Immobilization and utilization of the recombinant fusion proteins trypsin-streptavidin and streptavidin-transglutaminase for modification of whey protein isolate functionality

A method was developed for the production of a hydrolyzed/polymerized whey protein derivative with altered solution and gelation properties using a combination of recombinant DNA and immobilized enzyme technologies. The recombinant fusion proteins trypsin-streptavidin (TrypSA) and streptavidin-transglutaminase (cSAcTG) were produced in Escherichia coli, extracted, and then immobilized by selective adsorption on biotinylated controlled-pore glass. Recirculation through a TrypSA reactor induced limited proteolysis of whey proteins. Hydrolysates were then recirculated through a cSAcTG reactor for incremental periods of time to arrive at increasing degrees of polymerization. The polymers were subsequently analyzed for viscosity/flow behavior, gelation properties, and fracture properties using shear rate ramps/intrinsic viscosity, small-strain oscillatory rheology, and vane viscometry, respectively. By combining limited proteolysis with controlled cross-linking, it was possible to create derivatives of whey proteins with enhanced functional properties. Increases in the degree of whey protein modification were correlated with greater apparent viscosity and intrinsic viscosity, lowered gel point temperatures, and stronger, more brittle gels. This method allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of modification. Utilization of a similar process may allow for the production of designer proteins engineered with specific functionalities.     

Modification of the rheological properties of whey protein isolate through the use of an immobilized microbial transglutaminase

A process was developed in which calcium-independent, microbial transglutaminase (mTgase) was immobilized to controlled-pore glass. Avidin was adsorbed to glass beads that had been derivatized and biotinylated. The enzyme was biotinylated and adsorbed to the avidin affinity matrix. Solutions of 8% whey protein isolate (WPI) were then incubated with the mTgase beads, resulting in limited cross-linking of whey proteins. As incubation time increased, intrinsic viscosity increased, gelation temperature decreased, and stronger, more brittle gels were formed upon heating. This process allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of cross-linking. The functional properties of several batches of WPI were modified using <10 mg of the same enzyme, illustrating the capacity of immobilized enzymes to be used more frequently in applications of this nature.     

Interplanetary Magnetic Field Polarity and the Size of Low-Pressure Troughs Near 180{degrees}W Longitude

When the interplanetary magnetic field is directed away from the sun, the area of wintertime low-pressure (300-millibar) troughs near 180 degrees W longitude is significantly larger than when the field is toward the sun. This relation persists during most of the winters of 1951 to 1973.     

Crisis management: some opportunities

Modern crises present decision makers with many agonizing management choices. Very often a crisis manager is confronted with a plethora of conflicting information and given very little time to choose an appropriate course of action. Although contemporary methods of systems analysis have been used in attempts to organize data and clarify options, they have generally been of little use in presenting an accurate picture of an opponent's values and perceptions. Thus it is clear that we must now make use of the improved communications and technological devices at our disposal if crises are to be avoided or resolved with minimum damage. Our proposal to establish international model-oriented computer-assisted conferences is designed to promote greater cooperation and understanding among scientists and crisis managers of differing nations by enabling them to share images of themselves and one another. With better information and more rational options available, the chances of catastrophic misunderstanding or miscalculation can be meaningfully reduced. We have proposed a possible scenario for the initial implementation of such a system to combat famine, and hope that the same approach might be used in other areas over time. The ultimate goal is a system by which specialists of all persuasions cooperate so that international crises will be resolved on the bases of mutual benefits without resort to armed conflict.     

Sex Discrimination in Gerris remigis: Role of a Surface Wave Signal

Even when blinded with masks, adult male water striders (Gerris remigis) accurately ascertain the sex of other adult water striders in the laboratory. Freely moving females that were artificially made to play back computer-generated male surface wave and body-contact signals of about 90 waves per second were treated as males by the masked males and as females when no such playbacks were made. Thus, the males can use presence or absence of the male signal as the sole means for sex discrimination.     

Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Bali cattle (Bos javanicus), which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5'- and 3'-long terminal repeats (LTRs), 0.4kb of truncated gag and 1.1kb of 3'-env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences, including gag, pol, vif, tat and rev, but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped, disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2)x10(6)CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as well. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus.