Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus gamma-hemolysin and leukocidin, and their specific binding to the hlg2 and the LukS components of the toxins.

Staphylococcal gamma-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of gamma-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of gamma-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of gamma-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.     

The effects of food availability and distance to protective cover on the winter foraging behaviour of tits (Aves: Parus)

To maximize fitness, many animals must trade off their need to forage efficiently against their need to avoid predators. We studied such a trade-off in four species of tits (Paridae) in a forest near Oxford, UK. During winter, tits form flocks which increase feeding efficiency and reduce predation risk. These flocks feed extensively on beech (Fagus sylvatica) seeds, the abundance of which may be critical for winter survival. Because these seeds drop to the ground, where birds are exposed to sparrowhawk (Accipiter nisus) attack, tits need to trade off their need to find seeds against the proximity to protective cover, provided by dense clusters of hawthorn (Crataegus spp.). The quality of the beech crop differs markedly between trees and years. During a year of abundant beechmast, most tits searched for seeds close to protective cover. This 'safety-first' strategy precluded visits to superabundant food patches if they were too far from protective cover. Among beech trees near to cover, tits tended to prefer those with high seed density. Tits benefited from foraging under trees with high seed density because this correlated significantly with seed mass per square metre and because mean search times decreased with increasing seed density. Finally, we show experimentally that great tits, Parus major, can discriminate between edible (viable) and inedible (empty) seeds.     

Syntheses of (Z)-and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid and their inactivation of gamma-aminobutyric acid aminotransferase.

(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid (4 and 5, respectively) were synthesized and investigated as potential mechanism-based inactivators of gamma-aminobutyric acid aminotransferase (GABA-AT) in a continuing effort to map the active site of this enzyme. The core alpha-trifluoromethyl-alpha,beta-unsaturated ester moiety was prepared via a Reformatsky/reductive elimination coupling of the key intermediates tert-butyl 2,2-dichloro-3,3,3-trifluoropropionate and N,N-bis(tert-butoxy-carbonyl)glycinal. Both 4 and 5 inhibited GABA-AT in a time-dependent manner, but displayed non-pseudo-first-order inactivation kinetics; initially, the inactivation rate increased with time. Further investigation demonstrated that the actual inactivator is generated enzymatically from 4 or 5. This inactivating species is released from the active site prior to inactivation, and as a result, 4 and 5 cannot be defined as mechanism-based inactivators. Furthermore, 4 and 5 are alternate substrates for GABA-AT, transaminated by the enzyme with Km values of 0.74 and 20.5 mM, respectively. Transamination occurs approximately 276 and 305 times per inactivation event for 4 and 5, respectively. The enzyme also catalyzes the elimination of the fluoride ion from 4 and 5. A mechanism to account for these observations is proposed.     

Phorbol ester facilitates apoptosis in murine fibroblasts pretreated by mild ultraviolet radiation.

Although phorbol 12-myristate 13-acetate (PMA) inhibits apoptosis and promotes the growth of some types of cells, it induces apoptosis in other cells. We evaluated the apoptotic effects of PMA on murine fibroblasts (L-929) that had been exposed to ultraviolet-B (UV-B) radiation at 312 nm, which promotes tumor cell growth. Exposure to PMA alone did not induce Fas, Fas-L, or apoptosis. Cells exposed to mild UV-B irradiation (80 J/m(2)) alone exhibited a slight expression of Fas and Fas-L 36 to 48 h after the exposure, and exhibited apoptosis as evidenced by DNA fragmentation 72 h after exposure. The addition of PMA (0.8 x 10(-5) to 3.2 x 10(-5) M) to the medium 24 h after the UV-B exposure markedly and dose-dependently enhanced these cell responses. Confluent untreated cells, cells cocultured with PMA, and cells cocultured with PMA for 24 h after the UV-B exposure consistently expressed mRNAs for wild-type p53, bcl-2, and ICE. Expression of c-myc mRNA was initially observed, but became undetectable in the cells cocultured for 24 h with a high concentration of PMA (3.2 x 10(-5) M) following UV-B exposure. Such cells subsequently exhibited the maximal apoptotic response. We conclude that mild exposure to UV-B altered murine fibroblast cells in such a way as to facilitate their death by apoptosis upon addition of PMA.     

