Temperature dependence of the elastic modulus of alginate gels

The temperature dependence of the elastic modulus for alginate gels was studied using two different gel systems: covalently crosslinked Na-alginate gels and in-situ prepared Ca-alginate gels. The modulus of physically crosslinked gels showed a complex behaviour. The temperature coefficient of the modulus of covalently crosslink gels changed from positive for the lowest crosslinked gels to negative for the highest crosslinked gels. This suggests a change from rubberlike to enthalpy-driven elasticity with an increasing degree of crosslinking for these gel networks.     

Mechanical properties of a reversible, DNA-crosslinked polyacrylamide hydrogel

Mechanical properties of a polyacrylamide gel with reversible DNA crosslinks are presented. In this system, three DNA strands replace traditional chemical crosslinkers. In contrast to thermoset chemically crosslinked polyacrylamide, the new hydrogel is thermoreversible; crosslink dissociation without the addition of heat is also feasible by introducing a specific removal DNA strand. This hydrogel is characterized by a critical crosslink concentration at which gelation occurs. Below the critical point, a characteristic temperature exists at which a transition in viscosity is observed. Both temperature-dependent viscosity and elastic modulus of the material are functions of crosslink density.     

Swelling and de-swelling kinetics of gelatin hydrogels in ethanol-water marginal solvent

Controlled osmotic swelling and de-swelling measurements have been performed on gelatin, a polyampholyte, hydrogels suspended in water-ethanol marginal solvent at room temperature (20 degrees C) where the alcohol concentration was changed from 0 to 100% (v/v). The change in gel mass was monitored as function of time until osmotic equilibrium was established with the surrounding solvent. It was observed that osmotic pressure of polymer-solvent mixing, pi(m)相似文献   

Rigidity of fibrin gels as measured by quasielastic light scattering

The strength of fibrin gels has been investigated by a recently developed laser light scattering technique for determining the shear modulus of soft gels. By this method, changes in the modulus were monitored as a function of time without perturbing the material. Fibrin gels were crosslinked with blood coagulation factor XIII. Rigidity measurements and SDS–polyacrylamide gel electrophoresis were used to correlate gel strength with the number of covalently bonded subunit chains. The modulus was found to vary linearly with the number of crosslinks until maximum rigidity was achieved.     

A correlation between mechanical and electrical properties of the synthetic hydrogel chosen as an experimental model of cytoskeleton

The correlation between the electrochemical (Donnan) potential and volume swelling was studied for synthetic polyelectrolyte hydrogels considered as models of cytoskeleton gel-forming biopolymers. Hydrogels involving polyacrylic and polymethacrylic acids with varying network density were synthesized by a radical polymerization in aqueous solution. Electrical charge was introduced into the gel network by partial neutralization of monomer acids with several alkali and alkali earth (hydr)oxides. The electrochemical (Donnan) potential of synthetic gels was determined using conventional microelectrode tools for cell potential determination. It was demonstrated that the negative electrical potential of many anionic gels with various charges and network densities decreased with the decrease of equilibrium swelling, i.e., with the decrease in water content in the gel. It was shown that a drastic phase transition in the gel structure from a swollen to a compressed state induced by K⁺/Ca²⁺ exchange is accompanied by an analogous decrease in the absolute Donnan potential of the gels. A kinetic study demonstrated that the gel volume changed ahead of its electrical potential. This suggests that the volume phase transition in gel is the main cause of the electrical response. A similarity between the swelling/compression transition in synthetic gels and the volume changes in the cytoskeleton in the vicinity of the cell membrane was demonstrated. Based on the universal analogy between the properties of synthetic and natural polymer gels, a possible involvement of swelling of the gel-like cytoskeleton structures in electrical regulation in the cell was postulated.     

Counter-ion dynamics in crosslinked poly(styrene sulfonate) systems studied by NMR

The field dependence of the longitudinal and transverse nuclear magnetic relaxation rates of 23Na+ in aqueous crosslinked Na-poly(styrene sulfonate) (PSS) systems (ion exchange resins) has been obtained as a function of the degree of crosslinking. The relaxation is considerably enhanced relative to solutions of non-crosslinked NaPSS at equal ionizable group concentration. This is due to the dynamic constraints of the polymer chains, which render the averaging of the counter-ion chain interaction less efficient. The field dependence of the relaxation rates in the crosslinked NaPSS systems reveals two processes that are out of the extreme narrowing limit. This is in contrast to the relaxation behavior found in non-crosslinked NaPSS systems. To characterize these processes their correlation times were combined with constants of selfdiffusion to estimate the distances diffused by an ion in order to average the electric field gradient at its nucleus. These two distances are interpreted as characteristic length scales in the network. At all degrees of crosslinking it was found that the smallest of these length scales is roughly equal to the distance between two neighbouring crosslinks. The largest characteristic distance extends over several crosslinks and reflects inhomogeneities in the crosslink concentration. These conclusions were also reached from similar experiments on 7Li+ in LiPSS systems.     

