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.     

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.     

Anesthetic effects on pulmonary allergic responses in rats: changes in sensitivity to serotonin.

Subsequent to observations that pulmonary responses to antigen challenge are of different magnitudes in sensitized rats that are anesthetized with different drugs, we conducted studies to test whether the alterations in responses were due to changes in airway responsiveness to cholinergic or serotonergic challenge, opioid-receptor mediated events, or changes in mast cell mediator release. Immunoglobulin E-sensitized rats anesthetized with ketamine/urethan had larger changes in lung resistance and plasma histamine after pulmonary antigen challenge compared with rats anesthetized with fentanyl-droperidol. Blockade of opioid receptors with naloxone did not affect the responses. In unsensitized rats, airway responses to aerosolized methacholine were similar for the two anesthetics, indicating unchanged smooth muscle responsiveness; however, airway responses to intravenous serotonin were enhanced by ketamine and ablated by droperidol. We conclude that ketamine- and droperidol-induced alterations of pulmonary allergic responses are due to changes in sensitivity to serotonin and in mast cell mediator release. We speculate that mast cell mediator release may be modulated by a serotonin receptor-linked mechanism.     

Bone density parathyroid hormone and 25-hydroxyvitamin D concentrations in middle aged women.

OBJECTIVE--To examine the relation between bone density and indices of calcium metabolism including parathyroid hormone and 25-hydroxyvitamin D concentrations in middle aged women. DESIGN--A cross sectional study. SETTING AND SUBJECTS--138 women volunteers aged 45-65 with no known osteoporosis and unselected for disease status recruited for a dietary assessment study from the community using general practice registers. Volunteer rate was 20%. MAIN OUTCOME MEASURE--Bone mineral density measured with dual energy x ray absorptiometry. RESULTS--Bone density at the lumbar spine and neck and trochanteric regions of the femur was inversely related to serum intact parathyroid hormone concentrations and positively related to serum 25-hydroxyvitamin D concentrations. These associations were independent of possible confounding factors, including age, body mass index, cigarette smoking habit, menopausal status, and use of diuretics and postmenopausal hormone replacement therapy. These associations were apparent throughout the whole distribution of bone density and 25-hydroxyvitamin D and parathyroid hormone concentrations within the normal range, suggesting a physiological relation. CONCLUSIONS--The findings are consistent with the hypothesis that parathyroid hormone and 25-hydroxyvitamin D concentrations influence bone density in middle aged women. Findings from this study together with other work suggest that the role of vitamin D in osteoporosis should not be neglected. The associations with parathyroid hormone also indicate plausible biological mechanisms. The roughly 5-10% difference in bone density between top and bottom tertiles of serum 25-hydroxyvitamin D concentrations, though not large in magnitude, may have considerable public health implications in terms of prevention of osteoporosis and its sequelae, fractures.     

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.