Formation Of Cross-linked Products Research Articles

Neo-epitopes on methylglyoxal modified human serum albumin lead to aggressive autoimmune response in diabetes

Glyco-oxidation of proteins has implications in the progression of diabetes type 2. Human serum albumin is prone to glyco-oxidative attack by sugars and methylglyoxal being a strong glycating agent may have severe impact on its structure and consequent role in diabetes. This study has probed the methylglyoxal mediated modifications of HSA, the alterations in its immunological characteristics and possible role in autoantibody induction. We observed an exposure of chromophoric groups, loss in the fluorescence intensity, generation of AGEs, formation of cross-linked products, decrease in α-helical content, increase in hydrophobic clusters, FTIR band shift, attachment of methylglyoxal to HSA and the formation of Nε-(carboxyethyl) lysine in the modified HSA, when compared to the native albumin. MG-HSA was found to be highly immunogenic with additional immunogenicity invoking a highly specific immune response than its native counterpart. The binding characteristics of circulating autoantibodies in type 2 diabetes mellitus (DM) patients showed the generation of anti-MG-HSA auto-antibodies in the these patients, that are preferentially recognized by the modified albumin. We propose that MG induced structural perturbations in HSA, result in the generation of neo-epitopes leading to an aggressive auto-immune response and may contribute to the immunopathogenesis of diabetes type 2 associated complications.

Ring-opening polymerization of ɛ-caprolactone by base catalyst for synthesis of grafted polysilsesquioxane

The polysilsesquioxane having amino and phenyl groups (APSQ) was prepared from the corresponding trimethoxysilanes by co-condensation under basic conditions. The amino group on APSQ was utilized as an initiator for graft polymerization of ɛ-caprolactone (CL). The ring-opening polymerization of CL, in the presence of a catalytic amount of the base prepared from triazabicyclodecene, proceeded effectively to afford the polysilsesquioxane having poly(CL) as the graft chains (GrPSQ). In the grafting, no formation of cross-linked product was observed. The grafted poly(CL) component in GrPSQ showed improved durability for heat as an advantage of the hybrid polymer containing polysiloxane structure.

Cysteine-directed Cross-linking to Subunit B Suggests That Subunit E Forms Part of the Peripheral Stalk of the Vacuolar H+-ATPase

We have employed a combination of site-directed mutagenesis and covalent cross-linking to identify subunits in close proximity to subunit B in the vacuolar H(+)-ATPase (V-ATPase) complex. Unique cysteine residues were introduced into a Cys-less form of subunit B, and the V-ATPase complex in isolated vacuolar membranes from each mutant strain was reacted with the bifunctional, photoactivable maleimide reagent 4-(N-maleimido)benzophenone. Photoactivation resulted in cross-linking of the unique sulfhydryl groups on subunit B with other subunits in the complex. Four of the eight mutants constructed containing a unique cysteine residue at Ala(15), Lys(45), Glu(494), or Thr(501) resulted in the formation of cross-linked products, which were recognized by Western blot analysis using antibodies against both subunits B and E. These products had a molecular mass of 84 kDa, consistent with a cross-linked product of subunits B and E. Molecular modeling of subunit B places Ala(15) and Lys(45) near the top of the V(1) structure (i.e. farthest from the membrane), whereas Glu(494) and Thr(501) are predicted to reside near the bottom of V(1), with all four residues predicted to be oriented toward the external surface of the complex. A model incorporating these and previous data is presented in which subunit E exists in an extended conformation on the outer surface of the A(3)B(3) hexamer that forms the core of the V(1) domain. This location for subunit E suggests that this subunit forms part of the peripheral stalk of the V-ATPase that links the V(1) and V(0) domains.

Water-absorbing properties of hydrogels based on polymeric complexes

Colloidal and chemical properties of polymeric complexes based on polyacrylamide and polyvinyl alcohol have been investigated. Several factors affecting water absorption by polymeric complexes have been studied: concentration of initial aqueous polymer solutions; components ratio in the mixture; temperature and duration; sorbed aqueous solutions properties (pH; kind of ions).Macromolecular globules of polyacrylamide in aqueous solutions upon addition of polyvinyl alcohol exhibit interactions of hydroxyl and amide groups. The heat treatment of polycomplexes aqueous solutions leads to formation of cross-linked products (hydrogels) which are not soluble in water and show superabsorbents properties.Variation of conditions of polycomplex formation allows to regulate the properties of the resulting products such as hydrogel strength and the water absorption in wide ranges. This gives us possibilities for optimal use of the hydrogels in function of their specific applications.

