scholarly journals Effector-sensitive cross-linking of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-dione

1998 ◽  
Vol 331 (1) ◽  
pp. 137-141 ◽  
Author(s):  
Nancy A. AYERS ◽  
Owen W. NADEAU ◽  
Mark W. READ ◽  
Partha RAY ◽  
Gerald M. CARLSON
Keyword(s):  
Gel Electrophoresis ◽  
Conjugate Addition ◽  
Structural Features ◽  
Subunit Composition ◽  
Side Chain ◽  
Cross Linking ◽  
Initial Reaction ◽  
The Novel ◽  
Cross Linker ◽  
Novel Protein

The dienophile 4-phenyl-1,2,4-triazoline-3,5-dione (PTD) was identified as a novel protein cross-linker, and utilized as a conformational probe of phosphorylase b kinase (PhK), a hexadecameric enzyme with the subunit composition (αβγδ)4. In its reaction with this enzyme, PTD produced five major cross-linked conjugates as resolved by denaturing gel electrophoresis: αβ, βγγ, αγ and a doublet of differently migrating homodimers, ββ1 and ββ2. Cross-linking in the presence of six different activators of the kinase targeted to its various subunits caused substantial changes in the amounts of three of the conjugates. The formation of αγ was increased by all of the activators but the largest enhancement was caused by exogenous Ca2+/calmodulin. All except one of the activators decreased the amount of βγγ formed, with Mg2+ having the greatest effect, and all except two increased the amount of ββ1, with Mg2+ again having the largest influence. From the overall similarity of the changes in cross-linking by PTD induced by the various activators, we conclude that, even though they are targeted to different sites and subunits, they induce activated conformations of PhK that have certain structural features in common. Regarding the mechanism of cross-linking by PTD, its reaction with a model nucleophile suggests that its initial reaction with a side chain nucleophile of PhK involves a 1,4-conjugate addition to form a urazole adduct, with the secondary cross-linking reaction occurring through an as yet unknown pathway.

scholarly journals Anomalous electrophoretic behaviour of the glutathione S-transferase Ya and Yk subunits isolated from man and rodents. A potential pitfall for nomenclature

1986 ◽  
Vol 237 (3) ◽  
pp. 731-740 ◽  
Author(s):  
J D Hayes ◽  
T J Mantle
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Variable Cross ◽  
Glutathione S Transferase ◽  
Relative Electrophoretic Mobility ◽  
Low Concentrations ◽  
Cross Linker ◽  
Low Degrees

GSH S-transferases are dimeric enzymes. The subunits in the rat are resolved into six types, designated Yf, Yk, Ya, Yn, Yb and Yc, by discontinuous SDS/polyacrylamide-gel electrophoresis [Hayes (1986) Biochem. J. 233, 789-798]. The relative electrophoretic mobility of the Ya and Yk subunits is dependent on the amount of cross-linker (NN'-methylenebisacrylamide) in the resolving gel. At low degrees of cross-linking, CBis 0.6% (w/w), the Yk and Ya subunits possess a faster anodal mobility than do the Yf, Yn, Yb and Yc subunits (i.e. order of mobility Yk greater than Ya greater than Yf greater than Yn greater than Yb greater than Yc), whereas at higher degrees of cross-linking, CBis 5.0% (w/w), Yf subunits possess the fastest mobility (i.e. order of mobility Yf greater than Yk greater than or equal to Yn greater than Yb greater than or equal to Ya greater than Yc). Resolving gels that contain low concentrations of cross-linker [CBis 0.6% (w/w)] allow the resolution of a hitherto unrecognized polypeptide that is isolated by S-hexyl-GSH-Sepharose affinity chromatography. This new polypeptide, which we have designated Yb, is normally obscured by the main Yb band in resolving gels that comprise concentrations of cross-linker of at least CBis 1.6% (w/w). The Ya- and Yb-type subunits in guinea pig, mouse, hamster and man were identified by immuno-blotting and their apparent Mr values in different electrophoresis systems were determined. The Ya subunits in all species studied possess a variable cross-linker-dependent mobility during electrophoresis. Since the transferase subunits are currently classified according to their mobilities during SDS/polyacrylamide-gel electrophoresis, it is apparent that the variable electrophoretic behaviour of the Ya and Yk subunits may lead to the mis-identification of enzymes.