Quantitative Evaluation of Collagen Crosslinks and Corresponding Tensile Mechanical Properties in Mouse Cervical Tissue during Normal Pregnancy

The changes in the mechanical integrity of the cervix during pregnancy have implications for a successful delivery. Cervical collagens are known to remodel extensively in mice with progressing gestation leading to a soft cervix at term. During this process, mature crosslinked collagens are hypothesized to be replaced with immature less crosslinked collagens to facilitate cervical softening and ripening. To determine the mechanical role of collagen crosslinks during normal mouse cervical remodeling, tensile load-to-break tests were conducted for the following time points: nonpregnant (NP), gestation day (d) 6, 12, 15, 18 and 24 hr postpartum (PP) of the 19-day gestation period. Immature crosslinks (HLNL and DHLNL) and mature crosslinks (DPD and PYD) were measured using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). There were no significant changes in the total immature crosslink density (HLNL+DHLNL mol per collagen mol) throughout normal mouse gestation (range: 0.31–0.49). Total mature crosslink density (PYD+DPD mol per collagen mol) decreased significantly in early softening from d6 to d15 (d6: 0.17, d12: 0.097, d15: 0.026) and did not decrease with further gestation. The maturity ratio (total mature to total immature crosslinks) significantly decreased in early softening from d6 to d15 (d6: 0.2, d15: 0.074). All of the measured crosslinks correlated significantly with a measure of tissue stiffness and strength, with the exception of the immature crosslink HLNL. This data provides quantitative evidence to support the hypothesis that as mature crosslinked collagens decline, they are replaced by immature collagens to facilitate increased tissue compliance in the early softening period from d6 to d15.     

Thermally induced protein gelation: gelation and rheological characterization of highly concentrated ovalbumin and soybean protein gels

In order to optimize the use of proteins as functional ingredients in foods, one needs more insight into the effects of environmental conditions (pH, ionic strength, and temperature) on the functional properties of protein. This paper summarizes the results of an extensive study on heat-induced gelation of ovalbumin (egg-white protein) and soybean protein in the concentration range from 10 to 35 g/100 g. It was the aim of the study to relate the rheological properties of thermally induced protein gels to the microstructure of the gel and the physicochemical properties of the constituent protein. The gelling behavior of the protein was quantified with rheological techniques, and the physical properties of the gels were determined, at small and large deformations. From the swelling/dissolving behavior of the gels in various media, the nature of the crosslinks was determined qualitatively. The microstructure of the gels was determined with electron microscopy. Nmr-spectroscopy was applied in order to elucidate changes in conformation during heating. It was found that the formation of a continuous covalently crosslinked network is not a prerequisite for thermally-induced protein gelation. The properties of a gel strongly depend on the pH at which the gel is formed. When heat-set at high pH(pH～10), a homogeneous, strong, and almost transparent gel is formed, consisting of flexible crosslinked protein gels. Heat-setting at low pH (pH 5) leads to the formation of a heterogeneous and weak gel, which easily exudes water. This gel consists of crosslinked aggregated protein. The ionic strength of the solvent in which the protein is dissolved and heat-set has a much lower effect on gel properties.     

Ultraviolet light-induced crosslinking of mRNA to proteins.

Irradiation of intact or EDTA-dissociated L-cell polyribosomes with 254 nm UV light at doses of 1-2 x 10(5) ergs/mm2 extensively crosslinks mRNA to proteins. The crosslinked mRNA-protein complexes can be isolated on the basis of buoyant density in urea-containing CS2SO4 gradients that dissociate non-covalent complexes. Crosslinking of mRNA can also be assayed by phenolchloroform extraction. mRNA recovered from the crosslinked complexes by digestion with proteinase K has the same electrophoretic mobility in polyacrylamide gels as unirradiated mRNA. Therefore, irradiation does not either crosslink RNA molecules to RNA molecules or break phosphodiester bonds. With these methods it has been found that more than 70% of high molecular weight polydisperse mRNA, but only 25-40% of histone mRNA, can be crosslinked to protein. On the basis of buoyant density the histone mRNA-protein complex had a protein content of 26%, whereas the mean protein content of most non-histone mRNA-protein complexes was 65%. It is concluded that most mRNA in polyribosomes is in close contact with proteins, and that histone mRNA can be crosslinked to many fewer proteins that most other mRNAs.     