Structural analysis of the products of chymotryptic cleavage of the E1 form of Na,K-ATPase α-subunit: identification of the N-terminal fragments containing the transmembrane H 1-H 2 domain

Chymotryptic cleavage of the Na,K-ATPase in NaCl medium abolishes ATPase activity and alters other functional parameters. The structure of this modified enzyme is uncertain since only one product of selective proteolysis, the 83-kDa fragment of the α-subunit (Ala 267–C-terminus) has been identified previously. Here, we applied additional tryptic digestion followed by oxidative cross-linking to identify the products originating from the N-terminal part of the α-subunit. These fragments start at Ala 72 or Thr 74 and contain the transmembrane H 1-H 2 domain. Formation of cross-linked product between α-fragments containing H 1-H 2 and H 7-H 10 demonstrate that the structural integrity of the membrane moiety is preserved. We also determined that secondary cleavage of the 83-kDa fragment leads to the formation of C-terminal 48-kDa α-fragments with multiple N-termini at Ile 582, Ser 583, Met 584 and Ile 585.

Effect of nucleotides and actin on the intramolecular cross-linking of myosin subfragment-1.

The heavy chain of myosin subfragment-1 (S1) is cleaved by limited trypsinolysis into three fragments, 27, 50, and 20 kDa--aligned in this order from the N-terminus. The tertiary structure of the molecule is essentially not affected by trypsinolysis. The spatial relations between the various regions of the molecule and the nucleotide- and actin-induced intramolecular movements were studied by cross-linking tryptic S1 with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), phenylenediglyoxal (PDG), and glutaraldehyde. The formation of cross-linked products was monitored by SDS-PAGE, using the fluorescent probes 9-anthronitrile and N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS), which specifically label the 27- and 20-kDa fragments, respectively. The reaction with the cross-linkers leads to the formation of 50-kDa/20-kDa, 27-kDa/20-kDa, 27-kDa/50-kDa, and 20-kDa/light chain cross-linked products. Of these, the most intensive was the formation of the 50-kDa/20-kDa products, which appeared as a doublet on the SDS-PAGE with all the cross-linkers. This indicates that the interface between the two fragments is rather extended. The presence of MgATP or MgADP promoted the formation of the 20-kDa/50-kDa cross-linked products, especially with the lower electrophoretic mobility band, when EEDQ was used as a cross-linker. With PDG as a cross-linker, MgATP also affected the cross-link formation between the 20-kDa fragment and the light chains whereas it had no influence on the formation of other products. On the other hand, the effect of actin on the cross-linking with the various cross-linkers was quite extensive, and it was manifested in the reduction of cross-link formation between the various S1 domains. It is concluded that both nucleotides and actin induce intramolecular movements in S1 and that the nucleotide-induced movements are more restricted than those induced by actin, which extend to larger regions of the molecule.

Borasilazane Polymeric Precursors for Borosilicon Nitride

The reaction of H3B·S(CH3)2 with the (CH3SiHNH)n cyclic oligomers obtained by ammonolysis of methyldichlorosilane (CH3SiHCI2) results in evolution of hydrogen and formation of cross‐linked products that contain borazine rings as well as boron atoms that are linked to three nitrogen atoms. Pyrolysis of the products in a stream of argon gives a high yield of a black “borosilicon carbonitride,” whereas pyrolysis in a stream of ammonia gives white “borosilicon nitride” in high yield.

A new class of soluble basic protein precursors of the cornified envelope of mammalian epidermis

The cornified envelope hs been shown to be formed beneath the plasma membrane as a result of the cross-linking of soluble and membrane-associated precursor proteins by transglutaminase. We have obtained a monoclonal antibody which reacts with the periphery of cells in the upper layers of human epidermis by indirect immunofluorescence (IIF) following immunization of mice with cornified envelopes of cultured human keratinocytes. The antibody also stained the cell peripheries of bovine, rat and mouse epidermis as well as stratified epithelium. Neutral buffer extracts of human cultured keratinocytes and epidermis examined under denaturing conditions contained polypeptides of molecular weight 14 900 and 16 800 which reacted with the antibody, and an additional component of molecular weight 24 800 was found in cultured cells. The polypeptides were shown to have a p I of about 9.0. Under non-denaturing conditions the two lower-molecular-weight polypeptides had an apparent molecular weight of 30 000, while the 24 800 protein had one of 60 000. Incubation of the polypeptides under conditions that activate transglutaminase resulted in a disappearance of the polypeptides or the formation of cross-linked products. Basic polypeptides with somewhat different p I values and molecular weights were identified in neutral buffer extracts of bovine and rat epidermis. The HCE-2 antibody appears to identify a new class of basic protein precursors of mammalian cornified envelope.