scholarly journals Effects of thiolation on the immunoreactivity of the ribosome-inactivating protein gelonin

1989 ◽  
Vol 263 (2) ◽  
pp. 417-423 ◽  
Author(s):  
V Singh ◽  
M R Sairam
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Cross Linking ◽  
Carrier Protein ◽  
Ribosome Inactivating Protein ◽  
Reverse Phase ◽  
Specific Radioimmunoassay ◽  
Cross Linker

Gelonin purified from the seeds of Gelonium multiflorum using cation-exchange and gel-filtration chromatography was characterized for its purity, homogeneity and Mr by reverse-phase h.p.l.c. and SDS/polyacrylamide-gel electrophoresis analysis and judged to be 98% pure. As the cross-linking agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) has been used for linking gelonin via its epsilon-NH2 group to its carrier antibodies or hormones for immunotoxin or hormonotoxin respectively, an attempt was made to study the effect of this modification of gelonin on its immunoreactivity. A radioimmunoassay was developed for this purpose. By sequential modification, four categories of amino group modifications on immunoreactivity were observed. Even one or two modifications, representing one-twentieth to one-tenth of available epsilon-NH2 groups in the protein caused about 75% loss in immunoreactivity, with additional reactions contributing to further deteriorations. By using a gelonin radioimmunoassay, the immunoreactivity of gelonin in three hormonotoxins was determined with gelonin and modified gelonin as standards. The gelonin equivalent in our hormonotoxins was in agreement with the values determined by spectrophotometric and gel-electrophoresis methods. As the immunoreactivity of gelonin-SPDP was not further altered after conjugation to its carrier protein ovine lutropin, a specific radioimmunoassay of gelonin could be used to evaluate the molar ratio of the conjugates prepared by using SPDP as cross-linker and gelonin-SPDP as a standard.

ALS2CL, the novel protein highly homologous to the carboxy-terminal half of ALS2, binds to Rab5 and modulates endosome dynamics

FEBS Letters ◽  
2004 ◽  
Vol 575 (1-3) ◽  
pp. 64-70 ◽  
Author(s):  
Shinji Hadano ◽  
Asako Otomo ◽  
Kyoko Suzuki-Utsunomiya ◽  
Ryota Kunita ◽  
Yoshiko Yanagisawa ◽  
...  
Keyword(s):  
The Novel ◽  
Carboxy Terminal ◽  
Novel Protein

scholarly journals Unusual Structural Features in the Adduct of Dirhodium Tetraacetate with Lysozyme

2021 ◽  
Vol 22 (3) ◽  
pp. 1496
Author(s):  
Domenico Loreto ◽  
Giarita Ferraro ◽  
Antonello Merlino
Keyword(s):  
Anticancer Agents ◽  
Structural Features ◽  
Side Chain ◽  
Side Chains ◽  
Valuable Insight ◽  
Experimental Conditions ◽  
Egg White Lysozyme ◽  
Potential Applications ◽  
Model Protein ◽  
Hen Egg White

The structures of the adducts formed upon reaction of the cytotoxic paddlewheel dirhodium complex [Rh2(μ-O2CCH3)4] with the model protein hen egg white lysozyme (HEWL) under different experimental conditions are reported. Results indicate that [Rh2(μ-O2CCH3)4] extensively reacts with HEWL:it in part breaks down, at variance with what happens in reactions with other proteins. A Rh center coordinates the side chains of Arg14 and His15. Dimeric Rh–Rh units with Rh–Rh distances between 2.3 and 2.5 Å are bound to the side chains of Asp18, Asp101, Asn93, and Lys96, while a dirhodium unit with a Rh–Rh distance of 3.2–3.4 Å binds the C-terminal carboxylate and the side chain of Lys13 at the interface between two symmetry-related molecules. An additional monometallic fragment binds the side chain of Lys33. These data, which are supported by replicated structural determinations, shed light on the reactivity of dirhodium tetracarboxylates with proteins, providing useful information for the design of new Rh-containing biomaterials with an array of potential applications in the field of catalysis or of medicinal chemistry and valuable insight into the mechanism of action of these potential anticancer agents.

