Gelation Activity Research Articles

Real time monitoring of temperature variation and optical transmission during gelation of acrylamide (AAm) at various crosslinker contents

Free radical crosslinking copolymerization (FCC) of acrylamide (AAm) with various amounts of N,N′-methylenebis(acrylamide) (Bis) is performed at different onset temperatures (T on ). Variation of temperature, T , and optical transmission, I tr , are monitored in real time during the gelation process. The decrease in I tr is modeled by the monomer consumption rate, and the gelation activation energies, ΔE G , are measured at each Bis content. During FCC, temperature increases to a certain level then decreases to its initial value as the gelation time increases, which is explained by the heat released during the polymerization process. Decrease in I tr is attributed to the increase in scattered light intensity, I sc , during the formation of interconnected microgels in the system. It is observed that the number of microgels increases as the onset temperature, T on , is increased. At a given temperature, high Bis content samples produce larger size of microgel particles compared to low Bis content samples.

Gelation of Food Protein Induced by Recombinant Microbial Transglutaminase

ABSTRACT: The recombinant microbial transglutaminase from Streptoverticillium mobaraense var. (rMTGase) was expressed in Escherichia coli. Specific enzyme activity of rMTGase was comparable to native MTGase. However, the gelation of a sodium caseinate solution induced by rMTGase was slower than that induced by native MTGase. In addition, the mechanical property of kamaboko prepared with rMTGase was weaker than that with native MTGase. In SDS‐PAGE analysis, α‐casein monomers decreased more slowly during the incubation with rMTGase than MTGase. These results confirmed the difference of cross‐linking activity between the 2 enzymes. Furthermore, thermal stability of rMTGase was lower compared to native MTGase. These results suggest that the difference of cross‐linking activity and thermal stability between the 2 enzymes cause differences in gelation activity of protein.

Fast Transient Fluorescence Technique for Determination of Gelation Activation Energies in Free-Radical Cross-Linking Copolymerization

Gelation during free-radical cross-linking copolymerization (FCC) of methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDM) was studied using the fast transient fluorescence (FTRF) technique. Pyrene (Py) was used as a probe for the FTRF experiments. Fluorescence lifetimes of Py from its decay traces were measured and used to monitor the gelation processes in various EGDM contents at various temperatures. MMA consumption rates were measured during the gelation process by employing the Stern−Volmer kinetic model. Composite rate constants, kr, for FCC were determined for each EGDM content at different temperatures, and it is observed that as EGDM content and/or temperature is increased kr increased. Gelation activation energies, ΔEG, were measured for different EGDM contents, and it is observed that as ΔEG is decreased the EGDM content is increased.

Comparison of Filamin A-induced Cross-linking and Arp2/3 Complex-mediated Branching on the Mechanics of Actin Filaments

We compared the effects of human filamin A (FLNa) and the activated human Arp2/3 complex on mechanical properties of actin filaments. As little as 1 FLNa to 800 polymerizing actin monomers induces a sharp concentration-dependent increase in the apparent viscosity of 24 microm actin, a parameter classically defined as a gel point. The activated Arp2/3 complex, at concentrations up to 1:25 actins had no detectable actin gelation activity, even in the presence of phalloidin, to stabilize actin filaments against debranching. Increasing the activated Arp2/3 complex to actin ratio raises the FLNa concentration required to induce actin gelation, an effect ascribable to Arp2/3-mediated actin nucleation resulting in actin filament length diminution. Time lapse video microscopy of microparticles attached to actin filaments or photoactivation of fluorescence revealed actin filament immobilization by FLNa in contrast to diffusion of Arp2/3-branched actin filaments. The experimental results support theories predicting that polymer branching absent cross-linking does not lead to polymer gelation and are consistent with the observation that cells deficient in actin filament cross-linking activity have unstable surfaces. They suggest complementary roles for actin branching and cross-linking in cellular actin mechanics in vivo.

Comparison of Enzymatic Properties of Microbial Transglutaminase from Streptomyces sp.

