolling and intermediate monocytes producing a proinflammatory combination of cytokines compared with noncarriers (P = 0.037 and P = 0.046, respectively). Intermediate monocyte subset levels correlated with soluble interleukin-6 levels only in carriers (r = 0.89; P = 0.01). TLR4 Asp299Gly polymorphism is independently associated with the occurrence of CVDs in HIV-infected patients. The proinflammatory profile associated to this variant could be involved in the development of atherosclerotic pathologies.

  17. TLR4 Asp299Gly polymorphism may be protective against chronic periodontitis.

    PubMed

    Sellers, R M; Payne, J B; Yu, F; LeVan, T D; Walker, C; Mikuls, T R

    2016-04-01

    Periodontitis results from interplay between genetic and environmental factors. Single nucleotide polymorphisms (SNPs) in the coding region of the toll-like receptor 4 gene (TLR4) may be associated with periodontitis, although previous studies have been inconclusive. Moreover, the interaction between environmental factors, such as cigarette smoking (a major risk factor for periodontitis), and Porphyromonas gingivalis (a major periodontal pathogen) with the TLR4 coding region Asp299Gly SNP (rs4986790; a SNP associated with lipopolysaccharide-mediated inflammatory responses in periodontitis), have been largely ignored in previous reports. Therefore, the objective of this study was to examine the association between TLR4 Asp299Gly (rs4986790) with alveolar bone height loss (ABHL) and periodontitis, accounting for interactions between this SNP with smoking and P. gingivalis prevalence. The CD14/-260 SNP (rs2569190) served as a control, as a recent meta-analysis suggested no relationship between this SNP and periodontitis. This multicenter study included 617 participants who had rheumatoid arthritis or osteoarthritis. This report presents a secondary outcome from the primary case-control study examining the relationship of periodontitis with established rheumatoid arthritis. The Centers for Disease Control/American Academy of Periodontology case definitions of periodontitis were used for this analysis. Participants received a full-mouth clinical periodontal examination and panoramic radiograph. Percentage ABHL was measured on posterior teeth. The TLR4 Asp299Gly and CD14/-260 SNPs were selected a priori and genotypes were determined using the ImmunoChip array (Illumina(®) ). Minor allele frequencies and associations with periodontitis and ABHL did not differ according to rheumatoid arthritis vs. osteoarthritis status; therefore, data from these two groups were pooled. The presence of P. gingivalis was detected in subgingival plaque by PCR. Multivariate ordinal

  18. Solution nuclear magnetic resonance analyses of the anticodon arms of proteinogenic and nonproteinogenic tRNA(Gly).

    PubMed

    Chang, Andrew T; Nikonowicz, Edward P

    2012-05-01

    Although the fate of most tRNA molecules in the cell is aminoacylation and delivery to the ribosome, some tRNAs are destined to fulfill other functional roles. In addition to their central role in translation, tRNA molecules participate in processes such as regulation of gene expression, bacterial cell wall biosynthesis, viral replication, antibiotic biosynthesis, and suppression of alternative splicing. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extratranslational functions, including transcriptional regulation and cell wall biosynthesis. We have determined the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC. Two of the tRNA molecules are proteinogenic (tRNA(Gly,GCC) and tRNA(Gly,UCC)), and the third is nonproteinogenic (np-tRNA(Gly,UCC)) and participates in cell wall biosynthesis. The UV-monitored thermal melting curves show that the anticodon arm of tRNA(Gly,UCC) with a loop-closing C-A(+) base pair melts at a temperature 10 °C lower than those of tRNA(Gly,GCC) and np-tRNA(Gly,UCC). U-A and C-G pairs close the loops of the latter two molecules and enhance stem stability. Mg(2+) stabilizes the tRNA(Gly,UCC) anticodon arm and reduces the T(m) differential. The structures of the three tRNA(Gly) anticodon arms exhibit small differences among one another, but none of them form the classical U-turn motif. The anticodon loop of tRNA(Gly,GCC) becomes more dynamic and disordered in the presence of multivalent cations, whereas metal ion coordination in the anticodon loops of tRNA(Gly,UCC) and np-tRNA(Gly,UCC) establishes conformational homogeneity. The conformational similarity of the molecules is greater than their functional differences might suggest. Because aminoacylation of full-length tRNA molecules is accomplished by one tRNA synthetase, the similar structural context of the loop may facilitate efficient recognition of each of the anticodon sequences.

  19. PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

    PubMed Central

    Barrera, Susana P.; Castrejon-Tellez, Vicente; Trinidad, Margarita; Robles-Escajeda, Elisa; Vargas-Medrano, Javier; Varela-Ramirez, Armando; Miranda, Manuel

    2015-01-01

    Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40–50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications. PMID:26418248

  20. A Logical OR Redundancy within the Asx-Pro-Asx-Gly Type 1 {Beta}-Turn Motif

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Jihun; Dubey, Vikash Kumar; Longo, Lian M.

    2008-04-19

    Turn secondary structure is essential to the formation of globular protein architecture. Turn structures are, however, much more complex than either {alpha}-helix or {beta}-sheet, and the thermodynamics and folding kinetics are poorly understood. Type I {beta}-turns are the most common type of reverse turn, and they exhibit a statistical consensus sequence of Asx-Pro-Asx-Gly (where Asx is Asp or Asn). A comprehensive series of individual and combined Asx mutations has been constructed within three separate type I 3:5 G1 bulge {beta}-turns in human fibroblast growth factor-1, and their effects on structure, stability, and folding have been determined. The results show amore » fundamental logical OR relationship between the Asx residues in the motif, involving H-bond interactions with main-chain amides within the turn. These interactions can be modulated by additional interactions with residues adjacent to the turn at positions i + 4 and i + 6. The results show that the Asx residues in the turn motif make a substantial contribution to the overall stability of the protein, and the Asx logical OR relationship defines a redundant system that can compensate for deleterious point mutations. The results also show that the stability of the turn is unlikely to be the prime determinant of formation of turn structure in the folding transition state.« less

  1. Towards Understanding the Tandem Mass Spectra of Protonated Oligopeptides. 2: The Proline Effect in Collision-Induced Dissociation of Protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp)

    NASA Astrophysics Data System (ADS)

    Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G.; Paizs, Béla

    2011-06-01

    The product ion spectra of proline-containing peptides are commonly dominated by y n ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y 2 ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y 2 / b 3 abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y 2 / b 3 abundance ratio decreases.

  2. Towards understanding the tandem mass spectra of protonated oligopeptides. 2: The proline effect in collision-induced dissociation of protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp).

    PubMed

    Bleiholder, Christian; Suhai, Sándor; Harrison, Alex G; Paizs, Béla

    2011-06-01

    The product ion spectra of proline-containing peptides are commonly dominated by y(n) ions generated by cleavage at the N-terminal side of proline residues. This proline effect is investigated in the current work by collision-induced dissociation (CID) of protonated Ala-Ala-Xxx-Pro-Ala (Xxx includes Ala, Ser, Leu, Val, Phe, and Trp) in an electrospray/quadrupole/time-of-flight (QqTOF) mass spectrometer and by quantum chemical calculations on protonated Ala-Ala-Ala-Pro-Ala. The CID spectra of all investigated peptides show a dominant y(2) ion (Pro-Ala sequence). Our computational results show that the proline effect mainly arises from the particularly low threshold energy for the amide bond cleavage N-terminal to the proline residue, and from the high proton affinity of the proline-containing C-terminal fragment produced by this cleavage. These theoretical results are qualitatively supported by the experimentally observed y(2)/b(3) abundance ratios for protonated Ala-Ala-Xxx-Pro-Ala (Xxx = Ala, Ser, Leu, Val, Phe, and Trp). In the post-cleavage phase of fragmentation the N-terminal oxazolone fragment with the Ala-Ala-Xxx sequence and Pro-Ala compete for the ionizing proton for these peptides. As the proton affinity of the oxazolone fragment increases, the y(2)/b(3) abundance ratio decreases.

  3. Biphalin analogs containing β(3)-homo-amino acids at the 4,4' positions: Synthesis and opioid activity profiles.

    PubMed

    Frączak, Oliwia; Lasota, Anika; Kosson, Piotr; Leśniak, Anna; Muchowska, Adriana; Lipkowski, Andrzej W; Olma, Aleksandra

    2015-04-01

    Biphalin, a synthetic opioid octapeptide with a palindromic sequence has high analgesic activity. Biphalin displays a strong affinity for μ and δ-opioid receptors, and a significant to κ-receptor. The paper reports the synthesis of novel analogs of biphalin containing β(3)-homo-amino acid residues at the 4,4' positions and a hydrazine or 1,2-phenylenediamine linker. The potency and selectivity of the peptides were evaluated by a competitive receptor-binding assay in rat brain homogenate using [(3)H]DAMGO (a μ ligand) and [(3)H]DELT (a δ ligand). Analogs with β(3)-h-p-NO2Phe in positions 4 and 4' are the most active compounds. Selectivity depends on the degree of freedom between the two pharmacophore moieties. Analogs with a hydrazine linker show noticeable binding selectivity to μ receptors (IC50(μ)=0.72nM; IC50(δ)=4.66nM), while the peptides with a 1,2-phenylenediamine linker show slight δ selectivity (IC50(μ)=10.97nM; IC50(δ)=1.99nM). Tyr-d-Ala-Gly-β(3)-h-p-NO2PheNHNH-β(3)-h-p-NO2Phe (1) and (Tyr-d-Ala-Gly-β(3)-h-p-NO2PheNH)2 (2) produced greater antinociceptive effect compared to morphine after i.t. administration. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. May TLR4 Asp299Gly and IL17 His161Arg polymorphism be associated with progression of primary measles infection to subacute sclerosing panencephalitis?

    PubMed

    Karakas-Celik, Sevim; Piskin, Ibrahim Etem; Keni, Mehmet Fatih; Calık, Mustafa; Iscan, Akın; Dursun, Ahmet

    2014-09-01

    SSPE is a progressive neurological disorder of children. Only some of the children who are infected with measles virus develop SSPE, which supports individual variation. TLR-2 and TLR-4 play an important role in innate immunity by recognizing envelope proteins of MV. Another important cytokine that plays an important role in orchestrating innate immune function is IL-17. The purpose of our study is to elucidate whether the TLR2, TLR4, IL17F and IL17A gene polymorphisms are susceptibility genes for the development of SSPE. Using the PCR-RFLP methods, the single nucleotide polymorphisms of TLR2 (Arg753Gln, Arg677Trp, -194 to -174 del), TLR4 (Asp299Gly and Thr399Ile) IL17F (His161Arg, Glu126Gly) and IL17A were studied in 54 patients with SSPE and 81 healthy controls. For Asp299Gly polymorphism of the TLR4 gene we found that there were no control individuals who were homozygous carriers of the Gly/Gly genotype, and the risk for SSPE increased at approximately 4.7 fold for the heterozygous carriers of the Asp/Gly genotype (OR 4.727, 95%-CI 1.192-18.742; P=0.01), when compared to healthy controls. Also our findings demonstrate that homozygosity for the Arg161 variant of the IL17F His161Arg polymorphism is inversely associated with development of SSPE (OR 0.114 95%-CI 0.026-0.494; P<0.001). In conclusion, it is suggested that variation in susceptibility to SSPE disease may be in part due to variations in TLR4 and IL17 function resulting from polymorphisms of TLR4 Asp299Gly and IL17F His161Arg. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. New cytotoxic cyclic peptides and dianthramide from Dianthus superbus.

    PubMed

    Hsieh, Pei-Wen; Chang, Fang-Rong; Wu, Ching-Chung; Wu, Kuen-Yuh; Li, Chien-Ming; Chen, Su-Li; Wu, Yang-Chang

    2004-09-01

    Four new cyclic peptides, dianthins C-F (1-4), and a new dianthramide, 4-methoxydianthramide B (5), were isolated from the MeOH extract of the traditional Chinese medicinal plant Dianthus superbus. The sequences of cyclic peptides 1-4 were elucidated as cyclo(Gly(1)-Pro(2)-Phe(3)-Tyr(4)-Val(5)-Ile(6)-), cyclo(Gly(1)-Ser(2)-Leu(3)-Pro(4)-Pro(5)-Ile(6)-Phe(7)-), cyclo(Gly(1)-Pro(2)-Ile(3)-Ser(4)-Phe(5)-Val(6)-), and cyclo(Gly(1)-Pro(2)-Phe(3)-Val(4)-Phe(5)-) on the basis of ESI tandem mass fragmentation analysis, chemical evidence, and extensive 2D NMR methods. The conformation of compound 1 was established as an alpha-helix by CD analysis. Furthermore, compounds 3 and 5 showed cytotoxicities toward the Hep G2 cancer cell line with IC(50) values of 2.37 and 4.08, respectively.

  6. Characterization of the novel GlyT1 PET tracer [18F]MK-6577 in humans.

    PubMed

    Joshi, Aniket D; Sanabria-Bohórquez, Sandra M; Bormans, Guy; Koole, Michel; De Hoon, Jan; Van Hecken, Anne; Depre, Marleen; De Lepeleire, Inge; Van Laere, Koen; Sur, Cyrille; Hamill, Terence G

    2015-01-01

    Decreased glutamatergic neurotransmission is hypothesized to be involved in the pathophysiology of schizophrenia. Inhibition of glycine transporter Type-1 (GlyT1) reuptake is expected to increase the glutamatergic neurotransmission and may serve as treatment for cognitive and negative symptoms of schizophrenia. In this article, we present human data from a novel GlyT1 PET tracer, [(18) F]MK-6577. In the process of developing a GlyT1 inhibitor therapeutic, a PET tracer can assist in determining the dose with a high probability of sufficiently testing the mechanism of action. This article reports the human PET studies with [(18) F]MK-6577 for measuring GlyT1 receptor availability at baseline in normal human subjects and occupancy with a GlyT1 inhibitor, MK-2637. Studies were also performed to measure radiation burden and the baseline test-retest (T-RT) variability of the tracer. The effective dose from sequential whole-body dosimetry scans in three male subjects was estimated to be 24.5 ± 2.9 µSV/MBq (mean ± SD). The time-activity curves from T-RT scans modeled satisfactorily using a two tissue compartmental model. The tracer uptake was highest in the pons (VT  = 6.7 ± 0.9, BPND  = 4.1 ± 0.43) and lowest in the cortex (VT  = 2.1 ± 0.5, BPND  = 0.60 ± 0.23). VT T-RT variability measured in three subjects was <12% on average. The occupancy scans performed in a cohort of 15 subjects indicated absence of a reference region. The in vivo potency (Occ50 ) of MK-2637 was determined using two methods: A: Lassen plot with a population input function (Occ50  = 106 nM, SE = 20 nM) and B: pseudo reference tissue model using cortex as the pseudo reference region (Occ50  = 141 nM, SE = 21 nM). © 2014 Wiley Periodicals, Inc.

  7. New 2',6'-dimethyl-L-tyrosine (Dmt) opioid peptidomimetics based on the Aba-Gly scaffold. Development of unique mu-opioid receptor ligands.

    PubMed

    Ballet, Steven; Salvadori, Severo; Trapella, Claudio; Bryant, Sharon D; Jinsmaa, Yunden; Lazarus, Lawrence H; Negri, Lucia; Giannini, Elisa; Lattanzi, Roberta; Tourwé, Dirk; Balboni, Gianfranco

    2006-06-29

    The Aba-Gly scaffold, incorporated into Dmt-Tic ligands (H-Dmt-Tic-Gly-NH-CH2-Ph, H-Dmt-Tic-Gly-NH-Ph, H-Dmt-Tic-NH-CH2-Bid), exhibited mixed micro/delta or delta opioid receptor activities with micro agonism. Substitution of Tic by Aba-Gly coupled to -NH-CH2-Ph (1), -NH-Ph (2), or -Bid (Bid=1H-benzimidazole-2-yl) (3) shifted affinity (Ki(micro)=0.46, 1.48, and 19.9 nM, respectively), selectivity, and bioactivity to micro-opioid receptors. These compounds represent templates for a new class of lead opioid agonists that are easily synthesized and suitable for therapeutic pain relief.

  8. SPOP suppresses tumorigenesis by regulating Hedgehog/Gli2 signaling pathway in gastric cancer.

    PubMed

    Zeng, Chunyan; Wang, Yao; Lu, Quqin; Chen, Jiang; Zhang, Junyan; Liu, Tao; Lv, Nonghua; Luo, Shiwen

    2014-09-11

    Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including gastric cancer. Speckle-type POZ protein, SPOP, is an E3 ubiquitin ligase adaptor, and it is found to inhibit oncogenic signaling. However, the molecular mechanisms are largely unknown. In this study, we characterized the expression of SPOP in 88 pairs of gastric cancer tissues and adjacent tissues by immunohistochemical staining and Western blotting. The relationship between SPOP expression and clinical pathologic factors was analyzed. Transfected gastric cancer cell lines were used in cell viability, wound healing and colony formation assays. The interaction of SPOP with Gli2 and other related apoptotic proteins was assessed by immunoprecipitation, Western blotting, real-time PCR and dual luciferase reporter assays. Intracellular interaction of SPOP and Gli2 was visualized by immunofluorescent staining in gastric cancer cells. Immunohistochemical staining of SPOP can be detected in gastric cancer tissues but much less than adjacent gastric tissues (P < 0.01). High SPOP expression is negatively correlated with lymph node metastasis, poor histological differentiation, and tumor malignancy according to TNM staging. In vitro experiments revealed that over-expression of SPOP prevented tumor cells from proliferation, migration and colony formation in gastric cancer cell lines. Likewise, repression of SPOP promoted cell viability, migration, proliferation, and attenuated apoptosis. Mechanistic studies revealed that increasing SPOP accelerated Gli2 degradation but regardless of Gli2 synthesis. Furthermore, cytoplasmic Gli2 decreased markedly along with the abundant expression of SPOP in MKN45 cells. Our findings indicate that SPOP plays critical roles in suppressing gastric tumorigenesis through inhibiting Hh/Gli2 signaling pathway. It may provide an alternative strategy for developing

  9. Alanine and glycine conjugates of (2S,4R)-4-[18F]fluoroglutamine for tumor imaging.

    PubMed

    Zha, Zhihao; Ploessl, Karl; Lieberman, Brian P; Wang, Limin; Kung, Hank F

    2018-05-01

    Glutamine is an essential source of energy, metabolic substrates, and building block for supporting tumor proliferation. Previously, (2S,4R)-4-[ 18 F]fluoroglutamine (4F-Gln) was reported as a glutamine-related metabolic imaging agent. To improve the in vivo kinetics of this radiotracer, two new dipeptides, [ 18 F]Gly-(2S,4R)4-fluoroglutamine (Gly-4F-Gln) and [ 18 F]Ala-(2S,4R)4-fluoroglutamine (Ala-4F-Gln) were investigated. Radiolabeling was performed via 2-steps 18 F-fluorination. Cell uptake studies of Gly-4F-Gln and Ala-4F-Gln were investigated in 9 L cell lines. In vitro and in vivo metabolism studies were carried out in Fisher 344 rats. Biodistribution and microPET imaging studies were performed in 9 L tumor-bearing rats. In vitro incubation of these [ 18 F]dipeptides in rat and human blood showed a rapid conversion to (2S,4R)-4-[ 18 F]fluoroglutamine (t 1/2  = 2.3 and 0.2 min for [ 18 F]Gly-4F-Gln and [ 18 F]Ala-4F-Gln, respectively for human blood). Biodistribution and PET imaging in Fisher 344 rats bearing 9 L tumor xenografts showed that these dipeptides rapidly localized in the tumors, comparable to that of (2S,4R)-4-[ 18 F]fluoroglutamine (4F-Gln). The results support that these dipeptides, [ 18 F]Gly-4F-Gln and [ 18 F]Ala-4F-Gln, are prodrugs, which hydrolyze in the blood after an iv injection. They appear to be selectively taken up and trapped by tumor tissue in vivo. The dipeptide, [ 18 F]Ala-4F-Gln, may be suitable as a PET tracer for imaging glutaminolysis in tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. Purification and characterization of galanin and scyliorhinin I from the hybrid sturgeon, Scaphirhynchus platorynchus x Scaphirhynchus albus (Acipenseriformes).

    PubMed

    Wang, Y; Barton, B A; Thim, L; Nielsen, P F; Conlon, J M

    1999-01-01

    The sturgeons (order Acipenseriformes) are extant representatives of a group of ancient Actinopterygian (ray-finned) fish. Galanin and scyliorhinin I (a tachykinin with limited structural similarity to mammalian substance P) have been isolated from an extract of the gastrointestinal tract of a sturgeon (an F1 hybrid between the shovelnose sturgeon, Scaphirhynchus platorynchus, and the pallid sturgeon, Scaphirhynchus albus). The primary structure of sturgeon galanin (Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu10-Leu-Gly-Pro-His-Ala-Val -As p-Gly-His-Arg20-Ser-Leu-Ser-Asp-Lys-His-Gly-Leu-Pro.NH2) contains only two amino acid substitutions (Ser23 --> Asn and Pro29 --> Ala) compared with galanin from the bowfin, Amia calva (Amiiformes), but five amino acid substitutions compared with galanin from the trout (Teleostei). Similarly, the sturgeon tachykinin (Ser-Lys-Tyr-His-Gln-Phe-Tyr-Gly-Leu-Met.NH2) contains only one amino acid substitution (Tyr3 --> Ser) compared with scyliorhinin I previously isolated from bowfin stomach but five amino acid substitutions compared with trout substance P. The data support the hypothesis that the Acipenseriformes and the basal Neopterygians (gars and bowfin) share a close phylogenetic relationship. Copyright 1999 Academic Press.

  11. GLI inhibitor GANT-61 diminishes embryonal and alveolar rhabdomyosarcoma growth by inhibiting Shh/AKT-mTOR axis

    PubMed Central

    Srivastava, Ritesh K.; Kaylani, Samer Zaid; Edrees, Nayf; Li, Changzhao; Talwelkar, Sarang S.; Xu, Jianmin; Palle, Komaraiah; Pressey, Joseph G.; Athar, Mohammad

    2014-01-01

    Rhabdomyosarcoma (RMS) typically arises from skeletal muscle. Currently, RMS in patients with recurrent and metastatic disease have no successful treatment. The molecular pathogenesis of RMS varies based on cancer sub-types. Some embryonal RMS but not other sub-types are driven by sonic hedgehog (Shh) signaling pathway. However, Shh pathway inhibitors particularly smoothened inhibitors are not highly effective in animals. Here, we show that Shh pathway effectors GLI1 and/or GLI2 are over-expressed in the majority of RMS cells and that GANT-61, a specific GLI1/2 inhibitor dampens the proliferation of both embryonal and alveolar RMS cells-derived xenograft tumors thereby blocking their growth. As compared to vehicle-treated control, about 50% tumor growth inhibition occurs in mice receiving GANT-61 treatment. The proliferation inhibition was associated with slowing of cell cycle progression which was mediated by the reduced expression of cyclins D1/2/3 & E and the concomitant induction of p21. GANT-61 not only reduced expression of GLI1/2 in these RMS but also significantly diminished AKT/mTOR signaling. The therapeutic action of GANT-61 was significantly augmented when combined with chemotherapeutic agents employed for RMS therapy such as temsirolimus or vincristine. Finally, reduced expression of proteins driving epithelial mesenchymal transition (EMT) characterized the residual tumors. PMID:25432075

  12. A Distinct Malignant Epithelioid Neoplasm With GLI1 Gene Rearrangements, Frequent S100 Protein Expression, and Metastatic Potential: Expanding the Spectrum of Pathologic Entities With ACTB/MALAT1/PTCH1-GLI1 Fusions.

    PubMed

    Antonescu, Cristina R; Agaram, Narasimhan P; Sung, Yun-Shao; Zhang, Lei; Swanson, David; Dickson, Brendan C

    2018-04-01

    ACTB-GLI1 fusions have been reported as the pathognomonic genetic abnormality defining an unusual subset of actin-positive, perivascular myoid tumors, known as "pericytoma with the t(7;12) translocation." In addition, GLI1 oncogenic activation through a related MALAT1-GLI1 gene fusion has been recently reported in 2 unrelated gastric tumors, namely plexiform fibromyxoma and gastroblastoma. Triggered by unexpected targeted RNA-sequencing results detecting GLI1-related fusions in a group of malignant neoplasms with round to epithelioid morphology, and frequently strong S100 protein immunoreactivity, we investigated their clinicopathologic features in relation to other known pathologic entities sharing similar genetics. On the basis of a combined approach of targeted RNA sequencing and fluorescence in situ hybridization screening, we identified 6 cases with GLI1 gene fusions, including 4 fused to ACTB, 1 with MALAT1 and 1 with PTCH1 gene. Patients had a mean age of 36 years at diagnosis (range, 16 to 79 y) and slight female predilection all except 1 tumor originated in the soft tissue. Microscopically, the tumors had a monomorphic epithelioid phenotype arranged in a distinctive nested or cord-like architecture, separated by thin septae and delicate capillary network. All except 2 cases were strongly positive for S100 protein, whereas being negative for SOX10, SMA, and EMA. Only 1 tumor showed focal cytokeratin positivity in rare cells. Although the tumors showed some resemblance to pericytic/glomus tumors or myoepithelial tumors, the immunoprofile was not supportive of either lineage. Moreover, in contrast to the benign course of so-called pericytoma with t(7;12), 3 patients in this series developed metastatic disease to either lymph nodes or lung. In fact the only patient with lung metastases showed a novel PTCH1-GLI1 gene fusion. It remains to be determined whether these tumors represent a clinically and immunohistologically distinct subset of pericytoma, or an

  13. Expression profiling identifies novel Hh/Gli regulated genes in developing zebrafish embryos.

    PubMed Central

    Bergeron, Sadie A.; Milla, Luis A.; Villegas, Rosario; Shen, Meng-Chieh; Burgess, Shawn M.; Allende, Miguel L.; Karlstrom, Rolf O.; Palma, Verónica

    2008-01-01

    The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Mis-regulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants have been identified affecting different components of the Hh/Gli signaling system. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified several known (e.g. ptc1 and nkx2.2a) as well as a large number of novel Hh regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of mis-regulation during tumorogenesis. PMID:18055165

  14. Ruthenium-Nitrosyl Complexes with Glycine, l-Alanine, l-Valine, l-Proline, d-Proline, l-Serine, l-Threonine, and l-Tyrosine: Synthesis, X-ray Diffraction Structures, Spectroscopic and Electrochemical Properties, and Antiproliferative Activity

    PubMed Central

    2014-01-01

    The reactions of [Ru(NO)Cl5]2– with glycine (Gly), l-alanine (l-Ala), l-valine (l-Val), l-proline (l-Pro), d-proline (d-Pro), l-serine (l-Ser), l-threonine (l-Thr), and l-tyrosine (l-Tyr) in n-butanol or n-propanol afforded eight new complexes (1–8) of the general formula [RuCl3(AA–H)(NO)]−, where AA = Gly, l-Ala, l-Val, l-Pro, d-Pro, l-Ser, l-Thr, and l-Tyr, respectively. The compounds were characterized by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), 1H NMR, UV–visible and ATR IR spectroscopy, cyclic voltammetry, and X-ray crystallography. X-ray crystallography studies have revealed that in all cases the same isomer type (from three theoretically possible) was isolated, namely mer(Cl),trans(NO,O)-[RuCl3(AA–H)(NO)], as was also recently reported for osmium analogues with Gly, l-Pro, and d-Pro (see Z. Anorg. Allg. Chem.2013, 639, 1590–1597). Compounds 1, 4, 5, and 8 were investigated by ESI-MS with regard to their stability in aqueous solution and reactivity toward sodium ascorbate. In addition, cell culture experiments in three human cancer cell lines, namely, A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma), were performed, and the results are discussed in conjunction with the lipophilicity of compounds. PMID:24555845

  15. Glycyl-alanyl-histidine protects PC12 cells against hydrogen peroxide toxicity.

    PubMed

    Shimura, Hideki; Tanaka, Ryota; Shimada, Yoshiaki; Yamashiro, Kazuo; Hattori, Nobutaka; Urabe, Takao

    2017-11-22

    Peptides with cytoprotective functions, including antioxidants and anti-infectives, could be useful therapeutics. Carnosine, β-alanine-histidine, is a dipeptide with anti-oxidant properties. Tripeptides of Ala-His-Lys, Pro-His-His, or Tyr-His-Tyr are also of interest in this respect. We synthesized several histidine-containing peptides including glycine or alanine, and tested their cytoprotective effects on hydrogen peroxide toxicity for PC12 cells. Of all these peptides (Gly-His-His, Ala-His-His, Ala-His-Ala, Ala-Ala-His, Ala-Gly-His, Gly-Ala-His (GAH), Ala-His-Gly, His-Ala-Gly, His-His-His, Gly-His-Ala, and Gly-Gly-His), GAH was found to have the strongest cytoprotective activity. GAH decreased lactate dehydrogenase (LDH) leakage, apoptosis, morphological changes, and nuclear membrane permeability changes against hydrogen peroxide toxicity in PC12 cells. The cytoprotective activity of GAH was superior to that of carnosine against hydrogen peroxide toxicity in PC12 cells. GAH also protected PC12 cells against damage caused by actinomycin D and staurosporine. Additionally, it was found that GAH also protected SH-SY5Y and Jurkat cells from damage caused by hydrogen peroxide, as assessed by LDH leakage. Thus, a novel tripeptide, GAH, has been identified as having broad cytoprotective effects against hydrogen peroxide-induced cell damage.

  16. Inhibition of Gli1 mobilizes endogenous neural stem cells for remyelination

    PubMed Central

    Samanta, Jayshree; Grund, Ethan M.; Silva, Hernandez M.; Lafaille, Juan J.; Fishell, Gord; Salzer, James L.

    2016-01-01

    Summary Enhancing repair of myelin is an important, but still elusive therapeutic goal in many neurological disorders1. In Multiple Sclerosis (MS), an inflammatory demyelinating disease, endogenous remyelination does occur but is frequently insufficient to restore function. Both parenchymal oligodendrocyte progenitor cells (OPCs) and endogenous adult neural stem cells (NSCs) resident within the subventricular zone (SVZ) are known sources of remyelinating cells2. Here, we characterize the contribution to remyelination of a subset of adult NSCs, identified by their expression of Gli1, a transcriptional effector of the Sonic Hedgehog (Shh) pathway. We show that these cells are recruited from the SVZ to populate demyelinated lesions in the forebrain but never enter healthy, white matter tracts. Unexpectedly, recruitment of this pool of NSCs, and their differentiation into oligodendrocytes, is significantly enhanced by genetic or pharmacological inhibition of Gli1. Importantly, complete inhibition of canonical hedgehog signaling was ineffective indicating that Gli1’s role in both augmenting hedgehog signaling and retarding myelination is specialized. Indeed, inhibition of Gli1 improves the functional outcome in a relapsing/remitting model of experimental autoimmune encephalomyelitis (RR-EAE) and is neuroprotective. Thus, endogenous NSCs can be mobilized for the repair of demyelinated lesions by inhibiting Gli1, identifying a new therapeutic avenue for the treatment of demyelinating disorders. PMID:26416758

  17. The SHH/Gli axis regulates CD90-mediated liver cancer stem cell function by activating the IL6/JAK2 pathway.

    PubMed

    Zhang, Ketao; Che, Siyao; Pan, Chuzhi; Su, Zheng; Zheng, Shangyou; Yang, Shanglin; Zhang, Huayao; Li, Wenda; Wang, Weidong; Liu, Jianping

    2018-05-02

    The cell surface antigen CD90 has recently been established as a promising marker for liver cancer stem cells. This study aimed to investigate potential implications of SHH/Gli signalling in CD90+ liver cancer stem cells. Correlation of the expression of SHH signalling components and CD90 in liver cancer cells and clinical tissues, as well as in enriched CD90+ liver cancer stem cells and the TCGA database, were analysed by quantitative RT-PCR, Western blotting and flow cytometry. Functional analysis was conducted by siRNA-mediated CD90, Gli1 and Gli3 gene knockdown, SHH treatment and application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody in CD90+ liver cancer stem cells, followed by cell proliferation, migration, sphere formation and tumorigenicity assays. CD90 expression exhibited a high positive correlation with Gli1 and Gli3 in multiple liver cancer cell lines and human cancerous liver tissues, both of which showed a significant increase in liver cancer. Analysis of TCGA data revealed an association of CD90, Gli1 and Gli3 with a short overall survival and positive correlation between CD90 expression and Gli3 expression level. The stem cell potentials of CD90+ 97L liver cancer cells were greatly impaired by Gli1/3 knockdown with siRNA but enhanced by SHH treatment. Application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody showed the CD90 and SHH/Gli-regulated liver cancer stem cell functions were mediated by the IL6/JAK2/STAT3 pathway. The stem cell properties of CD90+ liver cancer cells are regulated by the downstream SHH/Gli and IL6/JAK2/STAT3 signalling pathways. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  18. Design and Synthesis of Novel and Selective Glycine Transporter-1 (GlyT1) Inhibitors with Memory Enhancing Properties.

    PubMed

    Santora, Vincent J; Almos, Theresa A; Barido, Richard; Basinger, Jillian; Bellows, Chris L; Bookser, Brett Carder; Breitenbucher, J Guy; Broadbent, Nicola J; Cabebe, Clifford; Chai, Chih-Kun; Chen, Mi; Chow, Stephine; Chung, De Michael; Crickard, Lindsay; Danks, Anne M; Freestone, Graeme; Gitnick, Dany; Gupta, Varsha; Hoffmaster, Christine; Hudson, Andrew R; Kaplan, Alan P; Kennedy, Michael R; Lee, Dong; Limberis, James; Ly, Kiev; Mak, Chi Ching; Masatsugu, Brittany; Morse, Andrew C; Na, Jim; Neul, David; Nikpur, John; Peters, Marco; Petroski, Robert E; Renick, Joel; Sebring, Kristen; Sevidal, Samantha; Tabatabaei, Ali; Wen, Jenny; Yan, Yingzhuo; Yoder, Zachary W; Zook, Douglas

    2018-06-11

    We report here the identification and optimization of a novel series of potent GlyT1 inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led to the identification of a novel series of pyrrolo[3,4-c]pyrazoles amides (21-50) with good in vitro potency. Subsequent optimization of physicochemical and in vitro ADME properties produced several compounds with promising pharmacokinetic profiles. In vivo inhibition of GlyT1 was demonstrated for select compounds within this series by measuring the elevation of glycine in the cerebrospinal fluid (CSF) of rats after a single oral dosing of 10 mg/kg. Ultimately, an optimized lead, compound 46, demonstrated in vivo efficacy in a rat Novel Object Recognition (NOR) assay after oral dosing at 0.1, 1, and 3 mg/kg.

  19. Basic Fibroblast Growth Factor Fused with Tandem Collagen-Binding Domains from Clostridium histolyticum Collagenase ColG Increases Bone Formation.

    PubMed

    Sekiguchi, Hiroyuki; Uchida, Kentaro; Matsushita, Osamu; Inoue, Gen; Nishi, Nozomu; Masuda, Ryo; Hamamoto, Nana; Koide, Takaki; Shoji, Shintaro; Takaso, Masashi

    2018-01-01

    Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly) 10 , induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly) 12 -NH 2 , a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly) 10 /bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

  20. Blocking Signaling at the Level of GLI Regulates Downstream Gene Expression and Inhibits Proliferation of Canine Osteosarcoma Cells

    PubMed Central

    Shahi, Mehdi Hayat; Holt, Roseline; Rebhun, Robert B.

    2014-01-01

    The Hedgehog-GLI signaling pathway is active in a variety of human malignancies and is known to contribute to the growth and survival of human osteosarcoma cells. In this study, we examined the expression and regulation of GLI transcription factors in multiple canine osteosarcoma cell lines and analyzed the effects of inhibiting GLI with GANT61, a GLI-specific inhibitor. Compared with normal canine osteoblasts, real-time PCR showed that GLI1 and GLI2 were highly expressed in two out of three cell lines and correlated with downstream target gene expression of PTCH1and PAX6. Treatment of canine osteosarcoma cells with GANT61 resulted in decreased expression of GLI1, GLI2, PTCH1, and PAX6. Furthermore, GANT61 inhibited proliferation and colony formation in all three canine osteosarcoma cell lines. The finding that GLI signaling activity is present and active in canine osteosarcoma cells suggests that spontaneously arising osteosarcoma in dogs might serve as a good model for future preclinical testing of GLI inhibitors. PMID:24810746

  1. Blocking signaling at the level of GLI regulates downstream gene expression and inhibits proliferation of canine osteosarcoma cells.

    PubMed

    Shahi, Mehdi Hayat; Holt, Roseline; Rebhun, Robert B

    2014-01-01

    The Hedgehog-GLI signaling pathway is active in a variety of human malignancies and is known to contribute to the growth and survival of human osteosarcoma cells. In this study, we examined the expression and regulation of GLI transcription factors in multiple canine osteosarcoma cell lines and analyzed the effects of inhibiting GLI with GANT61, a GLI-specific inhibitor. Compared with normal canine osteoblasts, real-time PCR showed that GLI1 and GLI2 were highly expressed in two out of three cell lines and correlated with downstream target gene expression of PTCH1and PAX6. Treatment of canine osteosarcoma cells with GANT61 resulted in decreased expression of GLI1, GLI2, PTCH1, and PAX6. Furthermore, GANT61 inhibited proliferation and colony formation in all three canine osteosarcoma cell lines. The finding that GLI signaling activity is present and active in canine osteosarcoma cells suggests that spontaneously arising osteosarcoma in dogs might serve as a good model for future preclinical testing of GLI inhibitors.

  2. Effect of Ala-Glu-Asp-Gly peptide on life span and development of spontaneous tumors in female rats exposed to different illumination regimes.

    PubMed

    Vinogradova, I A; Bukalev, A V; Zabezhinski, M A; Semenchenko, A V; Khavinson, V Kh; Anisimov, V N

    2007-12-01

    The effects of Ala-Glu-Asp-Gly peptide (Epithalon) on the life span and development of spontaneous tumors were studied in female rats exposed to standard, natural for North-Western Russia, and constant illumination. The mean life span of animals exposed to constant or natural illumination decreased by 13.5 and 25.5%, the maximum by 9 and 7 months, respectively, and spontaneous tumors developed much more rapidly than in animals living under conditions of the standard light regimen. Epithalon (0.1 microg daily 5 times a week from the age of 4 months) did not change the life span of rats living under conditions of standard day/night regimen, while in rats exposed to the natural and constant light it promoted prolongation of the maximum life span by 95 and 24 days, respectively. Epithalon prolonged the mean life span of the last 10% of rats exposed to natural and constant illumination, treated with Epithalon, by 137 and 43 days, respectively. This peptide exhibited virtually no effect on the development of spontaneous tumors in rats exposed to standard and constant illumination, but significantly inhibited their development in rats exposed to natural light.

  3. (/sup 3/H)-(Thr4,Gly7)OT: a highly selective ligand for central and peripheral OT receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Elands, J.; Barberis, C.; Jard, S.

    1988-01-01

    Oxytocin receptors in rat hippocampal synaptic plasma membranes were compared with mammary gland and uterine oxytocin receptors. For this purpose, a highly specific oxytocic agonist (Thr4,Gly7)oxytocin was tritiated. We demonstrated that this ligand labels oxytocin receptors selectively. Scatchard analyses revealed a high affinity for all the oxytocin receptors investigated, with equilibrium dissociation constants between 1.0 and 2.0 nM. Binding appeared to take place at a single population of receptor sites. Competition experiments confirmed the high affinity of arginine vasopressin for hippocampal oxytocin receptors but also revealed that mammary gland and uterine oxytocin receptors do not discriminate more efficiently between oxytocinmore » and arginine vasopressin. This lack in specificity is not affected by applying different concentrations of Mg ions.« less

  4. Efficacy and Safety of Indacaterol/Glycopyrronium (IND/GLY) Versus Salmeterol/Fluticasone in Chinese Patients with Moderate-to-Severe Chronic Obstructive Pulmonary Disease: The Chinese Cohort from the LANTERN Study.

    PubMed

    Zhong, Nanshan; Wang, Changzheng; Zhou, Xiangdong; Zhang, Nuofu; Humphries, Michael; Wang, Linda; Patalano, Francesco; Banerji, Donald

    2016-12-01

    Inhaled indacaterol/glycopyrronium fixed-dose combination (IND/GLY) is approved in over 80 countries, including the EU, Japan, Australia and Switzerland and the US. The LANTERN study evaluated the efficacy of IND/GLY compared with inhaled long-acting β 2 -agonist (LABA)/inhaled corticosteroid (ICS) or salmeterol/fluticasone (SFC) in patients with moderate-to-severe COPD with a history of ≤1 exacerbation in the previous year. Here we present the efficacy and safety of IND/GLY versus SFC in the Chinese cohort from the LANTERN study. LANTERN was a 26-week, multicenter, randomized, double-blind, double-dummy, parallel-group study conducted in patients with moderate-to-severe COPD with a history of ≤1 exacerbation in the previous year. The patients were randomized (1:1) to once-daily IND/GLY (110/50 μg) or twice-daily SFC (50/500 μg). The primary endpoint was non-inferiority of IND/GLY versus SFC in terms of trough FEV 1 . Of the total 744 patients randomized in the LANTERN study, 598 (80.4%) were from Mainland China and randomized to IND/GLY (n = 298) or SFC (n = 300), and 553 (92.5%) completed the study. IND/GLY showed superiority over SFC with a statistically significant and clinically meaningful improvement in trough FEV 1 , FEV 1 AUC 0-4h , peak FEV 1 and trough forced vital capacity (FVC) change from the baseline. Annualized rate of moderate or severe COPD exacerbations was significantly lower (43%) with IND/GLY compared with SFC (rate ratio: 0.57, p = 0.015). Overall, adverse events were lower for IND/GLY (34.6%) versus SFC (43.1%). IND/GLY was superior in achieving bronchodilation versus SFC in a Chinese subgroup of patients from this study. Clinicaltrials.gov identifier: NCT01709903.

  5. MiRNA-133b promotes the proliferation of human Sertoli cells through targeting GLI3

    PubMed Central

    Yao, Chencheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Chen, Zheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

    2016-01-01

    Sertoli cells play critical roles in regulating spermatogenesis and they can be reprogrammed to the cells of other lineages, highlighting that they have significant applications in reproductive and regenerative medicine. The fate determinations of Sertoli cells are regulated precisely by epigenetic factors. However, the expression, roles, and targets of microRNA (miRNA) in human Sertoli cells remain unknown. Here we have for the first time revealed that 174 miRNAs were distinctly expressed in human Sertoli cells between Sertoli-cell-only syndrome (SCOS) patients and obstructive azoospermia (OA) patients with normal spermatogenesis using miRNA microarrays and real time PCR, suggesting that these miRNAs may be associated with the pathogenesis of SCOS. MiR-133b is upregulated in Sertoli cells of SCOS patients compared to OA patients. Proliferation assays with miRNA mimics and inhibitors showed that miR-133b enhanced the proliferation of human Sertoli cells. Moreover, we demonstrated that GLI3 was a direct target of miR-133b and the expression of Cyclin B1 and Cyclin D1 was enhanced by miR-133b mimics but decreased by its inhibitors. Gene silencing of GLI3 using RNA inference stimulated the growth of human Sertoli cells. Collectively, miR-133b promoted the proliferation of human Sertoli cells by targeting GLI3. This study thus sheds novel insights into epigenetic regulation of human Sertoli cells and the etiology of azoospermia and offers new targets for treating male infertility PMID:26755652

  6. The Role of Limited Proteolysis of Thyrotropin-Releasing Hormone in Thermoregulation.

    DTIC Science & Technology

    1982-01-01

    exogenously. The limited proteolysis of TRH by pyroglutamate aminopeptidase from CNS results into formation of a new cyclic dipeptide, cyclo (His-Pro...amino acids (L-histidine and L-proline), and two analogues of cyclo (His-Pro), cyclo (Pro-Gly) and cyclo . (Ala-Gly). Cyclo (His-Pro) cross-reacted only...cyclo (His-Pro). Figure 3 shows the chromato- graphic profile obtained when a neutralized perchloric acid extract of rat brain was passed through DEAE

  7. Controlled self-assembling structures of ferrocene-dipeptide conjugates composed of Ala-Pro-NHCH2CH2SH chain.

    PubMed

    Moriuchi, Toshiyuki; Nishiyama, Taiki; Tayano, Yoshiki; Hirao, Toshikazu

    2017-12-01

    Bioorganometallic ferrocene-dipeptide conjugates with the Ala-Pro-cysteamine chain, Fc-L-Ala-L-Pro-NHCH 2 CH 2 SH (2) and Fc-L-Ala-D-Pro-NHCH 2 CH 2 SH (4) (Fc=ferrocenoyl), were prepared by the reduction of the ferrocene-dipeptide conjugates, Fc-L-Ala-L-Pro-cystamine-L-Pro-L-Ala-Fc (1) or Fc-L-Ala-D-Pro-cystamine-D-Pro-L-Ala-Fc (3), respectively. Control of the self-assembling structures of the ferrocene-dipeptide conjugates was demonstrated by changing the chirality of the amino acid. The molecular structure of 2 composed of the L-Ala-L-Pro-NHCH 2 CH 2 SH chain confirmed the formation of intramolecular hydrogen bond of N-H⋯N pattern between the NH of cysteamine moiety and the nitrogen of Pro moiety. Furthermore, intermolecular hydrogen bonds between NH (Ala) and CO (Pro of another molecule) and between NH (cysteamine) and CO (the ferrocenoyl moiety of another molecule) were formed, wherein each molecule is connected to four neighboring molecules by continuous intermolecular hydrogen bonds to form the hydrogen-bonded molecular assembling structure. On the contrary, the left-handed helical assembly through an intermolecular hydrogen-bonding network of 15-membered intermolecularly hydrogen-bonded ring between NH (Ala) and CO (the ferrocenoyl moiety of another molecule) and between NH (the cysteamine moiety of another molecule) and CO (Ala) was observed in the crystal packing of 4 composed of the L-Ala-D-Pro-NHCH 2 CH 2 SH chain. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Structure and receptor-binding activity of insulin from a holostean fish, the bowfin (Amia calva).

    PubMed

    Conlon, J M; Youson, J H; Whittaker, J

    1991-05-15

    The holostean fishes are the extant representatives of the primitive ray-finned fishes from which the present-day teleosts may have evolved. The primary structure of insulin from a holostean fish, the bowfin (Amia calva), was established as: A-chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Leu-Lys-Pro-Cys-Thr-Ile-Tyr-Glu-Met-Glu- Lys-Tyr-Cys-Asn B-chain: Ala-Ala-Ser-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Phe-Leu- Val-Cys-Gly-Glu-Ser-Gly-Phe-Phe-Tyr-Asn-Pro-Asn-Lys-Ser This amino acid sequence contains several substitutions (methionine at A16, phenylalanine at B16 and serine at B22) at sites that have been strongly conserved in other vertebrate species and that may be expected to influence biological activity. Consistent with this prediction, bowfin insulin was approx. 14-fold less potent than pig insulin in inhibiting the binding of [125I-Tyr-A14](human insulin) to transfected mouse NIH 3T3 cells expressing the human insulin receptor.

  9. Structure and receptor-binding activity of insulin from a holostean fish, the bowfin (Amia calva).

    PubMed Central

    Conlon, J M; Youson, J H; Whittaker, J

    1991-01-01

    The holostean fishes are the extant representatives of the primitive ray-finned fishes from which the present-day teleosts may have evolved. The primary structure of insulin from a holostean fish, the bowfin (Amia calva), was established as: A-chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Leu-Lys-Pro-Cys-Thr-Ile-Tyr-Glu-Met-Glu- Lys-Tyr-Cys-Asn B-chain: Ala-Ala-Ser-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Phe-Leu- Val-Cys-Gly-Glu-Ser-Gly-Phe-Phe-Tyr-Asn-Pro-Asn-Lys-Ser This amino acid sequence contains several substitutions (methionine at A16, phenylalanine at B16 and serine at B22) at sites that have been strongly conserved in other vertebrate species and that may be expected to influence biological activity. Consistent with this prediction, bowfin insulin was approx. 14-fold less potent than pig insulin in inhibiting the binding of [125I-Tyr-A14](human insulin) to transfected mouse NIH 3T3 cells expressing the human insulin receptor. PMID:2039477

  10. Pathogenetic role of Arg-Gly-Asp-recognizing integrins in acute renal failure. off.

    PubMed Central

    Goligorsky, M S; DiBona, G F

    1993-01-01

    Reorientation of the alpha 3 subunit of integrins from predominantly basal to the apical cell surface of cultured renal tubular epithelial cells subjected to oxidant stress has previously been demonstrated. The present study was designed to assess functional competence of ectopically expressed apical integrins. Cell-cell adhesion assay revealed enhanced cytoatractant properties of stressed cells. Stressed epithelial cells exhibited specific recognition and binding of laminin-coated latex beads. These processes were inhibited with the peptide Gly-Arg-Gly-Asp-Asn-Pro (GRGDNP) suggesting a role of RGD-recognizing integrins in augmented adhesion to stressed cells. Given that such enhanced adhesion in in vivo acute renal failure may govern tubular obstruction by desquamated epithelium, a physiological marker of patency of tubular lumen, proximal tubular pressure, was monitored in rats subjected to 60 min of renal ischemia followed by reperfusion. Proximal tubular pressure increased 2-fold after 2 hr of reperfusion in animals that had undergone 60 min of ischemia. Infusion of GRGDNP into the renal artery during reperfusion period virtually abolished an increase in proximal tubular pressure observed in ischemic acute renal failure. These in vitro and in vivo findings are consistent with the hypothesis that RGD-recognizing integrins play an important role in the pathogenesis of tubular obstruction in ischemic acute renal failure. Images Fig. 2 Fig. 3 PMID:8516318

  11. CCND2, CTNNB1, DDX3X, GLI2, SMARCA4, MYC, MYCN, PTCH1, TP53, and MLL2 gene variants and risk of childhood medulloblastoma.

    PubMed

    Dahlin, Anna M; Hollegaard, Mads V; Wibom, Carl; Andersson, Ulrika; Hougaard, David M; Deltour, Isabelle; Hjalmars, Ulf; Melin, Beatrice

    2015-10-01

    Recent studies have described a number of genes that are frequently altered in medulloblastoma tumors and that have putative key roles in the development of the disease. We hypothesized that common germline genetic variations in these genes may be associated with medulloblastoma development. Based on recent publications, we selected 10 genes that were frequently altered in medulloblastoma: CCND2, CTNNB1, DDX3X, GLI2, SMARCA4, MYC, MYCN, PTCH1, TP53, and MLL2 (now renamed as KMT2D). Common genetic variants (single nucleotide polymorphisms) annotating these genes (n = 221) were genotyped in germline DNA (neonatal dried blood spot samples) from 243 childhood medulloblastoma cases and 247 control subjects from Sweden and Denmark. Eight genetic variants annotating three genes in the sonic hedgehog signaling pathway; CCND2, PTCH1, and GLI2, were found to be associated with the risk of medulloblastoma (P(combined) < 0.05). The findings were however not statistically significant following correction for multiple testing by the very stringent Bonferroni method. The results do not support our hypothesis that common germline genetic variants in the ten studied genes are associated with the risk of developing medulloblastoma.

  12. The Gly972Arg polymorphism in insulin receptor substrate-1 is associated with decreased birth weight in a population-based sample of Brazilian newborns.

    PubMed

    Bezerra, Rosângela M N; de Castro, Vagner; Sales, Teresa; Passini, Renato; Marba, Sergio T M; Saad, Sara T O; Saad, Mario J A

    2002-03-01

    We studied the association between the Gly972Arg polymorphism in insulin receptor substrate-1 (IRS-1) and birth weight in a population-based sample of Brazilian newborns. We studied 194 newborn children with adequate gestational age to identify the association between the Gly972Arg polymorphism and birth weight using PCR-restriction fragment length polymorphism analysis. The data showed that the birth weight was lower in the newborns with the Gly972Arg polymorphism in IRS-1 compared with control subjects (3,141 +/- 31.8 vs. 3,373 +/- 80.3 g, P < 0.008). The results also showed that the frequency of this polymorphism was increased in newborns with a birth weight <3,000 g (P=0.041). These results suggest that the genotype Gly972Arg may influence birth weight, reinforcing the hypothesis that genetically determined insulin resistance and/or reduced insulin secretion can result in impaired insulin-mediated growth in the fetus.

  13. Energetics and Structure Prediction of the Network of Homo- and Hetero-Oligomers Formed by the Transmembrane Domains of the ErbB Receptor Family of Proteins

    DTIC Science & Technology

    2005-06-01

    acid residue motif, Small-x-x-Large-G/A, consist- complexes is critical to understanding the signal ing of a small residue (Gly, Ala , Ser, Thr, or Pro...in transduction process. Recent structures of the the zero position, a large aliphatic residue ( Ala , Val, ligand-binding domains of the erbB...receptors Leu, or Ile) in position 3, followed by Gly or Ala in have begun to provide insight into the mechanisms position four.’ This motif was identified

  14. Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

    PubMed Central

    Sanz, Yolanda; Toldrá, Fidel

    2001-01-01

    An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu2+, Hg2+, and Zn2+). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. Km s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed. PMID:11282638

  15. Maintenance of Taste Organs Is Strictly Dependent on Epithelial Hedgehog/GLI Signaling.

    PubMed

    Ermilov, Alexandre N; Kumari, Archana; Li, Libo; Joiner, Ariell M; Grachtchouk, Marina A; Allen, Benjamin L; Dlugosz, Andrzej A; Mistretta, Charlotte M

    2016-11-01

    For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste

  16. Maintenance of Taste Organs Is Strictly Dependent on Epithelial Hedgehog/GLI Signaling

    PubMed Central

    Mistretta, Charlotte M.

    2016-01-01

    For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste

  17. Sonic hedgehog (Shh)/Gli modulates the spatial organization of neuroepithelial cell proliferation in the developing chick optic tectum.

    PubMed

    Rapacioli, Melina; Botelho, Joao; Cerda, Gustavo; Duarte, Santiago; Elliot, Matías; Palma, Verónica; Flores, Vladimir

    2012-10-02

    Sonic hedgehog (Shh)/Gli pathway plays an important regulatory role on the neuroepithelial cells (NEc) proliferation in the dorsal regions of the developing vertebrate Central Nervous System. The aim of this paper was to analyze the effect of the Shh/Gli signaling pathway activation on the proliferation dynamics and/or the spatial organization of the NEc proliferation activity during early stages of the developing chick optic tectum (OT). In ovo pharmacological gain and loss of hedgehog function approaches were complemented with in vivo electroporation experiments in order to create ectopic sources of either Shh or Gli activator (GliA) proteins in the OT. NEc proliferating activity was analyzed at ED 4/4.5 by recording the spatial co-ordinates of the entire population of mitotic NEc (mNEc) located along OT dorsal-ventral sections. Several space signals (numerical sequences) were derived from the mNEc spatial co-ordinate records and analyzed by different standardized non-linear methods of signal analysis. In ovo pharmacologic treatment with cyclopamine resulted in dramatic failure in the OT expansion while the agonist purmorphamine produced the opposite result, a huge expansion of the OT vesicle. Besides, GliA and Shh misexpressions interfere with the formation of the intertectal fissure located along the dorsal midline. This morphogenetic alteration is accompanied by an increase in the mNEc density. There is a gradient in the response of NEcs to Shh and GliA: the increase in mNEc density is maximal near the dorsal regions and decrease towards the OT-tegmental boundary. Biomathematical analyses of the signals derived from the mNEc records show that both Shh and GliA electroporations change the proliferation dynamics and the spatial organization of the mNEc as revealed by the changes in the scaling index estimated by these methods. The present results show that the Shh/Gli signaling pathway plays a critical role in the OT expansion and modelling. This effect is

  18. GLI1-mediated regulation of side population is responsible for drug resistance in gastric cancer

    PubMed Central

    Yu, Beiqin; Gu, Dongsheng; Zhang, Xiaoli; Li, Jianfang; Liu, Bingya; Xie, Jingwu

    2017-01-01

    Gastric cancer is the third leading cause of cancer-related mortality worldwide. Chemotherapy is frequently used for gastric cancer treatment. Most patients with advanced gastric cancer eventually succumb to the disease despite some patients responded initially to chemotherapy. Thus, identifying molecular mechanisms responsible for cancer relapse following chemotherapy will help design new ways to treat gastric cancer. In this study, we revealed that the residual cancer cells following treatment with chemotherapeutic reagent cisplatin have elevated expression of hedgehog target genes GLI1, GLI2 and PTCH1, suggestive of hedgehog signaling activation. We showed that GLI1 knockdown sensitized gastric cancer cells to CDDP whereas ectopic GLI1 expression decreased the sensitivity. Further analyses indicate elevated GLI1 expression is associated with an increase in tumor sphere formation, side population and cell surface markers for putative cancer stem cells. We have evidence to support that GLI1 is critical for maintenance of putative cancer stem cells through direct regulation of ABCG2. In fact, GLI1 protein was shown to be associated with the promoter fragment of ABCG2 through a Gli-binding consensus site in gastric cancer cells. Disruption of ABCG2 function, through ectopic expression of an ABCG2 dominant negative construct or a specific ABCG2 inhibitor, increased drug sensitivity of cancer cells both in culture and in mice. The relevance of our studies to gastric cancer patient care is reflected by our discovery that high ABCG2 expression was associated with poor survival in the gastric cancer patients who underwent chemotherapy. Taken together, we have identified a molecular mechanism by which gastric cancer cells gain chemotherapy resistance. PMID:28404967

  19. In vitro DNA damage by Casiopeina II-gly in human blood cells.

    PubMed

    Rodríguez-Mercado, Juan José; Florín-Ramírez, Diana; Álvarez-Barrera, Lucila; Altamirano-Lozano, Mario Agustín

    2017-04-01

    A variety of metal ions have biological functions, and many researchers have not actively investigated copper compounds, based on the assumption that endogenous metals might be less toxic. In the present study, we used a dual fluorochrome method and a single cell gel electrophoresis (SCGE) assay at pH > 13 to evaluate the cell viability and DNA damage induced by a copper-based antineoplastic drug, Casiopeina II-gly®, at concentrations of 1.04, 2.08, 4.17, 8.35 or 16 μg/mL in human peripheral-blood leukocytes in vitro. We observed that Casiopeina II-gly® reduced cell viability at high concentrations (8.35 and 16 μg/mL) and induced DNA damage characterized by single-strand breaks and alkali labile sites at several concentrations from 2.08 to 16 μg/mL. This chemical clearly affected DNA migration in a concentration- and time-dependent manner and induced genotoxic effects in few minutes (>20 min), at which point the genotoxicity was followed by cytotoxicity.