Mechanically induced helix-coil transition in biopolymer networks

The quasi-equilibrium evolution of the helical fraction occurring in a biopolymer network (gelatin gel) under an applied stress has been investigated by observing modulation in its optical activity. Its variation with the imposed chain extension is distinctly nonmonotonic and corresponds to the transition of initially coiled strands to induced left-handed helices. The experimental results are in qualitative agreement with theoretical predictions of helices induced on chain extension. This new effect of mechanically stimulated helix-coil transition has been studied further as a function of the elastic properties of the polymer network: crosslink density and network aging.     

Thermodynamic aspects of the gelatinization and swelling of crosslinked starch

Samples of epichlorohydrin crosslinked potato starch were prepared by using a high ratio of starch to water and alkali concentration below the gelatinization level. This yielded crosslinked samples that were partially crystalline, and where the number of crosslinks could be varied between 1 and 20 crosslinks per 100 anhydroglucose units. The degree of swelling varied regularly with degree of crosslinking, and the molecular weight between crosslinks M_c as derived from swelling data in a good swelling agent compared favorably with M_c derived from chemical analysis. From the heat of gelatinization of the crosslinked starches, as observed in a differential scanning calorimeter, for gelatinization in glycerol, water, and dimethylsulfoxide, a model for the gel state of the crosslinked starch is proposed. It is concluded that the entropy of melting is the determining factor in establishing the temperature of gelatinization.     

Analysis of EDTA-chelatable proteins from DNA-protein crosslinks induced by a carcinogenic chromium(VI) in cultured intact human cells

DNA-protein crosslinks (DPCs) were induced in intact human leukemic T-lymphocyte MOLT4 cells or isolated nuclei by treatment with potassium chromate, chromium(III) chloride hexahydrate or x-rays. The proteins complexed to DNA were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE). A group of identical non-histone proteins was crosslinked to DNA by any of the three treatments, except that a 51 kDa basic protein was additionally complexed to DNA when either potassium chromate or chromium(III) chloride hexahydrate was the crosslinking agent. Treatment of chromate-induced DNA-protein crosslinks with EDTA or thiourea followed by ultracentifugation dissociated the major proteins from the complex indicating that these proteins were crosslinked to DNA by direct participation of a EDTA-chelatable form of chromium such as Cr(III) through sulfur containing amino acid residues. The 51 kDa protein was not seen in the post-EDTA pellet but was present in the post-thiourea pellet, indicating that it was also crosslinked to DNA by Cr(III) through non-sulfur-containing amino acids. Digestion of x-rays-induced DPCs by DNase I also revealed this protein on two-dimensional gels indicating that the same protein was also crosslinked by oxidative mechanisms. The involvement of oxidative mechanisms in the crosslinking process was indicated as the majority of the proteins in chromate-induced DPCs were resistant to EDTA and thiourea treatment, and were found to crosslink to DNA when x-rays were used as the crosslinking agent. These results suggest that the chromate-induced DPCs are formed by the generation of reactive oxygen species during the intracellular chromate reduction as well as by the biologically generated Cr(III). About 19% of DNA-protein crosslinks actually involve Cr(III) crosslinking DNA to proteins, about 14% involve Cr(III) crosslinking DNA to proteins through non-sulfhydryl containing moieties and about 5% involve Cr(III) crosslinking DNA to sulfhydryl groups on proteins. The remaining 81% of DNA-protein crosslinks appear to be oxidatively crosslinked out of which about 45% appear to be through sulfhydryl groups and another 36% appear to be through non-sulfhydryl groups.     