The microbial transglutaminase (TGase) from Streptomyces libani was purified from its culture broth and its enzymatic properties were compared with those of TGase from Streptoverticillium mobaraense var. TGase was purified by ion-exchange chromatography and size-exclusion chromatography. The specific activity of the main component was 10.7 unit/mg protein, lower than that of Streptoverticillium mobaraense var. (25 unit/mg). Several differences of enzymatic properties were found between the two enzymes. Optimum temperature, stability and gelation activity of TGase from Streptomyces libani were lower than those of TGase from Streptoverticillium mobaraense var, while, deamidation activity was higher. In addition, the existence of some TGases with different pI were suggested.

Gelation in free‐radical crosslinking copolymerization; fast transient fluorescence study

The fast transient fluorescence (FTRF) technique was used to study the sol‐gel phase transition in free‐radical crosslinking copolymerization (FCC) in two different monomeric systems. Pyrene (Py) was used as a fluorescence probe for the in situ polymerization experiments. The fluorescence lifetimes of Py from its decay traces were measured and used to monitor the gelation process. Monomer consumption profiles were determined during gelation process using Stern‐Volmer model. Gelations were performed at various temperatures and in various crosslinker contents. Composite rate constants, kr were determined and gelation activation energies, ΔEG were measured.

In situ monitoring of energy release in real time during gelation of acrylamide (AAm)

Temperature variation and optical transmission was monitored in real time during the free radical crosslinking copolymerization (FCC) of acrylamide (AAm) and N,N′-methylenebis(acrylamide) (Bis). Gelation experiments were initiated at various onset temperatures (Ton). During gelation the temperature rose then decreased to its initial value as the gelation time increased. That is explained with the energy release (Gelation activation energy) during the polymerization process. Gelation activation energies, ΔEG were measured and found to be increased as the onset temperature, Ton was increased. It was observed that transmitted photon intensities, Itr, decreased dramatically at the reaction time, which were attributed to the increase in scattered light intensity, Isc, during the formation of interconnected microgels in the system. Increase in Isc was modeled using Rayleigh's equation where reaction time was found to be proportional to the volume of the microgels. It was observed that a number of microgels increased as the onset temperature, Ton was increased.

Endotoxic properties of lipid A from Comamonas testosteroni.

The lipid A from Comamonas testosteroni has been isolated and its complete chemical structure determined [Iida, T., Haishima, Y., Tanaka, A., Nishijima, K., Saito, S. & Tanamoto, K. (1996). Eur J Biochem 237, 468-475]. In this work, the relationship between its chemical structure and biological activity was studied. The lipid A was highly homogeneous chemically and was characterized by the relatively short chain length (C(10)) of the 3-hydroxy fatty acid components directly bound to the glucosamine disaccharide backbone by either amide or ester linkages. The lipid A exhibited endotoxic activity in all of the assay systems tested (mitogenicity in mouse spleen cells; induction of tumour necrosis factor alpha release from both mouse peritoneal macrophages and mouse macrophage-like cell line J774-1, as well as from the human monocytic cell line THP-1; induction of nitric oxide release from J774-1 cells; Limulus gelation activity and lethal toxicity in galactosamine-sensitized mice) to the same extent as did 'Salmonella minnesota' lipid A or Escherichia coli LPS used as controls. The strong endotoxic activity of the C. testosteroni lipid A indicates that the composition of 3-hydroxydecanoic acid is not responsible for the low endotoxicity of the lipid A observed in members of the genus Rhodopseudomonas, as has previously been suggested. Furthermore, both the lack of a second acylation of the 3-hydroxy fatty acid attached at the 3' position, and the substitution of the hydroxyl group of the 3-hydroxy fatty acid attached at position 2, do not affect the manifestation of endotoxic activity or species specificity.

Biological properties of lipid A isolated from Flavobacterium meningosepticum.

