Immobilization of recombinant heparinase I fused to cellulose-binding domain.

Immobilization of biologically active proteins is of great importance to research and industry. Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins. We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD. Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD). The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies. The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column. The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin. C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose. CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity. By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled. The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared. The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization. Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step.     

Influence of Boar and Semen Parameters on Motility and Acrosome Integrity in Liquid Boar Semen Stored for Five Days

Ninety ejaculates from a total of 76 AI boars were extended in Beltsville Thawing Solution (BTS). Boar identity, breed, weight of the ejaculate and sperm concentration were registered. Motility and acrosome integrity were assessed after storage at 16–18°C for 6, 30, 54, 78, and 102 h. Storage time had a significant influence on both motility (p < 0.01) and acrosome integrity (p < 0.001). The Least Square Means for percentage of motility showed a small decline from 79.8% after 6 h of storage to 78.4% at 102 h. Motility at 78 and 102 h was significantly different from motility at 6 h (p < 0.05). The percentage of sperm cells with normal acrosomes declined throughout the experiment. The Least Square Means for 6, 30, 54, 78, and 102 h of storage were 93.9%, 90.6%, 88.0%, 84.8%, and 78.2%, respectively. The decrease in acrosome integrity from one storage time to the next was highly significant throughout the trial (p < 0.001). There was a significant influence of boar (p < 0.001) and sperm concentration (p < 0.01) on motility, while acrosome integrity was affected only by boar (p < 0.001). Breed of the boars and weight of the ejaculate did not influence the dependent variables.     

Seeing without being seen: a removal experiment with mixed flocks of Willow and Crested Tits Parus montanus and cristatus

This paper tests the hypothesis that foraging site selection reflects a trade-off between the various needs for concealment from predators, to find food, and for the individual to maintain some view of its surroundings. After removal of Crested Tits Parus cristatus (the dominant species in mixed flocks), Willow Tits P. montanus did not decrease their foraging heights as expected but remained in the most exposed parts of young pines. In contrast, after removal of Willow Tits, Crested Tits increased their foraging height from the sheltered lower canopy to sites previously occupied by Willow Tits. When flock size was reduced, the birds maintained the same high levels of vigilance without concealing themselves in dense vegetation. I suggest that flock members may benefit from foraging in sites that afford good anti-predator vigilance.     

Comparison of anti-inflammatory compounds in the carrageenan induced paw edema model and the reversed passive Arthus model utilizing the same animal

In order to better define antiinflammatory activity in new agents, a test was devised utilizing both carrageenan induced paw edema and the reversed passive Arthus reaction in the same animal. The model of carrageenan induced rat paw edema is a standard laboratory assay used to predict classical "aspirin-like" antiinflammatory molecules. The reversed passive cutaneous Arthus reaction involves precipitating antigen-antibody complexes, complement and infiltrating polymorphonuclear leukocytes (PMN's) and can be used to identify agents that affect one or more of these factors specifically. Antiinflammatory compounds were given orally one hour prior to the administration of carrageenan and goat anti-rat serum. Comparisons were made between several non-steroidal compounds and the steroid hydrocortisone. All of the compounds tested gave good carrageenan activity, but only hydrocortisone produced significant Arthus lesion inhibition in this assay.