  20. Proline oxidase silencing induces proline-dependent pro-survival pathways in MCF-7 cells

    PubMed Central

    Zareba, Ilona; Celinska-Janowicz, Katarzyna; Surazynski, Arkadiusz; Miltyk, Wojciech; Palka, Jerzy

    2018-01-01

    Proline degradation by proline dehydrogenase/proline oxidase (PRODH/POX) contributes to apoptosis or autophagy. The identification of specific pathway of apoptosis/survival regulation is the aim of this study. We generated knocked-down PRODH/POX MCF-7 breast cancer cells (MCF-7shPRODH/POX). PRODH/POX silencing did not affect cell viability. However, it contributed to decrease in DNA and collagen biosynthesis, increase in prolidase activity and intracellular proline concentration as well as increase in the expression of iNOS, NF-κB, mTOR, HIF-1α, COX-2, AMPK, Atg7 and Beclin-1 in MCF-7shPRODH/POX cells. In these cells, glycyl-proline (GlyPro, substrate for prolidase) further inhibited DNA and collagen biosynthesis, maintained high prolidase activity, intracellular concentration of proline and up-regulated HIF-1α, AMPK, Atg7 and Beclin-1, compared to GlyPro-treated MCF-7 cells. In MCF-7 cells, GlyPro increased collagen biosynthesis, concentration of proline and expression of caspase-3, cleaved caspases -3 and -9, iNOS, NF-κB, COX-2 and AMPKβ. PRODH/POX knock-down contributed to pro-survival autophagy pathways in MCF-7 cells and GlyPro-derived proline augmented this process. However, GlyPro induced apoptosis in PRODH/POX-expressing MCF-7 cells as detected by up-regulation of active caspases -3 and -9. The data suggest that PRODH/POX silencing induces autophagy in MCF-7 cells and GlyPro-derived proline supports this process. PMID:29568391

  1. DYRK1B as therapeutic target in Hedgehog/GLI-dependent cancer cells with Smoothened inhibitor resistance

    PubMed Central

    Gruber, Wolfgang; Hutzinger, Martin; Elmer, Dominik Patrick; Parigger, Thomas; Sternberg, Christina; Cegielkowski, Lukasz; Zaja, Mirko; Leban, Johann; Michel, Susanne; Hamm, Svetlana; Vitt, Daniel; Aberger, Fritz

    2016-01-01

    A wide range of human malignancies displays aberrant activation of Hedgehog (HH)/GLI signaling, including cancers of the skin, brain, gastrointestinal tract and hematopoietic system. Targeting oncogenic HH/GLI signaling with small molecule inhibitors of the essential pathway effector Smoothened (SMO) has shown remarkable therapeutic effects in patients with advanced and metastatic basal cell carcinoma. However, acquired and de novo resistance to SMO inhibitors poses severe limitations to the use of SMO antagonists and urgently calls for the identification of novel targets and compounds. Here we report on the identification of the Dual-Specificity-Tyrosine-Phosphorylation-Regulated Kinase 1B (DYRK1B) as critical positive regulator of HH/GLI signaling downstream of SMO. Genetic and chemical inhibition of DYRK1B in human and mouse cancer cells resulted in marked repression of HH signaling and GLI1 expression, respectively. Importantly, DYRK1B inhibition profoundly impaired GLI1 expression in both SMO-inhibitor sensitive and resistant settings. We further introduce a novel small molecule DYRK1B inhibitor, DYRKi, with suitable pharmacologic properties to impair SMO-dependent and SMO-independent oncogenic GLI activity. The results support the use of DYRK1B antagonists for the treatment of HH/GLI-associated cancers where SMO inhibitors fail to demonstrate therapeutic efficacy. PMID:26784250

  2. Mutations in the human GlyT2 gene define a presynaptic component of human startle disease

    PubMed Central

    Rees, Mark I.; Harvey, Kirsten; Pearce, Brian R.; Chung, Seo-Kyung; Duguid, Ian C.; Thomas, Philip; Beatty, Sarah; Graham, Gail E.; Armstrong, Linlea; Shiang, Rita; Abbott, Kim J.; Zuberi, Sameer M.; Stephenson, John B.P.; Owen, Michael J.; Tijssen, Marina A.J.; van den Maagdenberg, Arn M.J.M.; Smart, Trevor G.; Supplisson, Stéphane; Harvey, Robert J.

    2011-01-01

    Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) α1 subunit (GLRA1)1-3. Genetic heterogeneity has been confirmed in isolated sporadic cases with mutations in other postsynaptic glycinergic proteins including the GlyR β subunit (GLRB)4, gephyrin (GPHN)5 and RhoGEF collybistin (ARHGEF9)6. However, many sporadic patients diagnosed with hyperekplexia do not carry mutations in these genes2-7. Here we reveal that missense, nonsense and frameshift mutations in the presynaptic glycine transporter 2 (GlyT2) gene (SLC6A5)8 also cause hyperekplexia. Patients harbouring mutations in SLC6A5 presented with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnoea episodes. GlyT2 mutations result in defective subcellular localisation and/or decreased glycine uptake, with selected mutations affecting predicted glycine and Na+ binding sites. Our results demonstrate that SLC6A5 is a major gene for hyperekplexia and define the first neurological disorder linked to mutations in a Na+/Cl−-dependent transporter for a classical fast neurotransmitter. By analogy, we suggest that in other human disorders where defects in postsynaptic receptors have been identified, similar symptoms could result from defects in the cognate presynaptic neurotransmitter transporter. PMID:16751771

  3. Cathepsin B and uPAR regulate self-renewal of glioma-initiating cells through GLI-regulated Sox2 and Bmi1 expression

    PubMed Central

    Rao, Jasti S.

    2013-01-01

    Cancer-initiating cells comprise a heterogeneous population of undifferentiated cells with the capacity for self-renewal and high proliferative potential. We investigated the role of uPAR and cathepsin B in the maintenance of stem cell nature in glioma-initiating cells (GICs). Simultaneous knockdown of uPAR and cathepsin B significantly reduced the expression of CD133, Nestin, Sox2 and Bmi1 at the protein level and GLI1 and GLI2 at the messenger RNA level. Also, knockdown of uPAR and cathepsin B resulted in a reduction in the number of GICs as well as sphere size. These changes are mediated by Sox2 and Bmi1, downstream of hedgehog signaling. Addition of cyclopamine reduced the expression of Sox2 and Bmi1 along with GLI1 and GLI2 expression, induced differentiation and reduced subsphere formation of GICs thereby indicating that hedgehog signaling acts upstream of Sox2 and Bmi1. Further confirmation was obtained from increased luciferase expression under the control of a GLI-bound Sox2 and Bmi1 luciferase promoter. Simultaneous knockdown of uPAR and cathepsin B also reduced the expression of Nestin Sox2 and Bmi1 in vivo. Thus, our study highlights the importance of uPAR and cathepsin B in the regulation of malignant stem cell self-renewal through hedgehog components, Bmi1 and Sox2. PMID:23222817

  4. Energetics and Structure Prediction of the Network of Homo- and Hetero-Oligomers Formed by the Transmembrane Domains of the ErbReceptor Family of Proteins

    DTIC Science & Technology

    2006-06-01

    amino acid residue motif, Small-x-x-Large-G/A, consist- ing of a small residue (Gly, Ala , Ser, Thr, or Pro) in the zero position, a large aliphatic...residue ( Ala , Val, Leu, or Ile) in position 3, followed by Gly or Ala in position four.15 This motif was identified in a large number of receptor tyrosine...M. A., Codony-Servat, J., Albanell, J., Rojo, F., Arribas , J. & Baselga, J. (2001). Trastuzumab (her- ceptin), a humanized anti-Her2 receptor

  5. The sequential action of a dipeptidase and a beta-lyase is required for the release of the human body odorant 3-methyl-3-sulfanylhexan-1-ol from a secreted Cys-Gly-(S) conjugate by Corynebacteria.

    PubMed

    Emter, Roger; Natsch, Andreas

    2008-07-25

    Human axillary odor is formed by the action of Corynebacteria on odorless axilla secretions. Sulfanylalkanols, 3-methyl-3-sulfanylhexan-1-ol in particular, form one key class of the odoriferous compounds. A conjugate with the dipeptide Cys-Gly has been reported as the secreted precursor for 3-methyl-3-sulfanylhexan-1-ol. Here, we confirm the Cys-Gly-(S) conjugate as the major precursor of this odorant, with lower levels of the Cys-(S) conjugate being present in axilla secretions. The enzymatic release of 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate by the axilla isolate Corynebacterium Ax20 was thus investigated. Cellular extracts of Ax20 released 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate and from the Cys-(S) conjugate, whereas the previously isolated C-S lyase of this bacterial strain was only able to cleave the Cys-(S) conjugate. o-Phenanthroline blocked the release from the Cys-Gly-(S) conjugate but did not affect cleavage of the Cys-(S) conjugate, indicating that in a first step, a metal-dependent dipeptidase hydrolyzes the Cys-Gly bond. This enzyme was purified by four chromatographic steps and gel electrophoresis, and the partial amino acid sequence was determined. The corresponding gene was cloned and expressed in Escherichia coli. It codes for a novel dipeptidase with a high affinity toward the Cys-Gly-(S) conjugate of 3-methyl-3-sulfanylhexan-1-ol. Co-incubating either the synthetic Cys-Gly-(S) conjugate or fresh axilla secretions with both the C-S lyase and the novel dipeptidase did release 3-methyl-3-sulfanylhexan-1-ol, proving that the sequential action of these two enzymes from the skin bacterium Corynebacterium Ax20 does release the odorant from the key secreted precursor.

  6. GLI1+ progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic gonadal-like tissue.

    PubMed

    Dörner, Julia; Martinez Rodriguez, Verena; Ziegler, Ricarda; Röhrig, Theresa; Cochran, Rebecca S; Götz, Ronni M; Levin, Mark D; Pihlajoki, Marjut; Heikinheimo, Markku; Wilson, David B

    2017-02-05

    As certain strains of mice age, hyperplastic lesions resembling gonadal tissue accumulate beneath the adrenal capsule. Gonadectomy (GDX) accelerates this heterotopic differentiation, resulting in the formation of wedge-shaped adrenocortical neoplasms that produce sex steroids. Stem/progenitor cells that reside in the adrenal capsule and retain properties of the adrenogonadal primordium are thought to be the source of this heterotopic tissue. Here, we demonstrate that GLI1 + progenitors in the adrenal capsule give rise to gonadal-like cells that accumulate in the subcapsular region. A tamoxifen-inducible Cre driver (Gli1-creER T2 ) and two reporters (R26R-lacZ, R26R-confetti) were used to track the fate of GLI1 + cells in the adrenal glands of B6D2F2 mice, a strain that develops both GDX-induced adrenocortical neoplasms and age-dependent subcapsular cell hyperplasia. In gonadectomized B6D2F2 mice GLI1 + progenitors contributed to long-lived adrenal capsule cells and to adrenocortical neoplasms that expressed Gata4 and Foxl2, two prototypical gonadal markers. Pdgfra, a gene expressed in adrenocortical stromal cells, was upregulated in the GDX-induced neoplasms. In aged non-gonadectomized B6D2F2 mice GLI1 + progenitors gave rise to patches of subcapsular cell hyperplasia. Treatment with GANT61, a small-molecule GLI antagonist, attenuated the upregulation of gonadal-like markers (Gata4, Amhr2, Foxl2) in response to GDX. These findings support the premise that GLI1 + progenitor cells in the adrenal capsule of the adult mouse give rise to heterotopic tissue. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. The alyteserins: two families of antimicrobial peptides from the skin secretions of the midwife toad Alytes obstetricans (Alytidae).

    PubMed

    Conlon, J Michael; Demandt, Anni; Nielsen, Per F; Leprince, Jérôme; Vaudry, Hubert; Woodhams, Douglas C

    2009-06-01

    Two families of structurally related C-terminally alpha-amidated antimicrobial peptides have been identified in norepinephrine-stimulated skin secretions of the midwife toad Alytes obstetricans (Alytidae). The alyteserin-1 peptides (Gly-Leu-Lys-(Asp/Glu)-Ile-Phe-Lys-Ala-Gly-Leu-Gly-Ser-Leu-Val-Lys-(Gly/Asn)-Ile-Ala-Ala-His-Val-Ala-(Asn/Ser).NH(2)) show limited structural similarity to the ascaphins from the skins of frogs of the family Leiopelmatidae. Alyteserin-2a (Ile-Leu-Gly-Lys-Leu-Leu-Ser-Thr-Ala-Ala-Gly-Leu-Leu-Ser-Asn-Leu.NH(2)) and alyteserin-2b and -2c (Ile-Leu-Gly-Ala-Ile-Leu-Pro-Leu-Val-Ser-Gly-Leu-Leu-Ser-(Asn/Ser)-Lys-Leu x NH(2)) show limited sequence identity with bombinin H6, present in the skins of frogs of the family Bombinatoridae. The alyteserin-1 peptides show selective growth inhibitory activity against the Gram-negative bacteria Escherichia coli (MIC=25 microM) whereas alyteserin-2a is more potent against the Gram-positive bacteria Staphylococcus aureus (MIC=50 microM). The hemolytic activity against human erythrocytes of all peptides tested is relatively weak (LC(50)>100 microM). The data demonstrate that the frogs belonging to the family Alytidae are among those producing dermal antimicrobial peptides that may represent a component of the animal's system of innate immunity.

  8. The peptide transporter PepT2 mediates the uptake of the glutathione precursor CysGly in astroglia-rich primary cultures.

    PubMed

    Dringen, R; Hamprecht, B; Bröer, S

    1998-07-01

    The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cultures to utilize cysteinylglycine (CysGly) for glutathione synthesis. After a 24-h starvation period in the absence of glucose and amino acids, CysGly was able to substitute for cysteine plus glycine in the restoration of glutathione. Glutathione restoration from CysGly plus glutamate was only slightly affected by the dipeptides carnosine or serylglycine in a 200-fold excess. Captopril, a substrate of the peptide transporter PepT1, had almost no effect on glutathione restoration. In contrast, with increasing concentrations of alanylalanine or cefadroxil, known substrates of the peptide transporter PepT2, the amount of glutathione restored in the presence of CysGly and glutamate was strongly reduced. Cefadroxil in a 200-fold excess totally prevented the utilization of CysGly for glutathione restoration. The presence of mRNA for PepT2 in astroglia-rich primary cultures was demonstrated by application of RT-PCR. These results demonstrate that PepT2 is expressed in astroglia-rich primary cultures and that this transporter is highly likely to be responsible for the uptake of CysGly in these cultures.

  9. The transcription factor GLI1 modulates the inflammatory response during pancreatic tissue remodeling.

    PubMed

    Mathew, Esha; Collins, Meredith A; Fernandez-Barrena, Maite G; Holtz, Alexander M; Yan, Wei; Hogan, James O; Tata, Zachary; Allen, Benjamin L; Fernandez-Zapico, Martin E; di Magliano, Marina Pasca

    2014-10-03

    Pancreatic cancer, one of the deadliest human malignancies, is almost uniformly associated with a mutant, constitutively active form of the oncogene Kras. Studies in genetically engineered mouse models have defined a requirement for oncogenic KRAS in both the formation of pancreatic intraepithelial neoplasias, the most common precursor lesions to pancreatic cancer, and in the maintenance and progression of these lesions. Previous work using an inducible model allowing tissue-specific and reversible expression of oncogenic Kras in the pancreas indicates that inactivation of this GTPase at the pancreatic intraepithelial neoplasia stage promotes pancreatic tissue repair. Here, we extend these findings to identify GLI1, a transcriptional effector of the Hedgehog pathway, as a central player in pancreatic tissue repair upon Kras inactivation. Deletion of a single allele of Gli1 results in improper stromal remodeling and perdurance of the inflammatory infiltrate characteristic of pancreatic tumorigenesis. Strikingly, this partial loss of Gli1 affects activated fibroblasts in the pancreas and the recruitment of immune cells that are vital for tissue recovery. Analysis of the mechanism using expression and chromatin immunoprecipitation assays identified a subset of cytokines, including IL-6, mIL-8, Mcp-1, and M-csf (Csf1), as direct GLI1 target genes potentially mediating this phenomenon. Finally, we demonstrate that canonical Hedgehog signaling, a known regulator of Gli1 activity, is required for pancreas recovery. Collectively, these data delineate a new pathway controlling tissue repair and highlight the importance of GLI1 in regulation of the pancreatic microenvironment during this cellular process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The Gly-54-->Asp allelic form of human mannose-binding protein (MBP) fails to bind MBP-associated serine protease.

    PubMed Central

    Matsushita, M; Ezekowitz, R A; Fujita, T

    1995-01-01

    The human mannose-binding protein (MBP) is a pattern recognition molecule that appears to play a role in initial host defence. MBP activates the complement cascade and it may act as an opsonin both in the absence and in the presence of complement. A number of distinct MBP allelic forms exist in different population groups. An allele that occurs in 5-7% of Caucasians was identified by an inability to activate the complement system. A homozygous mutation at base pair 230 of the MBP gene results in a Gly-to-Asp substitution at the fifth collagen repeat. It appears that the resultant protein, MBPD, is able to form high-order multimers that bind bacteria but do not support complement activation. Recently a novel serine protease, the MBP-associated serine protease (MASP), has been described. MBP-MASP complexes circulate in serum and result in the direct activation of a novel complement pathway (lectin pathway) in the absence of the first complement components. In this study we demonstrate that MASP and its proenzyme proMASP are unable to bind to recombinant (r)MBPD. This lack of a MASP-rMBPD association corresponds to a failure of the Gly-54-->Asp form of MBP to activate complement. Our results provide a biochemical basis for the functional deficit in the Gly-54-->Asp allelic form of MBP and suggest that the proMASP/MASP binding site maps to the fifth collagen repeat of MBP. Images Figure 1 PMID:7487919

  11. Site-Specific Dynamics of β-Sheet Peptides with (D) Pro-Gly Turns Probed by Laser-Excited Temperature-Jump Infrared Spectroscopy.

    PubMed

    Popp, Alexander; Scheerer, David; Chi, Heng; Keiderling, Timothy A; Hauser, Karin

    2016-05-04

    Turn residues and side-chain interactions play an important role for the folding of β-sheets. We investigated the conformational dynamics of a three-stranded β-sheet peptide ((D) P(D) P) and a two-stranded β-hairpin (WVYY-(D) P) by time-resolved temperature-jump (T-jump) infrared spectroscopy. Both peptide sequences contain (D) Pro-Gly residues that favor a tight β-turn. The three-stranded β-sheet (Ac-VFITS(D) PGKTYTEV(D) PGOKILQ-NH2 ) is stabilized by the turn sequences, whereas the β-hairpin (SWTVE(D) PGKYTYK-NH2 ) folding is assisted by both the turn sequence and hydrophobic cross-strand interactions. Relaxation times after the T-jump were monitored as a function of temperature and occur on a sub-microsecond time scale, (D) P(D) P being faster than WVYY-(D) P. The Xxx-(D) Pro tertiary amide provides a detectable IR band, allowing us to probe the dynamics site-specifically. The relative importance of the turn versus the intrastrand stability in β-sheet formation is discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. GLI1 interferes with the DNA mismatch repair system in pancreatic cancer through BHLHE41-mediated suppression of MLH1.

    PubMed

    Inaguma, Shingo; Riku, Miho; Hashimoto, Mitsuyoshi; Murakami, Hideki; Saga, Shinsuke; Ikeda, Hiroshi; Kasai, Kenji

    2013-12-15

    The mismatch repair (MMR) system is indispensable for the fidelity of DNA replication, the impairment of which predisposes to the development and progression of many types of cancers. To date, GLI1 transcription factor, a key molecule of the Hedgehog signaling pathway, has been shown to regulate the expression of several genes crucial for a variety of cancer cell properties in many types of cancers, including pancreatic ductal adenocarcinoma (PDAC), but whether GLI1 could control the MMR system was not known. Here, we showed that GLI1 and GLI2 indirectly suppressed the expression of MLH1 in PDAC cells. Through GLI1 target gene screening, we found that GLI1 and GLI2 activated the expression of a basic helix-loop-helix type suppressor BHLHE41/DEC2/SHARP1 through a GLI-binding site in the promoter. Consistent with a previous report that BHLHE41 suppresses the MLH1 promoter activity, we found that the activation of GLI1 led to the BHLHE41-dependent suppression of MLH1, and a double knockdown of GLI1 and GLI2 conversely increased the MLH1 protein in PDAC cells. Using TALEN-based modification of the MLH1 gene, we further showed that GLI1 expression was indeed associated with an increased tolerance to a methylating agent, methylnitrosourea cooperatively with a lower copy number status of MLH1. Finally, GLI1 expression was immunohistochemically related positively with BHLHE41 and inversely with MLH1 in PDAC cells and precancerous lesions of the pancreas. On the basis of these results, we propose that GLI1 depresses the MMR activity and might contribute to the development and progression of PDAC. ©2013 AACR.

  13. The tRNA(Gly) T10003C mutation in mitochondrial haplogroup M11b in a Chinese family with diabetes decreases the steady-state level of tRNA(Gly), increases aberrant reactive oxygen species production, and reduces mitochondrial membrane potential.

    PubMed

    Li, Wei; Wen, Chaowei; Li, Weixing; Wang, Hailing; Guan, Xiaomin; Zhang, Wanlin; Ye, Wei; Lu, Jianxin

    2015-10-01

    Mitochondrial diabetes originates mainly from mutations located in maternally transmitted, mitochondrial tRNA-coding genes. In a genetic screening program of type 2 diabetes conducted with a Chinese Han population, we found one family with suggestive maternally transmitted diabetes. The proband's mitochondrial genome was analyzed using DNA sequencing. Total 42 known nucleoside changes and 1 novel variant were identified, and the entire mitochondrial DNA sequence was assigned to haplogroup M11b. Phylogenetic analysis showed that a homoplasmic mutation, 10003T>C transition, occurred at the highly conserved site in the gene encoding tRNA(Gly). Using a transmitochondrial cybrid cell line harboring this mutation, we observed that the steady-state level of tRNA(Gly) significantly affected and the amount of tRNA(Gly) decreased by 97%, production of reactive oxygen species was enhanced, and mitochondrial membrane potential, mtDNA copy number and cellular oxygen consumption rate were remarkably decreased compared with wild-type cybrid cells. The homoplasmic 10003T>C mutation in the mitochondrial tRNA(Gly) gene suggested to be as a pathogenesis-related mutation which might contribute to the maternal inherited diabetes in the Han Chinese family.

  14. The Sequential Action of a Dipeptidase and a β-Lyase Is Required for the Release of the Human Body Odorant 3-Methyl-3-sulfanylhexan-1-ol from a Secreted Cys-Gly-(S) Conjugate by Corynebacteria*S⃞

    PubMed Central

    Emter, Roger; Natsch, Andreas

    2008-01-01

    Human axillary odor is formed by the action of Corynebacteria on odorless axilla secretions. Sulfanylalkanols, 3-methyl-3-sulfanylhexan-1-ol in particular, form one key class of the odoriferous compounds. A conjugate with the dipeptide Cys-Gly has been reported as the secreted precursor for 3-methyl-3-sulfanylhexan-1-ol. Here, we confirm the Cys-Gly-(S) conjugate as the major precursor of this odorant, with lower levels of the Cys-(S) conjugate being present in axilla secretions. The enzymatic release of 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate by the axilla isolate Corynebacterium Ax20 was thus investigated. Cellular extracts of Ax20 released 3-methyl-3-sulfanylhexan-1-ol from the Cys-Gly-(S) conjugate and from the Cys-(S) conjugate, whereas the previously isolated C-S lyase of this bacterial strain was only able to cleave the Cys-(S) conjugate. o-Phenanthroline blocked the release from the Cys-Gly-(S) conjugate but did not affect cleavage of the Cys-(S) conjugate, indicating that in a first step, a metal-dependent dipeptidase hydrolyzes the Cys-Gly bond. This enzyme was purified by four chromatographic steps and gel electrophoresis, and the partial amino acid sequence was determined. The corresponding gene was cloned and expressed in Escherichia coli. It codes for a novel dipeptidase with a high affinity toward the Cys-Gly-(S) conjugate of 3-methyl-3-sulfanylhexan-1-ol. Co-incubating either the synthetic Cys-Gly-(S) conjugate or fresh axilla secretions with both the C-S lyase and the novel dipeptidase did release 3-methyl-3-sulfanylhexan-1-ol, proving that the sequential action of these two enzymes from the skin bacterium Corynebacterium Ax20 does release the odorant from the key secreted precursor. PMID:18515361

  15. Identification and Validation of Novel Hedgehog-Responsive Enhancers Predicted by Computational Analysis of Ci/Gli Binding Site Density

    PubMed Central

    Richards, Neil; Parker, David S.; Johnson, Lisa A.; Allen, Benjamin L.; Barolo, Scott; Gumucio, Deborah L.

    2015-01-01

    The Hedgehog (Hh) signaling pathway directs a multitude of cellular responses during embryogenesis and adult tissue homeostasis. Stimulation of the pathway results in activation of Hh target genes by the transcription factor Ci/Gli, which binds to specific motifs in genomic enhancers. In Drosophila, only a few enhancers (patched, decapentaplegic, wingless, stripe, knot, hairy, orthodenticle) have been shown by in vivo functional assays to depend on direct Ci/Gli regulation. All but one (orthodenticle) contain more than one Ci/Gli site, prompting us to directly test whether homotypic clustering of Ci/Gli binding sites is sufficient to define a Hh-regulated enhancer. We therefore developed a computational algorithm to identify Ci/Gli clusters that are enriched over random expectation, within a given region of the genome. Candidate genomic regions containing Ci/Gli clusters were functionally tested in chicken neural tube electroporation assays and in transgenic flies. Of the 22 Ci/Gli clusters tested, seven novel enhancers (and the previously known patched enhancer) were identified as Hh-responsive and Ci/Gli-dependent in one or both of these assays, including: Cuticular protein 100A (Cpr100A); invected (inv), which encodes an engrailed-related transcription factor expressed at the anterior/posterior wing disc boundary; roadkill (rdx), the fly homolog of vertebrate Spop; the segment polarity gene gooseberry (gsb); and two previously untested regions of the Hh receptor-encoding patched (ptc) gene. We conclude that homotypic Ci/Gli clustering is not sufficient information to ensure Hh-responsiveness; however, it can provide a clue for enhancer recognition within putative Hedgehog target gene loci. PMID:26710299

  16. Penultimate proline in neuropeptides.

    PubMed

    Glover, Matthew S; Bellinger, Earl P; Radivojac, Predrag; Clemmer, David E

    2015-08-18

    A recent ion mobility spectrometry-mass spectrometry (IMS-MS) study revealed that tryptic peptide ions containing a proline residue at the second position from the N-terminus (i.e., penultimate proline) frequently adopt multiple conformations, owing to the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds [J. Am. Soc. Mass Spectrom. 2015, 26, 444]. Here, we present a statistical analysis of a neuropeptide database that illustrates penultimate proline residues are frequently found in neuropeptides. In order to probe the effect of penultimate proline on neuropeptide conformations, IMS-MS experiments were performed on two model peptides in which penultimate proline residues were known to be important for biological activity: the N-terminal region of human neuropeptide Y (NPY1-9, Tyr(1)-Pro(2)-Ser(3)-Lys(4)-Pro(5)-Asp(6)-Asn(7)-Pro(8)-Gly(9)-NH2) and a tachykinin-related peptide (CabTRP Ia, Ala(1)-Pro(2)-Ser(3)-Gly(4)-Phe(5)-Leu(6)-Gly(7)-Met(8)-Arg(9)-NH2). From these studies, it appears that penultimate prolines allow neuropeptides to populate multiple conformations arising from the cis-trans isomerization of Xaa(1)-Pro(2) peptide bonds. Although it is commonly proposed that the role of penultimate proline residues is to protect peptides from enzymatic degradation, the present results indicate that penultimate proline residues also are an important means of increasing the conformational heterogeneity of neuropeptides.

  17. Peroxisome proliferator-activated receptor gamma co-activator 1 gene Gly482Ser polymorphism is associated with the response of low-density lipoprotein cholesterol concentrations to exercise training in elderly Japanese.

    PubMed

    Tobina, Takuro; Mori, Yukari; Doi, Yukiko; Nakayama, Fuki; Kiyonaga, Akira; Tanaka, Hiroaki

    2017-09-01

    Muscle peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1)α gene expression is influenced by the Gly482Ser gene polymorphism, which is a candidate genetic risk factor for diabetes mellitus and obesity. This study investigated the effects of PGC-1 gene Gly482Ser polymorphisms on alterations in glucose and lipid metabolism induced by exercise training. A 12-week intervention study was performed for 119 participants who were more than 65 years of age and completed exercise training at lactate threshold intensity. Total cholesterol and low-density lipoprotein cholesterol were significantly reduced in Gly/Gly but not in Gly/Ser and Ser/Ser participants after exercise. The Gly/Gly genotype of the PGC-1 gene Gly482Ser polymorphism influences the effects of moderate-intensity exercise training on low-density lipoprotein cholesterol and total cholesterol concentrations in older people.

  18. Sequences Flanking the Gephyrin-Binding Site of GlyRβ Tune Receptor Stabilization at Synapses

    PubMed Central

    Grünewald, Nora; Salvatico, Charlotte; Kress, Vanessa

    2018-01-01

    Abstract The efficacy of synaptic transmission is determined by the number of neurotransmitter receptors at synapses. Their recruitment depends upon the availability of postsynaptic scaffolding molecules that interact with specific binding sequences of the receptor. At inhibitory synapses, gephyrin is the major scaffold protein that mediates the accumulation of heteromeric glycine receptors (GlyRs) via the cytoplasmic loop in the β-subunit (β-loop). This binding involves high- and low-affinity interactions, but the molecular mechanism of this bimodal binding and its implication in GlyR stabilization at synapses remain unknown. We have approached this question using a combination of quantitative biochemical tools and high-density single molecule tracking in cultured rat spinal cord neurons. The high-affinity binding site could be identified and was shown to rely on the formation of a 310-helix C-terminal to the β-loop core gephyrin-binding motif. This site plays a structural role in shaping the core motif and represents the major contributor to the synaptic confinement of GlyRs by gephyrin. The N-terminal flanking sequence promotes lower affinity interactions by occupying newly identified binding sites on gephyrin. Despite its low affinity, this binding site plays a modulatory role in tuning the mobility of the receptor. Together, the GlyR β-loop sequences flanking the core-binding site differentially regulate the affinity of the receptor for gephyrin and its trapping at synapses. Our experimental approach thus bridges the gap between thermodynamic aspects of receptor-scaffold interactions and functional receptor stabilization at synapses in living cells. PMID:29464196

  19. An interdigit signalling centre instructs coordinate phalanx-joint formation governed by 5′Hoxd–Gli3 antagonism

    PubMed Central

    Huang, Bau-Lin; Trofka, Anna; Furusawa, Aki; Norrie, Jacqueline L.; Rabinowitz, Adam H.; Vokes, Steven A.; Mark Taketo, M.; Zakany, Jozsef; Mackem, Susan

    2016-01-01

    The number of phalanges and joints are key features of digit ‘identity' and are central to limb functionality and evolutionary adaptation. Prior chick work indicated that digit phalanges and their associated joints arise in a different manner than the more sparsely jointed long bones, and their identity is regulated by differential signalling from adjacent interdigits. Currently, there is no genetic evidence for this model, and the molecular mechanisms governing digit joint specification remain poorly understood. Using genetic approaches in mouse, here we show that functional 5′Hoxd–Gli3 antagonism acts indirectly, through Bmp signalling from the interdigital mesenchyme, to regulate specification of joint progenitors, which arise in conjunction with phalangeal precursors at the digit tip. Phalanx number, although co-regulated, can be uncoupled from joint specification. We propose that 5′Hoxd genes and Gli3 are part of an interdigital signalling centre that sets net Bmp signalling levels from different interdigits to coordinately regulate phalanx and joint formation. PMID:27713395

  20. Investigation of jumbo squid (Dosidicus gigas) skin gelatin peptides for their in vitro antioxidant effects.

    PubMed

    Mendis, Eresha; Rajapakse, Niranjan; Byun, Hee-Guk; Kim, Se-Kwon

    2005-09-09

    Peptides derived from tryptic hydrolysate of jumbo squid (Dosidicus gigas) skin gelatin were assessed for their antioxidant properties in different in vitro assay systems. The hydrolysate itself exhibited a strong lipid peroxidation inhibition and it was much higher than that of natural antioxidant, alpha-tocopherol. In addition, it could scavenge highly active free radicals in oxidative systems, in the order of hydroxyl and carbon-centered radicals. Two representative peptides with comparatively higher antioxidant potency were purified and characterized as Phe-Asp-Ser-Gly-Pro-Ala-Gly-Val-Leu (880.18 Da) and Asn-Gly-Pro-Leu-Gln-Ala-Gly-Gln-Pro-Gly-Glu-Arg (1241.59 Da). Furthermore, viability of radical-mediated oxidation-induced human lung fibroblasts was enhanced following the treatment of two peptides. However it did not exhibit substantial ion chelation, and we presumed that the observed radical scavenging potency of these peptides play a vital role for their strong antioxidant activity. Based on our results we suggest that hydrophobic amino acids present in peptide sequences contributed greatly for observed antioxidant activities.

  1. Atypical radiological findings in achondroplasia with uncommon mutation of the fibroblast growth factor receptor-3 (FGFR-3) gene (Gly to Cys transition at codon 375)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nishimuri, Gen; Fukushima, Yoshimitsu; Ohashi, Hirofumi

    1995-11-20

    The recent discovery of mutations in the FGFR-3 (fibroblast growth factor receptor-3) gene (FGFR3) as the cause of achondroplasia has provided new insight into understanding genetic diseases. It was surprising from the viewpoint of molecular genetics that most patients with achondroplasia showed the same mutation at nucleotide 1138, leading to a single amino acid substitution from glycine to arginine at codon 380 (Gly380Arg). All 39 patients examined by two groups had the Gly380Arg; 38 patients and the other demonstrated a G to A and a G to C transition at nucleotide 1138, respectively. Subsequently another group disclosed a G tomore » A transition at the same nucleotide 1138 in 21/23 patients of diverse ethnic origin, although mutations were not identified in two patients. To date, a total of 193 patients with the mutation of the G380Arg have been reported; a single patient with another mutation resulting in a substitution from glycine to cysteine at codon 375 (Gly375Cys) has been described. The presence of this common mutation is consistent with the clinical fact that achondroplastic individuals show less phenotypic variability than is unusual for autosomal dominant diseases. We encountered a Japanese boy with the Gly375Cys. His mother with achondroplasia has the same mutation. The molecular investigation of these patients was reported elsewhere. Here we report the clinical and radiological findings in this boy who demonstrated some atypical manifestations from those of typical achondroplasia. 8 refs., 1 fig.« less

  2. Synergistic effect with Phe-Gly-Leu-Met-NH2 of the C-terminal of substance P and insulin-like growth factor-1 on epithelial wound healing of rabbit cornea

    PubMed Central

    Nakamura, Masatsugu; Chikama, Tai-ichiro; Nishida, Teruo

    1999-01-01

    We previously reported that substance P and insulin-like growth factor-1 (IGF-1) synergistically stimulate corneal epithelial wound healing in vitro and in vivo. We wished to identify which portion of the amino acid sequence of substance P might be responsible for this synergism.Corneal epithelial migration was not affected by the addition of any one of the following factors: substance P; Phe-Gly-Leu-Met-NH2 (C-terminal of substance P); Val-Gly-Leu-Met-NH2 (C-terminal of neurokinin A, neurokinin B, and kassinin); Tyr-Gly-Leu-Met-NH2 (C-terminal of physalaemin); Ile-Gly-Leu-Met-NH2 (C-terminal of eledoisin); or Gly-Leu-Met-NH2 (common C-terminal of tachykinins).In the presence of IGF-1, only substance P and Phe-Gly-Leu-Met-NH2 were synergistic in stimulating corneal epithelial migration in a dose-dependent fashion.The combination of Phe-Gly-Leu-Met-NH2 and IGF-1 did not affect the incorporation of [3H]-thymidine into corneal epithelial cells.Treatment with Phe-Gly-Leu-Met-NH2 and IGF-1, but not with Phe-Gly-Leu-Met-NH2 or IGF-1 alone, increased attachment of corneal epithelial cells to a fibronectin matrix.The levels of α5 and β1 integrin were not affected by Phe-Gly-Leu-Met-NH2 or IGF-1 alone, but they were significantly increased by the combination of Phe-Gly-Leu-Met-NH2 and IGF-1.Topical application of the same combination facilitated corneal epithelial wound closure in vivo.These results demonstrated that Phe-Gly-Leu-Met-NH2, a sequence of 4 amino-acids of the C-terminal of substance P, is the minimum sequence necessary to produce the synergistic effects of substance P and IGF-1 on corneal epithelial wound healing. PMID:10385250

  3. Assessment of airborne soy-hull allergen (Gly m 1) in the Port of Ancona, Italy.

    PubMed

    Antonicelli, L; Ruello, M L; Monsalve, R I; González, R; Fava, G; Bonifazi, F

    2010-10-01

    Epidemic asthma outbreaks are potentially a very high-risk medical situation in seaport towns where large volumes of soybean are loaded and unloaded Airborne allergen assessment plays a pivotal role in evaluating the resulting environmental pollution. The aim of this study was to measure the airborne Gly m 1 allergen level in the seaport of Ancona in order assess the soybean-specific allergenic risk for the city. Allergen and PM10 were evaluated at progressive distances from the port area. Allergen analysis was performed by monoclonal antibody-based immunoassay on the sampled filters. Daily meteorological data were obtained from the local meteorological station. For estimating the assimilative capacity of the atmosphere, an approach based on dispersive ventilation coefficient was tried. The allergen concentrations detected were low (range = 0.4-171 ng/m3). A decreasing gradient of the airborne allergen from the unloading area (22.1 +/- 41.2 ng/m3) to the control area (0.6 +/- 0.7 ng/m3) was detected. The concentration of the airborne Gly m 1 was not coupled with the presence of the soy-carrying ships in the port. A statistically significant relationship between airborne allergen, PM10 and local meteorological parameters quantifies the association with the atmospheric condition. Airborne Gly m 1 is part of the atmospheric dust of Ancona. The low level of this allergen seems consistent with the absence of asthma epidemic outbreak.

  4. Toll receptor 4 Asp299Gly polymorphism and its association with preterm birth and premature rupture of membranes in a South American population

    PubMed Central

    Rey, G.; Skowronek, F.; Alciaturi, J.; Alonso, J.; Bertoni, B.; Sapiro, R.

    2008-01-01

    Preterm birth (PTB) is a worldwide health problem and remains the leading cause of perinatal morbidity and mortality. Systemic and local intrauterine infections have been implicated in the pathogenesis of preterm labor and delivery. Common pathways between PTB, premature rupture of ovular membranes (PROM) and altered molecular routes of inflammation have been proposed. There is evidence to support a genetic component in these conditions. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is thought to play a key role in eliciting an inflammatory response. LPS is recognized by proteins of the innate immune system, including Toll-like receptor 4 (TLR4). Individuals from some European countries carrying the variant alleles resulting in an amino acid substitution (Asp299Gly) are at increased risk of Gram-negative infections and premature birth. The objective of this study was to determine if preterm newborns have different allele frequency of the Asp299Gly TLR4 variant from healthy term neonates in Uruguay. The impact of PROM was also examined. There was an increase in the risk for fetuses carrying the Asp299Gly substitution in TLR4 of being severely premature (<33 weeks) and to present PROM at the same time. PMID:18723631

  5. Overexpression of a glyoxalase gene, OsGly I, improves abiotic stress tolerance and grain yield in rice (Oryza sativa L.).

    PubMed

    Zeng, Zhengming; Xiong, Fangjie; Yu, Xiaohong; Gong, Xiaoping; Luo, Juntao; Jiang, Yudong; Kuang, Haochi; Gao, Bijun; Niu, Xiangli; Liu, Yongsheng

    2016-12-01

    Glyoxalase I (Gly I) is a component of the glyoxalase system which is involved in the detoxification of methylglyoxal, a byproduct of glycolysis. In the present study, a gene of rice (Oryza sativa L., cv. Nipponbare) encoding Gly I was cloned and characterized. The quantitative real-time PCR analysis indicated that rice Gly I (OsGly I) was ubiquitously expressed in root, stem, leaf, leaf sheath and spikelet with varying abundance. OsGly I was markedly upregulated in response to NaCl, ZnCl 2 and mannitol in rice seedlings. For further functional investigation, OsGly I was overexpressed in rice using Agrobacterium-mediated transformation. Transgenic rice lines exhibited increased glyoxalase enzyme activity, decreased methylglyoxal level and improved tolerance to NaCl, ZnCl 2 and mannitol compared to wild-type plants. Enhancement of stress tolerance in transgenic lines was associated with reduction of malondialdehyde content which was derived from cellular lipid peroxidation. In addition, the OsGly I-overexpression transgenic plants performed higher seed setting rate and yield. Collectively, these results indicate the potential of bioengineering the Gly I gene in crops. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  6. Disturbed MEK/ERK signaling increases osteoclast activity via the Hedgehog-Gli pathway in postmenopausal osteoporosis.

    PubMed

    Li, Xiaojie; Jie, Qiang; Zhang, Hongyang; Zhao, Yantao; Lin, Yangjing; Du, Junjie; Shi, Jun; Wang, Long; Guo, Kai; Li, Yong; Wang, Chunhui; Gao, Bo; Huang, Qiang; Liu, Jian; Yang, Liu; Luo, Zhuojing

    2016-11-01

    Postmenopausal osteoporosis is a worldwide health problem and is characterized by increased and activated osteoclasts. However, the mechanism by which osteoclasts are dysregulated in postmenopausal osteoporosis is not fully understood. In this study, we found that the Hedgehog-Gli pathway was upregulated in postmenopausal osteoporotic osteoclasts and that 17β-estradiol both inhibited osteoclastogenesis and induced osteoclast apoptosis by downregulating Hedgehog-Gli signaling. Furthermore, we demonstrated that the Hedgehog-Gli pathway was negatively regulated by MEK/ERK signaling and that this effect was Sonic Hedgehog (SHH)-dependent and was partially blocked by an anti-SHH antibody. Moreover, we found that the stimulatory effect of Hedgehog signaling on osteoclastogenesis and the inhibitory effect on osteoclast apoptosis were dependent on the Gli family of transcription factors. The pathways and molecules that contribute to the regulation of osteoclastogenesis and apoptosis represent potential new strategies for designing molecular drugs for the treatment of postmenopausal osteoporosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. 14-3-3ζ turns TGF-β's function from tumor suppressor to metastasis promoter in breast cancer by contextual changes of Smad partners from p53 to Gli2.

    PubMed

    Xu, Jia; Acharya, Sunil; Sahin, Ozgur; Zhang, Qingling; Saito, Yohei; Yao, Jun; Wang, Hai; Li, Ping; Zhang, Lin; Lowery, Frank J; Kuo, Wen-Ling; Xiao, Yi; Ensor, Joe; Sahin, Aysegul A; Zhang, Xiang H-F; Hung, Mien-Chie; Zhang, Jitao David; Yu, Dihua

    2015-02-09

    Transforming growth factor β (TGF-β) functions as a tumor suppressor in premalignant cells but as a metastasis promoter in cancer cells. The dichotomous functions of TGF-β are proposed to be dictated by different partners of its downstream effector Smads. However, the mechanism for the contextual changes of Smad partners remained undefined. Here, we demonstrate that 14-3-3ζ destabilizes p53, a Smad partner in premalignant mammary epithelial cells, by downregulating 14-3-3σ, thus turning off TGF-β's tumor suppression function. Conversely, 14-3-3ζ stabilizes Gli2 in breast cancer cells, and Gli2 partners with Smads to activate PTHrP and promote TGF-β-induced bone metastasis. The 14-3-3ζ-driven contextual changes of Smad partners from p53 to Gli2 may serve as biomarkers and therapeutic targets of TGF-β-mediated cancer progression. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Prevention of Atrial Fibrillation by Bucindolol Is Dependent on the Beta1389 Arg/Gly Adrenergic Receptor Polymorphism

    PubMed Central

    Aleong, Ryan G.; Sauer, William H.; Davis, Gordon; Murphy, Guinevere A.; Port, J. David; Anand, Inder S.; Fiuzat, Mona; O’Connor, Christopher M.; Abraham, William T.; Liggett, Stephen B.; Bristow, Michael R.

    2013-01-01

    Objectives This study assessed the impact of bucindolol, a beta-blocker/sympatholytic agent, on the development of atrial fibrillation (AF) in advanced chronic heart failure with reduced left ventricular ejection fraction (HFREF) patients enrolled in the BEST (Beta-Blocker Evaluation of Survival Trial). Background β-Blockers have modest efficacy for AF prevention in HFREF patients. Bucindolol’s effects on HF and ventricular arrhythmic endpoints are genetically modulated by β1- and α2c-adrenergic receptor (AR) polymorphisms that can be used to subdivide HFREF populations into those with bucindolol effectiveness levels that are enhanced, unchanged, or lost. Methods BEST enrolled 2,708 New York Heart Association (NYHA) class III to IV HFREF patients. A substudy in which 1,040 patients’ DNA was genotyped for the β1-AR position 389 Arg/Gly and the α2c322–325 wild type (Wt)/deletion (Del) polymorphisms, and new-onset AF was assessed from adverse event case report forms or electrocardiograms at baseline and at 3 and 12 months. Results In the entire cohort, bucindolol reduced the rate of new-onset AF compared to placebo by 41% (hazard ratio [HR]: 0.59 [95% confidence interval (CI): 0.44 to 0.79], p = 0.0004). In the 493 β1389 arginine homozygotes (Arg/Arg) in the DNA substudy, bucindolol reduced new-onset AF by 74% (HR: 0.26 [95% CI: 0.12 to 0.57]), with no effect in β1389 Gly carriers (HR: 1.01 [95% CI: 0.56 to 1.84], interaction test = 0.008). When β1389 Gly carriers were subdivided by α2c Wt homozygotes (n = 413, HR: 0.94 [95% CI: 0.48 to 1.82], p = 0.84) or Del variant carriers (n = 134, HR: 1.33 [95% CI: 0.32 to 5.64], p = 0.70), there was a positive interaction test (p = 0.016) when analyzed with β1389 Arg homozygotes. Conclusions Bucindolol prevented new-onset AF; β1 and α2c polymorphisms predicted therapeutic response; and the 47% of patients who were β1389 Arg homozygotes had an enhanced effect size of 74%. (Beta-Blocker Evaluation in Survival

  9. Modulation of mitochondrial activity in HaCaT keratinocytes by the cell penetrating peptide Z-Gly-RGD(DPhe)-mitoparan.

    PubMed

    Richardson, Adam; Muir, Lewis; Mousdell, Sasha; Sexton, Darren; Jones, Sarah; Howl, John; Ross, Kehinde

    2018-01-30

    Biologically active cell penetrating peptides (CPPs) are an emerging class of therapeutic agent. The wasp venom peptide mastoparan is an established CPP that modulates mitochondrial activity and triggers caspase-dependent apoptosis in cancer cells, as does the mastoparan analogue mitoparan (mitP). Mitochondrial depolarisation and activation of the caspase cascade also underpins the action of dithranol, a topical agent for treatment of psoriasis. The effects of a potent mitP analogue on mitochondrial activity were therefore examined to assess its potential as a novel approach for targeting mitochondria for the treatment of psoriasis. In HaCaT keratinocytes treated with the mitP analogue Z-Gly-RGD(DPhe)-mitP for 24 h, a dose-dependent loss of mitochondrial activity was observed using the methyl-thiazolyl-tetrazolium (MTT) assay. At 10 μmol L -1 , MTT activity was less than 30% that observed in untreated cells. Staining with the cationic dye JC-1 suggested that Z-Gly-RGD(DPhe)-mitP also dissipated the mitochondrial membrane potential, with a threefold increase in mitochondrial depolarisation levels. However, caspase activity appeared to be reduced by 24 h exposure to Z-Gly-RGD(DPhe)-mitP treatment. Furthermore, Z-Gly-RGD(DPhe)-mitP treatment had little effect on overall cell viability. Our findings suggest Z-Gly-RGD(DPhe)-mitP promotes the loss of mitochondrial activity but does not appear to evoke apoptosis in HaCaT keratinocytes.

  10. Self-protection against gliotoxin--a component of the gliotoxin biosynthetic cluster, GliT, completely protects Aspergillus fumigatus against exogenous gliotoxin.

    PubMed

    Schrettl, Markus; Carberry, Stephen; Kavanagh, Kevin; Haas, Hubertus; Jones, Gary W; O'Brien, Jennifer; Nolan, Aine; Stephens, John; Fenelon, Orla; Doyle, Sean

    2010-06-10

    Gliotoxin, and other related molecules, are encoded by multi-gene clusters and biosynthesized by fungi using non-ribosomal biosynthetic mechanisms. Almost universally described in terms of its toxicity towards mammalian cells, gliotoxin has come to be considered as a component of the virulence arsenal of Aspergillus fumigatus. Here we show that deletion of a single gene, gliT, in the gliotoxin biosynthetic cluster of two A. fumigatus strains, rendered the organism highly sensitive to exogenous gliotoxin and completely disrupted gliotoxin secretion. Addition of glutathione to both A. fumigatus Delta gliT strains relieved gliotoxin inhibition. Moreover, expression of gliT appears to be independently regulated compared to all other cluster components and is up-regulated by exogenous gliotoxin presence, at both the transcript and protein level. Upon gliotoxin exposure, gliT is also expressed in A. fumigatus Delta gliZ, which cannot express any other genes in the gliotoxin biosynthetic cluster, indicating that gliT is primarily responsible for protecting this strain against exogenous gliotoxin. GliT exhibits a gliotoxin reductase activity up to 9 microM gliotoxin and appears to prevent irreversible depletion of intracellular glutathione stores by reduction of the oxidized form of gliotoxin. Cross-species resistance to exogenous gliotoxin is acquired by A. nidulans and Saccharomyces cerevisiae, respectively, when transformed with gliT. We hypothesise that the primary role of gliotoxin may be as an antioxidant and that in addition to GliT functionality, gliotoxin secretion may be a component of an auto-protective mechanism, deployed by A. fumigatus to protect itself against this potent biomolecule.

  11. Inhibitors of tripeptidyl peptidase II. 3. Derivation of butabindide by successive structure optimizations leading to a potential general approach to designing exopeptidase inhibitors.

    PubMed

    Ganellin, C Robin; Bishop, Paul B; Bambal, Ramesh B; Chan, Suzanne M T; Leblond, Bertrand; Moore, Andrew N J; Zhao, Lihua; Bourgeat, Pierre; Rose, Christiane; Vargas, Froylan; Schwartz, Jean-Charles

    2005-11-17

    The cholecystokinin-8 (CCK-8)-inactivating peptidase is a serine peptidase that has been shown to be a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10). It cleaves the neurotransmitter CCK-8 sulfate at the Met-Gly bond to give Asp-Tyr(SO3H)-Met-OH + Gly-Trp-Met-Asp-Phe-NH2. Starting from Val-Pro-NHBu, a dipeptide of submicromolar affinity that had previously been generated to serve as a lead, successive optimization at P3, P1, and then P2 gave Abu-Pro-NHBu (18, Ki = 80 nM). Further transformation (by making a benzologue) gave the indoline analogue, butabindide (33) as a reversible inhibitor having nanomolar affinity (Ki = 7 nM). Retrospective analysis suggested the possibility of a general approach to designing exopeptidase inhibitors starting from the structure of the first hydrolysis product. Application of this approach to CCK-8 led to Abu-Phe-NHBu (37), but this only had Ki = 9.4 microM. Molecular modeling, to determine the minimum energy conformations and explain the 1000-fold better affinity of butabindide, indicated that 37 cannot access the likely active conformation of butabindide.

  12. Characterization of Nα-Fmoc-protected dipeptide isomers by electrospray ionization tandem mass spectrometry (ESI-MS(n)): effect of protecting group on fragmentation of dipeptides.

    PubMed

    Ramesh, M; Raju, B; Srinivas, R; Sureshbabu, V V; Vishwanatha, T M; Hemantha, H P

    2011-07-30

    A series of positional isomeric pairs of Fmoc-protected dipeptides, Fmoc-Gly-Xxx-OY/Fmoc-Xxx-Gly-OY (Xxx=Ala, Val, Leu, Phe) and Fmoc-Ala-Xxx-OY/Fmoc-Xxx-Ala-OY (Xxx=Leu, Phe) (Fmoc=[(9-fluorenylmethyl)oxy]carbonyl) and Y=CH(3)/H), have been characterized and differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MS(n)). In contrast to the behavior of reported unprotected dipeptide isomers which mainly produce y(1)(+) and/or a(1)(+) ions, the protonated Fmoc-Xxx-Gly-OY, Fmoc-Ala-Xxx-OY and Fmoc-Xxx-Ala-OY yield significant b(1)(+) ions. These ions are formed, presumably with stable protonated aziridinone structures. However, the peptides with Gly- at the N-terminus do not form b(1)(+) ions. The [M+H](+) ions of all the peptides undergo a McLafferty-type rearrangement followed by loss of CO(2) to form [M+H-Fmoc+H](+). The MS(3) collision-induced dissociation (CID) of these ions helps distinguish the pairs of isomeric dipeptides studied in this work. Further, negative ion MS(3) CID has also been found to be useful for differentiating these isomeric peptide acids. The MS(3) of [M-H-Fmoc+H](-) of isomeric peptide acids produce c(1)(-), z(1)(-) and y(1)(-) ions. Thus the present study of Fmoc-protected peptides provides additional information on mass spectral characterization of the dipeptides and distinguishes the positional isomers. Copyright © 2011 John Wiley & Sons, Ltd.

  13. Preferential amino acid sequences in alumina-catalyzed peptide bond formation.

    PubMed

    Bujdák, J; Rode, B M

    2002-05-21

    The catalytic effect of activated alumina on amino acid condensation was investigated. The readiness of amino acids to form peptide sequences was estimated on the basis of the yield of dipeptides and was found to decrease in the order glycine (Gly), alanine (Ala), leucine (Leu), valine (Val), proline (Pro). For example, approximately 15% Gly was converted to the dipeptide (Gly(2)), 5% to cyclic anhydride (cyc(Gly(2))) and small amounts of tri- (Gly(3)) and tetrapeptide (Gly(4)) were formed after 28 days. On the other hand, only trace amounts of Pro(2) were formed from proline under the same conditions. Preferential formation of certain sequences was observed in the mixed reaction systems containing two amino acids. For example, almost ten times more Gly-Val than Val-Gly was formed in the Gly+Val reaction system. The preferred sequences can be explained on the basis of an inductive effect that side groups have on the nucleophilicity and electrophilicity, respectively, of the amino and carboxyl groups. A comparison with published data of amino acid reactions in other reaction systems revealed that the main trends of preferential sequence formation were the same as those described for the salt-induced peptide formation (SIPF) reaction. The results of this work and other previously published papers show that alumina and related mineral surfaces might have played a crucial role in the prebiotic formation of the first peptides on the primitive earth.

  14. Deficiencies in pro-thyrotropin-releasing hormone processing and abnormalities in thermoregulation in Cpefat/fat mice.

    PubMed

    Nillni, Eduardo A; Xie, Weihua; Mulcahy, Lawrence; Sanchez, Vanesa C; Wetsel, William C

    2002-12-13

    Cpe(fat/fat) mice are obese, diabetic, and infertile. They have a mutation in carboxypeptidase E (CPE), an enzyme that converts prohormone intermediates to bioactive peptides. The Cpe(fat) mutation leads to rapid degradation of the enzyme. To test whether pro-thyrotropin-releasing hormone (TRH) conversion to TRH involves CPE, processing was examined in the Cpe(fat/fat) mouse. Hypothalamic TRH is depressed by at least 75% compared with wild-type controls. Concentrations of pro-TRH forms are increased in homozygotes. TRH-[Gly(4)-Lys(5)-Arg(6)] and TRH-[Gly(4)-Lys(5)] represent approximately 45% of the total TRH-like immunoreactivity in Cpe(fat/fat) mice; they constitute approximately 1% in controls. Levels of TRH-[Gly(4)] were depressed in homozygotes. Because the hypothalamus contains some TRH, another carboxypeptidase must be responsible for processing. Immunocytochemical studies indicate that TRH neurons contain CPE- and carboxypeptidase D-like immunoreactivity. Recombinant CPE or carboxypeptidase D can convert synthetic TRH-[Gly(4)-Lys(5)] and TRH-[Gly(4)-Lys(5)-Arg(6)] to TRH-[Gly(4)]. When Cpe(fat/fat) mice are exposed to cold, they cannot maintain their body temperatures, and this loss is associated with hypothalamic TRH depletion and reduction in thyroid hormone. These findings demonstrate that the Cpe(fat) mutation can affect not only carboxypeptidase activity but also endoproteolysis. Because Cpe(fat/fat) mice cannot sustain a cold challenge, and because alterations in the hypothalamic-pituitary-thyroid axis can affect metabolism, deficits in pro-TRH processing may contribute to the obese and diabetic phenotype in these mice.

  15. 14-3-3ζ turns TGF-β’s function from tumor suppressor to metastasis promoter in breast cancer by contextual changes of Smad partners from p53 to Gli2

    PubMed Central

    Xu, Jia; Acharya, Sunil; Sahin, Ozgur; Zhang, Qingling; Saito, Yohei; Yao, Jun; Wang, Hai; Li, Ping; Zhang, Lin; Lowery, Frank J; Kuo, Wen-Ling; Xiao, Yi; Ensor, Joe; Sahin, Aysegul A; Zhang, Xiang H.-F.; Hung, Mien-Chie; Zhang, Jitao David; Yu, Dihua

    2015-01-01

    Summary Transforming growth factor-β (TGF-β) functions as a tumor suppressor in pre-malignant cells but as a metastasis promoter in cancer cells. The dichotomous functions of TGF-β are proposed to be dictated by different partners of its downstream effectors Smads. However, the mechanism for the contextual changes of Smad partners remained undefined. Here, we demonstrate that 14-3-3ζ destabilizes p53, a Smad partner in pre-malignant mammary epithelial cells, by downregulating 14-3-3σ, thus turning off TGF-β’s tumor suppression function. Conversely, 14-3-3ζ stabilizes Gli2 in breast cancer cells, and Gli2 partners with Smads to activate PTHrP and promote TGF-β-induced bone metastasis. The 14-3-3ζ-driven contextual changes of Smad partners from p53 to Gli2 may serve as biomarkers and therapeutic targets of TGF-β-mediated cancer progression. PMID:25670079

  16. Matrix Metalloproteinase-8 Facilitates Neutrophil Migration through the Corneal Stromal Matrix by Collagen Degradation and Production of the Chemotactic Peptide Pro-Gly-Pro

    PubMed Central

    Lin, Michelle; Jackson, Patricia; Tester, Angus M.; Diaconu, Eugenia; Overall, Christopher M.; Blalock, J. Edwin; Pearlman, Eric

    2008-01-01

    Matrix metalloproteinase (MMP)-8 and MMP-9 play several roles in inflammation, including degradation of extracellular matrix (ECM) components and regulation of cytokine activity. To determine the roles of MMP-8 and MMP-9 in a neutrophil-dependent inflammatory response, we used a murine model of corneal inflammation in which LPS is injected into the corneal stroma. In contrast to wild-type mice, we found that i) lipopolysaccharide (LPS)-injected CXCR2−/− corneas had impaired neutrophil infiltration and did not express either MMP-8 or MMP-9; ii) neutrophil migration through the central cornea was impaired in Mmp8−/−, but not Mmp9−/−, mice; iii) neutrophil migration was inhibited in collagenase-resistant mice; iv) the chemotactic Pro-Gly-Pro (PGP) tripeptide that binds CXCR2 was decreased in CXCR2−/− mice; v) PGP production was impaired in Mmp8−/− corneas; and vi) neutralizing anti-PGP antibody did not inhibit neutrophil infiltration in Mmp8−/− mice. We found no effects of MMP-8 on LPS-induced CXC chemokine (LIX, or CXCL5)-induced neutrophil recruitment or on LPS-induced CXC chemokine production. Together, these studies indicate that neutrophils contribute to the production of both MMP-8 and MMP-9 in LPS-injected corneas and that MMP-8 regulates neutrophil migration through the dense collagenous ECM of the corneal stroma by generating chemotactic PGP during inflammation. PMID:18556780

  17. The cytotoxic constituents from marine-derived streptomyces 3320#

    NASA Astrophysics Data System (ADS)

    Ren, Hong; Gu, Qianqun; Cui, Chengbin; Zhu, Weiming

    2006-01-01

    The present work studies the chemical constituents from marine-derived streptomyces 3320# and their antitumor activities. The n-BuOH extract of the ferment broth of 3320# was chromatographed on silica gel, Sephadex LH-20, ODS columns and HPLC to separate the compounds with antitoumor activities. Their structures were identified using IR, UV, NMR, MS spectroscopic techniques and compared with published data. The antitumor activities of the isolates were assayed using SRB method and flow cytometry assay, accompanied with the morphological observation of the cells under light microscope against mammalian tsFT210 cells. Ten compounds, cyclo-(Ala-Leu) 1, cyclo-(Ala-Ile) 2, cyclo-(Ala-Val) 3, cyclo-(Phe- Pro) 4, cyclo-(Phe-Gly) 5, cyclo-(Leu-Pro) 6, 1-methyl-1, 2, 3, 4-tetrahydro-β-carboline-3-carboxylic acid 7, N-(4-hydroxyphenethyl) acetamide 8, 4-methyoxy-1-(2-hydroxy) ethylbenzene 9 and uridine 10, were isolated from the ferment broth of streptomyces 3320#. Among them, compounds 6, 7, 8 and 10 showed potent cytotoxicity against the tsFT210 cell with the IC50 values of 3.6, 7.2, 5.2 and 1.6 mmol L-1, respectively. Compounds 8, 10 also exhibited apoptosis inducing activity under 2.0 mmol L-1. Compounds 6, 7, 8 and 10 are the principle bioactive constituents responsible for the antitumor activities of marine streptomyces 3320#. Compound 7 was isolated from this species for the first time.

  18. Response to Indacaterol/Glycopyrronium (IND/GLY) by Sex in Patients with COPD: A Pooled Analysis from the IGNITE Program.

    PubMed

    Tsiligianni, Ioanna; Mezzi, Karen; Fucile, Sebastian; Kostikas, Konstantinos; Shen, Steven; Banerji, Donald; Fogel, Robert

    2017-08-01

    In this pooled analysis, we compared the effect of indacaterol/glycopyrronium (IND/GLY) by sex versus other commonly used chronic obstructive pulmonary disease (COPD) treatments and placebo. Male and female patients with moderate-to-very-severe COPD who had participated in six randomized controlled trials were included in the analysis. Baseline demographics and disease characteristics were analyzed by sex, and any differences noted. The effects of IND/GLY versus salmeterol/fluticasone (SFC), glycopyrronium, tiotropium and placebo, on lung function and the patient-reported outcomes (health status, dyspnea, rescue medication use and symptoms) were assessed by sex after 26 weeks treatment. The analysis population comprised 4719 men and 1389 women. Most baseline parameters differed significantly between men and women. Nonetheless, despite these differences in baseline characteristics, IND/GLY significantly improved lung function versus placebo (p < 0.0001) and all active comparators (p < 0.01) in men and women. Overall, IND/GLY showed better improvement in dyspnea and health status compared with all other treatments in both sex. Greater reduction of rescue medication use was observed with IND/GLY versus placebo and other treatments (all p < 0.01 expect IND/GLY versus SFC). Although some variability was observed, improvements in health status, dyspnea, rescue medication use and symptoms were generally larger in women than in men. Irrespective of sex, IND/GLY provided superior efficacy to monotherapy or SFC in both men and women. Small differences in efficacy response by sex were observed, which should be evaluated further in prospective clinical studies. Nevertheless, the benefits observed with IND/GLY confirm dual bronchodilator as the preferred therapy in patients with moderate-to-very-severe COPD regardless of sex.

  19. Continuing evolution of canine parvovirus in China: Isolation of novel variants with an Ala5Gly mutation in the VP2 protein.

    PubMed

    Wang, Jianke; Lin, Peng; Zhao, Hang; Cheng, Yuening; Jiang, Zhong; Zhu, Hongwei; Wu, Hua; Cheng, Shipeng

    2016-03-01

    Canine parvovirus (CPV) type 2c is a new antigenic variant of CPV-2. Since the year 2000 it has spread to several countries, causing severe hemorrhagic enteritis in dogs. In 2014 and 2015, 58 fecal samples were collected from dogs in Beijing with suspected CPV infection. Regardless of the vaccination status of the dogs, 43 samples were found positive for CPV according to PCR results; i.e., 18, 7, and 18 respectively belonged to antigenic types new CPV-2a, new CPV-2b, and CPV-2c. A phylogenetic tree based on their VP2 gene sequences indicated that the Chinese CPV-2c strains form a separate cluster. In addition to synonymous mutations, the CPV-2c strains also contain a unique coding mutation in VP2 that leads to glycine at residue 5, instead of the highly conserved alanine at this position in all other CPV-2c strains sequenced to date. Using F81 cells, several novel isolates of CPV-2c, each with the Ala5Gly mutation, were obtained. One of these was used to infect experimentally beagle dogs, which subsequently developed the typical clinical symptoms of CPV infection. Hence, it appears that CPV-2c is still evolving in China, a finding that warrants continuous surveying and the eventual adaptation of current vaccines. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Evidence for greater oxidative substrate flexibility in male carriers of the Pro 12 Ala polymorphism in PPARgamma2.

    PubMed

    Thamer, C; Haap, M; Volk, A; Maerker, E; Becker, R; Bachmann, O; Machicao, F; Häring, H U; Stumvoll, M

    2002-03-01

    The Pro12Ala polymorphism of the peroxisome proliferator-activated receptor gamma2 (PPARgamma2) gene is associated with reduced type 2 diabetes risk and increased insulin sensitivity. It is possible that the oxidative shift from lipid to glucose as a fuel is more efficient in Ala allele carriers. To test this hypothesis, we examined carbohydrate and lipid oxidation by indirect calorimetry in lean, glucose tolerant subjects with (X/Ala, n = 25) and without the Pro12Ala polymorphism (Pro/Pro, n = 73) basally and after insulin stimulation during a 2-hour eugylcaemic hyperinsulinaemic clamp. Insulin sensitivity was non-significantly greater in X/Ala (0.13 +/- 0.01 micromol/kg/min/pM) than in Pro/Pro (0.12 +/- 0.01 micromol/kg/min/pM, p = 0.27). Basally, there were no lipid nor carbohydrate oxidation differences between the groups. Interestingly, the decrease in lipid oxidation during insulin stimulation was significantly greater in male X/Ala (- 0.51 +/- 0.06 mg/kg/min) than in male Pro/Pro (- 0.35 +/- 0.04 mg/kg/min, p = 0.03). No difference was observed in females. Analogously, the change in carbohydrate oxidation in male X/Ala (1.34 +/- 0.2 mg/kg/min) was significantly greater than in male Pro/Pro (1.03 +/- 0.12 mg/kg/min, p = 0.05). The respiratory quotient increased more, but not significantly more, in male X/Ala (0.11 +/- 0.01) than in male Pro/Pro subjects (0.08 +/- 0.01, p = 0.08) but similarly in females. These results indicate that the mechanism by which the Ala allele improves insulin sensitivity might involve enhanced suppression of lipid oxidation permitting more efficient (predominantly non-oxidative) glucose disposal. It is unclear why this could be demonstrated only in males, although gender differences in substrate oxidation are well documented.

  1. Dopamine D3 receptor Ser9Gly variant is associated with impulse control disorders in Parkinson's disease patients.

    PubMed

    Krishnamoorthy, Soumya; Rajan, Roopa; Banerjee, Moinak; Kumar, Hardeep; Sarma, Gangadhara; Krishnan, Syam; Sarma, Sankara; Kishore, Asha

    2016-09-01

    Impulse control disorders (ICD) are reported to occur at variable frequencies in different ethnic groups. Genetic vulnerability is suspected to underlie the individual risk for ICD. We investigated whether the allelic variants of dopamine (DRD3), glutamate (GRIN2B) and serotonin (HTR2A) receptors are linked to ICD in Indian Parkinson's disease (PD) patients. We conducted a prospective, case-control study which included PD patients (70 with ICD, 100 without ICD categorized after direct psychiatric interview of patient and caregiver) and 285 healthy controls. Single nucleotide polymorphism (SNP) variants of DRD3 p.S9G (rs6280), GRIN2B c.2664C>T (rs1806201) and HTR2A c.102T>C (rs6313) were genotyped. Multivariate regression analysis revealed that DRD3 p.Ser9Gly (rs6280) heterozygous variant CT (OR = 2.22, 95% CI: 1.03-4.86, p = 0.041), higher daily Levodopa equivalent doses (LED) of drugs (for 100 mg LED, OR = 1.14, 95% CI: 1.01-1.29, p = 0.041), current dopamine agonist but not Levodopa use (OR = 2.16, 95% CI: 1.03-4.55, p = 0.042) and age of onset of motor symptoms under 50 years (OR 2.09, 95% CI: 1.05-4.18, p = 0.035) were independently associated with ICD. DRD3 p.Ser9Gly (rs6280) CT genotype is associated with ICD in Indian PD patients and this association is novel. Enhanced D3 receptor affinity due to gain-of-function conferred by the glycine residues could impair reward-risk assessment in the mesolimbic system and contribute to development of impulsive behaviour, in carriers of this genotype. Copyright © 2016. Published by Elsevier Ltd.

  2. [New Retrieval Algorithms for Geophysical Products from GLI and MODIS Data

    NASA Technical Reports Server (NTRS)

    Dodge, James C.; Simpson, James J.

    2004-01-01

    Below is the 1st year progress report for NAG5-13435 "New Retrieval Algorithms for Geophysical Products from GLI and MODIS Data". Activity on this project has been coordinated with our NASA DB project NAG5-9604. For your convenience, this report has six sections and an Appendix. Sections I - III discuss specific activities undertaken during the past year to analyze/use MODIS data. Section IV formally states our intention to no longer pursue any research using JAXA's (formerly NASDA's) GLI instrument which catastrophically failed very early after launch (also see the Appendix). Section V provides some indications of directions for second year activities based on our January 2004 telephone discussions and email exchanges. A brief summary is given in Section VI.

  3. Correlation of Specific Mutations in Line Probe Assay (LPA) and Drug Susceptibility Test (DST) with respect to Fluoroquinolone Resistance in Drug Resistant Mycobacterium tuberculosis

    PubMed Central

    Agrawal, Umang; Savaj, Pratik; Davda, Kanishka; Rodrigues, Camilla; Soman, Rajeev

    2017-01-01

    Abstract Background This study was done to investigate the utility of specific fluoroquinolone mutations in LPA in predicting the susceptibility in DST at WHO recommended Critical Concentrations of 0.5 and 2 µg/dL of moxifloxacin within a short time frame as provided by LPA. Methods In a retrospective study performed at a tertiary care hospital of Mumbai, India from October 2015 to February 2017, consecutive samples demonstrating fluoroquinolone resistance by LPA were selected. The LPA kit used was Hain Lifescience Genotype MTBDRsl (Version 1). It detects the following mutations in gyrA gene: MUT1: Ala90Val, MUT2: Ser91Pro, MUT3A: Asp94Ala, MUT3B: Asp94Asn/Tyr, MUT3C: Asp94Gly, MUT3D: Asp94His. The causal mutation was noted. For 89 of these samples, DST had been requested and results with Critical Concentration of 0.5µg/dL and 2µg/dL for moxifloxacin were available Results The 89 samples studied were as follows: Sputum (n = 60), paravertebral soft tissue (n = 2), bronchoalveolar fluid (n = 2), cerebrospinal fluid (n = 1), endotracheal tube secretion (n = 1), pleural fluid (n = 1) and site not recorded (22). 3 of these samples had double mutations. Results are as follows. Mutation in gyrA gene Number of samples (n) Susceptible at 0.5 µg/dL [n (%)] Susceptible at 2 µg/dL [n (%)] MUT1 (Ala90Val) 18 6(33.33) 16(88.89) MUT2 (Ser91Pro) 2 0(0) 1(50) MUT3A (Asp94Asn) 13 3(23.07) 11(84.61) MUT3B (Asp94Asn/Tyr) 6 1(16.67) 4(66.67) MUT3C (Asp94Gly) 51 4(8) 43(84.31) MUT3D (Asp94His) 2 0(0) 2(100) Conclusion This study showed a higher proportion of M. tuberculosis susceptibility at 2 µg/dL rather than at 0.5 µg/dL, to moxifloxacin for gyrA mutations Ala90Val (MUT1), Asp94Ala (MUT3A), Asp94Gly (MUT3C), Asp94His (MUT3D) but not for Ser91Pro (MUT2) and Asp94Asn/Tyr (MUT3B). However, the number of samples with Ser91Pro (MUT2) and Asp94Asn/Tyr (MUT3B) mutations was too small for meaningful conclusion. This susceptibility at a higher critical concentration of

  4. The α Subunit of the G Protein G13 Regulates Activity of One or More Gli Transcription Factors Independently of Smoothened*

    PubMed Central

    Douglas, Andrew E.; Heim, Jennifer A.; Shen, Feng; Almada, Luciana L.; Riobo, Natalia A.; Fernández-Zapico, Martin E.; Manning, David R.

    2011-01-01

    Smoothened (Smo) is a seven-transmembrane (7-TM) receptor that is essential to most actions of the Hedgehog family of morphogens. We found previously that Smo couples to members of the Gi family of heterotrimeric G proteins, which in some cases are integral although alone insufficient in the activation of Gli transcription factors through Hedgehog signaling. In response to a report that the G12/13 family is relevant to Hedgehog signaling as well, we re-evaluated the coupling of Smo to one member of this family, G13, and investigated the capacity of this and other G proteins to activate one or more of forms of Gli. We found no evidence that Smo couples directly to G13. We found nonetheless that Gα13 and to some extent Gαq and Gα12 are able to effect activation of Gli(s). This capacity is realized in some cells, e.g. C3H10T1/2, MC3T3, and pancreatic cancer cells, but not all cells. The mechanism employed is distinct from that achieved through canonical Hedgehog signaling, as the activation does not involve autocrine signaling or in any other way require active Smo and does not necessarily involve enhanced transcription of Gli1. The activation by Gα13 can be replicated through a Gq/G12/13-coupled receptor, CCKA, and is attenuated by inhibitors of p38 mitogen-activated protein kinase and Tec tyrosine kinases. We posit that G proteins, and perhaps G13 in particular, provide access to Gli that is independent of Smo and that they thus establish a basis for control of at least some forms of Gli-mediated transcription apart from Hedgehogs. PMID:21757753

  5. Purification and structural characterization of insulin and glucagon from the bichir Polypterus senegalis (Actinopterygii: Polypteriformes).

    PubMed

    Conlon, J M; Fan, H; Fritzsch, B

    1998-01-01

    The Polypteriformes (bichirs and reedfish) are a family of ray-finned fishes of ancient lineage. Insulin has been isolated from an extract of the pancreas and upper gastrointestinal tract of the bichir Polypterus senegalis and its primary structure established as A-chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Asp-Thr-Pro10-Cys-Ser- Leu-Tyr-Asp-Leu-Glu-Asn-Tyr-Cys20-Asn: B-chain: Ala-Ala-Asn-Arg-His-Leu-Cys-Gly-Ser-His10-Leu-Val- Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly20-Asn-Arg-Gly-Phe- Phe-Tyr-Ile-Pro-Ser-Lys30-Met. Despite the fact that Polypterus insulin contains several unusual structural features that are not found in insulins from other jawed fish (Asp at A-8, Thr at A-9, Arg at B-4, Asn at B-21, Ile at B-27, Met at B-31), all the residues in human insulin that are involved in receptor binding, dimerization, and hexamerization have been conserved. A comparison of the structures of insulins from a range of species indicates that Polypterus insulin most closely resembles paddlefish insulin II (seven amino acid substitutions). In contrast, Polypterus glucagon (His-Ser- Gln-Gly-Thr-Phe-Thr-Asn-Asp-Tyr10-Thr-Lys-Tyr- Gln-Asp-Ser-Arg-Arg-Ala-Gln20-Asp-Phe-Val-Gln- Trp-Leu-Met-Ser-Asn) most closely resembles the glucagons from the gar Lepisosteus spatula and the bowfin Amia calva (four amino acid substitutions). The data are consistent with the conclusion based on comparison of morphological characteristics that the Polypterids are the most basal living group of the Actinopterygians with evolutionary connections to both the Acipenserids and the Neopterygians.

  6. Spectroscopic characterization of metal bound phytochelatin analogue (Glu-Cys)4-Gly.

    PubMed

    Cheng, Yongsheng; Yan, Yong-Bin; Liu, Jinyuan

    2005-10-01

    The metal ion binding properties of a phytochelatin (PC) analogue, (Glu-Cys)4-Gly (named as EC4), have been studied by a divalent metal ion binding assay monitored by UV-visible spectroscopy, circular dichroism and NMR spectroscopy. Spectro- photometric titration with different divalent metal ions have revealed that the stiochoimetry of metal-bound EC4 was 1:1, and its metal binding affinities with different divalent metal ions in the order of Cd(II)>Cu(II)>Zn(II)>Pb(II)>Ni(II)>Co(II). UV-visible spectroscopic analysis of metal complexes indicated that four sulfur atoms in cysteine residues are attributable to ligand-to-metal charge transfer (LMCT) between divalent metal ions and EC4, and further confirmed by 1D H1 NMR study and Circular Dichroism. In addition, Circular Dichroism spectra of both free and metal-bound forms of EC4 revealed that metal coordination drives the nonapeptide chain to fold into a turned conformation. The comprehensive analysis of spectroscopic properties of the nonapeptide complexed with metal ions not only provides a fundamental description of the metal ion binding properties of PC analogue, but also shows a correlation between metal binding affinity of PC analogue and the induction activity of metal ions.

  7. A Novel Dominant Hyperekplexia Mutation Y705C Alters Trafficking and Biochemical Properties of the Presynaptic Glycine Transporter GlyT2*

    PubMed Central

    Giménez, Cecilio; Pérez-Siles, Gonzalo; Martínez-Villarreal, Jaime; Arribas-González, Esther; Jiménez, Esperanza; Núñez, Enrique; de Juan-Sanz, Jaime; Fernández-Sánchez, Enrique; García-Tardón, Noemí; Ibáñez, Ignacio; Romanelli, Valeria; Nevado, Julián; James, Victoria M.; Topf, Maya; Chung, Seo-Kyung; Thomas, Rhys H.; Desviat, Lourdes R.; Aragón, Carmen; Zafra, Francisco; Rees, Mark I.; Lapunzina, Pablo; Harvey, Robert J.; López-Corcuera, Beatriz

    2012-01-01

    Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na+/Cl−-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [3H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311–Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H+ and Zn2+ dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo. PMID:22753417

  8. The Arg389Gly beta1-adrenoceptor polymorphism does not affect cardiac effects of exercise after parasympathetic inhibition by atropine.

    PubMed

    Leineweber, Kirsten; Bruck, Heike; Temme, Thomas; Heusch, Gerd; Philipp, Thomas; Brodde, Otto-Erich

    2006-01-01

    In vitro, Arg389Gly beta1-adrenoceptor (AR) polymorphism exhibits decreased beta-AR signalling. In vivo, beta1-AR-mediated cardiac effects of exercise showed no genotype-dependent differences in Arg389 vs. Gly389 beta1-AR subjects. We studied in 16 male subjects homozygous Arg389 or Gly389 beta1-AR, whether blockade of parasympathetic activity might unmask genotype-dependence of exercise effects. Subjects were infused with atropine (10 microg/kg i.v. loading dose followed by continuous i.v. infusion of 0.15 microg/kg/min throughout exercise-time); 20 min after start of atropine bicycle-exercise in supine position (25, 50, 75 and 100 W for 5 min each) was performed and heart rate, contractility, blood pressure, plasma noradrenaline and plasma-renin activity were assessed. Exercise-evoked increases in all but one parameters were not different between Arg389 and Gly389 beta1-AR subjects; only plasma noradrenaline increased slightly more in Gly389 vs. Arg389 beta1-AR subjects. It appears to be unlikely that lack of Arg389Gly beta1-AR genotype-dependence of exercise-effects can be explained by influences of parasympathetic activity.

  9. Toxicity of leucine-containing peptides in Escherichia coli caused by circumvention of leucine transport regulation.

    PubMed Central

    Tavori, H; Kimmel, Y; Barak, Z

    1981-01-01

    A variety of leucine-containing peptides (LCP), Phe-Leu, Gly-Leu, Pro-Leu, Ala-Leu, Ala-Leu-Lys, Leu-Phe-Ala, Leu-Leu-Leu, and Leu-Gly-Gly, inhibited the growth of a prototrophic strain of Escherichia coli K-12 at concentrations between 0.05 and 0.28 mM. Toxicity requires normal uptake of peptides. When peptide transport was impaired by mutations, strains became resistant to the respective LCP. Inhibition of growth occurred immediately after the addition of LCP, and was relieved when 0.4 mM isoleucine was added. The presence of Gly-Leu in the medium correlated with the inhibition of growth, and the bacteria began to grow at the normal rate 70 min after Gly-Leu became undetectable. Disappearance of the peptide corresponded with the appearance of free leucine and glycine in the medium. The concentration of leucine inside the LCP-treated bacteria was higher than that in the leucine-treated and the control cultures. We suggest that entry of LCP into the cells via peptide transport systems circumvents the regulation of leucine transport, thereby causing abnormality high concentrations of leucine inside the cells. This accumulation of leucine interferes with the biosynthesis of isoleucine and inhibits the growth of the bacteria. Images PMID:7012134

  10. Characterizing the Secondary Protein Structure of Black Widow Dragline Silk Using Solid-State NMR & X-ray Diffraction

    PubMed Central

    Jenkins, Janelle E.; Sampath, Sujatha; Butler, Emily; Kim, Jihyun; Henning, Robert W.; Holland, Gregory P.; Yarger, Jeffery L.

    2013-01-01

    This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked β-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) 13C-13C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and hence to the amino acids that make up the motifs. Specifically, alanine is incorporated in β-sheet (poly(Alan) and poly(Gly-Ala)), 31-helix (poly(Gly-Gly-Xaa), and α-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in β-sheet (poly(Gly-Ala)) and 31-helical (poly(Gly-Gly-Xaa)) regions, while serine is present in β-sheet (poly(Gly-Ala-Ser)), 31-helix (poly(Gly-Gly-Ser)), and β-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk. PMID:24024617

  11. Diarylheptanoids suppress proliferation of pancreatic cancer PANC-1 cells through modulating shh-Gli-FoxM1 pathway.

    PubMed

    Dong, Guang-Zhi; Jeong, Ji Hye; Lee, Yu-Ih; Lee, So Yoon; Zhao, Hui-Yuan; Jeon, Raok; Lee, Hwa Jin; Ryu, Jae-Ha

    2017-04-01

    Pancreatic cancer is one of the leading causes of cancer, and it has the lowest 5-year survival rates. It is necessary to develop more potent anti-pancreatic cancer drugs to overcome the fast metastasis and resistance to surgery, radiotherapy, chemotherapy, and combinations of these. We have identified several diarylheptanoids as anti-pancreatic cancer agents from Alpinia officinarum (lesser galangal) and Alnus japonica. These diarylheptanoids suppressed cell proliferation and induced the cell cycle arrest of pancreatic cancer cells (PANC-1). Among them, the most potent compounds 1 and 7 inhibited the shh-Gli-FoxM1 pathway and their target gene expression in PANC-1 cells. Furthermore, they suppressed the expression of the cell cycle associated genes that were rescued by the overexpression of exogenous FoxM1. Taken together, (E)-7-(4-hydroxy-3-methoxyphenyl)-1-phenylhept-4-en-3-one (1) from Alpinia officinarum (lesser galangal) and platyphyllenone (7) from Alnus japonica inhibit PANC-1 cell proliferation by suppressing the shh-Gli-FoxM1 pathway, and they can be potential candidates for anti-pancreatic cancer drug development.

  12. Binding Energies of the Proton-Bound Amino Acid Dimers Gly·Gly, Ala·Ala, Gly·Ala, and Lys·Lys Measured by Blackbody Infrared Radiative Dissociation

    PubMed Central

    Price, William D.; Schnier, Paul D.

    2005-01-01

    Arrhenius activation energies in the zero-pressure limit for dissociation of gas-phase proton-bound homodimers of N,N-dimethylacetamide (N,N-DMA), glycine, alanine, and lysine and the heterodimer alanine·glycine were measured using blackbody infrared radiative dissociation (BIRD). In combination with master equation modeling of the kinetic data, binding energies of these dimers were determined. A value of 1.25 ± 0.05 eV is obtained for N,N-DMA and is in excellent agreement with that reported in the literature. The value obtained from the truncated Boltzmann model is significantly higher, indicating that the assumptions of this model do not apply to these ions. This is due to the competitive rates of photon emission and dissociation for these relatively large ions. The binding energies of the amino acid dimers are ~1.15 ± 0.05 eV and are indistinguishable despite the difference in their gas-phase basicity and structure. The threshold dissociation energies can be accurately modeled using a range of dissociation parameters and absorption/emission rates. However, the absolute values of the dissociation rates depend more strongly on the absorption/emission rates. For N,N-DMA and glycine, an accurate fit was obtained using frequencies and transition dipole moments calculated at the ab initio RHF/2-31G* and MP2/2-31G* level, respectively. In order to obtain a similar accuracy using values obtained from AM1 semiempirical calculations, it was necessary to multiply the transition dipole moments by a factor of 3. These results demonstrate that in combination with master equation modeling, BIRD can be used to obtain accurate threshold dissociation energies of relatively small ions of biological interest. PMID:17235378

  13. N-Oleoyl-glycine reduces nicotine reward and withdrawal in mice.

    PubMed

    Donvito, Giulia; Piscitelli, Fabiana; Muldoon, Pretal; Jackson, Asti; Vitale, Rosa Maria; D'Aniello, Enrico; Giordano, Catia; Ignatowska-Jankowska, Bogna M; Mustafa, Mohammed A; Guida, Francesca; Petrie, Gavin N; Parker, Linda; Smoum, Reem; Sim-Selley, Laura; Maione, Sabatino; Lichtman, Aron H; Damaj, M Imad; Di Marzo, Vincenzo; Mechoulam, Raphael

    2018-03-19

    Cigarette smokers with brain damage involving the insular cortex display cessation of tobacco smoking, suggesting that this region may contribute to nicotine addiction. In the present study, we speculated that molecules in the insular cortex that are sensitive to experimental traumatic brain injury (TBI) in mice might provide leads to ameliorate nicotine addiction. Using targeted lipidomics, we found that TBI elicited substantial increases of a largely uncharacterized lipid, N-acyl-glycine, N-oleoyl-glycine (OlGly), in the insular cortex of mice. We then evaluated whether intraperitoneal administration of OlGly would alter withdrawal responses in nicotine-dependent mice as well as the rewarding effects of nicotine, as assessed in the conditioned place preference paradigm (CPP). Systemic administration of OlGly reduced mecamylamine-precipitated withdrawal responses in nicotine-dependent mice and prevented nicotine CPP. However, OlGly did not affect morphine CPP, demonstrating a degree of selectivity. Our respective in vitro and in vivo observations that OlGly activated peroxisome proliferator-activated receptor alpha (PPAR-α) and the PPAR-α antagonist GW6471 prevented the OlGly-induced reduction of nicotine CPP in mice suggests that this lipid acts as a functional PPAR-α agonist to attenuate nicotine reward. These findings raise the possibility that the long chain fatty acid amide OlGly may possess efficacy in treating nicotine addiction. Copyright © 2018. Published by Elsevier Ltd.

  14. Inhibition of angiogenesis in vitro by Arg-Gly-Asp-containing synthetic peptide.

    PubMed Central

    Nicosia, R. F.; Bonanno, E.

    1991-01-01

    This study was designed to evaluate the effect of the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) on angiogenesis in serum-free collagen gel culture of rat aorta. The GRGDS peptide contains the amino acid sequence Arg-Gly-Asp (RGD), which has been implicated as a recognition site in interactions between extracellular matrix (ECM) molecules and cell membrane receptors. RGD-containing synthetic peptides are known to inhibit attachment of endothelial cells to substrates, but their effect on angiogenesis has not been fully characterized. Aortic explants embedded in collagen gel in the absence of GRGDS generated branching microvessels through a process of endothelial migration and proliferation. Addition of GRGDS to the culture medium caused a marked inhibition of angiogenesis. In contrast, GRGES, a control peptide lacking the RGD sequence, failed to inhibit angiogenesis. The inhibitory effect of GRGDS was nontoxic and reversible. The angiogenic activity of aortic explants previously inhibited with GRGDS could be restored by incubating the cultures in GRGDS-free medium. These findings suggest that angiogenesis is an anchorage-dependent process that can be inhibited by interfering with the attachment of endothelial cells to the ECM. It also indicates that synthetic peptides can be used as probes to study the mechanisms by which the ECM regulates angiogenesis. Images Figure 1 PMID:1707235

  15. Molecular mechanisms for the reaction between (˙)OH radicals and proline: insights on the role as reactive oxygen species scavenger in plant stress.

    PubMed

    Signorelli, Santiago; Coitiño, E Laura; Borsani, Omar; Monza, Jorge

    2014-01-09

    The accumulation of proline (Pro) and overproduction of reactive oxygen species (ROS) by plants exposed to stress is well-documented. In vitro assays show that enzyme inactivation by hydroxyl radicals ((•)OH) can be avoided in the presence of Pro, suggesting this amino acid might act as a (•)OH scavenger. Although production of hydroxyproline (Hyp) has been hypothesized in connection with such antioxidant activity, no evidence on the detailed mechanism of scavenging has been reported. To elucidate whether and how Hyp might be produced, we used density functional theory calculations coupled to a polarizable continuum model to explore 27 reaction channels including H-abstraction by (•)OH and (•)OH/H2O addition. The structure and energetics of stable species and transition states for each reaction channel were characterized at the PCM-(U)M06/6-31G(d,p) level in aqueous solution. Evidence is found for a main pathway in which Pro scavenges (•)OH by successive H-abstractions (ΔG(‡,298) = 4.1 and 7.5 kcal mol(-1)) to yield 3,4-Δ-Pro. A companion pathway with low barriers yielding Δ(1)-pyrroline-5-carboxylate (P5C) is also supported, linking with 5-Hyp through hydration. However, this connection remains unlikely in stressed plants because P5C would be efficiently recycled to Pro (contributing to its accumulation) by P5C reductase, hypothesis coined here as the "Pro-Pro cycle".

  16. Preclinical evaluation of isostructural Tc-99m- and Re-188-folate-Gly-Gly-Cys-Glu for folate receptor-positive tumor targeting.

    PubMed

    Kim, Woo Hyoung; Kim, Chang Guhn; Kim, Myoung Hyoun; Kim, Dae-Weung; Park, Cho Rong; Park, Ji Yong; Lee, Yun-Sang; Youn, Hyewon; Kang, Keon Wook; Jeong, Jae Min; Chung, June-Key

    2016-06-01

    The purpose of the present study was to prepare isostructural Tc-99m- and Re-188-folate-Gly-Gly-Cys-Glu (folate-GGCE), and to evaluate the feasibility of their use for folate receptor (FR)-targeted molecular imaging and as theranostic agents in a mouse tumor model. Folate-GGCE was synthesized using solid-phase peptide synthesis and radiolabeled with Tc-99m or Re-188. Radiochemical characterization was performed by radio-high-performance liquid chromatography. The biodistribution of Tc-99m-folate-GGCE was studied, with or without co-injection of excess free folate, in mice bearing both FR-positive (KB cell) and FR-negative (HT1080 cell) tumors. Biodistribution of Re-188-folate-GGCE was studied in mice bearing KB tumors. Serial planar scintigraphy was performed in the dual tumor mouse model after intravenous injection of Tc-99m-folate-GGCE. Serial micro-single photon emission computed tomography/computed tomography (SPECT/CT) studies were performed, with or without co-injection of excess free folate, in the mouse tumor model after injection of Tc-99m-folate-GGCE or Re-188-folate-GGCE. The radiolabeling efficiency and radiochemical stability of Tc-99m- and Re-188-folate-GGCE were more than 95 % for up to 4 h after radiolabeling. Uptake of Tc-99m-folate-GGCE at 1, 2, and 4 h after injection in KB tumor was 16.4, 23.2, and 17.6 % injected dose per gram (%ID/g), respectively. This uptake was suppressed by 97.4 % when excess free folate was co-administered. Tumor:normal organ ratios at 4 h for blood, liver, lung, muscle, and kidney were 54.3, 25.2, 38.3, 97.8, and 0.3, respectively. Tumor uptake of Re-188-folate-GGCE at 2, 4, 8, and 16 h after injection was 17.4, 21.7, 24.1, and 15.6 %ID/g, respectively. Tumor:normal organ ratios at 8 h for blood, liver, lung, muscle, and kidney were 126.8, 21.9, 54.8, 80.3, and 0.4, respectively. KB tumors were clearly visualized at a high intensity using serial scintigraphy and micro-SPECT/CT in mice injected with Tc-99m- or Re

  17. Immunostimulatory activities of a decapeptide derived from Alcaligenes faecalis FY-3 to crucian carp.

    PubMed

    Wang, G-X; Li, F-Y; Cui, J; Wang, Y; Liu, Y-T; Han, J; Lei, Y

    2011-07-01

    A strain was isolated from a soil sample collected from Weihe river in Shaanxi province (108°03'E 34°14'N), which was identified as Alcaligenes faecalis by 16S rRNA analysis. A compound M showing potent immune activity was isolated from secondary metabolites of the strain through bioassay-guided isolation techniques. The structure of the compound M was elucidated using FT-IR, EI-MS, 1H NMR and 13C NMR spectra and identified as cyclo-(L-Pro-Gly)5 which was first time reported as a natural product. We evaluated the immune effects of the cyclo-(L-Pro-Gly)5 on the basis of serum lysozyme activity, bacterial agglutination titre assay, superoxide anion production and phagocytic activity assay, and they were found to be significantly increased by cyclo-(L-Pro-Gly)5. The effects of cyclo-(L-Pro-Gly)5 on immune-related gene expression were further investigated. The outcomes of real-time quantitative polymerase chain reaction (RQ-PCR) proved that the transcribing level of interleukin 6β (IL-6β) and inducible nitric oxide synthase 1β (iNOS-1β) mRNA in the blood leucocytes have been augmented by cyclo-(L-Pro-Gly)5. The challenge experiment showed that crucian carp injected the cyclo-(L-Pro-Gly)5 had significantly (P < 0.05) lower cumulative mortality (13.0%) compared with the control (45.4%) after infection with live Aeromonas hydrophila. These results suggested that cyclo-(L-Pro-Gly)5 is a possible immunostimulant and may strengthen the immune response and protect the heath status of crucian carp against A. hydrophila. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

  18. Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes.

    PubMed

    Veltmaat, Jacqueline M; Relaix, Frédéric; Le, Lendy T; Kratochwil, Klaus; Sala, Frédéric G; van Veelen, Wendy; Rice, Ritva; Spencer-Dene, Bradley; Mailleux, Arnaud A; Rice, David P; Thiery, Jean Paul; Bellusci, Saverio

    2006-06-01

    Little is known about the regulation of cell fate decisions that lead to the formation of five pairs of mammary placodes in the surface ectoderm of the mouse embryo. We have previously shown that fibroblast growth factor 10 (FGF10) is required for the formation of mammary placodes 1, 2, 3 and 5. Here, we have found that Fgf10 is expressed only in the somites underlying placodes 2 and 3, in gradients across and within these somites. To test whether somitic FGF10 is required for the formation of these two placodes, we analyzed a number of mutants with different perturbations of somitic Fgf10 gradients for the presence of WNT signals and ectodermal multilayering, markers for mammary line and placode formation. The mammary line is displaced dorsally, and formation of placode 3 is impaired in Pax3ILZ/ILZ mutants, which do not form ventral somitic buds. Mammary line formation is impaired and placode 3 is absent in Gli3Xt-J/Xt-J and hypomorphic Fgf10 mutants, in which the somitic Fgf10 gradient is shortened dorsally and less overall Fgf10 is expressed, respectively. Recombinant FGF10 rescued mammogenesis in Fgf10(-/-) and Gli3Xt-J/Xt-J flanks. We correlate increasing levels of somitic FGF10 with progressive maturation of the surface ectoderm, and show that full expression of somitic Fgf10, co-regulated by GLI3, is required for the anteroposterior pattern in which the flank ectoderm acquires a mammary epithelial identity. We propose that the intra-somitic Fgf10 gradient, together with ventral elongation of the somites, determines the correct dorsoventral position of mammary epithelium along the flank.

  19. From topical antidote against skin irritants to a novel counter-irritating and anti-inflammatory peptide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Brodsky, Berta; Erlanger-Rosengarten, Avigail; Proscura, Elena

    2008-06-15

    The primary purpose of the present study was to investigate the mechanism of the counter-irritating activity of topical iodine against skin lesions induced by chemical and thermal stimuli. The hypothesis that iodine exerts its activity by inducing an endogenous anti-inflammatory factor was confirmed by exposing guinea pig skin to heat stimulus followed by topical iodine treatment and skin extraction. Injection of the extract into naive guinea pigs reduced heat-induced irritation by 69%. The protective factor, identified as a new nonapeptide (histone H2A 36-44, H-Lys-Gly-Asn-Tyr-Ala-Glu-Arg-Ileu-Ala-OH), caused reduction of 40% in irritation score in heat-exposed guinea pigs. The murine analog (H-Lys-Gly-His-Tyr-Ala-Glu-Arg-Val-Gly-OH, termedmore » IIIM1) reduced sulfur mustard (SM)-induced ear swelling at a dose-dependent bell-shape manner reaching peak activity of 1 mg/kg. Cultured keratinocytes transfected with the peptide were more resistant towards SM than the control cells. The peptide suppressed oxidative burst in activated neutrophils in a concentration-dependent manner. In addition, the peptide reduced glucose oxidase-induced skin edema in mice at a dose-dependent bell-shape manner. Apart from thermal and chemical-induced skin irritation this novel peptide might be of potential use in chronic dermal disorders such as psoriasis and pemphigus as well as non-dermal inflammatory diseases like multiple sclerosis, arthritis and colitis.« less

  20. Physicochemical Properties of Glycine-Based Ionic Liquid [QuatGly-OEt][EtOSO3] (2-Ethoxy-1-ethyl-1,1-dimethyl-2-oxoethanaminium ethyl sulfate) and Its Binary Mixtures with Poly(ethylene glycol) (Mw = 200) at Various Temperatures

    PubMed Central

    Wu, Tzi-Yi; Chen, Bor-Kuan; Hao, Lin; Lin, Yuan-Chung; Wang, H. Paul; Kuo, Chung-Wen; Sun, I-Wen

    2011-01-01

    This work includes specific basic characterization of synthesized glycine-based Ionic Liquid (IL) [QuatGly-OEt][EtOSO3] by NMR, elementary analysis and water content. Thermophysical properties such as density, ρ, viscosity, η, refractive index, n, and conductivity, κ, for the binary mixture of [QuatGly-OEt][EtOSO3] with poly(ethylene glycol) (PEG) [Mw = 200] are measured over the whole composition range. The temperature dependence of density and dynamic viscosity for neat [QuatGly-OEt][EtOSO3] and its binary mixture can be described by an empirical polynomial equation and by the Vogel-Tammann-Fucher (VTF) equation, respectively. The thermal expansion coefficient of the ILs is ascertained using the experimental density results, and the excess volume expansivity is evaluated. The negative values of excess molar volume for the mixture indicate the ion-dipole interactions and packing between IL and PEG oligomer. The results of binary excess property (VmE ) and deviations (Δη, Δxn, ΔΨn, ΔxR, and ΔΨR) are discussed in terms of molecular interactions and molecular structures in the binary mixture. PMID:22272102

  1. Production of the angiotensin I converting enzyme inhibitory peptides and isolation of four novel peptides from jellyfish (Rhopilema esculentum) protein hydrolysate.

    PubMed

    Liu, Xin; Zhang, Miansong; Shi, Yaping; Qiao, Ruojin; Tang, Wei; Sun, Zhenliang

    2016-07-01

    Angiotensin I converting enzyme (ACE) plays an important role in regulating blood pressure in the human body. ACE inhibitory peptides derived from food proteins could exert antihypertensive effects without side effects. Jellyfish (Rhopilema esculentum) is an important fishery resource suitable for production of ACE inhibitory peptides. The objective of this study was to optimize the hydrolysis conditions for production of protein hydrolysate from R. esculentum (RPH) with ACE inhibitory activity, and to isolate and identify the ACE inhibitory peptides from RPH. Rhopilema esculentum protein was hydrolyzed with Compound proteinase AQ to produce protein hydrolysate with ACE inhibitory activity, and the hydrolysis conditions were optimized using response surface methodology. The optimum parameters for producing peptides with the highest ACE inhibitory activity were as follows: hydrolysis time 3.90 h, hydrolysis temperature 58 °C, enzyme:substrate ratio 2.8% and pH 7.60. Under these conditions, the ACE inhibitory rate reached 32.21%. In addition, four novel ACE inhibitory peptides were isolated, and their amino acids sequences were identified as Val-Gly-Pro-Tyr, Phe-Thr-Tyr-Val-Pro-Gly, Phe-Thr-Tyr-Val-Pro-Gly-Ala and Phe-Gln-Ala-Val-Trp-Ala-Gly, respectively. The IC50 value of the purified peptides for ACE inhibitory activity was 8.40, 23.42, 21.15 and 19.11 µmol L(-1) . These results indicate that the protein hydrolysate prepared from R. esculentum might be a commercial competitive source of ACE inhibitory ingredients to be used in functional foods. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  2. A novel c.1037C > G (p.Ala346Gly) mutation in TP63 as cause of the ectrodactyly-ectodermal dysplasia and cleft lip/palate (EEC) syndrome

    PubMed Central

    Alves, Leandro Ucela; Pardono, Eliete; Otto, Paulo A.; Mingroni Netto, Regina Célia

    2015-01-01

    Ectrodactyly – ectodermal dysplasia and cleft lip/palate (EEC) syndrome (OMIM 604292) is a rare disorder determined by mutations in the TP63 gene. Most cases of EEC syndrome are associated to mutations in the DNA binding domain (DBD) region of the p63 protein. Here we report on a three-generation Brazilian family with three individuals (mother, son and grandfather) affected by EEC syndrome, determined by a novel mutation c.1037C > G (p.Ala346Gly). The disorder in this family exhibits a broad spectrum of phenotypes: two individuals were personally examined, one presenting the complete constellation of EEC syndrome manifestations and the other presenting an intermediate phenotype; the third affected, a deceased individual not examined personally and referred to by his daughter, exhibited only the split-hand/foot malformation (SHFM). Our findings contribute to elucidate the complex phenotype-genotype correlations in EEC syndrome and other related TP63-mutation syndromes. The possibility of the mutation c.1037C > G being related both to acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome and SHFM is also raised by the findings here reported. PMID:25983622

  3. Circular RNA GLI2 promotes osteosarcoma cell proliferation, migration, and invasion by targeting miR-125b-5p.

    PubMed

    Li, Ji-Feng; Song, Yu-Ze

    2017-07-01

    Circular RNAs are novel identified type of endogenous non-coding RNAs, which exert vital functions in human and animals. However, the in-depth role of circular RNAs in the progression of tumorigenesis, especially osteosarcoma, is still undefined. Our preliminary study had found that cir-GLI2 was significantly upregulated in osteosarcoma tissues compared to adjacent non-tumor tissue. Moreover, cir-GLI2 silencing could effectively suppress the proliferation, migration, and invasion capacity of osteosarcoma cells, indicating the tumor-promoting role. Besides, bioinformatics analysis and luciferase reporter assay predicted the direct binding to miR-125b-5p, which has been reported to function as a tumor suppressor in osteosarcoma. Furthermore, functional experiments validated that cir-GLI2 exerted the tumor-promoting effects on osteosarcoma cells via negatively targeting miR-125b-5p. In conclusion, our study demonstrated that cir-GLI2 acts as an oncogenic circular RNA in osteosarcoma genesis, providing a novel diagnostic and therapeutic target for osteosarcoma.

  4. Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

    NASA Technical Reports Server (NTRS)

    Chang, D.; Haynes, J. I. Jr; Brady, J. N.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

  5. Hedgehog signaling pathway is active in GBM with GLI1 mRNA expression showing a single continuous distribution rather than discrete high/low clusters.

    PubMed

    Chandra, Vikas; Das, Tapojyoti; Gulati, Puneet; Biswas, Nidhan K; Rote, Sarang; Chatterjee, Uttara; Ghosh, Samarendra N; Deb, Sumit; Saha, Suniti K; Chowdhury, Anup K; Ghosh, Subhashish; Rudin, Charles M; Mukherjee, Ankur; Basu, Analabha; Dhara, Surajit

    2015-01-01

    Hedgehog (Hh) signaling pathway is a valid therapeutic target in a wide range of malignancies. We focus here on glioblastoma multiforme (GBM), a lethal malignancy of the central nervous system (CNS). By analyzing RNA-sequencing based transcriptomics data on 149 clinical cases of TCGA-GBM database we show here a strong correlation (r = 0.7) between GLI1 and PTCH1 mRNA expression--as a hallmark of the canonical Hh-pathway activity in this malignancy. GLI1 mRNA expression varied in 3 orders of magnitude among the GBM patients of the same cohort showing a single continuous distribution-unlike the discrete high/low-GLI1 mRNA expressing clusters of medulloblastoma (MB). When compared with MB as a reference, the median GLI1 mRNA expression in GBM appeared 14.8 fold lower than that of the "high-Hh" cluster of MB but 5.6 fold higher than that of the "low-Hh" cluster of MB. Next, we demonstrated statistically significant up- and down-regulation of GLI1 mRNA expressions in GBM patient-derived low-passage neurospheres in vitro by sonic hedgehog ligand-enriched conditioned media (shh-CM) and by Hh-inhibitor drug vismodegib respectively. We also showed clinically achievable dose (50 μM) of vismodegib alone to be sufficient to induce apoptosis and cell cycle arrest in these low-passage GBM neurospheres in vitro. Vismodegib showed an effect on the neurospheres, both by down-regulating GLI1 mRNA expression and by inducing apoptosis/cell cycle arrest, irrespective of their relative endogenous levels of GLI1 mRNA expression. We conclude from our study that this single continuous distribution pattern of GLI1 mRNA expression technically puts almost all GBM patients in a single group rather than discrete high- or low-clusters in terms of Hh-pathway activity. That is suggestive of therapies with Hh-pathway inhibitor drugs in this malignancy without a need for further stratification of patients on the basis of relative levels of Hh-pathway activity among them.

  6. [Secondary metabolites from a deep-sea-derived actinomycete Micrococcus sp. R21].

    PubMed

    Peng, Kun; Su, Rui-qiang; Zhang, Gai-yun; Cheng, Xuan-xuan; Yang, Quan; Liu, Yong-hong; Yang, Xian-wen

    2015-06-01

    To investigate cytotoxic secondary metabolites of Micrococcus sp. R21, an actinomycete isolated from a deep-sea sediment (-6 310 m; 142 degrees 19. 9' E, 10 degrees 54. 6' N) of the Western Pacific Ocean, column chromatography was introduced over silica gel, ODS, and Sephadex LH-20. As a result, eight compounds were obtained. By mainly detailed analysis of the NMR data, their structures were elucidated as cyclo(4-hydroxy-L-Pro-L-leu) (1), cyclo(L-Pro-L-Gly) (2), cyclo( L-Pro-L-Ala) (3), cyclo( D-Pro-L-Leu) (4), N-β-acetyltryptamine (5), 2-hydroxybenzoic acid (6), and phenylacetic acid (7). Compound 1 exhibited weak cytotoxic activity against RAW264. 7 cells with IC50 value of 9.1 μmol x L(-1).

  7. Changes of CFTR functional measurements and clinical improvements in cystic fibrosis patients with non p.Gly551Asp gating mutations treated with ivacaftor.

    PubMed

    Mesbahi, Myriam; Shteinberg, Michal; Wilschanski, Michael; Hatton, Aurelie; Nguyen-Khoa, Thao; Friedman, Hannah; Cohen, Michael; Escabasse, Virginie; Le Bourgeois, Muriel; Lucidi, Vicenzina; Sermet-Gaudelus, Isabelle; Bassinet, Laurence; Livnat, Galit

    2017-01-01

    Ivacaftor, a CFTR potentiator, has been found to improve CFTR function and clinical outcomes in patients with cystic fibrosis (CF) gating mutations. We investigated the effects of ivacaftor on CFTR functional measurement in CF patients carrying gating mutations other than p.Gly551Asp. Two siblings aged 13 and 12 carrying the p.Ser549Asn mutation, two sisters (45 and 43years old) compound heterozygotes for p.Asp1152His and p.Gly1244Glu, a 37year old man homozygous for the p.Gly1244Glu mutation, and a 7year old girl with p.Arg352Gln and p.Gly1244Glu mutations commenced treatment with ivacaftor. NPD was performed in all the patients and approached normal for four patients who had also clinical improvement (p.Ser549Asn compound heterozygotes, and p.Asp1152His/p.Gly1244Glu siblings). Beta-adrenergic sweat chloride secretion performed in thep.Asp1152His/p.Gly1244Glu patients improved significantly. The p.Gly1244Glu mutation homozygous patient, who had undergone an ileal resection with ileostomy and enterocutaneous fistula, did not respond clinically to ivacaftor and did not modify his sweat test. These results highlight the importance of different CFTR activity measurements to explore CFTR modulator efficacy. Copyright © 2016. Published by Elsevier B.V.

  8. Propensities of peptides containing the Asn-Gly segment to form β-turn and β-hairpin structures.

    PubMed

    Kang, Young Kee; Yoo, In Kee

    2016-09-01

    The propensities of peptides that contain the Asn-Gly segment to form β-turn and β-hairpin structures were explored using the density functional methods and the implicit solvation model in CH2 Cl2 and water. The populations of preferred β-turn structures varied depending on the sequence and solvent polarity. In solution, β-hairpin structures with βI' turn motifs were most preferred for the heptapeptides containing the Asn-Gly segment regardless of the sequence of the strands. These preferences in solution are consistent with the corresponding X-ray structures. The sequence, H-bond strengths, solvent polarity, and conformational flexibility appeared to interact to determine the preferred β-hairpin structure of each heptapeptide, although the β-turn segments played a role in promoting the formation of β-hairpin structures and the β-hairpin propensity varied. In the heptapeptides containing the Asn-Gly segment, the β-hairpin formation was enthalpically favored and entropically disfavored at 25°C in water. The calculated results for β-turns and β-hairpins containing the Asn-Gly segment imply that these structural preferences may be useful for the design of bioactive macrocyclic peptides containing β-hairpin mimics and the design of binding epitopes for protein-protein and protein-nucleic acid recognitions. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 653-664, 2016. © 2016 Wiley Periodicals, Inc.

  9. MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

    PubMed

    Constantin, Lena; Wainwright, Brandon J

    2015-12-01

    MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

  10. [Effects of epithalon and cortagene on immunity and hemostasis in neonatally hypophysectomized chicken and old birds].

    PubMed

    Kuznik, B I; Pateiuk, A V; Baranchugova, L M; Rusaeva, N S

    2008-01-01

    It has been found that chicken hypophysectomized early in the neonatal period develop anemia, cellular and humoral immune deficiency, hypercoagulation and inhibited fibrinolysis by their 45th postnatal day. An analogous operation performed on old birds produces less significant changes in erythrocytes, immunity and hemostasis. Injections of epithalon tetrapeptide (Ala-Glu-Asp-Gly) administered to either hypophysectomized chicken or old birds during a period of 40 days completely eliminate the shifts registered in erythrocytes, immunity and hemostasis, while injections of cortagene (Ala-Glu-Asp-Pro) which is distinguished from epithalon by a different terminal aminoacid (with Gly being replaced by Pro) do not affect the parameters studied.

  11. Silica, Alumina and Clay Catalyzed Peptide Bond Formation: Enhanced Efficiency of Alumina Catalyst

    NASA Astrophysics Data System (ADS)

    Bujdák, Juraj; Rode, Bernd M.

    1999-10-01

    Catalytic efficiencies of clay (hectorite), silica and alumina were tested in peptide bond formation reactions of glycine (Gly), alanine (Ala), proline (Pro), valine (Val) and leucine (Leu). The reactions were performed as drying/wetting (hectorite) and temperature fluctuation (silica and alumina) experiments at 85 °C. The reactivity of amino acids decreased in order Gly > Ala > Pro ~ Val ~ Leu. The highest catalytic efficiency was observed for alumina, the only catalyst producing oligopeptides in all investigated reaction systems. The peptide bond formation on alumina is probably catalyzed by the same sites and via similar reaction mechanisms as some alumina-catalyzed dehydration reactions used in industrial chemistry.

  12. Synthesis and antibacterial activity of new peptides from Alfalfa RuBisCO protein hydrolysates and mode of action via a membrane damage mechanism against Listeria innocua.

    PubMed

    Kobbi, Sabrine; Nedjar, Naima; Chihib, Nourdine; Balti, Rafik; Chevalier, Mickael; Silvain, Amandine; Chaabouni, Semia; Dhulster, Pascal; Bougatef, Ali

    2018-02-01

    In this work we evaluated the mode of action of six new synthesized peptides (Met-Asp-Asn; Glu-leu-Ala-Ala-Ala-Cys; Leu-Arg-Asp-Asp-Phe; Gly-Asn-Ala-Pro-Gly-Ala-Val-Ala; Ala-Leu-Arg-Met-Ser-Gly and Arg-Asp-Arg-Phe-Leu), previously identified, from the most active peptide fractions of RuBisCO peptic hydrolysate against Listeria innocua via a membrane damage mechanism. Antibacterial effect and the minimum inhibitory concentrations (MIC) of these peptides were evaluated against six strains and their hemolytic activities towards bovine erythrocytes were determined. Prediction of the secondary structure of peptides indicated that these new antibacterial peptides are characterized by a short peptide chains (3-8 amino acid) and a random coli structure. Moreover, it was observed that one key characteristic of antibacterial peptides is the presence of specific amino acids such as cysteine, glycine, arginine and aspartic acid. In addition the determination of the extracellular potassium concentration revealed that treatment with pure RuBisCO peptides could cause morphological changes of L. innocua and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from RuBisCO protein hydrolysates. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. SPR4-peptide Alters Bone Metabolism of Normal and HYP Mice

    PubMed Central

    Zelenchuk, Lesya V; Hedge, Anne-Marie; Rowe, Peter S N

    2015-01-01

    Context ASARM-peptides are substrates and ligands for PHEX, the gene responsible for X-linked hypophosphatemic rickets (HYP). PHEX binds to the DMP1-ASARM-motif to form a trimeric-complex with α5β3-integrin on the osteocyte surface and this suppresses FGF23 expression. ASARM-peptide disruption of this complex increases FGF23 expression. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide and ASARM-motif to study DMP1-PHEX interactions and to assess SPR4 for treating inherited hypophosphatemic rickets. Design Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle into wild-type mice (WT) and HYP-mice for 4 weeks. Results Asymmetrically distributed mineralization defects occurred with WT-SPR4 femurs. Specifically, SPR4 induced negative effects on trabecular bone and increased bone volume and mineralization in cortical-bone. Markedly increased sclerostin and reduced active β-catenin occurred with HYP mice. SPR4-infusion suppressed sclerostin and increased active β-catenin in WT and HYP mice and improved HYP-mice trabecular mineralization defects but not cortical mineralization defects. Conclusions SPR4-peptide has bimodal activity and acts by: (1) preventing DMP1 binding to PHEX and (2) sequestering an inhibitor of DMP1-PHEX binding, ASARM-peptide. In PHEX defective HYP-mice the second pathway predominates. Although SPR4-peptide improved trabecular calcification defects, decreased sclerostin and increased active β-catenin it did not correct HYP-mice cortical mineralization defects on a normal phosphate diet. Thus, for inherited hypophosphatemic rickets patients on a normal phosphate diet, SPR4-peptide is not a useful therapeutic. PMID:25460577

  14. Role of ASCA and the NOD2/CARD15 mutation Gly908Arg in predicting increased surgical costs in Crohn's disease patients: a project of the European Collaborative Study Group on Inflammatory Bowel Disease.

    PubMed

    Odes, Shmuel; Friger, Michael; Vardi, Hillel; Claessens, Greet; Bossuyt, Xavier; Riis, Lene; Munkholm, Pia; Wolters, Frank; Yona, Hagit; Hoie, Ole; Beltrami, Marina; Tsianos, Epameinondas; Katsanos, Kostas; Mouzas, Ioannis; Clofent, Juan; Monteiro, Estela; Messori, Andrea; Politi, Patrizia; O'Morain, Colm; Limonard, Charles; Russel, Maurice; Vatn, Morten; Moum, Bjorn; Stockbrugger, Reinhold; Vermeire, Severine

    2007-07-01

    NOD2/CARD15, the first identified susceptibility gene in Crohn's disease (CD), is associated with ileal stenosis and increased frequency of surgery. Anti-Saccharomyces cerevisiae antibody (ASCA), a serological marker for CD, is associated with ileal location and a high likelihood for surgery. We hypothesized that the presence of ASCA and NOD2/CARD15 mutations could predict increased health care cost in CD. CD patients in a prospectively designed community-based multinational European and Israeli cohort (n = 228) followed for mean 8.3 (SD 2.6) years had blood drawn for measurement of ASCA (IgG, IgA), Arg702Trp, Gly908Arg, and Leu1007fsinsC. Days spent in the hospital and the costs of medical and surgical hospitalizations and medications were calculated. The median duration of surgical hospitalizations was longer in Gly908Arg-positive than -negative patients, 3.5 and 1.5 days/patient-year (P < 0.01), and in ASCA-positive than -negative patients, 1.1 and 0 days/patient-year (P < 0.001). Median surgical hospitalization cost was 1,580 euro/patient-year in Gly908Arg-positive versus 0 euro/patient-year in -negative patients (P < 0.01), and 663 euro/patient-year in ASCA-positive versus 0 euro/patient-year in -negative patients (P < 0.001). Differences in cost of medications between groups were not significant. The effect of Gly908Arg was expressed in countries with higher Gly908Arg carriage rates. ASCA raised surgical costs independently of the age at diagnosis of disease. Arg702Trp and Leu1007fsinsC did not affect the cost of health care. Since CD patients positive for Gly908Arg and ASCA demonstrated higher health care costs, it is possible that measurement of Gly908Arg and ASCA at disease diagnosis can forecast the expensive CD patients.

  15. The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

    PubMed

    Rohrbeck, Astrid; Höltje, Markus; Adolf, Andrej; Oms, Elisabeth; Hagemann, Sandra; Ahnert-Hilger, Gudrun; Just, Ingo

    2017-10-27

    The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to β1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Active diuretic peptidomimetic insect kinin analogs that contain Beta-turn mimetic motif 4-aminopyroglutamate and lack native peptide bonds

    USDA-ARS?s Scientific Manuscript database

    The multifunctional arthropod 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide core sequence Phe-X1-X2-Trp-Gly-NH2, where X1 = His, Asn, Ser, or Tyr and X2 = Ser, Pro, or Ala. Insect kinins regulate diuresis in many species of insects, including the cricket. Insect kinins...

  17. Cloning of Novel Isoforms of the Human Gli2 Oncogene and Their Activities To Enhance Tax-Dependent Transcription of the Human T-Cell Leukemia Virus Type 1 Genome

    PubMed Central

    Tanimura, Akira; Dan, Shingo; Yoshida, Mitsuaki

    1998-01-01

    The expression of human T-cell leukemia virus type 1 (HTLV-1) is activated by interaction of a viral transactivator protein, Tax, and cellular transcription factor, CREB (cyclic AMP response element binding protein), which bind to a 21-bp enhancer in the long terminal repeats (LTR). THP (Tax-helping protein) was previously determined to enhance the transactivation by Tax protein. Here we report novel forms of the human homolog of a member of the Gli oncogene family, Gli2 (also termed Gli2/THP), an extended form of a zinc finger protein, THP, which was described previously. Four possible isoforms (hGli2 α, β, γ, and δ) are formed by combinations of two independent alternative splicings, and all the isoforms could bind to a DNA motif, TRE2S, in the LTR. The longer isoforms, α and β, were abundantly expressed in various cell lines including HTLV-1-infected T-cell lines. Fusion proteins of the hGli2 isoforms with the DNA-binding domain of Gal4 activated transcription when the reporter contained a Gal4-binding site and one copy of the 21-bp sequence, to which CREB binds. This activation was observed only in the presence of Tax. The 21-bp sequence in the reporter was also essential for the activation. These results suggest that simultaneous binding of hGli2 and CREB to the respective sites in the reporter seems to be critical for Tax protein to activate transcription. Consequently, it is probable that the LTR can be regulated by two independent signals through hGli2 and CREB, since the LTR contains the 21-bp and TRE2S sequences in the vicinity. PMID:9557682

  18. Analysis of the Gli-D2 locus identifies a genetic target for simultaneously improving the breadmaking and health-related traits of common wheat.