The biological properties of the lipid A from Flavobacterium meningosepticum, which we recently isolated and whose complete chemical structure has been determined (H. Kato, T. Iida, Y. Haishima, A. Tanaka, and K. Tanamoto. J. Bacteriol. 180:3891--3899, 1998), were studied. The lipid A exhibited generally moderate activity compared to Salmonella enterica subsp. enterica serovar abortus equi lipopolysaccharide (LPS) used as a control in the assay systems tested; lethal toxicity in galactosamine-sensitized mice, mitogenicity in mouse spleen cells, induction of tumor necrosis factor alpha (TNF-alpha) release from mouse peritoneal macrophages and J774-1 mouse macrophage-like and human THP-1 line cells, nitric oxide induction activity from J774-1 cells, and Limulus gelation activity. The moderate activity of the F. meningosepticum lipid A may be explained by its unique fatty acid composition and the lack of a phosphate group in position 4'. It is noteworthy that the lipid A apparently induced TNF-alpha release from peritoneal macrophages in LPS-unresponsive C3H/HeJ mice and that the activation was suppressed by the LPS-specific antagonist, succinylated lipid A precursor. Significant splenocyte mitogenicity in C3H/HeJ mice was also observed with the lipid A. Taken together with the previous results concerning Porphyromonas gingivalis lipid A, which has a high level of structural similarity to the lipid A of F. meningosepticum, and the induction of TNF-alpha release in macrophages from C3H/HeJ mice, the lipid A of F. meningosepticum, which has novel fatty acids, may possibly play an role for the activation of C3H/HeJ macrophages.

Monomer consumption rates during gelation at various temperatures: A fast transient fluorescence study

The gelation during free-radical crosslinking copolymerization (FCC) of methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDM) was studied by using fast transient fluorescence (FTRF) technique. Pyrene (Py) was used as a probe for the FTRF experiments. Fluorescence lifetimes of Py from its decay traces were measured and used to monitor the gelation process at various temperatures. MMA consumption rates were measured during gelation process by using the Stern–Volmer model. Gelation activation energy ΔEG was measured and found to be 15.6 kcal mol−1. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 3161–3168, 2001

Chemical and biological evaluation of endotoxin contamination on natural rubber latex products.

Relationship between pyrogenicity and bacterial endotoxin contamination on latex products was demonstrated by chemical analysis and biological assays. In commercially available latex products' surveillance, water extracts prepared from one surgical glove and two silicone elastomer-coated Foley catheters sterilized by gamma-irradiation were obviously pyrogenic in rabbits. The induced fever was monophasic at low dose of the pyrogenic extracts and biphasic at high dose. These extracts exhibited limulus amebocyte lysate gelation activity, and induced inflammatory cytokine (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) production from MM6-CA8 human monocytoid cells. These biological properties, including pyrogenicity, completely disappeared by treating the pyrogenic extracts with endotoxin-adsorbent affinity column. Limulus amebocyte lysate activity and cytokine production from MM6-CA8 cells induced by the extracts were significantly decreased by endotoxin inhibitors, an active fragment peptide of an 18-kDa cationic antimicrobial protein and a synthetic lipid A B464 analogue. Furthermore, very small amounts of 2-keto-3-deoxyoctonate and 3-hydroxy fatty acid, which are common constituents of bacterial endotoxins, were detected by gas chromatography-mass spectrometry analysis of the pyrogenic extracts. These findings clearly showed that the pyrogenicity found in these latex products originated from endotoxins contaminating the products.

Salmonella-type heptaacylated lipid A is inactive and acts as an antagonist of lipopolysaccharide action on human line cells.

The stimulation of both THP-1 and U937 human-derived cells by Salmonella lipid A preparations from various strains, as assessed by TNF-alpha induction and NF-kappaB activation, was found to be very low (almost inactive) compared with Escherichia coli lipid A, but all of the lipid As exerted strong activity on mouse cells and on Limulus gelation activity. Experiments using chemically synthesized E. coli-type hexaacylated lipid A (506) and Salmonella-type heptaacylated lipid A (516) yielded clearer results. Both lipid A preparations strongly induced TNF-alpha release and activated NF-kappaB in mouse peritoneal macrophages and mouse macrophage-like cell line J774-1 and induced Limulus gelation activity, although the activity of the latter was slightly weaker than that of the former. However, 516 was completely inactive on both THP-1 and U937 cells in terms of both induction of TNF-alpha and NF-kappaB activation, whereas 506 displayed strong activity on both cells, the same as natural E. coli LPS. In contrast to the action of the lipid A preparations, all the Salmonella LPSs also exhibited full activity on human cells. However, the polysaccharide portion of the LPS neither exhibited TNF-alpha induction activity on the cells when administered alone or together with lipid A nor inhibited the activity of the LPS. These results suggest that the mechanism of activation by LPS or the recognition of lipid A structure by human and mouse cells may differ. In addition, both 516 and lipid A from Salmonella were found to antagonize the 506 and E. coli LPS action that induced TNF-alpha release and NF-kappaB activation in THP-1 cells.

