Multiple Crosslinking Research Articles

Thermoreversible gelation of associating polymers

This paper studies sol/gel transition of associating polymers with multiple cross-link junctions. Paying special attention to the multiplicity and sequence length of the network junctions, we derive phase diagrams and analyze molecular structure of the networks. The conventional Eldridge–Ferry method is extended to evaluate multiplicity, as well as enthalpy, of the cross links, and applied to the experimental gel melting curves of poly(vinyl alcohol) in water. The effect of added surfactants on the formation of reversible gels in hydrophobically modified polymer solutions is also studied under the assumption of the existence of a minimum multiplicity required for stable cross-links. On the basis of the transient network model with temporal junctions, we calculate dynamic mechanical moduli, average lifetime of a junction, the number of elastically effective chains and of dangling ends as functions of the strength of the association constant, polymer concentration and the temperature. We compare the results with the experimentally measured moduli of hydrophobically modified poly(ethylene oxide).

Identification of the photocrosslinking sites in troponin-I with 4-maleimidobenzophenone labelled mutant troponin-Cs having single cysteines at positions 158 and 21.

Our previous studies have shown that 4-maleimidobenzophenone (BP-Mal) attached to troponin-C (TnC) mutants with single cysteines at positions 12, 57, 89 and 98 forms crosslinks to troponin-I (TnI), and the identified crosslinking regions indicate an antiparallel course of the two interacting polypeptide chains, in agreement with other studies using fragments of TnC and TnI. In this work we extended the mapping of the TnC-TnI interface by analysing photocrosslinking between TnI and BP-Mal labelled TnC mutants with single Cys residues at positions 21 (TnC21) and 158 (TnC158). We determined the sites of these photocrosslinks in TnI by progressive proteolysis of the crosslinked product, followed by N-terminal sequencing and mass spectrophotometric analyses. The results show that whereas TnC158 forms a specific crosslink with Met-21, TnC21 forms multiple crosslinks in the range of residues 96 to 134 of TnI. The results are discussed in light of the antiparallel model of the TnI-TnC complex and a structural model derived from low-angle X-ray and neutron scattering studies.

Effects of cyclization and pendant vinyl group reactivity on the swelling behavior of polyacrylamide gels

A series of polyacrylamide (PAAm) gels were prepared by free-radical crosslinking copolymerization of acrylamide and N,N′-methylenebis(acrylamide) (BAAm) in water at various crosslinker (BAAm) and chain transfer agent (isopropyl alcohol, IPA) concentrations. It was shown that only 5 % of the crosslinker used in the feed forms effective crosslinks in the final hydrogels. At BAAm contents as high as 3 mole %, the equilibrium swelling ratio of the gels in water is independent of the crosslinker content in the feed. This is due to the prevailing multiple crosslinking reactions during the gel formation process. At a fixed crosslinker content, the onset of gelation is shifted towards higher conversions and reaction times as the amount of IPA increases. Addition of IPA in the monomer mixture also increases the equilibrium swelling ratio of PAAm gels. It was shown that the gel crosslinking density increases on rising IPA concentration in the feed due to the increasing rate of intermolecular crosslinking reactions.

Mucin-1-related T cell infiltration in colorectal carcinoma.