    PubMed

    Li, Da; Jin, Huaibing; Zhang, Kunpu; Wang, Zhaojun; Wang, Faming; Zhao, Yue; Huo, Naxin; Liu, Xin; Gu, Yong Q; Wang, Daowen; Dong, Lingli

    2018-05-11

    Gliadins are a major component of wheat seed proteins. However, the complex homoeologous Gli-2 loci (Gli-A2, -B2 and -D2) that encode the α-gliadins in commercial wheat are still poorly understood. Here we analyzed the Gli-D2 locus of Xiaoyan 81 (Xy81), a winter wheat cultivar. A total of 421.091 kb of the Gli-D2 sequence was assembled from sequencing multiple bacterial artificial clones, and 10 α-gliadin genes were annotated. Comparative genomic analysis showed that Xy81 carried only eight of the α-gliadin genes of the D genome donor Aegilops tauschii, with two of them each experiencing a tandem duplication. A mutant line lacking Gli-D2 (DLGliD2) consistently exhibited better breadmaking quality and dough functionalities than its progenitor Xy81, but without penalties in other agronomic traits. It also had an elevated lysine content in the grains. Transcriptome analysis verified the lack of Gli-D2 α-gliadin gene expression in DLGliD2. Furthermore, the transcript and protein levels of protein disulfide isomerase were both upregulated in DLGliD2 grains. Consistent with this finding, DLGliD2 had increased disulfide content in the flour. Our work sheds light on the structure and function of Gli-D2 in commercial wheat, and suggests that the removal of Gli-D2 and the gliadins specified by it is likely to be useful for simultaneously enhancing the end-use and health-related traits of common wheat. Because gliadins and homologous proteins are widely present in grass species, the strategy and information reported here may be broadly useful for improving the quality traits of diverse cereal crops. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  19. GLI activation by atypical protein kinase C ι/λ regulates the growth of basal cell carcinomas.

    PubMed

    Atwood, Scott X; Li, Mischa; Lee, Alex; Tang, Jean Y; Oro, Anthony E

    2013-02-28

    Growth of basal cell carcinomas (BCCs) requires high levels of hedgehog (HH) signalling through the transcription factor GLI. Although inhibitors of membrane protein smoothened (SMO) effectively suppress HH signalling, early tumour resistance illustrates the need for additional downstream targets for therapy. Here we identify atypical protein kinase C ι/λ (aPKC-ι/λ) as a novel GLI regulator in mammals. aPKC-ι/λ and its polarity signalling partners co-localize at the centrosome and form a complex with missing-in-metastasis (MIM), a scaffolding protein that potentiates HH signalling. Genetic or pharmacological loss of aPKC-ι/λ function blocks HH signalling and proliferation of BCC cells. Prkci is a HH target gene that forms a positive feedback loop with GLI and exists at increased levels in BCCs. Genome-wide transcriptional profiling shows that aPKC-ι/λ and SMO control the expression of similar genes in tumour cells. aPKC-ι/λ functions downstream of SMO to phosphorylate and activate GLI1, resulting in maximal DNA binding and transcriptional activation. Activated aPKC-ι/λ is upregulated in SMO-inhibitor-resistant tumours and targeting aPKC-ι/λ suppresses signalling and growth of resistant BCC cell lines. These results demonstrate that aPKC-ι/λ is critical for HH-dependent processes and implicates aPKC-ι/λ as a new, tumour-selective therapeutic target for the treatment of SMO-inhibitor-resistant cancers.

  20. [Study on the secondary metabolites from the marine sponge Phakellia fusca fungi PF18].

    PubMed

    Tang, Li-Dan; Liang, Yuan-Wei; Liao, Xiao-Jian; Liang, Qiu; Xu, Shi-Hai; Li, Zhi-Yong

    2011-12-01

    To study the secondary metabolites from the marine sponge Phakellia fusca epiphytic fungi. The compounds were isolated by column chromatography over silica gel and purified by Sephadex LH-20 column chromatography and preparative TLC. The structures were elucidated by means of physiochemical properties and spectroscopic analyses. Four compounds were separated and identified as: cyclo-(L-Val-L-Pro) (1), cyclo-(L-Phe-L-Pro) (2), cyclo-(L-Tyr-L-Pro) (3), cyclo-(3-hydroxy-4-methyldecanoyl-Gly-L-Val-D-Leu-L-Ala-L-Phe) (4). Compounds 1-4 are obtained from the marine sponge Phakellia fusca epiphytic fungi for the first time.

  1. The C- and N-Terminal Residues of Synthetic Heptapeptide Ion Channels Influence Transport Efficacy Through Phospholipid Bilayers

    PubMed Central

    Djedovič, Natasha; Ferdani, Riccardo; Harder, Egan; Pajewska, Jolanta; Pajewski, Robert; Weber, Michelle E.; Schlesinger, Paul H.; Gokel, George W.

    2008-01-01

    The synthetic peptide, R2N-COCH2OCH2CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR’, was shown to be selective for Cl- over K+ when R is n-octadecyl and R’ is benzyl. Nineteen heptapeptides have now been prepared in which the N-terminal and C-terminal residues have been varied. All of the N-terminal residues are dialkyl but the C-terminal chains are esters, 2° amides, or 3° amides. The compounds having varied N-terminal anchors and C-terminal benzyl groups are as follows: 1, R = n-propyl; 2, R = n-hexyl; 3, R = n-octyl; 4, R = n-decyl; 5, R = n-dodecyl; 6, R = n-tetradecyl; 7, R = n-hexadecyl; 8, R = n-octadecyl. Compounds 9-19 have R = n-octadecyl and C-terminal residues as follows: 9, OR’ = OCH2CH3; 10, OR’ = OCH(CH3)2; 11, OR’ = O(CH2)6CH3; 12, OR’ = OCH2-c-C6H11; 13, OR’ = O(CH2)9CH3; 14, OR’ = O (CH2)17CH3; 15, NR’2 = N[(CH2)6CH3]2; 16, NHR’ = NH(CH2)9CH3; 17, NR’2 = N[(CH2)9CH3]2; 18, NHR’ = NH(CH2)17CH3; 19, NR’2 = N[(CH2)17CH3]2. The highest anion transport activities were observed as follows. For the benzyl esters whose N-terminal residues were varied, i.e. 1-8, compound 3 was most active. For the C18 anchored esters 10-14, n-heptyl ester 11 was most active. For the C18 anchored, C-terminal amides 15-19, di-n-decylamide 17 was most active. It was concluded that both the C- and N-terminal anchors were important for channel function in the bilayer but that activity was lost unless only one of the two anchoring groups was dominant. PMID:19633728

  2. Gli1-Mediated Regulation of Sox2 Facilitates Self-Renewal of Stem-Like Cells and Confers Resistance to EGFR Inhibitors in Non-Small Cell Lung Cancer.

    PubMed

    Bora-Singhal, Namrata; Perumal, Deepak; Nguyen, Jonathan; Chellappan, Srikumar

    2015-07-01

    Non-small cell lung cancer (NSCLC) patients have very low survival rates because the current therapeutic strategies are not fully effective. Although EGFR tyrosine kinase inhibitors are effective for NSCLC patients harboring EGFR mutations, patients invariably develop resistance to these agents. Alterations in multiple signaling cascades have been associated with the development of resistance to EGFR inhibitors. Sonic Hedgehog and associated Gli transcription factors play a major role in embryonic development and have recently been found to be reactivated in NSCLC, and elevated Gli1 levels correlate with poor prognosis. The Hedgehog pathway has been implicated in the functions of cancer stem cells, although the underlying molecular mechanisms are not clear. In this context, we demonstrate that Gli1 is a strong regulator of embryonic stem cell transcription factor Sox2. Depletion of Gli1 or inhibition of the Hedgehog signaling significantly abrogated the self-renewal of stem-like side-population cells from NSCLCs as well as vascular mimicry of such cells. Gli1 was found to transcriptionally regulate Sox2 through its promoter region, and Gli1 could be detected on the Sox2 promoter. Inhibition of Hedgehog signaling appeared to work cooperatively with EGFR inhibitors in markedly reducing the viability of NSCLC cells as well as the self-renewal of stem-like cells. Thus, our study demonstrates a cooperative functioning of the EGFR signaling and Hedgehog pathways in governing the stem-like functions of NSCLC cancer stem cells and presents a novel therapeutic strategy to combat NSCLC harboring EGFR mutations. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

    PubMed

    Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

    2016-06-01

    The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

  4. Gly25-Ser26 amyloid β-protein structural isomorphs produce distinct Aβ42 conformational dynamics and assembly characteristics.

    PubMed

    Roychaudhuri, Robin; Lomakin, Aleksey; Bernstein, Summer; Zheng, Xueyun; Condron, Margaret M; Benedek, George B; Bowers, Michael; Teplow, David B

    2014-06-26

    One of the earliest events in amyloid β-protein (Aβ) self-association is nucleation of Aβ monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aβ42 conformational dynamics and assembly. We used "click peptide" chemistry to quasi-synchronously create Aβ42 from 26-O-acyliso-Aβ42 (iAβ42) through a pH jump from 3 to 7.4. We also synthesized Nα-acetyl-Ser26-iAβ42 (Ac-iAβ42), which cannot undergo O→N acyl chemistry, to study the behavior of this ester form of Aβ42 itself at neutral pH. Data from experiments monitoring increases in β-sheet formation (thioflavin T, CD), hydrodynamic radius (RH), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy-mass spectrometry) were quite consistent. A rank order of Ac-iAβ42>iAβ42>Aβ42 was observed. Photochemically cross-linked iAβ42 displayed an oligomer distribution with a prominent dimer band that was not present with Aβ42. These dimers also were observed selectively in iAβ42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAβ42 and Aβ42 appear to be due to the conversion of iAβ42 into "pure" Aβ42 monomer, a nascent form of Aβ42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aβ42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aβ42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aβ oligomers and higher-order assemblies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness.

    PubMed

    Casas, Bárbara S; Adolphe, Christelle; Lois, Pablo; Navarrete, Nelson; Solís, Natalia; Bustamante, Eva; Gac, Patricio; Cabané, Patricio; Gallegos, Ivan; Wainwright, Brandon J; Palma, Verónica

    2017-10-13

    Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1 , the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1 lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes.

  6. Downregulation of the Sonic Hedgehog/Gli pathway transcriptional target Neogenin-1 is associated with basal cell carcinoma aggressiveness

    PubMed Central

    Casas, Bárbara S.; Adolphe, Christelle; Lois, Pablo; Navarrete, Nelson; Solís, Natalia; Bustamante, Eva; Gac, Patricio; Cabané, Patricio; Gallegos, Ivan; Wainwright, Brandon J.; Palma, Verónica

    2017-01-01

    Basal Cell Carcinoma (BCC) is one of the most diagnosed cancers worldwide. It develops due to an unrestrained Sonic Hedgehog (SHH) signaling activity in basal cells of the skin. Certain subtypes of BCC are more aggressive than others, although the molecular basis of this phenomenon remains unknown. We have previously reported that Neogenin-1 (NEO1) is a downstream target gene of the SHH/GLI pathway in neural tissue. Given that SHH participates in epidermal homeostasis, here we analyzed the epidermal expression of NEO1 in order to identify whether it plays a role in adult epidermis or BCC. We describe the mRNA and protein expression profile of NEO1 and its ligands (Netrin-1 and RGMA) in human and mouse control epidermis and in a broad range of human BCCs. We identify in human BCC a significant positive correlation in the levels of NEO1 receptor, NTN-1 and RGMA ligands with respect to GLI1, the main target gene of the canonical SHH pathway. Moreover, we show via cyclopamine inhibition of the SHH/GLI pathway of ex vivo cultures that NEO1 likely functions as a downstream target of SHH/GLI signaling in the skin. We also show how Neo1 expression decreases throughout BCC progression in the K14-Cre:Ptch1lox/lox mouse model and that aggressive subtypes of human BCC exhibit lower levels of NEO1 than non-aggressive BCC samples. Taken together, these data suggest that NEO1 is a SHH/GLI target in epidermis. We propose that NEO1 may be important in tumor onset and is then down-regulated in advanced BCC or aggressive subtypes. PMID:29137400

  7. Gallium-67-labeled lactam bridge-cyclized alpha-MSH peptides with enhanced melanoma uptake and reduced renal uptake.

    PubMed

    Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

    2012-06-20

    The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of (67)Ga-DOTA-GGNle-CycMSHhex {(67)Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and (67)Ga-NOTA-GGNle-CycMSHhex {(67)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and compare with (67)Ga-DOTA-GlyGlu-CycMSH {(67)Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM) in B16/F1 melanoma cells. Both (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than (67)Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, (67)Ga-NOTA-GGNle-CycMSHhex exhibited more favorable radiolabeling conditions (>85% radiolabeling yields started at 37 °C), as well as higher tumor/kidney uptake ratios than (67)Ga-DOTA-GGNle-CycMSHhex at 0.5, 2, and 24 h postinjection. High melanoma uptake coupled with low renal uptake highlighted the potential of (67)Ga-NOTA-GGNle-CycMSHhex for melanoma imaging and therapy.

  8. Gallium-67-Labeled Lactam Bridge-Cyclized Alpha-MSH Peptides with Enhanced Melanoma Uptake and Reduced Renal Uptake

    PubMed Central

    Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

    2012-01-01

    The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of 67Ga-DOTA-GGNle-CycMSHhex {67Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2} and 67Ga-NOTA-GGNle-CycMSHhex {67Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2} and compare with 67Ga-DOTA-GlyGlu-CycMSH {67Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-dPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of 67Ga-NOTA-GGNle-CycMSHhex and 67Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs. 2.1 nM) in B16/F1 melanoma cells. Both 67Ga-NOTA-GGNle-CycMSHhex and 67Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than 67Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, 67Ga-NOTA-GGNle-CycMSHhexexhibited more favorable radiolabeling conditions (> 85% radiolabeling yields started at 37°C), as well as higher tumor/kidney uptake ratios than 67Ga-DOTA-GGNle-CycMSHhex at 0.5, 2 and 24 h post-injection. High melanoma uptake coupled with low renal uptake highlighted the potential of 67Ga-NOTA-GGNle-CycMSHhexfor melanoma imaging and therapy. PMID:22621181

  9. Role of Thr399Ile and Asp299Gly polymorphisms of toll-like receptor-4 gene in acute dental abscess.

    PubMed

    Miri-Moghaddam, Ebrahim; Farhad-Mollashahi, Narges; Baghaee, Elnaz; Bazi, Ali; Garme, Yasaman

    2017-02-01

    Apical Periodontitis (AP) is an inflammatory disease that affects the tissues surrounding the root end of a tooth. The disease which is caused by endodontic infections presents in different clinical ways including development of an acute abscess. Recent studies have provided information suggesting role of a multitude of factors in pathogenesis of acute apical abscess (AAA). In this case-control study, our goal was to evaluate the frequency and potential role of two common polymorphisms of toll like receptor-4 (TLR-4) gene; Thr399Ile (1196 C>T) and Asp299Gly (+896 A>G), in 50 patients with AAA as cases and 50 patients with asymptomatic apical periodontitis (AAP) as controls. Saliva sample containing mucosal epithelial cells was used for DNA extraction. Polymorphisms were detected by Tetra-ARMS (Amplification Refractory Mutation System) PCR method. Statistical analyses were carried out in SPSS 21 software. Homozygous wild type (CC) and heterozygous (CT) genotypes of Thr399Ile polymorphism were detected in 84% and 16% of AAA patients respectively. In controls, respective ratios were 94% (CC) and 6% (CT). Observed difference was not statistically significant ( P >0.05) for distribution of these genotypes. The mutant homozygous (TT) genotype of this polymorphism was identified in neither of the participants. Overall, T allele frequency was obtained 8% in AAA and 3% in AAP (OR=2.6, 95% CI; 0. 6-10.6, p >0.05). For Asp299Gly polymorphism, no individual was detected with the mutant allele in case or control groups. Our results indicated a possible role for Thr399Ile polymorphism in acute presentations of abscess in AAA. However, the impact of this polymorphism needs to be more assessed in future studies. Key words: Genetic polymorphism, periapical abscess, periapical periodontitis, toll-like receptor 4.

  10. Role of Thr399Ile and Asp299Gly polymorphisms of toll-like receptor-4 gene in acute dental abscess

    PubMed Central

    Miri-Moghaddam, Ebrahim; Baghaee, Elnaz; Bazi, Ali; Garme, Yasaman

    2017-01-01

    Background Apical Periodontitis (AP) is an inflammatory disease that affects the tissues surrounding the root end of a tooth. The disease which is caused by endodontic infections presents in different clinical ways including development of an acute abscess. Recent studies have provided information suggesting role of a multitude of factors in pathogenesis of acute apical abscess (AAA). In this case-control study, our goal was to evaluate the frequency and potential role of two common polymorphisms of toll like receptor-4 (TLR-4) gene; Thr399Ile (1196 C>T) and Asp299Gly (+896 A>G), in 50 patients with AAA as cases and 50 patients with asymptomatic apical periodontitis (AAP) as controls. Material and Methods Saliva sample containing mucosal epithelial cells was used for DNA extraction. Polymorphisms were detected by Tetra-ARMS (Amplification Refractory Mutation System) PCR method. Statistical analyses were carried out in SPSS 21 software. Results Homozygous wild type (CC) and heterozygous (CT) genotypes of Thr399Ile polymorphism were detected in 84% and 16% of AAA patients respectively. In controls, respective ratios were 94% (CC) and 6% (CT). Observed difference was not statistically significant (P>0.05) for distribution of these genotypes. The mutant homozygous (TT) genotype of this polymorphism was identified in neither of the participants. Overall, T allele frequency was obtained 8% in AAA and 3% in AAP (OR=2.6, 95% CI; 0. 6-10.6, p>0.05). For Asp299Gly polymorphism, no individual was detected with the mutant allele in case or control groups. Conclusions Our results indicated a possible role for Thr399Ile polymorphism in acute presentations of abscess in AAA. However, the impact of this polymorphism needs to be more assessed in future studies. Key words:Genetic polymorphism, periapical abscess, periapical periodontitis, toll-like receptor 4. PMID:28210435

  11. Association of a Model Transmembrane Peptide Containing Gly in a Heptad Sequence Motif

    PubMed Central

    Lear, James D.; Stouffer, Amanda L.; Gratkowski, Holly; Nanda, Vikas; DeGrado, William F.

    2004-01-01

    A peptide containing glycine at a and d positions of a heptad motif was synthesized to investigate the possibility that membrane-soluble peptides with a Gly-based, left-handed helical packing motif would associate. Based on analytical ultracentrifugation in C14-betaine detergent micelles, the peptide did associate in a monomer-dimer equilibrium, although the association constant was significantly less than that reported for the right-handed dimer of the glycophorin A transmembrane peptide in similar detergents. Fluorescence resonance energy transfer (FRET) experiments conducted on peptides labeled at their N-termini with either tetramethylrhodamine (TMR) or 7-nitrobenz-2-oxa-1,3-diazole (NBD) also indicated association. However, analysis of the FRET data using the usual assumption of complete quenching for NBD-TMR pairs in the dimer could not be quantitatively reconciled with the analytical ultracentrifugation-measured dimerization constant. This led us to develop a general treatment for the association of helices to either parallel or antiparallel structures of any aggregation state. Applying this treatment to the FRET data, constraining the dimerization constant to be within experimental uncertainty of that measured by analytical ultracentrifugation, we found the data could be well described by a monomer-dimer equilibrium with only partial quenching of the dimer, suggesting that the helices are most probably antiparallel. These results also suggest that a left-handed Gly heptad repeat motif can drive membrane helix association, but the affinity is likely to be less strong than the previously reported right-handed motif described for glycophorin A. PMID:15315956

  12. Characterization of labelling and de-labelling reagents for detection and recovery of tyrosine residue in peptide.

    PubMed

    Toyo'oka, Toshimasa; Mantani, Tomomi; Kato, Masaru

    2003-01-01

    This paper characterized the labelling and de-labelling reagents for reversible labelling of tyrosine (Tyr)-containing peptide, which involves detection and recovery. The phenolic hydroxyl group (-OH) in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), and 1-fluoro-2,4-dinitrobenzene (DNFB) under mild conditions at room temperature at pH 9.3. The labels in the resulting derivatives were removed with the treatment of nucleophiles, such as thiols (cysteine, N-acetyl-L-cysteine and dithiothreitol) and amines (dimethylamine, methylamine, diethylamine, ethylamine and pyrrolidine). The de-labelling reactions of NBD-labelled N-acetyl-L-tyrosine (N-AcTyr) with the nucleophiles produced N-AcTyr, accompanied by NBD-nucleophile. Although DBD-F and DNFB also successfully labeled the -OH group in N-AcTyr, the efficiency of Cbond;O bond cleavage and recovery of N-AcTyr by the nucleophiles was relatively low compared with NBD-label. Among the de-labelling reagents, N-acetyl-L-cysteine and dimethylamine were recommended for the elimination of NBD moiety, with respect to the reaction rate, the side reaction, and the yield of recovery. The proposed procedure, which includes the labelling with NBD-F and the removal of NBD moiety by the nucleophiles, was successfully applied to the reversible labelling of N-terminal amine-blocked peptides, i.e. N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Copyright 2003 John Wiley & Sons, Ltd.

  13. Flexible xxx-asp/asn and gly-xxx residues of equine cytochrome C in matrix-assisted laser desorption/ionization in-source decay mass spectrometry.

    PubMed

    Takayama, Mitsuo

    2012-01-01

    The backbone flexibility of a protein has been studied from the standpoint of the susceptibility of amino acid residues to in-source decay (ISD) in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Residues more susceptible to MALDI-ISD, namely Xxx-Asp/Asn and Gly-Xxx, were identified from the discontinuous intense peak of c'-ions originating from specific cleavage at N-Cα bonds of the backbone of equine cytochrome c. The identity of the residues susceptible to ISD was consistent with the known flexible backbone amides as estimated by hydrogen/deuterium exchange (HDX) experiments. The identity of these flexible amino acid residues (Asp, Asn, and Gly) is consistent with the fact that these residues are preferred in flexible secondary structure free from intramolecular hydrogen-bonded structures such as α-helix and β-sheet. The MALDI-ISD spectrum of equine cytochrome c gave not only intense N-terminal side c'-ions originating from N-Cα bond cleavage at Xxx-Asp/Asn and Gly-Xxx residues, but also C-terminal side complement z'-ions originating from the same cleavage sites. The present study implies that MALDI-ISD can give information about backbone flexibility of proteins, comparable with the protection factors estimated by HDX.

  14. Impact of the Pro12Ala polymorphism of the PPARγ2 gene on diabetes and obesity in a highly consanguineous population

    PubMed Central

    Bener, Abdulbari; Zirie, M; Al-Hamaq, AOAA; Nawaz, Z; Samson, N; Mohammad, R

    2015-01-01

    Background: The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor subfamily of transcription factors. It has been reported that they play important roles in obesity and the development of type 2 diabetes mellitus (T2DM). Materials and Methods: This case-control study was carried out among 764 Qatari patients with diabetes and 764 healthy subjects above 20 years of age at Primary Healthcare Clinics (PHCs) from January 2011 to December 2012. Face-to-face interviews were based on a questionnaire that included variables such as age, sex, sociodemographic status, body mass index (BMI) and other clinical parameters. The Pro12Ala in the PPARγ2 gene was detected on the LightCycler using two specific probes. Univariate and multivariate statistical analysis were performed. Results: The study revealed that in the diabetes group, Pro/(10.2% vs 9.4%; P = 0.606) and Ala/Ala (1.4% vs 0.9%; P = 0.343) were higher than in controls, whereas Pro/Pro (88.4% vs 89.7%;P = 0.413) was lower in diabetes patients, but no significant difference was observed among the genotype groups. In obese patients with diabetes, Pro/Pro (89% vs 89.9%;P = 0.792) and Pro/Ala (8.9% vs 10.1%;P = 0.671) were lower than in obese healthy subjects. No homozygous Ala/Ala was found in obese healthy subjects, whereas 6 Ala/Ala homozygotes were in obese diabetes group. But in diabetes group, obese patients had higher homozygous of Pro/Pro (89.3% vs 87.8%;P = 0.523) and Ala/Ala (1.8% vs 1.2%;P = 0.771) compared to non-obese patients. Conclusion: The current study did not reveal an association between the Pro12Ala polymorphism of the PPAR γ2 gene and type 2 diabetes (T2D) in Qatari's population. PMID:25593831

  15. New features of the delta opioid receptor: conformational properties of deltorphin I analogues.

    PubMed

    Balboni, G; Marastoni, M; Picone, D; Salvadori, S; Tancredi, T; Temussi, P A; Tomatis, R

    1990-06-15

    Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.

  16. The Functional DRD3 Ser9Gly Polymorphism (rs6280) Is Pleiotropic, Affecting Reward as Well as Movement

    PubMed Central

    Savitz, Jonathan; Hodgkinson, Colin A.; Martin-Soelch, Chantal; Shen, Pei-Hong; Szczepanik, Joanna; Nugent, Allison; Herscovitch, Peter; Grace, Anthony A.; Goldman, David; Drevets, Wayne C.

    2013-01-01

    Abnormalities of motivation and behavior in the context of reward are a fundamental component of addiction and mood disorders. Here we test the effect of a functional missense mutation in the dopamine 3 receptor (DRD3) gene (ser9gly, rs6280) on reward-associated dopamine (DA) release in the striatum. Twenty-six healthy controls (HCs) and 10 unmedicated subjects with major depressive disorder (MDD) completed two positron emission tomography (PET) scans with [11C]raclopride using the bolus plus constant infusion method. On one occasion subjects completed a sensorimotor task (control condition) and on another occasion subjects completed a gambling task (reward condition). A linear regression analysis controlling for age, sex, diagnosis, and self-reported anhedonia indicated that during receipt of unpredictable monetary reward the glycine allele was associated with a greater reduction in D2/3 receptor binding (i.e., increased reward-related DA release) in the middle (anterior) caudate (p<0.01) and the ventral striatum (p<0.05). The possible functional effect of the ser9gly polymorphism on DA release is consistent with previous work demonstrating that the glycine allele yields D3 autoreceptors that have a higher affinity for DA and display more robust intracellular signaling. Preclinical evidence indicates that chronic stress and aversive stimulation induce activation of the DA system, raising the possibility that the glycine allele, by virtue of its facilitatory effect on striatal DA release, increases susceptibility to hyperdopaminergic responses that have previously been associated with stress, addiction, and psychosis. PMID:23365649

  17. Desipramine prevents cardiac gap junction uncoupling.

    PubMed

    Jozwiak, Joanna; Dietze, Anna; Grover, Rajiv; Savtschenko, Alex; Etz, Christian; Mohr, Friedrich W; Dhein, Stefan

    2012-11-01

    Uncoupling of cardiac gap junction channels is an important arrhythmogenic mechanism in ischemia/reperfusion. Antiarrhythmic peptide AAP10 (H-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH(2)) has been shown to prevent acidosis-induced uncoupling and ischemia-related increase in dispersion. Previous structure-effect investigations and subsequent computer modeling studies indicated that the tricyclic antidepressant desipramine may exert similar effects as AAP10. We assessed the binding of (14)C-AAP10 to membranes of rabbit cardiac ventricles and its displacement with desipramine in a classical radioligand binding and competition study. Gap junction currents were measured between isolated pairs of human atrial cardiomyocytes under normal and acidotic (pH 6.3) conditions with or without 1 μmol/l desipramine using dual whole-cell voltage clamp. The effect of 1 μmol/l desipramine was assessed in isolated rabbit hearts (Langendorff technique) undergoing local ischemia by coronary occlusion with 256-channel electrophysiological mapping and subsequent analysis of connexin43 (Cx43) expression, phosphorylation (Western blot), and subcellular localization (immunohistology). We found saturable (14)C-AAP10 binding to cardiac membranes (K (D), 0.29 ± 0.11 nmol/l; B (max), 42.5 ± 7.2 pmol/mg) which could be displaced by desipramine with a K (D.High) = 0.14 μmol/l and a K (D.Low) = 22 μmol/l. Acidosis reduced the gap junction conductance in human cardiomyocyte pairs from 24.1 ± 4.7 to 11.5 ± 2.5 nS, which could be significantly reversed by desipramine (26.6 ± 4.8 nS). In isolated hearts, ischemia resulted in significantly increased dispersion of activation-recovery intervals, loss of membrane Cx43, and dephosphorylation of Cx43, which all could be prevented by desipramine. Desipramine seems to prevent the uncoupling of cardiac gap junctions and ischemia-related increase in dispersion.

  18. Epigenomic study identifies a novel mesenchyme homeobox2-GLI1 transcription axis involved in cancer drug resistance, overall survival and therapy prognosis in lung cancer patients

    PubMed Central

    Armas-López, Leonel; Piña-Sánchez, Patricia; Arrieta, Oscar; de Alba, Enrique Guzman; Ortiz-Quintero, Blanca; Santillán-Doherty, Patricio; Christiani, David C.; Zúñiga, Joaquín; Ávila-Moreno, Federico

    2017-01-01

    Several homeobox-related gene (HOX) transcription factors such as mesenchyme HOX-2 (MEOX2) have previously been associated with cancer drug resistance, malignant progression and/or clinical prognostic responses in lung cancer patients; however, the mechanisms involved in these responses have yet to be elucidated. Here, an epigenomic strategy was implemented to identify novel MEOX2 gene promoter transcription targets and propose a new molecular mechanism underlying lung cancer drug resistance and poor clinical prognosis. Chromatin immunoprecipitation (ChIP) assays derived from non-small cell lung carcinomas (NSCLC) hybridized on gene promoter tiling arrays and bioinformatics analyses were performed, and quantitative, functional and clinical validation were also carried out. We statistically identified a common profile consisting of 78 gene promoter targets, including Hedgehog-GLI1 gene promoter sequences (FDR≤0.1 and FDR≤0.2). The GLI-1 gene promoter region from −2,192 to −109 was occupied by MEOX2, accompanied by transcriptionally active RNA Pol II and was epigenetically linked to the active histones H3K27Ac and H3K4me3; these associations were quantitatively validated. Moreover, siRNA genetic silencing assays identified a MEOX2-GLI1 axis involved in cellular cytotoxic resistance to cisplatinum in a dose-dependent manner, as well as cellular migration and proliferation. Finally, Kaplan-Maier survival analyses identified significant MEOX2-dependent GLI-1 protein expression associated with clinical progression and poorer overall survival using an independent cohort of NSCLC patients undergoing platinum-based oncological therapy with both epidermal growth factor receptor (EGFR)-non-mutated and EGFR-mutated status. In conclusion, this is the first study to investigate epigenome-wide MEOX2-transcription factor occupation identifying a novel overexpressed MEOX2-GLI1 axis and its clinical association with platinum-based cancer drug resistance and EGFR

  19. Adeno-associated virus-RNAi of GlyRα1 and characterization of its synapse-specific inhibition in OFF alpha transient retinal ganglion cells

    PubMed Central

    Zhang, C.; Rompani, S. B.; Roska, B.

    2014-01-01

    In the central nervous system, inhibition shapes neuronal excitation. In spinal cord glycinergic inhibition predominates, whereas GABAergic inhibition predominates in the brain. The retina uses GABA and glycine in approximately equal proportions. Glycinergic crossover inhibition, initiated in the On retinal pathway, controls glutamate release from presynaptic OFF cone bipolar cells (CBCs) and directly shapes temporal response properties of OFF retinal ganglion cells (RGCs). In the retina, four glycine receptor (GlyR) α-subunit isoforms are expressed in different sublaminae and their synaptic currents differ in decay kinetics. GlyRα1, expressed in both On and Off sublaminae of the inner plexiform layer, could be the glycinergic isoform that mediates On-to-Off crossover inhibition. However, subunit-selective glycine contributions remain unknown because we lack selective antagonists or cell class-specific subunit knockouts. To examine the role of GlyRα1 in direct inhibition in mature RGCs, we used retrogradely transported adeno-associated virus (AAV) that performed RNAi and eliminated almost all glycinergic spontaneous and visually evoked responses in PV5 (OFFαTransient) RGCs. Comparisons of responses in PV5 RGCs infected with AAV-scrambled-short hairpin RNA (shRNA) or AAV-Glra1-shRNA confirm a role for GlyRα1 in crossover inhibition in cone-driven circuits. Our results also define a role for direct GlyRα1 inhibition in setting the resting membrane potential of PV5 RGCs. The absence of GlyRα1 input unmasked a serial and a direct feedforward GABAAergic modulation in PV5 RGCs, reflecting a complex interaction between glycinergic and GABAAergic inhibition. PMID:25231618

  20. A new anti-MRSA antibiotic complex, WAP-8294A II. Structure characterization of minor components by ESI LCMS and MS/MS.

    PubMed

    Kato, Azusa; Hirata, Haruhisa; Ohashi, Yoshitami; Fujii, Kiyonaga; Mori, Kenji; Harada, Ken-ichi

    2011-05-01

    The anti-MRSA antibiotic, WAP-8294A, was isolated from the fermentation broth of Lysobacter sp. The major component, WAP-8294A2, is composed of 1 mol of Gly, L-Leu, L-Glu, D-Asn, D-Trp, D-threo-β-hydroxyasparagine, N-Me-D-Phe and N-Me-L-Val, and 2 mol of L-Ser, D-Orn and D-3-hydroxy-7-Me-octanoic acid. The structure of the WAP-8294A2 was mainly determined as a cyclic depsipeptide by 2D NMR experiments. However, it was difficult to use the NMR experiment to determine the minor components, A1, A4 and Ax13, isolated in small amounts. In the present study, ESI MS/MS was applied to the structure elucidation of these minor components. The structures of these minor components were determined on the basis of the fragmentation pattern of the product ions of WAP-8294A2 in the ESI MS/MS. As a result, it was confirmed that A1 and A4 had the same amino acid sequence as A2, while A1 and A4 had the 3-OH-octanoic acid and 3-OH-8-Me-nonanoic acid, respectively, in the place of the 3-OH-7-Me-octanoic acid in A2. In the structure of Ax13, it was found that Gly of A2 was changed to β-Ala of Ax13. © 2011 Japan Antibiotics Research Association All rights reserved

  1. DOE Office of Scientific and Technical Information (OSTI.GOV)

    James Graham, Robert Leslie; Graham, Ciaren; McClean, Stephen

    A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin actionmore » at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.« less

  2. Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors

    PubMed Central

    Whisenant, Thomas C.; Ho, David T.; Benz, Ryan W.; Rogers, Jeffrey S.; Kaake, Robyn M.; Gordon, Elizabeth A.; Huang, Lan; Baldi, Pierre; Bardwell, Lee

    2010-01-01

    In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new ‘D-site’ class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates. PMID:20865152

  3. Codon usage analysis of photolyase encoding genes of cyanobacteria inhabiting diverse habitats.

    PubMed

    Rajneesh; Pathak, Jainendra; Kannaujiya, Vinod K; Singh, Shailendra P; Sinha, Rajeshwar P

    2017-07-01

    Nucleotide and amino acid compositions were studied to determine the genomic and structural relationship of photolyase gene in freshwater, marine and hot spring cyanobacteria. Among three habitats, photolyase encoding genes from hot spring cyanobacteria were found to have highest GC content. The genomic GC content was found to influence the codon usage and amino acid variability in photolyases. The third position of codon was found to have more effect on amino acid variability in photolyases than the first and second positions of codon. The variation of amino acids Ala, Asp, Glu, Gly, His, Leu, Pro, Gln, Arg and Val in photolyases of three different habitats was found to be controlled by first position of codon (G1C1). However, second position (G2C2) of codon regulates variation of Ala, Cys, Gly, Pro, Arg, Ser, Thr and Tyr contents in photolyases. Third position (G3C3) of codon controls incorporation of amino acids such as Ala, Phe, Gly, Leu, Gln, Pro, Arg, Ser, Thr and Tyr in photolyases from three habitats. Photolyase encoding genes of hot spring cyanobacteria have 85% codons with G or C at third position, whereas marine and freshwater cyanobacteria showed 82 and 60% codons, respectively, with G or C at third position. Principal component analysis (PCA) showed that GC content has a profound effect in separating the genes along the first major axis according to their RSCU (relative synonymous codon usage) values, and neutrality analysis indicated that mutational pressure has resulted in codon bias in photolyase genes of cyanobacteria.

  4. Combination of Cyclopamine and Tamoxifen Promotes Survival and Migration of MCF-7 Breast Cancer Cells – Interaction of Hedgehog-Gli and Estrogen Receptor Signaling Pathways

    PubMed Central

    Uzarevic, Zvonimir; Ozretic, Petar; Musani, Vesna; Rafaj, Maja; Cindric, Mario; Levanat, Sonja

    2014-01-01

    Hedgehog-Gli (Hh-Gli) signaling pathway is one of the new molecular targets found upregulated in breast tumors. Estrogen receptor alpha (ERα) signaling has a key role in the development of hormone-dependent breast cancer. We aimed to investigate the effects of inhibiting both pathways simultaneously on breast cancer cell survival and the potential interactions between these two signaling pathways. ER-positive MCF-7 cells show decreased viability after treatment with cyclopamine, a Hh-Gli pathway inhibitor, as well as after tamoxifen (an ERα inhibitor) treatment. Simultaneous treatment with cyclopamine and tamoxifen on the other hand, causes short-term survival of cells, and increased migration. We found upregulated Hh-Gli signaling under these conditions and protein profiling revealed increased expression of proteins involved in cell proliferation and migration. Therefore, even though Hh-Gli signaling seems to be a good potential target for breast cancer therapy, caution must be advised, especially when combining therapies. In addition, we also show a potential direct interaction between the Shh protein and ERα in MCF-7 cells. Our data suggest that the Shh protein is able to activate ERα independently of the canonical Hh-Gli signaling pathway. Therefore, this may present an additional boost for ER-positive cells that express Shh, even in the absence of estrogen. PMID:25503972

  5. [The metabolites of cyclic peptides from three endophytic mangrove fungi].

    PubMed

    Guo, Zhi-yong; Huang, Zhong-jing; Wen, Lu; Wan, Qiao; Liu, Fan; She, Zhi-gang; Lin, Yong-cheng; Zhou, Shi-ning

    2007-12-01

    Nine secondary metaboites of cyclic peptide were isolated from three mangrove endophytic fungi Paecilomyces sp. (treel-7), 4557,ZZF65. They were viscumamide(1),cyclo(Pro-Iso)(2),cyclo(Phe-Gly)(3),cyclo(Phe-Ana)(4),cyclo(Gly-Pro) (5),cyclo(Gly-Leu)(6), cyclo(Trp-Ana)(7),neoechinulin A(8),cyclo(Pro-Thr)(9). The compounds 1,7,8,9 were firstly isolated from marine fungus.

  6. The electronegativity equalization method fused with molecular mechanics: a fluctuating charge and flexible body potential function for [Emim][Gly] ionic liquids.

    PubMed

    Wu, Yang; Li, Yao; Hu, Na; Hong, Mei

    2014-02-14

    Recently, experimental and theoretical studies on amino acid ionic liquid (AAIL) systems have attracted much attention. A transferable intermolecular potential approach that includes fluctuating charges and a flexible body based on a combination of the electronegativity equalization method and molecular mechanics (EEM/MM), and its application to an AAIL system containing 1-ethyl-3-methylimidazolium ([Emim](+)) and glycine ([Gly](-)) are explored and tested in this study. A consistent integration of EEM with MM requires the input of the EEM charges of all atoms into the MM intermolecular electrostatic interaction term. Compared with ionic liquid (IL) force fields, the EEM/MM model has an outstanding feature: the EEM/MM model not only presents the electrostatic interaction of atoms and their changes in response to different ambient environments but also introduces "the H-bond interaction region" in which a new parameter kHB(RHB) is used to describe the electrostatic interaction of hydrogen atoms in [Emim](+) and oxygen atoms in [Gly](-), which can form hydrogen bonds. The EEM/MM model gives quite accurate predictions for gas-phase state properties of [Emim](+), [Gly](-), and ion pairs, such as optimized geometries, dipole moments, vibrational frequencies, and cluster interaction energies. Due to its explicit description of charges and hydrogen bonds, the EEM/MM model also performs well for the liquid-phase properties of [Emim][Gly] under ambient conditions. The calculated properties, such as density, heat of vaporization, the self-diffusion coefficient, and ionic conductivity, are fairly consistent with available experimental results.

  7. Determination of 13C isotopic enrichment of valine and threonine by GC-C-IRMS after formation of the N(O,S)-ethoxycarbonyl ethyl ester derivatives of the amino acids.

    PubMed

    Godin, Jean-Philippe; Faure, Magali; Breuille, Denis; Hopfgartner, Gérard; Fay, Laurent-Bernard

    2007-06-01

    We describe a new method of assessing, in a single run, (13)C isotopic enrichment of both Val and Thr by gas chromatography-combustion-isotope-ratio mass spectrometry (GC-C-IRMS). This method characterised by a rapid one-step derivatisation procedure performed at room temperature to form the N(O,S)-ethoxycarbonyl ethyl ester derivatives, and a polar column for GC. The suitability of this method for Val and Thr in in-vivo samples (mucosal hydrolysate) was demonstrated by studying protein metabolism with two tracers ((13)C-valine or (13)C-threonine). The intra-day and inter-day repeatability were both assessed either with standards or with in-vivo samples at natural abundance and at low (13)C isotopic enrichment. For inter-day repeatability CVs were between 0.8 and 1.5% at natural abundance and lower than 5.5% at 0.112 and 0.190 atom% enrichment for Val and Thr, respectively. Overall isotopic precision was studied for eleven standard amino acid derivatives (those of Val, Ala, Leu, Iso, Gly, Pro, Asp, Thr, Ser, Met, and Phe) and was assessed at 0.32 per thousand. The (13)C isotopic measurement was then extended to the other amino acids (Ala, Val, Leu, Iso, Gly, Pro, Thr, and Phe) at natural abundance for in-vivo samples. The isotopic precision was better than 0.002 atom% per amino acid (for n = 4 rats). This analytical method was finally applied to an animal study to measure Thr utilization in protein synthesis.

  8. Experimental binding energies for the metal complexes [Mg(CH3OH)n](2+), [Ca(CH3OH)n](2+), and [Sr(CH3OH)n](2+) for n in the range 4-20.

    PubMed

    Bruzzi, E; Stace, A J

    2014-10-09

    A supersonic source of clusters has been used to prepare neutral complexes of methanol in association with an alkaline earth metal atom. From these complexes the following metal-containing dications have been generated through electron ionization: [Mg(CH3OH)n](2+), [Ca(CH3OH)n](2+), and [Sr(CH3OH)n](2+), and for n in the range 4-20, kinetic energy release measurements following the evaporation of a single molecule have been undertaken using a high resolution mass spectrometer. Using finite heat bath theory, these data have been transformed into binding energies for individual methanol molecules attached to each of the three cluster systems. In the larger complexes (n > 6) the results exhibit a consistent trend, whereby the experimental binding energy data for all three metal ions are similar, suggesting that the magnitude of the charge rather than charge density influences the strength of the interaction. From a comparison with data recorded previously for (CH3OH)nH(+) it is found that the 2+ charge on a metal ion has an effect on the binding energy of molecules in complexes containing up to 20 solvent molecules. The results recorded for [Ca(CH3OH)n](2+) show evidence of a very marked transition between n = 6 and 7, which is thought to coincide with the completion of a primary solvation shell and the onset of molecules starting to occupy a second and most probably a third shell.

  9. Relationship between ADD1 Gly460Trp gene polymorphism and essential hypertension in Madeira Island.

    PubMed

    Sousa, Ana Célia; Palma Dos Reis, Roberto; Pereira, Andreia; Borges, Sofia; Freitas, Ana Isabel; Guerra, Graça; Góis, Teresa; Rodrigues, Mariana; Henriques, Eva; Freitas, Sónia; Ornelas, Ilídio; Pereira, Décio; Brehm, António; Mendonça, Maria Isabel

    2017-10-01

    Essential hypertension (EH) is a complex disease in which physiological, environmental, and genetic factors are involved in its genesis. The genetic variant of the alpha-adducin gene (ADD1) has been described as a risk factor for EH, but with controversial results.The objective of this study was to evaluate the association of ADD1 (Gly460Trp) gene polymorphism with the EH risk in a population from Madeira Island.A case-control study with 1614 individuals of Caucasian origin was performed, including 817 individuals with EH and 797 controls. Cases and controls were matched for sex and age, by frequency-matching method. All participants collected blood for biochemical and genotypic analysis for the Gly460Trp polymorphism. We further investigated which variables were independently associated to EH, and, consequently, analyzed their interactions.In our study, we found a significant association between the ADD1 gene polymorphism and EH (odds ratio 2.484, P = .01). This association remained statistically significant after the multivariate analysis (odds ratio 2.548, P = .02).The ADD1 Gly460Trp gene polymorphism is significantly and independently associated with EH risk in our population. The knowledge of genetic polymorphisms associated with EH is of paramount importance because it leads to a better understanding of the etiology and pathophysiology of this pathology.

  10. Relationship between ADD1 Gly460Trp gene polymorphism and essential hypertension in Madeira Island

    PubMed Central

    Sousa, Ana Célia; Palma dos Reis, Roberto; Pereira, Andreia; Borges, Sofia; Freitas, Ana Isabel; Guerra, Graça; Góis, Teresa; Rodrigues, Mariana; Henriques, Eva; Freitas, Sónia; Ornelas, Ilídio; Pereira, Décio; Brehm, António; Mendonça, Maria Isabel

    2017-01-01

    Abstract Essential hypertension (EH) is a complex disease in which physiological, environmental, and genetic factors are involved in its genesis. The genetic variant of the alpha-adducin gene (ADD1) has been described as a risk factor for EH, but with controversial results. The objective of this study was to evaluate the association of ADD1 (Gly460Trp) gene polymorphism with the EH risk in a population from Madeira Island. A case-control study with 1614 individuals of Caucasian origin was performed, including 817 individuals with EH and 797 controls. Cases and controls were matched for sex and age, by frequency-matching method. All participants collected blood for biochemical and genotypic analysis for the Gly460Trp polymorphism. We further investigated which variables were independently associated to EH, and, consequently, analyzed their interactions. In our study, we found a significant association between the ADD1 gene polymorphism and EH (odds ratio 2.484, P = .01). This association remained statistically significant after the multivariate analysis (odds ratio 2.548, P = .02). The ADD1 Gly460Trp gene polymorphism is significantly and independently associated with EH risk in our population. The knowledge of genetic polymorphisms associated with EH is of paramount importance because it leads to a better understanding of the etiology and pathophysiology of this pathology. PMID:29049185

  11. Isolation and structural elucidation of antioxidant peptides from oyster (Saccostrea cucullata) protein hydrolysate.

    PubMed

    Umayaparvathi, S; Meenakshi, S; Vimalraj, V; Arumugam, M; Balasubramanian, T

    2014-01-01

    Protein derived from the oyster (Saccostrea cucullata) was hydrolyzed using protease from Bacillus cereus SU12 for isolation of antioxidant peptides. The oyster hydrolysate exhibited a strong antioxidant potential in DPPH (85.7±0.37%) followed by Hydrogen peroxide radical scavenging activity (81.6±0.3%), Hydroxyl radical-scavenging activity (79.32±0.6%), Reducing power assay (2.63±0.2 OD at 700nm). Due to the high antioxidant potential, hydrolysate was fractionated in Sephadex G-25 gel filtration chromatography. The active peptide fraction was further purified by UPLC-MS. Totally 7 antioxidant peptides were collected. Among 7 peptides (SCAP 1-7), 3 peptides (SCAP 1, 3 and 7) had highest scavenging ability on DPPH radicals. The amino acid sequence and molecular mass of purified antioxidant peptides (SCAP1, SCAP3 and SCAP7) were determined by Q-TOF ESI mass spectroscopy and structures of the peptides were Leu-Ala-Asn-Ala-Lys (MW=515.29Da), Pro-Ser-Leu-Val-Gly-Arg-Pro-Pro-Val-Gly-Lys-Leu-Thr-Leu (MW=1432.89Da) and Val-Lys-Val-Leu-Leu-Glu-His-Pro-Val-Leu (MW=1145.75Da), respectively. The unique amino acid composition and sequence in the peptides might play an important role in expression of their antioxidant activity. The results of this study suggest that oyster protein hydrolysate is good source of natural antioxidants.

  12. Acquisition of pro-oxidant activity of fALS-linked SOD1 mutants as revealed using circular dichroism and UV-resonance Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Fujimaki, Nobuhiro; Nishiya, Ken; Miura, Takashi; Nakabayashi, Takakazu

    2016-11-01

    The acquisition of pro-oxidant activity of the mutated form of human Cu, Zn-superoxide dismutase (SOD1) has been investigated to clarify the relationship between mutations in SOD1 and the pathogenesis of amyotrophic lateral sclerosis (ALS). Ala4 → Val (A4V) and Gly93 → Ala (G93A) mutants, which are representative ALS-linked SOD1 mutants, have been found to exhibit both the denaturation and the gain of pro-oxidant activity after incubation in the apo-form at a physiological condition of 37 °C and pH 7.4 and the rebinding of Cu2+. These characteristics are similar to those previously reported for the His43 → Arg (H43R) mutant. UV-resonance Raman spectra indicated that the coordination structure of the Cu-binding site catalyzing the oxidation reaction is the same among the denatured A4V, G93A, and H43R. Since wild-type SOD1 does not exhibit the denaturation in its apo-form at 37 °C and pH 7.4, the instability of the protein structure due to mutation can be considered as a significant factor that induces the denaturation and the subsequent pro-oxidant activity.

  13. An evaluation of the budget impact of a new 20% subcutaneous immunoglobulin (Ig20Gly) for the management of primary immunodeficiency diseases in Switzerland.

    PubMed

    Pollock, Richard F; Meckley, Lisa M

    2018-01-01

    While most individual primary immunodeficiency diseases (PID) are rare, the collective prevalence of PID results in a substantial economic and clinical burden. The aim of this study was to evaluate the budgetary implications of Ig20Gly (Immune Globulin Subcutaneous [human] 20% solution; CUVITRU ® , Baxalta US Inc, now part of Shire Plc, Westlake Village, CA, USA) as a treatment for PID relative to intravenous immunoglobulin (IVIG) and other subcutaneous immunoglobulin (SCIG) formulations in the Swiss health care setting. A budget impact model was developed in Microsoft Excel to capture the estimated prevalence of PID in Switzerland, the proportion of patients treated in different health care settings, and the costs of administering SCIG and IVIG in each setting. Unit costs were based on a recent cost-minimization analysis of SCIG in Lausanne, and drug costs were taken from the Spezialitätenliste. All costs were reported in 2016 Swiss Francs (CHF), and future costs were not discounted. The total cost of treating PID in Switzerland was estimated to be CHF 11.16 m over 3 years, comprising CHF 9.28 m of drug costs and CHF 1.87 m of ancillary costs, including health care professional time and other administration costs, such as pumps and needle sets. The analysis showed that using Ig20Gly in place of other SCIG formulations would be cost neutral, while using Ig20Gly in place of IVIG would result in savings of 4.0%. Ig20Gly would be cost neutral relative to existing SCIG products and would result in cost savings relative to IVIG in patients with PID in Switzerland, even with modest uptake.

  14. Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy.

    PubMed

    Yan, Si; Guo, Changmiao; Hou, Guangjin; Zhang, Huilan; Lu, Xingyu; Williams, John Charles; Polenova, Tatyana

    2015-11-24

    Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

  15. The pharmacological properties of the novel peptide BPC 157 (PL-10).

    PubMed

    Sikiric, P

    1999-01-01

    The reported beneficial effects of the gastric mucosal derived pentadecapeptide BPC 157 (Gly Glu Pro Pro Pro Gly Lys Pro Ala Asp Asp Ala Gly Leu Val, M.W. 1419) on different organ lesions are reviewed. Apart from the effects on various gastrointestinal lesions, the potentially beneficial effect on pancreas, liver injuries, endothelium and heart damage, i.e. dysrhythmias following reoxygenation, and blood pressure, along with effect on experimental acute/chronic inflammation, wound and fracture (pseudoarthrosis) healing are described. It appears that these beneficial effects all together provide a particular network reflecting activity of a special peptidergic defence system. In support of this concept, it appears that there are interactions of this pentadecapeptide with many important systems (namely, dopamine-, NO-, prostaglandin-, somatosensory neurone-systems), that could provide a basis for the observed protective effects. Moreover, since disturbance of these systems' functions (i.e. dopamine-, NO-, somatosensory neuronal-system) which manifest either over-activity or as inhibition, may contribute to the multiple lesions in different organs. The reported evidence that this pentadecapeptide is able to counteract both their over-action, and their inhibition, may suggest this pentadecapeptide as a new, but most probably essential physiological defence system and that should be further investigated.

  16. Bradykinin-related compounds as new drugs for cancer and inflammation.

    PubMed

    Stewart, John M; Gera, Lajos; Chan, Daniel C; Bunn, Paul A; York, Eunice J; Simkeviciene, Vitalija; Helfrich, Barbara

    2002-04-01

    Bradykinin (BK) (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) is an important growth factor for small-cell lung cancer (SCLC) and prostate cancer (PC). These cancers have cells of neuroendocrine origin and express receptors for a variety of neuropeptides. BK receptors are expressed on almost all lung cancer cell lines and on many PC cells. Our very potent BK antagonist B9430 (D-Arg-Arg-Pro-Hyp-Gly-lgl-Ser-D-Igl-Oic-Arg) (Hyp, trans-4-hydroxy-L-proline; Ig1, alpha-2-indanylglycine; Oic, octahydroindole-2-carboxylic acid) is a candidate anti-inflammatory drug but does not inhibit growth of SCLC or PC. When B9430 is dimerized by N-terminal cross-linking with a suberimide linker, the product B9870 is a potent growth inhibitor for SCLC both in vitro and in vivo in athymic nude mice. Daily i.p. injection at 5 mg x kg(-1) day(-1) beginning on day 8 after SCLC SHP-77 cell implantation gave 65% inhibition of tumor growth. B9870 stimulates apoptosis in SCLC by a novel "biased agonist" action. We have also developed new small mimetic antagonists. BKM-570 (F5C-OC2Y-Atmp) (F5C, pentafluorocinnamic acid; OC2Y, O-2,6-dichlorobenzyl tyrosine; Atmp, 4-amino-2,2,6,6-tetramethylpiperidine) is very potent for inhibition of SHP-77 growth in nude mice. When injected daily i.p. at 5 mg x kg(-1), M-570 gave 90% suppression of tumor growth. M-570 is more potent than the well-known anticancer drug cisPlatin (60% inhibition) or the recently developed SU5416 (40% inhibition) in this model. M-570 also showed activity against various other cancer cell lines in vitro (SCLC, non-SCLC, lung, prostate, colon, cervix) and inhibited growth of prostate cell line PC3 in nude mice. M-570 and related compounds evidently act in vivo through pathways other than BK receptors. These compounds have clinical potential for treatment of human lung and prostate cancers.

  17. Analysis of Arg-Gly-Asp mimetics and soluble receptor of tumour necrosis factor as therapeutic modalities for concanavalin A induced hepatitis in mice.

    PubMed Central

    Bruck, R; Shirin, H; Hershkoviz, R; Lider, O; Kenet, G; Aeed, H; Matas, Z; Zaidel, L; Halpern, Z

    1997-01-01

    BACKGROUND/AIMS: It has been shown that synthetic non-peptidic analogues of Arg-Gly-Asp, a major cell adhesive ligand of extracellular matrix, prevented an increase in serum aminotransferase activity, as a manifestation of concanavalin A induced liver damage in mice. This study examined the effects of an Arg-Gly-Asp mimetic on liver histology and cytokine release in response to concanavalin A administration, and the efficacy of soluble receptor of tumour necrosis factor (TNF) alpha in preventing hepatitis in this model of liver injury. METHODS: Mice were pretreated with either the Arg-Gly-Asp mimetic SF-6,5 or recombinant soluble receptor of TNF alpha before their inoculation with 10 mg/kg concanavalin A. Liver enzymes, histology, and the serum values of TNF alpha and interleukin (IL)6 were examined. RESULTS: The histopathological damage in the liver, and the concanavalin A induced release of TNF alpha and IL6 were significantly inhibited by the synthetic Arg-Gly-Asp mimetic (p < 0.001). Liver injury, manifested by the increase in serum aminotransferase and cytokines, as well as by histological manifestations of hepatic damage, was effectively prevented by pretreatment of the mice with the soluble TNF receptor (p < 0.001). CONCLUSIONS: This study confirms the efficacy of a synthetic Arg-Gly-Asp mimetic and soluble TNF receptor in the prevention of immune mediated liver damage in mice. Images PMID:9155591

  18. Phenotypic heterogeneity associated with a novel mutation (Gly112Glu) in the Norrie disease protein.

    PubMed

    Allen, R C; Russell, S R; Streb, L M; Alsheikheh, A; Stone, E M

    2006-02-01

    To determine the molecular pathology and clinical severity of two pedigrees with a history of early retinal detachment and peripheral retinal vascular abnormalities. Longitudinal cohort study. A longitudinal clinical study and DNA analysis was performed on 49 family members of two pedigrees. Nine individuals were found to be hemizygous for a mutation at codon 112 (Gly112Glu) of the Norrie disease protein (NDP) in one pedigree. Significant phenotypic heterogeneity was found. The proband presented with a unilateral subtotal retinal detachment at the age of 3 years, and subsequently developed a slowly progressive tractional retinal detachment involving the macula in the contralateral eye at the age of 4 years. One individual had only mild peripheral retinal pigmentary changes with normal vision at the age of 79 years. The remaining seven individuals had varying degrees of peripheral retinal vascular abnormalities and anterior segment findings. Seven affected members of a second pedigree affected by a previously reported mutation, Arg74Cys, also demonstrated wide ocular phenotypic variation. A novel mutation (Gly112Glu), which represents the most carboxy located, NDP mutation reported, results in significant phenotypic heterogeneity. These data support the contention that the spectrum of ocular disease severity associated with these NDP mutations is broad. Use of terms that characterize this entity by phenotypic appearance, such as familial exudative vitreoretinopathy, do not adequately communicate the potential spectrum of severity of this disorder to affected or carrier family members.

  19. Manipulations of extracellular Loop 2 in α1 GlyR ultra-sensitive ethanol receptors (USERs) enhance receptor sensitivity to isoflurane, ethanol, and lidocaine, but not propofol

    PubMed Central

    Naito, Anna; Muchhala, Karan H.; Trang, Janice; Asatryan, Liana; Trudell, James R.; Homanics, Gregg E.; Alkana, Ronald L.; Davies, Daryl L.

