Barnacle Settlement on Hydrogels

Settlement of cultured Balanus amphitrite cyprid larvae was tested on different non-solid hydrogel surfaces. Gels consisting of alginate (highly anionic), chitosan (highly cationic), polyvinyl alcohol substituted with light-sensitive stilbazolium groups (PVA-SbQ; very low cationic) and agarose (neutral) were applied in cell culture multi-well plates. Polystyrene served as a solid surface reference. Preliminary experiments were performed to determine whether any substances leaching out of the gels could inhibit barnacle settlement. Whilst leachate from the gels revealed no toxicity towards Artemia salina nauplius larvae, PVA-SbQ in solution at and above a concentration of 0.4 ppm inhibited B. amphitrite cyprid settlement. Gels were therefore washed to avoid such effects during further testing, and toxicity and settlement tests with B. amphitrite nauplii and cyprids, respectively, applied to verify that washing was effective. Settlement was tested directly on the different test materials, followed by a quality test of non-settled larvae. All gels inhibited barnacle settlement compared to the polystyrene controls. Gels consisting of 2.5% PVA-SbQ or 0.5% agarose showed promising antifouling properties. Although some settlement occurred on 2.5% PVA-SbQ gels, metamorphosis was clearly inhibited. Only 10% of the larvae had settled on 0.5% agarose gels after 8 d. Less than 40% settlement occurred on alginate gels, as well as on 2% chitosan gels. Quality testing showed that the majority of remaining non-settled larvae in all gel experiments were able to settle when offered a suitable solid substratum.     

An apparatus for the simultaneous casting of large numbers of uniform cylindrical polyacrylamide gels

An apparatus for the simultaneous casting of a large number of cylindrical polyacrylamide gels is described. Gels can be cast that are uniform with respect to length, loading-surface flatness, and internal polymerization properties. The basis of the method is casting the gels as an inverted single block which totally excludes oxygen from gel-loading surfaces during polymerization.     

Rheologic behavior of deoxyhemoglobin S gels

The physical properties of deoxyhemoglobin S gels formed from solutions at concentrations and temperatures approaching those in vivo have been characterized by stress relaxation using a rotational rheometer. Gels were annealed in the rheometer and then subjected to a constant shear strain; thereafter the stress sustained was followed with time. Gels with solid-like behavior held stress indefinitely, and were characterized by yield temperature (the temperature at which stress decreased). Gels with less solid behavior were unable to hold target stress, and were characterized by yield stress (maximum stress attained) and equilibrium stress (final stress held). The samples were ultracentrifuged to calculate pellet and polymer masses. The solidity of the gels, as measured by yield temperature or yield stress, was related to the initial hemoglobin concentration, pellet and polymer masses, shear history, temperature, and the temperature and time of annealing. Solidity increased significantly with time when gels were annealed at 37 degrees C, whereas, when annealed at 25 degrees C, no or minimal increases in solidity were noted. Studies suggest that polymerization occurs rapidly and is completed early in or before the gel annealing period and that the increase in solidity with time of annealing is mainly due to factors other than polymer mass, i.e. alignment, increasing bond strength, water loss. The chemical activity of deoxyhemoglobin S did not affect the solidity of the formed gels. When the resultant polymer masses were comparable, gels formed from samples with albumin present (higher initial total protein concentration, but lower initial deoxyhemoglobin S concentration), had the same behavior as gels formed from solutions with higher initial hemoglobin S concentration. These findings demonstrate that gel annealing conditions must be standardized when comparing the rheologic behaviors of deoxyhemoglobin S gels and indicate that the gel's physical properties (influenced by polymer mass, shear history, annealing time) must be considered in understanding pathophysiology of sickling disorders.     

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.     