Mucins (MUC) are highly glycosylated molecules widely expressed on epithelia of different origins, including colonic mucosa. Altered glycosylation processes in tumour cells result in the exposure of normally cryptic peptide epitopes, which may then be recognized as tumour-specific antigens. Recently, MUC1-specific antibodies were detected in the serum of a broad range of cancer patients, and from different tumours tumour-specific cytotoxic T lymphocytes (CTL) were isolated that recognized MUC1. Absence of HLA restriction in the recognition has been ascribed to the highly repetitive sequence of the polypeptide core, allowing simultaneous recognition of multiple identical epitopes and cross-linking and aggregation of T cell receptor on mucin-specific T cells. We investigated the expression of MUC1 epitopes in 56 cell suspensions from Dukes' B to D colorectal carcinomas using antibodies that recognize distinct peptide sequences on the glycosylated or deglycosylated MUC1 protein backbone. No relation was observed between MUC1 expression, or the extent of its glycosylation, and Dukes' stage, tumour location and tumour differentiation, but a positive correlation was detected between the percentages of tumour cells expressing mucin-1 and the numbers of CD3+ infiltrating cells. These tumour-infiltrating lymphocytes contained, however, only a few MUC1-specific T lymphocytes, as CTL showing preferential killing of MUC1-expressing target cells were only obtained from one tumour. Since, in addition, the majority of colorectal carcinomas were found to express the fully glycosylated MUC1 glycoprotein, its potential role as a target antigen for T-lymphocyte-mediated immunotherapy in this tumour type is probably limited.

Multistage Crosslinking of Nematic Networks

Nematic polymer melts, cross-linked in their isotropic phase, typically form polydomain nematic rubbers and gels. Such elastomers, if strained and then further cross-linked, form nematic monodomain elastomers with interesting mechanical and thermal properties. We calculate the elastic free energy of a network which has undergone multiple cross-linking and obtain the surprising result that it is identical in form to that for a sample once cross-linked from an oriented nematic monodomain. Gaussian elasticity thus predicts that it is impossible to permanently imprint complex structure (including, for instance, biaxiality) into a nematic elastomer by imposing a complex sequence of strains interspersed by cross-linking stages. A multistage cross-linked network should therefore exhibit the same behavior as a single-stage system, including recently predicted and observed nematic transitions in response to imposed strains.

A mechlorethamine-induced DNA interstrand cross-link bends duplex DNA.

The dG-to-dG, DNA-DNA interstrand cross-link at the duplex sequence 5'-d(GNC) formed by the antitumor drug mechlorethamine (bis(2-chloroethyl)methylamine) was studied both theoretically and experimentally. Computer models of cross-linked DNA were energy minimized using molecular mechanics. The energy minimized structures possessed local distortion of the DNA helix, especially propeller twisting and buckling, caused by the tether length being too small to bridge the spacing of N7 atoms of dG at the sequence 5'-d(GNC) in B DNA. Overwinding of 2-6 degrees was present at each of the two dinucleotide steps spanned by the cross-link. The predicted structural changes were compatible with the possibility that this cross-link would introduce a static bend into the DNA double helix axis. An experimental study provided evidence for this induced bending of the helix axis in interstrand cross-linked samples. DNAs containing multiple mechlorethamine-induced interstrand cross-links exhibited anomalously low electrophoretic mobility in polyacrylamide gels when the lesions were separated by one or two turns. From the degree of gel retardation, the cross-linked DNAs were estimated to be bent by 12.4-16.8 degrees per lesion; estimation of the extent to which this bend was induced by the lesion was complicated by a preexisting bend in the non-cross-linked DNAs used. The data did not allow distinction of a static from an anisotropic dynamic bend; "universal" and "hinge" joints were excluded. Anomalous mobility was maximal when the lesion spacing was 21 bp, suggesting a helical repeat of 10.5 bp per turn.

Chemical cross-linking of porcine luteinizing hormone: location of the cross-link and consequences for stability and biological activity.

To study the interaction between the subunits of LH and determine which amino acid residues are involved in this interaction, porcine and ovine LH (pLH and oLH) were cross-linked with 0.02 M 1-ethyl-(3-(3-dimethylaminopropyl)carbodiimide to generate one specific intersubunit cross-link. The cross-linked hormone was separated from the noncross-linked dissociated subunits by gel filtration on a Sephadex G-75 column. The position of the cross-link in cross-linked pLH (X-pLH) was determined by sequencing peptide fragments that were generated by digestion with endoproteinase Arg-C. In accordance with previous data for bovine LH, the position of the cross-link was between alpha-Lys49 and beta-Asp111, indicating that these residues are at the subunit-subunit interface. The biological activity of cross-linked hormone was tested by radioreceptor binding assay. The receptor-binding activity of X-pLH was slightly reduced to 84%, suggesting that the conformational stability of X-pLH is similar to that of pLH as a result of the introduction of the covalent cross-link. The receptor-binding activity of X-oLH was decreased by approximately 30%, which we attribute to the formation of multiple cross-links within the ovine molecule, making the molecule more rigid, and to oligomeric forms, resulting from multiple intermolecular cross-links, that are not able to bind to the testicular LH receptor. This observation implies that the oLH has more amino-carboxyl functional groups that are sterically susceptible to carbodiimide cross-linking than does pLH under the reaction conditions used.

