Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:P30536 (PBS)
9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

FPyME (1-[3-(2-fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione) was designed as a [(18)F]fluoropyridine-based maleimide reagent for the prosthetic labeling of peptides and proteins via selective conjugation with a thiol (sulfhydryl) function. Its pyridinyl moiety carries the radioactive halogen (fluorine-18) which can be efficiently incorporated via a nucleophilic heteroaromatic substitution, and its maleimido function ensures the efficient alkylation of a free thiol function as borne by cysteine residues. [(18)F]FPyME (HPLC-purified) was prepared in 17-20% non-decay-corrected yield, based on starting [(18)F]fluoride, in 110 min using a three-step radiochemical pathway. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination on [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as the fluorine-18 incorporation step, followed by (2) rapid and quantitative TFA-induced removal of the N-Boc-protective group and (3) optimized maleimide formation using N-methoxycarbonylmaleimide. Typically, 4.8-6.7 GBq (130-180 mCi) of radiochemically pure [(18)F]FPyME ([(18)F]-1) could be obtained after semipreparative HPLC in 110 min starting from a cyclotron production batch of 33.3 GBq (900 mCi) of [(18)F]fluoride (overall radiochemical yields, based on starting [(18)F]fluoride: 28-37% decay-corrected). [(18)F]FPyME ([(18)F]-1) was first conjugated with a small model hexapeptide ((N-Ac)KAAAAC), confirming the excellent chemoselectivity of the coupling reaction (CH(2)SH versus CH(2)NH(2)) and then conjugated with two 8-kDa proteins of interest, currently being developed as tumor imaging agents (c-AFIM-0 and c-STxB). Conjugation was achieved in high yields (60-70%, isolated and non-decay-corrected) and used optimized, short-time reaction conditions (a 1/9 (v/v) mixture of DMSO and 0.05 M aq Tris NaCl buffer (pH 7.4) or 0.1 M aq PBS (pH 8), at room temperature for 10 min) and purification conditions (a gel filtration using a Sephadex NAP-10 cartridge or a SuperDex Peptide HR 10/30 column), both compatible with the chemical stability of the proteins and the relatively short half-life of the radioisotope concerned. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the protein and the final purification took 130-140 min. [(18)F]FPyME ([(18)F]-1) represents a new, valuable, thiol-selective, fluorine-18-labeled reagent for the prosthetic labeling with fluorine-18 of peptides and proteins. Because of its excellent chemoselectivity, [(18)F]FPyME offers an interesting alternative to the use of the nonselective carboxylate and amine-reactive [(18)F]reagents and can therefore advantageously be used for the design and development of new peptide- and protein-based radiopharmaceuticals for PET.
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PMID:1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione: design, synthesis, and radiosynthesis of a new [18F]fluoropyridine-based maleimide reagent for the labeling of peptides and proteins. 1576 96

A glassy carbon electrode modified with platinum nanoparticle-decorated carbon nanotubes (Pt-CNT/GCE) was prepared. The electrochemical behaviors for the catalysis oxidations of hydrogen peroxide and cysteine were studied. The Pt-CNT/GCE showed catalytic activity for electro-oxidation of hydrogen peroxide at 0.6 V in PBS (pH = 7.0) and for that of cysteine at 0.55 V in sulfuric acid medium (pH <or = 2). The results indicated that the peak currents were proportional to the concentrations of both hydrogen peroxide and cysteine; the currents at Pt-CNT/GCE were almost 3-times higher than that at Pt electrode. The electrocatalytic oxidation mechanisms for hydrogen peroxide and cysteine at Pt-CNT/GCE surface were briefly studied.
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PMID:Electrocatalytic oxidation of hydrogen peroxide and cysteine at a glassy carbon electrode modified with platinum nanoparticle-deposited carbon nanotubes. 1584 29