Vertical distribution of zooplankton in the frontal zone of the Gulf Stream and Labrador Current

Zooplankton data collected during September 1995 in the NorthWest Atlantic at 4139'N, 4958'W (the location of the siteof the ‘Titanic’ wreck) were analysed. The regioninvestigated was characterized by a very sharp frontal zonebetween the Gulf Stream and the main stream of the LabradorCurrent. The total plankton biomass in the water column wasvery high. The macroplankton biomass values below the 600 mlayer were significantly higher as compared with the similarvalues measured before in other productive boreal regions ofthe Atlantic and Pacific oceans. A lot of dead mesoplanktonanimals occurred in the deep layers. The reason was that thecold-water mesoplankton advected by the Labrador Current diedoff intensively within the deep layers of the frontal zone andwere used as a food resource by the macroplankton carnivoresand scavengers that were very abundant there.     

A finite element based method to determine the properties of planar soft tissue.

A finite element based method to determine the incremental elastic material properties of planar membranes was developed and evaluated. The method is applicable to tissues that exhibit inhomogeneity, geometric and material nonlinearity, and anisotropy. Markers are placed on the tissue to form a four-node quadrilateral element. The specimen is loaded to an initial reference state, then three incremental loading sets are applied and the nodal displacements recorded. One of these loadings must include shear. These data are used to solve an over-determined system of equations for the tangent stiffness matrix. The method was first verified using analytical data. Next, data obtained from a latex rubber sheet were used to evaluate experimental procedures. Finally, experiments conducted on preconditioned rat skin revealed nonlinear orthotropic behavior. The vector norm comparing the applied and calculated nodal force vectors was used to evaluate the accuracy of the solutions.     

An implantable transducer for measuring tension in an anterior cruciate ligament graft.

The goal of this study was to develop a new implantable transducer for measuring anterior cruciate ligament (ACL) graft tension postoperatively in patients who have undergone ACL reconstructive surgery. A unique approach was taken of integrating the transducer into a femoral fixation device. To devise a practical in vivo calibration protocol for the fixation device transducer (FDT), several hypotheses were investigated: (1) The use of a cable versus the actual graft as the means for applying load to the FDT during calibration has no significant effect on the accuracy of the FDT tension measurements; (2) the number of flexion angles at which the device is calibrated has no significant effect on the accuracy of the FDT measurements; (3) the friction between the graft and femoral tunnel has no significant effect on measurement accuracy. To provide data for testing these hypotheses, the FDT was first calibrated with both a cable and a graft over the full range of flexion. Then graft tension was measured simultaneously with both the FDT on the femoral side and load cells, which were connected to the graft on the tibial side, as five cadaver knees were loaded externally. Measurements were made with both standard and overdrilled tunnels. The error in the FDT tension measurements was the difference between the graft tension measured by the FDT and the load cells. Results of the statistical analyses showed that neither the means of applying the calibration load, the number of flexion angles used for calibration, nor the tunnel size had a significant effect on the accuracy of the FDT. Thus a cable may be used instead of the graft to transmit loads to the FDT during calibration, thus simplifying the procedure. Accurate calibration requires data from just three flexion angles of 0, 45, and 90 deg and a curve fit to obtain a calibration curve over a continuous range of flexion within the limits of this angle group. Since friction did not adversely affect the measurement accuracy of the FDT, the femoral tunnel can be drilled to match the diameter of the graft and does not need to be overdrilled. Following these procedures, the error in measuring graft tension with the FDT averages less than 10 percent relative to a full-scale load of 257 N.