Ultrathin-layer isoelectric focusing in polyacrylamide gels on cellophane.

A method of ultrathin-layer isoelectric focusing in 0.12-, 0.24-, or 0.36-mm polyacrylamide gel layers polymerized on a sheet of cellophane as support is deseribed. The gel adheres firmly to the cellophane during all operation steps, is protected from fracture, and can be handied very conveniently. Resolution is markedly improved in ultrathin gels in comparison with the conventional 1- to 2-mm-thick gels. Staining and destaining are completed in a substantially shorter time than so far achieved. The ultrathin gels can be easily dried on the cellophane, a perfectly transparent record being obtained for future reference and for densitometric evaluation. Results are presented for a number of commercial proteins and legume seed proteins. The advantages of ultrathin-layer isoelectric focusing are discussed.     

Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing*

A method is described which allows the simultaneous separation of all polymorphic protein fractions in cow's milk in one single run. The separation could be achieved by isoelectric focusing in ultrathin-layer polyacrylamide gels. The method is especially suitable for screening purposes because it combines low costs, high resolution and short separation time.     

Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing

A method is described which allows the simultaneous separation of all polymorphic protein fractions in cow's milk in one single run. The separation could be achieved by isoelectric focusing in ultrathin-layer polyacrylamide gels. The method is especially suitable for screening purposes because it combines low costs, high resolution and short separation time.     

Use of immobilized cells of Rhizopus nigricans for the 11α-hydroxylation of progesterone

The filamentous fungus, Rhizopus nigricans, was immobilized in polyacrylamide, alginate, and agar gels and its ability to 11α-hydroxylate progesterone was examined. No activity was detected using polyacrylamide gel but both agar and alginate gels have proved capable of hydroxylation. Agar gels displayed faster rates and higher yields. It was possible to induce hydroxylase synthesis within agar and alginate gels, and microscopical examination provided evidence for hyphal growth within these gels. The concept of increased biomass was used to explain the observed increase in the rates of hydroxylase activity of the immobilized cells. Conversely, hyphal overcrowding was postulated for the rapid inactivation observed under some operating conditions.     

A New Brassinolide-related Steroid in the Leaves of Thea sinensis

Microscopic and dynamic mechanical properties for mixed aqueous gels of agar and gelatin have been studied. The microscopic observation showed formation of micro granules in the gels under the phase-contrast visual field. The constituent was recognized as agar by metachroma tic staining using a microspectrophotometer. Dynamic moduli of the gels were measured from 0.01 to 200 Hz by phase difference and by resonance. A minimum E' value was obtained for the mixed gel at a volume fraction of agar of 0.6. E′ of all gels and E″ of mixed gels were frequency dependent above 30 Hz.     

Molecular cloning in Escherichia coli of Erwinia chrysanthemi genes encoding multiple forms of pectate lyase.

The phytopathogenic enterobacterium Erwinia chrysanthemi excretes multiple isozymes of the plant tissue-disintegrating enzyme, pectate lyase (PL). Genes encoding PL were cloned from E. chrysanthemi CUCPB 1237 into Escherichia coli HB101 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322 and then screening recombinant transformants for the ability to sink into pectate semisolid agar. Restriction mapping of the cloned DNA in eight pectolytic transformants revealed overlapping portions of a 9.8-kilobase region of the E. chrysanthemi genome. Deletion derivatives of these plasmids were used to localize the pectolytic genotype to a 2.5-kilobase region of the cloned DNA. PL gene expression in E. coli was independent of vector promoters, repressed by glucose, and not induced by galacturonan. PL accumulated largely in the periplasmic space of E. coli. An activity stain used in conjunction with ultrathin-layer isoelectric focusing resolved the PL in E. chrysanthemi culture supernatants and shock fluids of E. coli clones into multiple forms. One isozyme with an apparent pI of 7.8 was produced at a far higher level in E. coli and was common to all of the pectolytic clones. Activity staining of renatured PL in sodium dodecyl sulfate-polyacrylamide gels revealed that this isozyme comigrated with the corresponding isozyme produced by E. chrysanthemi. The PL isozyme profiles produced by different clones and deletion derivative subclones suggest that the cloned region contains at least two PL isozyme structural genes. Pectolytic E. coli clones possessed a limited ability to macerate potato tuber tissues.     

