Computation-guided backbone grafting of a discontinuous motif onto a protein scaffold

The manipulation of protein backbone structure to control interaction and function is a challenge for protein engineering. We integrated computational design with experimental selection for grafting the backbone and side chains of a two-segment HIV gp120 epitope, targeted by the cross-neutralizing antibody b12, onto an unrelated scaffold protein. The final scaffolds bound b12 with high specificity and with affinity similar to that of gp120, and crystallographic analysis of a scaffold bound to b12 revealed high structural mimicry of the gp120-b12 complex structure. The method can be generalized to design other functional proteins through backbone grafting.     

Infestation in the dog by the paralysis tick, Ixodes holocyclus — 4. Cardiovascular effects

To determine the extent and significance of changes in heart rate and rhythm noticed previously in dogs paralysed with Ixodes holocyclus, two studies were undertaken. In one the electrocardiogram was recorded at stages throughout the disease and the traces analysed for changes, while in the second a detailed study of the effect of Ixodes holocyclus on the cardiovascular system was undertaken. The electrocardiographic changes were extremely variable between stages and between dogs. Generally, if a dysrhythmia occurred in stages 1, 2 or 3 it tended to be sinus tachycardia, ventricular tachycardia or sinus arrest. In stage 4 sinus arrest, sinus bradycardia, or sinus or ventricular tachycardia were the prominent dysrhythmias, whereas in stage 5 sinus bradycardia predominated. Cardiovascular measurements indicated an increase in peripheral vascular resistance leading to a significant elevation in mean arterial pressure at all stages of the disease. Cardiac output was decreased significantly only at stage 2, although it was below the control measurements at all stages. Pulmonary arterial pressure was significantly elevated at stages 2, 3 and 4 due most probably to an increase in pulmonary vascular resistance. Myocardial contractility was not significantly changed throughout the disease. The changes observed in the electrocardiogram and the cardiovascular system in stages 1, 2 and 3 are unlikely to be due to hypoxia and could represent dysfunction of the autonomic nervous system. During stages 4 and 5 oxygen levels were below normal and the bradycardia seen terminally is almost certainly due to hypoxaemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooled Storage of Canine Semen: in vitro Effects of Different Concentrations of an Antibiotic Combination on Growth of Mollicutes

The aim of this study was to examine effects of an antibiotic combination at different concentrations on growth of mycoplasma and ureaplasma during cooled storage of canine semen (n = 20). Semen aliquots were diluted with Tris–citric acid–fructose–egg yolk extender containing either 1.0 g/l streptomycin and 0.6 g/l benzylpenicillin (control) or a combination of gentamycin, tylosin, lincomycin and spectinomycin (GTLS‐1: 0.25, 0.05, 0.15 and 0.3; GTLS‐2: 0.5, 0.1, 0.3 and 0.6; GTLS‐3: 1.0, 0.2, 0.6 and 1.2 g/l). Samples were assessed for motility and membrane integrity by computer‐assisted sperm analysis immediately after dilution and at 24, 48 and 72 h of cooled storage. Morphologically, normal spermatozoa were determined, and bacterial culture was performed at 24 and 72 h. Mycoplasma spp. were detected in 14 of 20 ejaculates (70%) with severe growth in 12 samples. A reduction but not total elimination of mycoplasma growth occurred in all GTLS extenders with the most pronounced reduction in group GTLS‐3 (control vs GTLS‐1 and GTLS‐2 p < 0.05, control vs GTLS‐3 p < 0.001). Ureaplasmas were detected in four ejaculates, and growth was reduced to the same extent in GTLS and control extender. Progressive motility in all groups, total motility in groups GTLS 1–3 and percentage of membrane‐intact spermatozoa in groups GTLS 2 and 3 decreased slightly (p < 0.05) over time. In conclusion, dilution of canine semen with GTLS extender has no major detrimental effects on spermatozoa during cooled storage. It reduced the growth but did not totally eliminate mycoplasmas and ureaplasmas from cooled‐stored dog semen.