Crosslinking of phycobiliproteins from the cyanobacterium Mastigocladus laminosus with bis-imidates: localization of an intrasubunit and an intersubunit crosslink in C-phycocyanin

The light-harvesting pigment-protein complexes allophycocyanin (AP), C-phycocyanin (PC) and phycoerythrocyanin (PEC) of the cyanobacterium Mastigocladus laminosus consist of alpha- and beta-subunits containing about 170 amino-acid residues each. These two subunits form an alpha,beta-monomer, three of which build up a disc-shaped trimer. In this study these phycobiliproteins were crosslinked with bis-imidates. Various spacer lengths of the reagent and various aggregation states of the phycobiliprotein were tested. An intersubunit crosslink could be verified in all three phycobiliproteins. PC-trimers were crosslinked with the homobifunctional reagent dimethyl pimelimidate having a maximal crosslinking distance of 10 A. Two crosslinks could be identified: an intramonomer intersubunit crosslink with a yield of 48% and an intrasubunit crosslink within alpha PC (57%). These products were chemically and enzymatically fragmented and the small crosslinked peptides were isolated and then identified by amino-acid analysis. The following amino acids were crosslinked: alpha-Val 1 with beta-Ala 1 and alpha-Lys 62 with alpha-Lys 134. Both crosslinks could be localized within the known three-dimensional structure of PC.     

Ultracentrifugal compression of gels

It has been observed that the amount of water associated with casein micelles is markedly higher when measured by intrinsic viscosity than by water content of the pelleted casein. A possible explanation of this discrepancy is that water is squeezed out of the pellet by the ultracentrifugal field. We have calculated the potential magnitude of this effect by considering a simple model system: an elastic gel swollen by solvent and compressed by the centrifugal field. Equilibrium is reached when the sum of the ultracentrifugal, elastic, and mixing free energies is a minimum. The equilibrium degree of swelling is calculated as a function of rotor speed, thickness of the unsolvated gel material, and enthalpy of mixing of solvent and solute. Sizable compressions can occur for highly swollen gels, if the enthalpy of mixing is moderately positive. Casein micelles from intrinsic viscosity measurements have a “hydration” of about 3.7 g water/g protein, corresponding to a swelling ratio of 6.3 relative to the dry protein. The observed pellet hydration is 1.9 g/g. Under the conditions of the experiment, pelleting at 25,000 rpm is predicted to decrease the swelling ratio to 4.9, and the hydration to 2.7 g/g, about half the observed decrease, if the enthalpy of mixing is 0.5 RT/mol segment. These calculations may be relevent to the deterination by pelleting of the solvent content of other biopolymer gels.     

Cation effects on swelling of kappa-carrageenan: a photon transmission study

Swelling behavior of kappa-carrageenan gels in water and KCl solutions was investigated by photon transmission experiments following the preparation of gels in the presence and absence of externally added K+ ion as a gel promoting agent. Transmitted photon intensity, Itr, increased continuously during swelling depending on the carrageenan and ion content in the gel. This increase in Itr was modeled using the Li-Tanaka equation. Both the experimental work and the model showed that the swelling of low carrageenan and ion content gels took less time than that of high ion content gels. It is confirmed that double helices in a swollen gel move much faster in pure water than in KCl solution during swelling processes. Swelling time constants, tau1, and collective diffusion coefficients, Do, were measured for the gels swollen in water and KCl solutions.     

Crosslinking of the complementary strands of DNA by UV light: dependence on the oligonucleotide composition of the UV irradiated DNA

UV light crosslinks the complementary strands of DNA. The interstrand crosslinks may contribute to the biological and pathological effects that UV irradiation is known to bring about. Here alkaline agarose gel electrophoresis was used to assess the crosslinked fraction of 31 selected restriction fragments of six viral and plasmid DNA molecules exposed to UVC light irradiation. As many as 17 independent experiments were performed with the particular DNA fragments to get sufficiently precise data suitable for quantitative analyses. The data were used to determine how the crosslinked fraction depended on the dinucleotide, trinucleotide and tetranucleotide contents of the irradiated DNA fragments. This analysis demonstrated that DNA conformation and/or flexibility, rather than the local double helix thermostability, governed the phenomenon of crosslinking. For example, (GA).(TC) suppressed the crosslink formation in DNA more than any dinucleotide composed of only G and C. In addition, (CTAG).(CTAG) promoted crosslinking much more than any other tetranucleotide, including e.g. (TATA).(TATA), whereas the closely related (CATG).(CATG) belonged among the tetranucleotides that most suppressed the UV light induced crosslinks between the complementary strands of DNA. The present data reproduced crosslinking of the analyzed 31 restriction fragments with a correlation coefficient exceeding 0.90. This result will be useful to predict crosslinking along the whole human genome.     