A novel artificial substrate for cell culture: Effects of substrate flexibility/malleability on cell growth and morphology

Summary Gels of glyoxyl agarose (GA) are evaluated as a novel flexible substrate for cell culture with physical properties comparable to extracellular matrix (ECM) gels. We show here that cells adhere well to pure GA gels; in addition, specific interactions involving matrix receptors can be studied when individual matrix molecules are bound to the gel covalently. When cells are grown on such substrates, morphology is comparable to that observed on “natural” matrix gels (reconstituted gels of collagen type I or of Matrigel): rather than being flattened as in monolayer cultures on tissue culture plastic the cells assume a rounded morphology and tend to form tissue-like aggregates. The effects of the artificial matrix gels are discussed in the context of previous publications on cell interactions with the extracellular matrix, suggesting that in addition to specific recognition of matrix molecules the physical properties of ECM by themselves can be decisive for cell differentiation. We conclude that gels of glycoxyl agarose a) provide a useful model to mimic the physical properties of matrix gels without the presence of specific adhesion factors; b) may be useful as a general, non-specific ECM allowing cells to be cultured in vitro under conditions favorable for differentiation; and c) allow to design a variety of “synthetic” ECM models composed of a chemically defined gel matrix, which can be supplemented with covalently bound molecules to be recognized by cell surface receptors.     

Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEF

Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20-50 ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS.     

Peptides and proteins in a confined environment: NMR spectra at natural isotopic abundance.

Confinement of proteins and peptides in a small inert space mimics the natural environment of the cell, allowing structural studies in conditions that stabilize folded conformations. We have previously shown that confinement in polyacrylamide gels (PAGs) is sufficient to induce a change in the viscosity of the aqueous solution without changing the composition and temperature of the solvent. The main limitation of a PAG to run NMR experiments in a confined environment is the need for labelling the peptides. Here we report the use of the agarose gel to run the NMR spectra of proteins and peptides. We show that agarose gels are completely transparent in NMR experiments, relieving the need for labelling. Although it is necessary to expose biomolecules to fairly high temperatures during sample preparation, we believe that this is not generally an obstacle to the study of peptides, and found that the method is also compatible with temperature-resistant proteins. The mesh of agarose gels is too wide for direct effects of confinement on the stability of proteins but confinement can be easily exploited to interact the proteins with other reagents, including crowding macromolecules that can eventually lead to fold stabilization. The use of these gels is ideally suited for low-temperature studies; we show that a very flexible peptide at subzero temperatures is stabilized into a well-folded conformation.     

Electrophoresis of DNA fragments in gels from acrylamide-rich copolymers

Two polyacrylamide-rich, non-toxic, gelable copolymers have been developed to facilitate the formation of user-cast electrophoresis gels. Gel formation is accomplished with dithiothreitol as the chemical cross-linking agent. The higher molecular weight copolymer is suitable for casting gels of copolymer concentration less than or equal to 8%. Gels of 3% concentration are excellent for resolving dsDNA fragments up to approximately 3000 base pairs. Because the cross-linking chemistry is not thwarted by the presence of urea, it is also possible to cast denaturing gels with these copolymers.     

Immobilization of trypsin on chitosan gels: use of different activation protocols and comparison with other supports

Trypsin was immobilized on chitosan gels coagulated with 0.1 or 1 M NaOH and activated with glutaraldehyde or glycidol. The derivatives were characterized by their recovered activity, thermal (40, 55 and 70 degrees C) and alkaline (pH 11) stabilities, amount of enzyme immobilized on gels for several enzyme loads (8-14 mg(protein)/g(Gel)) and compared to agarose derivatives. Enzyme loads higher than 14 mg(protein)/g(Gel) can be immobilized on glutaraldehyde derivatives, which showed 100% immobilization yield and, for loads up to 8 mg(protein)/g(Gel), 100% recovered activity. Activation with glycidol led to lower immobilization yields than the ones obtained with glutaraldehyde, 61% for agarose-glyoxyl (AgGly) with low grade of activation and 16% for the chitosan-glyoxyl (ChGly), but allowed obtaining the most stable derivative (ChGly), that was 660-fold more stable than the soluble enzyme at 55 and 70 degrees C-approximately threefold more stable than AgGly. The ChGly derivative presented also the highest stability during incubation at pH 11. Analyses of lysine residue contents in soluble and immobilized trypsin indicated formation of multipoint bonds between enzyme and support, for glyoxyl derivatives.     

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.     

A technique for electrophoresis in multiple-concentration agarose gels

A technique has been developed for embedding several agarose gels (running gels), each of a different agarose concentration, within a single 1.5% agarose slab. Equal portions of a sample were placed at the origin of each running gel and were simultaneously subjected to electrophoresis. Protein within the running gels was detected by staining with Coomassie blue; 0.2% gels were the least concentrated gels that were stained without gel breakage. Using the above technique, the dependence of electrophoretic mobility on agarose concentration has been measured for bacteriophage T7 capsids and a capsid dimer.     