    2015-01-01

    We recently developed Ultra-Sensitive Ethanol Receptors (USERs) as a novel tool for investigation of single receptor subunit populations sensitized to extremely low ethanol concentrations that do not affect other receptors in the nervous system. To this end, we found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs) can significantly increase receptor sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase in ethanol sensitivity relative to wild type (WT) receptors. The current study investigated: 1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase receptor sensitivity to other anesthetics; and 2) If mutations exclusive to the C-terminal end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane (volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2 mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity, but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also found that structural manipulations in the C-terminal end of Loop 2 were sufficient and selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action. Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates isoflurane and lidocaine modulation. Moreover, the results identify important amino acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively, these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol action, and the structural requirements for allosteric modulation on

  20. GLI2 expression levels in radical nephrectomy specimens as a predictor of disease progression in patients with metastatic clear cell renal cell carcinoma following treatment with sunitinib

    PubMed Central

    Furukawa, Junya; Miyake, Hideaki; Fujisawa, Masato

    2016-01-01

    The aim of the present study was to investigate the role of the Hedgehog signaling pathway in the progression of metastatic clear cell renal cell carcinoma (m-ccRCC) as well as the molecular targets of sunitinib, an inhibitor of multiple tyrosine kinases. A total of 39 patients subjected to radical nephrectomy who were diagnosed with m-ccRCC and were subsequently treated with sunitinib were enrolled in the present study. The expression levels of the Hedgehog signaling proteins (GLI1, GLI2, cyclin D1, cyclin E and transforming growth factor-β) and major molecular targets of sunitinib [vascular endothelial growth factor receptor (VEGFR)-1 and −2, and platelet-derived growth factor receptor-α and -β] in primary RCC specimens were assessed by immunohistochemical staining. The expression levels of GLI2, VEGFR-1, VEGFR-2 and pre-treatment C-reactive protein as well as the Memorial Sloan-Kettering Cancer Center risk were identified as significant predictors of progression-free survival (PFS). Of these, only GLI2 expression was independently correlated to PFS according to multivariate analysis. Furthermore, treatment with sunitinib resulted in a marked inhibition of GLI2 expression in the parental human RCC ACHN cell line, but not in ACHN cells with acquired resistance to sunitinib. These findings suggested that GLI2 may be involved in the acquisition of resistance to sunitinib in RCC; thus, it may be useful to consider the expression levels of GLI2 in addition to conventional prognostic parameters when selecting m-ccRCC patients likely to benefit from treatment with sunitinib. PMID:27602218

  1. Structure-activity relationship of linear peptide Bu-His6-DPhe7-Arg8-Trp9-Gly10-NH2 at the human melanocortin-1 and -4 receptors: DPhe7 and Trp9 substitution.

    PubMed

    Danho, Waleed; Swistok, Joseph; Cheung, Adrian Wai-Hing; Kurylko, Grazyna; Franco, Lucia; Chu, Xin-Jie; Chen, Li; Yagaloff, Keith

    2003-02-24

    A series of pentapeptides, based on hMC4R pentapeptide agonist (Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2)), was prepared in which either DPhe(7) or Trp(9) residue was systematically substituted. A number of interesting DPhe surrogates (D-Thi, D-3-CF(3)Phe, D-2-Nal and D-3,4-diClPhe) as well as Trp surrogates (2-Nal and Bta) were identified in this study.

  2. The common Arg389gly ADRB1 polymorphism affects heart rate response to the ultra-short-acting β(1) adrenergic receptor antagonist esmolol in healthy individuals.

    PubMed

    Muszkat, Mordechai; Hoofien, Assaf; Orlanski-Meyer, Esther; Makhoul, Hani; Porat, Einav; Davidson, Eliad M; Blotnick, Simcha; Caraco, Yoseph

    2013-01-01

    The β1-adrenergic receptor (β1AR) Arg389Gly polymorphism affects responses to orally administered β1AR antagonists (β-blockers) in vivo. However, the effect of this polymorphism on the early heart rate response to β-blockers has not been evaluated. The aim of this study was to determine the effect of the Arg389Gly polymorphism on the inhibition of exercise-induced tachycardia by esmolol, an ultra-short-acting intravenously administered β1AR antagonist. Healthy nonsmoking White individuals were enrolled on the basis of their ADRB1 genotype, including carriers of 0, 1 or 2 Arg389 alleles (n=9 in each group, total 27, 18 men). Placebo and esmolol were infused consecutively for 10 min each, separated by 30 min. At the end of each infusion, participants performed dynamic handgrip exercise. Heart rate and blood pressure were compared among three ADRB1 genotypes. Carriers of 0, 1, or 2 Arg389 alleles varied significantly in both exercise-induced tachycardia during esmolol (P(ANOVA)=0.030) and esmolol inhibition of exercise-induced tachycardia [0.78±7.70, 5.11±4.05, 10.22±9.78 bpm, respectively (P=0.014)]. The early effect of esmolol on exercise-induced tachycardia was significantly greater among Arg389 than in Gly389 homozygote healthy individuals (NCT01388036). © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

  3. Insulin and proglucagon-derived peptides from the horned frog, Ceratophrys ornata (Anura:Leptodactylidae).

    PubMed

    White, A M; Secor, S M; Conlon, J M

    1999-07-01

    Insulin and peptides derived from the processing of proglucagon have been isolated from an extract of the pancreas of the South American horned frog, Ceratophrys ornata (Leptodactylidae). Ceratophrys insulin is identical to the insulin previously isolated from the toad, Bufo marinus (Bufonidae). Ceratophrys glucagon was isolated in two molecular forms with 29- and 36-amino acid residues in approximately equal amounts. Glucagon-29 is identical to glucagon from B. marinus and from the bullfrog, Rana catesbeiana (Ranidae) and contains only 1 amino acid substitution (Thr29 --> Ser) compared with glucagon from Xenopus laevis (Pipidae). Glucagon-36 comprises glucagon-29 extended from its C-terminus by Lys-Arg-Ser-Gly-Gly-Met-Ser. This extension is structurally dissimilar to the C-terminal octapeptide of mammalian oxyntomodulin and resembles more closely that found in C-terminally extended glucagons isolated from fish pancreata. Ceratophrys glucagon-like peptide-1 (GLP-1) (His-Ala-Asp-Gly-Thr-Tyr-Gln-Asn-Asp-Val10-Gln-Gln-Phe-Leu-Glu- Glu-Lys-Ala-Ala-Lys20-Glu-Phe-Ile-Asp-Trp-Leu-Ile-Lys-Gly- Lys30-Pro-Lys-Lys-Gln-Arg-Leu-Ser) contains 3 amino acid substitutions compared with the corresponding peptide from B. marinus, 8 substitutions compared with GLP-1 from R. catesbeiana, and between 4 and 11 substitutions compared with the three GLP-1 peptides identified in X. laevis proglucagon. GLP-2 was not identified in the extract of Ceratophrys pancreas. The data indicate that, despite its importance in the regulation of glucose metabolism, the primary structure of GLP-1 has been very poorly conserved during evolution, even among a single order such as the Anura. Copyright 1999 Academic Press.

  4. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.

    PubMed Central

    Kim, W; Choi, K; Kim, Y; Park, H; Choi, J; Lee, Y; Oh, H; Kwon, I; Lee, S

    1996-01-01

    Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis. PMID:8779587

  5. Involvement of PI3K/Akt pathway in the inhibition of hepatocarcinoma cell invasion and metastasis induced by SASH1 through downregulating Shh-Gli1 signaling.

    PubMed

    Sun, Changyu; Zhang, Zhihao; He, Ping; Zhou, Yan; Xie, Xuhua

    2017-08-01

    The SASH1 gene is discovered as a tumor suppressor recently. However, the molecular mechanisms of SASH1 in hepatocarcinoma (HCC) remain unclear. In present studies, we investigated the molecular mechanisms of SASH1 on cell invasion and metastasis of hepatocarcinoma in vivo and in vitro. In this study, SASH1 overexpression HCC cell lines were treated with purmorphamine (0, 0.5, 1, 2μmol/l). Western blot and qRT-PCR were used to examine the related gene expression of EMT markers and the Shh-Gli1 and PI3K/Akt-dependent pathway. Cell migration and invasion were assessed by Transwell assay. In addition, a mice SASH1 overexpression HCC orthotopic xenograft model was established and treated with purmorphamine or 740Y-P or PDGF. Tumor volume was assessed, and H&E staining was applied to histopathologic analysis. The results showed that purmorphamine exposure significantly increased the mRNA and protein expression levels of Shh and Gli1 in a dose-dependent manner in the SASH1 overexpression HepG2 and HCCLM3 cells. Besides, purmorphamine promoted the migration and invasion of SASH1 overexpression HCC cells, as well as the EMT progress. Moreover, purmorphamine significantly increased the synthesis of PI3K and pAkt in a dose-dependent manner. Furthermore, the invasion and migration abilities were also improved by treatment with 740Y-P or PDGF in the SASH1 overexpression HCC cells. Additionally, the agonists promoted tumor growth and intrahepatic and pulmonary metastasis of the orthotopic transplantation tumors grown from SASH1 overexpression HCC cells in vivo. In conclusion, SASH1 may inhibit hepatocarcinoma cell invasion and metastasis through down-regulating the Shh-Gli1 and PI3K-AKT pathways in vivo and in vitro. Copyright © 2017. Published by Elsevier Ltd.

  6. Frequencies of the Arg16Gly, Gln27Glu and Thr164Ile Adrenoceptor β2 Polymorphisms among Omanis

    PubMed Central

    Al-Balushi, Khalid; Zadjali, Fahad; Al-Sinani, Sawsan; Al-Zadjali, Al-Muatasim; Bayoumi, Riad

    2015-01-01

    Objectives: This study aimed to assess the distribution of missense mutations in the adrenoceptor β2 (ADRB2) gene in an Omani cohort. Methods: This study was carried out between May 2014 and March 2015 at the Sultan Qaboos University, Muscat, Oman. Blood samples were taken from 316 unrelated Omani subjects. Genotyping for rs1042713 (c.46A>G, p.Arg16Gly), rs1042714 (c.79C>G, p.Gln27Glu) and rs1800888 (c.491C>T, p.Thr164Ile) polymorphisms was performed by real-time polymerase chain reaction using single nucleotide polymorphism (SNP) genotyping assays. The allelic frequencies of these polymorphisms were estimated on the basis of the observed numbers of specific alleles from the genotype data for male and female subjects. The genotype frequencies for each polymorphism were tested for deviation from the Hardy-Weinberg equilibrium. Results: Gly16 and Glu27 were the most frequent variants found among the cohort (63% and 75%, respectively). The Ile164 variant was not detected in the study population. There was a significant linkage disequilibrium between the rs1042713 and rs1042714 SNPs (r2 = 0.209; P ≤0.001). The most observed haplotypes were Gly16-Gln27 and Arg16-Gln27 (0.37 and 0.38, respectively). The frequency of Gly16-Glu27 was 0.25, comprising all Glu27 carriers. Conclusion: The allelic distribution of variants in this Omani cohort was similar to distributions reported among Caucasian populations. PMID:26629374

  7. Structurally Ordered Nanowire Formation from Co-Assembly of DNA Origami and Collagen-Mimetic Peptides

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jiang, Tao; Meyer, Travis A.; Modlin, Charles

    In this paper, we describe the co-assembly of two different building units: collagen-mimetic peptides and DNA origami. Two peptides CP ++ and sCP ++ are designed with a sequence comprising a central block (Pro-Hyp-Gly) and two positively charged domains (Pro-Arg-Gly) at both N- and C-termini. Co-assembly of peptides and DNA origami two-layer (TL) nanosheets affords the formation of one-dimensional nanowires with repeating periodicity of similar to 10 nm. Structural analyses suggest a face-to-face stacking of DNA nanosheets with peptides aligned perpendicularly to the sheet surfaces. We demonstrate the potential of selective peptide-DNA association between face-to-face and edge-to-edge packing by tailoringmore » the size of DNA nanostructures. Finally, this study presents an attractive strategy to create hybrid biomolecular assemblies from peptide and DNA-based building blocks that takes advantage of the intrinsic chemical and physical properties of the respective components to encode structural and, potentially, functional complexity within readily accessible biomimetic materials.« less

  8. Structurally Ordered Nanowire Formation from Co-Assembly of DNA Origami and Collagen-Mimetic Peptides

    DOE PAGES

    Jiang, Tao; Meyer, Travis A.; Modlin, Charles; ...

    2017-09-26

    In this paper, we describe the co-assembly of two different building units: collagen-mimetic peptides and DNA origami. Two peptides CP ++ and sCP ++ are designed with a sequence comprising a central block (Pro-Hyp-Gly) and two positively charged domains (Pro-Arg-Gly) at both N- and C-termini. Co-assembly of peptides and DNA origami two-layer (TL) nanosheets affords the formation of one-dimensional nanowires with repeating periodicity of similar to 10 nm. Structural analyses suggest a face-to-face stacking of DNA nanosheets with peptides aligned perpendicularly to the sheet surfaces. We demonstrate the potential of selective peptide-DNA association between face-to-face and edge-to-edge packing by tailoringmore » the size of DNA nanostructures. Finally, this study presents an attractive strategy to create hybrid biomolecular assemblies from peptide and DNA-based building blocks that takes advantage of the intrinsic chemical and physical properties of the respective components to encode structural and, potentially, functional complexity within readily accessible biomimetic materials.« less

  9. Impact of the PPAR-γ2 Pro12Ala Polymorphism and ACE Inhibitor Therapy on New-Onset Microalbuminuria in Type 2 Diabetes: Evidence From BENEDICT

    PubMed Central

    De Cosmo, Salvatore; Motterlini, Nicola; Prudente, Sabrina; Pellegrini, Fabio; Trevisan, Roberto; Bossi, Antonio; Remuzzi, Giuseppe; Trischitta, Vincenzo; Ruggenenti, Piero

    2009-01-01

    OBJECTIVE Cross-sectional studies found less microalbuminuria in type 2 diabetic patients with the Ala12 allele of the peroxisome proliferator–activated receptor-γ2 (PPAR-γ2) Pro12Ala polymorphism. We prospectively evaluated the association between Pro12Ala polymorphism (rs1801282) and new-onset microalbuminuria. RESEARCH DESIGN AND METHODS Pro12Ala polymorphism was genotyped by TaqMan-based assay in genomic DNA of 1,119 consenting patients from BErgamo NEphrologic DIabetic Complications Trial (BENEDICT)—a prospective, randomized trial evaluating ACE inhibition effect on new-onset microalbuminuria (albuminuria 20–200 μg/min in at least two of three consecutive overnight urine collections in two consecutive visits) in hypertensive type 2 diabetes with albuminuria <20 μg/min at inclusion. RESULTS Baseline characteristics of Ala (Ala/Ala or Ala/Pro) carriers and Pro/Pro homozygotes were similar, with a nonsignificant trend to lower albuminuria (P = 0.1107) in the 177 Ala carriers. Over a median (interquartile range) of 44.0 (17.1–51.9) months, 7 (4%) Ala carriers and 86 (9.1%) Pro/Pro homozygotes developed microalbuminuria (hazard ratio [HR] 0.45 [95% CI 0.21–0.97]; P = 0.042). Final albuminuria was significantly lower in Ala carriers than Pro/Pro homozygotes (7.3 ± 9.1 vs. 10.5 ± 24.9 μg/min, respectively), even after adjustment for baseline albuminuria (P = 0.048). Baseline and follow-up blood pressure and metabolic control were similar in both groups. Incidence of microalbuminuria was significantly decreased by ACE versus non-ACE inhibitor therapy in Pro/Pro homozygotes (6.3 vs. 11.9%, respectively, HR 0.46 [0.29–0.72]; P < 0.001). CONCLUSIONS In type 2 diabetes, the Ala allele protects from worsening albuminuria and new-onset microalbuminuria, and ACE inhibition blunts the excess risk of microalbuminuria associated with the Pro/Pro genotype. Evaluating Pro12Ala polymorphism may help identifying patients at risk who may benefit the most from

  10. Consumption of peptide-included and free tryptophan induced by peroxyl radicals: A kinetic study.

    PubMed

    Fuentes, E; López-Alarcón, C

    2014-10-01

    It is well-known that tryptophan residues are efficiently oxidized by peroxyl radicals, generating kynurenine, and N-formyl kynurenine as well as hydroperoxide derivatives as products. In the present work we studied the kinetic of such reaction employing free and peptide-included tryptophan. Two azocompounds were used to produce peroxyl radicals: AAPH (2,2'-Azobis(2-methylpropionamidine) dihydrochloride) and ABCVA (4,4'-Azobis(4-cyanovaleric acid)), which generate cationic and anionic peroxyl radicals, respectively. Tryptophan consumption was assessed by fluorescence spectroscopy and the reactions were carried out in phosphate buffer (75mM, pH 7.4) at 45°C. Only a slight effect of the peroxyl radical charge was evidenced on the consumption of free tryptophan and the dipeptide Gly-Trp. Employing AAPH as peroxyl radical source, at low free tryptophan concentrations (1-10µM) near 0.3 mol of tryptophan were consumed per each mol of peroxyl radicals introduced into the system. However, at high free tryptophan concentrations (100µM-1mM) such stoichiometry increased in a tryptophan concentration-way. At 1mM three moles of tryptophan were consumed per mol of AAPH-derived peroxyl radicals, evidencing the presence of chain reactions. A similar behavior was observed when di and tri-peptides (Gly-Trp, Trp-Gly, Gly-Trp-Gly, Trp-Ala, Ala-Trp-Ala) were studied. Nonetheless, at low initial concentration (5µM), the initial consumption rate of tryptophan included in the peptides was two times higher than free tryptophan. In contrast, at high concentration (1mM) free and peptide-included tryptophan showed similar initial consumption rates. These results could be explained considering a disproportionation process of tryptophanyl radicals at low free tryptophan concentrations, a process that would be inhibited when tryptophan is included in peptides. Copyright © 2014. Published by Elsevier Inc.

  11. A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica.

    PubMed

    Hilbert, F; García-del Portillo, F; Groisman, E A

    1999-04-01

    The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.

  12. Isolation: analysis and properties of three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom.

    PubMed

    Ferreira, L A; Galle, A; Raida, M; Schrader, M; Lebrun, I; Habermehl, G

    1998-04-01

    In the course of systematic investigations on low-molecular-weight compounds from the venom of Crotalidae and Viperidae, we have isolated and characterized at least three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom by gel filtration on Sephadex G-25 M, Sephadex G-10 followed by HPLC. The peptides showed bradykinin-potentiating action on isolated guinea-pig ileum, for which the BPP-V was more active than of BPP-II, and BPP-III, rat arterial blood pressure, and a relevant angiotensin-converting enzyme (ACE) competitive inhibiting activity. The kinetic studies showed a Ki of the order of 9.7 x 10(-3) microM to BPP-II, 7 x 10(-3) microM to BPP-III, and 3.3 x 10(-3) microM to BPP-V. The amino acid sequence of the BPP-III has been determined to be pGlu-Gly-Gly-Trp-Pro-Arg-Pro-Gly-Pro-Glu-Ile-Pro-Pro, and the amino acid compositions of the BPP-II and BPP-V by amino acid analysis were 2Glu-2Gly-1Arg-4Pro-1Ile and 2Glu-2Gly-1Ser-3Pro-2Val-1Ile, with molecular weight of 1372, 1046, and 1078, respectively.

  13. Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2.

    PubMed

    Carland, Jane E; Thomas, Michael; Mostyn, Shannon N; Subramanian, Nandhitha; O'Mara, Megan L; Ryan, Renae M; Vandenberg, Robert J

    2018-03-21

    Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuT Aa , and more recently the Drosophila dopamine transporter and the human serotonin transporter. However, there is disputed evidence that the bacterial leucine transporter, LeuT Aa , contains two substrate binding sites that work cooperatively in the mechanism of transport, with the binding of a second substrate being required for the release of the substrate from the primary site. An alternate proposal is that there may be low affinity binding sites that serve to direct the flow of substrates to the primary site. We have used a combination of molecular dynamics simulations of substrate interactions with a homology model of GlyT2, together with radiolabeled amino acid uptake assays and electrophysiological analysis of wild-type and mutant transporters, to provide evidence that substrate selectivity of GlyT2 is determined entirely by the primary substrate binding site and, furthermore, if a secondary site exists then it is a low affinity nonselective amino acid binding site.

  14. Impaired coactivator activity of the Gly{sub 482} variant of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) on mitochondrial transcription factor A (Tfam) promoter

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Choi, Yon-Sik; Hong, Jung-Man; Lim, Sunny

    2006-06-09

    Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-{gamma} (PPAR-{gamma}) coactivator-1 {alpha} (PGC-1{alpha}) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1{alpha} coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1{alpha} affected the Tfam promoter activity. The cDNA of PGC-1{alpha} variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1{alpha} protein bearing glycine had impaired coactivatormore » activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1{alpha} genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1{alpha} genotypes in Koreans. These results suggest that PGC-1{alpha} variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication.« less

  15. An emerging, recognizable facial phenotype in association with mutations in GLI-similar 3 (GLIS3).

    PubMed

    Dimitri, Paul; De Franco, Elisa; Habeb, Abdelhadi M; Gurbuz, Fatih; Moussa, Khairya; Taha, Doris; Wales, Jerry K H; Hogue, Jacob; Slavotinek, Anne; Shetty, Ambika; Balasubramanian, Meena

    2016-07-01

    Neonatal diabetes and hypothyroidism (NDH) syndrome was first described in 2003 in a consanguineous Saudi Arabian family where two out of four siblings were reported to have presented with proportionate IUGR, neonatal non-autoimmune diabetes mellitus, severe congenital hypothyroidism, cholestasis, congenital glaucoma, and polycystic kidneys. Liver disease progressed to hepatic fibrosis. The renal disease was characterized by enlarged kidneys and multiple small cysts with deficient cortico-medullary junction differentiation and normal kidney function. There was minor facial dysmorphism (depressed nasal bridge, large anterior fontanelle, long philtrum) reported but no facial photographs were published. Mutations in the transcription factor GLI-similar 3 (GLIS3) gene in the original family and two other families were subsequently reported in 2006. All affected individuals had neonatal diabetes, congenital hypothyroidism but glaucoma and liver and kidney involvement were less consistent features. Detailed descriptions of the facial dysmorphism have not been reported previously. In this report, we describe the common facial dysmorphism consisting of bilateral low-set ears, depressed nasal bridge with overhanging columella, elongated, upslanted palpebral fissures, persistent long philtrum with a thin vermilion border of the upper lip in a cohort of seven patients with GLIS3 mutations and report the emergence of a distinct, probably recognizable facial gestalt in this group which evolves with age. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses.

    PubMed

    Preta, Giulio; de Klark, Rainier; Glas, Rickard

    2009-11-27

    Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to gamma-irradiation, and that nuclear expression of TPPII was present in most gamma-irradiated transformed cell lines. We used a panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after gamma-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following gamma-irradiation (at 1-4h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in gamma-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.

  17. Mutations in the Fusion Protein of Measles Virus That Confer Resistance to the Membrane Fusion Inhibitors Carbobenzoxy-d-Phe-l-Phe-Gly and 4-Nitro-2-Phenylacetyl Amino-Benzamide

    PubMed Central

    Ha, Michael N.; Delpeut, Sébastien; Noyce, Ryan S.; Sisson, Gary; Black, Karen M.; Lin, Liang-Tzung; Bilimoria, Darius; Plemper, Richard K.; Privé, Gilbert G.

    2017-01-01

    ABSTRACT The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein. IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with Me

  18. Mutation of Arg723Gly in beta-myosin heavy chain gene in five Chinese families with hypertrophic cardiomyopathy.

    PubMed

    Yang, Jun-hua; Zheng, Dong-dong; Dong, Ning-zheng; Yang, Xiang-jun; Song, Jian-ping; Jiang, Ting-bo; Cheng, Xu-jie; Li, Hong-xia; Zhou, Bing-yuan; Zhao, Cai-ming; Jiang, Wen-ping

    2006-11-05

    Hypertrophic cardiomyopathy (HCM) is a form of cardiomyopathy with an autosomal dominant inherited disease, which is caused by mutations in at least one of the sarcomeric protein genes. Mutations in the beta-myosin heavy chain (beta-MHC) are the most common cause of HCM. This study was to reveal the disease-causing gene mutations in Chinese population with HCM, and to analyze the correlation between the genotype and phenotype. The exons 3 to 26 of MYH7 were amplified by PCR, and the PCR products were sequenced in five non-kin HCM patients. A 17-year-old patient was detected to be an Arg723Gly mutation carrier. Then his family was gene-screened, and the correlation between genotype and phenotype was analyzed. The mutation of Arg723Gly in a Chinese family with HCM was detected for the first time. With a C-G transversion in nucleotide 13,619 of the MYH7 gene, located at the essential light chain interacting region in S1, the replacement of arginine by glycine took place at amino acid residue 723. A two-dimensional echocardiogram showed moderate asymmetrical septal hypertrophy with left atria enlargement. There was no obstruction in the left ventricular outflow tract. In his family, a total of 13 individuals were diagnosed HCM and 5 of them were dead of congestive heart failure at a mean age of 66-year-old. Eight living members were all detected to carry the mutation, in which 3 developed progressive heart failure. Moreover, the heart function of the people evidently deteriorates when their age are older than 50. The mutation and the disease show co-separated. The Arg723Gly mutation is a malignant type. In Chinese the mutation has the similar characters to the former report but has low degree malignant.

  19. Direct Assessment of the Effect of the Gly380Arg Achondroplasia Mutation on FGFR3 Dimerization Using Quantitative Imaging FRET

    PubMed Central

    Placone, Jesse; Hristova, Kalina

    2012-01-01

    The Gly380Arg mutation in FGFR3 is the genetic cause for achondroplasia (ACH), the most common form of human dwarfism. The mutation has been proposed to increase FGFR3 dimerization, but the dimerization propensities of wild-type and mutant FGFR3 have not been compared. Here we use quantitative imaging FRET to characterize the dimerization of wild-type FGFR3 and the ACH mutant in plasma membrane-derived vesicles from HEK293T cells. We demonstrate a small, but statistically significant increase in FGFR3 dimerization due to the ACH mutation. The data are consistent with the idea that the ACH mutation causes a structural change which affects both the stability and the activity of FGFR3 dimers in the absence of ligand. PMID:23056398

  20. Loss of the Arabidopsis thaliana P4-ATPase ALA3 Reduces Adaptability to Temperature Stresses and Impairs Vegetative, Pollen, and Ovule Development

    PubMed Central

    McDowell, Stephen C.; López-Marqués, Rosa L.; Poulsen, Lisbeth R.; Palmgren, Michael G.; Harper, Jeffrey F.

    2013-01-01

    Members of the P4 subfamily of P-type ATPases are thought to help create asymmetry in lipid bilayers by flipping specific lipids between the leaflets of a membrane. This asymmetry is believed to be central to the formation of vesicles in the secretory and endocytic pathways. In Arabidopsis thaliana, a P4-ATPase associated with the trans-Golgi network (ALA3) was previously reported to be important for vegetative growth and reproductive success. Here we show that multiple phenotypes for ala3 knockouts are sensitive to growth conditions. For example, ala3 rosette size was observed to be dependent upon both temperature and soil, and varied between 40% and 80% that of wild-type under different conditions. We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects. In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length. In genetic crosses under conditions of hot days and cold nights, pollen fitness was reduced by at least 90-fold; from ∼18% transmission efficiency (unstressed) to less than 0.2% (stressed). Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments. PMID:23667493

  1. Prohormone convertase 1 (PC1) when expressed with pro cholecystokinin (pro CCK) in L cells performs three endoproteolytic cleavages which are observed in rat brain and in CCK-expressing endocrine cells in culture, including the production of glycine and arginine extended CCK8.

    PubMed

    Wang, W; Birch, N P; Beinfeld, M C

    1998-07-30

    Pro CCK was expressed in an L cell line engineered to express PC1 and the products secreted into the media were characterized by a combination of RIA, gel filtration and HPLC. PC1 released from L cells, cleaved pro CCK generating the amino terminal pro peptide. PC1 also generated a peptide which after carboxypeptidase B treatment, was detected with an antiserum specific for CCK Gly. Neither of these peptides was found in media from L cells expressing pro CCK alone. This CCK Gly immunoreactive peptide was similar in size to CCK 8, and after treatment with arylsulfatase and carboxypeptidase B, it co-eluted on HPLC with unsulfated CCK 8 Gly. These results agree with previous studies which support a role for PC1 in generation of CCK 8. This is the first demonstration that PC1 acting alone is able to cleave pro CCK liberating the amino terminal pro peptide and a glycine and arginine extended CCK 8 which is the immediate precursor of CCK 8 amide.

  2. Electronic spectroscopy and photodissociation dynamics of Co(2+)-methanol clusters: Co2+ (CH3OH)n (n = 4-7).

    PubMed

    Thompson, Christopher J; Faherty, Kieron P; Stringer, Kay L; Metz, Ricardo B

    2005-03-07

    Solvated cluster ions Co2+ (CH3OH)n (n = 4-7) have been produced by electrospray and studied using photofragment spectroscopy. There are notable differences between the photodissociation spectra of these complexes and the analogous water complexes. Co2+ (CH3OH)6 absorbs significantly more strongly than Co2+ (H2O)6. The photodissociation spectra of Co2+ (CH3OH)n (n = 4, 5 and 6) are very similar, which suggests that they share the Co2+ (CH3OH)4 chromophore, with additional solvent molecules in the second shell. In contrast, our earlier studies indicate that Co2+ (H2O)6 is six coordinate, and its spectrum is significantly different from that of Co2+ (H2O)4. The larger clusters Co2+ (CH3OH)n (n = 5-7) dissociate by simple loss of one or more solvent molecules. Larger clusters tend to lose more solvent molecules, especially at higher photon energies. As with the corresponding water cluster, Co2+ (CH3OH)4 photodissociates by proton transfer through a salt-bridge intermediate. This is accompanied by a modest kinetic energy release of 170 kJ mol(-1) and occurs with a lifetime of 145 ns.

  3. An endo-acting proline-specific oligopeptidase from Treponema denticola ATCC 35405: evidence of hydrolysis of human bioactive peptides.

    PubMed Central

    Mäkinen, P L; Mäkinen, K K; Syed, S A

    1994-01-01

    An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides. Images PMID

  4. Thermo-chemotherapy Induced miR-218 upregulation inhibits the invasion of gastric cancer via targeting Gli2 and E-cadherin.

    PubMed

    Ruan, Qiang; Fang, Zhi-Yuan; Cui, Shu-Zhong; Zhang, Xiang-Liang; Wu, Yin-Bing; Tang, Hong-Sheng; Tu, Yi-Nuo; Ding, Yan

    2015-08-01

    Thermo-chemotherapy has been proven to reduce the invasion capability of cancer cells. However, the molecular mechanism underlying this anti-invasion effect is still unclear. In this study, the role of thermo-chemotherapy in the inhibition of tumor invasion was studied. The results demonstrated that expression of miR-218 was downregulated in gastric cancer tissues, which had a positive correlation with tumor invasion and metastasis. In vitro thermo-chemotherapy increased miR-218 expression in SGC7901 cells and inhibited both proliferation and invasion of cancer cells. Gli2 was identified as a downstream target of miR-218, and its expression was negatively regulated by miR-218. The thermo-chemotherapy induced miR-218 upregulation was also accompanied by increasing of E-cadherin expression. In conclusion, the present study indicates that thermo-chemotherapy can effectively decrease the invasion capability of cancer cells and increase cell-cell adhesion. miR-218 and its downstream target Gli2, as well as E-cadherin, participate in the anti-invasion process.

  5. Structure-activity relationship of linear peptide Bu-His-DPhe-Arg-Trp-Gly-NH(2) at the human melanocortin-1 and -4 receptors: arginine substitution.

    PubMed

    Cheung, Adrian Wai-Hing; Danho, Waleed; Swistok, Joseph; Qi, Lida; Kurylko, Grazyna; Franco, Lucia; Yagaloff, Keith; Chen, Li

    2002-09-02

    A series of pentapeptides, based on Bu-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-NH(2) and modified at the Arg(8) position, was prepared and pharmacologically characterized. Peptides containing either cyanoguanidine or acylguanidine, two substantially less basic arginine surrogates, were found to retain the agonist activity of the parent peptide at both hMC1R and hMC4R. This study unequivocally shows that the positive charge of Arg(8) is not essential for efficient interactions of our pentapeptide with both hMC1R and hMC4R.

  6. Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction.

    PubMed

    Hicks, Matt N; Gunasekara, Sanjiva; Serate, Jose; Park, Jin; Mosharaf, Pegah; Zhou, Yue; Lee, Jin-Won; Youn, Hwan

    2017-10-01

    The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP's recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and their in vitro DNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.

  7. Chronic Inhibition of ERK1/2 Signaling Improves Disordered Bone and Mineral Metabolism in Hypophosphatemic (Hyp) Mice

    PubMed Central

    Zhang, Martin Y. H.; Ranch, Daniel; Pereira, Renata C.; Armbrecht, Harvey J.; Portale, Anthony A.

    2012-01-01

    The X-linked hypophosphatemic (Hyp) mouse carries a loss-of-function mutation in the phex gene and is characterized by hypophosphatemia due to renal phosphate (Pi) wasting, inappropriately suppressed 1,25-dihydroxyvitamin D [1,25(OH)2D] production, and rachitic bone disease. Increased serum fibroblast growth factor-23 concentration is responsible for the disordered metabolism of Pi and 1,25(OH)2D. In the present study, we tested the hypothesis that chronic inhibition of fibroblast growth factor-23-induced activation of MAPK signaling in Hyp mice can reverse their metabolic derangements and rachitic bone disease. Hyp mice were administered the MAPK inhibitor, PD0325901 orally for 4 wk. PD0325901 induced a 15-fold and 2-fold increase in renal 1α-hydroxylase mRNA and protein abundance, respectively, and thereby higher serum 1,25(OH)2D concentrations (115 ± 13 vs. 70 ± 16 pg/ml, P < 0.05), compared with values in vehicle-treated Hyp mice. With PD0325901, serum Pi levels were higher (5.1 ± 0.5 vs. 3 ± 0.2 mg/dl, P < 0.05), and the protein abundance of sodium-dependent phosphate cotransporter Npt2a, was greater than in vehicle-treated mice. The rachitic bone disease in Hyp mice is characterized by abundant unmineralized osteoid bone volume, widened epiphyses, and disorganized growth plates. In PD0325901-treated Hyp mice, mineralization of cortical and trabecular bone increased significantly, accompanied by a decrease in unmineralized osteoid volume and thickness, as determined by histomorphometric analysis. The improvement in mineralization in PD0325901-treated Hyp mice was confirmed by microcomputed tomography analysis, which showed an increase in cortical bone volume and thickness. These findings provide evidence that in Hyp mice, chronic MAPK inhibition improves disordered Pi and 1,25(OH)2D metabolism and bone mineralization. PMID:22334725

  8. Chronic inhibition of ERK1/2 signaling improves disordered bone and mineral metabolism in hypophosphatemic (Hyp) mice.

    PubMed

    Zhang, Martin Y H; Ranch, Daniel; Pereira, Renata C; Armbrecht, Harvey J; Portale, Anthony A; Perwad, Farzana

    2012-04-01

    The X-linked hypophosphatemic (Hyp) mouse carries a loss-of-function mutation in the phex gene and is characterized by hypophosphatemia due to renal phosphate (Pi) wasting, inappropriately suppressed 1,25-dihydroxyvitamin D [1,25(OH)₂D] production, and rachitic bone disease. Increased serum fibroblast growth factor-23 concentration is responsible for the disordered metabolism of Pi and 1,25(OH)₂D. In the present study, we tested the hypothesis that chronic inhibition of fibroblast growth factor-23-induced activation of MAPK signaling in Hyp mice can reverse their metabolic derangements and rachitic bone disease. Hyp mice were administered the MAPK inhibitor, PD0325901 orally for 4 wk. PD0325901 induced a 15-fold and 2-fold increase in renal 1α-hydroxylase mRNA and protein abundance, respectively, and thereby higher serum 1,25(OH)₂D concentrations (115 ± 13 vs. 70 ± 16 pg/ml, P < 0.05), compared with values in vehicle-treated Hyp mice. With PD0325901, serum Pi levels were higher (5.1 ± 0.5 vs. 3 ± 0.2 mg/dl, P < 0.05), and the protein abundance of sodium-dependent phosphate cotransporter Npt2a, was greater than in vehicle-treated mice. The rachitic bone disease in Hyp mice is characterized by abundant unmineralized osteoid bone volume, widened epiphyses, and disorganized growth plates. In PD0325901-treated Hyp mice, mineralization of cortical and trabecular bone increased significantly, accompanied by a decrease in unmineralized osteoid volume and thickness, as determined by histomorphometric analysis. The improvement in mineralization in PD0325901-treated Hyp mice was confirmed by microcomputed tomography analysis, which showed an increase in cortical bone volume and thickness. These findings provide evidence that in Hyp mice, chronic MAPK inhibition improves disordered Pi and 1,25(OH)₂D metabolism and bone mineralization.

  9. Syntheses, characterization and antioxidant activity studies of mixed-ligand copper(II) complexes of 2,2‧-bipyridine and glycine: The X-ray crystal structure of [Cu(BPy)(Gly)]ClO4

    NASA Astrophysics Data System (ADS)

    Ibrahim, Mohamed M.; Ramadan, Abd El-Motaleb M.; Shaban, Shaban Y.; Mersal, Gaber A. M.; El-Shazly, Samir A.; Al-Juaid, Salih

    2017-04-01

    A series of mixed-ligand complexes, viz., [CuLL'X]Y {L = bipyridine; L' = glycine; X = 0, Y = ClO4- (1); X = Cl, Y = 2H2O (2); X = H2O, Y = NO3- (3); X = CH3COO-, Y = H2O (4)} and {[Cu(Gly)(BPy)]2-μ-(SO4)}(5)} have been synthesized and characterized by means of elemental analysis, spectroscopic (FT-IR, UV-Vis and ESR), and thermal analysis, as well as magnetic moment measurements. Spectral and X-ray structural features led to the conclusion that complexes 2-5 have square-pyramidal environments around copper(II) center with coordination chromophores CuN3OCl and CuN3O2, respectively. Whereas complex 1 displays square planar geometry. The quasi-reversible CuII/CuI redox couple slightly improves its reversibility with considerable decrease in current intensity. Additionally, the antioxidant (superoxide dismutase and catalase) biomimetic catalytic activities of the obtained complexes have been tested and found to be promising candidates as dual functional mimic enzyme to serve for complete reactive oxygen species (ROS) detoxification, both with respect to the superoxide radicals and the related peroxides.

  10. An Arg for Gly substitution at position 31 in the insulin receptor, linked to insulin resistance, inhibits receptor processing and transport.

    PubMed

    van der Vorm, E R; van der Zon, G C; Möller, W; Krans, H M; Lindhout, D; Maassen, J A

    1992-01-05

    In a patient with Leprechaunism, we have characterized a new mutation in the insulin receptor substituting Arg for Gly at position 31. The proband, the mother, and the maternal grandfather were heterozygous for the mutation. Fibroblasts of the proband show a strongly reduced number of high affinity insulin receptors on the cell surface, whereas fibroblasts of the healthy mother and grandfather show moderately reduced insulin receptor numbers. In the other family members neither the binding defect nor the Arg31 mutation was found. The Arg31-mutant receptor was overexpressed in Chinese hamster ovary cells. In these cells the mutant alpha beta-proreceptor was not proteolytically cleaved and no transport to the cell surface took place. The proreceptor was unable to bind insulin and to undergo autophosphorylation. In addition, the proreceptor was not recognized by monoclonal antibodies directed against conformation-dependent epitopes. These findings suggest that the Gly31 to Arg31 mutant is involved in the insulin receptor dysfunction seen in the Leprechaun patient. The mutation seems to alter the conformation of the receptor in such way that the transport of the proreceptor to the Golgi compartment, where proteolytical processing occurs, is inhibited.

  11. Dye adsorbates BrPDI, BrGly, and BrAsp on anatase TiO2(001) for dye-sensitized solar cell applications

    NASA Astrophysics Data System (ADS)

    Çakır, D.; Gülseren, O.; Mete, E.; Ellialtıoǧlu, Ş.

    2009-07-01

    Using the first-principles plane-wave pseudopotential method within density functional theory, we systematically investigated the interaction of perylenediimide (PDI)-based dye compounds (BrPDI, BrGly, and BrAsp) with both unreconstructed (UR) and reconstructed (RC) anatase TiO2(001) surfaces. All dye molecules form strong chemical bonds with surface in the most favorable adsorption structures. In UR-BrGly, RC-BrGly, and RC-BrAsp cases, we have observed that highest occupied molecular orbital and lowest unoccupied molecular orbital levels of molecules appear within band gap and conduction-band region, respectively. Moreover, we have obtained a gap narrowing upon adsorption of BrPDI on the RC surface. Because of the reduction in effective band gap of surface-dye system and possibly achieving the visible-light activity, these results are valuable for photovoltaic and photocatalytic applications. We have also considered the effects of hydration of surface to the binding of BrPDI. It has been found that the binding energy drops significantly for the completely hydrated surfaces.

  12. Evaluation of a novel Arg-Gly-Asp-conjugated α-melanocyte stimulating hormone hybrid peptide for potential melanoma therapy.

    PubMed

    Yang, Jianquan; Guo, Haixun; Gallazzi, Fabio; Berwick, Marianne; Padilla, R Steven; Miao, Yubin

    2009-08-19

    The purpose of this study was to determine whether Arg-Gly-Asp (RGD)-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptide could be employed to target melanocortin-1 (MC1) receptor for potential melanoma therapy. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys(3,4,10), DPhe(7), Arg(11)]α-MSH(3-13) {(Arg(11))CCMSH} to generate RGD-Lys-(Arg(11))CCMSH hybrid peptide. The MC1 receptor binding affinity of RGD-Lys-(Arg(11))CCMSH was determined in B16/F1 melanoma cells. The internalization and efflux, melanoma targeting and pharmacokinetic properties and single photon emission computed tomography/CT (SPECT/CT) imaging of (99m)Tc-RGD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma cells and melanoma-bearing C57 mice. Clonogenic cytotoxic effect of RGD-Lys-(Arg(11))CCMSH was examined in B16/F1 melanoma cells. RGD-Lys-(Arg(11))CCMSH displayed 2.1 nM MC1 receptor binding affinity. (99m)Tc-RGD-Lys-(Arg(11))CCMSH showed rapid internalization and extended retention in B16/F1 cells. The cellular uptake of (99m)Tc-RGD-Lys-(Arg(11))CCMSH was MC1 receptor-mediated. (99m)Tc-RGD-Lys-(Arg(11))CCMSH exhibited high tumor uptake (14.83 ± 2.94% ID/g 2 h postinjection) and prolonged tumor retention (7.59 ± 2.04% ID/g 24 h postinjection) in B16/F1 melanoma-bearing mice. Nontarget organ uptakes were generally low except for the kidneys. Whole-body clearance of (99m)Tc-RGD-Lys-(Arg(11))CCMSH was rapid, with approximately 62% of the injected radioactivity cleared through the urinary system by 2 h postinjection. Flank melanoma tumors were clearly imaged by small animal SPECT/CT using (99m)Tc-RGD-Lys-(Arg(11))CCMSH as an imaging probe 2 h postinjection. Single treatment (3 h incubation) with 100 nM of RGD-Lys-(Arg(11))CCMSH significantly (p < 0.05) decreased the clonogenic survival of B16/F1 cells by 65% compared to the untreated control cells. Favorable melanoma targeting property of (99m)Tc-RGD-Lys-(Arg(11))CCMSH and remarkable cytotoxic effect of RGD

  13. Effect of short peptides on expression of signaling molecules in organotypic pineal cell culture.

    PubMed

    Khavinson, V Kh; Linkova, N S; Chalisova, N I; Dudkov, A V; Koncevaya, E A

    2011-11-01

    We demonstrated the influence of short peptides on the expression of signaling molecules in organotypic culture of the pineal gland from 3-month-old rats. Peptides Ala-Glu-Asp-Gly and Lys-Glu-Asp stimulate the expression of proliferative protein Ki-67 in pineal gland culture. These peptides as well as Glu-Asp-Arg and Lys-Glu do not affect the expression of apoptosis marker AIF. The synthesis of transcription factor CGRP by pinealocytes was stimulated only by Ala-Glu-Asp-Gly. Thus, peptide Ala-Glu-Asp-Gly tissue-specifically stimulates proliferative and secretory activities of pinealocytes, which can be used for recovery of pineal gland functions at the molecular level.

  14. A new acylamidase from Rhodococcus erythropolis TA37 can hydrolyze N-substituted amides.

    PubMed

    Lavrov, K V; Zalunin, I A; Kotlova, E K; Yanenko, A S

    2010-08-01

    A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene-bis-acrylamide), acid para-nitroanilides (4'-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n-butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7-8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant (K(m)) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.

  15. Sonic hedgehog (SHH) and glioblastoma-2 (Gli-2) expressions are associated with poor jaundice-free survival in biliary atresia.

    PubMed

    Jung, Hae Yoen; Jing, Jin; Lee, Kyoung Bun; Jang, Ja-June

    2015-03-01

    Biliary atresia (BA) causes biliary obstruction in neonates. Although the Kasai operation can successfully treat certain BA cases, many patients exhibit recurrent jaundice and secondary biliary cirrhosis requiring liver transplantation. Consequently, studies of the prognostic factors of the Kasai operation are needed. Accordingly, sonic hedgehog (SHH) pathway expression at the extrahepatic bile duct (EHBD), an important bile duct repair mechanism, will be investigated via immunohistochemistry in patients with BA to examine the association with post-Kasai operation prognosis. Fifty-seven EHBD specimens were obtained during Kasai operations from 1992 to 2009. The SHH, patched (PTCH), and glioblastoma-2 (Gli-2) immunohistochemical staining results were analyzed quantitatively. Overall, 57.9% of patients had bile flow normalization after the Kasai operation; 43.1% did not. High preoperative serum total bilirubin, direct bilirubin, and aspartate aminotransferase levels were associated with sustained jaundice post-Kasai operation, as was an age ≥65days at the time of surgery (all p<0.05). High Gli-2 and SHH expression rates were significantly associated with early post-Kasai operation jaundice relapse. Strong Gli-2 and SHH expression in the EHBD might be a poor prognostic factor in Kasai operation-treated patients with BA. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. The single nucleotide polymorphism Gly482Ser in the PGC-1α gene impairs exercise-induced slow-twitch muscle fibre transformation in humans.

    PubMed

    Steinbacher, Peter; Feichtinger, René G; Kedenko, Lyudmyla; Kedenko, Igor; Reinhardt, Sandra; Schönauer, Anna-Lena; Leitner, Isabella; Sänger, Alexandra M; Stoiber, Walter; Kofler, Barbara; Förster, Holger; Paulweber, Bernhard; Ring-Dimitriou, Susanne

    2015-01-01

    PGC-1α (peroxisome proliferator-activated receptor γ co-activator 1α) is an important regulator of mitochondrial biogenesis and a master regulator of enzymes involved in oxidative phosphorylation. Recent evidence demonstrated that the Gly482Ser single nucleotide polymorphism (SNP) in the PGC-1α gene affects insulin sensitivity, blood lipid metabolism and binding to myocyte enhancer factor 2 (MEF2). Individuals carrying this SNP were shown to have a reduced cardiorespiratory fitness and a higher risk to develop type 2 diabetes. Here, we investigated the responses of untrained men with the Gly482Ser SNP to a 10 week programme of endurance training (cycling, 3 x 60 min/week, heart rate at 70-90% VO2peak). Quantitative data from analysis of biopsies from vastus lateralis muscle revealed that the SNP group, in contrast to the control group, lacked a training-induced increase in content of slow contracting oxidative fibres. Capillary supply, mitochondrial density, mitochondrial enzyme activities and intramyocellular lipid content increased similarly in both groups. These results indicate that the impaired binding of MEF2 to PGC-1α in humans with this SNP impedes exercise-induced fast-to-slow muscle fibre transformation.

  17. The Single Nucleotide Polymorphism Gly482Ser in the PGC-1α Gene Impairs Exercise-Induced Slow-Twitch Muscle Fibre Transformation in Humans

    PubMed Central

    Steinbacher, Peter; Feichtinger, René G.; Kedenko, Lyudmyla; Kedenko, Igor; Reinhardt, Sandra; Schönauer, Anna-Lena; Leitner, Isabella; Sänger, Alexandra M.; Stoiber, Walter; Kofler, Barbara; Förster, Holger; Paulweber, Bernhard; Ring-Dimitriou, Susanne

    2015-01-01

    PGC-1α (peroxisome proliferator-activated receptor γ co-activator 1α) is an important regulator of mitochondrial biogenesis and a master regulator of enzymes involved in oxidative phosphorylation. Recent evidence demonstrated that the Gly482Ser single nucleotide polymorphism (SNP) in the PGC-1α gene affects insulin sensitivity, blood lipid metabolism and binding to myocyte enhancer factor 2 (MEF2). Individuals carrying this SNP were shown to have a reduced cardiorespiratory fitness and a higher risk to develop type 2 diabetes. Here, we investigated the responses of untrained men with the Gly482Ser SNP to a 10 week programme of endurance training (cycling, 3 x 60 min/week, heart rate at 70-90% VO2peak). Quantitative data from analysis of biopsies from vastus lateralis muscle revealed that the SNP group, in contrast to the control group, lacked a training-induced increase in content of slow contracting oxidative fibres. Capillary supply, mitochondrial density, mitochondrial enzyme activities and intramyocellular lipid content increased similarly in both groups. These results indicate that the impaired binding of MEF2 to PGC-1α in humans with this SNP impedes exercise-induced fast-to-slow muscle fibre transformation. PMID:25886402

  18. Glycine formation in CO2:CH4:NH3 ices induced by 0-70 eV electrons

    NASA Astrophysics Data System (ADS)

    Esmaili, Sasan; Bass, Andrew D.; Cloutier, Pierre; Sanche, Léon; Huels, Michael A.

    2018-04-01

    Glycine (Gly), the simplest amino-acid building-block of proteins, has been identified on icy dust grains in the interstellar medium, icy comets, and ice covered meteorites. These astrophysical ices contain simple molecules (e.g., CO2, H2O, CH4, HCN, and NH3) and are exposed to complex radiation fields, e.g., UV, γ, or X-rays, stellar/solar wind particles, or cosmic rays. While much current effort is focused on understanding the radiochemistry induced in these ices by high energy radiation, the effects of the abundant secondary low energy electrons (LEEs) it produces have been mostly assumed rather than studied. Here we present the results for the exposure of multilayer CO2:CH4:NH3 ice mixtures to 0-70 eV electrons under simulated astrophysical conditions. Mass selected temperature programmed desorption (TPD) of our electron irradiated films reveals multiple products, most notably intact glycine, which is supported by control measurements of both irradiated or un-irradiated binary mixture films, and un-irradiated CO2:CH4:NH3 ices spiked with Gly. The threshold of Gly formation by LEEs is near 9 eV, while the TPD analysis of Gly film growth allows us to determine the "quantum" yield for 70 eV electrons to be about 0.004 Gly per incident electron. Our results show that simple amino acids can be formed directly from simple molecular ingredients, none of which possess preformed C—C or C—N bonds, by the copious secondary LEEs that are generated by ionizing radiation in astrophysical ices.

  19. Glycine formation in CO2:CH4:NH3 ices induced by 0-70 eV electrons.

    PubMed

    Esmaili, Sasan; Bass, Andrew D; Cloutier, Pierre; Sanche, Léon; Huels, Michael A

    2018-04-28

    Glycine (Gly), the simplest amino-acid building-block of proteins, has been identified on icy dust grains in the interstellar medium, icy comets, and ice covered meteorites. These astrophysical ices contain simple molecules (e.g., CO 2 , H 2 O, CH 4 , HCN, and NH 3 ) and are exposed to complex radiation fields, e.g., UV, γ, or X-rays, stellar/solar wind particles, or cosmic rays. While much current effort is focused on understanding the radiochemistry induced in these ices by high energy radiation, the effects of the abundant secondary low energy electrons (LEEs) it produces have been mostly assumed rather than studied. Here we present the results for the exposure of multilayer CO 2 :CH 4 :NH 3 ice mixtures to 0-70 eV electrons under simulated astrophysical conditions. Mass selected temperature programmed desorption (TPD) of our electron irradiated films reveals multiple products, most notably intact glycine, which is supported by control measurements of both irradiated or un-irradiated binary mixture films, and un-irradiated CO 2 :CH 4 :NH 3 ices spiked with Gly. The threshold of Gly formation by LEEs is near 9 eV, while the TPD analysis of Gly film growth allows us to determine the "quantum" yield for 70 eV electrons to be about 0.004 Gly per incident electron. Our results show that simple amino acids can be formed directly from simple molecular ingredients, none of which possess preformed C-C or C-N bonds, by the copious secondary LEEs that are generated by ionizing radiation in astrophysical ices.

  20. A genome-wide association scan implicates DCHS2, RUNX2, GLI3, PAX1 and EDAR in human facial variation

    PubMed Central

    Adhikari, Kaustubh; Fuentes-Guajardo, Macarena; Quinto-Sánchez, Mirsha; Mendoza-Revilla, Javier; Camilo Chacón-Duque, Juan; Acuña-Alonzo, Victor; Jaramillo, Claudia; Arias, William; Lozano, Rodrigo Barquera; Pérez, Gastón Macín; Gómez-Valdés, Jorge; Villamil-Ramírez, Hugo; Hunemeier, Tábita; Ramallo, Virginia; Silva de Cerqueira, Caio C.; Hurtado, Malena; Villegas, Valeria; Granja, Vanessa; Gallo, Carla; Poletti, Giovanni; Schuler-Faccini, Lavinia; Salzano, Francisco M.; Bortolini, Maria- Cátira; Canizales-Quinteros, Samuel; Cheeseman, Michael; Rosique, Javier; Bedoya, Gabriel; Rothhammer, Francisco; Headon, Denis; González-José, Rolando; Balding, David; Ruiz-Linares, Andrés

    2016-01-01

    We report a genome-wide association scan for facial features in ∼6,000 Latin Americans. We evaluated 14 traits on an ordinal scale and found significant association (P values<5 × 10−8) at single-nucleotide polymorphisms (SNPs) in four genomic regions for three nose-related traits: columella inclination (4q31), nose bridge breadth (6p21) and nose wing breadth (7p13 and 20p11). In a subsample of ∼3,000 individuals we obtained quantitative traits related to 9 of the ordinal phenotypes and, also, a measure of nasion position. Quantitative analyses confirmed the ordinal-based associations, identified SNPs in 2q12 associated to chin protrusion, and replicated the reported association of nasion position with SNPs in PAX3. Strongest association in 2q12, 4q31, 6p21 and 7p13 was observed for SNPs in the EDAR, DCHS2, RUNX2 and GLI3 genes, respectively. Associated SNPs in 20p11 extend to PAX1. Consistent with the effect of EDAR on chin protrusion, we documented alterations of mandible length in mice with modified Edar funtion. PMID:27193062

  1. A genome-wide association scan implicates DCHS2, RUNX2, GLI3, PAX1 and EDAR in human facial variation.

    PubMed

    Adhikari, Kaustubh; Fuentes-Guajardo, Macarena; Quinto-Sánchez, Mirsha; Mendoza-Revilla, Javier; Camilo Chacón-Duque, Juan; Acuña-Alonzo, Victor; Jaramillo, Claudia; Arias, William; Lozano, Rodrigo Barquera; Pérez, Gastón Macín; Gómez-Valdés, Jorge; Villamil-Ramírez, Hugo; Hunemeier, Tábita; Ramallo, Virginia; Silva de Cerqueira, Caio C; Hurtado, Malena; Villegas, Valeria; Granja, Vanessa; Gallo, Carla; Poletti, Giovanni; Schuler-Faccini, Lavinia; Salzano, Francisco M; Bortolini, Maria-Cátira; Canizales-Quinteros, Samuel; Cheeseman, Michael; Rosique, Javier; Bedoya, Gabriel; Rothhammer, Francisco; Headon, Denis; González-José, Rolando; Balding, David; Ruiz-Linares, Andrés

    2016-05-19

    We report a genome-wide association scan for facial features in ∼6,000 Latin Americans. We evaluated 14 traits on an ordinal scale and found significant association (P values<5 × 10(-8)) at single-nucleotide polymorphisms (SNPs) in four genomic regions for three nose-related traits: columella inclination (4q31), nose bridge breadth (6p21) and nose wing breadth (7p13 and 20p11). In a subsample of ∼3,000 individuals we obtained quantitative traits related to 9 of the ordinal phenotypes and, also, a measure of nasion position. Quantitative analyses confirmed the ordinal-based associations, identified SNPs in 2q12 associated to chin protrusion, and replicated the reported association of nasion position with SNPs in PAX3. Strongest association in 2q12, 4q31, 6p21 and 7p13 was observed for SNPs in the EDAR, DCHS2, RUNX2 and GLI3 genes, respectively. Associated SNPs in 20p11 extend to PAX1. Consistent with the effect of EDAR on chin protrusion, we documented alterations of mandible length in mice with modified Edar funtion.

  2. Stability assessment of a new antithrombotic small peptide, Arg-Gly-Asp-Trp-Arg (RGDWR), and its derivative.

    PubMed

    Yang, Lijun; Zhang, Litao; Yan, Lihong; Zheng, Haifeng; Lu, Peifen; Chen, Junjun; Dai, Jie; Sun, Haibiao; Xu, Yong; Yang, Tao

    2017-08-01

    To assess the stabilities of Arg-Gly-Asp-Trp-Arg (RGDWR, designated as RWR), a new patented antithrombotic small peptide, and its derivative with ω-aminocaprylic acid on its N-terminus (ωRWR). RWR in rat plasma was decreased by between 32 and 48% after 4 h incubation on ice, indicating its instability in plasma. In contrast, ωRWR in plasma remained at 96-107%. Concentration changes were within 6.2% for ωRWR after storage in various conditions. ωRWR is therefore stable in rat plasma, as well as under different storage methods. Furthermore, ω-aminocaprylic acid added onto the RWR peptide did not affect its antiplatelet aggregation activity. A novel small peptide, ωRWR, has been developed with a good stability for possible antithrombotic use.

  3. Association of Pro12Ala polymorphism in peroxisome proliferator activated receptor gamma with proliferative diabetic retinopathy

    PubMed Central

    Tariq, Khadija; Malik, Saira Bano; Ali, Syeda Hafiza Benish; Maqsood, Sundas Ejaz; Azam, Aisha; Muslim, Irfan; Khan, Muhammad Shakil; Azam, Maleeha; Waheed, Nadia Khalida

    2013-01-01

    Purpose The association of non-synonymous substitution polymorphism rs1801282 (c.34C>G, p.Pro12Ala) in exon 4 of the peroxisome proliferator activated receptor gamma gene with diabetic retinopathy (DR) has been reported inconsistently. Therefore, the purpose of the present study was to understand the population-specific role of the Pro12Ala polymorphism in DR susceptibility in Pakistani subjects. Methods A total of 180 subjects with DR, 193 subjects with type 2 diabetes mellitus (T2DM) with no diabetic retinopathy, and 200 healthy normoglycemic non-retinopathic Pakistani individuals were genotyped for the rs1801282 (c.34C>G) polymorphism using polymerase chain reaction-restriction fragment length polymorphism. Results We found the individuals with T2DM carrying 12Ala were at a reduced risk of developing DR (odds ratio [OR]=0.53; 95% confidence interval [CI]=0.33–0.87). Upon stratified analysis regarding disease severity, we observed this protective effect was confined to proliferative DR (OR=0.4; 95% CI=0.2–0.8) with non-significant effects on the susceptibility of non-proliferative DR (OR=0.67; 95% CI=0.37–1.19). Conclusions We report a protective role of the 12Ala polymorphism against proliferative DR in individuals with T2DM in Pakistan. PMID:23559865

  4. Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals.

    PubMed

    Liu, Fobang; Lai, Senchao; Tong, Haijie; Lakey, Pascale S J; Shiraiwa, Manabu; Weller, Michael G; Pöschl, Ulrich; Kampf, Christopher J

    2017-03-01

    Hydroxyl radical-induced oxidation of proteins and peptides can lead to the cleavage of the peptide, leading to a release of fragments. Here, we used high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and pre-column online ortho-phthalaldehyde (OPA) derivatization-based amino acid analysis by HPLC with diode array detection and fluorescence detection to identify and quantify free amino acids released upon oxidation of proteins and peptides by hydroxyl radicals. Bovine serum albumin (BSA), ovalbumin (OVA) as model proteins, and synthetic tripeptides (comprised of varying compositions of the amino acids Gly, Ala, Ser, and Met) were used for reactions with hydroxyl radicals, which were generated by the Fenton reaction of iron ions and hydrogen peroxide. The molar yields of free glycine, aspartic acid, asparagine, and alanine per peptide or protein varied between 4 and 55%. For protein oxidation reactions, the molar yields of Gly (∼32-55% for BSA, ∼10-21% for OVA) were substantially higher than those for the other identified amino acids (∼5-12% for BSA, ∼4-6% for OVA). Upon oxidation of tripeptides with Gly in C-terminal, mid-chain, or N-terminal positions, Gly was preferentially released when it was located at the C-terminal site. Overall, we observe evidence for a site-selective formation of free amino acids in the OH radical-induced oxidation of peptides and proteins, which may be due to a reaction pathway involving nitrogen-centered radicals.