Chemical cross-linking of porcine luteinizing hormone: location of the cross-link and consequences for stability and biological activity

To study the interaction between the subunits of LH and determine which amino acid residues are involved in this interaction, porcine and ovine LH (pLH and oLH) were cross-linked with 0.02 M 1-ethyl-(3-(3-dimethylaminopropyl)carbodiimide to generate one specific intersubunit cross-link. The cross-linked hormone was separated from the noncross-linked dissociated subunits by gel filtration on a Sephadex G-75 column. The position of the cross-link in cross-linked pLH (X-pLH) was determined by sequencing peptide fragments that were generated by digestion with endoproteinase Arg-C. In accordance with previous data for bovine LH, the position of the cross-link was between alpha-Lys49 and beta-Asp111, indicating that these residues are at the subunit-subunit interface. The biological activity of cross-linked hormone was tested by radioreceptor binding assay. The receptor-binding activity of X-pLH was slightly reduced to 84%, suggesting that the conformational stability of X-pLH is similar to that of pLH as a result of the introduction of the covalent cross-link. The receptor-binding activity of X-oLH was decreased by approximately 30%, which we attribute to the formation of multiple cross-links within the ovine molecule, making the molecule more rigid, and to oligomeric forms, resulting from multiple intermolecular cross-links, that are not able to bind to the testicular LH receptor. This observation implies that the oLH has more amino-carboxyl functional groups that are sterically susceptible to carbodiimide cross-linking than does pLH under the reaction conditions used.

Folding of DNA substrate-hairpin ribozyme domains: use of deoxy 4-thiouridine as an intrinsic photolabel.

Hairpin ribozymes derived from (-)sTRSV RNA exhibit substantial cleavage activity when wobble GU base pairs are introduced in place of the AU pairs normally involved in helices I and II between substrate and ribozyme. This finding prompted us to synthesize by in vitro transcription a new hairpin ribozyme, active against a 14-mer substrate derived from a conserved HIV sequence. Interactions of the canonical and anti-HIV hairpin ribozymes with non cleavable DNA substrate analogues containing the photoaffinity probe deoxy-4-thiouridine (ds4U) at a single site were investigated. Upon near-UV light irradiation (365 nm), all these substrate analogues were covalently attached to ribozyme via single or multiple crosslinks. In contrast, no crosslinks were detected using either a DNA substrate analogue lacking ds4U or a ds4U containing oligomer unrelated to the substrate sequence. As expected, if the dissociation constant is in the range of 5-15 microM, the yield of crosslinked ribozyme increased markedly with increasing the substrate analogue concentration. The ribozyme residues involved in the crosslinks were determined by RNA sequencing. The pattern of crosslinks obtained with the two ribozyme systems provides additional evidence in support of the consensus secondary structure proposed for the hairpin domain. Minor alternative conformations were detected in the case of the (-)sTRSV system.