Anti-endotoxin properties of a cinnamon bark-derived compound and its effect on the endotoxin shock model

An endotoxin inhibitor derived from cinnamon bark was characterized chemically and tested for anti-LPS properties. Chemical analysis suggested that the active center of the inhibitor was in the lipid portion. Upon incubation with LPS molecule, the inhibitor reduced the ability of LPS to induce TNFα and generate nitric oxide from various cells in vitro, and Limulus gelation activity. The lethal toxicity of LPS in galactosamine-sensitized mice and pyrogenicity of LPS in a rabbit model were reduced 1000- and 100-fold by pre-incubation with the inhibitor, respectively. Simultaneous but separate injection of the inhibitor with a lethal dose of LPS also protected the majority of mice. Protection against LPS was seen when the inhibitor was given to mice 1 h before the LPS challenge. Furthermore, the inhibitor significantly suppressed the induction of fever by simultaneous administration with LPS without prior mixing. These results suggest that the inhibitor may be a useful potent blocker of bacterial endotoxin.

The lipid A moiety of Porphyromonas gingivalis lipopolysaccharide specifically mediates the activation of C3H/HeJ mice.

The lipid A preparation isolated from Porphyromonas gingivalis was found to induce splenocyte mitogenicity and TNF-alpha release from peritoneal macrophages in LPS-unresponsive C3H/HeJ mice to the same extent as in LPS-responsive mice. In order to clarify whether the activation of C3H/HeJ mice was specifically caused by the lipid A and not by contaminating protein, two strategies were employed. The lipid A fraction from P. gingivalis was subjected to either hydrochloric acid or alkaline treatment to eliminate either glycosylated phosphate or O-acylated fatty acids from the lipid A structure, and the biologic activities of the derivatives were compared in both LPS-responsive and unresponsive C3H/HeJ mice. De-1-O-phosphorylated P. gingivalis lipid A showed partial loss, and de-O-acylated lipid A complete loss of splenocyte mitogenic and TNF-alpha-inductive activities from peritoneal macrophages in both LPS-responsive and unresponsive mice. The relative activities of the intact and treated lipid A compounds in splenocyte mitogenicity and TNF-alpha-inductive activity in macrophages were similar to the relative activities of these preparations in Limulus gelation activities. The LPS-specific antagonist, succinylated lipid A precursor, inhibited P. gingivalis lipid A-mediated splenocyte mitogenicity and TNF-alpha induction in macrophages in a similar manner in LPS-responsive and unresponsive mice. These results strongly suggest that the activation of LPS-unresponsive C3H/HeJ mice by P. gingivalis lipid A was specifically mediated by the lipid A portion and not by contaminating protein. The characteristic action of P. gingivalis lipid A on LPS-unresponsive C3H/HeJ mice was thought to reflect the unique chemical properties of this compound.

A Novel Inhibitor of Bacterial Endotoxin Derived from Cinnamon Bark

A substance that inhibits the activity of bacterial endotoxin (LPS) was found in cinnamon bark. The inhibitor, extracted from dry cinnamon bark with 67% ethanol/water, was purified by using Limulus gelation activity as an indicator of endotoxin activity. The inhibitor suppressed the activity of the LPS when it was mixed with the inhibitor prior to the assay. The reduction of the LPS activity depended on the concentration of both the inhibitor and LPS when mixed, and also on the incubation time. The inhibitor suppressed the activity of all LPS and lipid A preparations tested regardless of the origin of the bacteria. The inhibitor alone did not affect the Limulus system. These results indicate that the inhibition was caused by direct interaction of the inhibitor with the LPS molecule. Furthermore the inhibitor abrogated the pyrogenicity of the LPS. Although it is uncertain whether the inhibitor actually plays a role in the defense mechanism in cinnamon bark, this is the first report that an inhibitor of bacterial endotoxin exists in a plant.