  5. Analysis of the major epitope of the alpha2 chain of bovine type I collagen in children with bovine gelatin allergy.

    PubMed

    Hori, Hisae; Hattori, Shunji; Inouye, Sakae; Kimura, Akinori; Irie, Shinkichi; Miyazawa, Hiroshi; Sakaguchi, Masahiro

    2002-10-01

    Anaphylaxis to measles, mumps, and rubella vaccines has been reported. It has been found that most of these reactions to live vaccines are caused by type I allergy with the bovine gelatin present in the vaccines as an allergen. Gelatin mainly includes denatured type I collagen, which consists of alpha1 and alpha2 chains. We previously reported that allergic reactions to gelatin are caused by the type I collagen alpha2 (alpha2[I]) chain. To aid in the development of gelatin that has little or no allergenicity in human subjects, we investigated epitopes of bovine alpha2(I) chain with use of IgE in gelatin-sensitive children. Serum samples were collected from 15 patients who had systemic allergic reactions to vaccines and high levels of specific IgE to bovine gelatin. Eleven overlapping recombinant proteins that cover bovine alpha2(I) were prepared with a bacterial expression vector. We examined IgE reactivity to these recombinant proteins by means of ELISA. Fifteen peptides covering a major reactive recombinant protein were synthesized. The IgE-reacting epitope was identified by means of IgE-ELISA inhibition with these synthetic peptides and pooled serum from the patients. We found that of the 15 patients, 13 showed IgE reactivity to a recombinant protein (no. 3) spanning the central region of the collagenous domain ((418)Gly-(662)Pro). Furthermore, all 13 patients showed IgE reactivity to the 4-kd recombinant protein (no. 3a) spanning the region from (461)Pro to (500)Glu. In IgE-ELISA inhibition we found that a minimum IgE epitope of gelatin allergen was composed of the 10-amino-acid sequence (485)Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro(494). This sequence is not observed in the human type I collagen alpha1 and alpha2 chains, nor is it found in the bovine type I collagen alpha1 chain. We found that Ile-Pro-Gly-Glu-Phe-Gly-Leu-Pro-Gly-Pro is a major IgE epitope of the alpha2 chain of bovine type I collagen in patients with gelatin allergy. The degree of anaphylaxis

  6. PPARγ2Pro12Ala Polymorphism and Human Health

    PubMed Central

    He, Weimin

    2009-01-01

    The nuclear hormone receptor peroxisome proliferator activated receptor gamma (PPARγ) is an important transcription factor regulating adipocyte differentiation, lipid and glucose homeostasis, and insulin sensitivity. Numerous genetic mutations of PPARγ have been identified and these mutations positively or negatively regulate insulin sensitivity. Among these, a relatively common polymorphism of PPARγ, Pro12Ala of PPARγ2, the isoform expressed only in adipose tissue has been shown to be associated with lower body mass index, enhanced insulin sensitivity, and resistance to the risk of type 2 diabetes in human subjects carrying this mutation. Subsequent studies in different ethnic populations, however, have revealed conflicting results, suggesting a complex interaction between the PPARγ2 Pro12Ala polymorphism and environmental factors such as the ratio of dietary unsaturated fatty acids to saturated fatty acids and/or between the PPARγ2 Pro12Ala polymorphism and genetic factors such as polymorphic mutations in other genes. In addition, this polymorphic mutation in PPARγ2 is associated with other aspects of human diseases, including cancers, polycystic ovary syndrome, Alzheimer disease and aging. This review will highlight findings from recent studies. PMID:19390629

  7. Adsorption performance and mechanism of magnetic reduced graphene oxide in glyphosate contaminated water.

    PubMed

    Li, Yajuan; Zhao, Chuanqi; Wen, Yujuan; Wang, Yuanyuan; Yang, Yuesuo

    2018-05-16

    In this study, the magnetic reduced graphene oxide (RGO/Fe 3 O 4 ), with easy separation and high adsorption performance, was prepared and used to treat glyphosate (GLY) contaminated water. GLY adsorption performance of RGO/Fe 3 O 4 was investigated, and influences of pH, adsorption time, temperature, contaminant concentration, and competing anions were analyzed. Moreover, the adsorption mechanism was discussed in the light of several characterization methods, including scanning electron microscopy (SEM), energy dispersive spectrum (EDS), Fourier-transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). The results demonstrated that the RGO/Fe 3 O 4 presented a significant GLY adsorption capacity and acid condition was beneficial for this adsorption. The pseudo-second-order kinetic model and the Langmuir model correlated satisfactorily to the experimental data, indicating that this process was controlled by chemical adsorption and monolayer adsorption. Thermodynamic studies revealed that the adsorption of glyphosate onto RGO/Fe 3 O 4 was spontaneous, endothermic, and feasible process. High temperatures were beneficial to GLY adsorption. The GLY adsorption mechanism of RGO/Fe 3 O 4 was mainly attributed to hydrogen-bond interaction, electrostatic interaction, and coordination. Therefore, the RGO/Fe 3 O 4 investigated in this research may offer an attractive adsorbent candidate for treatment of glyphosate contaminated water and warrant further study as a mechanism for glyphosate efficient removal.

  8. Cholecystokinin octapeptide analogues stable to brain proteolysis.

    PubMed

    Knight, M; Barone, P; Tamminga, C A; Steardo, L; Chase, T N

    1985-01-01

    Based on recent findings identifying the initial degradative cleavage of CCK-8 at the Met3-Gly4 bond by a metalloendopeptidase, two analogues of CCK-8 with D-Ala and D-Trp substitutions at the Gly4 position were synthesized as stable analogues. Their stability to proteolysis by brain membranes and their binding potency at central CCK receptors were quantified. Both peptides are stable to degradation by peptidases in cortical synaptic membrane preparations. The analogues are nearly equipotent to CCK-8 in their affinities for inhibition of 125I-CCK-33 binding to guinea pig cortical membranes. L-Ala and L-Trp substituted peptides were synthesized for comparison. Both these peptides are degraded by synaptic membranes and the L-Trp substituted peptide possesses a greatly reduced affinity for central CCK receptors. Therefore, the structure of CCK due to the D conformation of Gly is more capable of interacting with brain CCK receptors. Further conformational analysis will establish whether the stabilized structure is a beta-bend or a beta-turn. Since these peptides are highly potent and stable to brain proteolysis they may be useful as stable CCK analogues for in vivo application.

  9. GlyCAM1 negatively regulates monocyte entry into the optic nerve head and contributes to radiation-based protection in glaucoma.

    PubMed

    Williams, Pete A; Braine, Catherine E; Foxworth, Nicole E; Cochran, Kelly E; John, Simon W M

    2017-04-26

    We previously reported a profound long-term neuroprotection subsequent to a single radiation-therapy in the DBA/2J mouse model of glaucoma. This neuroprotection prevents entry of monocyte-like immune cells into the optic nerve head during glaucoma. Gene expression studies in radiation-treated mice implicated Glycam1 in this protection. Glycam1 encodes a proteoglycan ligand for L-selectin and is an excellent candidate to modulate immune cell entry into the eye. Here, we experimentally test the hypothesis that radiation-induced over-expression of Glycam1 is a key component of the neuroprotection. We generated a null allele of Glycam1 on a DBA/2J background. Gene and protein expression of Glycam1, monocyte entry into the optic nerve head, retinal ganglion cell death, and axon loss in the optic nerve were assessed. Radiation therapy potently inhibits monocyte entry into the optic nerve head and prevents retinal ganglion cell death and axon loss. DBA/2J mice carrying a null allele of Glycam1 show increased monocyte entry and increased retinal ganglion cell death and axon loss following radiation therapy, but the majority of optic nerves were still protected by radiation therapy. Although GlyCAM1 is an L-selectin ligand, its roles in immunity are not yet fully defined. The current study demonstrates a partial role for GlyCAM1 in radiation-mediated protection. Furthermore, our results clearly show that GlyCAM1 levels modulate immune cell entry from the vasculature into neural tissues. As Glycam1 deficiency has a more profound effect on cell entry than on neurodegeneration, further experiments are needed to precisely define the role of monocyte entry in DBA/2J glaucoma. Nevertheless, GlyCAM1's function as a negative regulator of extravasation may lead to novel therapeutic strategies for an array of common conditions involving inflammation.

  10. Draft genome sequence of Raoultella terrigena R1Gly, a diazotrophic endophyte

    DOE PAGES

    Schicklberger, M.; Shapiro, N.; Loqué, D.; ...

    2015-06-11

    Raoultella terrigena R1Gly is a diazotrophic endophyte isolated from surface-sterilized roots of Nicotiana tabacum. The whole-genome sequence was obtained to investigate the endophytic characteristics of this organism at the genetic level, as well as to compare this strain with its close relatives. To our knowledge, this is the first genome obtained from the Raoultella terrigena species and only the third genome from the Raoultella genus, after Raoultella ornitholytic and Raoultella planticola. This genome will provide a foundation for further comparative genomic, metagenomic, and functional studies of this genus.

  11. DehydroalanylGly, a new post translational modification resulting from the breakdown of glutathione.

    PubMed

    Friedrich, Michael G; Wang, Zhen; Schey, Kevin L; Truscott, Roger J W

    2018-04-01

    The human body contains numerous long-lived proteins which deteriorate with age, typically by racemisation, deamidation, crosslinking and truncation. Previously we elucidated one reaction responsible for age-related crosslinking, the spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine and cysteine. This resulted in non-disulphide covalent crosslinks. The current paper outlines a novel posttranslational modification (PTM) in human proteins, which involves the addition of dehydroalanylglycine (DHAGly) to Lys residues. Human lens digests were examined by mass spectrometry for the presence of (DHA)Gly (+144.0535 Da) adducts to Lys residues. Peptide model studies were undertaken to elucidate the mechanism of formation. In the lens, this PTM was detected at 18 lysine sites in 7 proteins. Using model peptides, a pathway for its formation was found to involve initial formation of the glutathione degradation product, γ-Glu(DHA)Gly from oxidised glutathione (GSSG). Once the Lys adduct formed, the Glu residue was lost in a hydrolytic mechanism apparently catalysed by the ε-amino group of the Lys. This discovery suggests that within cells, the functional groups of amino acids in proteins may be susceptible to modification by reactive metabolites derived from GSSG. Our finding demonstrates a novel +144.0535 Da PTM arising from the breakdown of oxidised glutathione. Copyright © 2018. Published by Elsevier B.V.

  12. The frequent mutation Gly/Asp in CDR1H may determine a cross-reactive idiotope in anti-I cold agglutinins

    PubMed Central

    ABATANGELO, C; PLOTKIN, L; MATHOV, I; SQUIQUERA, L; LEONI, J

    1996-01-01

    Variable domains (VH) of all known anti i/I cold agglutinin (CA) heavy chains are codified by the VH4–21 gene. While anti-i CAs are the expression of gene rearrangement without mutations represented by amino acid changes, anti-I CAs present, among others, a frequent somatic mutation of Gly by Asp at position 31. The hydropathy profile calculated for the CDR1H (position 30 to position 35), as well as some adjacent positions of the heavy chain belonging to anti-i and anti-I antibodies, showed the conformational changes accompanying the replacement of Gly by Asp. A MoAb (LP91), which had been obtained in BALB/c mice immunized with a Fabμ fragment from a monoclonal IgMκIIIb anti-I CA (protein KAU), proved capable of inhibiting human adult erythrocyte cryoagglutination by anti-I CAs but not that of fetal erythrocytes by anti-i CAs. Western blot analysis disclosed that such MoAbs recognized a sequential epitope located in the Fd fragment of all anti-I CAs employed in this study. With the purpose of checking whether Asp31 was involved in the epitope recognized by the MoAb, two peptides, D and G, were synthesized which mimicked the CDR1H structure of anti-I and anti-i, respectively; the MoAb only reacted with peptide D by ELISA. Subsequent experimental results indicate that the Gly/Asp mutation could be associated with the diverse specificity presented by these autoantibodies, a change determining a characteristic epitope/idiotope, recognized by LP91 in the CDR1H. PMID:8603526

  13. [Gly482Ser polymorphism of the coactivator-1alpha of the activated receptor of peroxisome gamma proliferation in individuals from Maracaibo, Venezuela].

    PubMed

    Zambrano, Mariana; Fernández, Erika; López, Maciel; Rangel, Annell; de Romero, Pilar; Fernández, Virginia; Marina Morales, Luz; Molero-Conejo, Emperatriz; Connell, Lissett; Raleigh, Xiomara; Aranguren-Mendez, José

    2009-09-01

    The aim of this study was to determine the association of the Gly482Ser polymorphism of the PGC-1 gene with insulin resistance and type 2 diabetes mellitus in subjects from the city of Maracaibo. The study was performed on 64 no-diabetic subjects (36 without insulin resistance and 28 with insulin resistance) and 13 with type 2 diabetes. A clinical and nutritional history was carried out and the evaluation of anthropometric parameters was included. Fasting serum glucose, fasting serum insulin, total cholesterol, HDL-C and LDL-C, were measured. The Gly482Ser polymorphism was detected by PCR-RFLP. It was found that the allelic frequencies for A and G were 0.36 and 0.64, respectively. The population was found in genetic equilibrium of Hardy Weinberg. The genotypes of the polymorphism Gly482Ser were not associated with insulin resistance and type 2 diabetes mellitus (OR = 1.320, p = 0.74; OR = 2, p = 0.47 respectively). Nevertheless, the diabetic subjects with the genotype AA presented values of LDL-C higher (p < 0.05) than the individuals with the genotypes GG and GA. The diabetics with the genotype GA showed significantly higher concentrations of triglycerides (>150 mg/dL) compared with the genotype GG. According to the results, the polymorphism Gly482Ser of the PGC-1 gene would be able to contribute to the cardiovascular risk in type 2 diabetics, while in the insulin resistant individuals, this polymorphism was not associated with cardiovascular risk factors.

  14. Prevalence of the Pro12Ala missense mutation in the PPARG2 gene in Kuwaiti patients with primary knee osteoarthritis

    PubMed Central

    Al-Jarallah, Khaled F.; Shehab, Diaa K.; Haider, Mohammad Z.

    2011-01-01

    BACKGROUND AND OBJECTIVES: Peroxisome proliferator–activated receptors (PPARs) play an important role in a number of cellular and metabolic functions. This study was carried out to determine the prevalence of a missense mutation (Pro12Ala) in the PPARG2 gene in Kuwaiti Arab patients with primary knee osteoarthritis (OA) and healthy controls with the aim of identifying a possible association. DESIGN AND SETTING: A prospective cross-sectional study carried out at three major teaching hospitals (referral centers) in the country over a one-year period. PATIENTS AND METHODS: The prevalence of PPARG2 gene Pro12Ala missense mutation was determined in 104 Kuwaiti Arab patients with primary knee OA and 111 ethnically matched healthy controls. The prevalence of this Pro12Ala missense mutation was also determined in clinical subgroups of OA patients divided on the basis of age at onset, function and radiologic grading. RESULTS The Pro-Pro genotype of the PPARG2 gene Pro12Ala missense mutation was detected in 95/104 (91.3%) cases compared to 111/111 (100%) in the control subjects. The heterozygous Pro-Ala genotype was detected in 9/104 (8.7%) of the OA patients, while it was not detected in any of the controls. The Ala-Ala genotype was not detected in any of the OA patients or the controls. No significant differences were detected in the PPARG2 gene Pro12Ala genotypes in the subgroups of patients classified on the basis of age at onset, functional assessment using Lequesne’s functional index, and radiological grading using Kellgren-Lawrence (K-L) grading. CONCLUSIONS This study found no significant association between the PPARG2 gene Pro12Ala missense mutation and knee OA. However, the presence of the Pro-Pro genotype of the PPARG2 gene mutation has a protective effect against development of OA. PMID:21245597

  15. Evolution subverting essentiality: Dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus

    PubMed Central

    Martínez, Miguel A.; Verdaguer, Nuria; Mateu, Mauricio G.; Domingo, Esteban

    1997-01-01

    Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet—which is also a widespread motif involved in cell-to-cell adhesion—can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature. PMID:9192645

  16. Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum

    PubMed Central

    Vila-Aiub, Martin M.; Yu, Qin; Han, Heping; Powles, Stephen B.

    2015-01-01

    The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed. PMID:26019257

  17. A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Preta, Giulio; Klark, Rainier de; Glas, Rickard, E-mail: rickard.glas@ki.se

    2009-11-27

    Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to {gamma}-irradiation, and that nuclear expression of TPPII was present in most {gamma}-irradiated transformed cell lines. We used amore » panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after {gamma}-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following {gamma}-irradiation (at 1-4 h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in {gamma}-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.« less

  18. A novel prosthetic group for site-selective labeling of peptides for positron emission tomography.

    PubMed

    Olberg, Dag Erlend; Hjelstuen, Ole Kristian; Solbakken, Magne; Arukwe, Joseph; Karlsen, Hege; Cuthbertson, Alan

    2008-06-01

    Efficient methodologies for the radiolabeling of peptides with [(18)F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [(18)F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[(18)F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-Gly-Phe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 degrees C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 degrees C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated (18)F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.

  19. The delta-selective opioid peptide dermenkephalin and the mu-selective hybrid peptide dermenkephalin-[1-4]-dermophin-[5-7] display strikingly different conformations despite identical tetrapeptide N-termini. A quantitative 2-D NMR and molecular modeling analysis.

    PubMed

    Riand, J; Baron, D; Nicolas, P; Benajiba, A; Teng, Y; Naim, M

    1999-12-01

    The selective recognition of the aminoterminal binding pharmacophore Tyr-D-Xaa-Phe of the opioid heptapeptide dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM)1, and of dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), by the mu-opioid receptor and delta-opioid receptor, respectively, depends upon the constitution / conformation of the C-terminal tripeptide. The hybrid peptide DREK-[1-4]-DRM-[5-7] is very potent at, and exquisitely selective for the mu-opioid receptor, and differs only from dermenkephalin by its C-terminal tripeptide. Comparison of the structural features of DREK-[1-4]-DRM-[5-7] and dermenkephalin by nmr analysis and molecular modeling revealed striking differences, as well in the trans (Tyr5 - Pro6) isomer (population 75%) than in the cis isomer.. Whereas the folded C-terminal tail of dermenkephalin influenced the tertiary structure of the N-terminal tetrapeptide and placed the Tyr1 and Phe3 aromatic rings in definite orientations that are best suited for the delta-receptor, there were only weak contacts, as shown by NOE data, between the aminoterminal and carboxyterminal parts of the hybrid peptide. This promoted increased flexibility of the whole backbone and relaxed orientations for the side-chains of Tyr1 and Phe3 that are compatible with the mu-receptor but unsuitable for the delta-receptor. The steric hindrance introduced by Pro6 in DREK-[1-4]-DRM-[5-7], plus the absence of large hydrophobic side-chains in positions 5 and 6 may prevent close contacts between the N-terminal and C-terminal domains and reorientation of the main pharmacophoric elements Tyr1 and Phe3.

  20. Enhanced activity of human serotonin transporter variants associated with autism.

    PubMed

    Prasad, Harish C; Steiner, Jennifer A; Sutcliffe, James S; Blakely, Randy D

    2009-01-27

    Rare, functional, non-synonymous variants in the human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT) gene (SLC6A4) have been identified in both autism and obsessive-compulsive disorder (OCD). Within autism, rare hSERT coding variants associate with rigid-compulsive traits, suggesting both phenotypic overlap with OCD and a shared relationship with disrupted 5-HT signalling. Here, we document functional perturbations of three of these variants: Ile425Leu; Phe465Leu; and Leu550Val. In transiently transfected HeLa cells, the three variants confer a gain of 5-HT transport phenotype. Specifically, enhanced SERT activity was also observed in lymphoblastoid lines derived from mutation carriers. In contrast to previously characterized Gly56Ala, where increased transport activity derives from catalytic activation, the three novel variants exhibit elevated surface density as revealed through both surface antagonist-binding and biotinylation studies. Unlike Gly56Ala, mutants Ile425Leu, Phe465Leu and Leu550Val retain a capacity for acute PKG and p38 MAPK regulation. However, both Gly56Ala and Ile425Leu demonstrate markedly reduced sensitivity to PP2A antagonists, suggesting that deficits in trafficking and catalytic modulation may derive from a common basis in perturbed phosphatase regulation. When expressed stably from the same genomic locus in CHO cells, both Gly56Ala and Ile425Leu display catalytic activation, accompanied by a striking loss of SERT protein.

  1. A Novel Missense Mutation p.Gly162Glu of the Gene MYL2 Involved in Hypertrophic Cardiomyopathy: A Pedigree Analysis of a Proband.

    PubMed

    Renaudin, Pauline; Janin, Alexandre; Millat, Gilles; Chevalier, Philippe

    2018-04-01

    Hypertrophic cardiomyopathy (HCM), a common and clinically heterogeneous disease characterized by unexplained ventricular myocardial hypertrophy, is mostly caused by mutations in sarcomeric genes. Identifying the genetic cause is important for management, therapy, and genetic counseling. A molecular diagnosis was performed on a 51-year-old woman diagnosed with HCM using a next-generation sequencing workflow based on a panel designed for sequencing the most prevalent cardiomyopathy-causing genes. Segregation analysis was performed on the woman's family. A novel myosin regulatory light chain (MYL2) missense variant, NM_000432.3:c485G>A, p.Gly162Glu, was identified and firstly considered as a putative pathogenic mutation. Among the 27 family members tested, 16 were carriers for the MYL2-p.Gly162Glu mutation, of whom 12 with the phenotype were positive. None of the 11 family members without mutation had cardiomyopathy. Genetic analysis combined with a segregation study allowed us to classify this novel MYL2 variation, p.Gly162Glu, as a novel pathogenic mutation leading to a familial form of HCM. Due to absence of fast in vitro approaches to evaluate the functional impact of missense variants on HCM-causing genes, segregation studies remain, when possible, the easiest approach to evaluate the putative pathogenicity of novel gene variants, more particularly missense ones.

  2. Improvements in glucose homeostasis in response to regular exercise are influenced by the PPARG Pro12Ala variant: results from the HERITAGE Family Study.

    PubMed

    Ruchat, S-M; Rankinen, T; Weisnagel, S J; Rice, T; Rao, D C; Bergman, R N; Bouchard, C; Pérusse, L

    2010-04-01

    Exercise training improves glucose homeostasis, but large inter-individual differences are reported, suggesting a role of genetic factors. We investigated whether variants either confirmed or newly identified as diabetes susceptibility variants through genome-wide association studies (GWAS) modulate changes in phenotypes derived from an IVGTT in response to an endurance training programme. We analysed eight polymorphisms in seven type 2 diabetes genes (CDKAL1 rs7756992; CDKN2A and CDKN2B rs10811661 and rs564398; HHEX rs7923837; IGF2BP2 rs4402960; KCNJ11 rs5215; PPARG rs1801282; and TCF7L2 rs7903146) in a maximum of 481 sedentary, non-diabetic white individuals, who participated in a 20-week endurance training programme. Associations were tested between the variants and changes in IVGTT-derived phenotypes. The only evidence of association with training response was found with PPARG rs1801282 (Pro12Ala). We observed that Ala carriers experienced greater increase in overall glucose tolerance (Deltaglucose disappearance index Ala/Ala 0.22 +/- 0.22, Pro/Ala 0.14 +/- 0.06, Pro/Pro 0.004 +/- 0.03; p = 0.0008), glucose effectiveness (Ala/Ala 0.28 +/- 0.41, Pro/Ala 0.44 +/- 0.14, Pro/Pro 0.09 +/- 0.06; p = 0.004), acute insulin response to glucose (Ala/Ala 64.21 +/- 37.73, Pro/Ala -11.92 +/- 40.30, Pro/Pro -46.30 +/- 14.70; p = 0.03) and disposition index (Ala/Ala 551.8 +/- 448.5, Pro/Ala 534.6 +/- 218.3, Pro/Pro -7.44 +/- 88.18; p = 0.003). Compared with Pro/Pro individuals, PPARG Ala carriers experienced greater improvements in glucose and insulin metabolism in response to regular endurance training. However, we did not find evidence of association between type 2 diabetes susceptibility variants recently identified through GWAS and glucose homeostasis response to exercise. Our results extend those of previous studies showing that Ala carriers appear to be more responsive to beneficial health effects of lifestyle interventions.

  3. Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope.

    PubMed

    Endow, Joshua K; Rocha, Agostinho Gomes; Baldwin, Amy J; Roston, Rebecca L; Yamaguchi, Toshio; Kamikubo, Hironari; Inoue, Kentaro

    2016-01-01

    PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

  4. Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope

    DOE PAGES

    Endow, Joshua K.; Rocha, Agostinho Gomes; Baldwin, Amy J.; ...

    2016-12-09

    PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here in this study we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins,more » the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.« less

  5. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Endow, Joshua K.; Rocha, Agostinho Gomes; Baldwin, Amy J.

    PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here in this study we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins,more » the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.« less

  6. Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum.

    PubMed

    Vila-Aiub, Martin M; Yu, Qin; Han, Heping; Powles, Stephen B

    2015-08-01

    The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  7. The C-terminal ester of membrane anchored peptide ion channels affects anion transport.

    PubMed

    Djedovic, Natasha; Ferdani, Riccardo; Harder, Egan; Pajewska, Jolanta; Pajewski, Robert; Schlesinger, Paul H; Gokel, George W

    2003-12-07

    Five heptapeptide derivatives, [CH3(CH2)17]2NCOCH2OCH2CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR, in which R = ethyl, 2-propyl, heptyl, benzyl, and cyclohexylmethyl, were found to transport chloride anion through a phospholipid bilayer to varying extents dependent on the identity of R. It was concluded that the R group is a membrane anchor for the synthetic chloride channels.

  8. Calculation of boron-isotope fractionation between B(OH) 3(aq) and B(OH)4-(aq)

    NASA Astrophysics Data System (ADS)

    Rustad, James R.; Bylaska, Eric J.; Jackson, Virgil E.; Dixon, David A.

    2010-05-01

    Density functional and correlated molecular orbital calculations (MP2) are carried out on B(OH) 3· nH 2O clusters ( n = 0, 6, 32), and B(OH)4nH 2O ( n = 0, 8, 11, 32) to estimate the equilibrium distribution of 10B and 11B isotopes between boric acid and borate in aqueous solution. For the large 32-water clusters, multiple conformations are generated from ab initio molecular dynamics simulations to account for the effect of solvent fluctuations on the isotopic fractionation. We provide an extrapolated value of the equilibrium constant α34 for the isotope exchange reaction 10B(OH) 3(aq) + 11B(OH)4- (aq) = 11B(OH) 3(aq) + 11B(OH)4- (aq) of 1.026-1.028 near the MP2 complete basis set limit with 32 explicit waters of solvation. With some exchange-correlation functionals we find potentially important contributions from a tetrahedral neutral B(OH) 3·H 2O Lewis acid-base complex. The extrapolations presented here suggest that DFT calculations give a value for 10 3ln α34 about 15% higher than the MP2 calculations.

  9. Transport and metabolism of delta sleep-inducing peptide in cultured human intestinal epithelial cell monolayers.

    PubMed

    Augustijns, P F; Borchardt, R T

    1995-12-01

    A cultured human intestinal epithelial (Caco-2) cell monolayer was used to study the transport and metabolism of delta sleep-inducing peptide [DSIP (Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu)]. DSIP is of interest because it has been reported to be capable of permeating biological barriers (e.g. blood-brain barrier), and this property has been related to its solution conformation. When applied to the apical (AP) side of Caco-2 cell monolayers, DSIP was rapidly metabolized (8.2 +/- 1.1% remaining after a 2-hr incubation), affording Trp as the major metabolite and Trp-Ala as a minor metabolite. When DSIP was added to the basolateral (BL) side of the monolayer, the same metabolites were detected, but the peptide was more stable (70.6 +/- 3.0% remaining after a 2-hr incubation). Inclusion of bestatin, an inhibitor of aminopeptidases, at concentrations up to 0.29 mM with DSIP on the AP side of the Caco-2 cell monolayer increased the stability of the peptide only slightly but dramatically altered the distribution of the metabolites (Trp-Ala became the major metabolite, and Trp became the minor metabolite). Inclusion of other aminopeptidase inhibitors (e.g. amastatin, puromycin) alone, dipeptidylpeptidase IV inhibitors (e.g. diprotin A, Gly-Pro) alone, inhibitors of proteases that require heavy metals for proper activity (e.g. EDTA, 1,10-phenanthroline) alone, or cysteine protease inhibitors (e.g. leupeptin) alone did not lead to significant stabilization of the peptide. However, inclusion of a combination of 0.29 mM bestatin and 1 mM diprotin A with DSIP on the AP side of the monolayers resulted in a substantial increase in the stability of the peptide (83.2 +/- 3.7% remaining after a 2-hr incubation). However, under these conditions, a new metabolite (Trp-Ala-Gly-Gly-Asp-Ala-Ser) was observed with a formation that could be inhibited by inclusion of 1 mM captopril, an inhibitor of peptidyl dipeptidase A. Therefore, the stability of DSIP could be further increased (95.1 +/- 1

  10. Genotype-phenotype correlation in primary hyperoxaluria type 1: the p.Gly170Arg AGXT mutation is associated with a better outcome.

    PubMed

    Harambat, Jérôme; Fargue, Sonia; Acquaviva, Cécile; Gagnadoux, Marie-France; Janssen, Françoise; Liutkus, Aurélia; Mourani, Chebl; Macher, Marie-Alice; Abramowicz, Daniel; Legendre, Christophe; Durrbach, Antoine; Tsimaratos, Michel; Nivet, Hubert; Girardin, Eric; Schott, Anne-Marie; Rolland, Marie-Odile; Cochat, Pierre

    2010-03-01

    We sought to ascertain the long-term outcome and genotype-phenotype correlations available for primary hyperoxaluria type 1 in a large retrospective cohort study. We examined the clinical history of 155 patients (129 families primarily from Western Europe, North Africa, or the Middle East) as well as the enzymatic or genetic diagnosis. The median age at first symptom was 4 years, and at diagnosis 7.7 years, at which time 43% had reached end-stage renal disease. Presentations included: (1) early nephrocalcinosis and infantile renal failure, (2) recurrent urolithiasis and progressive renal failure diagnosed during childhood, (3) late onset with occasional stone passage diagnosed in adulthood, (4) diagnosis occurring on post-transplantation recurrence, and (5) family screening. The cumulative patient survival was 95, 86, and 74% at ages 10, 30, and 50 years, respectively, with the cumulative renal survival of 81, 59, 41, and 10% at ages 10, 20, 30, and 50 years, respectively; 72 patients had undergone a total of 97 transplantations. Among the 136 patients with DNA analysis, the most common mutation was p.Gly170Arg (allelic frequency 21.5%), with a median age at end-stage renal disease of 47 years for homozygotes, 35 years for heterozygotes, and 21 years for other mutations. Our results underscore the severe prognosis of primary hyperoxaluria type 1 and the necessity for early diagnosis and treatment, as well as confirm a better prognosis of the p.Gly170Arg mutation.

  11. Role of manganese oxides in peptide synthesis: implication in chemical evolution

    NASA Astrophysics Data System (ADS)

    Bhushan, Brij; Nayak, Arunima; Kamaluddin

    2017-10-01

    During the course of chemical evolution the role of metal oxides may have been very significant in catalysing the polymerization of biomonomers. The peptide bond formation of alanine (ala) and glycine (gly) in the presence of various oxides of manganese were performed for a period of 35 days at three different temperatures 50, 90 and 120°C without applying drying/wetting cycling. The reaction was monitored every week. The products formed were characterized by high-performance liquid chromatography and electrospray ionization-mass spectrometry techniques. Trace amount of oligomers was observed at 50°C. Maximum yield of peptides was found after 35 days at 90°C. It is important to note that very high temperatures of 120°C favoured the formation of diketopiperazine derivatives. Different types of manganese oxides [manganosite (MnO), bixbyite (Mn2O3), hausmannite (Mn3O4) and pyrolusite (MnO2)] were used as catalyst. The MnO catalysed glycine to cyclic (Gly)2, (Gly)2 and (Gly)3, and alanine, to cyclic (Ala)2 and (Ala)2. Mn3O4 also produced the same products but in lesser yield, while Mn2O3 and MnO2 produced cyclic anhydride of glycine and alanine with a trace amount of dimers and trimmers. Manganese of lower oxidation state is much more efficient in propagating the reaction than higher oxidation states. The possible mechanism of these reactions and the relevance of the results for the prebiotic chemistry are discussed.

  12. Peptide-Induced Antiviral Protection by Cytotoxic T Cells

    NASA Astrophysics Data System (ADS)

    Schulz, Manfred; Zinkernagel, Rolf M.; Hengartner, Hans

    1991-02-01

    A specific antiviral cytotoxic immune response in vivo could be induced by the subcutaneous injection of the T-cell epitope of the lymphocytic choriomeningitis virus (LCMV) nucleoprotein as an unmodified free synthetic peptide (Arg-Pro-Gln-Ala-Ser-Gly-Val-Tyr-Met-Gly-Asn-Leu-Thr-Ala-Gln) emulsified in incomplete Freund's adjuvant. This immunization rendered mice into a LCMV-specific protective state as shown by the inhibition of LCMV replication in spleens of such mice. The protection level of these mice correlated with the ability to respond to the peptide challenge by CD8^+ virus-specific cytotoxic T cells. This is a direct demonstration that peptide vaccines can be antivirally protective in vivo, thus encouraging further search for appropriate mixtures of stable peptides that may be used as T-cell vaccines.

  13. Association between PPAR-γ2 Pro12Ala genotype and insulin resistance is modified by circulating lipids in Mexican children

    PubMed Central

    Stryjecki, Carolina; Peralta-Romero, Jesus; Alyass, Akram; Karam-Araujo, Roberto; Suarez, Fernando; Gomez-Zamudio, Jaime; Burguete-Garcia, Ana; Cruz, Miguel; Meyre, David

    2016-01-01

    The Pro12Ala (rs1801282) polymorphism in peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) has been convincingly associated with insulin resistance (IR) and type 2 diabetes (T2D) among Europeans, in interaction with a high-fat diet. Mexico is disproportionally affected by obesity and T2D however, whether the Pro12Ala polymorphism is associated with early metabolic complications in this population is unknown. We assessed the association of PPAR-γ2 Pro12Ala with metabolic traits in 1457 Mexican children using linear regression models. Interactions between PPAR-γ2 Pro12Ala and circulating lipids on metabolic traits were determined by adding an interaction term to regression models. We observed a high prevalence of overweight/obesity (49.2%), dyslipidemia (34.9%) and IR (11.1%). We detected nominally significant/significant interactions between lipids (total cholesterol, HDL-cholesterol, LDL-cholesterol), the PPAR-γ2 Pro12Ala genotype and waist-to-hip ratio, fasting insulin, HOMA-IR and IR (9.30 × 10−4  ≤ Pinteraction ≤ 0.04). Post-hoc subgroup analyses evidenced that the association between the PPAR-γ2 Pro12Ala genotype and fasting insulin, HOMA-IR and IR was restricted to children with total cholesterol or LDL-cholesterol values higher than the median (0.02 ≤ P ≤ 0.03). Our data support an association of the Pro12Ala polymorphism with IR in Mexican children and suggest that this relationship is modified by dyslipidemia. PMID:27075119

  14. Combination cancer chemotherapy with one compound: pluripotent bradykinin antagonists.

    PubMed

    Stewart, John M; Gera, Lajos; Chan, Daniel C; York, Eunice J; Simkeviciene, Vitalija; Bunn, Paul A; Taraseviciene-Stewart, Laimute

    2005-08-01

    Lung and prostate cancers are major health problems worldwide. Treatments with standard chemotherapy agents are relatively ineffective. Combination chemotherapy gives better treatment than a single agent because the drugs can inhibit the cancer in different pathways, but new therapeutic agents are needed for the treatment of both tumor types. Bradykinin (BK) antagonists offer advantages of combination therapy in one compound. These promising multitargeted anti-cancer compounds selectively stimulate apoptosis in cancers and also inhibit both angiogenesis and matrix metalloprotease (MMP) action in treated lung and prostate tumors in nude mice. The highly potent, metabolism-resistant bradykinin antagonist peptide dimer, B-9870 [SUIM-(DArg-Arg-Pro-Hyp-Gly-Igl-Ser-DIgl-Oic-Arg)2] (SUIM=suberimidyl; Hyp=4-hydroxyproline; Igl=alpha-(2-indanyl)glycine; Oic=octahydroindole-2-carboxylic acid) and its non-peptide mimetic, BKM-570 [2,3,4,5,6-pentafluorocinnamoyl-(o-2,6-dichlorobenzyl)-L-tyrosine-N-(4-amino-2,2,6,6-tetramethylpiperidyl)amide] are superior to the widely used but toxic chemotherapeutic drugs cisplatin and taxotere. In certain combinations, they act synergistically with standard anti-cancer drugs. Due to its structure and biological activity, BKM-570 is an attractive lead compound for derivatization and evaluation for lung and prostate cancer drugs.

  15. Reactivity of Dimeric Tetrazirconium(IV) Wells-Dawson Polyoxometalate toward Dipeptide Hydrolysis Studied by a Combined Experimental and Density Functional Theory Approach.

    PubMed

    Ly, Hong Giang T; Mihaylov, Tzvetan; Absillis, Gregory; Pierloot, Kristine; Parac-Vogt, Tatjana N

    2015-12-07

    Detailed kinetic studies on the hydrolysis of glycylglycine (Gly-Gly) in the presence of the dimeric tetrazirconium(IV)-substituted Wells-Dawson-type polyoxometalate Na14[Zr4(P2W16O59)2(μ3-O)2(OH)2(H2O)4] · 57H2O (1) were performed by a combination of (1)H, (13)C, and (31)P NMR spectroscopies. The catalyst was shown to be stable under a broad range of reaction conditions. The effect of pD on the hydrolysis of Gly-Gly showed a bell-shaped profile with the fastest hydrolysis observed at pD 7.4. The observed rate constant for the hydrolysis of Gly-Gly at pD 7.4 and 60 °C was 4.67 × 10(-7) s(-1), representing a significant acceleration as compared to the uncatalyzed reaction. (13)C NMR data were indicative for coordination of Gly-Gly to 1 via its amide oxygen and amine nitrogen atoms, resulting in a hydrolytically active complex. Importantly, the effective hydrolysis of a series of Gly-X dipeptides with different X side chain amino acids in the presence of 1 was achieved, and the observed rate constant was shown to be dependent on the volume, chemical nature, and charge of the X amino acid side chain. To give a mechanistic explanation of the observed catalytic hydrolysis of Gly-Gly, a detailed quantum-chemical study was performed. The theoretical results confirmed the nature of the experimentally suggested binding mode in the hydrolytically active complex formed between Gly-Gly and 1. To elucidate the role of 1 in the hydrolytic process, both the uncatalyzed and the polyoxometalate-catalyzed reactions were examined. In the rate-determining step of the uncatalyzed Gly-Gly hydrolysis, a carboxylic oxygen atom abstracts a proton from a solvent water molecule and the nascent OH nucleophile attacks the peptide carbon atom. Analogous general-base activity of the free carboxylic group was found to take place also in the case of polyoxometalate-catalyzed hydrolysis as the main catalytic effect originates from the -C═O···Zr(IV) binding.

  16. Transient Receptor Potential Vanilloid 4 Activation-Induced Increase in Glycine-Activated Current in Mouse Hippocampal Pyramidal Neurons.

    PubMed

    Qi, Mengwen; Wu, Chunfeng; Wang, Zhouqing; Zhou, Li; Men, Chen; Du, Yimei; Huang, Songming; Chen, Lei; Chen, Ling

    2018-01-01

    Glycine plays an important role in regulating hippocampal inhibitory/ excitatory neurotransmission through activating glycine receptors (GlyRs) and acting as a co-agonist of N-methyl-d-aspartate-type glutamate receptors. Activation of transient receptor potential vanilloid 4 (TRPV4) is reported to inhibit hippocampal A-type γ-aminobutyric acid receptor, a ligand-gated chloride ion channel. GlyRs are also ligand-gated chloride ion channels and this paper aimed to explore whether activation of TRPV4 could modulate GlyRs. Whole-cell patch clamp recording was employed to record glycine-activated current (IGly) and Western blot was conducted to assess GlyRs subunits protein expression. Application of TRPV4 agonist (GSK1016790A or 5,6-EET) increased IGly in mouse hippocampal CA1 pyramidal neurons. This action was blocked by specific antagonists of TRPV4 (RN-1734 or HC-067047) and GlyR (strychnine), indicating that activation of TRPV4 increases strychnine-sensitive GlyR function in mouse hippocampal pyramidal neurons. GSK1016790A-induced increase in IGly was significantly attenuated by protein kinase C (PKC) (BIM II or D-sphingosine) or calcium/calmodulin-dependent protein kinase II (CaMKII) (KN-62 or KN-93) antagonists but was unaffected by protein kinase A or protein tyrosine kinase antagonists. Finally, hippocampal protein levels of GlyR α1 α2, α3 and β subunits were not changed by treatment with GSK1016790A for 30 min or 1 h, but GlyR α2, α3 and β subunits protein levels increased in mice that were intracerebroventricularly (icv.) injected with GSK1016790A for 5 d. Activation of TRPV4 increases GlyR function and expression, and PKC and CaMKII signaling pathways are involved in TRPV4 activation-induced increase in IGly. This study indicates that GlyRs may be effective targets for TRPV4-induced modulation of hippocampal inhibitory neurotransmission. © 2018 The Author(s). Published by S. Karger AG, Basel.

  17. Further screening of the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vaithinathan, R.; Berson, E.L.; Dryja, T.P.

    Here the authors report 8 novel mutations and 8 previously reported mutations found from further analysis of the rhodopsin gene in a large set of additional patients with autosomal dominant retinitis pigmentosa. Leukocyte DNA was purified from 122 unrelated patients with autosomal dominant retinitis pigmentosa who were not included in previous analyses. The coding region and splice donor and acceptor sites of the rhodopsin gene were screened for mutations using single-strand conformation polymorphism analysis and direct genomic sequencing. They found 29 patients with varient bands that were due to mutations. Sequence analysis showed that 20 cases each had 1 ofmore » 9 previously published mutations: Pro23His, Thr58Arg, Gly89Asp, Pro171Leu, Glu181Lys, Pro347Leu, Phe45Leu, Arg135Trp, and Lys296Glu. In 9 other cases, they found 8 novel mutations. One was a 3-bp deletion (Cys264-del), and the rest were point mutations resulting in an altered amino acid: Gly51Arg (GGC [yields] CGC), Cys110Tyr (TCG [yields] TAC), Gly114Asp (GGC [yields] GAC), Ala164Glu (GCG [yields] GAG), Pro171Ser (CCA [yields] TCA), Val345Leu (GTG [yields] CTG), and Pro347Gln (CCG [yields] CAG). Each of these novel mutations was found in only one family except for Gly51Arg, which was found in two. In every family tested, the mutation cosegregated with the disease. However, in pedigree D865 only one affected member was available for analysis. About two-thirds of the mutations affect amino acids in transmembrane domains, yet only one-half of opsin's residues are in these regions. One-third of the mutations alter residues in the extracellular/intradiscal space, which includes only 25% of the protein.« less

  18. Anticancer drugs and the regulation of Hedgehog genes GLI1 and PTCH1, a comparative study in nonmelanoma skin cancer cell lines.

    PubMed

    Olesen, Uffe H; Bojesen, Sophie; Gehl, Julie; Haedersdal, Merete

    2017-11-01

    Nonmelanoma skin cancer is the most common cancer in humans, comprising mainly basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). BCC proliferation is highly dependent on the Hedgehog signaling pathway. We aimed to investigate a panel of anticancer drugs with known activity against skin cancer for their therapeutic potential in localized, enhanced topical treatment of SCC and BCC. Cytotoxicity profiles for vismodegib, 5-fluorouracil (5-FU), methotrexate (MTX), cisplatin, bleomycin, and vorinostat were established in terms of half maximal inhibitory concentration values in a panel of immortalized keratinocytes (HaCaT), BCC (UWBCC1 and BCC77015), and SCC (A431 and SCC25) cell lines. The impact of treatment on the regulation of Hedgehog pathway target genes (GLI1 and PTCH1), measured by real-time PCR, was compared between UWBCC1 and HaCaT. Varying cell line sensitivity profiles to the examined anticancer drugs were observed. Generally, 24-h drug exposure was sufficient to reduce cell viability. We found that 5-FU, MTX, and cisplatin significantly downregulated the expression of two genes controlled by the Hedgehog pathway (≤25-, 2.9-, and 12.5-fold, respectively, for GLI1 in UWBCC1 cells at 48 h, P<0.0001). The gene regulation showed clear concentration dependence and correlated with cytotoxicity for both 5-FU and MTX. We find a potential for the use of anticancer drugs in localized and enhanced topical treatment of nonmelanoma skin cancer. Of importance in the clinical setting, 24-h drug exposure may be sufficient for significant cytotoxicity for vismodegib, 5-FU, cisplatin, and bleomycin. MTX, 5-FU, and cisplatin may offer particular promise through combined cytotoxicity and downregulation of Hedgehog pathway genes GLI1 and PTCH1.

  19. Purification, substrate specificity, and classification of tripeptidyl peptidase II.

    PubMed

    Bålöw, R M; Tomkinson, B; Ragnarsson, U; Zetterqvist, O

    1986-02-15

    An extralysosomal tripeptide-releasing aminopeptidase was recently discovered in rat liver (Bålöw, R.-M., Ragnarsson, U., and Zetterqvist, O. (1983) J. Biol. Chem. 258, 11622-11628). In the present work this tripeptidyl peptidase is shown to occur in several rat tissues and in human erythrocytes. The erythrocyte enzyme was purified about 80,000-fold from a hemolysate while the rat liver enzyme was purified about 4,000-fold from a homogenate. Upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate under reducing conditions more than 90% of the protein was represented by a polypeptide of Mr 135,000 in both cases. In addition, the two enzymes eluted at similar positions in the various chromatographic steps, showed similar specific activity, and had a pH optimum around 7.5. A tryptic pentadecapeptide from the alpha-chain of human hemoglobin, Val-Gly-Ala-His-Ala-Gly-Glu-Tyr-Gly-Ala-Glu-Ala-Leu-Glu-Arg, i.e. residues 17-31, was found to be sequentially cleaved by the erythrocyte enzyme into five tripeptides, beginning from the NH2 terminus. Chromogenic tripeptidylamides showed various rates of hydrolysis at pH 7.5. With Ala-Ala-Phe-4-methyl-7-coumarylamide, Km was 16 microM and Vmax 13 mumol min-1 . mg-1, comparable to the standard substrate Arg-Arg-Ala-Ser(32P)-Val-Ala values (Km 13 microM and Vmax 24 mumol . min-1 . mg-1). The tripeptidyl peptidase of human erythrocytes was classified as a serine peptidase from its irreversible inhibition by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The rate of inhibition was decreased by the presence of an efficient competitive inhibitor, Val-Leu-Arg-Arg-Ala-Ser-Val-Ala (Ki 1.5 microM). [3H]Diisopropylphosphate was incorporated to the extent of 0.7-0.9 mol/mol of Mr 135,000 subunit, which confirms the high purity of the enzyme.

  20. Pro12Ala PPAR γ2 gene polymorphism in PCOS women: the role of compounds regulating satiety.

    PubMed

    Bidzińska-Speichert, Bożena; Lenarcik, Agnieszka; Tworowska-Bardzińska, Urszula; Slęzak, Ryszard; Bednarek-Tupikowska, Grażyna; Milewicz, Andrzej

    2012-03-01

    Five to ten percent of women of reproductive age suffer from polycystic ovary syndrome (PCOS). Leptin, NPY, galanin, cholecystokinin (CCK) are involved in the regulation of eating behavior. PPARγ are receptors that are probably involved in hyperandrogenism. This study was designed to assess associations between the Pro12Ala PPARγ2 gene polymorphism and satiety factors in PCOS. Fifty-four PCOS women and 51 healthy women were studied. Leptin, NPY, galanin, CCK levels, and genetic studies to detect Pro12Ala PPARγ2 gene polymorphism were assessed. The leptin levels in the PCOS women carrying Pro12Ala genotype were higher than in those with Pro12Pro and Ala12Ala. The PCOS women had higher leptin and NPY levels and lower galanin levels. Obese PCOS patients had lower CCK levels. In the PCOS women, a single Ala allele may have a protective role as far as hyperleptinemia is concerned. The PCOS women may reveal a disrupted central leptin/NPY feedback loop with some shifts in food intake.

  1. Comparative proteomic analyses reveal that FlbA down-regulates gliT expression and SOD activity in Aspergillus fumigatus.

    PubMed

    Shin, Kwang-Soo; Park, Hee-Soo; Kim, Young-Hwan; Yu, Jae-Hyuk

    2013-07-11

    FlbA is a regulator of G-protein signaling protein that plays a central role in attenuating heterotrimeric G-protein mediated vegetative growth signaling in Aspergillus. The deletion of flbA (∆flbA) in the opportunistic human pathogen Aspergillus fumigatus results in accelerated cell death and autolysis in submerged culture. To further investigate the effects of ∆flbA on intracellular protein levels we carried out 2-D proteome analyses of 2-day old submerged cultures of ∆flbA and wild type (WT) strains and observed 160 differentially expressed proteins. Via nano-LC-ESI-MS/MS analyses, we revealed the identity of 10 and 2 proteins exhibiting high and low level accumulation, respectively, in ∆flbA strain. Notably, the GliT protein is accumulated at about 1800-fold higher levels in ∆flbA than WT. Moreover, GliT is secreted at high levels from ∆flbA strain, whereas Sod1 (superoxide dismutase) is secreted at a higher level in WT. Northern blot analyses reveal that ∆flbA results in elevated accumulation of gliT mRNA. Consequently, ∆flbA strain exhibits enhanced tolerance to gliotoxin toxicity. Finally, ∆flbA strain displayed enhanced SOD activity and elevated resistance to menadione and paraquat. In summary, FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and of exogenous gliotoxin in A. fumigatus. Regulator of G protein Signaling (RGS) proteins play crucial roles in fundamental biological processes in filamentous fungi. FlbA is the first studied filamentous fungal RGS protein, yet much remains to be understood about its roles in the opportunistic human pathogen Aspergillus fumigatus. In the present study, we examined the effects of the deletion of flbA using comprehensive analyses of the intra- and extracellular proteomes of A. fumigatus wild type and the flbA deletion mutant. Via MS analyses, we identified 10 proteins exhibiting high level accumulation in the flbA deletion

  2. Primary structure of pancreatic polypeptide from four species of Perissodactyla (Przewalski's horse, zebra, rhino, tapir).

    PubMed

    Henry, J S; Lance, V A; Conlon, J M

    1991-12-01

    Pancreatic polypeptide (PP) has been purified from extracts of the pancreas of four species of odd-toed ungulates (Perissodactyla): Przewalski's horse, mountain zebra, white rhinoceros, and mountain tapir. The amino acid sequence of Przewalski's horse pancreatic polypeptide was established as Ala-Pro-Met-Glu-Pro-Val-Tyr-Pro-Gly-Asp10-Asn- Ala-Thr-Pro-Glu-Gln-Met-Ala-Gln-Tyr20-Ala-Ala-Glu-Leu-Arg-Arg-Tyr- Ile-Asn-Met30 - Leu-Thr-Arg-Pro-Arg-Tyr.NH2. Zebra PP was identical to Przewalski's horse PP, rhinoceros PP contained three substitutions relative to the horse (Ser for Ala1, Leu for Met3, and Glu for Gln16), and tapir PP contained one substitution relative to the horse (Leu for Met3). On the basis of morphological characteristics and the fossil record, the rhinocerotids are classified with the tapirids in the suborder Ceratomorpha, whereas the horse and zebra belong to a separate suborder, Hippomorpha. On the basis of structural similarity of the PP molecules, however, it would appear that the tapir is more closely related to the horse than to the rhinoceros. These observations provide a further example of the need for extreme caution when inferring taxonomic or phylogenetic relationships between species from the structures of homologous peptides.

  3. The novel endomorphin degradation blockers Tyr-Pro-DClPhe-Phe-NH (EMDB-1) and Tyr-Pro-Ala-NH (EMDB-2) prolong endomorphin-2 action in rat ileum in vitro.

    PubMed

    Fichna, Jakub; Perlikowska, Renata; Gach, Katarzyna; do-Rego, Jean-Claude; Cravezic, Aurore; Janecka, Anna; Storr, Martin A

    2010-07-01

    The endogenous opioid system is involved in the control of gastrointestinal (GI) motility. The potential use of endogenous MOR ligands, endomorphins (EMs), as therapeutics is limited because of their rapid enzymatic degradation and short duration of action. Targeting enzymatic degradation is an approach to prolong EM activity. In the present study, we characterized the effects of novel blockers of EM degradation in GI tissue preparation in vitro. The effects of actinonin, diprotin A (DIP) and the novel peptide EM degradation blockers Tyr-Pro-DClPhe-Phe-NH(2) (EMDB-1), Tyr-Pro-Ala-NH(2) (EMDB-2) and Tyr-Pro-Ala-OH (EMDB-3) on EM-2-mediated inhibition of electrically induced cholinergic twitch contractions were compared in rat ileum in vitro using an organ bath. EMDB-1 and EMDB-2 significantly prolonged the inhibitory effect of EM-2 on smooth muscle contractility in rat ileum. EMDB-2 extended the EM-2 action for up to 60 min compared to 10 min in controls and was more potent than the conventional peptidase inhibitor DIP. EMDB-1 and EMDB-2 are potent EM degradation blockers, which prolong the inhibitory effects of EM-2 on smooth muscle contractility in rat ileum. These novel compounds may be of future use when targeting the endogenous opioid system in the treatment of GI motility disorders such as diarrhea.

  4. Real-time observation of formation and relaxation dynamics of NH4 in (CH3OH)m(NH3)n clusters.

    PubMed

    Yamada, Yuji; Nishino, Yoko; Fujihara, Akimasa; Ishikawa, Haruki; Fuke, Kiyokazu

    2009-03-26

    The formation and relaxation dynamics of NH4(CH3OH)m(NH3)n clusters produced by photolysis of ammonia-methanol mixed clusters has been observed by a time-resolved pump-probe method with femtosecond pulse lasers. From the detailed analysis of the time evolutions of the protonated cluster ions, NH4(+)(CH3OH)m(NH3)n, the kinetic model has been constructed, which consists of sequential three-step reaction: ultrafast hydrogen-atom transfer producing the radical pair (NH4-NH2)*, the relaxation process of radical-pair clusters, and dissociation of the solvated NH4 clusters. The initial hydrogen transfer hardly occurs between ammonia and methanol, implying the unfavorable formation of radical pair, (CH3OH2-NH2)*. The remarkable dependence of the time constants in each step on the number and composition of solvents has been explained by the following factors: hydrogen delocalization within the clusters, the internal conversion of the excited-state radical pair, and the stabilization of NH4 by solvation. The dependence of the time profiles on the probe wavelength is attributed to the different ionization efficiency of the NH4(CH3OH)m(NH3)n clusters.

  5. HOGA1 Gene Mutations of Primary Hyperoxaluria Type 3 in Tunisian Patients.

    PubMed

    M'dimegh, Saoussen; Aquaviva-Bourdain, Cécile; Omezzine, Asma; Souche, Geneviéve; M'barek, Ibtihel; Abidi, Kamel; Gargah, Tahar; Abroug, Saoussen; Bouslama, Ali

    2017-05-01

    Primary hyperoxaluria type 3 (PH3) is due to mutations in the recently identified 4-hydroxy-2-oxoglutarate aldolase (HOGA1) gene. PH3 might be the least severe form with a milder phenotype with good preservation of kidney function in most patients. The aim of this study was to report three PH3 cases carrying mutations in HOGA1. Genetic analysis of HOGA1 was performed in patients with a high clinical suspicion of PH after sequencing of AGXT and GRHPR genes, which was negative. Also, a complete AGXT/GRHPR MLPA was performed in these patients in order to detect large deletions/insertions. Two different HOGA1 gene mutations were identified: the p.Pro190Leu in a homozygous state and the p.Gly287Val in two patients in homozygous and heterozygous carriers. The median age at onset of clinical symptoms was 3.93 years. Most of the patients had a positive family history for recurrent urolithiasis. The p.Pro190Leu mutation was reported with impaired renal function at follow-up; however, the p.Gly287Val was presented with normal renal function. All patients were presented with urolithiasis, but only one had a nephrocalcinosis. This study expanded the number of PH3 patients from 63 to 66 cases. The p.Pro190Leu and the p.Gly287Val mutations found in this study can provide a first-line investigation in Tunisian PH1 patients. © 2016 Wiley Periodicals, Inc.

  6. Characterization of two peptides isolated from the venom of social wasp Chartergellus communis (Hymenoptera: Vespidae): Influence of multiple alanine residues and C-terminal amidation on biological effects.

    PubMed

    Lopes, Kamila Soares; Campos, Gabriel Avohay Alves; Camargo, Luana Cristina; de Souza, Adolfo Carlos Barros; Ibituruna, Beatriz Vasconcelos; Magalhães, Ana Carolina Martins; da Rocha, Lucas Ferreira; Garcia, Alessa Bembom; Rodrigues, Mosar Correa; Ribeiro, Dagon Manoel; Costa, Michelle Cruz; López, Manuel Humberto Mera; Nolli, Luciana Marangni; Zamudio-Zuniga, Fernando; Possani, Lourival Domingos; Schwartz, Elisabeth Ferroni; Mortari, Márcia Renata

    2017-09-01

    Chatergellus communis is a wasp species endemic to the neotropical region and its venom constituents have never been described. In this study, two peptides from C. communis venom, denominated Communis and Communis-AAAA, were chemically and biologically characterized. In respect to the chemical characterization, the following amino acid sequences and molecular masses were identified: Communis: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-COOH (1340.9Da) Communis-AAAA: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-Ala-Ala-Ala-Ala-Val-Xle-NH 2 (1836.3Da). Furthermore, their biological effects were compared, accounting for the differences in structural characteristics between the two peptides. To this end, three biological assays were performed in order to evaluate the hyperalgesic, edematogenic and hemolytic effects of these molecules. Communis-AAAA, unlike Communis, showed a potent hemolytic activity with EC 50 =142.6μM. Moreover, the highest dose of Communis-AAAA (2nmol/animal) induced hyperalgesia in mice. On the other hand, Communis (10nmol/animal) was able to induce edema but did not present hemolytic or hyperalgesic activity. Although both peptides have similarities in linear structures, we demonstrated the distinct biological effects of Communis and Communis-AAAA. This is the first study with Chartegellus communis venom, and both Communis and Communis-AAAA are unpublished peptides. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Rational Design of Multilayer Collagen Nanosheets with Compositional and Structural Control.

