1H NMR investigation of distal mutant deoxy myoglobins. Interpretation of proximal His contact shifts in terms of a localized distal water molecule

1H NMR spectra of a series of distal point mutants of human and sperm whale deoxy myoglobin have been recorded and their spectral parameters compared with those of wild type. The substitutions investigated include His64(E7)-->Gly, Ala, Val, Leu, Ile, and Gln and Val68(E11)-->Ala, Ile. The three resonances from the proximal His F8 imidazole ring, as well as two heme methyl signals, are identified in each of the proteins. Significant perturbations of the NMR spectra of mutant deoxy myoglobins (Mbs) occurred only upon substitution of His64(E7) by any non-polar residue, with only minor variation in parameters throughout the range of side chains. These spectral changes are attributed to the elimination of a non-coordinated ordered water molecule in the distal pocket found hydrogen bonded to His64(E7) in crystals of wild-type deoxy Mb, but abolished in the His64(E7)-->Leu mutant deoxy Mb crystal (Quillin, M. L., Arduini, R. M., Olson, J. S., and Philips, G. N., Jr. (1993) J. Mol. Biol. 234, 140-155). The observed spectral changes, increased His F8 ring spin delocalization, and decreased heme in-plane asymmetry, can be directly attributed to the weakening of the effective axial field and a decrease in the asymmetry in the rhombic ligand field resulting from removal of the water molecule. The hyperfine shift patterns for the mutants His64(E7)-->Gln and Val68(E11)-->Ile deoxy Mbs are minimally perturbed from that of wild type and are interpreted to reflect a conserved distal water-binding site. In the point mutant Val68(E11)-->Ala, the decreased covalency to the axial His F8 is interpreted as reflecting a conserved distal water molecule that can interact more strongly with the iron due to the reduced steric bulk of the E11 side chain. The differential 1H NMR spectral parameters for the His F8 resonances in the two subunits of T state deoxy Hb A are shown to be similarly consistent with the known occupation of the distal water binding site in the alpha-, but not beta-subunit.     

Defibrillation efficacy with endocardial electrodes is influenced by reductions in cardiac preload

Several studies were undertaken to develop three-dimensional (3-D) cell culture models that allow conditions closer to the in vivo situation. To this end, alginate gels were tested as a 3-D cell culture model that might be useful in the study of the effects of UVA on human dermal fibroblasts. Cell culture in alginate gels and the irradiation conditions were optimized. Results showed that optimized cultures in alginate gels experienced considerable cell death on UVA irradiation compared to the classical monolayer cell culture. Viability tests (cell counting and neutral red assay) were performed to show that only UVA-irradiated alginate gels were responsible for this cytotoxicity. The implication of oxygen species in the phototoxicity induced by ultraviolet light has already been described; for this reason we investigated whether oxygen species were involved in the cytotoxicity induced by alginate upon UVA irradiation. It appeared that superoxide anion is not implicated.     

The quality of friendships between children with and without learning problems

The nature and quality of preadolescent friendships between children with and without learning problems due to mental retardation or mild cognitive difficulties were investigated. Based on an assessment of the reciprocal relationship status of 373 children, including 54 with learning problems, 33 friend and 32 acquaintance dyads were identified. Of these dyads, half included a child with learning problems and half consisted of 2 children without learning problems. The dyads were observed performing a play task. Unlike friendships between children without disabilities, friendships between children with and without learning problems were marked by limited collaboration and shared decision-making, a low level of cooperative play and shared laughter, and an asymmetrical, hierarchical division of roles. The importance of advancing beyond the study of the social acceptance of children with learning problems to study the qualitative aspects of their friendships was discussed.     

241Am removal by DTPA vs. occurrence of skeletal malignancy

PURPOSE: The distribution of hyaluronan (HA) and the cellular response after photokeratitis induced by different ultraviolet (UV) wavelengths in the rabbit cornea was examined to help understand the mechanism of corneal injury and repair after UV damage. HA is a high molecular weight disaccharide polymer capable of binding considerable amounts of water. It is not normally found in the rabbit corneal stroma. The production of HA represents a generalized corneal response to injury. METHODS: Twenty-four albino rabbit corneas were exposed to 270, 290, and 310 nm of UV radiant energy in 8-nm full wavebands in doses producing biomicroscopically significant keratitis (three corneal thresholds for keratitis (Hc): 0.016 J/cm2 for 270 nm, 0.04 J/cm2 for 290 nm, and 0.14 J/cm2 for 310 nm) and in subkeratitis doses (0.7 Hc: 0.004 J/cm2 for 270 nm, 0.008 J/cm2 for 290 nm, and 0.03 J/cm2 for 310 nm). The rabbits exposed to 270 and 290 nm of UV radiation were sacrificed 3 days after exposure. The rabbits exposed to 310 nm of UV radiation were sacrificed 3, 7, and 14 days after exposure, respectively. The corneal tissue specimens were double stained and examined morphologically and histochemically for HA by light microscopy. RESULTS: Evaluation of corneas exposed to 270 and 290 nm of UV radiant energy in both subkeratitis and keratitis doses and those corneas exposed to 310 nm of radiant energy in subkeratitis dose showed neither stromal changes nor production of HA by corneal cells. Corneas exposed to 310 nm of UV radiant energy in keratitis dose at 3 days after exposure showed disappearance of keratocytes in entire thickness of central cornea. Cells bordering this damaged area were staining for HA. By 7 days after exposure almost the whole damaged area, except one fourth of anterior stroma, was repopulated by new keratocytes staining positive for HA. The corneal structures became normal and HA almost completely disappeared 14 days after exposure. CONCLUSIONS: A keratitis dose of 310 nm of UV light irradiation is needed to cause keratocyte damage. A keratitis dose of the shorter wavelengths does not cause keratocyte cell damage at the light microscopic level. The keratocyte production of HA appears to be a sign of cell readiness to repopulate the damaged stroma devoid of keratocytes.