Effects of common components on hardness of culture media prepared with gelrite™

Summary Tissue cultures on properly solidified Gelrite media generally showed superior shoot proliferation and rooting, as well as shoot and root vigor and callus development to those on TC agar. Vitrification, or hyperhydricity, was observed in both Gelrite and agar media and minimized by increasing the gel concentrations. Rigidity of Gelrite media depended on combined levels of MS macrosalts, basal nutrient formulations, sucrose concentration, pH, and Gelrite concentration. Most MS macrosalts increased hardness of Gelrite gels; NH₄NO₃ had a decreasing effect. Rigidity of TC agar gels increased with reductions of MS macrosalts. A slightly softer Gelrite medium resulted when sucrose was excluded. Both Gelrite and agar media were softer at lower pHs and harder at higher pHs. Activated charcoal and mannitol increased gel hardness, and more noticeably of agar gels. NaCl addenda reduced rigidity, with their effects being more pronounced on Gelrite than on agar gels. Gelrite is a trademark of Kelco Division of Merck & Co. (San Diego, CA), manufacturer of the gellan gum. Phytagel is a trademark of Sigma Chemical Co. (St. Louis, MO) for repackaged Gelrite. TC agar used for comparisons in this investigation is plant tissue culture tested agar obtained from JRH Biosciences (Lenexa, KS).     

The effects of culture medium rigidity on adventitious bud production and tissue vitrification in needle cultures of Sitka spruce [Picea sitchensis (Bong.) Carr.]

Adventitious bud formation on Sitka spruce [ Picca sitchensis (Bong.) Carr.] needle explants was strongly dependent upon the rigidity of the culture medium. In general, of organogenesis was greatest on weak gels and poorest on more rigid gels resulting from increased medium pH or agar strength. There was a significant interaction between agar strength and pH, with the optimum pH for organogenesis declining with increasing agar strength. Poor organogenesis at high agar concentrations was not due to toxic impurities since increased adventitious bud production could be stimulated by decreasing the medium pH whilst maintaining a high agar strength and an agar washing treatment had no significant effect. Although high levels of organogenesis could be sustained on weak gels the resultant adventitious shoots often showed severe vitrification. The frequency of shoots showing vitrification could be reduced by growing the tissues on harder media but this resulted in reduced shoot elongation. Vitrification of needle tissues did not stimulate the formation of adventitious buds in the absence of cytokinins.     

Mechanical properties of hydrocolloid gels filled with internally produced CO2 gas bubbles.

Agar (2%), alginate (1% algin), and kappa-carrageenan (1.5%) gel specimens were prepared from mother solutions that contained 0-2.5% sodium bicarbonate (agar and carrageenan) or calcium carbonate (alginate). Upon immersion in a citric acid bath (0-2%), the carbonate reacted with the diffusing acid to produce numerous carbon dioxide bubbles. The compressive strength and deformability of the gas-filled gels so produced were determined using a Universal testing machine and compared with those of pure gels and gels containing the carbonate but not subjected to the process after various immersion times. While the agar and alginate gels retained considerable mechanical integrity even after several hours, the carrageenan gels disintegrated after about 2-5 h. Under similar conditions, the number of bubbles produced in the agar gels was about twice that in the alginate gels, an observation that cannot be explained solely by stoichiometric considerations.     