    PubMed

    Jiang, Tao; Vail, Owen A; Jiang, Zhigang; Zuo, Xiaobing; Conticello, Vincent P

    2015-06-24

    Two collagen-mimetic peptides, CP(+) and CP(-), are reported in which the sequences comprise a multiblock architecture having positively charged N-terminal (Pro-Arg-Gly)3 and negatively charged C-terminal (Glu-Hyp-Gly)3 triad extensions, respectively. CP(+) rapidly self-associates into positively charged nanosheets based on a monolayer structure. In contrast, CP(-) self-assembles to form negatively charged monolayer nanosheets at a much slower rate, which can be accelerated in the presence of calcium(II) ion. A 2:1 mixture of unassociated CP(-) peptide with preformed CP(+) nanosheets generates structurally defined triple-layer nanosheets in which two CP(-) monolayers have formed on the identical surfaces of the CP(+) nanosheet template. Experimental data from electrostatic force microscopy (EFM) image analysis, zeta potential measurements, and charged nanoparticle binding assays support a negative surface charge state for the triple-layer nanosheets, which is the reverse of the positive surface charge state observed for the CP(+) monolayer nanosheets. The electrostatic complementarity between the CP(+) and CP(-) triple helical cohesive ends at the layer interfaces promotes a (CP(-)/CP(+)/CP(-)) compositional gradient along the z-direction of the nanosheet. This structurally informed approach represents an attractive strategy for the fabrication of two-dimensional nanostructures with compositional control.

  8. The Hinge Region as a Key Regulatory Element of Androgen Receptor Dimerization, DNA Binding, and Transactivation

    DTIC Science & Technology

    2005-05-01

    an impaired activity (see report of 2003). We obtained an EGFP fusion from Dr. Karen Knudsen (Ohio University, Cincinatti) in which a Gly-Ala linker ... Smad3 after its acetylation. The mutation of this lysine to glutamine or threonine (mimics acetylation), when expressed in DU145 cells promoted cell...forms. A Gly-Ala linker between the two proteins is necessary, since a direct fusion protein was largely impaired in its activity (not shown). 6. The

  9. The conformation of cyclo(-D-Pro-Ala4-) as a model for cyclic pentapeptides of the DL4 type.

    PubMed

    Heller, Markus; Sukopp, Martin; Tsomaia, Natia; John, Michael; Mierke, Dale F; Reif, Bernd; Kessler, Horst

    2006-10-25

    The conformation of the cyclic pentapeptide cyclo(-D-Pro-Ala(4)-) in solution and in the solid state was reinvestigated using modern NMR techniques. To allow unequivocal characterization of hydrogen bonds, relaxation behavior, and intramolecular distances, differently labeled isotopomers were synthesized. The NMR results, supported by extensive MD simulations, demonstrate unambiguously that the preferred conformation previously described by us, but recently questioned, is indeed correct. The validation of the conformational preferences of this cyclic peptide is important given that this system is a template for several bioactive compounds and for controlled "spatial screening" for the search of bioactive conformations.

  10. Revisiting the ALA/N (alpha-lipoic acid/low-dose naltrexone) protocol for people with metastatic and nonmetastatic pancreatic cancer: a report of 3 new cases.

    PubMed

    Berkson, Burton M; Rubin, Daniel M; Berkson, Arthur J

    2009-12-01

    The authors, in a previous article, described the long-term survival of a man with pancreatic cancer and metastases to the liver, treated with intravenous alpha-lipoic acid and oral low-dose naltrexone (ALA/N) without any adverse effects. He is alive and well 78 months after initial presentation. Three additional pancreatic cancer case studies are presented in this article. At the time of this writing, the first patient, GB, is alive and well 39 months after presenting with adenocarcinoma of the pancreas with metastases to the liver. The second patient, JK, who presented to the clinic with the same diagnosis was treated with the ALA/N protocol and after 5 months of therapy, PET scan demonstrated no evidence of disease. The third patient, RC, in addition to his pancreatic cancer with liver and retroperitoneal metastases, has a history of B-cell lymphoma and prostate adenocarcinoma. After 4 months of the ALA/N protocol his PET scan demonstrated no signs of cancer. In this article, the authors discuss the poly activity of ALA: as an agent that reduces oxidative stress, its ability to stabilize NF(k)B, its ability to stimulate pro-oxidant apoptosic activity, and its discriminative ability to discourage the proliferation of malignant cells. In addition, the ability of lowdose naltrexone to modulate an endogenous immune response is discussed. This is the second article published on the ALA/N protocol and the authors believe the protocol warrants clinical trial.

  11. Association between the Pro12Ala polymorphism of peroxisome proliferator-activated receptor gamma 2 and inflammatory bowel disease: a meta-analysis.

    PubMed

    Zhang, Zhi-Feng; Yang, Ning; Zhao, Gang; Zhu, Lei; Wang, Li-Xia

    2012-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor, has been implicated playing a role in the development of inflammatory bowel disease (IBD). However, previous studies evaluating the association between the PPARγ2 Pro12Ala polymorphism and IBD are inconsistent. We performed a meta-analysis to determine whether the PPARγ2 Pro12Ala mutation was associated with the presence of IBD. Electronic databases were searched for case-control studies evaluating the association between the Pro12Ala mutation and the presence of IBD. Effects were summarized with the methods recommended by the Cochrane Collaboration. A total of 7 studies including 1002 ulcerative colitis (UC) cases, 1090 Crohǹs disease (CD) cases and 1983 controls were involved in this meta-analysis. In the overall analysis, no significant association of this polymorphism with UC or CD was found. In the subgroup analyses in different populations, AlaAla genotype seemed to protect the European Caucasian population against the development of CD (Pro vs Ala: OR = 1.135, 95%CI = 0.951-1.354, P = 0.162, Bon = 1.000; ProPro vs ProAla: OR = 1.042, 95%CI = 0.852-1.273, P = 0.690, Bon = 1.000; ProPro vs AlaAla: OR = 2.379, 95%CI = 1.110-5.100, P = 0.026, Bon = 0.156; ProAla vs AlaAla: OR = 2.315, 95%CI = 1.064-5.037, P = 0.034, Bon = 0.204; Pro homozygotes vs Ala positives: OR = 1.094, 95%CI = 0.899-1.330, P = 0.371, Bon = 1.000; Pro positives vs Ala homozygotes: OR = 2.360, 95%CI = 1.103-5.053, P = 0.027, Bon = 0.162; heterozygotes vs all homozygotes: OR = 0.976, 95%CI = 0.799-1.192, P = 0.809, Bon = 1.000). There was no significant association of this polymorphism with UC or CD in the East Asian population and the Turkish population. AlaAla genotype may be a protective factor in the European Caucasian population against the development of CD in a recessive way.

  12. Conformational analysis of α-helical polypeptide included L-proline residue by high-resolution solid-state NMR measurement and quantum chemical calculation

    NASA Astrophysics Data System (ADS)

    Souma, Hiroyuki; Shoji, Akira; Kurosu, Hiromichi

    2008-10-01

    We challenged the problem about the stabilization mechanism of an α-helix formation for polypeptides containing L-proline (Pro) residue. We computed the optimized structure of α-helical poly( L-alanine) molecules including a Pro residue, H-(Ala) 8-Pro-(Ala) 9-OH, based on the molecular orbital calculation with density functional theory, B3LYP/6-31G(d) and the 13C and 15N chemical shift values based on the GIAO-CHF method with B3LYP/6-311G(d,p), respectively. It was found that two kinds of optimized structures, 'Bent structure' and 'Included α-helix structure', were preferred structures in H-(Ala) 8-Pro-(Ala) 9-OH. In addition, based on the precise 13C and 15N chemical shift data of the simple model, we successfully analyzed the secondary structure of well-defined synthetic polypeptide H-(Phe-Leu-Ala) 3-Phe C-Pro-Ala N-(Phe-Leu-Ala) 2-OH (FLA-11P), the secondary structure of which was proven to the 'Included α-helix structure'.

  13. Novel angiotensin I-converting enzyme inhibitory peptides isolated from Alcalase hydrolysate of mung bean protein.

    PubMed

    Li, Guan-Hong; Wan, Ju-Zhen; Le, Guo-Wei; Shi, Yong-Hui

    2006-08-01

    Mung bean protein isolates were hydrolyzed for 2 h by Alcalase. The generated hydrolysate showed angiotensin I-converting enzyme (ACE) inhibitory activity with the IC(50) value of 0.64 mg protein/ml. Three kinds of novel ACE inhibitory peptides were isolated from the hydrolysate by Sephadex G-15 and reverse-phase high performance liquid chromatography (RP-HPLC). These peptides were identified by amino acid composition analysis and matrix assisted-laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), as Lys-Asp-Tyr-Arg-Leu, Val-Thr-Pro-Ala-Leu-Arg and Lys-Leu-Pro-Ala-Gly-Thr-Leu-Phe with the IC(50) values of 26.5 microM, 82.4 microM and 13.4 microM, respectively. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

  14. Targeting and retention enhancement of quantum dots decorated with amino acids in an invertebrate model organism

    NASA Astrophysics Data System (ADS)

    Xing, Rui; Chen, Xue-Dong; Zhou, Yan-Feng; Zhang, Jue; Su, Yuan-Yuan; Qiu, Jian-Feng; Sima, Yang-Hu; Zhang, Ke-Qin; He, Yao; Xu, Shi-Qing

    2016-01-01

    The use of quantum dots (QDs) in biological imaging applications and targeted drug delivery is expected to increase. However, the efficiency of QDs in drug targeting needs to be improved. Here, we show that amino acids linked to CdTe QDs significantly increased the targeted transfer efficiency and biological safety in the invertebrate model Bombyx mori. Compared with bare QDs530, the transfer efficiency of Ala- and Gly-conjugated QDs (QDs530-Ala and QDs530-Gly) in circulatory system increased by 2.6 ± 0.3 and 1.5 ± 0.3 times, and increased by 7.8 ± 0.9 and 2.9 ± 0.2 times in target tissue silk glands, respectively, after 24 h of QDs exposure. Meanwhile, the amount of conjugated QDs decreased by (68.4 ± 4.4)% and (46.7 ± 9.1)% in the non-target tissue fat body, and the speed at which they entered non-target circulating blood cells significantly decreased. The resultant QDs530-Ala revealed a better structural integrity in tissues and a longer retention time in hemolymph than that of QDs530 after exposure via the dorsal vessel. On the other hand, QDs530-Ala significantly reduced the toxicity to hemocytes, silk gland, and fat body, and reduced the amount of reactive oxygen species (ROS) in tissues.

  15. Effects of VU0410120, a novel GlyT1 inhibitor, on measures of sociability, cognition and stereotypic behaviors in a mouse model of autism.

    PubMed

    Burket, Jessica A; Benson, Andrew D; Green, Torrian L; Rook, Jerri M; Lindsley, Craig W; Conn, P Jeffrey; Deutsch, Stephen I

    2015-08-03

    The NMDA receptor is a highly regulated glutamate-gated cationic channel receptor that has an important role in the regulation of sociability and cognition. The genetically-inbred Balb/c mouse has altered endogenous tone of NMDA receptor-mediated neurotransmission and is a model of impaired sociability, relevant to Autism Spectrum Disorders (ASDs). Because glycine is an obligatory co-agonist that works cooperatively with glutamate to promote opening of the ion channel, one prominent strategy to promote NMDA receptor-mediated neurotransmission involves inhibition of the glycine type 1 transporter (GlyT1). The current study evaluated the dose-dependent effects of VU0410120, a selective, high-affinity competitive GlyT1 inhibitor, on measures of sociability, cognition and stereotypic behaviors in Balb/c and Swiss Webster mice. The data show that doses of VU0410120 (i.e., 18 and 30mg/kg) that improve measures of sociability and spatial working memory in the Balb/c mouse strain elicit intense stereotypic behaviors in the Swiss Webster comparator strain (i.e., burrowing and jumping). Furthermore, the data suggest that selective GlyT1 inhibition improves sociability and spatial working memory at doses that do not worsen or elicit stereotypic behaviors in a social situation in the Balb/c strain. However, the elicitation of stereotypic behaviors in the Swiss Webster comparator strain at therapeutically relevant doses of VU0410120 suggest that genetic factors (i.e., mouse strain differences) influence sensitivity to GlyT1-elicited stereotypic behaviors, and emergence of intense stereotypic behaviors may be dose-limiting side effects of this interventional strategy. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Effects of Arg-Gly-Asp sequence peptide and hyperosmolarity on the permeability of interstitial matrix and fenestrated endothelium in joints.

    PubMed

    Poli, A; Mason, R M; Levick, J R

    2004-09-01

    The aims were to assess the contribution of arg-gly-asp (RGD) mediated cell integrin-matrix bonds to interstitial hydraulic resistance and to fenestrated endothelial permeability in joints. Joint fluid is generated by filtration from fenestrated capillaries and drains through a fibronectin-rich synovial intercellular matrix. The role of parenchymal cell-matrix bonding in determining tissue hydraulic resistance is unknown. The knee cavity of anesthetized rabbits was infused with saline or the competitive hexapeptide blocker GRGDTP, with or without added osmotic stress (600 mosm saline). Intra-articular pressure Pj, net trans-synovial drainage rate s, and the permeation of Evans blue-labeled albumin (EVA) from plasma into the joint cavity were measured. GRGDTP increased the hydraulic conductance of the synovial drainage pathway, ds/dPj, by 71% (p =.02, paired t test, n = 6 animals). Synovial plasma EVA clearance (control 7.1 +/- 0.8 microL h-1, mean +/- SEM, n = 15) was unaffected by GRGDTP (7.0 +/- 2.3 microL h(-1), n = 6) or hyperosmolarity (4.9 +/- 1.5 microL h(-1), n = 8) but was increased by GRGDTP and hyperosmolarity together (15.9 +/- 4.8 microL h(-1), n = 5) (p =.01, ANOVA). Changes in dPj/dt evoked by GRGDTP plus hyperosmolarity, but neither alone, demonstrated increased microvascular filtration into the joint cavity (p <.001, ANOVA), as did changes in fluid absorption from the infusion system at fixed Pj. RGD-mediated bonds between the parenchymal cells and interstitial polymers reduce the interstitial hydraulic conductance by 42%. This helps to retain the lubricating fluid inside a joint cavity. RGD-mediated bonds also support the macromolecular barrier function of fenestrated endothelium, but in vivo this is evident only in stressed endothelium (cf. in vitro).

  17. The Successive OH Binding Energies of Sc(OH)n+ for n=1-3

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Partridge, Harry; Arnold, James O. (Technical Monitor)

    1996-01-01

    The geometries of Sc(OH)n+, for n = 1-3, have been optimized using density functional theory, in conjunction with the B3LYP hybrid functional. The zero-point energies are computed at the same level of theory. The successive OH bond energies have been computed at the CCSD(T) level for ScOH+ and Sc(OH)2+. The computed result for ScOD+ is in excellent agreement with the recent experiment of Armentrout and co-workers. There is a dramatic drop for the third OH, because Sc+ has only two valence electrons and therefore the bonding changes when the third OH is added. The difference between the B3LYP and CCSD(T) OH binding energies for the first two OH groups is discussed.

  18. Longicalycinin A, a new cytotoxic cyclic peptide from Dianthus superbus var. longicalycinus (MAXIM.) WILL.

    PubMed

    Hsieh, Pei-Wen; Chang, Fang-Rong; Wu, Ching-Chung; Li, Chien-Ming; Wu, Kuen-Yuh; Chen, Su-Li; Yen, Hsin-Fu; Wu, Yang-Chang

    2005-03-01

    A new cyclic peptide, longicalycinin A (1), and six known compounds, vaccaroside A, dianoside A, dianoside G, 3-(4-hydroxy-3-methoxy-phenyl)propionic acid methyl ester, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde were isolated from the MeOH extract of Dianthus superbus var. longicalycinus. The amino acid sequences of 1 was elucidated as cyclo(Gly(1)-Phe(2)-Tyr(3)-Pro(4)-Phe(5)-) on the basis of ESI tandem mass fragmentation analysis, chemical evidence, and extensive 2D NMR methods. Furthermore, compound 1 showed cytotoxicity to Hep G2 cancer cell line.

  19. Characterization of PepB, a group B streptococcal oligopeptidase.

    PubMed Central

    Lin, B; Averett, W F; Novak, J; Chatham, W W; Hollingshead, S K; Coligan, J E; Egan, M L; Pritchard, D G

    1996-01-01

    Group B streptococci were recently reported to possess a cell-associated collagenase. Although the enzyme hydrolyzed the synthetic collagen-like substrate N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala, we found that neither the highly purified enzyme nor crude group B streptococcal cell lysate solubilized a film of reconstituted rat tail collagen, an activity regarded as obligatory for a true collagenase. We cloned and sequenced the gene for the enzyme (pepB). The deduced amino acid sequence showed 66.4% identity to the PepF oligopeptidase from Lactococcus lactis, a member of the M3 or thimet family of zinc metallopeptidases. The group B streptococcal enzyme also showed oligopeptidase activity and degraded a variety of small bioactive peptides, including bradykinin, neurotensin, and peptide fragments of substance P and adrenocorticotropin. PMID:8757883

  20. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Waksman, G.; Hamel, E.; Fournie-Zaluski, M.C.

    The neutral endopeptidase EC 3.4.24.11, also designated enkephalinase, has been visualized by in vitro autoradiography using the tritiated inhibitor (/sup 3/H)-N-((2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl)glycine, ((/sup 3/H)HACBO-Gly). Specific binding of (/sup 3/H)HACBO-Gly corresponding to 85% of the total binding to brain slices was inhibited by 1 ..mu..M thiorphan, a selective inhibitor of enkephalinase, but remained unchanged in the presence of captopril, a selective inhibitor of angiotensin-converting enzyme. Very high levels of (/sup 3/H)HACBO-Gly binding were found in the choroid plexus and the substantia nigra. High levels were present in the caudate putamen, globus pallidus, nucleus accumbens, olfactory tubercle, and in the substantia gelatinosa ofmore » the spinal cord. The distribution of enkephalinase was compared to that of ..mu.. and delta opioid receptors, selectively labeled with (/sup 3/H)Tyr-D-Ala-Gly-MePhe-glycinol and (/sup 3/H)Try-D-Thr-Gly-Phe-Leu-Thr, respectively. In the caudate putamen, (/sup 3/H)HACBO-Gly binding overlapped the clustered ..mu.. sites but appeared more closely related to the diffusely distributed delta sites. The association of enkephalinase with delta and ..mu.. opioid receptors in these areas is consistent with the observed role of the enzyme in regulating the effects of opioid peptides in striatal dopamine release and analgesia, respectively. Except for the choroid plexus and the cerebellum, the close similarity observed in numerous rat brain areas between the distribution of enkephalinase and that of ..mu.. and/ or delta opioid binding sites could account for most of the pharmacological effects elicited by enkephalinase inhibitors.« less

  1. Exendin-4 and GLP-1 decreases induced expression of ICAM-1, VCAM-1 and RAGE in human retinal pigment epithelial cells.

    PubMed

    Dorecka, Mariola; Siemianowicz, Krzysztof; Francuz, Tomasz; Garczorz, Wojciech; Chyra, Agnieszka; Klych, Agnieszka; Romaniuk, Wanda

    2013-01-01

    Advanced glycation end products (AGEs) take part in the development of diabetic retinopathy. Hyperglycemia triggers an inflammatory response in the retina. These mechanisms may lead to an enhanced expression of adhesion molecules (ICAM-1 and VCAM-1) in human retinal pigment epithelium (HRPE). Glucagon-like peptide 1 (GLP-1) functions as an incretin hormone with antidiabetogenic properties. GLP-1 also possesses vasoprotective properties. The aim of our study was to evaluate the influence of glycated albumin (GlyAlb; 100; 500 and 1000 mg/l) and pro-inflammatory cytokine, TNF-α (2.5 and 10 ng/ml), on expression of RAGE, ICAM-1 and VCAM-1 and to evaluate the influence of GLP-1 (100 nM) and its analogue, exendin-4 (10 nM), on the expression of RAGE, ICAM-1 and VCAM-1 in stimulated HRPE. TNF-α increased RAGE expression in HRPE cells. The addition of GlyAlb (500 and 1000 mg/l) resulted in a decrease of RAGE expression. Both TNF-α and GlyAlb increased the secretion of both adhesion molecules. In cells co-treated with GLP-1 or exendin-4 both incretins decreased RAGE expression in TNF-α treated cells, and in GlyAlb group. The ICAM-1 expression was lowered by exendin-4 and GLP-1 in cells stimulated by TNF-α and GlyAlb. The similar results were obtained for VCAM-1. All observed alterations were statistically significant. The obtained results indicate that both GLP-1 and exendin-4 by decreasing the expression of RAGE in HRPE can make these cells more resistant to circulating AGEs, and decreased expression of circulating VCAM-1 and ICAM-1, can be the result of anti-inflammatory properties of incretins and decreased expression of RAGE.

  2. Cu(II) Binding to the Peptide Ala-His-His, a Chimera of the Canonical Cu(II)-Binding Motifs Xxx-His and Xxx-Zzz-His.

    PubMed

    Gonzalez, Paulina; Vileno, Bertrand; Bossak, Karolina; El Khoury, Youssef; Hellwig, Petra; Bal, Wojciech; Hureau, Christelle; Faller, Peter

    2017-12-18

    Peptides and proteins with the N-terminal motifs NH 2 -Xxx-His and NH 2 -Xxx-Zzz-His form well-established Cu(II) complexes. The canonical peptides are Gly-His-Lys and Asp-Ala-His-Lys (from the wound healing factor and human serum albumin, respectively). Cu(II) is bound to NH 2 -Xxx-His via three nitrogens from the peptide and an external ligand in the equatorial plane (called 3N form here). In contrast, Cu(II) is bound to NH 2 -Xxx-Zzz-His via four nitrogens from the peptide in the equatorial plane (called 4N form here). These two motifs are not mutually exclusive, as the peptides with the sequence NH 2 -Xxx-His-His contain both of them. However, this chimera has never been fully explored. In this work, we use a multispectroscopic approach to analyze the Cu(II) binding to the chimeric peptide Ala-His-His (AHH). AHH is capable of forming the 3N- and 4N-type complexes in a pH dependent manner. The 3N form predominates at pH ∼ 4-6.5 and the 4N form at ∼ pH 6.5-10. NMR experiments showed that at pH 8.5, where Cu(II) is almost exclusively bound in the 4N form, the Cu(II)-exchange between AHH or the amidated AHH-NH 2 is fast, in comparison to the nonchimeric 4N form (AAH). Together, the results show that the chimeric AHH can access both Cu(II) coordination types, that minor changes in the second (or further) coordination sphere can impact considerably the equilibrium between the forms, and that Cu kinetic exchange is fast even when Cu-AHH is mainly in the 4N form.

  3. Potent μ-Opioid Receptor Agonists from Cyclic Peptides Tyr-c[D-Lys-Xxx-Tyr-Gly]: Synthesis, Biological, and Structural Evaluation.

    PubMed

    Li, Yangmei; Cazares, Margret; Wu, Jinhua; Houghten, Richard A; Toll, Laurence; Dooley, Colette

    2016-02-11

    To optimize the structure of a μ-opioid receptor ligand, analogs H-Tyr-c[D-Lys-Xxx-Tyr-Gly] were synthesized and their biological activity was tested. The analog containing a Phe(3) was identified as not only exhibiting binding affinity 14-fold higher than the original hit but also producing agonist activity 3-fold more potent than morphine. NMR study suggested that a trans conformation at D-Lys(2)-Xxx(3) is crucial for these cyclic peptides to maintain high affinity, selectivity, and functional activity toward the μ-opioid receptor.

  4. Correction of the mineralization defect in hyp mice treated with protease inhibitors CA074 and pepstatin

    PubMed Central

    Rowe, Peter S.N.; Matsumoto, Naoko; Jo, Oak D.; Shih, Remi N.J.; Oconnor, Jeannine; Roudier, Martine P.; Bain, Steve; Liu, Shiguang; Harrison, Jody; Yanagawa, Norimoto

    2012-01-01

    Increased expression of several osteoblastic proteases and MEPE (a bone matrix protein) occurs in X-linked hypophosphatemic rickets (hyp). This is associated with an increased release of a protease-resistant MEPE peptide (ASARM peptide), a potent inhibitor of mineralization. Cathepsin B cleaves MEPE releasing ASARM peptide and hyp osteoblast/osteocyte cells hypersecrete cathepsin D, an activator of cathepsin B. Our aims were to determine whether cathepsin inhibitors correct the mineralization defect in vivo and whether hyp-bone ASARM peptide levels are reduced after protease treatment. Normal littermates and hyp mice (n = 6) were injected intraperitoneally once a day for 4 weeks with pepstatin, CAO74 or vehicle. Animals were then sacrificed and bones plus serum removed for comprehensive analysis. All hyp mice groups (treated and untreated) remained hypophosphatemic with serum 1,25 vitamin D3 inappropriately normal. Serum PTH was significantly elevated in all hyp mice groups relative to normal mice (P = 0.0017). Untreated hyp mice had six-fold elevated levels of serum alkaline-phosphatase and two-fold elevated levels of ASARM peptides relative to normal mice (P < 0.001). In contrast, serum alkaline phosphatase and serum ASARM peptides were significantly reduced (normalized) in hyp mice treated with CA074 or pepstatin. Serum FGF23 levels remained high in all hyp animal groups (P < 0.0001). Hyp mice treated with protease inhibitors showed dramatic reductions in unmineralized osteoid (femurs) compared to control hyp mice (Goldner staining). Also, hyp animals treated with protease inhibitors showed marked and significant improvements in growth plate width (42%), osteoid thickness (40%) and cortical area (40%) (P < 0.002). The mineralization apposition rate, bone formation rate and mineralization surface were normalized by protease-treatment. High-resolution pQCT mineral histomorphometry measurements and uCT also confirmed a marked mineralization improvement. Finally

  5. Angiotensin-converting enzyme: I. New strategies for assay

    PubMed Central

    Ryan, James W.; Chung, Alfred; Ryan, Una S.

    1980-01-01

    The disposition of converting enzyme (kininase II) on the luminal surface of pulmonary endothelial cells is well established. Further, it is known that there is a net conversion of angiotensin I into angiotensin II as blood passes through the lungs. However, little is known about modulations of converting enzyme activity that may arise through, e.g., changes in the quality of inhalants, blood flow, or blood oxygenation. There are few data on the effects of lung disease. A major barrier to studies to examine for pathophysiologic modulations of converting enzyme is that of assay. The enzyme can be measured in terms of the rate of formation of angiotensin II from a known quantity of angiotensin I. However, both peptides are biologically active, and lungs contain other enzymes capable of degrading them. We have developed a series of radiolabeled, acylated tripeptides to improve our ability to examine for changes in the net converting enzyme of intact lungs. The enzyme, a dipeptidyl carboxypeptidase, is capable of removing C-terminal dipeptides from a variety of oligopeptides. We have prepared benzoyl-Gly-Gly-Gly (I), benzoyl-Pro-Phe-Arg (II), benzoyl-Gly-His-Leu (III), benzoyl-Phe-Ala-Pro (IV), and benzoyl-Phe-His-Leu (V), each containing a 3H-atom in the para position of the benzoyl moiety. Substrates I and III have been used previously in photometric assays of low sensitivity. II is the acylated C-terminal tripeptide of bradykinin, IV is an acylated tripeptide analog of BPP5a (Ala-Pro) and V is the acylated C-terminal tripeptide of angiotensin I. These substrates can be used in vitro or in vivo to measure converting enzyme. The 3H-labeled product is separable by partitioning between an organic solvent and acidified aqueous solution. The product is quantified by scintillation counting of the organic phase. The choice of substrate depends on the goals of the experiment: substrate I or III when wide variations in substrate concentrations are needed but high

  6. Haloperidol-stomach lesions attenuation by pentadecapeptide BPC 157, omeprazole, bromocriptine, but not atropine, lansoprazole, pantoprazole, ranitidine, cimetidine and misoprostol in mice.

    PubMed

    Bilic, I; Zoricic, I; Anic, T; Separovic, J; Stancic-Rokotov, D; Mikus, D; Buljat, G; Ivankovic, D; Aralica, G; Prkacin, I; Perovic, D; Mise, S; Rotkvic, I; Petek, M; Rucman, R; Seiwerth, S; Sikiric, P

    2001-03-09

    The focus was on haloperidol (central dopamine antagonist)-stomach lesion, a longly described suitable counterpart of dopamine blocker cysteamine-duodenal lesion. In this, the contribution of blockade of central/peripheral dopamine receptors and prostaglandins synthesis, along with influence of antiulcer agents was evaluated in mice. Male NMRI Hannnover mice were sacrificed 24 h after haloperidol (25 mg/kg b.w. i.p., given alone or with saline (haloperidol+saline) (i) or in combination (ii,iii)). Supporting central dopamine predominance for haloperidol stomach lesion induction, co-administration of peripheral dopamine receptor antagonist domperidone (5 mg/kg i.p.) (haloperidol+ domperidone) (ii), or prostaglandin synthesis inhibitor indomethacin (10 mg/kg s.c.) (haloperidol+ indomethacin) (iii) did not aggravate this lesion. (i) In haloperidol+saline challenged mice the lesions were inhibited by co-administration (/kg i.p.) of a gastric pentadecapeptide BPC 157, GlyGluProProProGlyLysProAlaAspAspAlaGlyLeuVal, M.W. 1419 (10 microg, 10 ng, 10 pg, but not 1 pg, 100 fg, 10 fg), bromocriptine (10 mg), omeprazole (10 mg, 100 mg, but not 1 mg). Atropine (10, 100, 200 mg), pirenzepine (10, 100, 200 mg), misoprostol (10, 100, 200 microg), pantoprazole (1, 10, 100 mg), lansoprazole (0.1, 1, 10 mg), cimetidine (10, 100, 200 mg) and ranitidine (10, 100, 200 mg) were not effective. (ii) Dopamine peripheral blockade influence: in haloperidol+domperidone mice, previously effective bromocriptine, pentadecapeptide BPC 157 (10 microg) or omeprazole (10 mg) did not attenuate stomach lesions. (iii) Prostaglandins synthesis blockade effect: in haloperidol+indomethacin mice, previously effective agents, bromocriptine or omeprazole were not active, while BPC 157 effect was only lessened.

  7. In vitro effect of short peptides on expression of interleukin-2 gene in splenocytes.

    PubMed

    Kazakova, T B; Barabanova, S V; Khavinson, V Kh; Glushikhina, M S; Parkhomenko, E P; Malinin, V V; Korneva, E A

    2002-06-01

    Synthetic peptides Vilon (Lys-Glu), Epithalon (Ala-Glu-Asp-Gly), and Cortagen (Ala-Glu-Asp-Pro) in vitro activated interleukin-2 mRNA synthesis in splenocytes from CBA mice in the absence of specific inductors. The intensity of interleukin-2 mRNA synthesis in splenocytes depended on the type, concentration, and duration of treatment with the peptides. Vilon and Epithalon were most potent, while Cortagen produced a less pronounced effect on interleukin-2 mRNA synthesis.

  8. Association between the Pro12Ala Polymorphism of Peroxisome Proliferator-Activated Receptor Gamma 2 and Inflammatory Bowel Disease: A Meta-Analysis

    PubMed Central

    Zhao, Gang; Zhu, Lei; Wang, Li-Xia

    2012-01-01

    Background Peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor, has been implicated playing a role in the development of inflammatory bowel disease (IBD). However, previous studies evaluating the association between the PPARγ2 Pro12Ala polymorphism and IBD are inconsistent. We performed a meta-analysis to determine whether the PPARγ2 Pro12Ala mutation was associated with the presence of IBD. Methods and Findings Electronic databases were searched for case-control studies evaluating the association between the Pro12Ala mutation and the presence of IBD. Effects were summarized with the methods recommended by the Cochrane Collaboration. A total of 7 studies including 1002 ulcerative colitis (UC) cases, 1090 Crohǹs disease (CD) cases and 1983 controls were involved in this meta-analysis. In the overall analysis, no significant association of this polymorphism with UC or CD was found. In the subgroup analyses in different populations, AlaAla genotype seemed to protect the European Caucasian population against the development of CD (Pro vs Ala: OR = 1.135, 95%CI = 0.951–1.354, P = 0.162, Bon = 1.000; ProPro vs ProAla: OR = 1.042, 95%CI = 0.852–1.273, P = 0.690, Bon = 1.000; ProPro vs AlaAla: OR = 2.379, 95%CI = 1.110–5.100, P = 0.026, Bon = 0.156; ProAla vs AlaAla: OR = 2.315, 95%CI = 1.064–5.037, P = 0.034, Bon = 0.204; Pro homozygotes vs Ala positives: OR = 1.094, 95%CI = 0.899–1.330, P = 0.371, Bon = 1.000; Pro positives vs Ala homozygotes: OR = 2.360, 95%CI = 1.103–5.053, P = 0.027, Bon = 0.162; heterozygotes vs all homozygotes: OR = 0.976, 95%CI = 0.799–1.192, P = 0.809, Bon = 1.000). There was no significant association of this polymorphism with UC or CD in the East Asian population and the Turkish population. Conclusion AlaAla genotype may be a protective factor in the European Caucasian population against the

  9. The directed migration of gonadal distal tip cells in Caenorhabditis elegans requires NGAT-1, a ß1,4-N-acetylgalactosaminyltransferase enzyme

    PubMed Central

    Veyhl, Joseph; Dunn, Robert J.; Johnston, Wendy L.; Bennett, Alexa; Zhang, Lijia W.; Dennis, James W.; Schachter, Harry

    2017-01-01

    Glycoproteins such as growth factor receptors and extracellular matrix have well-known functions in development and cancer progression, however, the glycans at sites of modification are often heterogeneous molecular populations which makes their functional characterization challenging. Here we provide evidence for a specific, discrete, well-defined glycan modification and regulation of a stage-specific cell migration in Caenorhabditis elegans. We show that a chain-terminating, putative null mutation in the gene encoding a predicted β1,4-N-acetylgalactosaminyltransferase, named ngat-1, causes a maternally rescued temperature sensitive (ts) defect in the second phase of the three phase migration pattern of the posterior, but not the anterior, hermaphrodite Distal Tip Cell (DTC). An amino-terminal partial deletion of ngat-1 causes a similar but lower penetrance ts phenotype. The existence of multiple ts alleles with distinctly different molecular DNA lesions, neither of which is likely to encode a ts protein, indicates that NGAT-1 normally prevents innate temperature sensitivity for phase 2 DTC pathfinding. Temperature shift analyses indicate that the ts period for the ngat-1 mutant defect ends by the beginning of post-embryonic development–nearly 3 full larval stages prior to the defective phase 2 migration affected by ngat-1 mutations. NGAT-1 homologs generate glycan-terminal GalNAc-β1-4GlcNAc, referred to as LacdiNAc modifications, on glycoproteins and glycolipids. We also found that the absence of the GnT1/Mgat1 activity [UDP-N-acetyl-D-glucosamine:α-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase 1 (encoded by C. elegans gly-12, gly-13, and gly-14 and homologous to vertebrate GnT1/Mgat1)], causes a similar spectrum of DTC phenotypes as ngat-1 mutations–primarily affecting posterior DTC phase 2 migration and preventing manifestation of the same innate ts period as ngat-1. GnT1/Mgat1 is a medial Golgi enzyme known to modify mannose residues and initiate

  10. Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo.

    PubMed

    Reinhardt, Robert; Centanin, Lázaro; Tavhelidse, Tinatini; Inoue, Daigo; Wittbrodt, Beate; Concordet, Jean-Paul; Martinez-Morales, Juan Ramón; Wittbrodt, Joachim

    2015-06-03

    Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina. © 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  11. Hb Hope [beta136(H14)Gly-->Asp (GGT-->GAT)]: interactions with Hb S [beta6(A3)Glu-->Val (GAG-->GTG)], other variant hemoglobins and thalassemia.

    PubMed

    Ingle, John; Adewoye, Adeboye; Dewan, Robert; Okoli, Michael; Rollins, Lamarr; Eung, Shawn H; Luo, Hong-Yuan; Chui, David H K; Steinberg, Martin H

    2004-01-01

    Hb Hope [beta136(H14)Gly-->Asp (GGT-->GAT)] was first described in an African-American family in 1965. Since then, it has been found in combination with several different globin gene mutations in many other families of divergent ethnic backgrounds. The basis for its relatively frequent occurrences remains unexplained. This variant hemoglobin (Hb) is mildly unstable and has reduced oxygen affinity, but is generally innocuous clinically. This variant Hb can present as a confounding factor in arriving at a correct diagnosis by either electrophoresis or high performance liquid chromatography (HPLC), particularly during the neonatal period. DNA-based diagnostics can help solve this potential problem.

  12. A novel p. Gly630Ser mutation of COL2A1 in a Chinese family with presentations of Legg-Calvé-Perthes disease or avascular necrosis of the femoral head.

    PubMed

    Li, Na; Yu, Jian; Cao, Xiang; Wu, Qiu-Yue; Li, Wei-Wei; Li, Tian-Fu; Zhang, Cui; Cui, Ying-Xia; Li, Xiao-Jun; Yin, Zhi-Min; Xia, Xin-Yi

    2014-01-01

    Mutations in the type II collagen gene are associated with certain human disorders, collectively termed type II collagenopathies. They include Legg-Calvé-Perthes disease (LCPD) and avascular necrosis of the femoral head (ANFH). These two diseases are skeletal dysplasias, inherited in an autosomal dominant fashion, characterized by groin pain, dislocation of the hip and diminished joint mobility. Coxa vara and elevation of the greater trochanter of the femur comprise the typical phenotype of LCPD, but do not occur in ANFH. Lack of synthesis of type II collagen and structural defects are responsible for the major clinical outcomes, because collagen is the essential matrix protein of all connective tissues. Type II collagen, encoded by the COL2A1 gene, contains N- and C- terminal regions that are cleaved after secretion into the extracellular matrix, and the core area is composed of a triple helical (Gly-X-Y) domain. If the Gly in this specific region is replaced by other amino acids, the structure of type II collagen will be destroyed. Forty-five members of a four-generation family were recruited and investigated. Diagnosis was made by independent orthopedic surgeons and radiologists. A mutation of the COL2A1 gene was detected. In our research, we identify a heterozygous mutation (c.1888 G>A, p. Gly630Ser) in exon 29 of COL2A1 in the Gly-X-Y domain, in a Chinese family affected by LCPD and ANFH. Our findings provide significant clues to the phenotype-genotype relationships in these syndromes and may be helpful in clinical diagnosis. Furthermore, these results should assist further studies of the mechanisms underlying collagen diseases. Our data add new variants to the repertoire of COL2A1 mutation resulting in related collagenopathies.

  13. Impact of Cyanidin-3-Glucoside on Glycated LDL-Induced NADPH Oxidase Activation, Mitochondrial Dysfunction and Cell Viability in Cultured Vascular Endothelial Cells

    PubMed Central

    Xie, Xueping; Zhao, Ruozhi; Shen, Garry X.

    2012-01-01

    Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC. PMID:23443099

  14. Impact of cyanidin-3-glucoside on glycated LDL-induced NADPH oxidase activation, mitochondrial dysfunction and cell viability in cultured vascular endothelial cells.

    PubMed

    Xie, Xueping; Zhao, Ruozhi; Shen, Garry X

    2012-11-27

    Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC.

  15. Antibacterial activity of Pd(II) complexes with salicylaldehyde-amino acids Schiff bases ligands.

    PubMed

    Rîmbu, Cristina; Danac, Ramona; Pui, Aurel

    2014-01-01

    Palladium(II) complexes with Schiff bases ligands derived from salicylaldehyde and amino acids (Ala, Gly, Met, Ser, Val) have been synthesized and characterized by Fourier transform (FT)-IR, UV-Vis and (1)H-NMR spectroscopy. The electrospray mass spectrometry (ES-MS) spectrometry confirms the formation of palladium(II) complexes in 1/2 (M/L) molar ratio. All the Pd(II) complexes 1, [Pd(SalAla)2]Cl2; 2, [Pd(SalGly)2]Cl2; 3, [Pd(SalMet)2]Cl2; 4, [Pd(SalSer)2]Cl2; 5, [Pd(SalVal)2]Cl2; have shown antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli.

  16. R(-)-4-(3-Isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole, a fluorescent chiral tagging reagent: sensitive resolution of chiral amines and amino acids by reversed-phase liquid chromatography.

    PubMed

    Toyo'oka, T; Jin, D; Tomoi, N; Oe, T; Hiranuma, H

    2001-02-01

    The usefulness of R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with beta-blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in beta-blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed-phase chromatography. R(-)-DBD-PyNCS was also applied to the determination of DL-amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL-amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D-amino acids (D-Ala, D-Ser, D-Asp and/or D-Glu) was identified in the samples tested. The structures and the purities were elucidated with on-line HPLC-MS. The chiral reagent possessing an isothiocyanate group (-NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D-amino acids. The thiocarbamoyl derivatives obtained from the reaction with DL-amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations were sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D- and

  17. Cyclopropyl glycine and proline-containing preparation noopept evoke two types of membrane potential responses in synaptoneurosomes.

    PubMed

    Lutsenko, V K; Vukolova, M N; Gudasheva, T A

    2003-06-01

    Proline, cyclo(Pro-Gly), and acyl-prolyl-containing dipeptide GVS-111 decreased synaptoneurosome membrane potential in a Ca2+-free medium. The efficiency of these preparations decreased in the following order: GVS>cyclo(Pro-Gly)>proline. Depolarization responses induced by endogenous nootropic agent cyclo(Pro-Gly) was dose-dependent and saturable; the threshold concentration of cyclo(Pro-Gly) was 10(-9) M. In a Ca2+-containing medium GVS and cyclo(Pro-Gly) induced both hyperpolarizing and depolarizing membrane responses of synaptoneurosomes. Possible mechanisms underlying changes in the membrane potential of synaptoneurosomes induced by nootropic agents are discussed. It was interesting whether modulation of electrogenesis can improve memory and potentiate the neuroprotective effect of the test nootropic agents.

  18. The Role of Water in the Stability of Wild Type and Mutant Insulin Dimers.

    PubMed

    Raghunathan, Shampa; El Hage, Krystel; Desmond, Jasmine; Zhang, Lixian; Meuwly, Markus

    2018-06-19

    Insulin dimerization and aggregation play important roles in the endogenous delivery of the hormone. One of the important residues at the insulin dimer interface is Phe B24 which is an invariant aromatic anchor that packs towards its own monomer inside a hydrophobic cavity formed by Val B12 , Leu B15 , Tyr B16 , Cys B19 and Tyr B26 . Using molecular dynamics and free energy simulations in explicit solvent, the structural and dynamical consequences of mutations of Phe at position B24 to Gly, Ala, and d-Ala and the des-PheB25 variant are quantified. Consistent with experiments it is found that the Gly and Ala modifications lead to insulin dimers with reduced stability by 4 and 5 kcal/mol from thermodynamic integration and 4 and 8 kcal/mol from results using MM-GBSA, respectively. Given the experimental difficulties to quantify the thermodynamic stability of modified insulin dimers, such computations provide a valuable complement. Interestingly, the Gly-mutant exists as a strongly and a weakly interacting dimer. Analysis of the molecular dynamics simulations shows that this can be explained by water molecules that replace direct monomer-monomer H-bonding contacts at the dimerization interface involving residues B24 to B26. It is concluded that such solvent molecules play an essential role and must be included in future insulin dimerization studies.

  19. Daidzein suppresses tumor necrosis factor-α induced migration and invasion by inhibiting hedgehog/Gli1 signaling in human breast cancer cells.

    PubMed

    Bao, Cheng; Namgung, Hyeju; Lee, Jaehoo; Park, Hyun-Chang; Ko, Jiwon; Moon, Heejung; Ko, Hyuk Wan; Lee, Hong Jin

    2014-04-30

    In breast cancer, the cytokine tumor necrosis factor-α (TNF-α) induces cell invasion, although the molecular basis of it has not been clearly elucidated. In this study, we investigated the role of daidzein in regulating TNF-α induced cell invasion and the underlying molecular mechanisms. Daidzein inhibited TNF-α induced cellular migration and invasion in estrogen receptor (ER) negative MCF10DCIS.com human breast cancer cells. TNF-α activated Hedgehog (Hh) signaling by enhancing Gli1 nuclear translocation and transcriptional activity, which resulted in increased invasiveness; these effects were blocked by daidzein and the Hh signaling inhibitors, cyclopamine and vismodegib. Moreover, these compounds suppressed TNF-α induced matrix metalloproteinase (MMP)-9 mRNA expression and activity. Taken together, mammary tumor cell invasiveness was stimulated by TNF-α induced activation of Hh signaling; these effects were abrogated by daidzein, which suppressed Gli1 activation, thereby inhibiting migration and invasion.

  20. C-glycoside mimetics inhibit glioma stem cell proliferation, migration, and invasion.

    PubMed

    Clarion, Ludovic; Jacquard, Carine; Sainte-Catherine, Odile; Decoux, Marc; Loiseau, Séverine; Rolland, Marc; Lecouvey, Marc; Hugnot, Jean-Philippe; Volle, Jean-Noël; Virieux, David; Pirat, Jean-Luc; Bakalara, Norbert

    2014-10-23

    This paper reports the design and synthesis of C-glycoside mimetics (d-glycero-d-talo- and d-glycero-d-galactopyranose analogues), a subset of the recently published phostines, belonging to the [1,2]oxaphosphinane core. Eighteen new compounds were tested against 11 cancer cell types belonging to six categories of tumor tissues and three different species. The hit compound 5.3d inhibited invasion and migration of both GBM stem cells (Gli7 and Gli4) and GBM cancer cell lines (C6, SNB75) on fibronectin, vitronectin, and laminin. Ki values for Gli7 and Gli4 migration inhibition on fibronectin were 16 and 31 nM respectively. Ki values for invasion inhibition in a 3D system were 46 nM for Gli7 and 290 nM for Gli4. These activities were associated with an antiproliferative effect on Gli4 (EC50 = 5.20 μM) and Gli7 (EC50 = 2.33 μM). In conclusion, the heptopyranose mimetic 5.3d, devoid of toxicity on astrocyte and cortical neuron cultures at concentrations below 100 μM, opens new therapeutic perspectives against glioblastoma.

  1. Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor*

    PubMed Central

    An, Bo; Abbonante, Vittorio; Xu, Huifang; Gavriilidou, Despoina; Yoshizumi, Ayumi; Bihan, Dominique; Farndale, Richard W.; Kaplan, David L.; Balduini, Alessandra; Leitinger, Birgit; Brodsky, Barbara

    2016-01-01

    A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities. PMID:26702058

  2. Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure.

    PubMed

    Lakshmi, Balasubramanian; Sinduja, Chandrasekaran; Archunan, Govind; Srinivasan, Narayanaswamy

    2014-06-01

    High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety-six of these alignment positions correspond to conserved Gly residue with (φ, ψ) values allowed for non-glycyl residues. Reasons for this observation were investigated by in-silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C(β) atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (φ, ψ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (φ, ψ) values which are disallowed for Ala. In-silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C(β) atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins. © 2014 The Protein Society.

  3. Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori.

    PubMed

    Hu, Heidi Q; Johnson, Ryan C; Merrell, D Scott; Maroney, Michael J

    2017-02-28

    The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic six-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA, while another is a diamagnetic four-coordinate planar site characterized by solution nuclear magnetic resonance in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant, L2*-HypA, in which a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using XAS showed a coordination change from six-coordinate in wild-type HypA (WT-HypA) to five-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni sites in WT-HypA compared to those of the Ni sites in L2*-HypA confirmed that a spin-state change from high to low spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA-UreE interaction that is essential for urease maturation but was at least 20-fold weaker in Ni binding than WT-HypA. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.

  4. Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hu, Heidi Q.; Johnson, Ryan C.; Merrell, D. Scott

    The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic six-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA, while another is a diamagnetic four-coordinate planar site characterized by solution nuclear magnetic resonance in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant,more » L2*-HypA, in which a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using XAS showed a coordination change from six-coordinate in wild-type HypA (WT-HypA) to five-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni sites in WT-HypA compared to those of the Ni sites in L2*-HypA confirmed that a spin-state change from high to low spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA–UreE interaction that is essential for urease maturation but was at least 20-fold weaker in Ni binding than WT-HypA. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.« less

  5. PAX3 mutations and clinical characteristics in Chinese patients with Waardenburg syndrome type 1

    PubMed Central

    Wang, Juan; Li, Shiqiang; Xiao, Xueshan; Wang, Panfeng; Guo, Xiangming

    2010-01-01

    Purpose To detect paired box gene 3 (PAX3) mutations and associated phenotypes in Chinese patients with Waardenburg syndrome type 1 (WS1). Methods Five unrelated families with suspected WS1 were selected from our Genomic DNA Repository for Hereditary Eye Diseases. The coding and adjacent intronic regions of PAX3 were amplified by polymerase chain reaction and the amplicons were then analyzed by cycle sequencing. Variations detected were further evaluated in available family members as well as one hundred controls with heteroduplex-single strand conformational polymorphism (heteroduplex-SSCP) analysis and/or clone sequencing. Results Three novel and two known mutations in PAX3 were detected in five patients, respectively: c.567_586+17del (p.Asp189_Gln505delinsGluGlyGlyAlaLeuAlaGly), c.456_459dupTTCC (p.Ile154PhefsX162), c.795_800delCTGGTT (p.Trp266_Phe267del), c.799T>A (p.Phe267Ile), and c.667C>T (p.Arg223X). Two novel mutations proved to be de novo as their parents did not carry the mutations. All five patients with PAX3 mutations had dystopia canthorum and different iris color and fundi between their two eyes. However, none had white forelock, skin hypopigmentation, and deafness. Conclusions Our findings expand the frequency and spectrum of PAX3 mutations and ethnic-related phenotypes in Chinese patients with WS1. De novo mutations in PAX3 have not been reported before. PMID:20664692

  6. PAX3 mutations and clinical characteristics in Chinese patients with Waardenburg syndrome type 1.

    PubMed

    Wang, Juan; Li, Shiqiang; Xiao, Xueshan; Wang, Panfeng; Guo, Xiangming; Zhang, Qingjiong

    2010-06-22

    To detect paired box gene 3 (PAX3) mutations and associated phenotypes in Chinese patients with Waardenburg syndrome type 1 (WS1). Five unrelated families with suspected WS1 were selected from our Genomic DNA Repository for Hereditary Eye Diseases. The coding and adjacent intronic regions of PAX3 were amplified by polymerase chain reaction and the amplicons were then analyzed by cycle sequencing. Variations detected were further evaluated in available family members as well as one hundred controls with heteroduplex-single strand conformational polymorphism (heteroduplex-SSCP) analysis and/or clone sequencing. Three novel and two known mutations in PAX3 were detected in five patients, respectively: c.567_586+17del (p.Asp189_Gln505delinsGluGlyGlyAlaLeuAlaGly), c.456_459dupTTCC (p.Ile154PhefsX162), c.795_800delCTGGTT (p.Trp266_Phe267del), c.799T>A (p.Phe267Ile), and c.667C>T (p.Arg223X). Two novel mutations proved to be de novo as their parents did not carry the mutations. All five patients with PAX3 mutations had dystopia canthorum and different iris color and fundi between their two eyes. However, none had white forelock, skin hypopigmentation, and deafness. Our findings expand the frequency and spectrum of PAX3 mutations and ethnic-related phenotypes in Chinese patients with WS1. De novo mutations in PAX3 have not been reported before.

  7. Effects of short peptides on thymocyte blast transformation and signal transduction along the sphingomyelin pathway.

    PubMed

    Khavinson, V Kh; Rybakina, E G; Malinin, V V; Pivanovich, I Yu; Shanin, S N; Korneva, E A

    2002-05-01

    Immunomodulating effects of synthetic peptides Vilon (Lys-Glu), Epithalon (Ala-Glu-Asp-Gly), and Cortagen (Ala-Glu-Asp-Pro) and possible involvement of the sphingomyelin signal transduction pathway in their effects in mouse thymocytes were studied. Vilon produced the most potent comitogenic effect on thymocyte proliferation and modulated comitogenic activity of interleukin-1b. Epithalon was less potent, while Cortagen produced no such effects. Vilon produced a more pronounced stimulatory effect on sphingomyelinase activity in mouse thymocyte membranes compared to Epithalon and Cortagen.

  8. 1α,25(OH)2-dihydroxyvitamin D3/VDR protects the skin from UVB-induced tumor formation by interacting with the β-catenin pathway.

    PubMed

    Jiang, Yan J; Teichert, Arnaud E; Fong, Frankie; Oda, Yuko; Bikle, Daniel D

    2013-07-01

    Ultra violet (UV) irradiation, in particular UVB, is the single most important carcinogen for skin tumor formation. UVB induces genetic mutations and immune suppression, which lead to abnormal cell proliferation and eventually tumor formation. Previously studies from our group and others demonstrated that both global and epidermal specific VDR knock out mice are predisposed to either chemical (DMBA)- or long-term UVB-induced skin tumor formation, paralleled by an increase in β-catenin signaling. Using primary cultured human keratinocytes, we further demonstrated that 1,25(OH)2-dihydroxyvitamin D3 (1,25(OH)2D3) suppresses cyclin D1 and Gli1 which are regulated by β-catenin/TCF signaling and have a critical role in epidermal carcinogenesis. Blockage of VDR by siRNA resulted in hyperproliferation of keratinocytes, and increased expression of cyclin D1 and Gli1. In addition, we also showed that 1,25(OH)2D3/VDR directly regulates transcriptional activity of β-catenin/TCF signaling using the -catenin reporter TopGlow. Using K14 driven tamoxifen-induced cre recombinase to delete both VDR and β-catenin in keratinocytes of mice following the first hair follicle cycle, we found that ablation of epidermal specific β-catenin cannot rescue VDR null mice from UVB-induced skin tumor formation. Further study using VDR or β-catenin single null mice is necessary to compare with the data from double null mice. This article is part of a Special Issue entitled 'Vitamin D Workshop'. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Purification and characterization of a tripeptidyl peptidase I from human osteoclastomas: evidence for its role in bone resorption.

    PubMed

    Page, A E; Fuller, K; Chambers, T J; Warburton, M J

    1993-11-01

    Tripeptidyl peptidase I (EC 3.4.14.9), which cleaves tripeptides from the N-terminus of synthetic substrates, has been purified from human osteoclastomas (a bone tumor containing large numbers of normal osteoclasts). The enzyme has an M(r) of 48 kDa but forms aggregates with an M(r) of about 700 kDa. The tripeptidyl peptidase has an acidic pH optimum (approximately pH 5.0), suggesting that it has a lysosomal localization and prefers substrates with a hydrophobic amino acid in the P1 position. There is an absolute requirement for a nonsubstituted N-terminus. The enzyme is inhibited by reagents which modify serine and histidine residues. Lysosomal tripeptidyl peptidase is known to be capable of cleaving Gly-Pro-X triplets from synthetic collagen-like polypeptides. Ala-Ala-Phe-CH2Cl, a potent inhibitor of osteoclastoma tripeptidyl peptidase, inhibits osteoclastic bone resorption in an in vitro test system. This suggests that tripeptidyl peptidase I, secreted by osteoclasts, is involved at some stage in the degradation of bone collagen.

  10. Codon-Anticodon Recognition in the Bacillus subtilis glyQS T Box Riboswitch

    PubMed Central

    Caserta, Enrico; Liu, Liang-Chun; Grundy, Frank J.; Henkin, Tina M.

    2015-01-01

    Many amino acid-related genes in Gram-positive bacteria are regulated by the T box riboswitch. The leader RNA of genes in the T box family controls the expression of downstream genes by monitoring the aminoacylation status of the cognate tRNA. Previous studies identified a three-nucleotide codon, termed the “Specifier Sequence,” in the riboswitch that corresponds to the amino acid identity of the downstream genes. Pairing of the Specifier Sequence with the anticodon of the cognate tRNA is the primary determinant of specific tRNA recognition. This interaction mimics codon-anticodon pairing in translation but occurs in the absence of the ribosome. The goal of the current study was to determine the effect of a full range of mismatches for comparison with codon recognition in translation. Mutations were individually introduced into the Specifier Sequence of the glyQS leader RNA and tRNAGly anticodon to test the effect of all possible pairing combinations on tRNA binding affinity and antitermination efficiency. The functional role of the conserved purine 3′ of the Specifier Sequence was also verifiedin this study. We found that substitutions at the Specifier Sequence resulted in reduced binding, the magnitude of which correlates well with the predicted stability of the RNA-RNA pairing. However, the tolerance for specific mismatches in antitermination was generally different from that during decoding, which reveals a unique tRNA recognition pattern in the T box antitermination system. PMID:26229106

  11. Computational and experimental investigations into the conformations of cyclic tetra-α/β-peptides.

    PubMed

    Oakley, Mark T; Oheix, Emmanuel; Peacock, Anna F A; Johnston, Roy L

    2013-07-11

    We present a combined computational and experimental study of the energy landscapes of cyclic tetra-α/β-peptides. We have performed discrete path sampling calculations on a series of cyclic tetra-α/β-peptides to obtain the relative free energies and barriers to interconversion of their conformers. The most stable conformers of cyclo-[(β-Ala-Gly)2] contain all-trans peptide groups. The relative energies of the cis isomers and the cis-trans barriers are lower than in acyclic peptides but not as low as in the highly strained cyclic α-peptides. For cyclic tetra-α/β-peptides containing a single proline residue, of the type cyclo-[β-Ala-Xaa-β-Ala-Pro], the energy landscapes show that the most stable isomers containing cis and trans β-Ala-Pro have similar free energies and are separated by barriers of approximately 15 kcal mol(-1). We show that the underlying energy landscapes of cyclo-[β-Ala-Lys-β-Ala-Pro] and cyclo-[β-Ala-Ala-β-Ala-Pro] are similar, allowing the substitution of the flexible side chain of Lys with Ala to reduce the computational demand of our calculations. However, the steric bulk of the Val side chain in cyclo-[β-Ala-Val-β-Ala-Pro] affects the conformations of the ring, leading to significant differences between its energy landscape and that of cyclo-[β-Ala-Ala-β-Ala-Pro]. We have synthesized the cyclic peptide cyclo-[β-Ala-Lys-β-Ala-Pro], and NMR spectroscopy shows the presence of conformers that interconvert slowly on the NMR time scale at temperatures up to 80 °C. Calculated circular dichroism (CD) spectra for the proposed major isomer of cyclo-[β-Ala-Ala-β-Ala-Pro] are in good agreement with the experimental spectra of cyclo-[β-Ala-Lys-β-Ala-Pro], suggesting that the Ala cyclic tetrapeptide is a viable model for the Lys analogue.

  12. Synthesis and pharmacology of halogenated δ-opioid-selective [d-Ala(2)]deltorphin II peptide analogues.

    PubMed

    Pescatore, Robyn; Marrone, Gina F; Sedberry, Seth; Vinton, Daniel; Finkelstein, Netanel; Katlowitz, Yitzchak E; Pasternak, Gavril W; Wilson, Krista R; Majumdar, Susruta

    2015-06-17

    Deltorphins are naturally occurring peptides produced by the skin of the giant monkey frog (Phyllomedusa bicolor). They are δ-opioid receptor-selective agonists. Herein, we report the design and synthesis of a peptide, Tyr-d-Ala-(pI)Phe-Glu-Ile-Ile-Gly-NH2 3 (GATE3-8), based on the [d-Ala(2)]deltorphin II template, which is δ-selective in in vitro radioligand binding assays over the μ- and κ-opioid receptors. It is a full agonist in [(35)S]GTPγS functional assays and analgesic when administered supraspinally to mice. Analgesia of 3 (GATE3-8) is blocked by the selective δ receptor antagonist naltrindole, indicating that the analgesic action of 3 is mediated by the δ-opioid receptor. We have established a radioligand in which (125)I is incorporated into 3 (GATE3-8). The radioligand has a KD of 0.1 nM in Chinese hamster ovary (CHO) cells expressing the δ receptor. Additionally, a series of peptides based on 3 (GATE3-8) was synthesized by incorporating various halogens in the para position on the aromatic ring of Phe(3). The peptides were characterized for binding affinity at the μ-, δ-, and κ-opioid receptors, which showed a linear correlation between binding affinity and the size of the halogen substituent. These peptides may be interesting tools for probing δ-opioid receptor pharmacology.