Flexibility of the Bulge Formed Between a Hairpin Ribozyme and Deoxy-Substrate Analogues

The conformation of the bulge formed between the hairpin ribozyme R derived from (−)sTRSV and noncleavable all-deoxy-substrate analogues dS was studied by photoaffinity labelling. The photolabel deoxy-6-thioinosine was inserted in place of residue G+1 or A−1, located immediately 3′ and 5′ to the cleavage site, respectively. Upon 335 nm irradiation both substrate analogues were linked to ribozyme at multiple sites. Formation of the R-dS complex is absolutely required for the generation of the crosslinks, since they were detected neither in the absence of Mg2+ nor upon using a ds6l containing 14-mer, unable to interact with the ribozyme. The fraction of ribozyme crosslinked at completion of the reaction increased with increasing analogue concentrations, yielding apparent KD values for the R-dS complex in the range of 5 ± 2 μM. Multiple crosslinks between ribozyme and each one of the substrate analogues provide clear evidence for a large flexibility of the bulge region.

Enhancement of durability of chymotrypsin through cross-linking with poly(allylamine)

α-Chymotrypsin (ChT) is covalently linked to various derivatives of poly(allylamine) (PAA) by using a carbodiimide as a coupling reagent. The PAA derivatives contain cationic, anionic, or hydrophobic microdomains. The ChTs linked to the PAA derivatives exhibit considerable stability against denaturing agents such as 0.5 m sodium dodecylsulfate or 4 m guanidinium chloride. This suggests multiple intramolecular cross-linking of ChT by the linear PAA derivatives. Stabilization of ChT by cross-linking with PAA derivatives is also reflected in much greater resistance of the PAA-bound ChTs to thermoinactivation. The multiple attachment of ChT to PAA derivatives does not significantly damage the active site as indicated by the reactivity of the modified ChTs at optimum pHs.

Biophysical characterization of involucrin reveals a molecule ideally suited to function as an intermolecular cross-bridge of the keratinocyte cornified envelope.

Involucrin is a 68-kDa precursor of the keratinocyte cornified envelope. During keratinocyte terminal differentiation glutamine residues of involucrin become covalently cross-linked to other envelope precursors via covalent epsilon-(gamma-glutamyl)lysine bonds. In the present study we examine the secondary and tertiary structure of human involucrin using computer algorithms, circular dichroism, and electron microscopy. Our results indicate that involucrin is an extended, flexible, rod-shaped molecule that has a length of 460 A, an axial ratio of 30:1 and possesses between 50 and 75% alpha-helical content. Glutamine residues are circumferentially distributed along the length of the alpha-helical segments of the molecule, a distribution that is conserved in all species. We hypothesize that this distribution of glutamine residues together with the elongated shape of the molecule permits optimal interaction of involucrin glutamyl side chains with the lysine residues of other para-membranous proteins during transglutaminase-mediated cross-linking. Moreover, its long length allows involucrin to cross-link molecules that are separated by substantial distances in the cornified envelope. These properties allow a single involucrin molecule to form multiple cross-links, in multiple spatial planes, with other envelope precursors. Thus, the structure of involucrin is that of an ideal intermolecular cross-bridge.

Synthesis and structure of a new type of polycyclic peptide

The synthesis of a ‘polycyclic peptide’ is described, in which the Iµ-nitrogens of cyclo-(Lys-Lys-Gly)2 are interconnected by four p-xylyl moieties; this is the first example of the use of multiple cross-linking to generate highly constrained peptides, and NMR spectroscopic evidence indicates the presence of two unusual β-turns that are predicted from molecular modelling studies.

Preclinical studies and future directions in the development of new hematologic growth factors

Normal hematopoiesis is controlled by a cascade of interacting hormones collectively referred to as cytokines. These growth factors have been studied both individually and in specific combinations to determine their optimal clinical use. In some cases, the combination of certain cytokines produces a synergistic effect enhancing their efficacy. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated the ability to stimulate early- and late-phase granulocyte and macrophage progenitor cells, activate mature neutrophils and macrophages, and enhance their peripheral infection fighting performance. Interleukin-3 (IL-3), currently undergoing clinical evaluation, acts early in the development of multiple types of white blood cells and, when used in combination with GM-CSF, also produces a synergistic effect in raising white blood cell and platelet levels. A recombinant protein, PIXY321, has recently been developed that contains both IL-3 and GM-CSF domains. The development of this molecule was supported by the discovery of a dual IL-3-GM-CSF receptor on the surface of hematopoietic progenitor cells. PIXY321 provides a significantly enhanced biologic effect (10-fold greater proliferation) via multiple cross-linking of GM-CSF, IL-3, and dual receptor binding sites. PIXY321 has the same molecular weight as the equivalent molar concentrations of GM-CSF and IL-3 combined and offers the advantage of combination therapy in an easy-to-administer regimen. Another recombinant cytokine, mast cell growth factor (MGF), has shown profound hematopoietic activity in vitro and has the ability to enhance proliferation of hematopoietic stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-linking and O-acetylation of peptidoglycan in Staphylococcus aureus (strains H and MR-1) grown in the presence of sub-growth-inhibitory concentrations of -lactam antibiotic

Staphylococcus aureus H was grown for 4 generation times with various sub-growth-inhibitory concentrations of beta-lactam antibiotics specific for particular penicillin-binding proteins (PBPs) - PBP2, clavulanic acid; PBP3, methicillin; PBP4, cefoxitin - and also with the non-specific benzylpenicillin. Isolated cell walls were digested with Chalaropsis muramidase and the resulting peptidoglycan fragments were fractionated by HPLC into disaccharide-peptide monomers and cross-linked dimers, trimers, tetramers and greater oligomers. The pattern of relative fragment concentrations with increasing amounts of drug was roughly the same regardless of the antibiotic used, monomers and dimers increasing while trimers and tetramers changed little and oligomers decreased rapidly. The patterns resembled closely those predicted by the 'random addition' model for multiple cross-link formation and not at all those predicted by the 'monomer addition' model. The O-acetylation of the peptidoglycan remained essentially unaffected under all these conditions. S. aureus MR-1, a constitutive producer of PBP2', gave similar results when treated with methicillin.

FcγRII-Mediated Superoxide Production by Phagocytes Is Augmented by GM-CSF Without a Change in FCγRII Expression

Freshly purified neutrophils and monocytes respond to multiple cross-linking of Fc gamma RII with the IgG1 monoclonal antibody, CIKM5, with a rapid rise in Ca(2+)i, but not with a respiratory burst, although superoxide is generated by these cells when stimulated with the chemotactic peptide, FMLP, or phorbol ester (TPA). Incubation in vitro for 30-60 min at 37 degrees C in medium + 0.1% FCS had no effect on the neutrophil superoxide response to CIKM5 but induced a weak monocyte response in 11/13 experiments. However, incubation with rhGM-CSF (10 ng/ml) under similar conditions induced a neutrophil respiratory burst in response to cross-linking Fc gamma RII in 12/14 experiments and enhanced the monocyte response by 181%. GM-CSF also enhanced the response of neutrophils and monocytes to FMLP by 308% and 165%, respectively. The response to TPA was not significantly enhanced by GM-CSF. rhIFN-gamma (100 mu/ml) was ineffective as a priming agent for all agonists tested in short-term incubations but augmented the monocyte response to CIKM5 after 5 d exposure in vitro. Whilst GM-CSF induced neutrophil superoxide production in response to cross-linking Fc gamma RII, there was no concomitant change in Fc gamma RII expression either in in vitro studies of neutrophils from healthy individuals or in in vivo studies of patients receiving GM-CSF. Stimulation of unprimed neutrophils with CIKM5 induced a rapid transient increase in intracellular calcium levels to 181% of resting levels. However, incubation with GM-CSF did not further augment the calcium transients above the stimulated level. The mechanism by which GM-CSF induces an enhanced respiratory burst in response to cross-linking of Fc gamma RII remains to be elucidated, but is not related to receptor expression or increases in receptor mediated calcium mobilization.