Multipoint covalent immobilization of microbial lipase on chitosan and agarose activated by different methods

In this work Candida antarctica lipase type B (CALB) was immobilized on agarose and chitosan. The influence of activation agents (glycidol, glutaraldehyde and epichlorohydrin) and immobilization time (5, 24 and 72 h) on hydrolytic activity, thermal and alkaline stabilities of the biocatalyst was evaluated. Protein concentration and enzymatic activity in the supernatant were determined during the immobilization process. More active derivatives were attained when the enzymatic extract was first purified through dialysis. The highest activities achieved were: for agarose-glyoxyl (with glycidol), 845 U/g of gel, after 72 h of immobilization; for chitosan-glutaraldehyde and agarose-glutaraldehyde, respectively, 1209 U/g of gel and 2716 U/g of gel, after 5 h of immobilization. Thermal stability was significantly increased, when compared to the soluble enzyme: 20-fold for agarose-glyoxyl (with glycidol)-CALB, 18-fold for chitosan-glutaraldehyde-CALB and 21-fold for agarose-glutaraldehyde. The best derivative, 58-fold more stable than the soluble enzyme, was obtained when CALB was immobilized on chitosan activated in two steps, using glycidol and glutaraldehyde, 72 h immobilization time. The stabilization degree of the derivative increased with the immobilization time, an indication that a multipoint covalent attachment between enzyme and the support had really occurred.     

In vivo measurement of flavour release from mixed phase gels

Flavour release was investigated from pure gelatin, pure agarose and mixed gelatin-agarose gels, all containing 25% sucrose and flavoured with p-cymene, ethyl butyrate, pyrazine and ethanol. Gels were characterised by optical microscopy, and rheological techniques to determine phase separation, elastic modulus and melting temperature. Volatile release was measured by monitoring the four volatiles in the expired air from one individual eating the gels, using Atmospheric Pressure Chemical Ionisation-Mass Spectrometry. The release pattern of p-cymene was not affected by gel type. The release of ethanol, ethyl butyrate and pyrazine was affected to different extents by the matrix suggesting that both the properties of the volatile and the matrix determine volatile release in vivo.     

The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis

1. Gels were prepared with recrystallized acrylamide and bisacrylamide. Electrophoresis was in tris-sodium acetate-EDTA buffer for 0.5 to 3hr. Gels were scanned at 280 or 265mmu. Techniques are described for slicing and radioactive counting. 2. The mobility of RNA was inversely related to the sedimentation coefficient and varied with gel concentration. Electrophoresis in 2.2-2.6% gels gives a fractionation similar to density-gradient centrifugation. It shows the two ribosomal RNA components, the 45s precursor, transfer RNA and minor components. In 5% and 7.5% gels, 4s and 5s RNA separated and ribosomal RNA was excluded. 3. The resolution is greater and more detailed than by centrifugation, and many samples can be analysed simultaneously and rapidly.     

Stereocontrolled Synthesis of an Octasaccharide Part of I-Active Glycolipids

The effects of glycerin and ethylene glycol on the elastic modulus and DSC thermograms of agarose and kappa-carrageenan gels were examined to clarify the relation between structure and properties. The elastic modulus of these gels as a function of the concentration of polyols increased up to a certain concentration and then decreased with increasing concentration of polyols. These polyols shifted the melting temperature of the gel to higher temperatures in kappa-carrageenan gels but to lower temperatures in agarose gels. The temperature dependence of elastic modulus was changed in opposite directions in agarose and kappa-carrageenan gels by the addition of polyols, and this is discussed on the basis of model consisting of junction zones which are connected by Langevin chains. It was suggested that the mean distance between junction zones became shorter in the presence of a small amount of polyols.     

Effects of Ca, Cu, Al and La on pectin gel strength: implications for plant cell walls

Rheology of Ca-pectate gels is widely studied, but the behaviour of pectate gels formed by Cu, Al and La is largely unknown. It is well known that gel strength increases with increasing Ca concentration, and it is hypothesised that this would also be the case for other cations. Pectins are a critical component of plant cell walls, imparting various physicochemical properties. Furthermore, the mechanism of metal toxicity in plants is hypothesised to be, in the short term, related to metal interactions with cell wall pectin. This study investigated the influence of Ca, Cu, Al and La ion concentrations at pH 4 on the storage modulus as a function of frequency for metal-pectin gels prepared from pectin (1%) with a degree of esterification of 30%. Gels were formed in situ over 6 d in metal chloride solution adjusted daily to pH 4. Cation concentration was varied to develop a relationship between gel strength and cation concentration. At similar levels of cation saturation, gel strength increased in the order of La < Ca ? Al ? Cu. The swelling of the gels also varied between cations with Ca gels being the most swollen.     