The role of free radicals in protein modification and the importance of anti-4-hydroxy-2-nonenal (HNE) antibodies as marker of HNE-mediated cell toxicity has been well documented. Proteins modified by HNE in vitro, prior to immobilization on ELISA plates, have served as substrates for assaying these antibodies. We found preferential binding of HNE-modified versus unmodified proteins to ELISA plates and this prompted us to seek a more reliable assay. We report a method to HNE-modify any cysteine/histidine/lysine-containing protein or multiple antigenic peptide (MAP) following their immobilization on an ELISA plate. To a set of wells, HNE (200 microM) dissolved in PBS is added and incubated for 4 h, followed by regular ELISA. Since HNE was supplied dissolved in ethanol, PBS with appropriate amount of ethanol added was used as control. For inhibition experiments, HNE is incubated with or without inhibitors and then added to the wells. The commercial anti-HNE serum bound only to HNE-modified antigens. Sera from rabbits and mice immunized with HNE-modified 60 kDa Ro autoantigen preferentially bound the modified antigens. Modification of solid phase antigens in this manner makes assaying anti-HNE antibodies unambiguous. Lengthy dialysis procedures or the use of spin columns that lead to antigen loss becomes unnecessary for the separation of free HNE. We were able to HNE-modify various antigens (BSA, the autoantigens Ro, La and Sm/nRNP, 60 kDa Ro and Sm MAPs) using this procedure. Using MAPs, we confirmed the importance of histidine, lysine and cysteine residues in HNE modification. In addition, this method allowed identification of inhibitors of HNE-modification. We obtained 61%, 70% and 74% inhibition of HNE-modification of solid phase Ro MAP 166 substrate using BSA, Ro MAP 482 and Ro MAP 166, respectively. Glycyl-proline dipeptide and a MAP from the Sm autoantigen (PPPGMRPP) showed 0% inhibition of HNE-modification.
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PMID:In vitro modification of solid phase multiple antigenic peptides/autoantigens with 4-hydroxy-2-nonenal (HNE) provide ideal substrates for detection of anti-HNE antibodies and peptide antioxidants. 1605 45

A new family of heterobifunctional linkers (L1-L9) containing a terminus consisting of a tridentate donor set for coordination of the {M(CO)(3)}(+) core (M = Tc, Re), and a thiol reactive maleimide group has been prepared conveniently and in high yield under Mitsunobu reaction conditions by the coupling of an appropriate alcohol derivative with maleimide. The rhenium complexes [Re(CO)(3)(Lx)]Br (x= 1-9) were prepared in good yields from the reactions of the ligands and (NEt(4))(2)[Re(CO)(3)Br(3)] in refluxing methanol. The ligands and their Re complexes were characterized by (1)H and (13)C NMR, IR, and ESI-MS. Ligand L4 and [Re(CO)(3)(L5)]Br have been structurally characterized by X-ray crystallography. Photoexcitation of solutions of the complexes [Re(CO)(3)(Lx)]Br (x= 4-6) gives rise to intense and prolonged luminescence at room temperature (fluorescence lifetimes of ca. 16 micros). The ligands and their Re complexes react smoothly at the maleimide linker with sulfhydryl groups of peptides and proteins at room temperature in phosphate-buffered saline (PBS, pH 7.4) to form stable thioether bioconjugates. The photoluminescence properties of the labeled conjugates are similar to those of the parent complexes, but with even longer lifetimes. The ligands can also be labeled at room temperature with (99m)Tc to give chemically robust complexes. The corresponding hydrazinonicotinamide derivative N-[5-(6'-hydrazinopyridine-3'-carbonyl)aminopentyl]maleimide (L10) was also prepared. While coupling of L10 to cysteine ethylester and synthesis of the rhenium derivative [ReCl(3)(HYNIC-maleimide)(2)] were successfully accomplished, attempts to couple [ReCl(3)(HYNIC-maleimide)(2)] to glutathione or BSA yielded intractable mixtures.
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PMID:Design and synthesis of site directed maleimide bifunctional chelators for technetium and rhenium. 1631 43