scholarly journals Chemically-Boosted Corneal Cross-Linking for the Treatment of Keratoconus through a Riboflavin 0.25% Optimized Solution with High Superoxide Anion Release

2021 ◽  
Vol 10 (6) ◽  
pp. 1324
Author(s):  
Cosimo Mazzotta ◽  
Marco Ferrise ◽  
Guido Gabriele ◽  
Paolo Gennaro ◽  
Alessandro Meduri
Keyword(s):  
Superoxide Anion ◽  
Hydroxypropyl Methylcellulose ◽  
Anterior Segment ◽  
Preclinical Study ◽  
Cross Linking ◽  
Specular Microscopy ◽  
The Novel ◽  
Power Setting ◽  
Contralateral Eye

The purpose of this study was to evaluate the effectiveness and safety of a novel buffered riboflavin solution approved for corneal cross-linking (CXL) in progressive keratoconus and secondary corneal ectasia. Following the in vivo preclinical study performed on New Zealand rabbits comparing the novel 0.25% riboflavin solution (Safecross®) containing 1% hydroxypropyl methylcellulose (HPMC) with a 0.25% riboflavin solution containing 0.10% EDTA, accelerated epithelium-off CXL was performed on 10 patients (10 eyes treated, with the contralateral eye used as control) through UV-A at a power setting of 9 mW/cm2 with a total dose of 5.4 J/cm2. Re-epithelialization was evaluated in the postoperative 7 days by fluorescein dye test at biomicroscopy; endothelial cell count and morphology (ECD) were analyzed by specular microscopy at the 1st and 6th month of follow-up and demarcation line depth (DLD) measured by anterior segment optical coherence tomography (AS-OCT) one month after the treatment. We observed complete re-epithelization in all eyes between 72 and 96 h after surgery (88 h on average). ECD and morphology remained unchanged in all eyes. DLD was detected at a mean depth of 362 ± 50 µm, 20% over solutions with equivalent dosage. SafeCross® riboflavin solution chemically-boosted corneal cross-linking seems to optimize CXL oxidative reaction by higher superoxide anion release, improving DLD by a factor of 20%, without adverse events for corneal endothelium.

scholarly journals Effect of the Cross-Linking Density on the Swelling and Rheological Behavior of Ester-Bridged β-Cyclodextrin Nanosponges

Materials ◽  
2021 ◽  
Vol 14 (3) ◽  
pp. 478
Author(s):  
Gjylije Hoti ◽  
Fabrizio Caldera ◽  
Claudio Cecone ◽  
Alberto Rubin Pedrazzo ◽  
Anastasia Anceschi ◽  
...  
Keyword(s):  
Scale Up ◽  
Future Application ◽  
Cross Linking ◽  
Synthesis Reaction ◽  
Dependent Behavior ◽  
Cyclodextrin Nanosponges ◽  
Potential Applications ◽  
Cross Linker ◽  
The Cross ◽  
Shed Light

The cross-linking density influences the physicochemical properties of cyclodextrin-based nanosponges (CD-NSs). Although the effect of the cross-linker type and content on the NSs performance has been investigated, a detailed study of the cross-linking density has never been performed. In this contribution, nine ester-bridged NSs based on β-cyclodextrin (β-CD) and different quantities of pyromellitic dianhydride (PMDA), used as a cross-linking agent in stoichiometric proportions of 2, 3, 4, 5, 6, 7, 8, 9, and 10 moles of PMDA for each mole of CD, were synthesized and characterized in terms of swelling and rheological properties. The results, from the swelling experiments, exploiting Flory–Rehner theory, and rheology, strongly showed a cross-linker content-dependent behavior. The study of cross-linking density allowed to shed light on the efficiency of the synthesis reaction methods. Overall, our study demonstrates that by varying the amount of cross-linking agent, the cross-linked structure of the NSs matrix can be controlled effectively. As PMDA βCD-NSs have emerged over the years as a highly versatile class of materials with potential applications in various fields, this study represents the first step towards a full understanding of the correlation between their structure and properties, which is a key requirement to effectively tune their synthesis reaction in view of any specific future application or industrial scale-up.