Identification of a Phosphatidylinositol 4,5-Bisphosphate-binding Site in Chicken Skeletal Muscle α-Actinin

We previously reported that phosphatidylinositol 4,5-bisphosphate (PIP2) dramatically increases the gelating activity of smooth muscle alpha-actinin (Fukami, K., Furuhashi, K., Inagaki, M., Endo, T., Hatano, S., and Takenawa, T. (1992) Nature 359, 150-152) and that the hydrolysis of PIP2 on alpha-actinin by tyrosine kinase activation may be important in cytoskeletal reorganization (Fukami, K., Endo, T., Imamura, M., and Takenawa, T. (1994) J. Biol. Chem. 269, 1518-1522). Here we report that a proteolytic fragment with lysylendopeptidase comprising amino acids 168-184 (TAPYRNVNIQNFHLSWK) from striated muscle alpha-actinin contains a PIP2-binding site. A synthetic peptide composed of the 17 amino acids remarkably inhibited the activities of phospholipase C (PLC)-gamma 1 and -delta 1. Furthermore, we detected an interaction between PIP2 and a bacterially expressed alpha-actinin fragment (amino acids 137-259) by PLC inhibition assay. Point mutants in which arginine 172 or lysine 184 of alpha-actinin were replaced by isoleucine reduced the inhibitory effect on PLC activity by nearly half. Direct interactions between PIP2 and the peptide (amino acids 168-184) or the bacterially expressed protein (amino acids 137-259) were confirmed by enzyme-linked immunosorvent assay. We also found this region homologous to the sequence of the PIP2-binding site in spectrin and the pleckstrin homology domains of PLC-delta 1 and Grb7. Synthetic peptides from the homologous regions in spectrin and PLC-delta 1 inhibited PLC activities. These results indicate that residues 168-184 comprise a binding site for PIP2 in alpha-actinin and that similar sequences found in spectrin and PLC-delta 1 may be involved in the interaction with PIP2.

Chemically detoxified lipid A precursor derivatives antagonize the TNF-alpha-inducing action of LPS in both murine macrophages and a human macrophage cell line.

The chemically synthesized disaccharide precursor of lipid A (406) has been demonstrated to reduce its activity in mice by an order of 10(5) or more by replacing the hydroxyl groups with succinyl or acetyl residues. In the present study, these chemically detoxified synthetic lipid A precursors were found to antagonize the LPS action of inducing TNF-alpha in both murine peritoneal macrophages and in human monocyte-macrophage cell line THP-1. These preparations were found to antagonize the induction of TNF-alpha in murine macrophages by both stimulants of LPS and untreated 406, which acts as an agonist in the murine system, in a dose-dependent manner. Complete inhibition by succinylated 406 occurred at 10-fold excesses of the antagonist. Succinylated precursor also antagonized the TNF-alpha-inducing action of LPS in human cell line THP-1. The precursor itself also exhibited antagonism in this assay. The activity of the succinylated 406, however, was much more potent than that of the unmodified precursor. These derivatives do not inhibit either the Limulus gelation activity of LPS, or the induction of TNF-alpha in macrophages by zymosan, indicating that the inhibition is not caused by molecular interaction and that the inhibition is specific to LPS action. These findings suggest common features in murine macrophage and human monocytic cell receptors. They also suggest the importance of the substituents on the hydroxyl groups of 3-hydroxy fatty acids of the nonreducing glucosamine of lipid A for the activity and for transformation to the antagonistic structure.