Influence of agar on in vitro cultures: I. Physicochemical properties of agar and agar gelled media

Summary The success of in vitro culture is related to several factors. Beside factors associated with the plant material or the medium composition, the physicochemical characteristics of gelled media can play an important role. In this paper, the latter aspect has been considered and the nature of agar powders has been investigated. Moreover, the process of gel formation for three different media and the availability of water and minerals for the corresponding gels have been studied. Analysis of agar powders showed that they can contain different amounts of impurities and the dialysis of these powders suggested that the impurities might be available to the tissues. Thermal analysis on the hygroscopic properties of the agar brands suggest the importance of these data to obtain comparable and reproducible gelled media. The study on the process of formation of gelled media indicates that there is a critical temperature T_ss which can be used to control the gel processing. In fact, at this temperature, agar powders in water transform into a sol status through a rapid shift of electrical conductivity. Water potential of the medium, water loss from gels over the culture period, and the ease of releasing liquid from gels under pressure were shown to be different for different agar brands. A different availability of water and minerals in Murashige and Skoog medium was deduced from the gels prepared with three agar brands (Oxoid, Merck, and Roth).     

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.     

Biocatalysis using microemulsion-based polymer gels containing lipase

Natural gelling agents such as gelatin, agar and κ-carrageenan have been tested for the formation of lecithin microemulsion-based gels as well as hydrogels (without surfactant and oil). The results presented in this work provide information concerning the utility of these solid gels as lipase immobilization matrices. It was found that lipase from Pseudomonas cepacia keeps its catalytic function after entrapment in the gels, catalyzing the esterification reaction of propanol with lauric acid in various hydrocarbons at room temperature. Various parameters which affect the lipase catalytic behavior such as the nature and the concentration of the gelling agent, as well as the concentration of the biocatalyst and the mole ratio of the substrates have been examined. High yields (80%) were obtained with agar and κ-carrageenan organogels in isooctane. The remaining lipase activity, in repeated syntheses was found to depend on the nature of the biopolymer used for the formation of the organogels. Gelatin and agar microemulsion-based gels showed the highest operational stability.     

Cell wall polysaccharides from Gelidium species: physico-chemical studies using MRI techniques

Magnetic resonance imaging (MRI) has already been successively used to investigate polysaccharide matrices. In particular, MRI at microscopic resolution (MR microscopy) is now one of the most powerful techniques for studying the physical properties of natural hydrogels. To contribute to a better understanding of the correlation between chemical and physical properties of agar gels, we report here the measurement of the water magnetic parameters for agar gels extracted from different species of Gelidium: T1 and T2 relaxation times, magnetisation transfer (Ms /M0) and diffusion (D) were measured to evaluate their use for studying the gel characteristics. MR microscopic images were acquired at 7.05 Tesla using various pulse sequences. The results obtained confirmed the possibility to use quantitative MRI for the characterisation of physical parameters correlated with the type of agar chemical structure. In particular, T2 data obtained for gels at different concentrations indicate that this magnetic parameter is very sensitive to the agar concentration and hence particularly useful for the gel strength determination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of Biomass Dry Weights and Radial Growth Rates of Fungal Colonies on Media Solidified with Different Gelling Compounds

Penicillium commune, Aureobasidium pullulans, and Paecilomyces farinosus were grown on two different media solidified with agar, Pluronic F-127, Carrageenan X-4910, or Carrageenan X-4910 overlaid with cellophane. Growth on Carrageenan X-4910 was generally the same as that on agar, as was the visual appearance of the colonies, e.g., the pigmentation. The Carrageenan X-4910 gels had a melting point, depending on the medium, of 41 to 46(deg)C, and the dry weights of the colonies were readily determined at 60(deg)C. To determine the dry weights of the colonies grown on agar plates, the gels were boiled for 10 min to melt the agar. Comparison of these two procedures showed that the boiling procedure resulted in a 22% reduction of the biomass dry weight. Cellophane membranes did not affect the radial growth rate profoundly. The biomass density was almost halved for P. commune and P. farinosus grown with membranes, whereas the presence of the membrane did not affect the biomass density of A. pullulans. The biomass densities of the colonies grown on Pluronic F-127 were significantly reduced, while in most cases, the radial growth rates of colonies grown on Pluronic F-127 were significantly higher than those obtained on agar or Carrageenan X-4910. Furthermore, the morphology of the leading hyphae was altered, and the hyphal growth unit length was more than twice that obtained on agar and Carrageenan X-4910. Carrageenan X-4910 is a valuable gelling compound for the study of the growth of fungi, as the biomass dry weight is readily determined and growth is similar to that obtained on agar gels.     