scholarly journals Dihydroquinolines, Dihydronaphthyridines and Quinolones by Domino Reactions of Morita-Baylis-Hillman Acetates

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 890
Author(s):  
Joel K. Annor-Gyamfi ◽  
Ebenezer Ametsetor ◽  
Kevin Meraz ◽  
Richard A. Bunce
Keyword(s):  
Aromatic Ring ◽  
Aromatic Amines ◽  
Control Experiment ◽  
Ring Closure ◽  
Domino Reaction ◽  
Side Chain ◽  
Initial Reaction ◽  
Dual Reactivity ◽  
Michael Acceptor ◽  
Nitrogen Nucleophile

An efficient synthetic route to highly substituted dihydroquinolines and dihydronaphthyridines has been developed using a domino reaction of Morita-Baylis-Hillman (MBH) acetates with primary aliphatic and aromatic amines in DMF at 50–90 °C. The MBH substrates incorporate a side chain acetate positioned adjacent to an acrylate or acrylonitrile aza-Michael acceptor as well as an aromatic ring activated toward SNAr ring closure. A control experiment established that the initial reaction was an SN2′-type displacement of the side chain acetate by the amine to generate the alkene product with the added nitrogen nucleophile positioned trans to the SNAr aromatic ring acceptor. Thus, equilibration of the initial alkene geometry is required prior to cyclization. A further double bond migration was observed for several reactions targeting dihydronaphthyridines from substrates with a side chain acrylonitrile moiety. MBH acetates incorporating a 2,5-difluorophenyl moiety were found to have dual reactivity in these annulations. In the absence of O2, the expected dihydroquinolines were formed, while in the presence of O2, quinolones were produced. All of the products were isolated in good to excellent yields (72–93%). Numerous cases (42) are reported, and mechanisms are discussed.

scholarly journals FLIm and Raman Spectroscopy for Investigating Biochemical Changes of Bovine Pericardium upon Genipin Cross-Linking

Molecules ◽  
2020 ◽  
Vol 25 (17) ◽  
pp. 3857
Author(s):  
Tanveer Ahmed Shaik ◽  
Alba Alfonso-Garcia ◽  
Martin Richter ◽  
Florian Korinth ◽  
Christoph Krafft ◽  
...  
Keyword(s):  
Raman Spectroscopy ◽  
Fluorescence Lifetime ◽  
Density Functional ◽  
Bovine Pericardium ◽  
Biochemical Changes ◽  
Blue Pigment ◽  
Cross Linking ◽  
Fluorescence Lifetime Imaging ◽  
Fluorescence Intensity Ratio ◽  
Cross Linker

Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm−1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively.

scholarly journals Preparation and properties of hydrogel based on sawdust cellulose for environmentally friendly slow release fertilizers

2020 ◽  
Vol 9 (1) ◽  
pp. 139-152 ◽  
Author(s):  
Xiao Zhang ◽  
Yanlu Liu ◽  
Panfang Lu ◽  
Min Zhang
Keyword(s):  
Slow Release ◽  
Environmentally Friendly ◽  
Water Absorbency ◽  
The Novel ◽  
Structure And Properties ◽  
Nitrogen Release ◽  
Release Property ◽  
Slow Release Fertilizers ◽  
Grafting Copolymerization ◽  
Cross Linker

AbstractA novel hydrogel slow-release nitrogen fertilizer based on sawdust with water absorbency was prepared using grafting copolymerization. Urea was incorporated as nitrogen source in a hydrogel fertilizer. Potassium persulfate (KPS) and N,N᾽-methylenebis acrylamide (MBA) were used as the initiator and cross-linker, respectively. The structure and properties of the samples were characterized by XPS, EDS, SEM, XRD and FTIR. The effects of various salt solutions, ionic strength and pH on swelling behavior were discussed. The results showed that the largest water absorbency of the sample reached 210 g/g in distilled water. In addition, the sample had the good nitrogen release property. Thus, the novel environmentally friendly hydrogel fertilizer may be widely applied to agricultural and horticultural fields.

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