Staining of glycoproteins in polyacrylamide and agarose gels with fluorescent lectins.

We describe a simple and sensitive method for staining of the carbohydrate moiety of glycoproteins in polyacrylamide or agarose electrophoretic gels. Gels are incubated in a solution of fluorescein-labeled concanavalin A. Following destaining with a neutral buffer, glycoproteins exhibit fluorescence under long-range ultraviolet light. Thus, the glucose/mannose containing β- and γ-chains of human fibrinogen give fluorescent bands, whereas the carbohydrate-free α-chain does not react. Less than 100 ng of hexose bound to fibrinogen β- or γ-chains could be detected. The procedure is suitable for staining of other carbohydrate residues in glycoproteins, which can be recognised by specific agglutinins, as shown by binding of fluorescein-labeled lectins from Ricinus communis to galactose residues of fibrinogen.     

Evaluation of agar and agarose gels for studying mechanical impedance in rice roots

Agar and agarose gels were evaluated as systems to mechanically impede roots of rice (Oryza sativa L.). Two-layer gels were used so that seedlings established in a layer of weak gel (0.35% weight/volume) and then grew downwards to encounter a treatment gel of up to 5.0% (w/v). Agarose gels were stronger than agar gels of the same concentration, reaching a maximum penetrometer resistance of 1.2 MPa at a concentration of 5.0%, compared to 0.3 MPa with agar. The 5.0% agar gel stimulated elongation of the seminal axis by 40% in seedlings of variety TN1 (compared with elongation in the 0.2% gel), but decreased it by 15% in the variety Lac 23. Although increasing agarose concentration decreased seminal axis elongation in both varieties, the seminal axis did not reach the lower layer of treatment gel when the concentration of the treatment gel was greater than 2.0%. The decreased root elongation was therefore a non-mechanical inhibition. In experiments conducted using a different batch of agarose, these inhibitory effects were not seen and strong agarose gels stimulated seminal axis elongation. It was concluded that the agar and agarose gel systems studied were unsuitable for studying the effect of mechanical impedance on the elongation of rice roots and that great care should be taken in interpreting the results of experiments using gels as a growth medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Hyaluronate-alginate gel as a novel biomaterial: mechanical properties and formation mechanism

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated to combine the gel-forming properties of alginate with the healing properties of hyaluronate. Gels were prepared by diffusion of calcium into alginate-hyaluronate mixtures, with an alginate content of 20 mg/mL. The hyaluronate source was shown to have significant effect on the aspect and the properties of the gels. The gels have viscoelastic behaviour and the transient measurements carried out in creep mode could be interpreted through a Kelvin-Voigt generalised model: experimental data led to the steady state hardness and a characteristic viscosity of the gel. Gels prepared from Na rooster comb hyaluronate with weight ratio up to 0.50 have satisfactory mechanical properties, and fully stable gels are obtained after a few days; on the contrary, use of lower molecular weight hyaluronate led to loose gels for hyaluronate contents over 0.25. Gel formation was investigated by measurements of the exchange fluxes between the calcium chloride solution and the forming gel, which allowed thorough investigations of the occuring diffusion phenomena of water, calcium ion and hyaluronate. Strong interactions of water with hyaluronate reduce significantly the rate of weight loss from the gel beads and allows higher water content in steady-state gels. Calcium content in the gel samples could be correlated to the actual alginate concentration, whatever the nature and the weight ratio of hyaluronate.     

Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels

We have studied with a number of bacterial restriction enzymes the conditions for digestion of DNA in agarose and polyacrylamide gels. The restriction endonucleases HpaII, MspI, HaeIII, HindIII, TaqI, HhaI, AluI, BamHI, EcoRI and SalI are capable of digesting DNA in agarose gels of low electroendosmosis and low sulfate concentration. All enzymes, except BamHI, are also capable of digesting DNA in polyacrylamide gels. With this method, rapid two-dimensional restriction mapping of genomes with low and high sequence complexity is possible.