Key Aspects of Novel Catalyst Systems in all-Water Blown Integral Skin Foams-Related Catalytic Activities in the Isocyanate Reaction

This paper will focus on the generalization of catalytic activities of a variety of typical amine and tin catalysts in the isocyanate reaction of gelation, blowing and crosslinking Secondly the relationship between catalytic activities and foaming behavior, as well as foam properties, will be discussed from the stand-point of developing novel catalyst systems for all-water blown integral skin foams (ISF). Catalysts play an important role in the control and balance between the gelation and blowing reactions. Especially in ISF systems, this control becomes increasingly important for the improvement of surface skin formation in all-water blown systems, which are now required for eliminating CFC. That is to say, skin formation and cure become inferior when using water as a blowing agent, primarily because the role of CFC is to aid in skin formation. Moreover, other than the two primary reactions, it will be considered that the catalytic activity of the crosslinking reactions such as allophanate, biuret and isocyanurate formation will have some effect on foaming behavior as well as foam properties. Therefore, it will be important to know the crosslinking activities for the design of novel catalyst systems. Concerning the catalytic activities for the primary gelation and blowing reactions, many reports have been presented in the past where the relationships between catalytic activities and foaming behaviors, as well as foam properties, have been reported. On the other hand, several reports have been recently presented on the crosslinking activities, based on the method using HPLC analysis techniques in a model reaction. Few reports, however, describe the reaction kinetic analysis of the catalytic crosslinking activities and the systematic examination of its relation to foaming behavior and foam properties. In this paper, the catalytic activity of a variety of typical amine and tin catalysts will be generalized with a review on gelation and blowing activities and a reaction kinetic analysis of the crosslinking activities using HPLC analysis in a model reaction. In addition, the relationship between these catalytic activities and foaming behaviors, as well as foam properties, will be discussed in an all-water blown ISF system. As to the foaming reaction, the relationship between viscosity rise profiles, which may have a large relation to cure, will also be discussed. Concerning foam properties, the relation to cure and skin formation will be discussed. As a result, it will be shown that strong gelling catalysts with high crosslinking activities provide improvement in skin formation. This report will intro-duce novel catalyst systems in all-water blown ISF systems by suggesting the effectiveness of catalytic activities in the selection of a suitable catalyst system.

Dissociation of endotoxic activities in a chemically synthesized lipid A precursor after acetylation.

In a previous study, a chemically synthesized disaccharide precursor of lipid A (406; identical to lipid IVA) was shown to have dramatically reduced lethality, B-cell mitogenicity, and tumor necrosis factor induction in macrophages when its hydroxyl groups were replaced with either succinyl or acetyl residues (K. Tanamoto, FEBS Lett. 351:325-329, 1994). Succinylated 406 was found to lose Limulus amoebocyte lysate gelation activity completely as a result of the modification (about 10(5)-fold), too, as expected. However, acetyl 406, surprisingly, exhibited activity comparable to that of the original 406. Both succinylated and acetylated 406 lost pyrogenicity completely. These results indicate that one of the typical endotoxic activities was dissociated from the others and that the ability to induce Limulus amoebocyte lysate gelation is not always representative of endotoxin activity.

Free hydroxyl groups are not required for endotoxic activity of lipid A.

Previous studies demonstrated that lipid A from Salmonella abortusequi loses its B-cell mitogenicity for murine spleen cells as a result of the introduction of succinyl residues on hydroxyl groups and that the inactivated lipid A specifically antagonizes the mitogenicity of endotoxin. Hypothesizing that the hydroxyl groups are essential both for its biological activity and for producing nontoxic preparations having antagonistic activity, I tested the role of the hydroxyl groups in its activities by using well-characterized biologically active lipid A preparations synthesized chemically (Escherichia coli and Salmonella types 506 and 516, respectively) by the introduction of either succinyl or acetyl residues at the hydroxyl groups of each of these lipid A preparations. However, the biological activities of neither lipid A preparation were reduced at all after succinylation; in fact, succinylated 516 became much more potent than the original molecule with respect to most activities tested, i.e., lethal toxicity, Limulus gelation activity, and the induction of tumor necrosis factor release. On the other hand, when the hydroxyl groups were replaced with acetyl residues, the lethality and tumor necrosis factor-inducing activity of both lipid A preparations were decreased, whereas their Limulus gelation activity was increased. Mitogenicity was not affected much by the chemical modifications of either lipid A preparation. These findings indicate that although the residues introduced into the free hydroxyl groups of lipid A modulate its activities, the hydroxyl groups in lipid A need not exist in free form.