Cross-linking of actin to myosin subfragment 1: course of reaction and stoichiometry of products.

The cross-linking of actin to myosin subfragment 1 (S-1) with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide was reexamined by using two cross-linking procedures [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306; Sutoh, K. (1983) Biochemistry 22, 1579-1585] and two independent methods for quantitating the reaction products. In the first approach, the cross-linked acto-S-1 complexes were cleaved with elastase at the 25K/50K and 50K/22K junctions in S-1. This enabled direct measurements of the cross-linked and un-cross-linked fractions of the 50K and 22K fragments of S-1. We found that in all cases actin was preferentially cross-linked to the 22K fragment and that the overall stoichiometry of the main cross-linked products was that of a 1:1 complex of actin and S-1. In the second approach, actin was cross-linked to tryptically cleaved S-1, and the course of these reactions was monitored by measuring the decay of the free 50K and 20K fragments and the formation of cross-linked products. After selecting the optimal cross-linking procedure and conditions, we determined that the rate of actin cross-linking to the 20K fragment of S-1 was 3-fold faster than the reaction with the 50K peptide. The overall rate of cross-linking actin to S-1 corresponded to the sum of the individual reactions of the 50K and 20K fragments, indicating their mutually exclusive cross-linking to actin. Thus, the reactions with tryptically cleaved S-1 were consistent with the 1:1 stoichiometry of actin and S-1 in the main cross-linked products and verified the preferential cross-linking of actin to the 20K fragment of S-1. These results are discussed in the context of the binding of actin to S-1.

Reactions of spin-label crosslinking reagents with red blood cell proteins

The reactions of two spin-label cross-linking reagents with components of red blood cells have been studied. A bifunctional bis(N-hydroxysuccinimide ester) reagent passes readily through the membrane of intact red cells and reacts almost exclusively with hemoglobin. The rotational motion of hemoglobin inside of red cells may be studied by electron paramagnetic resonance (EPR) by using this spin-labeling method. A second cross-linking molecule, a negatively charged, disulfide-exchange reagent, reacts both with mem- brane components and with hemoglobin when intact red cells are labeled. Upon reaction with membranes stripped of components other than band 3, this reagent readily produces dimers and higher oligomers of this protein. The EPR spec- trum of band 3 labeled in this way shows that most of the spin-label molecules are highly immobilized on the protein. When the same reagent reacts with intact red cells, the cross-linked membrane products include components identified as band 3 dimers and a high molecular weight product con- sisting primarily of bands 1 and 2 but also containing lesser amounts of most of the other red cell membrane components and a slight amount of band 3. The reagent was made ra- dioactive to demonstrate that the spin-label disulfide exchange reagent is indeed involved in formation of cross-linked products. Comparison of Coomassie Blue staining and radioactivity in the region where band 3 dimers are expected on a one-di- mensional sodium dodecyl sulfate-polyacrylamide gel shows that there is approximately 0.5 cross-linking reagent per protein Te reactions of chemical reagents with components of the red blood cell have been used in studies of the geometry of erythrocyte membrane proteins, of the mobility of the band 3 protein in membranes, and of potential antisickling agents. Questions pertaining to the geometry of erythrocyte mem- branes have been addressed by using cross-linking reagents,

Synthesis and reactions of vinyl ethers of ethanolamines Communication 10. Free-radical polymerization and copolymerization of the trivinyl ether of triethanolamine

1. A study was made of the polymerization of the trivinyl ether of triethanolamine and of its copolymerization with methyl methacrylate in presence of benzoyl peroxide or 2,2′-azobis[2-methylpropionitrile]. 2. Benzoyl peroxide is unable to initiate the polymerization of this vinyl ether and its copolymerization with methyl methacrylate. 3. The polymerization of the trivinyl ether of triethanolamine and its copolymerization with methyl methacrylate will proceed in presence of 2, 2′-zobis [2-methylpropionitrile] with preferential formation of cross-linked products. 4. The polymer and copolymers were hydrolyzed, and the results indicated the presence of free vinyl groups. The hydrolysis product had a three-dimensional structure.