Dynamic rheology of agar gels: theory and experiments. Part I. Development of a rheological model

A theoretical rheological model for agar gels is proposed, based on the bead and spring model for linear flexible random coils and the model for crosslinked polymers. The model introduces the concept of a temperature dependence of the monomeric friction coefficient, ζ₀, of the agar molecule. The model has a random coil-like behavior at high temperatures (close to 373 K), and contributions from a three-dimensional network at low temperatures (close to 273 K). A proposed temperature dependence of the net association rate allows the calculation of the fraction of associated molecules as a function of time and temperature. The proposed model predicts the gelation behavior of agar gels utilizing time–temperature data (cooling curves).     

Effect of "stacking" on the resolving power of ultrathin-layer two-dimensional gel electrophoresis

The resolving power of two-dimensional ultrathin-layer polyacrylamide gel electrophoresis with and without "stacking" was investigated. Side-by-side analysis shows that the use of a properly adjusted upper gel improves the resolution and reproducibility of this sensitive analytical method. The effects of various detergents (Nonidet-P40, Zwittergent, urea) on the ultrathin-layer polyacrylamide gel electrophoresis were also investigated. For this case, whole cell proteins of Pseudomonas aeruginosa and Staphylococcus aureus treated with different detergents were electrofocused in the presence of the same detergents.     

The agar component of the red seaweed Gelidium purpurascens

Chemical composition and rheological properties of agar isolated from Gelidium purpurascens, the agar after alkaline treatment, and a commercial agar are presented. The agar and alkali-treated agar gave weaker gels, as measured with an Instron 1122, than those of commercial agar. Xylose, glucose and glucuronic acid in the agar were removed together with 86% of the nitrogen content on alkaline treatment, indicating occurrence of these residues as carbohydrate-protein complexes. Sequential extraction of the alga accounted for low yields of agar as losses incurred on ethanol precipitation. Acid treatment of the residue from exhaustive aqueous extraction of the alga liberated a further 10% agar with increased gel strengths despite increased glucose inclusion, suggesting a lack of involvement of these ‘contaminant’ carbohydrate-protein residues in helix coil formation during gelling.     

Densitometric determination of nucleic acids in gels after electrophoresis

A technique for the quantitative determining of nucleic acids after electrophoresis by double-wave densitometry in UV spectrum by scanning agar gel along both axes was developed. Optical characteristics of agar and acrylamide-agar gels are given. The range of quantities measured is 6 divided by 30 micrograms of DNA per gel; the standard deviation adjusted to 1 microgram is less than +/- 3%.     

Sensitive detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in gels

A simple, highly sensitive zymogram technique for detection of endo-1,4-beta-glucanases and endo-1,4-beta-xylanases in polyacrylamide gels after electrophoresis or isoelectric focusing was developed. The detection employs transparent agar replicas containing soluble covalently dyed polysaccharides, hydroxyethylcellulose dyed with Ostazin brilliant red H-3B and beechwood 4-O-methyl-D-glucurono-D-xylan dyed with Remazol brilliant blue R, as the respective substrates. The high sensitivity of the detection is achieved by selective removal of depolymerized dyed substrates from the agar replicas by solvents which neither solubilize nor precipitate the original nondegraded dyed polysaccharides present in the agar gel.     

Dynamic rheology of agar gels: theory and experiments. Part II: gelation behavior of agar sols and fitting of a theoretical rheological model

A theoretical rheological model for agar gels, based on the bead spring model for linear flexible random coils and the model for crosslinked polymers, is successfully fitted to experimental gelation curves obtained over a wide range of cooling rates (0.5–20 °C min⁻¹) and agar concentration (1–3 wt%). The theoretical gelation temperature, T_gel^model increases with increasing agar concentration and decreasing cooling rate. The intrinsic net association rate increases significantly with increasing cooling rate. This increase is related to the higher probability of association of the non-associated agar molecules at higher cooling rates.