Physical characteristics of calcium induced κ- carrageenan networks

The presence of an optimum counter-ion concentration in calcium-induced κ-carrageenan gels at low polymer concentrations of 5 and 10 g/l is observed. At approximately the stoicheometric molar ratio of 1 calcium per carrageenan sulphate, a gel with high elastic modulus, high optical clarity and fine network structure is observed. On further increase of counter-ion concentration beyond this optimum, elastic modulus decreases significantly associated with sharp increase in the gels turbidity together with a network characterised with coarse and large-pore mesh.

The quite complete characterisation of the various gel networks both mechanically by ways of oscillatory and static rheology and optically by turbidimetry and cryo-SEM shows that the extensive structural charge neutralisation of the polysaccharide by divalent calcium ions is responsible for a marked aggregation of the polymer strands reminiscent of precipitation. At lower counter-ion to polymer ratios, onset of gelation might prevent such phase separation.     

Structure of E. coli 16S RNA elucidated by psoralen crosslinking

E. coli 16S RNA in solution was photoreacted with hydroxymethyltrimethylpsoralen and long wave ultraviolet light. Positions of crosslinks were determined to high resolution by partially digesting the RNA with T1 RNase, separating the crosslinked fragments by two-dimensional gel electrophoresis, reversing the crosslink, and sequencing the separated fragments. This method yielded the locations of crosslinks to +/- 15 nucleotides. Even finer placement has been made on the basis of our knowledge of psoralen reactivity. Thirteen unique crosslinks were mapped. Seven crosslinks confirmed regions of secondary structure which had been predicted in published phylogenetic models, three crosslinks discriminated between phylogenetic models, and three proved the existence of new structures. The new structures were all long range interactions which appear to be in dynamic equilibrium with local secondary structure. Because this technique yields direct information about the secondary structure of large RNAs, it should prove invaluable in studying the structure of other RNAs of all sizes.     

Crosslinking of proteins to ribosomal RNA in HeLa cell polysomes by sodium periodate.

HeLa cell polysomes were oxidized with sodium periodate and reduced with sodium borohydride to induce covalent crosslinks between ribosomal RNA and nearby proteins. We proved that RNA was tryly crosslinked to protein in oxidized, and not in control, samples using denaturing cesium trichloroacetate density gradients and phenol extraction. By both one- and two-dimensional gel analysis, we found that protein S3a can be crosslinked to 18S RNA, protein L3 to 28S RNA, and proteins L7′ and L23′ to 5.8S RNA. Because of the specificity of the periodate reaction, and since we were able to crosslink protein S1 to 16S RNA in $\text{Escherichia}$,$\text{coli}$ 30S ribosomal subunits, it is likely that we have crosslinked proteins to the 3′OH ends of HeLa polysomal RNAs.     

Participation of the alpha 2(I) chain of bovine skin collagen in the formation of mature crosslinks

It is shown that regions of unreduced, insoluble cow hide collagen, represented by the peptides alpha 1(I)-CB6, alpha 2(I)-CB4 and the alpha 2(I)-CB3,5, are involved in the formation of unreducible acid-stable and mature-type crosslinks. The characteristic ratio of the CNBr peptides in soluble type I collagen was found to be changed in the insoluble collagen of cow hides. The intensity of the bands of alpha 1(I)-CB6, alpha 2(I)-CB4 and alpha 2(I)-CB3,5, shown by dodecyl sulfate polyacrylamide gel electrophoresis, is significantly reduced in such samples, which indicates an involvement of these peptides in crosslink formation. The purified highly polymeric CNBr peptide fraction was also investigated to confirm the participation of the alpha 2 chain of type I collagen in mature crosslink formation. Chymotryptic digests of such material contain peptides which originate from alpha 2(I)-CB4, alpha 2(I)-CB3,5, and alpha 1(I)-CB6. Finally, acid hydrolysates of crosslinked material were screened carefully for crosslinks down to concentrations of 1 in 1000 amino acids. Only two compounds were detected, one identified as "hydroxyaldol-histidine" and the other an as yet unknown compound. These results indicate that both the alpha 1(I) and the alpha 2(I) chains are involved in mature crosslink formation and that the polymeric CNBr peptide fraction contains components crosslinked by so far uncharacterized, nonreducible crosslinks.