A novel technique for entrapment of hybridoma cells in synthetic thermally reversible polymers

Summary A new technique for entrapment of cells in thermally reversible polymer (poly-N-vinylcaprolactam - PVCl) has been developed. Using stabilizers, such as ovalbumin, sodium carboxymethylcellulose or egg powder were necessary to give structures that were stable upon stirring carriers. Two hybridoma lines, in particular, 3B4 and 1A10 cells were entrapped in gel and produced monoclonal antibodies IgG and IgM classes, respectively, against Arabis mosaic virus.     

Water-in-ionic liquid microemulsion-based organogels as novel matrices for enzyme immobilization

The use of water-in-ionic liquid microemulsion-based organogels (w/IL MBGs) as novel supports for the immobilization of lipase B from Candida antarctica and lipase from Chromobacterium viscosum was investigated. These novel lipase-containing w/IL MBGs can be effectively used as solid phase biocatalysts in various polar and non-polar organic solvents or ILs, exhibiting up to 4.4-fold higher esterification activity compared to water-in-oil microemulsion-based organogels. The immobilized lipases retain their activity for several hours at 70°C, while their half life time is up to 25-fold higher compared to that observed in w/IL microemulsions. Fourier-transform infrared spectroscopy data indicate that immobilized lipases adopt a more rigid structure, referring to the structure in aqueous solution, which is in correlation with their enhanced catalytic behavior observed.     

Studies on immobilization of the thermophilic bacterium Thermus aquaticus YT-1 by entrapment in various matrices

Summary Immobilization of the thermophilic bacterium Thermus aquaticus YT-1 has been studied using various entrapment techniques. Alginate, -carrageenan, agar, agarose and polyacrylamide were tested as supports during operation at 65°C at which the cells are known to produce protease when grown free in solution. Alginate showed toxic effects and no viability was observed after entrapment in Ca alginate or even after exposure of free living cells to sodium alginate. Polyacrylamide was observed to be the best support. Protease activity was closely related to the appearance of free cells in the medium.     

A novel technique for measuring heart rate in a free swimming marine vertebrate

A mouth opening sensor incorporating a magnet and Hall sensor attached to a data logging unit was used to monitor the breathing and foraging behavior of a free-swimming leatherback sea turtle (Dermochelys coriacea). Analysis of these data revealed a rhythmic low amplitude oscillation. Further investigation of the frequency of this signal lead us to believe that the movements (< 0.1 mm) are caused by the movement of blood through the nearby blood vessels. Putative heart rate decreased during dive descent and increased considerably during dive ascent reflecting the bradycardia and anticipatory tachycardia recorded by other means in other air-breathing divers. Oscillation frequencies were also comparable to the heart rate recorded in leatherbacks by means of implanted electrodes. We therefore propose that this device which was already known to reliably record behaviour such as breathing, feeding and buccal oscillations in sea turtles also has potential for recording other signals which cause movement on the external surface of an animal.     

A novel micromanipulation technique for measuring the bursting strength of single mammalian cells

Summary Information about the bursting strength of animal cells is essential if the mechanisms of cell damage in bioreactors are to be understood, and if cell mechanical properties are ever to be related to cell structure and physiology. We have developed a novel cell compression technique that makes it possible to directly measure the bursting strength of single mammalian cells, and to infer information about cell mechanical properties. Offprint requests to: C. R. Thomas     

Microemulsion-based organogels as matrices for lipase immobilization

Organogels based on water-in-oil microemulsions can be formed using various natural polymers such as gelatin, agar or cellulose derivatives. Enzymes entrapped in the water core of the microemulsion can keep their activity and enhance their stability within the gel matrix. The importance of the microemulsion based organogels (MBGs) leans on their numerous potential biotechnological applications. An important example is the use of various lipase microemulsion systems for hydrolytic or synthetic reactions. In this review, several MBGs are being evaluated as immobilization matrices for various enzymes. The main subject focuses on the parameters that affect the use of MBGs as media for bioorganic reactions using lipases as catalysts.     

Cell growth patterns in immobilization matrices

Within an immobilized cell matrix, mass transfer limitations on substrate delivery or product removal can often lead to a wide range of local chemical environments. As immobilized living cell populations actively grow and adapt to their surroundings, these mass transfer effects often lead to strong, time-dependent spatial variations in substrate concentration and biomass densities and growth rates. This review focuses on the methods that have been devised, both experimentally and theoretically, to study the non-uniform growth patterns that arise in the mass transfer limited environment of an immobilization matrix, with particular attention being paid to cell growth in polysaccharide gels.     

A novel organic-inorganic hybrid monolith for trypsin immobilization

In proteomics, attention has focused on various immobilized enzyme reactors (IMERs) for the realization of high throughput digestion. In this report, a novel organic-inorganic hybrid monolith based IMER was prepared in a 100 μm i.d. capillary with 3-glycidoxypropyltrimethoxysilane (GLYMO) as the monomer and tetraethoxysilane (TEOS) as the crosslinker. Trypsin immobilization was achieved via the reaction between vicinal diol groups, which were obtained from hydrolysis of epoxy groups, and the amino groups of trypsin. Bovine serum albumin was digested thoroughly by this IMER in 47 s. After micro-reverse phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS) analysis and database searching, beyond 35% sequence coverage was obtained, and the result was comparable to that of 12 h in solution digestion. The present IMER has potential for high throughput digestion.     

A novel matrix for the immobilization of acetylcholinesterase

In this study, a new matrix for immobilization of acetylcholinesterase was investigated by using alginate and kappa-carrageenan. The effects of pH, temperature, storage and thermal stability on the free and immobilized acetylcholinesterase activity were examined. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) was also investigated for free and immobilized enzymes. For free and immobilized enzymes into Ca-alginate and alginate/kappa-carrageenan polymer blends, optimum pH and temperature was found to be 7 and 30 degrees C, respectively. For free enzyme, maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) values were found to be 6.35 mM and 50 mM min(-1), respectively, the same values for immobilized enzymes were determined as 8.68, 12.7 mM and 39.7, 52.9 mM min(-1), respectively. Storage and thermal stability of acetylcholinesterase was increased by as a result of immobilization.     

Poly-N-vinylcaprolactam gel: A novel matrix for entrapment of microorganisms

Summary A new gel-type support poly-N-vinylcaprolactam for microbial cell immobilization is presented. The method allows one to obtain beads of biocatalyst in a single step. The properties of beads obtained using different types of gel stabilizers were compared; the best stabilizer was found to be tannin. The method developed was used for entrapment of viable bacterial cells and fungal spores. The biocatalysts obtained were used for transformations of both hydrophilic (sorbitol, indolyl-3-acetic acid) and lipophilic (cortexolone, hydrocortisone) substrates.Abbreviations PVC poly-N-vinylcaprolactam - ImC immobilized cells - IAA indolyl-3-acetic acid - TLC thin layer chromatography     

A facile radiometric technique for measuring ATP

A facile radiometric technique for measuring ATP in samples of biologic origin is presented. The d-glucose-6-phosphate produced by the phosphorylation of excess tritiated d-glucose with crystalline hexokinase is stoichiometrically equivalent to the ATP present. Product is selectively precipitated with ethanolic barium acetate, washed with ethanol and the tritium label counted in a scintillation spectrometer. The technique is especially suitable for the measurement of ATP in large numbers of samples (50–100) and offers acceptable sensitivity down to 62 fmoles.     