  13. Synthesis and Pharmacology of Halogenated δ-Opioid-Selective [D-Ala2]Deltorphin II Peptide Analogues

    PubMed Central

    Pescatore, Robyn; Marrone, Gina F.; Sedberry, Seth; Vinton, Daniel; Finkelstein, Netanel; Katlowitz, Yitzchak E.; Pasternak, Gavril W.; Wilson, Krista R.; Majumdar, Susruta

    2015-01-01

    Deltorphins are naturally occurring peptides produced by the skin of the giant monkey frog (Phyllomedusa bicolor). They are δ-opioid receptor-selective agonists. Herein, we report the design and synthesis of a peptide, Tyr-D-Ala-(pI)Phe-Glu-Ile-Ile-Gly-NH2 3 (GATE3-8), based on the [D-Ala2]deltorphin II template, which is δ-selective in in vitro radioligand binding assays over the μ- and κ-opioid receptors. It is a full agonist in [35S]GTPγS functional assays and analgesic when administered supraspinally to mice. Analgesia of 3 (GATE3-8) is blocked by the selective δ receptor antagonist naltrindole, indicating that the analgesic action of 3 is mediated by the δ-opioid receptor. We have established a radioligand in which 125I isincorporated into 3 (GATE3-8). The radioligand has a KD of 0.1 nM in Chinese hamster ovary (CHO) cells expressing the δ receptor. Additionally, a series of peptides based on 3 (GATE3-8) was synthesized by incorporating various halogens in the para position on the aromatic ring of Phe3. The peptides were characterized for binding affinity at the μ-, δ-, and κ-opioid receptors, which showed a linear correlation between binding affinity and the size of the halogen substituent. These peptides may be interesting tools for probing δ-opioid receptor pharmacology. PMID:25844930

  14. Polymorphism of MSH2 Gly322Asp and MLH1 –93G>A in non-familial colon cancer – a case-controlled study

    PubMed Central

    Dziki, Lukasz; Malinowska, Katarzyna; Trzcinski, Radzislaw; Majsterek, Ireneusz; Dziki, Adam

    2017-01-01

    Introduction Our aim was to determine the effect of the single nucleotide polymorphisms (SNP) –93G>A of the MLH1 gene (rs1800734) and Gly322Asp of the MSH2 gene (rs4987188) on the risk of colon cancer (CC) and identify any relationship with clinical factors. Material and methods The study included 144 unrelated patients with sporadic CC (71 males; mean age: 61.7 ±11 years) and 151 control patients (74 males; mean age: 63 ±11 years). DNA was extracted from peripheral blood lymphocytes, and genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism. Results In our population, the homozygous G/G genotype of the –93G>AMLH1 gene increased the risk of sporadic CC (OR = 2.07; 95% CI: 1.11–3.83; p < 0.02). For A/G and A/A genotypes, the MLH1-93G>A polymorphism was significantly more common in women (p = 0.034). The SNP demonstrated differences in allele distribution according to the location of the tumor, i.e. right vs. left side (p = 0.014), and disease recurrence (p = 0.022). Significant differences were found in the occurrence of Gly322Asp of MSH2 with regard to primary and recurrent disease (p = 0.001). Conclusions The –93G>AMLH1 polymorphism plays an important role in evaluating the risk of sporadic CC. It can also be used as an indicator in some patients with left-sided and recurrent tumors. MSH2 Gly322Asp is a potential marker in patients with risk of recurrence. PMID:29181059

  15. Antitumor and antimicrobial activity of some cyclic tetrapeptides and tripeptides derived from marine bacteria.

    PubMed

    Chakraborty, Subrata; Tai, Dar-Fu; Lin, Yi-Chun; Chiou, Tzyy-Wen

    2015-05-15

    Marine derived cyclo(Gly-l-Ser-l-Pro-l-Glu) was selected as a lead to evaluate antitumor-antibiotic activity. Histidine was chosen to replace the serine residue to form cyclo(Gly-l-His-l-Pro-l-Glu). Cyclic tetrapeptides (CtetPs) were then synthesized using a solution phase method, and subjected to antitumor and antibiotic assays. The benzyl group protected CtetPs derivatives, showed better activity against antibiotic-resistant Staphylococcus aureus in the range of 60-120 μM. Benzyl group protected CtetPs 3 and 4, exhibited antitumor activity against several cell lines at a concentration of 80-108 μM. However, shortening the size of the ring to the cyclic tripeptide (CtriP) scaffold, cyclo(Gly-l-Ser-l-Pro), cyclo(Ser-l-Pro-l-Glu) and their analogues showed no antibiotic or antitumor activity. This phenomenon can be explained from their backbone structures.

  16. Purification and Characterization of Peptides Inhibiting MMP-1 Activity with C Terminate of Gly-Leu from Simulated Gastrointestinal Digestion Hydrolysates of Tilapia (Oreochromis niloticus) Skin Gelatin.

    PubMed

    Liping, Sun; Qiuming, Liu; Jian, Fan; Xiao, Li; Yongliang, Zhuang

    2018-01-24

    Tilapia skin gelatin hydrolysates (TSGHs) were prepared by simulated gastrointestinal digestion and separated by gel filtration and semi-preparative reversed-phase high-performance liquid chromatography. The anti-photoaging effects were evaluated using an ultraviolet radiation B (UVB)-induced mouse embryonic fibroblast (MEF) photoaging model in vitro. Three fractions from TSGHs with high inhibitory intercellular matrix metalloproteinase-1 (MMP-1) activities and reactive oxygen species (ROS) production were obtained. Three key peptides, GYTGL, LGATGL, and VLGL, were identified, and their C terminate was Gly-Leu. Three peptides were synthesized and exhibited a significant inhibition of intercellular MMP-1 activity and ROS production. Furthermore, three peptides inhibiting MMP-1 activities were evaluated through their docking of S 1 ' and S 3 ' active pockets of MMP-1. Hydrogen bonds and C terminate Gly-Leu played important roles. Finally, the protective effects of three peptides on intercellular collagen in UVB-induced MEFs were compared. Our results indicated that tilapia gelatin peptides exhibited potential activities to prevent and regulate photoaging.

  17. Toll like receptor-4 gene polymorphisms in patients with solitary cysticercus granuloma.

    PubMed

    Singh, Akhilesh; Garg, Ravindra Kumar; Jain, Amita; Malhotra, Hardeep Singh; Prakash, Shantanu; Verma, Rajesh; Sharma, Praveen Kumar

    2015-08-15

    Solitary cysticercus granuloma (SCG) of the brain is the most common type of neurocysticercosis in India. In this study, we evaluated TLR4 polymorphisms in patients with SCG. One-hundred-forty-three patients with SCG and 134 controls were enrolled. Assessment for TLR4 Asp299Gly and Thr399Ile polymorphism was done. TLR4 genotype was determined by PCR-sequencing chain termination method. The patients were followed for 6 months. Asp/Gly (P=0.024) and Thr/Ile (P=0.004) genotypes were significantly associated with the SCG. The Gly (Asp/Gly plus Gly/Gly) genotype (P=0.025) and Ile (Thr/Ile plus Ile/Ile) genotype (P=0.008) were significantly associated with the SCG. Gly/Gly and Ile/Ile genotypes were not significantly associated with SCG (P=0.767 for Gly/Gly, P=0.936 for Ile/Ile). At 6 months, TLR4 299Asp/Gly (P=0.02) and 399Ile/Thr (P=0.023) polymorphisms were significantly associated with the calcification or persistence of SCG. TLR4 polymorphisms are associated with the susceptibility to infection with SCG. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kimira, Yoshifumi, E-mail: kimira@josai.ac.jp; Ogura, Kana; Taniuchi, Yuri

    Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicatemore » that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.« less

  19. New cyclic peptides with osteoblastic proliferative activity from Dianthus superbus.

    PubMed

    Tong, Yun; Luo, Jian-Guang; Wang, Rui; Wang, Xiao-Bing; Kong, Ling-Yi

    2012-03-01

    Two new cyclic peptides, dianthins G-H (1 and 2), together with the known dianthin E (3), were isolated from the traditional Chinese medicinal plant Dianthus superbus. The sequences of cyclic peptides 1 and 2 were elucidated as cyclo (-Gly(1)-Pro(2)-Leu(3)-Thr(4)-Leu(5)-Phe(6)-) and cyclo (-Gly(1)-Pro(2)-Val(3)-Thr(4)-Ile(5)-Phe(6)-), on the basis of ESI tandem mass fragmentation analysis, extensive 2D NMR methods and X-ray diffraction. The isolated three compounds all increase proliferation of MC3T3-E1 cells in vitro using MTT method. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. The adsorption of amino acids and cations onto goethite: a prebiotic chemistry experiment.

    PubMed

    Farias, Ana Paula S F; Carneiro, Cristine E A; de Batista Fonseca, Inês C; Zaia, Cássia T B V; Zaia, Dimas A M

    2016-06-01

    Few prebiotic chemistry experiments have assessed the adsorption of biomolecules by iron oxide-hydroxides. The present work investigated the effects of cations in artificial seawaters on the adsorption of Gly, α-Ala and β-Ala onto goethite, and vice versa. Goethite served to concentrate K and Mg cations from solution; these effects could have played important roles in peptide nucleoside formation. Goethite showed low adsorption of Gly and α-Ala. On the other hand, β-Ala (a non-protein amino acid) was highly adsorbed by goethite. Because Gly and α-Ala are the most common amino acids in living beings, and iron oxide-hydroxides are widespread on Earth, additional iron oxides should be studied. Increased ionic strength in artificial seawaters decreased the adsorption of amino acids by goethite. Because Na was highly abundant in the artificial seawater, it showed the highest effect on amino acid adsorption. β-Ala increased the adsorption of K and Ca by goethite, this effect could have been important for peptide synthesis.

  1. Germline missense pathogenic variants in the BRCA1 BRCT domain, p.Gly1706Glu and p.Ala1708Glu, increase cellular sensitivity to PARP inhibitor olaparib by a dominant negative effect

    PubMed Central

    Vaclová, Tereza; Woods, Nicholas T.; Megías, Diego; Gomez-Lopez, Sergio; Setién, Fernando; García Bueno, José María; Macías, José Antonio; Barroso, Alicia; Urioste, Miguel; Esteller, Manel; Monteiro, Alvaro N.A.; Benítez, Javier; Osorio, Ana

    2016-01-01

    Abstract BRCA1-deficient cells show defects in DNA repair and rely on other members of the DNA repair machinery, which makes them sensitive to PARP inhibitors (PARPi). Although carrying a germline pathogenic variant in BRCA1/2 is the best determinant of response to PARPi, a significant percentage of the patients do not show sensitivity and/or display increased toxicity to the agent. Considering previously suggested mutation-specific BRCA1 haploinsufficiency, we aimed to investigate whether there are any differences in cellular response to PARPi olaparib depending on the BRCA1 mutation type. Lymphoblastoid cell lines derived from carriers of missense pathogenic variants in the BRCA1 BRCT domain (c.5117G > A, p.Gly1706Glu and c.5123C > A, p.Ala1708Glu) showed higher sensitivity to olaparib than cells with truncating variants or wild types (WT). Response to olaparib depended on a basal PARP enzymatic activity, but did not correlate with PARP1 expression. Interestingly, cellular sensitivity to the agent was associated with the level of BRCA1 recruitment into γH2AX foci, being the lowest in cells with missense variants. Since these variants lead to partially stable protein mutants, we propose a model in which the mutant protein acts in a dominant negative manner on the WT BRCA1, impairing the recruitment of BRCA1 into DNA damage sites and, consequently, increasing cellular sensitivity to PARPi. Taken together, our results indicate that carriers of different BRCA1 mutations could benefit from olaparib in a distinct way and show different toxicities to the agent, which could be especially relevant for a potential future use of PARPi as prophylactic agents in BRCA1 mutation carriers. PMID:27742776

  2. Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases.

    PubMed

    Crabbe, T; Willenbrock, F; Eaton, D; Hynds, P; Carne, A F; Murphy, G; Docherty, A J

    1992-09-15

    The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.

  3. Inhibition of polyphenol oxidases activity by various dipeptides.

    PubMed

    Girelli, Anna M; Mattei, Enrico; Messina, Antonella; Tarola, Anna M

    2004-05-19

    In an effort to develop natural and nontoxic inhibitors on the activity of mushroom polyphenol oxidase (PPO) the effect of various glycyl-dipeptides (GlyAsp, GlyGly, GlyHis, GlyLeu, GlyLys, GlyPhe, GlyPro, GlyTyr) was investigated. The inhibition study with dihydroxyphenylalanine (DOPA) as substrate is based on separation of the enzymatic reaction components by reversed phase HPLC and the UV detection of the dopachrome formed. The results have evidenced that several of tested dipeptides inhibited PPO activity in the range of 20-40% while GlyPro and GlyLeu had no effect. The study has also permitted the characterization of the following kinetic pattern: a linear-mixed-type mechanism for GlyAsp, GlyGly, GlyLys, and GlyPhe and a hyperbolic-mixed-type for GlyTyr. It was not possible to identify the inhibition mechanism for GlyHis, although it affects PPO activity. In addition the effects of GlyAsp, GlyLys and GlyHis were evaluated for lessening the browning of fresh Golden Delicious apple and Irish White Skinned potato. The effectiveness of such inhibitors was determined by the difference between the colors observed in the dipeptide-treated sample and the controls using the color space CIE-Lab system. The % browning inhibition on potato (20-50%) was greater than of apple (20-30%) by the all tested dipeptides. Only GlyLys presented the significant value of 50%.

  4. X-ray diffraction analysis of 4- and 4'-substituted C n H2 n + 1O-C6H3(OH)-CH=N-C6H4-C m H2 m + 1 ( n/ m = 2/1 and 3/4) salicylideneanilines

    NASA Astrophysics Data System (ADS)

    Kuz'mina, L. G.; Navasardyan, M. A.; Mikhailov, A. A.

    2017-11-01

    X-ray diffraction study of two crystalline modifications of C2H5O-C6H3(OH)-CH=N-C6H4-CH3 ( 1a, sp. gr. P21/ n, and 1b, sp. gr. C2/c) and C3H7O-C6H3(OH)-CH=N-C6H4-C4H9 ( 2, sp. gr. P212121) has been performed. The 1a crystal structure contains two independent molecules. The molecules are conformationally nonrigid with respect to the mutual rotation of benzene rings; the dihedral angles between their planes are 29.19° and 26.00° in the independent molecules of 1a, 18.72° in the molecule of 1b, and 50.35° in the molecule of 2. The crystal packing of the compounds is discussed.

  5. GANT61, a GLI inhibitor, sensitizes glioma cells to the temozolomide treatment.

    PubMed

    Li, Jianlong; Cai, Jinquan; Zhao, Shihong; Yao, Kun; Sun, Ying; Li, Yongli; Chen, Lingchao; Li, Ruiyan; Zhai, Xiuwei; Zhang, Junhe; Jiang, Chuanlu

    2016-11-28

    The aim of this study was to investigate the effect of downregulating Hedgehog pathway by GANT61 on human glioma cells, examine the consequent changes of temozolomide (TMZ)-induced effects and explore the molecular mechanisms. The cytotoxicity of a Gli1/2 inhibitor, GANT61 was examined both alone and in combination with TMZ in human glioma cell lines. The mRNA and protein expression alterations were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. CCK-8 assay detected the cell proliferative capability. Apoptotic cell number was measured by flow cytometry. The transwell assay was used to test the cell invasive capability. DNA damage effect was identified by COMET assay and γH2AX expression. Proliferation of tumor cells treated with GANT61 in combination with TMZ was significantly suppressed compared with those treated with either drug used alone. The combination treatment induced a higher rate of apoptosis, DNA damage and reduced the invasive capability of glioma cells. DNA damage repair enzyme MGMT and the Notch1 pathway increased in the cells treated by TMZ treatment. However, GANT61 could abrogated the protein increasing. GANT61 sensitizes glioma cells to TMZ treatment by enhancing DNA damage effect, decreasing MGMT expression and the Notch1 pathway.

  6. Structure determination of a peptide model of the repeated helical domain in Samia cynthia ricini silk fibroin before spinning by a combination of advanced solid-state NMR methods.

    PubMed

    Nakazawa, Yasumoto; Asakura, Tetsuo

    2003-06-18

    Fibrous proteins unlike globular proteins, contain repetitive amino acid sequences, giving rise to very regular secondary protein structures. Silk fibroin from a wild silkworm, Samia cynthia ricini, consists of about 100 repeats of alternating polyalanine (poly-Ala) regions of 12-13 residues in length and Gly-rich regions. In this paper, the precise structure of the model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, was determined using a combination of three kinds of solid-state NMR studies; a quantitative use of (13)C CP/MAS NMR chemical shift with conformation-dependent (13)C chemical shift contour plots, 2D spin diffusion (13)C solid-state NMR under off magic angle spinning and rotational echo double resonance. The structure of the model peptide corresponding to the silk fibroin structure before spinning was determined. The torsion angles of the central Ala residue, Ala(19), in the poly-Ala region were determined to be (phi, psi) = (-59 degrees, -48 degrees ) which are values typically associated with alpha-helical structures. However, the torsion angles of the Gly(25) residue adjacent to the C-terminal side of the poly-Ala chain were determined to be (phi, psi) = (-66 degrees, -22 degrees ) and those of Gly(12) and Ala(13) residues at the N-terminal of the poly-Ala chain to be (phi, psi) = (-70 degrees, -30 degrees ). In addition, REDOR experiments indicate that the torsion angles of the two C-terminal Ala residues, Ala(23) and Ala(24), are (phi, psi) = (-66 degrees, -22 degrees ) and those of N-terminal two Ala residues, Ala(13) and Ala(14) are (phi, psi) = (-70 degrees, -30 degrees ). Thus, the local structure of N-terminal and C-terminal residues, and also the neighboring residues of alpha-helical poly-Ala chain in the model peptide is a more strongly wound structure than found in typical alpha-helix structures.

  7. Spontaneous resolution of binary copper(II) complexes with racemic dipeptides: crystal structures of glycyl-L-alpha-amino-n-butyrato copper(II) monohydrate, glycyl-D-valinato copper(II) hemihydrate, and glycyl-L-valinato copper(II) hemihydrate.

    PubMed

    Inomata, Yoshie; Yamaguchi, Takeshi; Tomita, Airi; Yamada, Dai; Howell, F Scott

    2005-08-01

    Copper(II) complexes with glycyl-DL-alpha-amino-n-butyric acid (H2gly-DL-but), glycyl-DL-valine (H2gly-DL-val), glycyl-DL-norleucine (H2gly-DL-norleu), glycyl-DL-threonine (H2gly-DL-thr), glycyl-DL-serine (H2gly-DL-ser), glycyl-DL-phenylalanine (H2gly-DL-phe), and glycyl-L-valine (H2gly-L-val), have been prepared and characterized by IR, powder diffuse reflection, CD and ORD spectra, and magnetic susceptibility measurements, and by single-crystal X-ray diffraction. The crystal structures of the copper complex with H2gly-DL-but, the copper complex with H2gly-DL-val, and [Cu(gly-L-val)]n.0.5nH2O have been determined by a single-crystal X-ray diffraction method. As for the structure of the copper complex with H2gly-DL-but, the configuration around the asymmetric carbon atom is similar to that of [Cu(gly-L-val)]n.0.5nH2O. Therefore it is concluded that the copper complex with H2gly-DL-but is [Cu(gly-L-but)]n.nH2O. On the contrary, as for the structure of the copper complex with H2gly-DL-val, the configuration around the asymmetric carbon atom is different from that of [Cu(gly-L-val)]n.0.5nH2O. Therefore it is concluded that the copper complex with H2gly-dl-val is [Cu(gly-D-val)]n.0.5nH2O. So during the crystallization of the copper(II) complexes with H2gly-DL-but and H2gly-DL-val, spontaneous resolution has been observed; the four complexes have separated as [Cu(gly-D-but)]n.nH2O, [Cu(gly-L-but)]n.nH2O, [Cu(gly-D-val)]n.0.5nH2O, and [Cu(gly-L-val)]n.0.5nH2O, respectively. [Cu(gly-L-but)]n.nH2O is orthorhombic with the space group P2(1)2(1)2(1). [Cu(gly-D-val)]n.0.5nH2O and [Cu(gly-L-val)]n.0.5nH2O are monoclinic with the space group C2. In these complexes, the copper atom is in a square-pyramidal geometry, ligated by a peptide nitrogen atom, an amino nitrogen atom, a carboxyl oxygen atom, and a carboxyl oxygen atom and a peptide oxygen atom from neighboring molecules. So these complexes consist of a two-dimensional polymer chain bridged by a carboxyl oxygen atom and a

  8. Accumbal strychnine-sensitive glycine receptors: an access point for ethanol to the brain reward system.

    PubMed

    Molander, Anna; Söderpalm, Bo

    2005-01-01

    Ethanol (EtOH), like other drugs of abuse, increases extracellular dopamine (DA) levels in the nucleus accumbens (nAc) of the brain reward system, an effect that may be of importance for alcohol addiction. How this DA increase is produced is not fully understood, although previous studies from the present laboratories indicate that nicotinic acetylcholine receptors in the ventral tegmental area play an important role in mediating this effect. Furthermore, activation of these receptors may be secondary to some priming effect produced by EtOH in the nAc. We recently demonstrated that strychnine-sensitive glycine receptors (GlyRs) are present in the nAc and that they are involved in regulating extracellular DA levels. Here we examine the tentative role of these accumbal GlyRs in the above-mentioned priming mechanism of EtOH. In vivo microdialysis (coupled to high pressure liquid chromatography with electrochemical detection) and reversed microdialysis, in awake, freely moving adult male Wistar rats. Local perfusion of strychnine decreased accumbal DA levels per se and completely prevented the increase of accumbal DA levels after both local and systemic EtOH administration. Accumbal perfusion of the GlyR agonist glycine instead increased DA levels in a subpopulation of rats and prevented the EtOH-induced increase after local but not systemic EtOH in all animals. The present results suggest that GlyRs in the nAc might constitute targets for EtOH in its mesolimbic DA-activating effect. Gene polymorphism and drug developmental studies that focus on this receptor population and its relation to alcohol dependence are warranted.

  9. Experimental determination of torsion angles in the polypeptide backbone of the gramicidin A channel by solid state nuclear magnetic resonance.

    PubMed

    Teng, Q; Nicholson, L K; Cross, T A

    1991-04-05

    An analytical method for the determination of torsion angles from solid state 15N nuclear magnetic resonance (n.m.r.) spectroscopic data is demonstrated. Advantage is taken of the 15N-1H and 15N-13C dipolar interactions as well as the 15N chemical shift interaction in oriented samples. The membrane-bound channel conformation of gramicidin A has eluded an atomic resolution structure determination by more traditional approaches. Here, the torsion angles for the Ala3 site are determined by obtaining the n.m.r. data for both the Gly2-Ala3 and Ala3-Leu4 peptide linkages. Complete utilization of the orientational constraints derived from these orientation-dependent nuclear spin interactions in restricting the conformational space is most effectively achieved by utilizing spherical trigonometry. Two possible sets of torsion angles for the Ala3 site are obtained (phi, psi = -129 degrees, 153 degrees and -129 degrees, 122 degrees), both of which are consistent with a right-handed beta-helix. Other functional and computational evidence strongly supports the set for which the carbonyl oxygen atom of the Ala3-Leu4 linkage is rotated into the channel lumen.

  10. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonylmore » amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.« less

  11. Severe coagulation factor VII deficiency caused by a novel homozygous mutation (p. Trp284Gly) in loop 140s.

    PubMed

    Hao, Xiuping; Cheng, XiaoLi; Ye, Jiajia; Wang, Yingyu; Yang, LiHong; Wang, Mingshan; Jin, Yanhui

    2016-06-01

    Congenital coagulation factor VII (FVII) deficiency is a rare disorder caused by mutation in F7 gene. Herein, we reported a patient who had unexplained hematuria and vertigo with consanguineous parents. He has been diagnosed as having FVII deficiency based on the results of reduced FVII activity (2.0%) and antigen (12.8%). The thrombin generation tests verified that the proband has obstacles in producing thrombin. Direct sequencing analysis revealed a novel homozygous missense mutation p.Trp284Gly. Also noteworthy is the fact that the mutational residue belongs to structurally conserved loop 140s, which majorly undergo rearrangement after FVII activation. Model analysis indicated that the substitution disrupts these native hydrophobic interactions, which are of great importance to the conformation in the activation domain of FVIIa.

  12. Simultaneous determination of three dipeptides (JBP485, Gly-Sar and JBP923) in the cell lysates by liquid chromatography-tandem mass spectrometry: application to identify the function of the PEPT1 transfected cell.

    PubMed

    Guo, Xinjin; Meng, Qiang; Liu, Qi; Wang, Changyuan; Huo, Xiaokui; Zhang, Zhe; Kaku, Taiichi; Liu, Kexin

    2014-12-01

    A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates using methanol as a deproteinization solvent was developed and validated. Detection was performed by turbo ionspray ionization in multiple reaction monitoring mode using the transitions of m/z 147.1 → m/z 90.1 for Gly-Sar, m/z 201.1 → m/z 86.1 for JBP485, m/z 219.1 → m/z 86.1 for JBP923 and m/z 152.0 → m/z 110.0 for paracetamol (internal standard). The analytes were separated on a Hypersil ODS C18 HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water-methanol (97:3, v/v) at a flow rate of 0.2 mL/min. The calibration curves were demonstrated to be linear over the concentration range of 5.00-5000 nm with coefficient of 0.9968 for Gly-Sar, 0.9975 for JBP485 and 0.9952 for JBP923. The intra- and inter-day precisions were <10.2% for each quality contro; level, and the accuracy was within ±5.6% for each analyte. The matrix effect, the extraction recovery and stabilities of LC-MS/MS analysis were also investigated. This validated method was successfully applied to the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates for identification of stably transfected HeLa cells with human PEPT1. Copyright © 2014 John Wiley & Sons, Ltd.

  13. Antitumor and Antimicrobial Activity of Some Cyclic Tetrapeptides and Tripeptides Derived from Marine Bacteria

    PubMed Central

    Chakraborty, Subrata; Tai, Dar-Fu; Lin, Yi-Chun; Chiou, Tzyy-Wen

    2015-01-01

    Marine derived cyclo(Gly-l-Ser-l-Pro-l-Glu) was selected as a lead to evaluate antitumor-antibiotic activity. Histidine was chosen to replace the serine residue to form cyclo(Gly-l-His-l-Pro-l-Glu). Cyclic tetrapeptides (CtetPs) were then synthesized using a solution phase method, and subjected to antitumor and antibiotic assays. The benzyl group protected CtetPs derivatives, showed better activity against antibiotic-resistant Staphylococcus aureus in the range of 60–120 μM. Benzyl group protected CtetPs 3 and 4, exhibited antitumor activity against several cell lines at a concentration of 80–108 μM. However, shortening the size of the ring to the cyclic tripeptide (CtriP) scaffold, cyclo(Gly-l-Ser-l-Pro), cyclo(Ser-l-Pro-l-Glu) and their analogues showed no antibiotic or antitumor activity. This phenomenon can be explained from their backbone structures. PMID:25988520

  14. Glyoxal and methylglyoxal in Atlantic seawater and marine aerosol particles: method development and first application during the Polarstern cruise ANT XXVII/4

    NASA Astrophysics Data System (ADS)

    van Pinxteren, M.; Herrmann, H.

    2013-06-01

    An analytical method for the determination of the alpha dicarbonyls glyoxal (GLY) and methylglyoxal (MGLY) from seawater and marine aerosol samples is presented. The method is based on derivatisation with o-(2,3,4,5,6-Pentafluorobenzyl)-hydroxylamine (PFBHA) reagent, solvent extraction and GC-MS (SIM) analysis. The method showed good precision (RSD <10%), sensitivity (detection limits in the low ng L-1 range), and accuracy (good agreement between external calibration and standard addition). The method was applied to determine GLY and MGLY in oceanic water sampled during the POLARSTERN cruise ANT XXVII/4 from Capetown to Bremerhaven in spring 2011. GLY and MGLY were determined in the sea surface microlayer (SML) of the ocean and corresponding bulkwater (BW) with average concentrations of 228 ng L-1 (GLY) and 196 ng L-1 (MGLY). The results show a significant enrichment (factor of 4) of GLY and MGLY in the SML. Furthermore, marine aerosol particles (PM1) were sampled during the cruise and analyzed for GLY (average concentration 0.19 ng m-3) and MGLY (average concentration 0.15 ng m-3). On aerosol particles, both carbonyls show a very good correlation with oxalate, supporting the idea of a secondary formation of oxalic acid via GLY and MGLY. Concentrations of GLY and MGLY in seawater and on aerosol particles were correlated to environmental parameters such as global radiation, temperature, distance to the coastline and biological activity. There are slight hints for a photochemical production of GLY and MGLY in the SML (significant enrichment in the SML, higher enrichment at higher temperature). However, a clear connection of GLY and MGLY to global radiation as well as to biological activity cannot be concluded from the data. A slight correlation between GLY and MGLY in the SML and in aerosols could be a hint for interactions of especially GLY between seawater and the atmosphere.

  15. Phosphorylation of α3 Glycine Receptors Induces a Conformational Change in the Glycine-Binding Site

    PubMed Central

    2013-01-01

    Inflammatory pain sensitization is initiated by prostaglandin-induced phosphorylation of α3 glycine receptors (GlyRs) that are specifically located in inhibitory synapses on spinal pain sensory neurons. Phosphorylation reduces the magnitude of glycinergic synaptic currents, thereby disinhibiting nociceptive neurons. Although α1 and α3 subunits are both expressed on spinal nociceptive neurons, α3 is a more promising therapeutic target as its sparse expression elsewhere implies a reduced risk of side-effects. Here we compared glycine-mediated conformational changes in α1 and α3 GlyRs to identify structural differences that might be exploited in designing α3-specific analgesics. Using voltage-clamp fluorometry, we show that glycine-mediated conformational changes in the extracellular M2-M3 domain were significantly different between the two GlyR isoforms. Using a chimeric approach, we found that structural variations in the intracellular M3-M4 domain were responsible for this difference. This prompted us to test the hypothesis that phosphorylation of S346 in α3 GlyR might also induce extracellular conformation changes. We show using both voltage-clamp fluorometry and pharmacology that Ser346 phosphorylation elicits structural changes in the α3 glycine-binding site. These results provide the first direct evidence for phosphorylation-mediated extracellular conformational changes in pentameric ligand-gated ion channels, and thus suggest new loci for investigating how phosphorylation modulates structure and function in this receptor family. More importantly, by demonstrating that phosphorylation alters α3 GlyR glycine-binding site structure, they raise the possibility of developing analgesics that selectively target inflammation-modulated GlyRs. PMID:23834509

  16. Is there a tripeptidyl peptidase in the renal brush-border membrane?

    PubMed Central

    Kenny, A J; Ingram, J

    1988-01-01

    A recent claim that the renal brush border contains a tripeptidyl peptidase [Andersen & McDonald (1987) Am. J. Physiol. 253, F649-F655] was examined. In a fluorescent assay, the hydrolysis of Gly-Pro-Met-2-naphthylamide (-NH-Nap) and Gly-Pro-Leu-NH-Nap by pig kidney microvilli was strongly inhibited by amastatin or di-isopropyl phosphorofluoridate (inhibitors of aminopeptidases and dipeptidyl peptidase IV). The products formed were shown to be Gly-Pro and Met-NH-Nap (or Leu-NH-Nap) and free 2-naphthylamine. Specific antibodies to pig and rat aminopeptidase N abolished the apparent tripeptidyl peptidase activity. We conclude that these substrates are hydrolysed by the sequential attack of dipeptidyl peptidase IV and aminopeptidase N and that pig and rat brush borders lack a detectable tripeptidyl peptidase. Images Fig. 1. PMID:3058122

  17. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ward, G.E.

    When intact Arbacia punctulata spermatozoa are exposed to solubilized egg jelly, the electrophoretic mobility of an abundant sperm flagellar membrane protein changes from an apparent molecular mass of 160 kDa to 150 kDa. A. punctulata spermatozoa can be labeled in vivo with /sup 32/P-labeled cells it was demonstrated that the mobility shift of the 160-kDa protein is due to dephosphorylation. The peptide resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH/sub 2/) is the component of egg jelly which is responsible for inducing the dephosphorylation. The 160/150-kdal sperm membrane protein has been purified to homogeneity by affinity chromatography on concanavalin A-agarose, and identified as sperm guanylate cyclase.more » The enzymatic activity of the guanylate cyclase is tightly coupled to its phosphorylation state. Resact has been shown to act as a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration-dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for external calcium. This work represents the first demonstration of animal sperm chemotaxis in response to a precisely-defined molecule of egg origin. The results established a new, biologically meaningful function for resact, and may implicate sperm guanylate cyclase and cGMP in flagellar function and the chemotactic response.« less

  18. Autosomal dominant retinitis pigmentosa with macular involvement associated with a disease haplotype that included a novel PRPH2 variant (p.Cys250Gly).

    PubMed

    Katagiri, Satoshi; Hayashi, Takaaki; Mizobuchi, Kei; Yoshitake, Kazutoshi; Iwata, Takeshi; Nakano, Tadashi

    2018-06-01

    It is known that PRPH2 variants appear to be rare causes of retinitis pigmentosa (RP) in the Japanese population. The purpose of this study was to describe clinical and genetic features in autosomal dominant RP (adRP) patients with a novel disease-causing variant in the PRHP2 gene. A total of 57 unrelated Japanese probands with adRP were investigated in this study. Comprehensive ophthalmic examinations include fundus photography, fundus autofluorescence imaging, spectral-domain optical coherence tomography, and electroretinography. Whole exome sequencing or Sanger sequencing for 25 targeted exons of multiple genes causing adRP was performed to identify disease-causing variants. Co-segregation and haplotype analyses were performed to determine a disease-causing gene variant and its haplotype. Genetic analysis identified a novel heterozygous PRPH2 variant (c.748T>G, p.Cys250Gly) as disease causing in four probands from four families. The variant co-segregated with the RP phenotype in the eight affected patients in all families. At least three of the four families shared the same haplotype for the variant allele. Clinically, seven of the eight affected patients exhibited typical RP presentation, as well as variable macular involvement including cystoid macular change, vitelliform-like appearance, choroidal neovascularization, and macular atrophy. The same disease haplotype that included a novel PRPH2 variant (p.Cys250Gly) was identified in three of the four Japanese families with adRP, suggesting a founder effect. Our clinical findings indicate that adRP caused by the p.Cys250Gly variant may accompany macular involvement with high frequency.

  19. The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues

    PubMed Central

    Slatter, David A.; Bihan, Dominique G.; Jarvis, Gavin E.; Stone, Rachael; Pugh, Nicholas; Giddu, Sumana; Farndale, Richard W.

    2012-01-01

    Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)10. With Gly-Pro-Cys triplets appended to both of its termini, designated CRPcys, chemical cross-linking using heterobifunctional reagents generates CRPcys-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRPcys antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze–thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays. PMID:22555281

  20. The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues.

    PubMed

    Slatter, David A; Bihan, Dominique G; Jarvis, Gavin E; Stone, Rachael; Pugh, Nicholas; Giddu, Sumana; Farndale, Richard W

    2012-07-01

    Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)(10). With Gly-Pro-Cys triplets appended to both of its termini, designated CRP(cys), chemical cross-linking using heterobifunctional reagents generates CRP(cys)-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP(cys) antagonizes Glycoprotein VI. Here, we describe how cysteine in these triplets may also undergo random air-induced oxidation, especially upon prolonged storage or repeated freeze-thawing, to form disulphide bonds, resulting in a lesser degree of polymerization than with chemical cross-linking. We investigated the monomeric and polymeric states of these and other cysteine-containing collagen-derived peptides, using gel filtration and dynamic light scattering, allowing the size of a CRP-XL aggregate to be estimated. The effect of cysteine thiols upon peptide adsorption to surfaces and subsequent platelet responses was investigated. This demonstrated that cysteine is required for strong binding to glass coverslips and to plastic plates used in ELISA assays. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Breast-Feeding Modulates the Influence of the Peroxisome Proliferator–Activated Receptor-γ (PPARG2) Pro12Ala Polymorphism on Adiposity in Adolescents

    PubMed Central

    Verier, Caroline; Meirhaeghe, Aline; Bokor, Szilvia; Breidenassel, Christina; Manios, Yannis; Molnár, Dénes; Artero, Enrique G.; Nova, Esther; De Henauw, Stefaan; Moreno, Luis A.; Amouyel, Philippe; Labayen, Idoia; Bevilacqua, Noemi; Turck, Dominique; Béghin, Laurent; Dallongeville, Jean; Gottrand, Frédéric

    2010-01-01

    OBJECTIVE The peroxisome proliferator–activated receptor-γ2 (PPARG2) Pro12Ala polymorphism has been associated with a higher BMI and a lower risk of type 2 diabetes in adulthood. The association between adiposity and PPARG variants can be influenced by environmental factors such as early growth, dietary fat, and (as recently shown) breast-feeding. The objectives of this study were to assess 1) the influence of the PPARG2 Pro12Ala polymorphism on adiposity markers in adolescents and 2) a possible modulating effect of breast-feeding on these associations. RESEARCH DESIGN AND METHODS Data on breast-feeding duration, BMI, and genotypes for the Pro12Ala polymorphism were available for 945 adolescents (mean age 14.7 years). The breast-feeding duration was obtained from parental records. We measured weight, height, waist circumference, and six skinfold thicknesses. RESULTS No significant associations between the Pro12Ala polymorphism and any of the above-mentioned anthropometric parameters were found. There were significant interactions between the PPARG2 Pro12Ala polymorphism and breast-feeding with regard to adiposity measurements (all adjusted P < 0.05). Indeed, in children who had not been breast-fed, Ala12 allele carriers had higher adiposity parameters (e.g., Δ BMI +1.88 kg/m2, adjusted for age, sex, and center, P = 0.007) than Pro12Pro adolescents. In contrast, in breast-fed subjects, there was no significant difference between Ala12 allele carriers and Pro12Pro children in terms of adiposity measurements, whatever the duration of breast-feeding. CONCLUSIONS Breast-feeding appears to counter the deleterious effect of the PPARG2 Pro12Ala polymorphism on anthropometric parameters in adolescents. PMID:19846795

  2. Substitutions of cysteine residues of Escherichia coli heat-stable enterotoxin by oligonucleotide-directed mutagenesis.

    PubMed Central

    Okamoto, K; Okamoto, K; Yukitake, J; Kawamoto, Y; Miyama, A

    1987-01-01

    The Escherichia coli 18-amino-acid, heat-stable enterotoxin STp has six cysteine residues linked intramolecularly by three disulfide bonds. These disulfide bonds are important for toxic activity, but the precise role of each bond is not clear. We substituted cysteine residues of STp in vivo by oligonucleotide-directed site-specific mutagenesis to dissociate each disulfide bond and examined the biological activities of the resulting mutants. The Cys-6----Ala and Cys-17----Ala mutations caused a complete loss of toxic activity. The Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17 mutations caused a large decrease in toxic activity. These results mean that all three disulfide bonds formed at fixed positions are required for full expression of the biological activity of STp. However, a weak but significant toxicity still remained after three mutations, Cys-5----Ala, Cys-10----Ser, and Gly-16, Cys-17----Cys-16, Gly-17. This indicates that STp has some flexibilities in its conformation to exert toxic activity and that the role of each disulfide bond exerting toxic activity is not quite the same. Images PMID:3305364

  3. Femtomolar Ln(III) affinity in peptide-based ligands containing unnatural chelating amino acids.

    PubMed

    Niedźwiecka, Agnieszka; Cisnetti, Federico; Lebrun, Colette; Delangle, Pascale

    2012-05-07

    The incorporation of unnatural chelating amino acids in short peptide sequences leads to lanthanide-binding peptides with a higher stability than sequences built exclusively from natural residues. In particular, the hexadentate peptide P(22), which incorporates two unnatural amino acids Ada(2) with aminodiacetate chelating arms, showed picomolar affinity for Tb(3+). To design peptides with higher denticity, expected to show higher affinity for Ln(3+), we synthesized the novel unnatural amino acid Ed3a(2) which carries an ethylenediamine triacetate side-chain and affords a pentadentate coordination site. The synthesis of the derivative Fmoc-Ed3a(2)(tBu)(3)-OH, with appropriate protecting groups for direct use in the solid phase peptide synthesis (Fmoc strategy), is described. The two high denticity peptides P(HD2) (Ac-Trp-Ed3a(2)-Pro-Gly-Ada(2)-Gly-NH(2)) and P(HD5) (Ac-Trp-Ada(2)-Pro-Gly-Ed3a(2)-Gly-NH(2)) led to octadentate Tb(3+) complexes with femtomolar stability in water. The position of the high denticity amino acid Ed3a(2) in the hexapeptide sequence appears to be critical for the control of the metal complex speciation. Whereas P(HD5) promotes the formation of polymetallic species in excess of Ln(3+), P(HD2) forms exclusively the mononuclear complex. The octadentate coordination of Tb(3+) by both P(HD) leads to total dehydration of the metal ion in the mononuclear complexes with long luminescence lifetimes (>2 ms). Hence, we demonstrated that unnatural amino acids carrying polyaminocarboxylate side-chains are interesting building blocks to design high affinity Ln-binding peptides. In particular the novel peptide P(HD2) forms a unique octadentate Tb(3+) complex with femtomolar stability in water and an improvement of the luminescence properties with respect to the trisaquo TbP(22) complex by a factor of 4.

  4. Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1.

    PubMed Central

    Slepkov, Emily R; Chow, Signy; Lemieux, M Joanne; Fliegel, Larry

    2004-01-01

    NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167-->Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE. PMID:14680478

  5. Fluorimetric assay of the neurotensin-degrading metalloendopeptidase, endopeptidase 24.16.

    PubMed

    Dauch, P; Barelli, H; Vincent, J P; Checler, F

    1991-12-01

    Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp (Mcc = 3-carboxy-7-methoxycoumarin; Dnp = dinitrophenyl), a quenched fluorimetric substrate originally designed as a probe to measure Pz-peptidase (also called endopeptidase 24.15), was examined as a putative substrate of the neurotensin-degrading neutral metalloendopeptidase, endopeptidase 24.16. During the purification of endopeptidase 24.16 the neurotensin(1-10) and neurotensin(11-13) formation due to this enzyme was coeluted with Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp-hydrolysing activity. By both fluorimetric and h.p.l.c. analyses, we observed that the latter activity was dose-dependently and completely abolished by neurotensin with an IC50 value (2.6 microM) that closely corresponds to the affinity of purified endopeptidase 24.16 for neurotensin (Km = 2.5 microM). Furthermore, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp hydrolysis was inhibited by a series of dipeptides with a rank of order of potencies that parallels that observed in competition experiments of tritiated neurotensin hydrolysis by brain and intestinal endopeptidase 24.16. Altogether, these data clearly demonstrate that, in addition to Pz-peptidase, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp also behaves as a substrate of endopeptidase 24.16, with a Km of about 26 microM. In addition, we show that, even in crude membrane preparations, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp behaves as a useful tool to monitor and accurately quantify endopeptidase 24.16.

  6. Fluorimetric assay of the neurotensin-degrading metalloendopeptidase, endopeptidase 24.16.

    PubMed Central

    Dauch, P; Barelli, H; Vincent, J P; Checler, F

    1991-01-01

    Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp (Mcc = 3-carboxy-7-methoxycoumarin; Dnp = dinitrophenyl), a quenched fluorimetric substrate originally designed as a probe to measure Pz-peptidase (also called endopeptidase 24.15), was examined as a putative substrate of the neurotensin-degrading neutral metalloendopeptidase, endopeptidase 24.16. During the purification of endopeptidase 24.16 the neurotensin(1-10) and neurotensin(11-13) formation due to this enzyme was coeluted with Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp-hydrolysing activity. By both fluorimetric and h.p.l.c. analyses, we observed that the latter activity was dose-dependently and completely abolished by neurotensin with an IC50 value (2.6 microM) that closely corresponds to the affinity of purified endopeptidase 24.16 for neurotensin (Km = 2.5 microM). Furthermore, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp hydrolysis was inhibited by a series of dipeptides with a rank of order of potencies that parallels that observed in competition experiments of tritiated neurotensin hydrolysis by brain and intestinal endopeptidase 24.16. Altogether, these data clearly demonstrate that, in addition to Pz-peptidase, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp also behaves as a substrate of endopeptidase 24.16, with a Km of about 26 microM. In addition, we show that, even in crude membrane preparations, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp behaves as a useful tool to monitor and accurately quantify endopeptidase 24.16. PMID:1747117

  7. Impact of GNB3-C825T, ADRB3-Trp64Arg, UCP2-3′UTR 45 bp del/ins, and PPARγ-Pro12Ala Polymorphisms on Bofutsushosan Response in Obese Subjects: A Randomized, Double-Blind, Placebo-Controlled Trial

    PubMed Central

    Park, Junghyun; Bose, Shambhunath; Hong, Sun-Woo; Lee, Dong-Ki; Yoo, Jae-Wook; Lim, Chi-Yeon; Lee, Myeongjong

    2014-01-01

    Abstract Obesity is known to be influenced by a number of genes, including the β3 subunit of G protein (GNB3), β3-adrenergic receptor (ADRB3), uncoupling protein 2 (UCP2), and peroxisome proliferator activated receptor gamma (PPARγ). The single nucleotide polymorphisms (SNPs) of the above genes, such as GNB3-C825T, ADRB3-Trp64Arg, UCP2-3′UTR 45 bp del/ins, and PPARγ-Pro12Ala, are associated with obesity and body mass index. The present study evaluates the impact of Bofutsushosan, a traditional Eastern Asian herbal medicine with known anti-obesity properties, on obese subjects according to the presence of the above-mentioned SNPs. Upon randomization, the volunteers were allocated to receive Bofutsushosan (n=55) or placebo (n=56) treatments for 8 weeks. Following the treatment schedule, significant reductions in total cholesterol and significant improvement in the Korean version of obesity-related quality of life scale were seen in the Bofutsushosan-treated group, but not in placebo. Bofutsushosan exerted significant anti-obesity effects on a number of parameters in the carriers of the GNB3-825T allele, but only on waist circumference in the GNB3-C/C homozygote. Significant anti-obesity impact of Bofutsushosan was also seen on a number of obesity-indices in both ADRB3-Arg64 carriers and ADRB3-Trp64 homozygotes, as well as in UCP2-D/D carriers, but not in UCP2-D/I+I/I variants. The effect of Bofutsushosan was more pronounced in PPARγ-Pro/Pro genotype compared to PPARγ-Pro/Ala variants. Thus, the results revealed differential responses of the subjects to the anti-obesity effects of Bofutsushosan treatment according to the polymorphism of the vital obesity-related genes. Our study provides new insight into individualized clinical applications of Bofutsushosan for obesity. PMID:24827746

  8. Loss of the Arabidopsis thaliana P₄-ATPase ALA3 reduces adaptability to temperature stresses and impairs vegetative, pollen, and ovule development.

    PubMed

    McDowell, Stephen C; López-Marqués, Rosa L; Poulsen, Lisbeth R; Palmgren, Michael G; Harper, Jeffrey F

    2013-01-01

    Members of the P4 subfamily of P-type ATPases are thought to help create asymmetry in lipid bilayers by flipping specific lipids between the leaflets of a membrane. This asymmetry is believed to be central to the formation of vesicles in the secretory and endocytic pathways. In Arabidopsis thaliana, a P4-ATPase associated with the trans-Golgi network (ALA3) was previously reported to be important for vegetative growth and reproductive success. Here we show that multiple phenotypes for ala3 knockouts are sensitive to growth conditions. For example, ala3 rosette size was observed to be dependent upon both temperature and soil, and varied between 40% and 80% that of wild-type under different conditions. We also demonstrate that ala3 mutants have reduced fecundity resulting from a combination of decreased ovule production and pollen tube growth defects. In-vitro pollen tube growth assays showed that ala3 pollen germinated ∼2 h slower than wild-type and had approximately 2-fold reductions in both maximal growth rate and overall length. In genetic crosses under conditions of hot days and cold nights, pollen fitness was reduced by at least 90-fold; from ∼18% transmission efficiency (unstressed) to less than 0.2% (stressed). Together, these results support a model in which ALA3 functions to modify endomembranes in multiple cell types, enabling structural changes, or signaling functions that are critical in plants for normal development and adaptation to varied growth environments.

  9. Processing of an anglerfish somatostatin precursor to a hydroxylysine-containing somatostatin 28.

    PubMed Central

    Spiess, J; Noe, B D

    1985-01-01

    A novel 28-residue somatostatin (SS) has been isolated from anglerfish pancreatic islets and characterized by complete Edman degradation, peptide mapping, and amino acid analysis. The primary structure of this anglerfish SS-28 (aSS-28) containing hydroxylysine (Hyl) was established to be H-Ser-Val-Asp-Ser-Thr-Asn-Asn-Leu-Pro-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys- Lys-Asn-Phe-Tyr-Trp-Hyl-Gly-Phe-Thr-Ser-Cys-OH. This sequence (with the exception of hydroxylysine-23, which is replaced by lysine) is identical to the sequence of the COOH-terminal 28 residues of prepro-SS II predicted on the basis of cDNA analysis [Hobart, P., Crawford, R., Shen, L., Pictet, R. & Rutter, W. J. (1980) Nature (London) 288, 137-141]. This is the first instance in which hydroxylysine (to date characteristically observed in collagen or collagen-like structures) has been found in a potential regulatory peptide. Chromatographic characterization of peptides, radiolabeled in islet culture, revealed that aSS-28 contained 10-12% of the radioactivity incorporated into the 8000- to 1000-dalton SS-like polypeptides, whereas 88-90% of this radioactivity was detected in anglerfish SS-14. It appears probable that aSS-28 represents the predominant primary cleavage product derived from prepro-SS II by cleavage at the COOH-terminal side of a single arginine. Based on knowledge of the collagen biosynthesis, it is speculated that hydroxylation may take place as an early post-translational event. Images PMID:2857489

  10. PPARγ Pro12Ala interacts with fat intake for obesity and weight loss in a behavioural treatment based on the Mediterranean diet

    PubMed Central

    Garaulet, Marta; Smith, Caren E; Hernández-González, Teresa; Lee, Yu-Chi; Ordovás, Jose M.

    2014-01-01

    Scope The goal of this study was to examine whether the Pro12Ala polymorphism of peroxisome proliferator-activated receptor γ (PPARγ) is associated with insulin resistance, obesity and weight loss and to analyze potential interactions between fat intake and PPARγ polymorphism in a Spanish overweight/obese population. Materials and methods We recruited 1465 subjects enrolled in a behavioural treatment program for obesity based on a Mediterranean diet, which included the following: dietary treatment, physical activity, nutritional education and behavioral techniques. A significant association was found between PPARγ2 Pro12Ala genotype and plasma insulin concentration and homeostasis model assessment insulin resistance. Subjects with the Ala12 genotype had lower insulin levels than those with the Pro12Pro genotype. We detected a gene–diet interaction between the PPARγ Pro12Ala polymorphism and MUFA for BMI and body fat. Furthermore, we detected an interaction between the PPARγ Pro12Ala polymorphism and fat intake for total weight loss (p<0.001). When total fat intake was high, Ala12-carriers exhibited a significantly lower percentage of total weight loss than major-allele-carriers (p=0.037). Conclusion Data are consistent with previous results showing a protective role for the Ala12 allele against insulin resistance, and replicate an earlier study that detected an interaction between dietary MUFA and PPARγ2 for BMI. Our detection of a gene–diet interaction between PPARγ Pro12Ala and fat intake for weight loss may explain previous discrepancies among different studies. PMID:22102511

  11. Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem.

    PubMed

    Patin, Delphine; Bostock, Julieanne; Chopra, Ian; Mengin-Lecreulx, Dominique; Blanot, Didier

    2012-06-01

    Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.

  12. Formation of g-C3N4@Ni(OH)2 Honeycomb Nanostructure and Asymmetric Supercapacitor with High Energy and Power Density.

    PubMed

    Dong, Bitao; Li, Mingyan; Chen, Sheng; Ding, Dawei; Wei, Wei; Gao, Guoxin; Ding, Shujiang

    2017-05-31

    Nickel hydroxide (Ni(OH) 2 ) has been regarded as a potential next-generation electrode material for supercapacitor owing to its attractive high theoretical capacitance. However, practical application of Ni(OH) 2 is hindered by its lower cycling life. To overcome the inherent defects, herein we demonstrate a unique interconnected honeycomb structure of g-C 3 N 4 and Ni(OH) 2 synthesized by an environmentally friendly one-step method. In this work, g-C 3 N 4 has excellent chemical stability and supports a perpendicular charge-transporting direction in charge-discharge process, facilitating electron transportation along that direction. The as-prepared composite exhibits higher specific capacities (1768.7 F g -1 at 7 A g -1 and 2667 F g -1 at 3 mV s -1 , respectively) compared to Ni(OH) 2 aggregations (968.9 F g -1 at 7 A g -1 ) and g-C 3 N 4 (416.5 F g -1 at 7 A g -1 ), as well as better cycling performance (∼84% retentions after 4000 cycles). As asymmetric supercapacitor, g-C 3 N 4 @Ni(OH) 2 //graphene exhibits high capacitance (51 F g -1 ) and long cycle life (72% retentions after 8000 cycles). Moreover, high energy density of 43.1 Wh kg -1 and power density of 9126 W kg -1 has been achieved. This attractive performance reveals that g-C 3 N 4 @Ni(OH) 2 with honeycomb architecture could find potential application as an electrode material for high-performance supercapacitors.

  13. Hydroxyapatite-binding peptides for bone growth and inhibition

    DOEpatents

    Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

    2011-09-20

    Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

  14. Targeting a Cross-Reactive Gly m 5 Soy Peptide as Responsible for Hypersensitivity Reactions in a Milk Allergy Mouse Model

    PubMed Central

    Curciarello, Renata; Smaldini, Paola L.; Candreva, Angela M.; González, Virginia; Parisi, Gustavo; Cauerhff, Ana; Barrios, Ivana; Blanch, Luis Bruno; Fossati, Carlos A.

    2014-01-01

    Background Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity. Methods Cow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy. Results Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice. Conclusions Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide. PMID:24416141

  15. Targeting a cross-reactive Gly m 5 soy peptide as responsible for hypersensitivity reactions in a milk allergy mouse model.

    PubMed

    Curciarello, Renata; Smaldini, Paola L; Candreva, Angela M; González, Virginia; Parisi, Gustavo; Cauerhff, Ana; Barrios, Ivana; Blanch, Luis Bruno; Fossati, Carlos A; Petruccelli, Silvana; Docena, Guillermo H

    2014-01-01

    Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity. Cow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy. Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice. Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide.

  16. Eco-Friendly Insecticide Discovery via Peptidomimetics: Design, Synthesis, and Aphicidal Activity of Novel Insect Kinin Analogues.

    PubMed

    Zhang, Chuanliang; Qu, Yanyan; Wu, Xiaoqing; Song, Dunlun; Ling, Yun; Yang, Xinling

    2015-05-13

    Insect kinin neuropeptides are pleiotropic peptides that are involved in the regulation of hindgut contraction, diuresis, and digestive enzyme release. They share a common C-terminal pentapeptide sequence of Phe(1)-Xaa(2)-Yaa(3)-Trp(4)-Gly(5)-NH2 (where Xaa(2) = His, Asn, Phe, Ser, or Tyr; Yaa(3) = Pro, Ser, or Ala). Recently, the aphicidal activity of insect kinin analogues has attracted the attention of researchers. Our previous work demonstrated that the sequence-simplified insect kinin pentapeptide analogue Phe-Phe-[Aib]-Trp-Gly-NH2 could retain good aphicidal activity and be the lead compound for the further discovery of eco-friendly insecticides which encompassed a broad array of biochemicals derived from micro-organisms and other natural sources. Using the peptidomimetics strategy, we chose Phe-Phe-[Aib]-Trp-Gly-NH2 as the lead compound, and we designed and synthesized three series, including 31 novel insect kinin analogues. The aphicidal activity of the new analogues against soybean aphid was determined. The results showed that all of the analogues exhibited aphicidal activity. Of particular interest was the analogue II-1, which exhibited improved aphicidal activity with an LC50 of 0.019 mmol/L compared with the lead compound (LC50 = 0.045 mmol/L) or the commercial insecticide pymetrozine (LC50 = 0.034 mmol/L). This suggests that the analogue II-1 could be used as a new lead for the discovery of potential eco-friendly insecticides.

  17. Phenotype of hepatocyte spheroids in Arg-GLY-Asp (RGD) containing a thermo-reversible extracellular matrix.

    PubMed

    Park, Keun-Hong; Bae, You Han

    2002-07-01

    The spheroid of specific cells is often regarded as the better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions. In this study, freshly harvested primary rat hepatocytes, which had been cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined for differentiated morphology and enhanced liver-specific functions as compared to a control set (hepatocytes in single-cell form). A copolymer of N-isopropylacrylamide (98 mole % in the feed) and acrylic acid (poly(NiPAAm-co-AAc)), and the adhesion molecule, an Arg-Gly-Asp (RGD)-incorporated thermo-reversible matrix, were used to entrap hepatocytes in the form of either spheroids or single cells. In a 28-day culture period, the spheroids in the RGD-incorporated gel maintained higher viability and produced albumin and urea at constant rates, while there was lower cell viability and less albumin secretion by the spheroids in p(NiPAAm-co-AAc). Hepatocytes cultured as spheroids in the RGD-incorporated gel would constitute a potentially useful three-dimensional cell system for application in a bio-artificial liver device.

  18. Primary structure of a guanyl-specific ribonuclease from the fungus Penicillium brevicompactum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kulikov, V.A.; Shlyapnikov, S.V.; Yakovlev, G.I.

    1986-01-01

    By the automatic Edman degradation of the intact S-carboxymethylated protein and a mixture of the products of its proteolytic cleavage at Arg, Lys, and Glu residues, together with results on the kinetics of the proteolysis of the protein under the action of carboxypeptidase Y, the primary structure of the extracellular guanyl-specific RNase of the fungus Penicillium brevicompactum has been determined. The RNase contains 102 amino acid residues: 7 Asp, 7 Asn, 9 Thr, 11 Ser, 4 Glu, 1 Gln, 4 Pro, 10 Gly, 11 Ala, 4 Cys, 7 Val, 4 Ile, 3 Leu, 9 Tyr, 5 Phe, 2 Lys, 3more » His, 1 Arg (M/sub r/ 10,801). It has been established that four hemicystine residues of the P. compactum RNase form, in pairs, two disulfide bonds« less

  19. Vascular Ehlers-Danlos syndrome mutations in type III collagen differently stall the triple helical folding.

    PubMed

    Mizuno, Kazunori; Boudko, Sergei; Engel, Jürgen; Bächinger, Hans Peter

    2013-06-28

    Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)7 with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)7-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.

  20. Evaluation of glucose metabolism and reproductive hormones in polycystic ovary syndrome on the basis of peroxisome proliferator-activated receptor (PPAR)-gamma2 Pro12Ala genotype.

    PubMed

    Tok, E C; Aktas, A; Ertunc, D; Erdal, E M; Dilek, S

    2005-06-01

    Peroxisome proliferator-activated receptor (PPAR)-gamma2 Pro12Ala polymorphism has been suggested as a protective factor for polycystic ovary syndrome (PCOS). In this study, we aimed to investigate metabolic features and reproductive hormones in women with PCOS and compare these features with control women on the basis of Pro12Ala genotype. This study involved 60 randomly selected women with PCOS and 60 controls. Main outcome measures were anthropometric measures, variables of glucose metabolism and reproductive hormones. All the patients were genotyped for Pro12Ala variant of PPAR-gamma2 gene. Patients with Pro12Ala polymorphism were more obese in both groups. Furthermore, they had lower fasting insulin levels, were less insulin-resistant and were less glucose-intolerant as demonstrated by 2 h glucose concentrations. However, there was no difference in reproductive hormone levels on the basis of Pro12Ala genotype. Both control women and women with PCOS had significant differences in glucose metabolism on the basis of PPAR-gamma2 Pro12Ala polymorphism. Pro12Ala variant may break the process that leads to PCOS in susceptible women, instead of being a direct causal relationship between Pro12Ala polymorphism and PCOS.