Analysis of rhodamine and fluorescein-labeled F-actin diffusion in vitro by fluorescence photobleaching recovery

Properties of filamentous acetamidofluorescein-labeled actin and acetamidotetramethylrhodamine-labeled actin (AF and ATR-actin, respectively) were examined to resolve discrepancies in the reported translational diffusion coefficients of F-actin measured in vitro by FPR and other techniques. Using falling-ball viscometry and two independent versions of fluorescence photobleaching recovery (FPR), the present data indicate that several factors are responsible for these discrepancies. Gel filtration chromatography profoundly affects the viscosity of actin solutions and filament diffusion coefficients. ATR-actin and, to a lesser degree, AF-actin show a reduction in viscosity in proportion to the fraction labeled, presumably due to filament shortening. Actin filaments containing AF-actin or ATR-actin are susceptible to photoinduced damage, including a covalent cross-linking of actin protomers within filaments and an apparent cleavage of filaments detected by a decrease of the measured viscosity and an increase in the measured filament diffusion coefficients. Quantum yields of the two photoinduced effects are quite different. Multiple cross-links are produced relative to each photobleaching event, whereas less than 1% filament cleavage occurs. Substantial differences in the filament diffusion coefficients measured by FPR are also the result of differences in illumination geometry and sampling time. However, under controlled conditions, FPR can be used as a quantitative tool for measuring the hydrodynamic properties of actin filaments. Incremented filament shortening caused by photoinduced cleavage or incremental addition of filament capping proteins produces a continuous and approximately linear increase of filament diffusion coefficients, indicating that filaments are not associated in solution. Our results indicate that actin filaments exhibit low mobilities and it is inferred that actin filaments formed in vitro by column-purified actin, under standard conditions, are much longer than has conventionally been presumed.

Protein topography of the 40 S ribosomal subunit from Saccharomyces cerevisiae as shown by chemical cross-linking.

Protein-protein cross-linking was used to examine the spatial arrangement of proteins within the 40 S ribosomal subunits of Saccharomyces cerevisiae. Purified ribosomal subunits were treated with either 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate under conditions such that the ribosomal particle was intact and that formation of 40 S subunit dimers was minimized. Proteins were extracted from the treated subunits and fractionated on Sephadex G-150 or by acid-urea-polyacrylamide gel electrophoresis. Cross-linked proteins in these fractions were analyzed by two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Constituent members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers. Forty-two pairs involving 25 of the 32 40 S subunit proteins were identified. Many proteins were detected in several cross-linked dimers. These proteins with multiple cross-links form foci for the construction of a schematic model of the spatial arrangement of proteins within the 40 S subunit.

Multiple crosslinks of proteins S7 and S9 to domains 3 and 4 of 16S ribosomal RNA in the Escherichia coli 30S particle

RNA-protein cross-links were introduced into Escherichia coli 30S subunits by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. 16S rRNA, cross-linked to 30S ribosomal proteins, was isolated and hybridized with seven single-stranded bacteriophage M13-DNA probes. These probes, each carrying an inserted rDNA fragment, were used to select contiguous RNA sections covering domains 3 and 4 (starting at nucleotide 868 and ending at the 3'OH terminus) of the 16S rRNA. The proteins covalently linked to each selected RNA section were identified by two-dimensional polyacrylamide gel electrophoresis. Proteins S7 and S9 were shown to be efficiently cross-linked to multiple sites belonging to both domains.

Multiple crosslinks of proteins S7, S9, S13 to domains 3 and 4 of 16S RNA in the 30S particle

Functionally active 70S ribosomes containing 4-thiouracil in place of uracil (substitution level 2%) were prepared by an in vivo substitution method. RNA-protein crosslinks were introduced by 366 nm photoactivation of 4-thiouracil in the purified 30S subunits. Seven single stranded M13 probes containing rDNA inserts complementary to domains 3 and 4 of 16S RNA were constructed. These inserts approximately 100 nucleotides long starting at nucleotide 868 and ending at the 3' OH terminus were used to select contiguous RNA sections. The proteins covalently linked to each selected RNA section were identified by 2D gel electrophoresis. Proteins S7, S9, S13 were shown to be efficiently crosslinked to multiple sites belonging to both domains.