A radiometric technique for measuring l-asparagine in picomol quantities

1. HeLa cells were cultured in the presence of heterologous immunoglobulin G and guinea-pig serum together with [(32)P]phosphate. 2. Incorporation of [(32)P]phosphate was significantly stimulated by anti-HeLa immunoglobulin G and complement-sufficient serum compared with immunoglobulin G from unimmunized rabbits and complement. Within 2.5h heat-inactivated guinea-pig serum and anti-HeLa immunoglobulin G stimulated [(32)P]phosphate incorporation to the same extent as heat-inactivated complement and immunoglobulin G from unimmunized rabbits. 3. Compared with cells exposed to immunoglobulin G from unimmunized rabbits together with complement, anti-HeLa immunoglobulin G with complement increased the phospholipid content of HeLa cells twofold within 5h of incubation. 4. Exposure of HeLa cells to anti-HeLa immunoglobulin G and complement for 5-22h resulted in a twofold increase in the net accumulation of [(32)P]phosphate in sphingomyelin and phosphatidylcholine and a 50% increase in the net accumulation of [(32)P]phosphate in phosphatidylethanolamine, compared with cultures exposed to immunoglobulin G from unimmunized rabbits and complement. 5. A transient accumulation of (32)P-labelled lysophosphoglycerides in HeLa cells exposed to antibody and complement was detected, confirming previous findings (Güttler & Clausen, 1969b). 6. The stimulation of [(32)P]phosphate turnover occurred in cells filling up their cytoplasma with vacuoles. This supports the suggestion that the accumulation of phospholipid in these cells may be concerned with the synthesis and function of cytomembranes.     

A stirred bath technique for diffusivity measurements in cell matrices

A stirred bath technique was developed for determining effective diffusivities in cell matrices. The technique involves cell immobilization in a dilute gel which has negligible effect on solute diffusion. Agar and collagen were tested as immobilizing gels. Agar gel was shown to have minor interactions with the diffusion of various biological molecules, and was used for immobilization of Ehrlich Ascites Tumor (EAT) cells. Diffusivities of glucose and lactic acid were measured in EAT matrices for cell loadings between 20 and 45 vol %. Treatment with glutaraldehyde was effective in quenching the metabolic activity of the cells while preserving their physical properties and diffusive resistance. The measured data agree favorably with predictions based on Maxwell's equation for effective diffusion in a periodic composite material. The stirred bath technique is useful for diffusivity determinations in immobilized matrices or free slurries, and is applicable to both microbial and mammalian cell systems.     

Efficient immobilization of lipases by entrapment in hydrophobic sol-gel materials

The commercial application of lipases as biocatalysts for organic synthesis requires simple but efficient methods to immobilize the enzyme, yielding highly stable and active biocatalysts which are easy to recover. In this study, we present a novel method to achieve lipase immobilization by entrapment in chemically inert hydrophobic silica gels which are prepared by hydrolysis of alkyl-substituted silanes in the presence of the enzyme. A typical immobilization procedure uses: an aqueous solution of lipase; sodium fluoride as a catalyst; and additives like polyvinyl alcohol or proteins and alkoxysilane derivatives like RSi-(OMe)(3) with R = alkyl, aryl, or alkoxy as gel precursors. The effect of various immobilization parameters like stoichiometric ratio of water, silane, type and amount of additive, type and amount of catalyst, and type of silane has been carefully studied. The new method is applicable for a wide variety of lipases, yielding immobilized lipases with esterification activities enhanced by a factor of up to 88, compared to the commercial enzyme powders under identical conditions. Studies on the stability of sol-gel immobilized lipases under reaction conditions or storage (dry, in aqueous or organic medium) revealed an excellent retention of enzymatic activity. The possible reasons for the increased enzyme activities are discussed. (c) 1996 John Wiley & Sons, Inc.     

A novel and in situ technique for the quantitative detection of MTBE and benzene degrading bacteria in contaminated matrices

A novel and in situ technique is presented here as a better alternative to culture-dependent and PCR-based techniques for the quantitative detection of predominant bacterial species involved in the bioremediation of contaminants. It allowed rapid, specific and in situ identification of Biosep-immobilized eubacteria from MTBE- and benzene-contaminated matrices.     

A stroboscopic technique for measuring Daphnia heart-beat rate

The variation of Daphnia heart-beat rate with temperature is described. The temperature can be changed and monitored continuously while the heartbeat rate is measured using stroboscopic techniques.