Pseudallescheria boydii is an opportunistic filamentous fungus that causes serious infections in humans. Virulence attributes expressed by P. boydii are unknown. Conversely, peptidases are incriminated as virulence factors in several pathogenic fungi. Here we investigated the extracellular peptidase profile in P. boydii. After growth on Sabouraud for 7 days, mycelia of P. boydii were incubated for 20 h in PBS-glucose. The cell-free PBS-glucose supernatant was submitted to SDS-PAGE and 12 secretory polypeptides were observed. Two of these polypeptides (28 and 35 kD) presented proteolytic activity when BSA was used as a copolymerized substrate. The extracellular peptidases were most active in acidic pH (5.5) and fully inhibited by 1,10-phenanthroline, a zinc-metallopeptidase inhibitor. Other metallo-, cysteine, serine and aspartic proteolytic inhibitors did not significantly alter these activities. To confirm that these enzymes belong to the metallo-type peptidases, the apoenzymes were obtained by dialysis against chelating agents, and supplementation with different cations, especially Cu(2+) and Zn(2+), restored their activities. Except for gelatin, both metallopeptidases hydrolyzed various co-polymerized substrates, including human serum albumin, casein, hemoglobin and IgG. Additionally, the metallopeptidases were able to cleave different soluble proteinaceous substrates such as extracellular matrix components and sialylated proteins. All these hydrolyses were inhibited by 1,10-phenanthroline. Interestingly, Scedosporium apiospermum (the anamorph of P. boydii) produced a distinct extracellular peptidase profile. Collectively, our results demonstrated for the first time the expression of acidic extracellular metallopeptidases in P. boydii capable of degrading several proteinaceous compounds that could help the fungus to escape from natural human barriers and defenses.
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PMID:Pseudallescheria boydii releases metallopeptidases capable of cleaving several proteinaceous compounds. 1648 86

A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen was diluted with PBS or defined milk protein extender and was either stored directly or 90% of seminal plasma was removed by centrifugation and replaced by defined milk protein extender before storage. In both experiments, eight stallions were available for semen collections. Motility, velocity and membrane integrity of spermatozoa were determined by CASA immediately after semen processing and after 24, 48 and 72 h of storage at 5 degrees C. Total motility after 24 h of storage was lowest in semen diluted with PBS (p<0.05 versus all extenders). At 48 and 72 h, motility of spermatozoa in defined milk protein extender was significantly (p<0.05) higher than in PBS or skim milk extender. Velocity of spermatozoa after storage was highest in defined milk protein extender. Membrane integrity after storage was significantly (p<0.05) lower in semen diluted with PBS than in semen diluted with both extenders. Addition of NAC was without effect on the examined parameters. Centrifugation further increased the percentage of motile and membrane-intact spermatozoa in the defined milk protein extender (p<0.05). Velocity of spermatozoa in this extender was not negatively affected by centrifugation.
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PMID:Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees C. 1662 Sep 43

Bilirubin, the final product of heme catabolism, plays a crucial role in the cellular defense against oxidative and nitrosative stress. This study investigated the interaction of albumin-bound bilirubin, the circulating form of the bile pigment, with nitric oxide (NO), a gaseous modulator involved in many physiological functions but able to induce cytotoxicity and cell death if produced in excess. A short-lived endogenous S-nitrosothiol such as S-nitroso-cysteine was used as NO donor. In PBS without chelators, bilirubin was bound to human serum albumin with an apparent affinity of 1.6 +/- 0.2 microM (n = 4). Furthermore, albumin (2-20 microM) dose-dependently increased the half-life of BR (10 microM) exposed to S-nitroso-cysteine (100 microM) of 2.4 +/- 0.4 times (n = 4). Albumin-bound bilirubin was almost completely oxidized by S-nitroso-cysteine-derived NO, and biliverdin was the major product formed; this reaction seemed to be rather specific for albumin-bound bilirubin because when free bilirubin was reacted with S-nitroso-cysteine the formation of biliverdin was significantly lower. Uric acid and reduced glutathione, two well-known plasma antioxidants, at physiological concentrations protected albumin-bound bilirubin from NO-mediated oxidation. Taken together, these data suggest that albumin-bound bilirubin maintains its ability to interact with NO also in the bloodstream counteracting extracellular nitrosative reactions.
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PMID:Albumin-bound bilirubin interacts with nitric oxide by a redox mechanism. 1667 92

Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (beta-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to beta-lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with beta-lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops.
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PMID:Mapping epitopes and antigenicity by site-directed masking. 1675 78

Previously we established the hypotensive action of nitric oxide donors, dinitrosyl-iron complexes (DNIC) with thiol-containing ligands, stored in frozen solution at 77K. In the present study, we tested recently designed water soluble dry powder preparations of DNICs keeping their characteristics in dry air for a long time. The complexes dissolved in PBS were injected intravenously into normotensive Wistar and spontaneously hypertensive SHR rats. The average arterial pressure (AAP) was recorded through preliminary implanted catheter in a carotid artery. The initial hypotensive action of DNIC with cysteine (DNIC-cys) was comparable to action of nitroprusside (SNP) but, in contrast to the latter, lasted for 20-120min depending on a doze. The blood DNIC content as detected by electronic paramagnetic resonance steadily decreased at this time. The hypotensive action of S-nitrosocysteine was similar to SNP while binding of iron in DNIC by batophenantroline-disulphonate prevented its hypotensive effect. These data suggest that long-lasting hypotensive action of DNICs may be caused by stable protein-bound DNICs forming in the process of transfer of Fe(+)(NO(+))(2) moieties from low-molecular DNICs to thiol protein ligands. The relative initial dose-dependent effect of DNIC-cys was similar in Wistar and SHR but secondary AAP reduction was more profound in SHR. A substitution of cysteine in DNIC by thiosulphate resulted in markedly less initial AAP reduction while long-lasting effect was similar and substitution by glutathione smoothed initial AAP decline and stabilized AAP level in the second phase. Prolonged AAP reduction induced by DNIC-cys was considerably shortened in narcotized rats. Thus, dry preparations of DNICs preserve prolonged hypotensive activity.
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PMID:Long-lasting hypotensive action of stable preparations of dinitrosyl-iron complexes with thiol-containing ligands in conscious normotensive and hypertensive rats. 1747 15

A new group of hybrid nitric oxide-releasing anti-inflammatory drugs wherein an O(2)-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (11a-d), or 2-nitrooxyethyl (12a-d), (*)NO-donor moiety is attached directly to the carboxylic acid group of (E)-3-(4-methanesulfonylphenyl)-2-(phenyl)acrylic acids were synthesized. The 2-nitrooxyethyl ester prodrugs (12a-d) all exhibited in vitro inhibitory activity against the cyclooxygenase-2 (COX-2) isozyme (IC(50)=0.07-2.8 microM range). All compounds released a low amount of (*)NO upon incubation with phosphate buffer (PBS) at pH 7.4 (1.0-4.8% range). In comparison, the percentage (*)NO released was significantly higher (76.2-83.0% range) when the diazen-1-ium-1,2-diolate ester prodrugs were incubated in the presence of rat serum, or moderately higher (7.6-10.1% range) when the nitrooxyethyl ester prodrugs were incubated in the presence of L-cysteine. These incubation studies suggest that both (*)NO and the parent anti-inflammatory (E)-3-(4-methanesulfonylphenyl)-2-(phenyl)acrylic acid would be released upon in vivo cleavage by non-specific serum esterases in the case of the diazen-1-ium-1,2-diolate esters (11a-d), or interaction with systemic thiols in the case of the nitrate esters (12a-d). O(2)-Acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (E)-3-(4-methanesulfonylphenyl)-2-phenylacrylate (11a) released 83% of the theoretical maximal release of 2 molecules of (*)NO/molecule of the parent hybrid ester prodrug upon incubation with rat serum. Hybrid ester anti-inflammatory/(*)NO donor prodrugs offer a potential drug design concept targeted toward the development of anti-inflammatory drugs that are devoid of adverse ulcerogenic and/or cardiovascular effects.
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PMID:Diazen-1-ium-1,2-diolated and nitrooxyethyl nitric oxide donor ester prodrugs of anti-inflammatory (E)-2-(aryl)-3-(4-methanesulfonylphenyl)acrylic acids: synthesis, cyclooxygenase inhibition, and nitric oxide release studies. 1809 94


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